National Cancer Institute 

CARCIIMOGENESIS 

Technical Report Series 

No. 44 

1978 

IN VITRO CARCINOGENESIS 

GUIDE TO THE LITERATURE, RECENT ADVANCES 

AND LABORATORY PROCEDURES 

NCI-CG-TR-44 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 

IN VITRO CARCINOGENESIS 

GUIDE TO THE LITERATURE, RECENT ADVANCES 

AND LABORATORY PROCEDURES 

Edited by: 

UMBERTO SAFFIOTTI, M.D. 

HERMAN AUTRUP, Ph.D. 

Experimental Pathology Branch, 

Carcinogenesis Program, Division of Cancer Cause and Prevention 

National Cancer Institute, NIH, Bethesda, MD 20014 

Based on presentations made at the Seminar and Workshop on 

"In Vitro Carcinogenesis", held at the Given Institute of Pathobiology, 

Aspen, Colorado, July 18-23, 1976, U. Saffiotti and D.W. King, Chairmen 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service National Institutes of Health 

DHEW Publication No.(NIH) 78-844 

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 

ERRAT A SHEE T
 

IN VITRO CARCINOGENESIS
 

Guide to the Literature, Recent Advances and Laboratory Procedures
 

DHEW Publication No. (NIH) 78-844
 

Index, page xiv, 5.7 and 5.8
 

change Andrew R. Peterson to Bruce C. Casto
 

Page 314 and 315
 

change ANDREW R. PETERSON
 

Los Angeles County—University of Southern California Comprehensive
 

Cancer Center, Los Angeles, CA
 

to BRUCE C. CASTO
 

Biolabs, Inc.
 Northbrook, IL
 

CONTRIBUTORS TO THIS VOLUME

 

Herman Autrup, Ph.D.
 Experimental Pathology Branch
 

Carcinogenesis, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

J.Carl Barrett, Ph.D.
 Division of Biophysics
 

School of Hygiene and Public Health
 

Johns Hopkins University
 

615 North Wolfe Street
 

Baltimore, MD 21205
 

Lucy A. Barrett, Ph.D.
 Department of Pathology
 

University of Maryland
 

School of Medicine
 

Baltimore, MD 21201
 

William F. Benedict, M.D.
 Division of Hematology-Oncology
 

Children's Hospital of Los Angeles
 

4650 Sunset Blvd.
 

Los Angeles, CA 90027
 

John Bertram, Ph.D.
 Department of Experimental
 

Therapeutics
 

Roswell Park Memorial Institute
 

666 Elm Street
 

Buffalo, NY 14263
 

Noel Bouck, Ph.D.
 Department of Microbiology &
 

Immunology
 

School of Basic Medical Sciences
 

University of Illinois at the
 

Medical Center
 

835 South Wolcott Street
 

Chicago, IL 60612
 

Bruce C. Casto, Sc.D.
 BioLabs, Inc.
 2910 MacArthur Blvd.
 

Northbrook, IL 60062
 

Sara M. Clarke, M.A.
 Department of Pathology
 

University of Colorado Medical
 

Center
 

4200 E. 9th Street
 

Denver, CO 80220
 

iii 
Nancy H. Colburn, Ph.D.
 Experimental Pathology Branch
 

Carcinogenesis, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Giampiero di Mayorca, M.D.
 Department of Microbiology &
 

Immunology
 

School of Basic Medical Sciences
 

University of Illinois at the
 

Medical Center
 

835 South Wolcott Street
 

Chicago, IL 60612
 

Charles H. Evans, M.D., Ph.D.
 Biology Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute
 

Bethesda, MD 20014
 

Robert M. Evans
 Department of Pathology
 

University of Colorado Medical
 

Center
 

4200 E. 9th Street
 

Denver, CO 80220
 

Elliott Fineman, M.S.
 Litton-Bionetics, Inc.
 5516 Nicholson Lane
 

Kensington, MD 20795
 

Louis M. Fink, M.D.
 Department of Pathology
 

University of Colorado Medical
 

Center
 

4200 E. 9th Avenue
 

Denver, CO 80220
 

Aaron E. Freeman, Ph.D.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Present address;
 

Torrey Pines Research Center
 

Microbiological Associates
 

2945 Science Park Road
 

La Jolla, CA 92037
 

Rosalind D. Friedman, M.S.
 Lung Cancer Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

iv 
Lisa Gernand, B.S.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Donald A. Glaser, Ph.D.
 Department of Molecular Biology
 

Wendell M. Stanley Hall
 

University of California, Berkeley
 

Berkeley, CA 94720
 

Curtis C. Harris, M.D.
 Experimental Pathology Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Charles Heidelberger, Ph.D.
 Los Angeles County-University of
 

Southern California
 

Comprehensive Cancer Center
 

1303 North Mission Road
 

Los Angeles, CA 90033
 

Howard J. Igel, M.D.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Takeo Kakunaga, Ph.D.
 Chemistry Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Melvin L. Kropko, B.S., M.T.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Robert S. Lake, Ph.D.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Bernhard P. Lane, M.D.
 Department of Pathology
 

Health Sciences Center
 

State University of New York at
 

Stony Brook
 

Stony Brook, NY 11794
 

Stephen Lesko, Ph.D.
 Division of Biophysics
 

School of Hygiene and Public
 

Health
 

Johns Hopkins University
 

615 North Wolfe Street
 

Baltimore, MD 21205
 

V 
John B. Little, M.D.
 Department of Physiology
 

Harvard University
 

School of Public Health
 

665 Huntington Avenue
 

Boston, MA 02115
 

Peter N. Magee, M.B., M.R.C.P.,
 Pels Research Institute
 

F.R.C. Path.
 Temple University
 

School of Medicine
 

Philadelphia, PA 19122
 

Veronica M. Maher, Ph.D
 Carcinogenesis Laboratory
 

College of Osteopathic Medicine
 

Michigan State University
 

East Lansing, MI 48824
 

James M. Malone, M.S.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Ann C. Marchok, Ph.D.
 Biology Division
 

Oak Ridge National Laboratory
 

Oak Ridge, TN 37830
 

Corey Mark, B.S.
 Division of Hematology-Oncology
 

Children's Hospital of Los Angeles
 

Los Angeles, CA 90027
 

J. Justin McCormick, Ph.D.
 Carcinogenesis Laboratory
 

College of Osteopathic Medicine
 

Michigan State University
 

East Lansing, MI 48824
 

Elizabeth M. McDowell, DVM, Ph.D.
 Department of Pathology
 

University of Maryland
 

School of Medicine
 

Baltimore, MD 21201
 

Mortimer L. Mendelsohn, M.D., Ph.D.
 Biomedical and Environmental
 

Research Program
 

Lawrence Livermore Laboratory
 

Livermore, CA 94550
 

Sukdeb Mondal, M.D., Ph.D.
 Los Angeles County-University of
 

Southern California
 

Comprehensive Cancer Center
 

1303 North Mission Road
 

Los Angeles, CA 90033
 

vi 
Robert Moyzis, Ph.D.
 Division of Biophysics
 

School of Hygiene and Public
 

Health
 

Johns Hopkins University
 

614 North Wolfe Street
 

Baltimore, MD 21205
 

Andrew R. Peterson, Ph.D.
 Los Angeles County-University of
 

Southern California
 

Comprehensive Cancer Center
 

1303 North Mission Road
 

Los Angeles, CA 90033
 

Mary Rose Pezzutti, B.S., M.T.
 Department of Pathology
 

Children's Hospital of Akron
 

Akron, OH 44308
 

Roman J. Pienta, Ph.D.
 Chemical Carcinogenesis Program
 

National Cancer Institute's
 

Frederick Cancer Research Center
 

Frederick, MD 21701
 

David H. Reese, Ph.D.
 Lung Cancer Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Umberto Saffiotti, M.D.
 Experimental Pathology Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Paul W. Schafer, M.D.
 Department of Surgery
 

Veterans Administration Hospital
 

50 Irving Street, NE
 

Washington, DC 20422
 

Gary D. Stoner, Ph.D.
 Experimental Pathology Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

Margaret Terzaghi, Ph.D.
 Biology Division
 

Oak Ridge National Laboratory
 

Oak Ridge, TN 37830
 

vii 
Benjamin F. Trump, M.D.
 Department of Pathology
 

University of Maryland
 

School of Medicine
 

Baltimore, MD 21201
 

Paul O.P. Ts'o, Ph.D.
 Division of Biophysics
 

Johns Hopkins University
 

School of Hygiene and Public
 

Health
 

615 North Wolfe Street
 

Baltimore, MD 21205
 

Marion Valeric, M.D.
 Litton-Bionetics, Inc.
 5516 Nicholson Lane
 

Kensington, MD 20795
 

Stuart H. Yuspa, M.D.
 Experimental Pathology Branch
 

Carcinogenesis Program, DCCP
 

National Cancer Institute, NIH
 

Bethesda, MD 20014
 

viii
 
PREFACE
 

In the Fall 1974, plans for a "Workshop on In Vitro Chemical
 

Carcinogenesis" were initiated at the suggestion of Drs. Umberto
 

Saffiotti and I. Bernard Weinstein and developed together with Dr.
 Donald W. King as part of the conferences held at the Given Institute
 

of Pathobiology, University of Colorado, Aspen. It was felt that this
 

was an important research area on which to hold an initial small
 

workshop that would bring together most of the leading investigators
 

in the field, at a time when several laboratories were attempting to
 

establish criteria for in vitro neoplastic cell transformation
 

following chemical treatment.
 

The workshop, chaired by Drs. Saffiotti and Weinstein, took place
 

from July 27 to August 1, 1975, and it included 33 participants. It
 

was supported by the National Cancer Institute and consisted of
 

lectures, laboratory demonstrations, and laboratory work actively
 

involving all participants. The workshop was primarily devoted to
 

reviewing laboratory methods, transformation criteria, and markers.
 The unique environment and intellectual atmosphere of the Given
 

Institute at Aspen were particularly stimulating and the workshop
 

succeeded in fostering friendly, uninhibited, and constructive
 

discussion and cooperation among investigators from different
 

laboratories. The participants found it a unique opportunity to
 

review each other's experimental material and to resolve controversial
 

questions of interpretation directly on site in the laboratory.
 

The participants in the 1975 workshop expressed their desire to
 

meet again in 1976 for a conference that would allow the findings of
 

the leading research groups to be demonstrated to a larger audience.
 

On this basis, a "Seminar and Workshop on In Vitro
 

Carcinogenesis" was planned and organized by Drs. Umberto Saffiotti
 

and Donald W. King with advice from several participants, and it was
 

held at the Given Institute of Pathobiology in Aspen from July 18 to
 

23, 1976. Drs. U. Saffiotti and D. W. King served as chairmen of the
 

conference and Dr. Paul 0. P. Ts'o as chairman of the laboratory
 

workshop. This seminar was part of the series held at the Given
 

Institute entitled "Advances in Cancer Biology" and supported by a
 

grant (No. 5R13CA15961) from the National Cancer Institute.
 

The present publication is based on the presentations made at
 

this conference. The members of the seminar's faculty agreed that it
 

would not be desirable to publish the detailed papers which were
 

presented orally, since many involved discussion of previously
 

published results. Several good reviews were also recently published
 

by some of the participants. A need was recognized, however, to
 

ix 
collect in a single publication the extensive literature that has
 

developed in the area of neoplastic transformation of cells in culture
 

by chemical and physical agents. The editors suggested and the
 

faculty agreed that this publication be devoted to an extensive
 

bibliography on the subject, with brief papers serving as a guide to
 

the literature on different topics. Selected papers give more


extensive reports of previously unpublished new advances. In

addition, a section is devoted to the publication of detailed

laboratory procedures which are not available in the current

literature. 

The editors have arranged the sequences of papers around main
 

topics and edited their format and bibliography for consistency. They


wish to express their appreciation to the authors for their


cooperation; to Dr. D. W. King, Director of the Given Institute of
 

Pathobiology, for his sponsorship of the Conference; and to both Dr.
 D. W. King and Dr. Cecilia Fenoglio, Department of Pathology, Columbia
 

University, for their editorial advice.
 

The National Cancer Institute Carcinogenesis Technical Report
 

Series, in which this volume appears, was established early in 1976,
 at the initiative of Drs. U. Saffiotti and N. P. Page, then


respectively responsible for the NCI Carcinogenesis Program and for
 

its Carcinogen Bioassays and Program Resources Branch, as a means to
 

publish documentation methods, procedures, and findings in the field
 

of Carcinogenesis bioassays and research, particularly when format
 

limitations make it difficult to report detailed data through the
 

conventional scientific journals.
 

It is hoped that this volume will be a useful contribution to the
 

rapidly developing field of Carcinogenesis studies by in vitro culture


methods.
 

Of the participants in the Symposium, four were unable to con­ tribute directly to this volume: J.A. DiPaolo, M.W. Lieberman, F.H.
 Ruddle, and I.E. Weinstein.
 

The Editors
 

x
 
INDEX

 

Page
 

1. INTRODUCTION 

1.1	 IN VITRO CARCINOGENESIS METHODS IN RELATION TO THE
 

DEVELOPMENT OF CARCINOGENESIS RESEARCH
 

Umberto Saffiotti 1
 

2.	 CARCINOGENESIS STUDIES IN CELL CULTURE SYSTEMS
 

2.1	 REVIEWS ON IN VITRO CARCINOGENESIS AND SELECTED GENERAL
 

REFERENCES 21
 

2.1.1	 A Bibliography 21
 

2.2	 MOUSE FIBROBLASTS 25
 

2.2.1	 A Bibliography 25
 

2.2.2	 Promotion of Oncogenesis in Cell Culture by
 

Phorbolesters
 

S. Mondal	 31
 

2.2.3	 Radiation Transformation In Vitro
 

John B. Little . . 38
 

2.2.4	 Factors Modifying Transformation Frequency
 

in the C3H/10T1/2 in CL8 Cell Line
 

John S. Bertram 43
 

2.3	 MOUSE EPITHELIAL CELLS 47
 

2.3-1 Mouse Epidermal Cell Cultures as an In Vitro
 

Model for the Study of Chemical Carcinogenesis
 

Stuart H. Yuspa 47
 

2.3.2	 Chemical Transformation of Epidermal Cell
 

Cultures
 

Nancy H. Colburn 57
 

2.4	 RAT CELLS 65
 

2.4.1	 A Bibliography 65
 

xi
 
Page
 

2.5 HAMSTER CELLS	 69
 

2.5.1	 The Use of Early Passage Hamster Embryo Cells
 

for the Identification of Chemical Carcinogens
 

Roman J. Pienta 69
 

2.5.2	 Somatic Mutation as the Basis for In Vitro Trans­ formation of BHK Cells by Chemical Carcinogens
 

Noel Bouck and Giampiero di Mayorca 76
 

2.6 GUINEA PIG CELLS	 82
 

2.6.1	 Neoplastic Transformation by Chemicals of
 

Guinea Pig Fetal Cells in Culture
 

Charles H. Evans 82
 

2.7 HUMAN CELLS	 84
 

2.7.1	 Chemical Transformation of Human and Rodent
 

Cells
 

Takeo Kakunaga 84
 

2.7.2	 Heteroploid Conversio on f Human Skin Cells by
 

Methylcholanthrene: Possible Role of Hydrocarbon
 

Metabolizing Epithelial Cells
 

Aaron E. Freeman, Robert S. Lake, Howard J. Igel,
 Lisa Gernand, Mary Rose Pezzutti, James M. Malone
 

Corey Mark and William F. Benedict 100
 

3.	 CARCINOGENESIS STUDIES IN ORGAN CULTURE SYSTEMS
 

3.1 ANIMAL TISSUES	 123
 

3.1.1	 In Vitro Carcinogenesis in Tracheal Organ
 

Culture
 

Bernard P. Lane 123
 

3.1.2	 In Vivo-In Vitro Studies with Respiratory Tract
 

Epithelium Exposed to Carcinogen
 

Ann C. Marchok and Margaret Terzaghi 132
 

3.1.3	 Induction and Suppression of Dysplastic Growth in
 

Organ-Cultured Urinary Bladder Epithelium
 

David H. Reese and Rosalind D. Friedman 139
 

xii
 
Page 

3.2	 HUMAN TISSUES 144

 

3.2.1 Explant Culture and Xenotransplantation of Human 

Bronchi 

Curtis C. Harris, Herman Autrup, Gary D. Stoner, 

Marion Valeric, Elliott Fineman, Paul W. Schafer, 

Lucy A. Barrett, Elizabeth M. McDowell, 

and Benjamin F. Trump 144

 

4. MECHANISMS, METABOLISM, CO-FACTORS AND MARKERS 

4.1 Metabolic Activation of Chemical Carcinogens 

Peter N. Magee 153

 

4.2 Certain Aspects of Chemical Carcinogenesis 

Paul O.P. Ts'o, J. Carl Barrett, Stephen Lesko, 

and Robert Moyzis 179

 

4.3 A Strategem for Experiments on Oncogenesis In Vitro 

Andrew R. Peterson 205

 

4.4 The Use of Diploid Human Fibroblasts to Measure the 

Effect of DMA Repair on the Cytotoxic and Mutagenic 

Action of Carcinogens

 

Veronica M. Maher and J. Justin McCormick 212

 

4.5 Enhancement of DNA Viral Transformation by Chemical 

Carcinogens and Mutagens 

Bruce C. Casto 226

 

4.6 Morphological Transformation, Chromosonal Changes 

and Increases in Plasminogen Activator in 

In Vitro Carcinogenesis 

William F. Benedict 231

 

4.7 Cell Surface Antigens as Markers for the Transformed 

Phenotype 

Louis M. Fink, Robert M. Evans, J. Carl Barrett 

and Sara M. Clarke 238

 

4.8 Biophysical Cytological Approaches to In Vitro 

Carcinogenesis 

Mortimer L. Mendelsohn 255 

xiii 
Page 

4.9 Automatic Techniques In Cell Biology: Possible 

Applications to In Vitro Carcinogenesis 

Donald A. Glaser 267 

5. WORKSHOP ON LABORATORY PROCEDURES 

5.1 The use of Early Passage Hamster Embryo Cells for 

the identification of Chemical Carcinogens 

Roman J. Pienta 273 

5.2 Quantitative Focus Assay for Transformation of Syrian 

Hamster Embryo Cells by Chemical Carcinogens 

Bruce C. Casto 278 

5.3 In Vitro Culture of Diploid Human Skin Epithelial 

Cells for Chemical Transformation Studies 

Robert S. Lake, Aaron E. Freeman, Howard J. Igel, Mary Rose Pezzutti, and Melvin L. Kropko 283 

5.4 Procedures and Protocols for Using Diploid Human 

Fibroblasts to Measure the Effect of DNA Repair on the 

Cytotoxic and Mutagenic Action of Carcinogens 

Veronica M. Maher and J. Justin McCormick 293 

5.5 Fibrinolytic Activities and Quantitative Soft Agar 

Assays 

J. Carl Barrett 307 

5.6 Assays for Somatic Mutation Studies on Syrian Hamster 

Embryonic Cells 

J. Carl Barrett 311 

5.7 Analysis of DNA Strand Breaks by Alkaline Sucrose 

Gradients 

Andrew R. Peterson 313 

5.8 Analysis of DNA Repair Synthesis by Auto- Radiography 

Andrew R. Peterson 314 

xiv 
1.

 INTRODUCTION 

1.1. IN VITRO CARCINOGENESIS METHODS IN RELATION TO THE DEVELOPMENT OF CARCINOGENESIS RESEARCH

 

UMBERTOSAFFIOTTI 

Experimental Pathology Branch, Carcinogenesis Program 

Division of Cancer Cause and Prevention, National Cancer 

Institute, NIH, Bethesda, MD 

A. EFFECTS OF CHEMICAL AND PHYSICAL CARCINOGENS 

Evidence of the effects of chemical and physical carcinogens is 

rapidly expanding from studies on several target levels of biological 

organization as outlined in the following paragraphs. 

1. Human cancers and precancers 

The effects of chemical and physical agents are becoming
 

well recognized in the causation of most cancers in the human
 

population. Epidemiologic evidence, even with its limitations, has
 

pointed to clear trends which indicate the major role of environmental
 

agents in the causation of human cancer. Geographic and epidemiologic
 

pathology studies have identified high risk populations and
 

particularly population subgroups, linking them to differences in
 

environmental exposures (6,9,10,15,31,33,34). The vast majority of
 

human cancers throughout the world originates from epithelial tissues,
 particularly lining epithelia (skin, respiratory tract, digestive
 

tract, urinary tract, pancreas, mammary gland, uterus, prostate).
 

Research on the histopathogenesis of human cancers has made
 

considerable progress, particularly in the study of the early lesions
 

which can often become useful markers of the initial stages of the
 

response of target tissues to carcinogens, both at the morphologic and
 

at the biochemical levels. A recent symposium organized by Drs. E.
 

Farber and M. B. Sporn for the NCI Carcinogenesis Program reviewed
 

these important developments (63)­

Human cancer types have been well defined, characterized and
 

classified and good documentation has been assembled through national
 

and international collaborative efforts, particularly in the Atlas of
 

Tumor Pathology of the Armed Forces Institute of Pathology (3), in the
 

1
 
series of the International Histological Classification of Tumors of
 

the World Health Organization (25) and in several excellent textbooks.
 

2. Whole animal models 

The understanding of the processes by which carcinogens
 

induce neoplasia in their target tissues is largely based on
 

experimentation in animal models. These have allowed us to establish
 

a close similarity between the development of cancers in the
 

laboratory animals and in their human counterpart. The development of
 

animal models for carcinogenesis represents a major methodological
 

step in the investigation of the effects of carcinogens on their
 

target tissues, as well as a powerful tool in defining their metabolic
 

pathways and the permissive host conditions required for a
 

carcinogenic response (51).
 

Many new animal models have been established in the last
 

decade. Their development was stimulated by the discovery of several
 

new classes of multipotential carcinogens, (e.g. nitrosamines,
 nitrosamides and substituted hydrazines), by the investigation of new
 

modes of exposure (e.g. respiratory exposure to carcinogens absorbed
 

on carrier dust particles (29,56)) and of new species of laboratory
 

animals (e.g. hamsters (22,61)).
 

By now, most of the major types of human cancers can be
 

reproduced in animals by chemical induction. Their pathologic
 

characteristics closely resemble those known from human pathology.
 This close correlation by itself strongly supports the evidence for a
 

chemical origin of a large proportion of human cancers (55).
 

Examples of recently developed chemically-induced animal
 

models of carcinogenesis include: bronchogenic lung cancer
 

(29,30,41,56,60); carcinoma of the larynx (58); large bowel cancer
 

(11,66,70-72); carcinoma of the pancreas (32,43,46-48); kidney
 

carcinoma (8,19,49,50); urinary bladder carcinoma (20,21,57,67) and
 

mammary carcinoma (16).
 

Such advances provide us now with a large body of methods
 

for inducing in experimental animals cancers of many specific
 

histologic characteristics, which we can then learn to correlate with
 

functional pathogenetic mechanisms.
 

The use of animal models for bioassay screening of
 

carcinogens will be discussed below.
 

The definition, characterization and classification of tumor
 

types by morphological criteria in experimental animals are so far
 

much less extensively documented than their human counterparts. The
 

International Agency for Research on Cancer has undertaken the
 

2
 
publication of a series of volumes entitled "Pathology of Tumours in
 

Laboratory Animals" which are expected to provide extensive pathology
 

reference standards (69). It is hoped that when an even wider range
 

of tumors will be experimentally induced and tumor types in experi­ mental animals will be more precisely defined, the already close
 

correlation between human and experimental tumor pathology will be
 

further strengthened.
 

3. Cellular level studies 

The next step is to correlate the carcinogenic events seen
 

at the tissue level in humans and animals with the events at the
 

cellular and molecular level. A new dimension in carcinogenesis
 

studies has been brought about by the great progress made in the last
 

decade in the development of models for the study of neoplastic
 

transformation induced by chemical and physical agents in cells in
 

culture. The present volume provides an extensive documentation of
 

this progress.
 

Presently a major task we face is that of evaluating the
 

methods for neoplastic cell transformation in culture to determine
 

their reliability for carcinogenesis research and for the screening of
 

chemicals for carcinogenic activity. The complex issues of
 

developing, defining, and validating short-term screening methods for
 

carcinogenesis were effectively reviewed and debated at a conference
 

organized by the International Agency for Research on Cancer in
 

Brussels in June 1975, the proceedings of which were recently
 

published (36). The report of a previous IARC conference was also
 

published (37).
 

The research methods needed to provide reliable validation
 

are dependent on several factors: e.g., level of critical quality
 

control of the methods under consideration; extent of their
 

reproducibility; adequacy and accuracy of the in vivo carcinogenesis
 

data against which the in vitro methods are supposed to be validated;
 

number and type of chemicals and chemical groups used in the process
 

of validation.
 

Much work in this direction has been developed by the NCI
 

Carcinogenesis Program with the collaboration of many investigators
 

and advisors in the United States and abroad. A summary review was
 

given at the Brussels meeting (53). The present volume covers
 

extensive additional bibliography.
 

4. Mutagenesis and molecular interaction studies 

The studies on molecular events in carcinogenesis can be
 

considered under two broad categories:
 
(a) Events that lead to a critical DNA damage and can be
 

expressed as mutations. They represent the postulated basis of the
 

correlation between mutagenesis and carcinogenesis. The study of the
 

mutagenic effects of carcinogens has rapidly expanded in recent years
 

with studies on different biological targets ranging from bacterial to
 

mammalian systems. We must remain aware of the respective limitations
 

of the mutagenesis and carcinogenesis models used for such
 

correlations, particularly in terms of metabolic activation of
 

carcinogens and susceptibility of the target organisms.
 

(b) Molecular mechanisms. Particularly important are those
 

affecting the metabolic activation and detoxification of carcinogens,
 their interaction with target macromolecules and their binding to
 

them, the damage and repair of DNA as well as the effects on cell
 

regulatory control mechanisms.
 

There is a great need now to correlate what we learn about
 

the effects of carcinogens through all the levels of observation, from
 

the human and the whole animal level to the tissue and cellular level
 

and to the molecular level.
 

B. DEFINITIONS OF CARCINOGENS AND RELATED AREAS OF UNCERTAINTY 

1. Quality of the evidence 

We can learn that an agent can be carcinogenic in mammalian 

species from two main sources, each of which requires critical 

evaluation of the findings: (a) epidemiologic observations, which 

require populations of adequate size, exposed under accurately 

definable conditions and observed over a long period of time; they 

provide therefore essentially an analysis "after the fact"; and (b) 

experimental investigations, which require properly conducted 

long-term animal bioassays with adequate experimental design and well 

defined experimental exposures (54,55). The experimental method is 

used for predictive tests capable of detecting the carcinogenic 

effects of an agent before a large population is exposed. 

The concept of pretesting chemicals before a large 

population is exposed had been recommended by scientists in 

carcinogenesis over the last two decades, and has been finally 

established in the United States' law with the passage of the Toxic 

Substances Control Act of 1976 (68). For carcinogenesis bioassays, the use of long-term animal tests is still the recommended definitive 

procedure. Growing consideration is being given to a battery of 

short-term tests as effective prescreening methods. 

i\ 
Much of the bioassay data developed from the 1930s through
 

the 1960s were obtained from small scale bioassays, whose design,
 conduct, analysis and reporting were often poor. The use of animal
 

models for carcinogenesis bioassays has been recently made much more
 

rigorous by detailed requirements for carefully defined methodogies in
 

the selection of test animals, in their maintenance conditions, in the
 

experimental design, in the clinical observations of the test animals,
 in the procedures adopted for gross and microscopic pathology and in
 

the collection, analysis and publication of data. These procedures,
 initially outlined by several expert committees (4,12-14,26,65) were
 

subsequently further developed and specified in much greater detail by
 

the NCI Carcinogenesis Program as guidelines for carcinogen bioassays
 

(62). A model for the fully detailed publication of the reports of
 

such bioassays was also established (38).
 

I have personally devoted considerable time and effort in
 

the last decade to the development of these standards for conducting
 

and reporting carcinogenesis bioassays, because good bioassays
 

represent our almost exclusive source of direct evidence on the
 

carcinogenicity of chemicals. This kind of evidence is the foundation
 

on which we have to rely for all subsequent studies based on the
 

carcinogenicity or noncarcinogenicity of an agent in terms of
 

mechanisms, predictiveness of structure-activity relationships,
 development of new biological models, evaluation of susceptibility
 

factors and development of inhibitory and preventive measures.
 

2. Documentation sources of chemical carcinogenicity data 

The following sources provide this information: 

(a)

of Chemicals

 "IARC Monographs on the Evaluation of Carcin

 to Man," published by the International

ogenic

 Agency

 Risk 

 for 

Research on Cancer (24). So far, 14 volumes have been published
 

containing 292 monographs covering a total of about 350 individual
 

chemicals. These monographs are the result of a systematic review by
 

experts of the data on the carcinogenicity of environmental chemicals,
 and of related data on their characteristics, environmental
 

distribution, human exposure and metabolism. At present, these
 

monographs are the only extensive reference source of carcinogenicity
 

data evaluated by a systematic process of critical review of the
 

experimental conditions, bioassay protocols, standards of pathology
 

and results. The NCI Carcinogenesis Program has extensively
 

collaborated in this initiative, providing extensive data bases for it
 

and participating in the reviews.
 

(b) "Survey of Compounds Which Have Been Tested for
 

Carcinogenic Activity," published by the National Cancer Institute
 

(39). This is a comprehensive compilation of literature data on
 

5 
experimental design and results of bioassays on chemicals. It 

contains no critical evaluation of the information and each entry 

should, therefore, be evaluated by the reader from the original 

literature articles. It has become a key documentation source, and 

has been used to provide a data base for the IARC Monographs. 

(c) "Suspected Carcinogens. A Subfile of the NIOSH Toxic 

Substances List," published by the National Institute for Occupational 

Safety and Health (40). This is a listing of about 1500 chemicals 

which have been reported in the literature as having "neoplastic" or 

"carcinogenic" effects. It gives no critical evaluation of the 

information, which in many cases derives from totally inadequate 

reports. 

(d) Other lists of carcinogens compiled by various 

individual authors or groups in several books on carcinogenesis 

(1,2,5,7,23,64) 

I believe that it is very important that these sources be 

used with great caution, particularly when they have not been 

assembled through a critical review process examining all the relevant 

data. In the matter of carcinogenicity data, qualifying the data 

critically is essential. As I have pointed out before (52,53), many 

chemicals have been badly or inadequately tested, and yet they have 

been reported in the literature as being "carcinogens" or 

"non-carcinogens" (the latter obviously even more difficult to 

establish). They have been sometimes reported in summary tables and 

their alleged carcinogenicity has been classified by plus or minus 

signs: this information has been used in turn as a basis to quote the 

activity of reference compounds without further questioning the 

validity of the data. 

Particularly in the evaluation of the predictiveness of 

short-term methods in relation to the animal carcinogenicity data, it 

is essential that the latter be critically evaluated from the original 

reports. 

The number of "known carcinogen" will vary considerably, 

depending on the degree of critical stringency adopted for accepting 

evidence of carcinogencity. 

3. Classification of results of carcinogenicity studies 

It is important to recognize that the evidence of carcinogenicity for an agent, obtained from epidemiologic observations and/or 

from experimental bioassays, does not fall necessarily into two 

sharply distinct categories of positive and negative. In many cases 

the evidence is insufficient for a definitive assessment. I have 

6 
therefore proposed (54,55) the following classification for the 

evidence of carcinogen!city. 

(a) Positive, when exposure to the test agent is 

conclusively shown to cause the formation of malignant or benign 

neoplasms, or a combination thereof, in humans or in animals of one or 

more mammalian species, by adequately designed and conducted studies. 

(b) Negative under the conditions of observation, when no 

indication is obtained that there is any positive influence of the 

exposure on the induction of tumors; such negative results are only 

valid within the conditions of the observation. However, the extent 

and quality of the observations can add predictive value to the 

findings. If the observations meet a minimum standard of adequacy 

with negative results, then operationally the test agent can be 

considered negative. The currently recommended standard is for the 

agent to be tested in animals of both sexes, in two species, at levels 

including the maximum tolerated dose (62). 

(c) Inconclusive, when neither of the two preceding 

classifications applies either because adequate observations are 

lacking, or because they are of poor quality, or of excessively 

limited extent. This gray area includes results which range from 

uninformative to highly suggestive. I believe that a similar 

three-way classification on the results should be used for the 

evauation of in vitro studies. 

4. The so-called "potency "of carcinogens 

Estimating the so-called "potency" of an individual 

carcinogen is in my view, at least at the present time, still an 

unreliable exercise, unless it is taken in a limited context of well 

defined experimental conditions. The difficulty stems from the fact 

that "potency" implies a measurement of a biological interaction and 

is therefore dependent on different biological variables. 

We know several classic examples of chemicals that can be 

rated either as "negative" or as "very potent" depending on the 

biological test system used (e.g., aflatoxin in adult mice and rats; 

2-fluorenylacetamide in different strains of rats; 2-naphthylamine in 

rats and humans). The "potency" is only a measure of the intensity of 

the response, which is in turn conditioned in most cases by host 

factors such as metabolic activation systems, tissue susceptibility, 

age and diet. 

At present I therefore recommend great caution in any 

attempt to extrapolate "potency" estimates from one biological system 

to another, We need much more data and more work to analyze 

7 
dose-response patterns and to define control mechanisms, in order to 

approach the issue of "potency" on a more quantifiable and reliable 

basis. 

Organ and tissue specificity for carcinogens are highly 

variable in different species and exposure conditions, so that we 

cannot predict that a tissue or a cell type will respond in a given 

way to a chemical simply on the basis of the fact that the same tissue 

responds in another species or set of conditions. 

The rapidly expanding knowledge on metabolic

requirements and pathways is expected to bring this whole

much sharper focus. 

 activation 

 area into 

C. CORRLEATIONS AMONG DIFFERENT BIOLOGICAL SYSTEMS 

What has been said above is meant to stress how important it is 

to establish well defined correlations on the effects of carcinogens 

as observed in several different biological systems. 

I would like to repeat some points I made at the IARC Brussels 

conference (53). A revolutionary advance has occurred in chemical 

carcinogenesis studies with the development of biological models in 

which a response to chemical carcinogens can be identified by 

short-term observations of specific biochemical reactions, mutational 

events or neoplastic cells transformation. 

I hope that the extensive development, thorough evaluation and 

careful validation of short-term test systems will lead to the 

establishment of procedures by which positive results, obtained in a 

battery of short-term tests, will be considered equivalent or better 

in predictive value to positive results now obtained in a long-term 

animal bioassay, for the purpose of predicting possible 

carcinogenicity in humans. The future short-term tests may include 

bioassays directly on human cells or tissues in culture. 

If these short-term systems are to be accepted as valid 

indicators of a carcinogenic event, they must show a high degree of 

positive correlation with the results of observations on long-term 

systems for tumor induction. This requires an extensive process of 

validation or "testing the test systems," to determine whether they 

consistently and reliably provide a response analogous to the response 

obtained in long-term bioassays in animals and, whenever possible, to 

the observation of carcinogenic effect directly in human populations. 

Direct observations of carcinogenic effects in humans remain 

severely limited by the requirement for long observation periods, well 

defined exposures, and adequate control populations. Therefore, in 

8 
assessing the carcinogenic potential of the thousands of chemicals to 

which humans are now exposed and of the thousands of new products 

proposed for use, we must rely on experimental bioassay systems. The 

consideration, which will determine the acceptability of the shortterm systems as indicators of carcinogenicity, will be their 

correlation with the animal bioassay results. Therefore, the whole 

process of validating the predict!veness of the short-term systems 

will depend largely on how valid and reliable are the results obtained 

in long-term animal bioassays. In other words, if we are willing to 

accept or reject the validity of the short-term test systems, 

depending on whether or not they match the results of long-term 

bioassays, then we must first of all accept reliable standards for the 

performance and evaluation of long-term bioassay systems. 

Until the predictive value of the short-term methods 

(individually or as a battery) will have been sufficiently validated, 

the finding of positive results in short-term tests, in the absence of 

adequate long-term results, may give rise to controversial 

interpretations. The controversy may become particularly intense with 

chemicals of major economic value and involving major human exposure. 

Positive results obtained with the short-term tests that have 

been more extensively studied may now be considered as useful 

indications of priority for further testing and possibly as warning 

signals suggesting that the test compound may be found carcinogenic 

when tested by long-term bioassays. Such warnings, in the absence of 

definitive bioassays, will provide an opportunity for planning 

long-term bioassays and related studies, such as metabolic studies. An 

opportunity will also be provided for planning the long-term bioassays 

of chemicals representing technological alternatives. 

On the side of the technological uses of the substances in 

question, the time between the first positive findings in short-term 

tests and the completion of subsequent long-term tests (several years) 

can be effectively used in studying and preparing for technological 

alternatives and technical approaches to the reduction of exposure 

(53). Identifying and evaluating technological alternatives should 

become an integral phase of the process of assessing preventive 

approaches against the effects of environmental carcinogens (54). 

I believe that the process of testing the short-term systems for 

their predictive value in identifying carcinogens should include the 

following approaches: 

(a) Selection of several short-term test systems, including 

tests for mutagenesis and for neoplastic transformation of cells in 

culture. 

9 
(b) Definition and standardization of laboratory procedures 

and measurement of their reproducibility, using a small number of test 

chemicals for each test method. 

(c) Selection of a large list of chemicals as reference 

compounds, including direct carcinogens, procarcinogens and 

noncarcinogens. About 100 compounds have been used by the NCI 

Carcinogenesis Program in the evaluation of mutagenesis test systems 

(35,44,45); the Ames test has been evaluated using an additional 200 

chemicals in various laboratories (35). In the hamster embryo cell 

transformation system, a list of about 75 chemicals has been tested so 

far (42). The lists of reference chemicals need to be evaluated for 

their reliability in terms of in vivo carcinogenesis data. It is 

important to include a sufficiently large number of noncarcinogenic 

reference chemicals. These lists should be periodically extended and 

revised to include a representative range of chemical classes. I 

believe that, in a second phase, each biological test system should be 

screened using nearly all the available chemicals for which extensive 

reliable data exist on carcinogenesis bioassays in animals (perhaps 

500 to 600 compounds). 

(d) Definition of biological metabolic

and of the metabolic requirements for each of the

reference list. 

 activation

 chemicals

 systems 

 of the 

(e) Systematic screening of the selected

selected bioassay systems, including tests for repr

a double blind procedure. 

 chemicals

oducibility

 in

 and

 the 

 by 

(f) Comparison of the response of different test systems, 

with particular consideration for the susceptibility of different cell 

systems with regards to host and tissue of origin. 

(g) Concurrently, much new imaginative research is needed 

in the following areas: development and definition of new cell 

transformation systems, with emphasis on epithelial cells and on human 

cells; metabolic activation mechanisms; relation of mutagenic to 

carcinogenic events in the same target cells; biochemical mechanisms 

involved in transformation; markers of the transformed state; and 

definition of culture conditions. 

The NCI Carcinogenesis Program has undertaken an extensive effort 

towards the development and validation of short-term methods for 

carcinogenesis screening, and towards their definition and 

interpretation along these lines. 

10 
The Aspen Workshop held in July 1975 and the Seminar and Workshop 

on which the present volume is based have provided an exciting 

analysis of the rapid and remarkable progress made in this field. 

A particularly exciting advance in the field of in vitro chemical 

carcinogenesis is the development of methods for studying the effects 

of carcinogens directly on human cells and tissues in culture. A 

method for the effective induction of neoplastic transformation in 

human cells in culture by chemical carcinogens was first reported at 

this conference by Kakunaga (27,28). 

Other studies with human cells and tissues, also reported here, show that methods are being developed for defining the effects of 

carcinogens directly by using human target tissues. 

The establishment of organ culture systems for various human 

epithelial tissues in our laboratory by Harris and co-workers (17,18) 

is, in my view, a major further step toward providing a basis for a 

much closer correlation between animals and humans in their response 

to carcinogens. Thus the study of chemical carcinogenesis can now be 

visualized at four levels of biological targets: 

The problem of correlating the in vivo responses to carcinogens 

in animals and in humans ("extrapolating from mouse to man," as 

toxicologists say) can now be approached much more directly by selecting measurable parameters that can be defined in closely 

comparable conditions in animal and human tissues. 

This approach will, I expect, bring us to a much better under­ standing of the response of human tissues to carcinogens. Therefore 

it will give us a much more precise basis to attain two main research 

goals: a) to identify carcinogens representing particularly important 

hazards for the exposed populations; and b) to identify individuals in 

the population who are particularly susceptible to given types of 

carcinogens. Knowledge of both these factors will represent a solid 

basis for a most effective effort toward cancer prevention. 

11
 
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17 
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 U. Saffiotti, and B. Toth. "Factors Determining the Neoplastic
 

Response Induced by Carcinogens." In: Biological Interactions
 

in Normal and Neoplastic Growth. M.G. Brennan and W.L. Simpson
 

(Eds.). Boston, Mass., Little, Brown and Co., 1962, 285-297.
 

62.	 Sontag, J.M., N.P. Page, and U. Saffiotti. "Guidelines for
 

Carcinogen Bioassay in Small Rodents." NCI Carcinogenesis
 

Technical Report Series No. 1. DHEW Publ. No. (NIH) 76-801.
 Washington, D.C., U.S. Government Printing Office, 1976, 65 pp.
 

63.	 Symposium. Early Lesions and The Development of Epithelial
 

Cancer. Cancer Res., 36, No. 7, Part 2 (1976), 2475-2706.
 

64.	 Teichmann, B., and T. Schramm. "Substanzen mit kanzerogener
 

Wirkung." Zentralinstitut fur Krebsforschung der Wissenschaften
 

der DDR. Berlin-Buch, East Germany, 1973, 124 pp.
 

65.	 Th Testin e g of Chemicals for Carcinogenicity, Mutagenicity, and
 

Teratogenicity. Ottawa, Canada, Ministry of Health and Welfare,
 1973, 183 PP.
 

66.	 Thurnherr, N., E. Deschner, E. Stonehill, and M. Lipkin.
 "Induction of Adenocarcinomas of the Colon in Mice by Weekly
 

Injections of 1,2-Dimethylhydrazine." Cancer Res., 33 (1973),
 940-945.
 

67.	 Tiltman, A.J., and G.H. Friedell. "The Histogenesis of
 

Experimental Bladder Cancer." Invest. Urol., 9 (1971), 218-2126.
 

68.	 Toxic Substances Control Act. Public Law 94-469, Oct. 11, 1976,
 94th Congress. Washington, D.C., U.S. Congress.
 

69.	 Turusov, V.S. (Ed.). "Pathology of Tumours in Laboratory
 

Animals. Vol. 1. Tumours of the Rat. Part 1." IARC Scientific
 

Publ. No. 5. International Agency for Research on Cancer, Lyon,
 1973, 214 pp. "-Part 2." IARC Scientific Publ. No. 6, ibid.,
 1976, 319 PP.
 

70.	 Ward, J.M. "Morphogenesis of Chemically-Induced Neoplasms of the
 

Colon and Small Intestine in Rats." Lab. Invest., 30 (1974),
 

505-513.
 

71.	 Ward, J.M., R.S. Yamamoto, and C.S. Brown. "Pathology of
 

Intestinal Neoplasms and Other Lesions in Rats Exposed to
 

Axoxymethane." J. Natl. Cancer Inst., 51 (1973), 1029-1039­

72.	 Weisburger, J.H. "Chemical Carcinogens and Their Mode of Action
 

in Colonic Neoplasia." Pis. Colon Rectum, 16 (1973), 431-437.
 

18
 
ADDITIONAL REFERENCES: 

73- Saffiotti, U., and N.P. Page. "Releasing Carcinogenesis Test 

Results: Timing and Extent of Reporting." Med. Pediatr. Oncol., 

3 (1977), 159-167. 

74. Saffiotti, U. "Scientific Bases of Environmental Carcinogenesis 

and Cancer Prevention: Developing an Interdisciplinary Science 

and Facing Its Ethical Implications." J. Toxicol. Environ. 

Health (1977) (In Press). 

75. Saffiotti, U. "Carcinogenesis 1957-77: Notes for a Historical 

Review." J. Natl. Cancer Inst. (1977) (In Press). 

76. Saffiotti, U. "Experimental Identification of Chemical 

Carcinogens, Risk Evaluation and Animal-to-Human Correlations." 

Environm. Health Perspectives (1977) (In Press). 

77. NOTE: A series of studies for the validation of mutagenesis 

assays as prescreens for carcinogens was developed by the 

National Cancer Institute Carcinogenesis Collaborative Program 

and coordinated by Dr. L.A. Poirier. These studies are reported 

in the following publications: 

77-1. Poirier, L.A. , and E.K. Weisburger. "Carcinogens and related 

compounds selected for mutagenicity screens." Submitted to £._ 

Natl. Cancer Inst. 

77-2. Brown, M.M. , J.S. Wassom, H.V. Mailing, M.D. Shelby, and E.S. 

von Halle. "Microbial assay systems used in the evaluation of 

potential carcinogenic chemicals for mutagenic activity - A 

guide to the literature." Submitted to J. Natl. Cancer Inst. 

77-3- Rosenkranz, H., and L.A. Poirier. "An evaluation of the 

mutagenicity and DNA modifying activity in microbial systems 

of carcinogens and noncarcinogens." Submitted to J. Natl. 

Cancer Inst. 

77-^. Simmon, V.F. "In vitro mutagenicity assays with Salmonella 

typhimurium." Submitted to J. Natl. Cancer Inst. 

77-5. Simmon, V.F. "In vitro mutagenicity assays with Saccharomyces 

cerevisiae D-3." Submitted to J. Natl. Cancer Inst. 

77-6. Simmon, V.F., H. Rosenkranz, E. Zeiger, and L.A. Poirier. "The 

mutagenic activities of chemical carcinogens and related 

compounds in the host-mediated assay." Manuscript in 

preparation. 

19 
77-7. Carter, J.J., P.S. Thayer, G.A. Fischer, and M.L. Sturgeon. "Use of lymphoma cells in vitro and in hose-mediated bioassays 

as a potential prescreen for chemical carcinogens." Submitted 

to J. Natl. Cancer Inst. 

20 
2.

 CARCIIMOGENESIS STUDIES IN CELL CULTURE 

SYSTEMS 

2.1	 REVIEWS ON IN VITRO CARCINOGENESIS AND SELECTED GENERAL
 

REFERENCES
 

2.1.1	 A BIBLIOGRAPHY
 

The following references, suggested by C. Heidelberger,
 I. B. Weinstein, and others, provide a list of reviews and selected
 

papers of general interest.
 

1.	 Berwald, Y., and L. Sachs. "In Vitro Transformation of Normal
 

Cells to Tumor Cells by Carcinogenic Hydrocarbons." J. Natl.
 Cancer Inst., 35 (1965), 641-661.
 

2.	 Bertram, J.S., and C. Heidelberger. "Cell-Cycle Dependency of
 

Chemical Oncogenic Transformation in Culture." Proc. Am. Assoc.
 

Cancer Res., 14 (1973), 70.
 

3.	 Casto, B.C., and J.A. DiPaolo. "Virus, Chemicals and Cancer."
 

Progr. Med. Virol., 16 (1973), 1-^7.
 

4.	 Chen, T.T., and C. Heidelberger. "Quantitative Studies on the
 

Malignant Transformation of Mouse Prostate Cells by Carcinogenic
 

Hydrocarbons In Vitro." Int. J. Cancer, H (1969), 166-178.
 

5.	 Dao, T.L., and D. Sinha. "Mammary Adenocarcinoma Induced in
 

Organ Culture by 7,12-dimethylbenz(a)anthracene." J. Natl.
 Cancer Inst., 49 (1972), 591-593.
 

6.	 DiPaolo, J.A., and B.C. Casto. "Chemical Carcinogenesis." In:
 

Recent Advances in Cancer Research: Cell Biology, Molecular
 

Biology and Tumor Virology. R.C. Gallo (Ed.). CRC Press, Inc.,
 Cleveland, Ohio (in press).
 

7.	 DiPaolo, J.A., R.L. Nelson, P.J. Donovan, and C.H. Evans.
 

"Host-mediated In_ Vivo - In Vitro Assay for Chemical Carcinogenesis." Arch7~Path., 95 (1973), 380-385.
 

8.	 DiPaolo, J.A., K. Takano, and N.C. Popescu. "Quantitation of
 

Chemically Induced Neoplastic Transformation of Balb/3T3 Cloned
 

Cell Lines." Cancer Res., 32 (1972), 2686-2695.
 

21
 
9.

 DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "Quantitative 

Studies of In Vitro Transformation by Chemical Carcinogens." J. Natl. Cancer Inst., 42 (1969), 867-876. 

10. Freeman, A.E. , H.J. Igel, and P.T. Price. "Carcinogenesis In 

Vitro I. In Vitro Transformation of Rat Embryo Cells: 

Correlations with the Known Tumorigenic Activities of Chemicals 

in Rodents." In Vitro, 11 (1975), 107-116. 

11. Freeman, A.E. , P.J. Price, H.J. Igel, J.M. Maryak, and R.J. Huebner. "Morphologic Transformation of Rat Embryo Cells Induced 

by Diethylnitrosamine and Murine Leukemia Viruses." J. Natl. Cancer Inst., 44 (1970), 65-78. 

12. Freeman, A.E. , E.K. Weisburger, J.H. Weisburger, R.G. Wolford, J.M. Maryak, and R.J. Huebner. "Transformation of Cell Cultures 

as an Indication of the Carcinogenic Potential of Chemicals." J_._ Natl. Cancer Inst.. 51 (1973), 799-808. 

13- Heidelberger, C. "Cell Culture Studies on the Mechanisms of 

Hydrocarbon Oncogenesis." In: Chemical Carcinogenesis, Part B, P.O.P. Ts'o and J.A. DiPaolo, (Eds.). Marcel Dekker, Inc., New 

York (1974), 457-462. 

14. Heidelberger, C. "Chemical Carcinogenesis." Ann. Rev. Biochem., 44 (1975), 79-121. 

15. Heidelberger, C. "Chemical Carcinogenesis, Chemotherapy: 

Cancer's Continuing Core Challenges." G.H.A . Clowes Memorial 

Lecture. Cancer Res., 30 (1970), 1549-1569. 

16. Heidelberger, C. "Chemical Oncogenesis in Culture." Adv. Cancer 

Res., 18 (1973), 317-366. 

17. Heidelberger, C., and P.F. Boshell. "Chemical Oncogenesis in 

Cultures." In: Recent Topics in Chemical Carcinogenesis. Gann 

Monograph on Cancer Research, 17 (1975), 39-58. 

18. Heidelberger, C. "Chemically and Metabolically Induced DNA 

Adducts: Relationship to Chemical Carcinogenesis." In: Aging, Carcinogenesis, and Radiation Biology. The Role of Nucleic Acid 

Addition Reactions. K.D. Smith (Ed.). Plenum Press, New York, 

(1975), 341-371. 

19. Heidelberger, C. "Current Trends in Chemical Carcinogenesis." 

Fed. Proc., 32 (1973), 317-366. 

22 
20.	 Heidelberger, C. "In Vitro Studies on the Role of Epoxides in
 

Carcinogenic Hydrocarbon Activation." In: Topics in Chemical
 

Carcinogenesis. W. Nakahara, S. Takyama, T. Sugimura, and S.
 

Odashima (Eds.). Univ. of Tokyo Press, Tokyo, Japan (1972)
 

371-386 (discussion).
 

21.	 Heidelberger, C., C.A. Reznikoff, and D.F. Krahn. "Malignant
 

Transformation of Cells in Culture Using Oncogenic Chemicals."
 

In: Tissue Culture Methods and Applications. P.F. Kruse Jr.,
 and M.K. Patterson, Jr. (Eds.). Academic Press, Inc., New York
 

(1973) 644-653.
 

22.	 Heidelberger, C. "Studies on the Cellular Mechanisms of Chemical
 

Oncogenesis in Culture." In: Fundamental Aspects of Neoplasia.
 

A.A. Gottlieb, O.J. Plescia, and D.H.L. Bishop (Eds.).
 

Springer-Verlag, New York (1975) 357-363.
 

23.	 Huberman, E. "Mammalian Cell Transformation and Cell-Mediated
 

Mutagenesis by Carcinogenic Polycyclic Hydrocarbons." Mutation
 

Res., 29 (1975), 285-291.
 

24.	 Katsuta, H., and T.T. Takaoka. "Carcinogenesis in Tissue
 

Culture. XII. Transformation in Culture of Rat Liver Cells with
 

4-Nitroquinoline-1-oxide." Proceedings of the 27th General
 

Meeting of Japanese Cancer Association, Tokyo (1968), 95.
 

25.	 Kakunaga, T., and J. Kamahova. "Properties of Hamster Embryonic
 

Cells Transformed by 4-Nitroquinoline-1-oxide In Vitro and Their
 

Correlations with the Malignant Properties of the Cells." Biken
 

J., 11 (1968), 313-332.
 

26.	 Macpherson, I. "The Characteristics of Animal Cells
 

Transformation In Vitro." Advances in Cancer Research, 13
 

(1970), 169-215.
 

27.	 Marquardt, H., T. Kuroki, E. Huberman, J.K. Selkirk, C.
 

Heidelberger, P.L. Grover, and P. Sims. "Malignant
 

Transformation of Cells Derived from Mouse Prostate by Epoxides
 

and other Derivatives of Polycyclic Hydrocarbons." Cancer Res.,
 32 (1972), 716-720.
 

26.	 Mishra, N.K., and G. DiMayorca. "In Vitro Malignant
 

Transformation of Cells by Chemical Carcinogens." Biochim.
 

Biophys. Acta, 335 (1974), 205-219.
 

29.	 Montesano, R., and L. Tomatis (Eds.). "Chemical Carcinogenesis
 

Essays." International Agency for Research on Cancer, Lyon, IARC
 

Scientific Publication No. 10 (1974), 230 pp.
 

23
 
30.	 Montesano, R., L. Saint Vincent, and L. Tomatis. "Malignant
 

Transformation In Vitro of Rat Liver Cells by Dimethylnitrosamine
 

and N-methyl-N'-nitro-nitrosoguanidine." Br. J. Cancer, 28
 

(1973), 215-220.
 

31.	 Montesano, R., H. Bartsch, and L. Tomatis (Eds.). "Screening
 

Tests in Chemical Carcinogenesis." International Agency for
 

Research on Cancer, Lyon, IARC Scientific Publ. No. 12 (1976),
 661 pp.
 

32.	 Namba, M., H. Masuji, and J. Sato. "Carcinogenesis in Tissue
 

Culture. IX: Malignant Transformation of Cultured Rat Cells
 

Treated with 4-Nitroquinoline-1-oxide." Japan J. Exptl. Med., 39
 

(1969), 253-365.
 

33- Sato, J., and T. Yabe. "Carcinogenesis in Tissue Culture. V.
 Effect on Long-Term Addition of 4-Dimethylaminoazobenzene and
 

3'-Methyl-4-dimethylaminoazobenzene on Liver Cells in Culture."
 

Jap. J. Exptl. Med., 35 (1965), 445-462.
 

34.	 Sivak, A., and B.L. Van Duuren. "Phenotypic Expression of
 

Transformation: Induction in Cell Culture by Phorbol Ester."
 

Science, 157 (1967), 1433-1434.
 

35.	 Sivak, A., and B.L. Van Duuren. "A Cell Culture System for the
 

Assessment of Tumor Promoting Activity." J. Natl. Cancer Inst.,
 

44 (1970), 1091-1097.
 

36.	 Ts'o, P.O.P., and J.A. DiPaolo, (Eds.). "Chemical
 

Carcinogenesis." Marcel Dekker, Inc., New York (1974) Part A,
 1-440, and Part B, 441-794.
 

37.	 Unkeless, J., K. Dano, G.M. Kellerman, and E. Reich.
 "Fibrinolysis Associated with Oncogenic Transformation." J_._
 Biol. Chem., 249 (1974), 4295-4305.
 

38.	 Williams, G.M., J.M. Elliott, and J.H. Weisburger. "Carcinoma
 

after Malignant Conversion In Vitro of Epithelial-like Cells from
 

Rat Liver Following Exposure to Chemical Carcinogens." Cancer
 

Res., 33 (1973), 606-612.
 

39.	 Weinstein, I.E., J.M. Orenstein, R. Gebert, M.E. Kaighn, and
 

U.C. Stadler. "Growth and Structural Properties of Epithelial
 

Cell Cultures Established from Normal Rat Liver and Chemically
 

Induced Hepatomas." Cancer Res., 35 (1975), 253-263.
 

24 
2.2 MOUSE FIBROBLASTS 

2.2.1 A BIBLIOGRAPHY 

The following references were suggested by C. Heidelberger. 

A. MOUSE PROSTATE FIBROBLASTS 

1. Chen, T.T., and C. Heidelberger. "Cultivation In Vitro of Cells 

Derived from Adult C3H Mouse Ventral Prostate." J. Natl. Cancer 

Inst., 42 (1969), 915-925. 

2. Chen, T.T., and C. Heidelberger. "In Vitro Malignant 

Transformation of Cells Derived from Mouse Prostate in the 

Presence of 3-methylcholanthrene." J. Natl Cancer Inst., 42 

(1969), 915-925. 

3- Chen, T.T., and C. Heidelberger. "Quantitative Studies on the 

Malignant Transformation of Mouse Prostate Cells by Carcinogenic 

Hydrocarbons In Vitro." Int. J. Cancer, 4 (1969), 166-178. 

4. Embleton, M.J. , and C. Heidelberger. "Antigenicity of Clones of 

Mouse Prostate Cells Transformed In Vitro." Int. J. Cancer, 9 

(1972), 8-18. 

5. Grover, P.L., P. Sims, E. Huberman, H. Marquardt, T. Kuroki, and 

C. Heidelberger. "In Vitro Transformation of Rodent Cells by 

K-Region Derivatives of Polycyclic Hydrocarbons." Proc. Nat. 

Acad. Sci. USA, fa8 (197D, 1098-1101. 

6. Heidelberger, C., T.T. Chen, and P.T. lype. "Malignant 

Transformation In Vitro with Carcinogenic Hydrocarbons." Adv. 

Enzyme Regulation, 7 (1969), 339-349­

7. Heidelberger, C. "Quantitative Studies on Hydrocarbon 

Carcinogenesis In Vitro." In: Physico-Chemical Mechanisms of 

Carcinogenesis, The Jersusalem Symposium on Quantum Chemistry and 

Biology, 1 (1969), 45-58. 

8. Heidelberger, C. , P.T. lype, M.R. Roller, and T.T. Chen. 

"Studies of Hydrocarbon Carcinogenesis in Organ and Cell 

Culture." In: Proliferation and Spread of Neoplastic Cells. 

21st Annual Symposium on Fundamental Cancer Research, University 

of Texas M. D. Anderson Hospital and Tumor Institute, Williams 

and Wilkins, Baltimore, MD (196S), 137-156. 

25 
9.

 Huberman, E., J.K. Selkirk, and C. Heidelberger. "Metabolism of 

Polycyclic Aromatic Hydrocarbons in Cell Cultures." Cancer Res., 31 (1971), 2161-2167. 

10. Kuroki, T., and C. Heidelberger. "Determination of the H-Protein 

in Transformable and Transformed Cells in Culture." Biochemistry, 11 (1972), 2116-2124. 

11. Kuroki, T., E. Huberman, H. Marquardt, J.K. Selkirk, C. Heidelberger, P. L. Grover, and P. Sims. "Binding of K-Region 

Epoxides and Other Derivatives of Benz(a)anthracene and 

Dibenz(a,h)anthracene to DNA, RNA, and Proteins of Transformable 

Cells." Chem.-Biol. Interactions, 4 (1971/72), 389-397. 

12. Kuroki, T., and C. Heidelberger. "The Binding of Polycyclic 

Aromatic Hydrocarbons to the DNA, RNA, and Proteins of 

Transformable Cells in Culture." Cancer Res., 31 (1971), 2168-2176. 

13- Marquardt, H. "Cell Cycle Dependence of Chemically Induced 

Malignant Transformation Iri Vitro." Cancer Res., 34 (197*0, 1612-1615. 

14. Marquardt, H., and C. Heidelberger. "Influence of Feeder Cells 

and Inducers and Inhibitors of Microsomal Mixed-Function Oxidases 

on Hydrocarbon-Induced Malignant Transformation of Cells Derived 

from C3H Mouse Prostate." Cancer Res., 32 (1972), 721-725. 

15. Marquardt, H., P.L. Grover, and P. Sims. "In Vitro Malignant 

Transformation of Mouse Fibroblasts by Non-K-Region Dihydrodiols 

Derived from 7-Methylbenz(a)anthracene, 7,12-Dimethylbenz(a)an­ thracene, and Benzo(a)pyrene." Cancer Res., 36 (1976), 2059-2064. 

16. Marquardt, H., T.E. Sodergren, P. Sims, and P.L. Grover. "Malignant Transformation In Vitro of Mouse Fibroblasts by 7,12­ Dimethylbenz(a)anthracene and 7-Hydroxymethylbenz(a)anthracene 

and by Their K-Region Derivatives." Int. J. Cancer, 13 (1974), 304-310. 

17. Marquardt, H., T. Kuroki, E. Huberman, J.K. Selkirk, C. Heidelberger, P.L. Grover, and P. Sims. "Malignant 

Transformation of Cells Derived from Mouse Prostate by Epoxides 

and other Derivatives of Polycyclic Hydrocarbons." Cancer Res., 32 (1972), 716-720. 

1«. Marquardt, H.. "Polyriboinosinic-Polyribocytidylic Acid Prevents 

Chemically-Induced Malignant Transformation In Vitro." Nature 

New Biol., 246 (1973), 228-229. 

26 
19.

 Marquardt, H. "The Effect of X-Irradiation on Hydrocarbon 

Methabolism and on Hydrocarbon-Induced Lethality and 

Transformation in Cells Derived from Mouse Prostate." Z. Krebsforsch, 80 (1973), 223-228. 

20. Marquardt, H., F.S. Philips, andS.S. Sternberg. "Tumorigenicity 

In Vivo and Induction of Malignant Transformation and Mutagenesis 

in Cell Cultures by Adriamycin and Daunomycin." Cancer Res., 36 

(1976), 2065-2069. 

21. Mondal, S., P.T. lype, L.M. Griesbach, and C. Heidelberger. "Antigenicity of Cells Derived from Mouse Prostate Cells After 

Malignant Transformation In Vitro by Carcinogenic Hydrocarbons." 

Cancer Res., 20 (1970), 1502-1507. 

22. Mondal, S., and C. Heidelberger. "In Vitro Malignant 

Transformation by Methylcholanthrene of the Progeny of Single 

Cells Derived from C3H Mouse Prostate." Proc. Nat. Acad. Sci. USA, 65 (1970), 219-225. 

23. Mondal, S., M.J. Embleton, H. Marquardt, and C. Heidelberger. "Production of Variants of Decreased Malignancy and Antigenicity 

from Clones Transformed In Vitro by Methylcholanthrene." Int. J. Cancer, 8 (1971), 410-420. 

B. C3H/10T1/2 MOUSE EMBRYO FIBROBLASTS 

1. Bairstow, F., and C. Heidelberger. "Increased Thymidine Uptake 

by Methylcholanthrene-Treated C3H/10T1/2 Cells." Int. J. Cancer, 16 (1975), 370-375. 

2. Benedict, W.F. , N. Ricker, J. Faust, and R.E. Kouri. "Malignant 

Transformation of Mouse Cells by Cigarette Smoke Condensate." 

Cancer Res., 35 (1975), 857-860. 

3. Benedict, W.F. "Morphological Transformation and Chromosomal 

Abberrations Produced by Two Hair Dye Components." Nature, 260 

(1976), 368-369. 

4. Bertram, J.S., and C. Heidelberger. "Cell Cycle Dependency of 

Oncogenic Transformation Induced by N-Methyl-N'-nitro-N­ nitrosoguanidine in Culture." Cancer Res., 34 (1974), 526-537. 

5. Bertram, J.S., A.R. Peterson, and C. Heidelberger. "Chemical 

Oncogenesis in Cultured Mouse Embryo Cells in Relation to the 

Cell Cycle." In Vitro, 11 (1975), 96-106. 

27 
6.

 Embleton, M.J., and C. Heidelberger. "Neoantigens on Chemically 

Transformed Cloned C3H Mouse Embryo Cells." Capper Res., 35 

(1975), 2049-2055. 

7. Heidelberger, C., C.A. Reznikoff, and D.F. Krahn. "Malignant 

Transformation of Cells in Culture Using Oncogenic Chemicals." 

In: Tissue Culture Methods and Applications. P.R. Kruse, Jr. and 

M.K. Patterson, Jr. (Eds.). Academic Press, Inc., New York, 1973, 644-653. 

8. Jones, P.A., W.F. Benedict, S. Strickland, and E. Reich. "Fibrin 

Overlay Methods for the Detection of Single Transformed Cells and 

Colonies of Transformed Cells." Cell, 5 (1975), 323-329. 

9- Jones, P.A. , W.E. Lang, A. Gardner, C.A. Nye, L.M. Fink, and W.F. Benedict. "In Vitro Correlates of Transformation in 

C3H/10T1/2 Clone 8 Mouse Cells." Cancer Res., 36 (1976), 2863-2867. 

10. Jones, P.A., W.F. Benedict, M.S. Baker, S. Mondal, U. Rapp, and 

C. Heidelberger. "Oncogenic Transformation of C3H/10T1/2 Clone 8 

Mouse Embryo Cells by Halogenated Pyrimidine Nucleosides." Cancer 

Res., 36 (1976), 101-107. 

11. Malick, L.E., and R. Langenbach. "Scanning Electron Microscopy 

of In Vitro Chemically Transformed Mouse Embryo Cells." J. Cell 

Biol., 68 (1976), 654-664. 

12. Mondal, S., and C. Heidelberger. "Transformation of C3H/10T1/2 

CL8 Mouse Embryo Fibroblasts by Ultraviolet Irradiation and 

Phorbol Ester." Nature, 260 (1976), 710-711. 

13. Mondal, S., D.W. Brankow, and C. Heidelberger. "Two-Stage 

Chemical Oncogenesis in Cultures of C3H/10T1/2 Cells." Cancer 

Res., 36 (1976), 2254-2260. 

14. Nesnow, S., and C. Heidelberger. "The Effect of Modifiers of 

Microsomal Enzymes on Chemical Oncogenesis in Cultures of C3H 

Mouse Cell Lines." Cancer Res., 36 (1976), 1801-1808. 

15. Peterson, A.R. , J.S. Bertram, and C. Heidelberger. "Cell Cycle 

Dependency of DNA Damage and Repair in Transformable Mouse 

Fibroblasts Treated with N-methyl-N'-nitro-N-nitrosoguanidine." 

Cancer Res., 34 (1974), 1600-1607. 

16. Peterson, A.R., J.S. Bertram, and C. Heidelberger. "DNA Damage 

and Its Repair in Transformable Mouse Fibroblasts Treated with 

N-methyl-N'-nitro-N-nitrosoguanidine." Cancer Res., 34 (1974), 

1592-1599. 

28 
17.

 Rapp, U.K. , R.C. Nowinski, C.A . Reznikoff, and C. Heidelberger. "The Role of Endogenous Oncornaviruses in Chemically-Induced 

Transformation. 1. Transformation Independent ' of Virus 

Production." Virology, 65 (1975), 392-409. 

18. Reznikoff, C.A. , D.W. Brankow, and C. Heidelberger. 

"Establishment and Characterization of a Cloned Line of C3H Mouse 

Embryo Cells Sensitive to Post confluence Inhibition of 

Division." Cancer Res., 33 (1973), 3231-3238. 

19. Reznikoff, C.A. , J.S. Bertram, D.W. Brankow, and C. Heidelberger. "Quantitative and Qualitative Studies of Chemical 

Tranformation of Cloned C3H Mouse Embryo Cells Sensitive to 

Postconfluence Inhibition of Cell Division." Cancer Res., 33 

(1973), 3239-3249. 

20. Terzaghi, M., and J.B. Little. "Repair of Potentially Lethal 

Radiation Damage in Mammalian Cells is Associated with 

Enhancement of Malignant Transformation." Nature, 235 (1975), 548-549. 

21. Terzaghi, M. , and J.B. Little. "X-Radiation-Induced 

Transformation in a C3H Mouse Embryo-Derived Cell Line." Cancer 

Res., 36 (1976), 1367-1374. 

C. BALB/3T3 CELLS 

1. Aaronson, S.A., andG.J. Todaro. "Development of 3T3-Like Lines 

From BALB/C Mouse Embryo Cultures: Transformation Susceptibility 

to SV40." J. Cell. Physiol., 72 (1968), 141-148. 

2. Boone, C.W. "Malignant Hemangioendotheliomas Produced By 

Subcutaneous Inoculation of BALB/3T3 Cells Attached to Glass 

Beads." Science, 188 (1975), 68-70. 

3. Boone, C.W. , and N. Takeichi, M. Paranjpe, and R. Gil den. "Vasoformative Sarcomas Arising From BALB/3T3 Cells Attached to 

Solid Substrates." Cancer Res., 36 (1976), 1626-1633. 

4. DiPaolo, J.A. , K. Takano, and N.C. Popescu. "Quantitation of 

Chemically Induced Neoplastic Transformation of BALB/3T3 Cloned 

Cell Lines." Cancer Res., 32 (1972), 2686-2695. 

5. Kakunaga, T. "Caffeine Inhibits Cell Transformation By 

4-nitroquinoline-1-oxide." Nature, 258 (1975), 248-250. 

29 
6.

 Kakunaga, T. "Quantitative System For Assay of Malignant 

Transformation By Chemical Carcinogens Using a Clone Derived From 

BALB/3T3." Int. J. Cancer, 12 (1973), 463-473. 

7. Kakunaga, T. "Requirement For Cell Replication in the Fixation 

and Expression of the Transformed State in Mouse Cells 

Treated With 4-Nitroquinoline-1-Oxide." Int. J. Cancer, 14 

(1974), 736-742. 

8. Kakunaga, T. "The Role of Cell Division in the Malignant 

Transformation of Mouse Cells Treated With 3-Methylcholan­ threne." Cancer Res., 35 (1975), 1637-1642. 

9. Kuroki, T., S.Y. Miyashita, and Y. Yuasa. "Development of a 

3T3-Like Line From an Embryo Culture of an Inbred Strain of 

Syrian Golden Hamster." Cancer Res., 35 (1975), 1819-1825. 

10. Kuroki, T., and S. Yamakawa. "Kinetics of Uptake of 

2-Deoxy-D-Glucose and 2-Aminoisobutyric Acid in Chemically 

Transformed Cells." Int. J. Cancer, 13 (1974), 240-245. 

11. Olinici, C.D. , C.H. Evans, and J.A. DiPaolo. "Chromosome 

Patterns of Non-transformed Variants From Chemically Transformed 

BALB/3T3 Cells." J. Cell. Physiol., 83 (1974), 401-408. 

12. Oshiro, Y. , L.E. Gerschenson, and J.A. DiPaolo. "Carcinomas From 

Rat Liver Cells Transformed Spontaneously in Culture." Cancer 

Res., 32 (1972), 877-879. 

13. Quarles, J.M., and R.W . Tennant. "Effects of Nitrosocarbaryl On 

BALB/3T3 Cells." Cancer Res., 35 (1975), 2637-2645. 

14. Yoshida, M.D. , M. Sasaki, N. Takeichi, and C.W. Boone. "Karyotypes of Vasoformative Sarcomas Arising From BALB/3T3 Cells 

Attached To Polycarbonate Plates." Cancer Res., 36 (1976), 2235-2240. 

30 
2.2.2 PROMOTION OF ONCOGENESIS IN CELL CULTURE BY PHORBOLESTERS 

SUKDEB MONDAL 

Los Angeles County - University of Southern California, Comprehensive Cancer Research Center, Los Angeles, CA 

Berenblum (5) and later Mottram (27) demonstrated two stage 

carcinogenesis of mouse skin by polycyclic hydrocarbons followed by 

croton oil. They initiated the process of carcinogenesis by 

application of 7, 12-dimenthylbenz(a)anthracene (DMBA) or benzo(a) 

pyrene (BP), and promoted the process with repeated application of 

croton oil. Later Hecker (17) isolated compounds from croton oil, some of which are active promoters, e.g., TPA (12-0-tetradecanoyl 

phorbol-13-acetate), FDD (Phorbol-12, 13-didecanoated) and some of 

which are inactive as promoters, e.g., phorbol. These pure compounds 

facilitated investigations of the two stage carcinogenesis: "initi­ ation" and "promotion." Since the original work, many laboratories 

have studied the two stage process of carcinogenesis and the subject 

has been reviewed periodically (5,10,11,35,42). 

For the study of carcinogenesis in a well-controlled uniform 

environment, various quantitative systems for obtaining chemical 

oncogenesis in cell cultures have been developed, and important 

mechanistic informations have been obtained, as reviewed by 

Heidelberger (18,19) and Casto and DiPaolo (12). Sivak and Van Duuren 

(37,3^) demonstrated some effects of phorbolestors on cultured 3T3 

cells and Lasne et al. (23) reported on experiments suggesting a 

promotion effect by TPA on oncogenic transformation of rat embryo 

cells in culture. 

We have demonstrated two stages of chemical oncogenesis in 

culture C3H/10T1/2 mouse embryo cells (25,26). This cell line has 

been developed in our laboratory by Rezinkoff et al. (32,33). Treatment of the cells with a subeffective concentration (0.1 ug/ml) 

of 3-methylcholanthrene (MCA), DMBA, or BP, followed 4 days later by a 

nontransforming and nontoxic amount (0.1 ug/ml) of TPA or FDD pro­ duced transformation. Phorbol was inactive. When TPA was added 

immediately after hydrocarbon treatment there was a significant 

inhibition of transformation. When TPA treatment preceded MCA 

treatment, no enhancement of transformation was observed, corroborating the in vivo experience (7,3*0. Another experiment 

showed that the promoting action of TPA could not be attributed to a 

stimulation of cell division, though cell division was essential but 

TPA had a property specific for promotion. In mouse skin it has been 

found that all inducers of hyperplasia are not promoters (6,11,17). Inhibition of transformation, when TPA was added immediately after 

31 
hydrocarbon treatment was attributed to the initial inhibition of cell
 

division by TPA preventing the transformed state to get "fixed" which
 

requires at least 2 cell divisions (21).
 

UV light was shown to be carcinogenic by repeated exposures
 

of rat and mouse skin (15). When UV was given in a single exposure
 

followed by repeated application of croton oil, there was high
 

incidence of tumor development in mouse skin, whereas the single UV
 

exposure was ineffective (3D- We showed that UV in single or
 

repeated exposures at different dose levels did not transform
 

C3H/10T1/2 cells (25), but acted solely as initiator, i.e., when UV
 

treated cells were further cultured in the presence of TPA, there was
 

high frequency of transformation.
 

So, it is clear that the "initiation" and "promotion" stages
 

of carcinogenesis occur in our mouse embryo C3H/10T1/2 cells in
 

culture, just as in mouse skin. When the higher concentration of MCA,
 BP or DMBA were used, which by themselves produced considerable
 

transformation, no further enhancement of transformation by TPA was
 

observed. This is in agreement with mouse skin experiments (6,8,10).
 It was also observed that initial treatment with the carcinogenic
 

hydrocarbon, i.e., "initiation" is essentially irreversible (8,10).
 When the hydrocarbon or its metabolites are not oncogenic to a
 

particular species or cells, there is no "initiation" and so there is
 

no tumor development after repeated application of "promoters." This
 

was evident in the experiments where the cells were treated with the
 

MCA11, 12 oxide (an active metabolite of MCA) and then with TPA, there
 

was no transformation. This epoxide is highly oncogenic in mouse
 

prostate and hamster embryo cells (20,24) but completely ineffective
 

in C3H/10T1/2 cells. Bereblum et al. have recently proposed that such
 

a 2 stage process occurs in the production of mammary tumors and
 

leukemia in female Wistar rats (2) and of liver and lung tumors in AKR
 

mice (1). Peranino et al. have also produced evidence that the
 

chemical induction of rat hepatomas is a 2 stage process (29).
 

The mechanisms of "promotion" is still unknown. After
 

application of TPA to the adult mouse skin it was found that there was
 

an initial depression of DNA synthesis for first 10-12 hours followed
 

by an increased extent of synthesis (3,9,22). Similar effect was
 

found in the growth rate of different cell lines in culture (14). We
 

have also similar observations (unpublished data). TPA has been found
 

to inhibit semiconservative and repair DNA synthesis (3,9,22). Sivak
 

and Van Duuren showed that it enhanced RNA synthesis in stationary
 

cultures of 3T3 mouse fibroblasts (30) and Baird et al. found the same
 

in mouse skin (4). The protein synthesis was also increased in the
 

cells of mouse skin after TPA application (3). These increases in the
 

maceromolucular synthesis was similar as observed following a single
 

32
 
application of polycyclic hydrocarbons in sufficient quantity to
 

result in tumor development (40). TPA binds weakly with skin proteins
 

and such binding has been considered to be important for its promoting
 

activity (36,41). O'Brien et al. found a good correlation between the
 

stimulatory action on the induction of ornithine decarbonxylase by a
 

series of phorbol esters and their promoting activity after topical
 

application on the mouse skin (28). Wigler and Weinstein (43)
 

reported that TPA is a potent inducer of plasminogen activator in
 

chick embryo fibroblasts and hela cells.
 

None of the above work explains clearly the biochemical
 

pathway of "initiation" and "promotion." Most of the work was done on
 

mouse skin which is not an ideal tissue for the study of the cellular
 

events that occur at each stage because of the obvious difficulty in
 

obtaining a homogenous population of target cells. Our study of 2
 

stage oncogenesis in cell culture opens up new possibilities for
 

investigating the cellular and molecular mechanisms of both
 

"initiation" and "promotion" and for developing a relatively rapid
 

prescreening system for environmental promoting agents.
 

REFERENCES
 

1.	 Armuth, V., and I. Berenblum. "Systemic Promoting Action of
 

Phorbol in Liver and Lung Carcinogenesis in AK Mice." Cancer
 

Res., 32 (1972), 2259-2262.
 

2.	 Armuth, V., and I. Berenblum. "Promotion of Mammary
 

Carcinogenesis and Leukemogenic Action by Phorbol in Virgin
 

Female Wistar Rats." Cancer Res., 34 (1974), 2704-2707.
 

3.	 Baird, W.M., J.A. Sedgwick, and R.K. Boutwell. "Effects of
 

Phorbol and Four Diesters of Phorbol in the Incorporation of
 

Tritiated Precursors Into DNA, RNA and Protein in Mouse
 

Epidermis." Cancer Res.. 31 U97D, 1434-1439­

4.	 Baird, W.M., P.W. Melera, and R.K. Boutwell. "Acrylamide Gel
 

Electrophoresis Studies of the Incorporation of Cytidine-3H Into
 

Mouse Skin RNA at Early Times After Treatment." Cancer Res., 32
 

(1972), 781-788.
 

5.	 Berenblum, I. "The Carcinogenic Action of Croton Resin." Cancer
 

Res., 1 (1941), 44-48.
 

6.	 Berenblum, I. "A Speculative Review: The Probable Nature of
 

Promoting Action and Its Significance in the Understanding of the
 

Mechanism of Carcinogenesis." "Cancer Res., 14 (1954), 471-477.
 

33
 
7.

 Berenblum, I., and N, Haran-Ghera. "The Significance of the 

Sequence of Initiating and Promoting Actions in the Process of 

Skin Carcinogenesis in Mouse." Brit. J. Cancer, 9 (1955), 268-271. 

8. Berenblum, I., and P. Shubik. "The Persistance of Latent Tumor 

Cells Induced in the Mouse's Skin By Single Application of 

9,10-Dimethyl-1,2-benzanthracene." Brit. J. Cancer, 3 (1949), 384-386. 

9. Bertsch, S., and F. Marks. "Lack of An Effect of Tumor Promoting 

Phorbol Esters and of Epidermal G1 Chalone on DMA Synthesis in 

the Epidermis of New Born Mice." Cancer Res., 34 (1974), 3283-3288. 

10. Boutwell, B.K. "Some Biological Aspects of Skin 

Carcinogenesis." Prog. Exp. Tumor Res., 4 (1964), 207-250. 

11. Boutwell, R.K. "The Function and Mechanism of Promoters of 

Carcinogensis." CRC Critical Reviews in Toxicology, 2 (1974), 419-443. 

12. Casto, B.C., and J.A. DiPaolo. "Virus, Chemicals and Cancer." 

Prog. Med. Virol., 16 (1973), 1-47. 

13. Cleaver, J.E., and R.B. Painter. "Absence of Specificity in 

Inhibition of DNA Repair Replication by DNA-binding Agents, Cocarcinogens, and Steroids in Human Cells." Cancer Res., 35 

(1975), 1773-1778. 

14. Diamond, L., S. O'Brien, C. Donaldson, and Y. Shimizu. "Growth 

Stimulation of Human Diploid Fibroblasts By the Tumor Promoter, 12-0-Tetradecanoyl-phorbol-13-acetate." Int. J. Cancer, 13 

(1974), 721-730. 

15. Findlay, G.M. "Ultraviolet Light and Skin Cancer." Lancet, 215 

(1928), 1070. 

16. Gaudin, D., R.S. Gregg, and K.L. Yielding. "Inhibition of DNA 

Repair Replication By Cocarcinogens." Biochm. Biophys. Res. Commun., 48 (1972), 945-949. 

17- Hecker, E. "Isolation and Characterization of the Cocarcinogenie 

Principles From Croton Oil." Methods Cancer Res., 6 (1971), 439-484. 

18. Heidelberger, C. "Chemical Oncogenesis in Culture." Adv. Cancer 

Res., 18 (1974), 317-366. 

34 
19.

 Heidelbeger, C. "Chemical Carcinogenesis." Ann. Rev. Biochem., 44 (1975), 79-121. 

20. Huberman, E., T. Kuroki, H. Marquardt, J.K. Selkirk, C. Heidelberger, P.L. Grover, and P. Sims. "Transformation of 

Hamster Embryo Cells By Epoxides and Other Derivatives of 

Polycyclic Hydrocarbons." Cancer Res., 32 (1972), 1391-1396. 

21. Kakunaga, T. "Requirement For Cell Replication in the Fixation 

and Expression of the Transformed State in Mouse Cells Treated 

With 4-Nitroquinoline-1-oxide." Int. J. Cancer, 14 (1974), 736-742. 

22. Krieg, L., I. Kuhlman, and F. Marks. "Effect of Tumor Promoting 

Phorbol Esters and of Acetic Acid on Mechanisms Controlling DNA 

Synthesis and Mitosis (Chalones) and on the Biosynthesis of 

Histidine-rich Protein in Mouse Epidermis." Cancer Res., 34 

(1974), 3135-3146. 

23. Lasne, C., A. Gentil, and I. Chouroulinkov. "Two Stage Malignant 

Transformation of Rat Fibroblasts in Tissue Culture." Nature, 247 (1974), 490-491. 

24. Marquardt, H., T. Kuroki, E. Huberman, J.K. Selkirk, C. Heidelberger, P.L. Grover, and P. Sims. "Malignant 

Transformation of Cells Derived From Mouse Prostate By Epoxides 

and Other Derivatives of Polycyclic Hydrocarbons." Cancer Res., 32 (1972), 716-720. 

25. Mondal, S., and C. Heidelberger. "Transformation of C3H/10T1/2 

C18 Mouse Fibroblasts By Ultraviolet Irradiation and Phorbol 

Ester." Nature, 260 (1976), 710. 

26. Mondal, S., D.W. Brankow, and C. Heidelberger. "Two Stage 

Chemical Oncogenesis In Cultures of C3H/10T1/2 Cells." Cancer 

Res., 36 (1976), 2254-2260. 

27. Mottran, J.C. "A Developing Factor in Experimental 

Blastogenesis." J. Pathol. Bacterio., 56 (1944), 181-187. 

28. O'Brien, T.G., R.C. Simsiman, andR.K . Boutwell. "Induction of 

the Polyamine-bi©synthetic Enzymes in Mouse Epidermis and Their 

Specificity for Tumor Promotion." Cancer Res., 35 (1975), 2426-2433. 

29. Peraino, C., R.J.M. Fry, E. Staffeldt, and J.P. Christopher. "Comparative Enchancing Effects of Phenobarbital, Aminobarbital, 

35 
Diphenylhydantoin and Dichlorodiphenyltrichloroethane on 

2-Acetylaminofluorene-induced Hepatic Tumorigenesis in the Rat." 

Cancer Res., 35 (1975), 2884-2890. 

30. Poirier, M.C. , B.T. DeCicco, and M.W. Lieberman. "Nonspecific 

Inhibition of DNA Repair Synthesis By Tumor Promoters in Human 

Diploid Fibroblasts Damaged With N-Acetoxy-2-acetylaminofluorene." 

Cancer Res., 35 (1975), 1392-1397. 

31. Pound, A.W. "Induced Cell Proliferation and the Initiation of 

Skin Tumor Formation in Mice By Ultraviolet Light." Pathology, 2 

(1970), 269-275. 

32. Reznikoff, C.A., D.W. Brankow, and C. Heidelberger. "Establish­ ment and Characterization of a Cloned Line of C3H Mouse Embryo 

Cells Sensitive to Post confluence Inhibition of Division." Cancer 

Res., 33 (1973), 3231-3238. 

33. Reznikoff, C.A. , J.S. Bertram, D.W. Brankow, and C. Heidelberger. "Quantitative and Qualitative Studies of Chemical Transformation 

of Cloned C3H Mouse Embryo Cells Sensitive €o Post confluence 

Inhibition of Cell Division." Cancer Res., 33 (1973), 3239-3249. 

34. Roe, F.J.C. "The Effect of Applying Croton Oil Before a Single 

Application of 9,10-Dimenthyl-1,2-benzanthracene (DMBA)." Brit. J. Cancer, 13 (1959), 89-91. 

35. Saffiotti, U., and P. Shubik. "Studies On Promoting Action in 

Skin Carcinogenesis." Natl. Cancer Inst. Monograph, 10 (1963), 489-507. 

36. Scribner, J.D., and R.K. Boutwell. "Inflammation and Tumor 

Promotion-Selective Protein Induction in Mouse Skin by 

Promoters." Europ. J. Cancer, 8 (1972), 617-621. 

37. Sivak, A. , and B.L. Van Duuren. "Phenotypic Expression of 

Transformation: Induction in Cell Culture By a Phorbol Ester." 

Science, 157 (1967), 1443-1444. 

38. Sivak, A., and B.L. Van Duuren. "A Cell Culture System For the 

Assessment of Tumor Promoting Activity." J. Nat. Cancer Inst., 44 (1970), 1091-1097. 

39. Sivak, A., and B.L. Van Duuren. "RNA Synthesis Induction in Cell 

Culture By a Tumor Promoter." Cancer Res., 30 (1970), 1203-1205. 

36 
40.

 Slaga, T.J., G.T. Bowden, B.C. Shapas, and R.K . Boutwell. "Macromolecular Synthesis Following a Single Application of 

Polycyclic Hydrocarbons Used As Initiators of Mouse Skin 

Tumorigenesis." Cancer Res., 3^ (1974), 771-777. 

41. Traut, M., G. Kreibich, and E. Hecker. "Uber Die Protein Bindung 

Carcinogener Kohlenwasserstoffe Und Cocarcinogener Phorbol Ester." 

Aktuelle Problem Aus Den Gebiet Per Cancerologie, 3 (1971), 91-96. 

42. Van Duuren, B.L. "Tumor Promoting Agents in Two-stage 

Carcinogenesis." In: F. Homberger (Ed.), Prog. Exptl. Tumor 

Res., 11 (1969), 31-68. 

43. Wigler, M. , and B.I. Weinstein. "Tumor Promoter Induces 

Plasminogen Activator." Nature, 259 (1976), 232-233­

37 
2.2.3 RADIATION TRANSFORMATION IN VITRO 

JOHN B. LITTLE 

Laboratory of Radiobiology, Harvard University, School of 

Public Health, Boston, MA 

In the past there has been conflicting evidence with respect 

to the induction by X-rays of oncogenic transformation in mammalian 

cells in vitro. Borek and Sachs (1,2) and later Borek and Hall (3) 

reported inducing malignant transformation in primary hamster embryo 

cells with X-ray doses as low as 1 rad. Their work has been 

questioned by some owing to the lack of firm evidence that morpho­ logically transformed cells formed persistent viable tumors upon 

reinjection into hamsters. Pollack et al. (16) observed X-ray-induced 

transformation in 3T3 cultures following exposure to 1500 rads; 

turnorigenicity of transformed clones was found to be associated with 

the appearance of C-type viral particles. A number of other investi­ gators, however, were unable to induce oncogenic transformation in 

vitro by radiation alone (8,11,17), although radiation enhanced the 

transformation induced by chemical or viral agents. Klein (12,13 ) 

presented evidence that the morphologic transformation she observed in 

vitro was an indirect effect, due to the close contact of the 

radiation killed and damaged cells with viable cells and presumably 

mediated by an as yet unidentified cellular transforming factor. Recent studies utilizing the 10T1/2 C1 8 mouse embryo fibroblast cell 

system (18,19) support those of Borek et al. Direct X-ray induced 

malignant transformation has been induced in this cell line. Transformation is independent of the presence of dead cells or feeder 

layers, and upon injection in syngeneic hosts transformed colonies 

form large viable tumors which will eventually kill the animal. 

Studies of the dose-response relationship for in vitro 

transformation (3,19) indicate that an exponential rise in the trans­ formation frequency per viable cell occurs with increasing radiation 

dose up to 300-400 rads. At this point a plateau is reached and the 

transformation frequency per surviving cell remains constant with 

doses up to 1500 rads (19). The incorporation of the thymidine 

analogue BUdR into cellular DNA enhanced their sensitivity to 

X-ray-induced transformation (Terzaghi and Little, unpublished) 

implicating DNA as the target molecule in the cell. Splitting the 

radiation dose into two equal fractions separated by several hours 

enhanced the transformation frequency at low total doses (50-75 rads) 

(4) , but led to a decline in the transformation frequency at higher 

total doses (300-800 rads) (20). Neutron irradiation appears to be 

more efficient per rad of dose than x-irradiation in inducing 

transformation in vitro (5). 

38 
The development of malignant transformation in vitro can be 

thought of as involving at least two steps: 1) The fixation of the 

transformed state (presumably irreversible DNA damage); and 2) its 

subsequent expression in terms of morphologically altered cells. The 

fixation of X-ray damage appears to require that one cell division 

occurs within 2M to 48 hours after exposure (1,2,18). The expression 

of the transformed state in 10T1/2 cells, on the other hand, requires 

approximately 12 subsequent cell divisions (19). Recent evidence 

implicates DNA repair processes as an important factor in fixation of 

transformational damage (18). In liquid holding recovery type 

experiments, confluent 10T1/2 cell cultures were x-irradiated, then 

held in confluency for varying periods of time (thus preventing cell 

division) before subculture at low density. Survival was markedly 

enhanced, reaching a maximum with a repair interval of 3-6 hours. 

Transformation was initially enhanced reaching maximum at 3 hours, but 

declined with longer repair times. This result has been interpreted 

as offering evidence for the action of two repair processes after 

x-irradiation: The first a rapid, error producing (error-prone) 

process; and the second a slower error correcting mechanism (18). We 

are currently investigating the nature of these processes. 

Transformation has also been induced in Syrian hamster cells 

(9) and in 10T1/2 mouse embryo fibroblasts (7) by exposure to 254 nM 

ultraviolet light. The shape of the dose-trans form at ion frequency 

curve in 10T1/2 cells was similar to that for X-rays, reaching the 

plateau at a dose of 75-100 ergs/mm2 (7). During the exponential rise 

at lower doses, the transformation frequency per viable cell was 

similar to that seen following X-ray exposures which yielded similar 

survival levels, but the frequency on the plateau (100-300 ergs/mm2 

was lower by a factor of 2-3 than that for X-rays. Several reports 

have indicated that treatment with caffeine following exposure to UV 

light or UV-like chemical agents suppresses the induction of tumors in 

animals. Caffeine is thought to block a specific DNA repair process 

in UV-irradiated cells. We have very preliminary data (Chan and 

Little, unpublished) which suggest that post-irradiation incubation 

with 1 mM caffeine significantly suppressed UV-induced transformation 

in 10T1/2 cells. 

There have been a number of investigations of the 

interactions between x-rays and other agents in the induction of 

transformation in vitro. These agents include viruses (11,15,17), 

chemical carcinogens (8,10,21), and noncarcinogenic compounds 

(6,14,19). DiPaolo et al. (8) demonstrated clear synergistic interaction between X-rays and benzo(a)pyrene (BP) in primary hamster 

embryo cell cultures. X-irradiation alone did not induce transforma­ tion in these cells, and the enhancement of BP-induced transformation 

was maximal when x-irradiation preceded BP exposure by an interval of 

39 
48 hours. This finding has since been confirmed with mouse 10T1/2 

cells (21) though maximal enhancement occurred with an interval of 18 

hours between exposures. Such enhancement did not occur when exposure 

to BP was preceded by UV irradiation (9). Treatment of cells with 

Amphoterecin-B after x-irradiation suppressed transformation (19). Mondal and Heidelberger (14) showed that a high frequency of transfor­ mation resulted when cells were exposed to the phorbol ester TPA after 

irradiation with 254 nM UV light in doses which yielded no 

transformation by UV alone. TPA is a classical promoting agent which 

by itself does not induce transformation. Brouty-Boye and Little (6) 

showed that post irradiation treatment with interferon greatly 

enhanced X-ray induced transformation. They hypothesize that this 

effect may be due to a selective action of interferon in suppressing 

cell proliferation among the normal cells, allowing potential 

transformants to multiply preferentially and be expressed as 

transformed foci. 

These results stress the importance of the proliferative state of 

the cell population, and of the interactions between agents in the 

induction of transformation. 

REFERENCES 

1. Borek, C., and L. Sachs. "In Vitro Cell Transformation By 

X-irradiation." Nature, 210 (1966), 276-278. 

2. Borek, C., and L. Sachs. "Cell Susceptibility to Transformation 

by X-irradiation and Fixation of the Transformed State." Proc. Natl. Acad. Sci. U.S.A., 57 (1967), 1522-1527­

3. Borek, C., and E. Hall. "Transformation of Mammalian Cells In 

Vitro by Low Doses of X-rays." Nature, 243 (1973), 450-453­

4. Borek, C., and E. Hall. "Effect of Split Doses of X-rays on 

Neoplastic Transformation of Single Cells." Nature, 252 (1974), 499-501. 

5. Borek, C. "In Vitro Cell Transformation By Low Doses of 

X-irradiation and Neutrons." In: Biology of Radiation 

Carcinogenesis. J.M. Yuhas, R.W. Tennant, and J.D. Regan (Eds.). Raven Press, New York, 1976, 309-326. 

6. Brouty-Boye, D. , and J.B. Little. "Enhancement of X-ray Induced 

Transformation In Vitro By Interferon." Cancer Res. (1977) in 

press. 

40 
7.

 Chan, G.C. , and J.B. Little. "Induct!an of Oncogenic Transformation In Vitro By Ultraviolet Light." Nature. 264 (1976), 

442-444. 

8. DiPaolo, J.A., P.J. Donovan, and R.L . Nelson. "X-irradiation 

Enhancement of Transformation By Benzo(a)pyrene in Hamster Embryo 

Cells." Proc. Natl. Acad. Sci. U.S.A., 68 (1971), 1734-1737. 

9. DiPaolo, J.A., and P.J. Donovan. "In Vitro Transformation of 

Syrian Hamster Cells by UV Irradiation is Enhanced By 

X-irradiation and Unaffected By Chemical Carcinogens." Int. J. 

Radiat. Biol., 30 (1976), 41-53. 

10. DiPaolo, J.A. , P.J. Donovan, and N.C. Popescu. "Kinetics of 

Syrian Hamster Cells During X-irradiation Enhancement of 

Transformation In Vitro By Chemical Carcinogen." Radiation Res., 

66 (1976), 310-325. 

11. Coggin, J.H. "Enhanced Virus Transformation of Hamster Embryo 

Cells In Vitro." J. Virol., 3 (1969), 458-462. 

12. Klein, J.C. "Absence of Malignant Transformation After Weekly 

Irradiation of a Mouse Spleen in Culture." J. Natl. Cancer 

Inst., 37 (1966), 655-661. 

13. Klein, J.C. "Evidence Against a Direct Carcinogenic Effect of 

X-rays In Vitro." J. Natl. Cancer Inst., 52 (1974), 1111-1116. 

14. Mondal, S., and C. Heidelberger. "Transformation of C3H/10T1/2 

C18 Mouse Embryo Fibroblasts By Ultraviolet Irradiation and a 

Phorbol Ester." Nature, 260 (1976), 710-711. 

15. Pollock, E.J., and G.J. Todaro. "Radiation Enhancement of SV40 

Transformation in 3T3 and Human Cells." Nature, 219 (1968), 

520-521. 

16. Pollock, E.J., S.A. Aaronson, and G. Todaro. "X-irradiation of 

Balb 3T3: Sarcoma Forming Ability and Virus Induction." Intern. 

J. Radiation Biol., 17 (1970), 97-100. 

17. Stoker, M. "Effect of X-irradiation on Susceptibility of Cells 

to Transformation By Polyoma Virus." Nature, 200 (1963), 756-758. 

18. Terzaghi, M. , and J.B. Little. "Repair of Potentially Lethal 

Radiation Damage in Mammalian Cells is Associated With 

Enhancement of Malignant Transformation." Nature, 253 (1975), 

548-549. 

41 
19.

 Terzaghi, M. , and J.B. Little. "X-Radiation Transformation of 

C3H Mouse Embryo-derived Cell Line." Cancer Res., 36 (1976), 1367-1374. 

20. Terzaghi, M. , and J.B. Little. "Oncogenic Transformation In 

Vitro Following Split-dose X-irradiation." Int. J. Radiat. Biol., 

29 (1976), 583-587. 

21. Terzaghi, M. , and J.B. Little. "Interactions Between Radiation 

and Benzo(a)pyrene in an In Vitro Model for Malignant 

Transformation." In: Experimental Lung Cancer. Carcinogenesis 

and Bioassays. E. Karbe and J.F. Parks, (Eds.) Springer-Verlag, Berlin, 1974, 497-506. 

42 
Carcinogens are drugs since they alter the behavior of 

biological systems. As such their mechanism of action can be 

investigated by pharmacological methodology similar to that 

successfully used for other more conventional drugs. Just as many 

studies of conventional pharmacology required the development of in 

vitro systems, so too detailed investigations into the pharmacology of 

carcinogens required the availability of in vitro systems which can be 

closely controlled and quantitated. Several such systems are now 

available and have been described at this Conference. This paper is a 

review of factors modifying transformation frequency in the 

C3H/10T1/2/CL8 cell line, developed as an assay system for chemically 

induced neoplastic transformation in culture by Reznikoff et al. (14,15). Transformation frequency (TF) is here defined as the number 

of transformed foci per dish divided by the number of surviving cells 

in that dish x 100 (5). This value is thus normalized for variations 

in cell survival from treatment group to treatment group. 

A. PHARMACODYNAMIC MODI FICATIONS OF TRANSFORMATION FREQUENCY 

1. Dose. Straight line log dose response relationships have 

been obtained for malignant transformation induced by polycyclic 

hydrocarbons (14 ) X-rays (16) and FUdR (8). TF's of up to 10% have 

been obtained (14). 

2. Time. Within limits, increasing the time of exposure to the 

carcinogen increases the TF (8,14). 

3. Cell Cycle Dependency. The TF decreases progressively if 

cells are held in the non-cycling state for up to 48 hours 

post-treatment with X-rays. A transient rise in TF after brief (2-4 

hours) holding periods has been attributed to the introduction of 

errors during repair of potentially lethal damage (17). 

4. Cell Cycle Phase Dependency. The methylating carcinogen 

N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) induces maximum TF in 

cells exposed in late G1 of the cycle (5), whereas the 

chemotherapeutic agents FUdR (8) and ara-C (1,7) exhibit maximum 

transformation when applied to S phase cells. No direct correlation 

was observed among the rate of repair of MNNG induced DMA damage and 

TF or cytotoxicity (6,12,13). 

43 
5. Cell Density at the Time of Treatment. Increasing the
 

number of cells from 500 to 10,000 cells/Petri dish reduces the TF
 

resulting from a standard dose of methylcholanthrene. Furthermore,
 relatively more of the foci appearing in heavily seeded dishes develop
 

at the edges of those dishes (14). Studies with X-rays have indicated
 

a 10-fold decrease in TF on increasing the number of viable cells/100
 

mm Petri dish from 400 to 1000 cells (16). This finding requires
 

confirmation with other transforming agents.
 

6. Cell Density Post-treatment. Expression of the malignant
 

phenotype can be reversibly inhibited by an increase in the
 

post-treatment saturation density as a result of exposure to high
 

serum levels. Conversely low saturation densities obtained with low
 

concentrations of serum (2.5 - 5.0$) allow maximum expression of the
 

malignant phenotype (2,3).
 

B. CARCINOGEN/DRUG INTERACTIONS
 

1. Actinomycin D. Exposure of 10T1/2 cells to low
 

concentrations of Actinomycin D (1 ng - 10 ng/ml), prior to,
 simultaneously with, or after, treatment with dimethylbenzanthracene
 

caused an enhancement of TF over the entire concentration range
 

studied (0.1 - 10.0 ug/ml). DMBA induced cytotoxicity was enhanced in
 

cultures treated with the higher concentrations, but not the lowest
 

concentration of Actinomycin D (4, J. S. Bertram, paper in
 

preparation).
 

2. Tumor Promoters. Marked enhancement of transformation was
 

observed in cultures treated with non-transforming concentrations of
 

methylchlolanthrene or benzpyrene, and treated 4 days later with a
 

concentration of tetradecanoylphorbol acetate (TPA) which by itself
 

produced little transformation. Treatment earlier than 4 days, or
 

prior to, carcinogen treatment reduced this response (9). A similar
 

enhancement of transformation has also been observed for ultraviolet
 

radiation (10).
 

3. Modifiers of Carcinogen Metabolism. Inhibition of aryl
 

hydrocarbon hydroxylase inhibits methylcholanthrene transformation,
 while stimulation of this enzyme • enhances the TF. Inhibition of
 

epoxide hydrase enhances TF. Simultaneous enhancement of aryl
 

hydrocarbon hydroxylase activity and inhibition of epoxide hydrase
 

activity gave the expected large increase in TF (11).
 

4. Interaction Between Two Carcinogens. In 10T1/2 cells
 

treated with benzpyrene an increase in TF was observed when cultures
 

were subsequently exposed to X-rays. The enhancement was maximal when
 

about 20 hours separated the two treatments, and was approximately 5
 

fold higher than that to be expected from the sum of the individual
 

exposures (18).
 

44
 
5. Amphotericin B. The polyene antibiotic has been used as an 

antimycotic agent in cell culture. It has been found to inhibit TF 

induced by both methylcholanthrene (14) and X-rays (16). Its 

mechanism of action in this respect is not understood. 

REFERENCES 

1. Benedict, W.F. , M.S. Baker, and A. Gardner. "Malignant 

Tranformation With Ara-C, FUdR, MIX and Bleomycin in 10T1/2 CL8 

Cells." Proc. Am. Assoc. Cancer Res., 16 (1975), 40. 

2. Bertram, J.S. "Effects of Serum Concentration on Expression of 

Chemically Induced Neoplastic Transformation in Culture." Proc. Am. Assoc. Cancer Res., 17 (1976), 51. 

3. Bertram, J.S. "Effects of Serum Concentration on the Expression 

of Carcinogen-induced Transformation in the C3H/10T1/2CL8 Cell 

Line." Cancer Res., 37 (1977), 514-523. 

4. Bertram, J.S. "Enhancement By Actinomycin D of Dimethyl­ benzanthracene Induced Cell Killing and Malignant Transformation 

in Culture C3H/10T1/2 CL8 Cells." Fed. Proc., 35 (1976) (abs), 410. 

5. Bertram, J.S., and C. Heidelberger. "Cell Cycle Dependency of 

Oncogenic Transformation Induced by N-methyl N'-nitro­ N-nitrosoguanidine." Cancer Res., 34 (1974), 526-537. 

6. Bertram, J.S., A.R. Peterson, and C. Heidelberger. "Chemical 

Oncogenesis in Cultured Mouse Embryo Cells in Relation to the 

Cell Cycle." In Vitro, 11 (1975), 97-106. 

7. Jones, P.A. , M.S. Baker, J.S. Bertram, and W.F. Benedict. "Cell 

Cycle Specific Oncogenic Transformation of C3H/10T1/2CL8 Mouse 

Embryo Cells by 1-g-D-arabinofuranosylcytosine." Cancer Res., 37 

(1977), in press. 

8. Jones, P.A. , W.F. Benedict, M.S. Baker, S. Mondal, U. Rapp, and 

C. Heidelberger. "Oncogenic Transforma- tion of C3H/10T1/2 Clone 

8 Mouse Embryo Cells by Halogenated Pyrimidine Nucleosides." 

Cancer Res., 36 (1976), 101-107. 

9. Mondal, S., D.W. Brankow, and C. Heidelberger. "Two Stage 

Chemical Oncogenesis In Cultures of C3H/10T1/2 Cells. Cancer 

Res., 36 (1976), 2254-2260. 

45 
10.

 Mondal, S., and C. Heidelberger. "Transformation of C3H/10T1/2 

C18 Mouse Embryo Fibroblasts By Ultraviolet Irradiation and a 

Phorbol Ester." Nature, 260 (1976), 710-711. 

11. Nesnow, S., and C. Heidelberger. "The Effect of Modifiers of 

Microsomal Enzymes on Chemical Oncogenesis in Cultures of C3H 

Mouse Cell Lines." Cancer Res.. 36 (1976), 1801-1808. 

12. Peterson, A.R. , J.S. Bertram, and C. Heidelberger. "DNA Damage 

and Its Repair in Transformable Mouse Fibroblasts Treated With 

N-methyl-N'-nitro-N-nitrosoguanidine." Cancer Res., 34 (1974), 1592-1599. 

13. Peterson, A.R. , J.S. Bertram, and C. Heidelberger. "Cell Cycle 

Dependency of DNA Damage and Its Repair in Transformable Mouse 

Fibroblasts Treated With N-methyl-N'-nitro-N-nitrosoguanidine." 

Cancer Res., 34 (1974), 1600-1607. 

14. Reznikoff, C.A. , J.S. Bertram, D.W. Brankow, and C. Heidelberger. "Quantitative and Qualitative Studies on Chemical 

Transformation of Cloned C3H Mouse Embryo Cells Sensitive to 

Postconfluence Inhibition of Cell Division." Cancer Res., 33 

(1973), 3239-3249. 

15. Reznikoff, C.A. , D.W. Brankow, and C. Heidelberger. "Establishment and Characterization of a Cloned Line of C3H Mouse 

Embryo Cells Sensitive to Postconfluence Inhibition of Cell 

Division." Cancer Res., 33 (1973), 3231-3238. 

16. Terzaghi, M., and J.B. Little. "X-Radiation-Induced Transforma­ tion in a C3H Mouse Embryo-Derived Cell Line." Cancer Res., 36 

(1976), 1367-1374. 

17. Terzaghi, M. , and J.B. Little. "Repair of Potentially Lethal 

Radiation Damage in Mammalian Cells is Associated With 

Enhancement of Malignant Transformation." Nature, (1975), 

548-549. 

18. Terzaghi, M. , and J.B. Little. "Interactions Between Radiation 

and Benzo(a)pyrene in An In Vitro Model For Malignant 

Transformation." In: Experimental Lung Cancer Carcinogenesis 

and Bioassays. E. Karbe and J.F. Park (Eds.), Springer-Verlag, Berlin, Heidelberg, New York, 1974, 497-506. 

46 
2.3	 MOUSE EPITHELIAL CELLS
 

2.3.1	 MOUSE EPIDERMAL CELL CULTURES AS AN IN VITRO MODEL FOR
 

THE STUDY OF CHEMICAL CARCINOGENESIS
 

STUART H. YUSPA
 

Experimental Pathology Branch, Carcinogenesis Program,
 Division of Cancer Cause and Prevention, National Cancer
 

Institute, N.I.H., Bethesda, MD
 

The use of cell culture systems for the study of chemical
 

carcinogenesis has been an important advance in recent years. Much of
 

the exciting information which has rapidly accumulated using such
 

models has been recently reviewed (7). In general most of the
 

existing models have concentrated on the use of cell strains or cell
 

lines of fibroblastic origin. These have the obvious advantages
 

related to their ability to adapt and grow well in monolayer culture.
 However the majority of human cancers are of epithelial origin as are
 

most of the malignancies derived from animal models for chemical
 

carcinogenesis. Thus the development of epithelial cell systems for
 

in vitro transformation would have the distinct advantage of a large
 

in vivo data base upon which to validate results obtained under the
 

highly artificial conditions of in vitro growth. Additionally
 

epithelial cell model systems may divulge specialized markers
 

associated with the transformed state which could be useful as
 

experimental tools as well as diagnostic aids for the early detection
 

of disease in human populations. Such specialized culture systems
 

also promise a potential approach to understanding the relationship
 

between normal differentiation and malignant transformation.
 

Certain problems are posed by the use of epithelial cultures
 

for carcinogenesis studies. The specialized cells may have strict
 

nutritional requirements for successful growth in vitro. The
 

differentiated state may be difficult to maintain. Adequate criteria
 

for recognizing the transformed state in vitro have been difficult to
 

establish for epithelial cells (25,26).
 

The use of mouse epidermal cell cultures seems especially
 

promising for in vitro studies since many of our current concepts
 

concerning mechanisms of chemical carcinogenesis were derived from
 

studies of mouse skin. Thus there is an enormous in vivo data base
 

(27) to validate findings in vitro. Assuming a parallel exists, one
 

should be able to separate and study at least two distinct stages of
 

carcinogenesis, initiation and promotion. A single dose of carcinogen
 

should be sufficient to induce transformation. Both premalignant and
 

47
 
malignant lesions should be produced and available for study. The
 

information gained might give insight toward developing an analogous
 

human cell model system using a tissue which also appears readily
 

transformable in vivo, the human epidermis.
 

«
 

This report will serve as an overview of the progress made
 

in the development, characterization and application of mouse
 

epidermal cell cultures for the study of chemical carcinogenesis. The
 

culture techniques and biological behavior of the cells in vitro will
 

first be outlined. The response of cells to both initiating and
 

promoting agents will be reviewed and compared to similar studies in
 

vivo. Finally data will be presented which may shed light on the
 

cellular mechanisms involved in response to carcinogenic or promoting
 

chemicals and the relationship between differentiation and
 

carcinogenesis.
 

CELL ISOLATION AND GROWTH CONDITIONS
 

We have modified a trypsin-flotation technique first
 

described by Szabo (23) to isolate epidermal cells from newborn Balb/c
 

mice (28). Such cells are isolated as small cell aggregates, probably
 

representing superficial hair follicles, and single cells which
 

migrate into colonies to form large sheets of round to polygonal
 

epidermal cells (28). Cell attachment is completed by 4-6 hours after
 

plating with a plating efficiency of about 40-50?. The standard
 

culture conditions employ Medium 199 plus 11$ fetal calf serum and 1$
 

antimycotic antibiotic solution (GIBCO) and growth is maintained in a
 

humidified atmosphere of 5% C02 at 37oC. In certain experiments it is
 

useful to have dermal fibroblasts or hair follicles as controls. To
 

isolate these fractions the dermis, left behind after separation of
 

epidermis, is dissociated by stirring in 0.35$ crude collagenase. The
 

cell suspension containing fibroblasts and intact hair follicles can
 

be separated into its component parts using a linear 4-10$ Ficoll
 

gradient (29).
 

CHARACTERISTICS OF EPIDERMAL CELLS IN VITRO
 

Within 72 hours of culture initiation, the central areas of
 

epidermal foci begin to accumulate birefringent amorphous material
 

which stains red with Kreyberg stain (12,28) and is presumed to be
 

keratin. If the cells are heavily seeded, this material forms a
 

second layer above the cell monolayer and sheds into the medium by day
 

6 or 7 leaving behind larger cells with poorly delineated borders.
 Electron micrographs of such cultures reveal many typical epidermal
 

markers including desmosomes, cytoplasmic filaments, thickened cell
 

membranes and keratohyaline granules (28). When attached cells or
 

sloughed material is extracted with urea buffers (20) and the extracts
 

48
 
electrophoresed on SDS polyaorylamide gels, several bands in the 

50,000-60,000 molecular weight range are detected in extracts fron 

attached cells by 1-1/2 days in culture which disappear by 3 days. These bands are identical to those found in urea extracts of stratum 

corneum (21). The sloughed material, on the other hand, displays this 

stratum corneum pattern at all times studied indicating that 

keratinization is proceeding in a relatively normal way in these 

cultures. Of interest also is the appearance in the attached cells of 

a band which co-migrates with mouse muscle aotin and comprises up to 

15-2056 of the extracted proteins by the 6th day of culture (22). The 

functional integrity of these epidermal cell cultures can also be 

demonstrated by grafting cells grown in vitro for 4 days to syngeneic 

hosts. Such grafts result in the formation of donor skin at the graft 

site (30). Similar results have been reported by Worst et al. (24). 

The maturation and terminal differentiation occurring in 

epidermal cultures results in a limited lifespan in vitro under 

standard conditions. Thus growth curves reveal a rather stable 

population density for the first 5 days in culture while there is a 

balance between proliferation and differentiation, followed by a 

considerable drop in population density associated with the rapid 

keratinization and sloughing on day 7 or 8 (31). A stable low 

population density is maintained for 1-2 months but ultimately most 

cultures die. If 1.255& DMSO is included in the culture medium, a 

similar growth curve is produced but the stable population achieved 

after 7 days is 4-5 times higher than in untreated cultures and 

remains intact for 6 months (28,32). Likewise, 12.5 yg/ml retinyl 

acetate in the medium prevents keratinization and increases the 

population density (28,32). Lowering the growth temperature from 37 

to 31° without altering medium constituents also seems to delay the 

maturation process (8). Jepsen reported that conditions of low 

temperature prolonged the growth of rat oral epithelium in vitro (11). 

Under standard culture conditions rather unusual growth 

kinetics are observed which make these cells valuable for the study of 

proliferation control as well as carcinogenesis. When DNA synthesis 

is studied every 24 hours by a one hour pulse with (methyl-3H) 

thymidine (3H-TdR) and subsequent autoradiography, a pattern of 

partially synchronous growth is noted with peaks of DNA synthetic 

activity on the 2nd and 8th day in culture (3D- A more intensive 

study utilizing multiple 3H-TdR pulses (every 2 hours) during the 

first 96 hours in vitro demonstrated a natural cell cycle synchrony 

with peaks of DNA synthesis occurring about 20 hours apart, starting 

20 hours after culture (5). Mitotic peaks occur approximately 9-12 

hours after synthesis peaks. Labeling continuously with 3H-TdR 

demonstrated that the growth fraction represents initially about 

10-15/1 of the attached cells and is a single cohort of cycling cells 

which at its maximum after 8 days in culture comprises about 7056 of 

49 
the attached population (5). Thus it appears that the epidermal cell
 

model system could be useful for studying the effects of chemicals on
 

the cell cycle without further manipulation. This synchrony however
 

varies somewhat in timing and degree from experiment to experiment so
 

that cell cycle studies on any primary isolate would have to include a
 

validation of synchrony in that population. When cells are grown at
 

31o in 2% serum, the first peak of DNA synthesis is delayed but an
 

increased number of cells (1.5-2 times) are cycling suggesting either
 

that lower temperature enhances the degree of synchrony or allows a
 

group of non-cycling cells to enter the proliferating pool.
 

ENZYMATIC INTEGRITY OF CULTURED CELLS
 

Several enzyme systems believed to be involved in cellular
 

growth control and chemical carcinogenesis have been studied in mouse
 

epidermal cell cultures. A remarkable and transient rise in activity
 

of ornithine decarboxylase (ODC), the first enzyme in the polyamine
 

biosynthetic pathway, has been demonstrated in a number of biological
 

systems that are stimulated to proliferate (18). ODC activity in
 

freshly isolated uncultured epidermal cells is almost undetectable low
 

but begins to rise by 4 hours after plating, peaking at 12 hours, with
 

a return to almost undetectable levels by 24 hours. These low values
 

remain unchanged for at least the next 3 days even after a medium
 

change (14). Whether this early activity is related to the
 

synchronous proliferation observed subsequently remains to be
 

established.
 

Carcinogen methabolism reflects an enzymatic activity which
 

must be present in a model system to be used for chemical
 

carcinogenesis studies. One enzymatic pathway involved in such
 

metabolism is the aryl hydrocarbon hydroxylase (AHH) system (6).
 During their first several days in culture, epidermal cells have
 

easily measurable levels of AHH; moreover, when the cells are exposed
 

to 3 ug/ml benz-(a)anthracene (BA) for 20 hours before harvest and
 

assay, enzyme activity increases 12-15 fold (3D. After the cells
 

have been cultured for 10 days, the activity of both the constitutive
 

and induced enzymes decreases significantly. This decline can be
 

prevented by growing the cells in the presence of 1.25$ DMSO (3D, a
 

condition which also appears to prolong the lifespan of these
 

cultures. The presence of 12.5 ug/ml retinyl acetate in the medium
 

for 10 days appears to preserve the levels of constitutive AHH seen in
 

younger cultures but prevents the induction of this enyzme by
 

polycyclic hydrocarbons (32).
 

50
 
RESPONSE OF EPIDERMAL CELLS TO CARCINOGENS
 

In vivo, chemical carcinogens have been shown to effect the
 

state of proliferation of mouse epidermis and to bind to epidermal
 

macromolecules and induce DNA repair. Similar studies can be
 

performed in vitro. When epidermal cultures in vitro for 24 hours are
 

exposed to 0.1 yg/ml or 1 yg/ml 7,12 dimenthylbenz(a)anthracene (DMBA)
 

for 20 hours there is an inhibition of DNA synthesis which increases
 

gradually during the exposure time. DNA synthetic activity returns to
 

control levels by 3 days after treatment and is followed by a
 

stimulation of proliferation for the subsequent 48 hours prior to
 

returning toward normal on day 6 (Yuspa - unpublished results). When
 

epidermal cells in vitro for 24 hours are exposed to
 

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG - 1 yg/ml and 5 yg/ml) for
 

1 hour, an immediate inhibition of DNA synthesis occurs which returns
 

to control values by day 4 for the lower dose but the higher dose
 

suppression persists for at least 6 days. MNNG induces no stimulation
 

of proliferation (Yuspa - unpublished results). These effects on
 

epidermal proliferation kinetics are identical to those reported in
 

mouse skin in vivo for DMBA (1) or MNNG (4).
 

Binding of DMBA to epidermal cell DNA can be measured by
 

exposing the cells to 14C-DMBA for 20 hours, isolating DNA and
 

determining specific activity (31). Such studies reveal that
 

carcinogen binding is 2 to 4 times greater in 3 day old cells than in
 

10 day old cells and is in the range (53 molecules DMBA per 106
 

nucleotides) seen for binding of this carcinogen in other
 

nontransformed mammalian cells in culture (13). Inclusion of 1.25$
 

DMSO in the culture medium increases the amount of DNA bound
 

carcinogen in 10 day old cultures back to the 3 day level. The
 

presence of 12.5 yg/ml retinyl acetate in the culture medium for 10
 

days decreases the amount of carcinogen bound to DNA but increases the
 

amount bound to protein (32) relative to control cultures.
 

Using mouse skin cell cultures we have been able to show
 

that DMBA binding to DNA was not increased when DNA was in the
 

replicative phase during exposure (33). In addition DNA which had the
 

carcinogen bound'to it was still capable of functioning as a template
 

for new DNA synthesis and the parent strand to which the carcinogen
 

was attached was carried over into the daughter cell (2). These cells
 

also respond to carcinogen damage to the genome with an induction of
 

DNA repair synthesis. At least two types of repair occur, excision
 

repair after ultraviolet light or high doses of MNNG and
 

B-propiolation (BPL) and guanine specific repair after low doses of
 

MNNG and BPL (9,10).
 

51
 
RESPONSE OF EPIDERMAL CELLS TO PROMOTING AGENTS 

The concept of tumor promotion as a distinct entity during 

car ci no genesis was derived from studies on mouse skin. The epidermal 

culture system should be a useful model to extend the in vivo studies 

on mechanisms of tumor promotion. All promoters have in common the 

ability to stimulate proliferation (3). When epidermal cell cultures 

are exposed to the phorbol ester and strong tumor promoter 

12-0-tetradecanoyl phorbol-13-acetate (TPA), DNA synthesis is 

stimulated 5-15 fold 72 to 96 hours after initiation of treatment 

(29). This response is dose dependent between 10~5 and 10~9 M and 

occurs even after a TPA exposure of only 1 hour's duration. The 

stimulation is preceded by an inhibition of synthesis for 24 hours. The maximum response is observed when cells are treated after 24 hours 

in culture, a time when many cells are in the late G-| phase of the 

cell cycle (5). Dermal fibroblasts are not stimulated to proliferate 

by TPA. There is an excellent correlation between the in vivo tumor 

promoting potency of a series of phorbol esters and their ability to 

stimulate epidermal cell proliferation in vitro (34). The addition of 

the antiinflammatory steroid fluocinolone acetonide to the culture 

medium prior to or during TPA exposure inhibits the DNA synthetic 

response (15). This steroid has also been shown to inhibit stimulated 

proliferation and tumor promotion when applied with TPA on mouse skin 

in1 vivo (19). 

TPA treatment of epidermal cell cultures at 24 hours after 

plating results in a transient 5-15 fold rise in ODC activity compared 

to untreated controls (14). The peak occurs between 6-10 hours after 

treatment and the magnitude of the response is dose dependent. A 10 

minute exposure to TPA is sufficient to stimulate ODC maximally. The 

degree of ODC responsiveness varies with the time exposure begins 

after plating. In cells older than 3 days in culture the response is 

minimal. However, in cultures previously treated with TPA on day 1, the responsiveness is maintained in older cultures suggesting a 

selection of a sensitive cell type by the initial treatment. There is 

an excellent correlation between the skin tumor promoter potency in 

vivo of a series of phorbol esters and their ability to stimulate ODC 

activity in epidermal cells in vitro (3^). Recent data from mouse 

skin in vivo (16) show that TPA induces ODC activity within 6 hours of 

painting and that the degree of stimulation correlates well with the 

promoter potency of phorbol esters. Similarly repeated TPA treatments 

in vivo increased the magnitude of the ODC response (17). 

The mouse epidermal cell culture system appears to be a 

valid and valuable tool to extend studies on chemical car ci no genesis 

to the cellular level. These cells appear to differentiate in vitro 

in a near normal manner. They grow synchronously, at least for several 

52 
days, without exogenous treatment. Media additives such as DMSO and
 

retinyl acetate appear to modulate both growth and differentiation.
 

The cells can metobolize polycyclic aromatic hydrocarbon carcinogens
 

and activated products bind to cellular macromolecules. Carcinogen
 

induced DNA damage can be repaired by at least two mechanisms,
 

excision repair and guanine specific repair. These cells also respond
 

to promoting agents in vitro in a fashion highly analogous to mouse
 

skin in vivo. Thus the in vivo-in vitro comparisons necessary to
 

validate this culture system to date are highly positive. The
 

development of a quantitive transformation assay seems to be the next
 

logical refinement necessary for maximum usefulness of the model, and
 

progress is being made in that direction.
 

REFERENCES
 

1.	 Bates, R.R., J.S. Wortham, W.B. Counts, C.W. Dingman, and
 

H.V. Gelboin. "Inhibition by Actinomycin D of DNA Synthesis and
 

Skin Tumorigenesis Induced by 7,12-dimethylbenz(a)anthracene."
 

Cancer Res., 28 (1968), 27-34.
 

2.	 Bates, R.R., S. Eaton, D.L. Morgan, and S.H. Yuspa. "Replication
 

of DNA after Binding of the Carcinogen 7,12-dimethylbenz­

(a)anthracene." J. Natl. Cancer Inst., 45 (1970), 1223-1228.
 

3.	 Boutwell, R.K. "The Function and Mechanism of Promoters of
 

Carcinogenesis." CRC Crit. Rev. Toxicol. (1974), 419-443­

4.	 Bowden, G.T., and R.K. Boutwell. "Studies on the Role of
 

Stimulated Epidermal DNA Synthesis in the Initiation of Skin
 

Tumors in Mice by N-methyl-N'-nitro-N-nitrosoguanidine." Cancer
 

Res., 34 (1974), 1552-1563.
 

5.	 Elgjo, J., H. Hennings, D. Michael, and S.H. Yuspa. "Natural
 

Synchrony of Newborn Mouse Epidermal Cells In Vitro." J. Invest.
 

Dermatol., 66 (1976), 292-296.
 

6.	 Gelboin H.V., N. Kinoshita, and F.J. Wiebel. "Microsomal
 

Hydroxylases: Induction and Role in Polycyclic Hydrocarbon
 

Carcinogenesis and Toxicity." Fed. Proc., 31 (1972), 1298-1309­

7.	 Heidelberger, C. "Chemical Oncogenesis in Culture." Advan.
 

Cancer Res., 18 (1973), 317-366.
 

53
 
8.

 Hennings H. , D.M. Michael, K. Elgjo, and S.H. Yuspa. "Optimal 

Growth of Mouse Epidermal Cells in Culture." In Vitro, 12 

(1976), 303. 

9. Hennings, H. , D.M. Michael, S. Eaton, and D.L. Morgan. "Purine 

Specific Repair of 3-Propiolactone Induced DNA Damage in Mouse 

Skin Cells." J. Invest. Dermatol., 62 (1971), 480-484. 

10. Hennings, H. , and D. Michael. "Guanine-Specific DNA Repair After 

Treatment of Mouse Skin Cells with N-methyl-N'-nitro-N­ nitrosoguanidine." Cancer Res., 36 (1976), 2321-2325. 

11. Jepsen, A. "An In Vitro Model of an Oral Keratinizing Squamous 

Epithelium." Scand. J. Dent. Res., 82 (1974), 144-146. 

12. Kreyburg, L. "Histologic Typing of Lung Tumors." World Health 

Organization, Geneva, 1967. 

13. Kuroki, T., and C. Heidelberger. "The Binding of Polycyclic 

Aromatic Hydrocarbons to the DNA, RNA, and Proteins of 

Transformable Cells in Culture." Cancer Res., 31 (1971), 2168-2176. 

14. Lichti U., S.H. Yuspa, and H. Hennings. "Ornithine and 

S-Adenosylmethionine Decarboxylase in Mouse Epidermal Cell 

Cultures Treated with Tumor Promoters." In: Mechanisms of Tumor 

Promotion and Cocarcinogenesis." T. Slaga (Ed), Raven Press, New 

York, 1977, in press. 

15. Lichti, U. , T.S. Slaga, T. Ben, E. Patterson, H. Hennings, and 

S.H. Yuspa. "Dissociation of Tumor Promoter Stimulated Ornithine 

Decarboxylase Activity and DNA Synthesis in Mouse Epidermis In 

Vivo and In Vitro By the Inhibitor of Tumor Promotion, Fluocino­ lone Acetonide." Submitted for publication. 

16. O'Brien T.G., R.C. Simsiman, and R.K. Boutwell. "Induction of 

Polyamine-Biosynthetic Enzymes in Mouse Epidermis by 

Tumor-Promoting Agents." Cancer Res., 35 (1975), 1662-1670. 

17. O'Brien, T.G., R.C. Simsiman, and R.K . Boutwell. "Induction of 

the Polyamine-Biosynthetic Enzymes in Mouse Epidermis and their 

Specificity for Tumor Promotion." Cancer Res., 35 (1975), 

2426-2433­

18. Russel, D.H. , (Ed.). "Polyamines in Normal and Neoplastic 

Growth." New York, Raven Press, Publishers, 1973. 

54 
19.

 Slaga, T.J., S.H. Yuspa, H. Hennings, K. Elgjo, J.A. Schwartz, and A. Viaje. "Effects of a Steroidal Anti-Inflammatory Agent on 

DNA Synthesis in Adult and Newborn Mouse Epidermis." Proc. Amer. Ass. Cancer Res., 17 (1976), 39. 

20. Steinert, P.M. "The Extraction and Characterization of Bovine 

Epidermal a-Keratin." Biochem. J.. 149 (1975), 39-48. 

21. Steinert, P.M. , and S.H. Yuspa. "Kinetics of Production of 

Keratin-Like Proteins in Mouse Epidermal Cell Cultures." In 

preparation. 

22. Steinert, P., G. Peck, S.H. Yuspa, J. McGuire, and A. Tasquale. "Isolation of an Actin-Like Protein from Epidermal Tumors and 

Cultured Epidermal Cells." J. Invest. Dermatol., 66 (1976), 276. 

23. Szabo, G. "A Modification of the Technique of Skin Splitting 

with Trypsin." J. Patho. Bacteriol., 70 (1955), 545. 

24. Worst, R.K.M., E.A. Valentine, and N.E. Fusenig. "Formation of 

Epidermis After Reimplantaticn of Pure Primary Epidermal Cell 

Cultures from Perinatal Mouse Skin." J. Natl. Cancer Inst., 53 

(1974), 1061-1064. 

25. Weinstein, I.E., J.M. Orenstein, R. Gebert, M.E. Kaighn, and 

U. C. Stadler. "Growth and Structural Properties of Epithelial 

Cell Cultures Established from Normal Rat Liver and Chemically 

Induced Hepatcmas." Cancer Res., 35 (1975), 253-263. 

26. Yuspa, S.H., D.L. Morgan, and J.A. Levy. "In Vitro Cultivation 

of a Chemically Induced Epidermal Carcinoma: Establishment of 

Three Cell Lines and Isolation of Murine Leukemia Virus." £. Natl. Cancer Instit.. 50 (1973), 1561-1570. 

27. Yuspa, S.H., H.H. Hennings, and U. Saffiotti. "Cutaneous 

Chemical Carcinogenesis: Past, Present and Future." J. Invest. Dermatol., 67 (1976), 199-208. 

28. Yuspa, S.H., and C.C. Harris. "Altered Differentiation of Mouse 

Epidermal Cells Treated with Retinyl Acetate in vitro." Exp. Cell Res.. 86 (1974), 95-105. 

29. Yuspa, S.H., T. Ben, E. Patterson, D.M. Michael, K. Elgjo, and H. Hennings. "Stimulated DNA Synthesis in Mouse Epidermal Cell 

Cultures Treated with 12-0-Tetradecanoyl Phorbol-13-acetate." 

Cancer Res., 36 (1977), 4062-4068. 

55 
30.	 Yuspa, S.H., D.L. Morgan, R.J. Walker, and R.R. Bates. "The
 

Growth of Fetal Mouse Skin in Cell Cultures and Transplantation
 

to F-| Mice." J. Invest Dermatol., 55 (1970), 379-389­

31.	 Yuspa, S.H., H. Hennings, P. Dermer, and D. Michael. "DMSO
 

Induced Enhancement of 7, 12 Dimethylbenz(a)anthracene Metabolism
 

and DNA Binding in Differentiating Mouse Epidermal Cell
 

Cultures." Cancer Res., 36 (1976), 9*17-951.
 

32.	 Yuspa, S.H., K. Elgjo, M.A. Morse, and F.J. Wiebel. "Retinyl
 

Acetate Modulation of Cell Growth Kinetics and Carcinogen-
 Cellular Interaction in Mouse Epidermal Cell Cultures."
 

Chem.-Biol. Interactions, 16 (1977), 251-264.
 

33.	 Yuspa, S.H., S. Eaton, D.L. Morgan, and R.R. Bates. "The Binding
 

of 7, 12 Dimethylbenz(a)anthracene to Replicating and Nonrepli­ cating DNA in Cell Culture." Chem. Biol. Interact., 1 (1969),
 

223-233.
 

34.	 Yuspa, S.H., U. Lichti, T. Ben, E. Patterson, H. Hennings, T.J.
 Slaga, N. Colburn, and W. Kelsey. "Phorbol-Ester Tumor Promoters
 

Stimulate DNA Synthesis and Ornithine Decarboxylase Activity in
 

Mouse Epidermal Cell Cultures." Nature, 262 (1976), 402-404.
 

56
 
2.3.2 CHEMICAL TRANSFORMATION OF EPIDERMAL CELL CULTURES 

NANCY COLBURN 

Experimental Pathology Branch, Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer 

Institute, N.I.H., Bethesda, MD 

The newborn mouse primary epidermal cell cultures currently 

being used as model systems for in vitro carcinogenesis studies are 

characterized by 1) the capacity to undergo spontaneous vertical 

stratification analogous to keratinization, 2) the capacity to 

produce stratum corneum as indicated by histochemical staining and 

electrophoretic detection of keratin proteins, 3) a cell population 

which decreases with time in culture, 4) the lack of capacity to be 

subcultured, and 5) responses to carcinogens and tumor promoters 

which mimic in vivo responses (10,11,31,41,42,43). The conditions 

used in these studies include growth on plastic dishes in the absence 

of fibroblast feeder layers or collagen. 

Although growth of epidermal cells in contact with dermis or 

dermal components has been shown to enhance the organization, growth 

rate, or time in culture of skin epithelial cells (2,3,8,14,15,20,29), attempts to subculture mouse epidermal cells have been largely 

unsuccessful. Recently Rheinwald and Green (29) have reported that 

human epidermal cells grown on irradiated fibroblasts form Rhodamin B 

staining material, show ultrastructural evidence for keratinization 

and can be subcultured for 50 to 100 generations (100 or more in the 

subultivation. 

presence of epidermal growth factor; H. Green personal 

communication). Epithelial cells of human (8,29) and guinea pig 

(16,26,28) skin appear to be unique in their amenability to 

Although a number of laboratories have reported neoplastic 

transformation of liver (17,24,37,40) and bladder (12) epithelial 

cells in culture after treatment with chemical carcinogens, there have 

been relatively few reports of chemical transformation of skin 

epithelial cells. Fusenig and coworkers (9) have reported on 2 

morphologically transformed cell strains obtained after treatment of 

primary newborn mouse epidermal cell cultures with 7,12-dimethylbenz­ (a)anthracene (DMBA). These cell strains can be subcultured 

indefinitely, produce carcinomas (one keratinizing and one anaplastic) 

on injection into syngeneic hosts, and show a substantial decrease in 

the levels of an epidermal cell surface antigen (38,39). These 

workers found no spontaneous transformation, but they used conditions 

in which the transformation of treated cultures was rare (N. Fusenig 

57 
and W. Thon, personal communication). These reports contained only 

brief preliminary descriptions of tumorigenicity data, histopathology, and quantitation of epidermal antigen in tumorigenic cells. 

Elias et al. (5) have treated mixed cultures of epidermal 

and dermal cells from newborn mouse skin with DMBA to obtain 

morphologically transformed epithelial-like cell strains. These cells 

produced anaplastio tumors on injection. 

Recently Col burn et al. (4) have obtained some 10 

morphologically transformed continuously subculturable cell strains 3 

months after treatment of mouse epidermal cell cultures with 

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) with and without 

additional phorbol ester treatment. Two spontaneously transformed 

strains have also been obtained. These cells along with 3 other 

strains provided by S. Yuspa et al. (2 produced after DMBA treatment 

and one spontaneously produced) have been assayed for growth rate, colony formation in 0.33$ agar medium (21,22), fibrinolytic activity 

(19,33) and tumorigenicity. All strains which were tumorigenic also 

formed colonies in soft agar while all non-tumorigenic strains did not 

form colonies. Two spontaneously transformed strains were tumorigenic 

while one was not. The tumorigenic cells grew in soft agar with 

colony forming efficiencies (CFE) which varied from 7-75%. Doubling 

times varied from 29 to 78 hrs. Preliminary studies in collaboration 

with W. Benedict and coworkers indicate that 2 tumorigenic epidermal 

strains have high fibrinolytic activity while 2 nontumorigenic strains 

show no or very low activity. Although growth in soft agar is a 

highly reliable correlate of tumorigenicity for epidermal cells, as it 

is for liver epithelial cells (25,34 ,40), it appears that high 

fibrinolytic activity, a parameter which is not a reliable 

tumorigeniciity correlate for liver epithelial cells (40), may be a 

second useful tumorigenicity indicator for transformed epidermal cells. 

Growth in semi-sold medium (anchorage independent growth) 

appears to be the only consistently reliable in vitro correlate of 

tumorigenicity for a wide variety of cell types (7,13,30,32,35). At 

this writing we are aware of no reports in the literature in which a 

false negative has been obtained. There have been reports of apparent 

false positives (i.e., growth in soft agar of cells that could not be 

shown to be tumorigenic (19) and DiPaolo and coworkers, personal 

communication). However, failure to produce a tumor could occur for 

reasons other than lack of tumorigenicity such as antigenicity (even 

in nude mice) host response, etc. 

Another recent result of our studies (4, and manuscript in 

preparation) is the production by chemical carcinogen treatment of 

transformed cell strains which in early passages failed to grow in 

soft agar or to produce tumors, but which in later passages shifted in 

58 
both properties. The transitional passage(s) was characterized by low 

colony forming efficiency in soft agar ( < 1$) while subsequent 

passages showed much higher CFE's. These strains in early passages 

can then be said to be preneoplastic and may be useful for studying 

late stages in the conversion to the neoplastic phenotype. The 

observation that a number of passages may be required for manifesta­ tion of tumorigenicity has been reported for chemically transformed 

guinea pig fibroblasts by Evans and DiPaolo (6) for rat fetal brain 

cells by Laerum and Rajewsky (18) and more recently for hamster embryo 

fibroblasts by J.C. Barrett (this volume). These findings imply a 

multistage process of carcinogenesis involving some minimum number of 

cell divisions and suggest that it may be difficult to test directly 

the induction (vs selection) hypothesis in so far as this entails 

limiting the number of cell divisions. 

Most of the questions which have been posed regarding the 

molecular and cellular mechanism of carcinogenesis have not been 

answered in experiments using epithelial cell culture systems. With 

the exception of the report of Yamaguchi and Weinstein (MO) on the 

production of conditional mutants for growth in soft agar, the 

induction theory, the somatic mutation theory, and the 2-stage 

hypothesis have not been tested. Other unanswered questions for 

epithelial cells include the cell cycle specificity of transformation, the role of cell proliferation, the role of DNA repair, and the role 

of differentiation. The possible role of viruses in liver epithelial 

transformation has been discussed by Weinstein and coworkers (36) who 

isolated A-type and C-type viruses from N-acetoxy acetylaminofluorene 

transformed liver cells and cell lines established from heptomas. Virus particles have also been found by Yuspa and coworkers (4M ) in 

cell cultures established from DMBA-induced epidermal carcinomas, and 

by us (Colburn et al., unpublished) in some MNNG and spontaneously 

transformed mouse epidermal cell strains. These viruses have not been 

tested for their ability to produce neoplastic transformation. However, the results of Rapp et al. (27) indicate that at least for 

10T1/2 mouse embryo fibroblasts, virus production and chemical 

transformation are independent processes. Virus production in 

tumorigenic epithelial cells as well as in most non-epithelial cells 

may turn out to be symptomatic, i.e., occurring concommitantly but 

inert in the process of transformation as suggested by Weinstein et 

al. (36). 

Efforts are underway to produce modifications in the 

epidermal cell culture system which will permit one to ask more 

questions about the mechanism of chemical carcinogenesis. These 

include attempts to increase the transformation frequency and decrease 

the latent period for transformation in carcinogen treated cells while 

decreasing the spontaneous transformation frequency. Efforts are also 

directed at increasing the size and stability of the proliferative 

59 
(stem cell) population and developing a quantitative transformation
 

assay. Regarding the issue of decreased latent period the possibility
 

exists that a long lag time may be inherent in the process of
 

carcinogenesis in differentiating epithelial systems since carcinoma
 

production in mouse skin in vivo requires some 4 to 10 months (1) and
 

transformation of liver and bladder epithelial cells in culture thus
 

far reported requires 2 to 9 months (12,35).
 

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3.	 Briggaman, R.A., and C.E. Wheeler, Jr. "Epidermal-dermal Inter­ action in Adult Human Skin. II. The Nature of The Dermal Influ­ ence." J. Invest. Dermatol., 56 (1971), 18-26.
 

4.	 Colburn, N.H., J. Bates, W. VorderBruegge, and J. Rossen.
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Proc. Amer. Assoc. Cancer Res., 17 (1976), 74.
 

5.	 Elias, P.M., S.H. Yuspa, and M. Gullino, et al. "In Vitro
 

Neoplastic Transformation of Mouse Skin Cells: Morphology and
 

Ultrastructure of Cells and Tumors." J. Invest. Derm., 62
 

(1974), 569-581.
 

6.	 Evans, C.H., and J.A. DiPaolo. "Neoplastic Transformation of
 

Guinea Pig Fetal Cells in Culture Induced by Chemical
 

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7.	 Freedman, V.H., and S. Shin. "Cellular Tumorigenicity in Nude
 

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8.	 Freeman, A.E., H.J. Igel, B.J. Herrman, and K.L. Kleinfield.
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9.	 Fusenig, N.E., W. Samsel, W. Thon, and P.K.M. Worst. "Malignant
 

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19 (1973), 219-228.
 

60
 
10.	 Fusenig, N.E. "Isolation and Cultivation of Epidermal Cells from
 

Embryonic Mouse Skin." Die Naturwissenschaften, 8 (1971), 421.
 

11.	 Fusenig, N.E., and P.K.M. Worst. "Mouse Epidermal Cell Culture,
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from Perinatal Mouse Skin." Exptl. Cell Res., 93 (1975), 443-457.
 

12.	 Hashimoto, Y., and H. Kitagawa. "In Vitro Neoplastic
 

Transformation of Epithelial Cells of Rat Urinary Bladder by
 

Nitrosamines." Nature, 252 (1974), H97-499.
 

13.	 Kakunaga, T., and J. Kamahova. "Properties of Hamster Embryonic
 

Cells Transformed by 4-Nitroquinoline-1-Oxide In Vitro and their
 

Correlations with the Malignant Properties of the Cells." Biken
 

J., 11 (1968), 313-332.
 

14.	 Karasek, M., and M. Charlton. "Postembryonic Skin Epithelial
 

Cells on Collagen Gels." J. Invest. Dermatol., 56 (1968),
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15.	 Karasek, M.A. "In Vitro Growth and Maturation of Epithelial
 

Cells from Postembryonic Skin." J. Invest. Dermatol., 65 (1975),
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16.	 Karasek, M., S. Liu, and M. Charlton. "Comparative Growth and
 

Differentiation of Skin Epithelial Cells in Cell Culture." In
 

Vitro, 9 (1974), 359.
 

17.	 Katsuta, H., and T. Takaoka. "Carcinogenesis in Tissue Culture,
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Treated with 4-Nitroquinoline 1-Oxide in Tissue Culture." £.
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18.	 Laerum, O.D., and M.F. Rajewsky. "Neoplastic Transformation of
 

Fetal Rat Brain Cells in Culture After Exposure to
 

Ethylnitrosourea In Vivo." J. Natl. Cancer Inst., 55 (1975),
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19- Laug, W.E., P.A. Jones, and W.F. Benedict. "Relationship between
 

Fibrinolysis of Cultured Cells and Malignancy." J. Natl. Cancer
 

Inst., 54 (1975), 173-179.
 

20.	 Liu, S., and M. Karasek. "Biochemistry of Dermal-Epidermal
 

Interrelationships." In Vitro, 9 (1974), 368.
 

61
 
21.	 MacPherson, I., and I. Montagnier. "Agar Suspension Culture for
 

the Selective Assay of Cells Transformed by Polyoma Virus."
 

Virology, 23 (1964), 291-294.
 

22.	 MacPherson, I. "Agar Suspension Culture for Quantitation of
 

Transformed Cells in: Fundamental Techniques in Virology." K.
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23.	 Montagnier, L. "Factors Controlling the Multiplication of
 

Untransformed and Transformed BHK21 Cells Under Various
 

Environmental Conditions." G.E.W. Wostenholme, and J. Knight
 

(Eds.). Ciba Symposium, Growth Control in Cell Cultures, London,
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24.	 Montesano, R., L. Saint Vincent, and L. Tomatis. "Malignant
 

Transformation In Vitro of Rat Liver Cells By
 

Dimethyl-nitrosamine and N-Methyl-N'-Nitro-N-Nitrosoguanidine."
 

Br. J. Cancer, 28 (1973), 214-220.
 

25.	 Oshiro, Y., L.E. Gerschenson, and J.A. DiPaolo. "Carcinomas from
 

Rat Liver Cells Transformed Spontaneously in Culture." Cancer
 

Res., 32 (1972) 877-879.
 

26.	 Prunieras, M., C. Delescluse, and M. Regnier. "Culture de longue
 

duree de cellules issues de 1'epiderme de cobaye adulte. I
 

Potentiel de croissance caryotype et carcteres morphologiques."
 

Path. Biol., 17 (1969), 235-241,243-249,251-259.
 

27.	 Rapp, U., R.C. Nowinski, C.A. Reznikoff, and C. Heidelberger.
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(1975), 392-409.
 

28.	 Regnier, M., C. Delescluse, and M. Prunieras. "Studies on Guinea
 

Pig Skin Cell Cultures I Separate Cultures of Keratinocytes and
 

Dermal Fibroblasts." Acta Dermatovener (Stockholm), 53 (1973),
 241-247.
 

29.	 Rheinwald, J.G., and H. Green. "Serial Cultivation of Strains of
 

Human Epidermal Keratinocytes. The Formation of Keratinizing
 

Colonies from Single Cells." Cell, 6 (1975), 331-344.
 

30.	 Shin, S., V.H. Freedman, R. Risser, and R. Pollack.
 "Tumorigenicity of Virus-Transformed Cells in Nude Mice is
 

Correlated Specifically with Anchorage Independent Growth in
 

Vitro." Proc. Natl. Acad. Sci.. 72 (1975), 4435-4439.
 

62
 
31.	 Steinert, P.M., and S.H. Yuspa. "Kinetics of Production of
 

Keratin-like Proteins in Mouse Epidermal Cell Cultures." In
 

preparation.
 

32.	 Stoker, M., C. O'Neill, S. Berryman, and V. Maxman. "Anchorage
 

and Growth Regulation in Normal and Virus-Transformed Cells."
 

Int. J. Cancer, 3 (1968), 683-693.
 

33.	 Unkeless, J., K. Dano, G.M. Kellerman, and E. Reich.
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Chem., 249 (197*0, 4395-4305.
 

34.	 Weinstein, I.E., J.M. Orenstein, R. Gebert, M.E. Kaighn, et al.
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Established from Normal Rat Liver and Chemically Induced
 

Hepatomas." Cancer Res., 35 (1975), 263.
 

35.	 Weinstein, I.E., N. Wigler, and U. Stadler. "Analysis of the
 

Mechanism of Chemical Carcinogenesis in Epithelial Cell
 

Cultures." In: Screening Tests in Chemical Carcinogenesis.
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(Eds.), (1976).
 

36.	 Weinstein, I.E., N. Yamaguchi, and R. Gebert. "Use of Epithelial
 

Cell Cultures for Studies on the Mechanism of Transformation by
 

Chemical Carcinogens." In Vitro, 11 (1975), 1130-141.
 

37.	 Williams, G.M., J.M. Elliott, and J.H. Weisburger. "Carcinoma
 

After Malignant Conversion In Vitro of Epithelial-like cells from
 

Rat Liver Following Exposure to Chemical Carcinogens." Cancer
 

Res, 33 (1973), 606-612.
 

38.	 Worst, P.K.M., and N.E. Fusenig. "Specific Surface Antigens of
 

Mouse Epidermal Cells in Culture." Inserm., 19 (1973), 245-252.
 

39.	 Worst, P.K.M., and N.E. Fusenig. "A Tissue Antigen on the
 

Surface of Cultivated Mouse Epidermal Cells Detected by Mixed
 

Hemadsorption Technique." J. Invest. Dermatol., 61 (1973)>
 

277-281.
 

40.	 Yamaguchi, N., and I.E. Weinstein. "Temperature-Sensitive
 

Mutants of Chemically Transformed Epithelial Cells." Proc. Natl.
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41.	 Yuspa, S.H., and C.C. Harris. "Altered Differentiation of Mouse
 

Epidermal Cells Treated with Retinyl Acetate In Vitro." Exptl.
 

Cell Res., 86 (1974), 95-105.
 

63
 
42.	 Yuspa, S.H., H. Hennings, P. Dermer, and D. Michael. "DMSO
 

Induced Enhancement of 7, 12 Dimethylbenz(a)-anthracene
 

Metabolism and DNA Binding in Differentiating Mouse Epidermal
 

Cell Cultures." Cancer Res., 36 (1976), 947-951.
 

43.	 Yuspa, S.H., U. Lichti, T. Ben, E. Patterson, H. Hennings, T.J.
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Stimulate DNA Synthesis and Oroithine Decarboxylase Activity in
 

Mouse Epidermal Cell Cultures." Nature, 262 (1976), 402-404.
 

44.	 Yuspa, S.H., D.L. Morgan, and J.A. Levy. "In Vitro Cultivation
 

of a Chemically Induced Epidermal Carcinoma: Establishment of
 

Three Cell Lines and Isolation of Murine Leukemia Virus." J_._
 Natl. Cancer Inst.. 50 (1973), 1561-1570.
 

64 
2.4 RAT CELLS 

2.4.1 A BIBLIOGRAPHY 

The following references were suggested by C. Heidelberger
 

and others.
 

A.	 HIGH PASSAGE RAT EMBRYO CELLS
 

1.	 Freeman, A.E., P.J. Price, R.J. Bryan, R.J. Gordon, R.V. Gilden,
 

G.J. Kelloff, and R.J. Huebner. "Transformation of Rat and
 

Hamster Embryo Cells by Extracts of City Smog." Proc. Nat. Acad.
 

Sci. USA, 68 (1971), 445-449.
 

2.	 Freeman, A.E., P.J. Price, H.J. Igel, J.C. Young, J.M. Maryak,
 and R.J. Huebner. "Morphological Transformation of Rat Embryo
 

Cells Induced by Diethylnitrosamine and Murine Leukemia
 

Viruses." J. Natl. Cancer Inst., 44 (1970), 65-78.
 

3.	 Freeman, A.E., E.K. Weisburger, J.H. Weisburger, R.G. Wolford,
 J.M. Maryak, and R.J. Huebner. "Transformation of Cell Cultures
 

as an Indication of the Carcinogenic Potential of Chemicals." J_._
 

Natl. Cancer Inst., 51 (1973), 799-808.
 

4.	 Price, P.J., A.E. Freeman, W.T. Lane, and R.J. Huebner.
 "Morphological Transformation of Rat Embryo Cells by the Combined
 

Action of 3-Methylcholanthrene and Rauscher Leukemia Virus."
 

Nature New Biol., 230 (197D, 144-146.
 

5.	 Price, P.J., W.A. Suk, R.L. Peters, C.E. Martin, T.M. Bellew, and
 

R.J. Huebner. "Cordycepin Inhibition of 3-Methycholanthrene­ induced Transformation In Vitro." Proc. Soc. Exp. Biol. Med, 150
 

(1975), 650-653.
 

6.	 Price, P.J., W.A. Suk, P.C. Skeen, M.A. Chirigos, and R.J.
 Huebner. "Transforming Potential of the Anticancer Drug
 

Adriamycin." Science, 187 (1975), 1200-1201.
 

7.	 Rhim, J.S., H.Y. Cho, M.H. Joglekar, and R.J. Huebner.
 

"Comparison of the Transforming Effect of Benzo(a)pyrene in
 

Mammalian Cell Lines In Vitro." J. Natl. Cancer Inst., 48
 

(1972), 949-957.
 

65
 
B. LEUKEMIA VIRUS-INFECTED MOUSE AND RAT FIBROBLASTS 

1. Gordon, R.J., R.J. Bryan, J.S. Rhim, C. Demoise, R.G. Wolford, 

A.E. Freeman, and R.J. Huebner. "Transformaticn of Rat and Mouse 

Embryo Cells by a New Class of Carcinogenic Compounds Isolated 

from Particles in City Air." Int. J. Cancer. 12 (1973), 223-232. 

2. Price, P.J., T.M. Bellow, M.P. King, A.E. Freeman, R.V. Gilden, 

and R.J. Huebner. "Prevention of Viral-Chemical Cocarcinogenesis 

In Vitro by Type-Specific Anti-Viral Antibody." Proc. Nat. Acad. 

Sci. USA. 73 (1976), 152-155. 

3. Price, P.J., W.A. Suk, and A.E. Freeman. "Type C RNA Viruses as 

Determinants of Chemical Carcinogenesis: Effects of Sequence of 

Treatment." Science. 177 (1972), 1003-1004. 

4. Price, P.J., W.A. Suk, G.J. Spahn, and A.E. Freeman. "Transformation of Fischer Rat Embryo Cells by the Combined Action of 

Murine Leukemia Virus and (-)-Trans-A 9-Tetrahydrocannabinol." 

Proc. Soc. Exp. Biol. Med.. 140 (1972), 454-456. 

5. Rhim, J.S., H.Y. Cho, L. Rabstein, R.J. Gordon, R.J. Gryan, 

M.B. Gardner, and R.J. Huebner. "Transformation of Mouse Cells 

Infected with AKR Leukemia Virus Induced by Smog Extracts." 

Nature, 239 (1972), 103-107. 

6. Rhim, J.S., B. Creasy, and R.J. Huebner. "Production of Altered 

Cell Foci by 3-Methylcholanthrene in Mouse Cells Infected with 

AKR Leukemia Virus." Proc. Nat. Acad. Sci. USA. 68 (1971), 

2212-2216. 

7. Rhim, J.S., R.J. Gordon, R.J. Bryan, and R.J. Huebner. "Trans­ formation of Mouse Cells Infected with AKR Leukemia Virus by 

Benzene Extract Fractions of City Air Particles." Int. J. 

Cancer. 12 (1973), 485-492. 

8. Rhim, J.S., D.K. Park, E.K. Weisburger, and J.H. Weisburger. 

"Evaluation of an ^n Vitro Assay System for Carcinogens Based on 

Prior Infection of Rodent Cells with Non-transforming RNA Tumor 

Virus." J. Natl. Cancer Inst., 52 (1974), 1167-1173. 

9. Zimmerman, E.M., R.E. Kouri, K. Higuchi, F. Laird, and A.E. 

Freeman. "Uptake, Metabolism, and Persistence of 3-Methyl­ cholanthrene in Rat Embryo Cells Infected with Murine Leukemia 

Virus." Cancer Res.. 35 (1975), 139-143. 

66 
C.

 RAT EPITHELIAL CELLS 

1. Abrahams, P.J., C. Mulder, A. Van de Voorde, S.O. Warnaar, and 

A.J. Van der Eb. "Transformation of Primary Rat Kidney Cells by 

Fragments of Simian Virus-40 DNA." J. Virol.. 16 (1975), 818­

823­

2. Altaner, C., and E. Hlavoyova. "Transformation of Rat Liver 

Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus." 

J. Virol., 11 (1973), 177-182. 

3. Bartsch, H. , A. Camus, and C. Malaveille. "Comparative 

Mutagenicity of N-nitrosamines in a Semi-solid and in a. Liquid 

Incubation System in the Presence of Rat or Human Tissue 

Fractions." Mutation Research, 37 (1976), 149-156. 

4. Bomford, R. , and I.B. Weinstein. "Transformation of a Rat 

Epithelial Like Cell Line by Murine Sarcoma Virus." J. Natl. Cancer Inst., 49 (1972), 379-386. 

5. Borek, C. "Neoplastic Transformation In Vitro of a Clone of 

Adult Epithelial Cells into Differentiated Keystone-Like Cells 

Under Conditions of Nutritional Stress." Proc. Natl. Acad. Sci., 

69 (1972), 956-959. 

6. Borek, C. , M. Grob, and M.M. Bargor. "Surface Alterations in 

Transformed Epithelial and Fibroblastic Cells in Culture. A 

Disturbance of Membrane Degradation Versus Biosynthesis." Exptl. 

Cell Res., 77 (1973), 207-215. 

7. Borenfreund, E., P.J. Higgins, M. Steinglass, and A. Bendich. "Properties and Malignant Transformation of Established Rate 

Liver Parenchymal Cells in Culture." J. Natl. Cancer Inst. , 55 

(1975), 375-3.84. 

8. Hashimoto, Y. , Kitagawa, H.S., and Ogura, K. "In Vivo and In 

Vitro Aspects of the Urinary Bladder by Nitrosamines." In: 

Magee, P.N., Takayama, S., Sugimura, T., and Matsushima, T. (Eds). Fundamentals in Cancer Prevention. University of Tokyo 

Press. Tokyo (1976) 267-279. 

9. Katsuta, H., and T. Takaoka. "Carcinogenesis in Tissue Culture. XIV Malignant Transformation of Rat Liver Parenchymal Cells 

Treated with 4-Nitroquinoline-1-Oxide in Tissue Culture." J. Natl. Cancer Inst., 49 (1972), 1563-1576. 

10. Lasne, C. , A. Gentil, and I. Chouroulinkov. "Two-Stage Malignant 

Transformation of Rat Fibroblasts in Tissue Culture." Nature, 

247 (1974), 490-491. 

67 
11.

 Montesano, R. , L. Saint Vincent, C. Draven, and L. Tomatis. "Production of Epithelial and Mesenchymal Timers with Rat Liver 

Cells Transformed In Vitro." Int. J. Cancer, 16 (1975), 550-558. 

12. Montesano, R. , L. Saint Vincent, and L. Tonatis. "Malignant 

Transformation In Vitro of Rat Cells by Dimethylnitrosamine and 

N-methyl-N'-nitro-nitrosoguanidine." Br. J. Cancer, 28 (1973), 215-220. 

13. Pars a, I. and W.H. Marsch. "An In Vitro Model of Pancreatic 

Carcinoma Morphology and I_n Vivo Growth." Am. J. Pathol., 84 

(1976), 469-478. 

14. Sato, J., and T. Yabe. "Carcinogenesis in Tissue Culture. V. Effects of Long-Term Additions of 4-dimethyl-aminoazobenzene on 

Liver Cells in Cultures." Jap. J. Exp. Med., 35 (1965), 445. 

15. Schwartz, A.G. "The Protective Effect of Benzoflavone and 

Estrogen Against 7,12-dimethylbenz(a)anthracene and Aflatoxin- Induced Cytotoxicity in Cultured Liver Cells." Cancer Res., 34 

(1974), 10-15. 

16. Toyoshima, K. , Y. Hiasa, N. Ito, and Y. Tsubura. "In vitro 

Malignant Transformation of Cells Derived from Rat Liver by Means 

of Aflatoxin B| ____ " Gann_

 , 61 (1970), 557-562. 

17. Williams, G.M. "Carcinogen-Induced DNA Repair in Primary Rat 

Liver Cells Cultures; a Possible Screen for Chemical 

Carcinogens." Cancer Letters, 1 (1975), 231-235. 

18. Williams, G.M. , J.M. Elliott, and J.H. Weisburger. "Carcinoma 

after Malignant Conversion In Vitro of Epithelial-Like Cells from 

Rat Liver Following Exposure to Chemical Carcinogens." Cancer 

Res., (1973), 606-612. 

68 
2.5 HAMSTER CELLS 

2.5.1 THE USE OF EARLY PASSAGE HAMSTER EMBRYO CELLS FOR THE 

IDENTIFICATION OF CHEMICAL CARCINOGENS 

ROMAN J. PIENTA 

Chemical Carcinogenesis Program, NCI Frederick Cancer 

Research Center, Frederick, MD 

A number of model systems for studying in vitro 

carcinogenesis have been described (43). Several of these lend 

themselves to quantitation and may have potential use for the routine 

bioassay or identification of chemical carcinogens. In general, two 

morphological endpoints for activity are measured, i.e., the formation 

of altered colonies when small numbers of cells are seeded and 

challenged with carcinogen or the formation of foci of altered cells 

when mass cultures are employed. Models employing altered colonies 

have been limited to the use of early passage cells derived from 

hamster embryos (3,4,10-14,24,32,33). Cells from various strains of 

mouse (8,15,26,35,36), guinea pig (16) and rat (28,42) have exploited 

the use of focus formation in carcinogenesis studies. 

In vitro systems employing cells derived from rat 

(17,18,34,39), mouse (37,38) and hamster cells have been used for 

studying the cocarcinogenic interactions of viruses and chemicals. Parameters other than abnormal morphology, such as the growth of 

temperature sensitive mutants in semisolid agar (9), may serve as 

useful tools for studying carcinogenesis in vitro. The system 

described here, which we are evaluating for use as a routine bioassay, is a modification of the system described by Berwald and Sachs (3), DiPaolo (10,14) and their associates. The system relies upon the use 

of early passage cultures derived from golden Syrian hamster embryos. Approximately 500 target cells are seeded onto a sparse lawn of 

x-irradiated feeder cells. The cells are then challenged with 

carcinogen and monitored for the appearance of morphologically altered 

colonies indicative of transformation. Cultures derived from 

morphologically transformed colonies induce sarcomas in hamsters. Variation in response among experiments, which can result in false 

negative results with standard control carcinogens, can be minimized 

by using, aliquot samples of cells stored frozen in liquid nitrogen as 

the source of target and feeder cells (32,33). 

69 
The reproducibility and reliability of the standard bioassy 

requires stringent control of the quality of culture medium, serum and 

other reagents to insure the absence of mycoplasmas or other 

adventitious agents that may interfere. Samples of these as well as 

the plastic culture vessels must be pretested for their capacity to 

support optimum growth. 

To reduce the observed subjectivity when altered morphology 

is used as the endpoint parameter for activity, a number of properties 

of transformed cells are available for study (1, 5,6,19,22,23,26,27, 29-31t40). Results with hamster embryo cells indicate that the 

ability of morphologically transformed cells to grow in semisolid agar 

correlates completely with their tumorigenic potential in hamsters. Normal cells failed to grow in agar or induce tumors. Other criteria 

showing good correlation include the increase in cloning efficiency, extended life span, loss of contact inhibition, and relative growth in 

factor-free medium or medium supplemented with serum substitute. However, varying degrees of activity with these criteria were seen 

with normal cell cultures. Similar subjective results were seen with 

plasminogen activation or cytotoxicity by activated macrophages. 

To verify the reliability of the system to identify 

carcinogens, a large number of standard reference chemicals with known 

carcinogenic activity were bioassayed using morphologic transformation 

as the endpoint for activity. These included carcinogenic and 

noncarcinogenic analogs of direct alkylating agents, polycyclic 

hydrocarbons, nitrosamines, aromatic amines and aminoazo dyes, metal 

carcinogens and a number of miscellaneous compounds. A very high 

correlation (89$) with reported carcinogenic activities was obtained 

when 77 chemicals were bioassayed. Transformation was not observed 

when cells were treated with a few carcinogens requiring metabolic 

activitation. No false positive results were obtained when known 

noncarcinogens were bioassayed and no spontaneous transformation was 

observed in untreated control cultures. 

Dose responses for transformation have been obtained with many of 

the chemicals (10), however, this was not always the case. 

In the standard bioassay, run in the absence of liver microsome 

enzymes, N-2-acetylaminofluorene routinely failed to transform hamster 

cells. However, the inclusion of hamster liver enzymes to the assay 

resulted in the metabolic conversion of AAF to an active carcinogen 

which transformed the cells. Preliminary evidence indicates that 

2-aminofluorene is the major active metabolite in this case. 

70 
REFERENCES 

1. Abercrombie, M. "Contact Inhibition in Tissue Culture." _In 

Vitro, 6 (1970), 128-142. 

2. Ames, B.N. , W.E. Durston, E. Yamasaki, and F.D. Lee. "Carcinogens are Mutagens: A Simple Test System Combining Liver 

Honogenates for Activation and Bacteria for Detection." Proc. Natl. Academy Sci. U.S., 70 (1973), 2281-2285. 

3. Berwald, Y., and L. Sachs. "In Vitro Cell Transformation with 

Chemical Carcinogens." Nature, 200 (1963), 1182-1184. 

4. Borenfreund, E., M. Krim, and F.K. Sanders, et al. "Malignant 

Conversion of Cells In Vitro by Carcinogens and Viruses." Proc.

 Natl. Acad. of Sci. US. 56 (1969), 672-679.

 

5. Burger, M.M. "Lectin Monitored Surface Changes in Transformed 

and Mitotic Normal Cells." In Seventh National Cancer 

Conference, 1973, 405-413. 

6. Burger, M.M. "Surface Changes in Transformed Cells Detected by 

Lectins." Fed. Proc.. 32 (1973), 91-101. 

7. Casto, B.C., W.J. Pieczynski, and J.A. DiPaolo. "Enhancement of 

Advenovirus Transformation by Pretreatment of Hamster Cells with 

Carcinogenic Polycyclic Hydrocarbons." Cancer Res., 33 (1973), 819-824.

 

8. Chen, T.T., and C. Heidelberger. "Quantitative Studies on the 

Malignant Transformation of Mouse Prostate Cells by Carcinogenic 

Hydrocarbons In Vitro." Intern. J. Cancer, 4 (1969), 166-178. 

9. DiMayorca, G., M. Greenblatt, and T. Franthen, et al. "Malignant 

Transformation of BHK21 Clone 13 Cells In Vitro by Nitrosamines ­ A Conditional State." Proc. Natl. Acad. Sci. US, 70 (1973),

 46-49.

 

10. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "Quantitative 

Studies of In Vitro Transformation by Chemical Carcinogens." ^J. Natl. Cancer Inst., 42 (1969), 867-876. 

11. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "In Vitro 

Transformation of Hamster Cells by Polycyclic Hydrocarbons: 

Factors Influencing the Number of Cells Transformed." Nature New 

Biology, 230 (1971), 240-242. 

71 
12.

 DiPaolo, J.A., R.L. Nelson, and P.J. Donovan. "Morphological, Oncogenic, and Karyological Characteristics of Syrian Hamster 

Embryo Cells Transformed In Vitro by Carcinogenic Polycyclic 

Hydrocarbons." Cancer Res.. 31 (1971), 1118-1127. 

13. DiPaolo, J.A., R.L. Nelson, P.J. Donovan, and C.H. Evans. "Host-Mediated In Vivo-In Vitro Assay for Chemical 

Carcinogenesis." Arch. Pathol.. 95 (1973), 380-385. 

14. DiPaolo, J.A., R.L. Nelson, and P.J. Donovan. "In Vitro 

Transformation of Syrian Hamster Embryo Cells by Diverse Chemical 

Carcinogens." Nature. 235 (1972), 278-280. 

15. DiPaolo, J.A., K. Takano, and N.C. Popescu. "Quantitation of 

Chemically Induced Neoplastic Transformation of BALB/3T3 Cloned 

Cell Lines." Cancer Res., 32 (1972), 2686-2695. 

16. Evans, C.H. , and J.A. DiPaolo. "Chemical Carcinogen-Induced 

Neoplastic Transformation In Vitro. In Vitro. 8 (1973), 431. 

17. Freeman, A.E. , P.J. Price, and H.J. Igel, et al. "Morphological 

Transformation of Rat Embryo Cells Induced by Diethylnitrosamine 

and Murine Leukemia Viruses." J. Natl. Cancer Inst., 44 (1970), 65-78. 

18. Freeman, A.E. , E.K. Weisburger, and J.H. Weisburger, et al. "Transformation of Cell Cultures as an Indication of the 

Carcinogenic Potential of Chemicals." J. Natl. Cancer Inst., 51 

(1973), 799-808. 

19. Goldberg, A.R. "Increased Protease Levels in Transformed Cells: 

A Casein Overlay Assay for the Detection of Plasminogen Activator 

Production." Cell, 2 (1974), 95-102. 

20. Heidelberger, C. "Chemical Oncogenesis in Culture." Adv. Cancer 

Res.. 18 (1973), 317-366. 

21. Heidelberger, C., C.A. Reznikoff, and D.F. Krahn. "Malignant 

Transformation of Cells in Culture Using Oncogenic Chemicals." 

In: Tissue Culture, Methods, and Applications. P. F. Kruse, Jr. and M.K . Patterson, Jr. (Eds.). Academic Press, N.Y. , 644-653. 

22. Hibbs, J.B. "Discrimination Between Neoplastic and 

Non-Neoplastic Cells In Vitro by Activated Macrophages." J_. 

Natl. Cancer Inst.. 53 (1974), 1487-1492. 

23. Hibbs, J.B., J.H. Lambert, and J.S. Remington. "In Vitro 

Nonimmunologic Destruction of Cells with Abnormal Growth 

72 
Characteristics by Adjuvant Activated Macrophages." Proc. Soc. Exptl. Biol. Med., 139 (1972), 1049-1052. 

24. Huberman, E., P.J. Donovan, and J.A. DiPaolo. "Mutation and 

Transformation of Cultured Mamnalian Cells by 

N-Acetoxy-N-2-Fluorenylacetamide." J. Natl. Cancer Inst., 48 

(1972), 837-840. 

25. Kakunaga, T. "A Quantitative System for Assay of Malignant 

Transformation by Chemical Carcinogens Using a Clone Derived from 

BALB/3T3." Intern. J. Cancer, 12 (1973), 463-473. 

26. Laug, W.E. , P.A. Jones, and W.F. Benedict. "Relationship between 

Fibrinolysis of Cultured Cells and Malignancy." J. Natl. Cancer 

Inst.. 54 (1975), 173-179. 

27. Macpherson I. "The Characteristics of Animal Cells Transformed 

In Vitro." In: Advances in Cancer Research, 13 (1970), 169-215. 

28. Namba, M., H. Masuji, and J. Sata. "Carcinogenesis in Tissue 

Culture IX. Malignant Transformation of Cultured Rat Cells 

Treated with 4-Nitroquinoline-1-Oxide." Japan J. Exptl. Med., 39 

(1969), 253-269. 

29. Ossowski, L, J.P. Quigley, G.M. Kellerman, and E. Reich. "Fibrinolysis Associated with Oncogenic Transformation." J. 

Expl. Med., 138 (1973), 1056-1064. 

30. Ossowski, L., J.P. Quigley, and E. Reich. "Fibrinolysis 

Associated with Oncogenic Transformation." J. Biol. Chem., 249 

(1974), 4312-4320. 

31. Paul, D., M. Henahan, and S. Walter. "Changes in Growth Control 

and Growth Requirements Associated with Neoplastic Transformation 

In Vitro." J. Natl. Cancer Inst., 53 (1974), 1499-1503. 

32. Pienta, R.J. , J.A. Poiley, and W.B. Lebherz, III. "Standardiza­ tion of a Hamster Cell In Vitro Carcinogenesis Bioassay Using 

Cells Stored in Liquid Nitrogen." Proc. Amer. Assoc. Cancer 

Res.. 16 (1975), 24. 

33. Pienta, R.J., J.A. Poiley, and W.B. Lebherz, III. "Further 

Evaluation of a Hamster Embryo Cell Carcinogenesis Bioassay." 

Proc. of Third International Symposium on Detection and 

Prevention of Cancer, N.Y. , N.Y. (1976) (in press). 

34. Price, P.J., A.E. Freeman, W.T. Lane, and R.J. Huebner. "Morphological Transformation of Rat Embryo Cells by the Combined 

73 
Action of 3-Methylcholantrene and Rauscher Leukemia Virus."
 

Nature New Biol., 230 (1971), 144-146.
 

35.	 Reznikoff, C.A., D.W. Brankow, and C. Heidelberger.
 

"Establishment and Characterization of a Coned Line of C3H Mouse
 

Embryo Cells Sensitive to Post Confluence Inhibition of
 

Division." Cancer Res., 33 (1973), 3231-3233.
 

36.	 Reznikoff, C.A., J.S. Bertram, D.W. Brankow, and C. Heidelberger.
 

"Quantitative and Qualitative Studies of Chemical Transformation
 

of Cloned C3H Mouse Embryo Cells Sensitive to Post Confluence
 

Inhibition of Cell Division." Cancer Res.. 33 (1973), 3239-3249.
 

37.	 Rhim, J.S., B. Creasy, and R.J. Huebner. "Production of Altered
 

Cell Foci by 3-Methylcholanthrene in Mouse Cells Infected with
 

AKR Leukemia Virus." Proc. Natl. Acad. Sci. U.S., 68 (1971),
 2212-2216.
 

38.	 Rhim, J.S., O.K. Parks, E.K. Weisburger, and J.H. Weisburger.
 

"Evaulation of an In Vitro Assay System for Carcinogens Based on
 

Prior Infection of Rodent Cells with Non-Transforming RNA Tumor
 

Virus." J. Natl. Cancer Inst., 52 (1974), 1167-1173­

39.	 Rhim, J.S., W. Vass, H.Y. Cho, and R.J. Huebner. "Malignant
 

Transformation Induced by 7,12-Dimethylbenz(a)anthracene in Rat
 

Embryo Cells Infected with Rauscher Leukemia Virus." Intern. J.
 

Cancer, 7 (197D, 65-74.
 

40.	 Sanford, K.K. "Biologic Manifestations of Oncogenesis In Vitro:
 

A Critique." J. Natl. Cancer Inst., 53 (1974), 1481-1485.
 

41.	 Scher, C.D., and G.J. Todaro. "Selective Growth of Human
 

Neoplastic Cells in Medium Lacking Serum Growth Factor." Exptl.
 

Cell Res., 68 (1971), 479-481.
 

42.	 Sekely, L.I., D. Malejka-Giganti, H.R. Gutmann, and R.E. Rydell.
 "Malignant Transformation of Rat Embryo Fibroblasts by Carcino­ genic Fluorenylhydroxamic Acids In Vitro." J. Natl. Cancer
 

Inst., 50 (1973), 1137-1345.
 

43.	 Stoltz, D.R., L.A. Poirier, and C.C. Irving, et al. "Evaluation
 

of Short-Term Tests for Carcinogenicity." Toxicology Appl.
 

Pharm., 29 (1974), 157-180.
 

74
 
ADDITIONAL REFERENCE:
 

MM. Pienta, R.J., J.A. Poiley, and W.B. Lebherz, III. "Morphological
 

Transformation of Early Passage Golden Syrian Hamster Embryo
 

Cells Derived from Cryopreserved Primary Cultures as a Reliable
 

In Vitro Bioassay for Identifying Diverse Carcinogens." Int. J.
 

Cancer. 19 (1977), 642-655.
 

75
 
2.5.2

 SOMATIC MUTATION AS THE BASIS FOR IN VITRO TRANSFORMATION 

OF BHK CELLS BY CHEMICAL CARCINOGENS 

NOEL BOUCK AND GIAMPIERO Dl MAYORCA1 

Department of Microbiology & Inmunology, University of 

Illinois at the Medical Center, Chicago, IL 

The extensive mutagenio activity of carcinogens which has 

recently been demonstrated in a number of systems (4,16,17,25,26), has 

been interpreted by many (2,5,25) but not all (31,37) investigators as 

supportive of the mutational origin of chemically induced cancers. The alternative explanation, that these cancers result from an 

epigenetic change in gene function rather than from a mutational 

change in gene structure is also supported by a number of experiments 

(see 31,37) including the demonstration that markers from embryonic 

tumor cells can reappear in normal adult tissues (28). To 

differentiate between these two hypotheses we have studied the 

transformation process itself and asked if it is or is not a 

mutational event. 

Using a subclone of the quasidiploid line BHK21/cl 13 (38), BHK Supernormal clone 10, which was selected for the exceedingly 

normal phenotype it displays when cultured in liquid and in semi-solid 

media, we have investigated in vitro transformation by the chemical 

carcinogens nitrosomethylurea (NMU) and 4-nitroquinoline-1-oxide 

(NQO). To insure that in the in vitro study we are truly measuring 

malignant changes, we have used the soft agar assay for transformation 

(23), which is the only selective characteristic of transformed cells 

which shows nearly complete correlation with in vivo tumorigenicity 

(12,35). To determine if the change induced by the carcinogens in the 

BHK line from a normal to a transformed phenotype indeed represents a 

genetic mutation it is not possible to apply the techniques of 

classical genetics (11). Rather one must rely, as do others 

(22,36,39), on a set of criteria for mutagenesis which are 

operationally applicable to somatic cells to determine if a new 

phenotypic variant is a mutant. These criteria are as follows: 

a. The new variant must arise with low frequency 

spontaneously. Transformed variants arise in the BHK Supernormal line 

at the 7th passage following cloning with a frequency of 2.2 x 10"? 

per viable cell. This frequency is well within the range observed for 

spontaneous mutations at other loci in mammalian cells (6,15). 

1

This research supported by Contract #NIH NIC N01 CP 23303 

76 
b. The frequency of the new variant must increase in response 

to mutagenic agents. When the BHK Supernormal line is treated with 

either NMJ or NQO, both potent mutagens in bacterial systems (3,26), the frequency of transformants per survivor increases in a dose- dependent manner to a maximum of 4 x 10"** and 2 x 10"^ respectively. These frequencies are also within the range commonly encountered for 

the mutagenesis of other better characterized mammalian cell loci 

(6,15). In similar experiments Kakanuga has found NQO to be even more 

effective at the in vitro transformation of his BALB/3T3 mouse cell 

line (19,20,21). In vitro transformation by

previously by Sanders and Burford (33). NMU has been reported 

c. The new variant phenotype should be associated with an 

altered gene product. When the transformants induced by NMU and NQO 

are isolated, recloned, and grown and tested for the expression of the 

transformed phenotype at high and low temperatures, it is found that 

44$ (17/39) of the independently arising clones express the 

transformed phenotype at both temperatures. The remaining clones are 

either normal at low temperature and transformed at high temperature 

or are transformed at low temperature and normal at high temperature. These temperature-restricted phenotypes are indicative of a 

temperature-restricted gene product which appears to have resulted 

from the initial treatment with the mutagenic carcinogens. 

A similar high frequency of temperature sensitive mutants is 

found at a few loci in other organisms (1,13,18,34) and also among 

revertents of both bacterial and mammalian cell mutants (7,9,24), and 

presumably reflects an unusually high proportion of temperature 

sensitive sites among the recoverable or permissible lesions. Temperature limited transformed clones similar in some respects to 

those reported here have occasionally been isolated before, both this 

laboratory (12), and in others (30,32,40) although in the latter cases 

data is not yet available to show that it is transformation and not 

just cloning ability in general which is modulating with temperature. 

d. The variant phenotype should be stable. The BHK 

transformants have been extensively tested and found to stably retain 

their original temperature-limited transformed phenotype both in vitro 

and in vivo. 

e. Reversion of variants to the normal phenotype should be low 

but demonstrable. Reversion frequency has been measured for one of 

the temperature restricted transformants, Me2N4 (12), by using lethal 

growth in methylcellulose containing flurodeoxyuridine and uridine to 

enrich for normal reverants after ethylmethylsufonate mutagenesis. The induced reversion rate is 1 x 10""^ revertants per survivor, similar to reversion frequencies observed for other temperature 

sensitive mutants in the BHK cell line (27). 

77 
f. The variant phenotype should be localized on a chromosome. This final criteria has not yet been approached using the BHK 

transform ants. 

Although a chromosomal location remains to be demonstrated, the above evidence strongly suggests that chemically induced BHK 

transformants originate as somatic mutations. Epigenetic alterations 

can be demonstrated to occur in hybrids of cultured cells (10), but 

most epigenetic explanations for somatic cell variants have been found 

unnecessary (8) and remain uproven (11,36). Our observation in this 

work that temperature sensitive, cold sensitive, and unrestricted 

transformants can all arise simultaneously in a cloned line in 

response to the same single short carcinogen treatment is hard to 

explain by epigenetic mechanisms whereas it is easily understood as 

mutations induced at different sites in the gene or genes controlling 

normal growth. 

As recent reviews demonstrate (14,29) chemical 

carcinogenesis is far from being clearly understood, but the evidence 

now available in the case of BHK cells is consistent with the 

hypothesis that a somatic mutation is the primary event by which 

chemicals transform normal cells into ones capable of malignant growth. 

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28. Mintz, B., and K. Illmensee. "Normal Genetically Mosaic Mice 

Produced from Malignant Teratocar cinema Cells." Proc. Natl. Acad. Sci. U.S.. 72 (1975), 3585-3589. 

29. Mishra, N.K. , and G. di Mayorca. "In Vitro Malignant 

Transformation of Cells by Chemical Carcinogens." Biochim. Biophys. Acta. 335 (1974), 205-219. 

80 
30.

 Miyashita, K. , and T. Kakunaga. "Isolation of Heat - and Cold ­ Sensitive Mutants of Chinese Hamster Lung Cells Affected in Their 

Ability to Express the Transformated State." Cell, 5 (1975), 131-138. 

31. Rubin, H. "Letters: Carcinogenicity Tests." Science, 191 

(1976), 241. 

32. Rudland, P.S., E. Pearlstein, D. Kamely, M. Nutt, and 

W. Eckhart. "Independent Regulation of Cellular Properties in 

Therm os ens itive Transformation Mutants of Mouse Fibroblasts." 

Nature. 256 (1975), 43-46. 

33. Sanders, F.K., and B.O. Burford. "Morphological Conversion of 

Cells In Vitro by N-Nitroscmethylurea." Nature. 213 (1967), 

1171-1173. 

34. Shell enbarger, D.L. , and J.D. Mohler. "Temperature-Sensitive 

Mutations of the Notch Locus in Drosophila melanogaster." 

Genetics. 81 (1975), 143-162. 

35. Shin, S., V.H. Freedman, R. Risser, and R. Pollack. "Tumorigenicity of Virus-Transformed Cells in Nude Mice is 

Correlated Specifically with Anchorage Independent Growth In 

Vitro. "Proc. Natl. Acad. Sci. U.S., 72 (1975), 4435-4439. 

36. Siminovitch, L. "On the Nature of Hereditable Variation in 

Cultured Sonatic Cells." Cell. 7 (1976), 1-11. 

37. Sivak, A. "Letters: The Ames Assay." Science, 193 (1976), 272-274. 

38. Stoker, M. , and I. Macpherson. "Syrian Hamster Fibroblast Cell 

Line BHK21 and Its Derivatives." Nature. 203 (1964), 1355-1357. 

39. Thompson, L.H., and R.M. Baker. "Isolation of Mutants of 

Cultured Mammalian Cells." In: Methods in Cell Biology VI. , D.M.

 Prescott (Ed.). Academic Press, N.Y. , 1973, 209-281. 

40. Yamaguchi, N. , and I.E. Weinstein. "Temperature-Sensitive 

Mutants of Chemically Transformed Epithelial Cells." Proc. Natl. Acad. Sci. U.S.. 72 (1975), 214-218. 

81 
2.6

 GUINEA PIG CELLS 

2.6.1 NEOPLASTIC TRANSFORMATION BY CHEMICALS OF GUINEA PIG FETAL 

CELLS IN CULTURE 

CHARLES H. EVANS 

Biology Branch, Carcinogenesis Program, Division of Cancer 

Cause and Prevention, National Cancer Institute, NIH, 

Bethesda, MD 

Carcinogens from diverse chemical classes induce neoplastic 

transformation of guinea pig fetal cells in culture. The transition 

to the neoplastic state often occurs in a step-wise manner (1). 

Morphological alterations in carcinogen treated cultures appear 

shortly after treatment, but transformation, a loss of cell orientation does not appear for four or more months, is not seen in cultures 

treated with a noncarcinogenic chemical and may precede by 2-6 months 

development of the capacity of transformed cells to grow as tumors in 

irradiated syngeneic newborn guinea pigs or in nude mice (2). Alterations in plating efficiency, growth rate, serum dependence, cell 

surface antigens (4), endogenous virus expression (5), and chromosome 

number and structure (6) are not necessary for the development of 

neoplastic transformation. The ability of transformed cells to grow 

as colonies in agar (1), susceptibility of the cells to the cytotoxic 

effects of lymphokines produced by nonimmune syngeneic leukocytes 

(3,7), intradermal skin reactivity of transformed cells in nonimmune 

syngeneic guinea pigs (3), and secretion of large amounts of 

plasminogen activator (I. B. Weinstein, personal communication) are 

characteristics that correlate well with neoplastic transformation. 

These cell properties are useful indicators of the tumorigenic 

potential of guinea pig cells transformed by chemical carcinogens. 

Development of this model system offers an additional avenue for 

studying the process of carcinogenesis using a species with 

established tumor biology in which spontaneous transformation has not 

been seen, which possesses well defined immunological parameters, and 

in which discrete stages in the transition from the growth controlled 

to the neoplastic state may be studied. 

REFERENCES 

1. Evans, C.H. , and J.A. DiPaolo. "Neoplastic Transformation of 

Guinea Pig Fetal Cells in Culture Induced by Chemical 

Carcinogens." Cancer Res., 35 (1975), 1035-1044. 

82 
2.

 Evans, C.H., and J.A. DiPaolo. "Comparison of Nude Mice with the 

Host Species for Evaluation of the Tumorigenicity of Guinea Pig 

and Hamster Cells Transformed In Vitro by Chemical Carcinogens." 

Cancer Res., 36 (1976), 128-131. 

3. Evans, C.H. , A.M. Cooney, and J.A. DiPaolo. "Colony Inhibition 

Mediated by Nonimmune Leukocytes In Vitro and Skin Reactivity In 

Vivo as Indices of Tumorigenicity of Guniea Pig Cultures 

Transformed by Chemical Carcinogens." Cancer Res., 35 (1975), 1045-1052. 

4. Evans, C.H. , S.H. Ohanian, and J.A. DiPaolo. "Immunogenicity of 

Guinea Pig Cells Transformed by Chemical Carcinogens in 

Culture." Fed. Proceed, 31* (1975), 828. 

5. Hsiung, G.D., C.K.Y. Fong, and C.H. Evans. "Prevalence of 

Endogenous Oncornavirus in Guinea Pigs." Intervirology, 3 

(1974), 319-331. 

6. Popescu, N.C., C.H. Evans, and J.A. DiPaolo. "Chromosome 

Patterns (G and C Bands) of In Vitro Chemical Carcinogen- Transformed Guinea Pig Cells." Cancer Res.. 36 (1976), 1404-1413. 

7. Zwilling, B.S., M.A. Meltzer, and C.H. Evans. "Differential 

Cytogoxicity of Tumorigenic and Nontumorigenic Strain-2 Guinea 

Pig Cells Mediated by Syngeneic Phytohemagglutinin Stimulated 

Peritoneal Exudate Cells." J. Natl. Cancer Inst., 54 (1975), 743-747. 

83 
2.7 HUMAN CELLS
 

2.7.1 CHEMICAL TRANSFORMATION OF HUMAN AND RODENT CELLS
 

TAKEO KAKUNAGA
 

Chemistry Branch, Carcinogenesis Program, Division of Cancer
 

Cause and Prevention, National Cancer Institute, NIH,
 Bethesda, MD
 

The human risk to environmental carcinogens has been
 

assessed mainly by epidemiological methods and by carcinogenesis
 

experiments in animals. Recently other rapid assay systems have also
 

been developed using various species such as prokaryote and methods
 

such as mutagenesis and transformation of cultured rodent cells.
 However, the evolutionary aspects of biological functions such as
 

repair activity and endogenous virus indicate that there may be some
 

discrepancy between the results obtained with animals or prokaryotes
 

and the actual carcinogenicity of substances in human. In order to
 

solve this problem of the extrapolation of the experimental results
 

into human risk, i-t would be very useful to develop a system for the
 

transformation of human diploid cells by chemical substances (see:
 

Saffiotti, in this Volume).
 

The system of cultured human diploid cells has many
 

advantages for genetic and biochemical studies such as the stability
 

of the ploidy and the abundant availability of well analyzed cell
 

mutants, i.e., the cultured cells derived from genetic disease
 

patients. Development of a system for the transformation of human
 

diploid cells by chemicals is very important to establish a better
 

method for understanding the mechanisms of carcinogenesis as well as
 

for the assessment of human risk to enviromental carcinogens.
 

Although two cases of chemical transformation have been
 

reported by using human cell strains derived from tumor tissues
 

(7,13), there has been no success in the chemical transformation of
 

normal human diploid cells. Many explanations were considered to
 

explain this difficulty before starting my experiments. Most of these
 

explanations were based on the problems dealing with the methods and
 

the conditions of the experiments such as the following: 1) Usual
 

culture conditions used for mammalian cells are not suitable for human
 

cells. This was generally observed by the low plating efficiency of
 

human cells. 2) The transformed phenotype of human cells may be
 

different from those of the experimental animals. 3) Human diploid
 

cells, especially fibroblasts, lack the enzyme activity needed to
 

convert the chemicals into active forms.
 

84 
First, we examined the culture conditions and established a
 

good system which routinely produced a cloning efficiency of 80 to 95?
 

with most of the human diploid fibroblast strains available. The
 

second explanation seemed unlikely, because it had been reported that
 

all the cells in culture obtained from human tumor tissues show
 

similar biological properties to the transformed rodent cells (M,5).
 The third problem could be easily solved by using directly acting
 

chemicals or the chemicals which are activated in the cells of all
 

species and all tissues. Thus, these three problems were satisfied,
 and then we had a chance to try to transform human diploid cells by
 

chemical carcinogens. Here I described the outline of our
 

experimental procedures and results of the chemical transformation.
 

CHEMICALS
 

4-nitorquinoline-1-oxide (UNQO) and 4-amino-quinoline­ 1-oxide (4AQO) were obtained from Daiichi Pure Chemical Co. (Tokyo),
 N-methyl-N1

-nitro-N-nitrosoguanidine (MNNG) from City Chemical Co.
 (New York), and 3-methylcholanthrene (MCA) from Eastman Kodak Co.
 (Rochester). All chemicals were dissolved in dimethylsulfoxide (DMSO)
 

spectranalyzed (Fisher Scientific Co. (Pittsburgh)) at the appropriate
 

concentrations and then added to culture medium in the dark. The
 

chemical solution was prepared immmediately before usage. Final
 

concentration of DMSO in culture medium was 0.2 - 1.0/J.
 

TYPE OF CELLS
 

A human diploid cell strain, KD, derived from a skin biopsy
 

taken from a female adult was used in this experiment. The following
 

points were considered: 1. It is desirable that the cells are the
 

most available type of human diploid cells taken from the individual.
 This would be an advantage for the further development of a
 

comparative study of the sensitivity to chemical carcinogenesis of
 

cells derived from normal patients and various genetic disease
 

patients who are predisposed to the higher incidence of cancer. 2.
 The normality or abnormality of the individual from whom the cells are
 

taken is demonstrable. 3- The cells of the same genetic background
 

can be obtained repeatedly from the same individual. This strain was
 

initiated by Dr. R. Day (2) from a skin biopsy taken from the lip of
 

an adult female who has not shown any genetic disease. The primary
 

culture was made by plating the cells dissociated by stirring the
 

biopsy in a trypsin-collagenase solution. The grown cells were
 

subcultured at 1:2 dilution and stored in LN? freezer as stock cells.
 For the transformation experiment, the cells were used at the 6th to
 

34th passage after isolation.
 

Although the original biopsy contained epidermal tissue, the
 

KD cells used in the experiment ahowed the typical morphology of
 

fibroblast in mass culture (Figure 1) and did not produce any colony
 

85
 
of epithelial-like morphology. Throughout the experiment a total of 

more than 2 x 10^ colonies of KD cells were observed. The major 

population of the KD cell stock seemed to be fibroblastic in 

morphology and preliminary examination of the karyotype indicated that 

the KD cells are diploid and that their diploidy is stable as

they grow actively. 

 long as 

CULTURE CONDITIONS 

The cells were cultured in 100 plastic dishes (Falcon 

Plastics (Oxnard)) containing 10 ml culture medium in a humidified CC>2 

- incubator at 37°C. The regular culture medium consisted of Eagle's 

minimum essential medium (MEM) (Grand Island Biological Co. (Grand 

Island)) supplemented with '\0% fetal calf serum (Flow Laboratories 

(Rockville)). The serum used was selected by pre-screening the serum 

samples from various sources for the cloning efficiency and the effect 

on the morphology of cultured KD cells. The culture medium was 

changed 2 or 3 times a week. The subcultivation was made by adding 

0.2556 trypsin solution (Grand Island Biological Co. (Grand Island)) to 

86 
the culture for 3-5 min, removing the trypsin solution, letting the 

culture

in

medium, morp

 stand at room temperature until

 a sign of digestion, the

 aspirating and dispensing into

hology,

 the cells

n adding fre

 new dishes. 

 showed

sh reg

 some

ular

 change 

 culture 

MARKER OF TRANSFORMATION 

As described before and will be discussed later, many 

diagnoses of neoplastic transformation of cultured cells have been 

reported. Among them, the morphological change in growth pattern and 

the anchorage independence of cell growth seem to be the most common 

markers of the neoplastic fibroblast in culture as well as the 

pathological diagnosis of in vivo tumors. As an initial step in the 

development of the transformation system, any kind of change in the 

morphology of the gowth pattern was taken as a primary diagnosis to be 

observed. 

ASSAY PROCEDURE 

Using the established assay system of the chemical 

transformation of rodent cells as reference, several methods were 

applied to detect the chemical transformaticn of KD cells. 

First, the simplest assay method was tried. That is, the 

growing cells were treated with 4NQO (0.01 - 1.0 yg/ml) or MNNG (0.1 ­

1.0 yg/ml) for 30 or 60 min in suspension, and then plated at an 

appropriate concentration of cells so that each surviving cell formed 

a separate colony. Ten to 16 days after plating, the cultures were 

fixed, stained, and the morphology of the colonies was examined under 

the stereomicroscope. No change has been observed in more than 

200,000 colonies observed so far. 

Second, the focus assay method was used. The growing cells 

were treated with chemicals and the surviving cells were allowed to 

divide 4 or 5 times before attaining a confluent state. After 

reaching confluence, the cultures were maintained without subculturing 

for 3 to 4 months until they were fixed and stained. About 5 x 10^ 

cells were treated with 4NQO or MNNG and 600 dishes containing 

approximately 10° surviving cells were tested. No transformed focus 

has ever been found in these experiments. 

Colony formation of the cells in soft agar was used as an 

indicator of transf ormaticn in a third assay method. The cells were 

allowed to divide for 4 or 5 cell generations after the chemical 

treatment and then were plated into a soft agar layer. No colony 

formation was observed in the soft agar layer into which a total of 

3-3 x 10? surviving cells were inoculated. 

87 
The fourth was a classical method to obtain transformation
 

in culture. Actively growing cells were treated with various
 

concentrations of 4NQO or MNNG for 30 min and were then subcultured at
 

1:4 dilution ratio whenever they became confluent. Because of the
 

limitation of capacity of the incubator, the experiment was designed
 

so that only one of four subcultured dishes was used for the
 

subsequent subculturing. All the cultures were examined for
 

morphologic change under the microscope at least once a week.
 

Seven to 14 weeks and 5 to 10 passages after the treatment
 

with carcinogen, morphologically altered cells were found in small
 

area of a very small number of dishes. The main morphologic
 

alteration was the formation of an area with a high density of cells
 

showing frequent mitosis. When these cultures containing the morpho­ logically altered cells were maintained without subculturing, the
 

dense area became visible by the naked eye as a focus (Figure 2). The
 

shape of the focus was dependent on the stream of the surrounding un­ altered cell. The edge of the focus followed the direction of the
 

surrounding cells (Figure 3)• Disorganization and a criss-cross
 

pattern of cell arrangement, which is the typical morphology in most
 

of the transformed cells in culture, was not distinctive although some
 

disorder of cell arrangement was easily found by careful observation
 

(Figure 4). It appears that the density-dependent inhibition of "cell
 

growth" was lost whereas the loss of the sensitivity to the density
 

dependent inhibition of "cell movement" is not marked in these
 

morphologically altered cells. When the cells were picked up from the
 

dense area and transferred into new dishes, they retained the property
 

of dense growth and formed heavily multilayered sheets (Figures 5 and
 

6).
 

Five out of 6 separate experiments resulted in the
 

development of morphological alteration in subcultures derived from
 

the chemical-treated cultures. The morphologically altered cells
 

appeared only in the cultures treated with about 30% survival dose of
 

4NQO or MNNG and did not appear in the other cultures including those
 

which were untreated, treated with MCA or 4AOO, and treated with a low
 

concentration of 4NQO, as far as tested.
 

REQUIREMENT OF CELL DIVISION FOR THE DEVELOPMENT OF
 

TRANSFORMATION
 

In the transformation of mouse or hamster embryonic cells by
 

4NQO, we found that approximately 4 or 5 cell generations after chemi­ cal treatment are required for the development of the transformation
 

(10,11). As described above, however, no morophologically altered
 

88
 
Figure 2. Photograph after fixation and Giemsa-staining of dishes as 

an example of the focus formation by the morphologically altered 

cells. They were derived from the cultures which were treated for 30 

min with 0.3$ DMSO (left) and 0.1 yg/ml of 4NQO (right), then subcultured 11 times at 1:4 dilution ratio whenever they became confluent, and maintained for 24 days without further subculturing. 

Figure 3- Focal area of morphologically altered cells 

89 
Figure H. Higher magnification of Figure 3­
Figure 6. Photography after fixation and Giemsa-staining of dishes as
 

an example of the formation of dense cultures by the morphologically
 

altered cells. The culture of untreated KD cells (left) and the
 

culture in which the cells picked up from foci area were grown (right).
 

91 
cells were found in tests using the first three assay procedures, 

i.e., clonal morphology assay, focus assay, and soft agar assay. In 

these three methods, the surviving cells were allowed to divide 4 to 8 

times after chemical treatment. The morphologically altered cells 

appeared only after extensive subcultivation. When a small number of 

the morphologically altered cell lines were plated on the monolayer 

sheet of untreated KD cells, they grew and formed clear foci at the 

efficiency of approximately 80%. These findings indicate the 

possibility that an increased number of cell divisions is necessary 

for the development of the transformation of human diploid cells. In 

order to test this possibility and to confirm the chemically-induced 

transformation, the experiments were done by using cloned KD cell. Many clones were isolated from the original KD cell strain and then 

each clone was increased to have a sufficient number of cells to start 

the transformation experiment. All the clones isolated showed the 

fibroblastic morphology. Each clone was treated with 30$ survival 

dose of MNQO, and subcultured as described above. Regardless of the 

duration of time the cultures were maintained in the confluent state, the morphologically altered cells were found only after many repeated 

subcultivations. The average number of cell generations required for 

the development of morphological alteration was calculated to be at 

least 13. This is based on the assumption that all the surviving 

cells divided equally. No morphological alteration was observed in 

the control cultures in which the same clone was treated with DMSO 

only and subcultured (until it degenerated) in the same manner as the 

treated cultures. This supports the idea that morphological 

alteration was induced by treatment with l|NQO in the human diploid 

fibroblast. 

There was a variation in the time of the first observation 

of morphological alteration between cultures of separate experiments 

in which a different clone of KD cells was used as well as between 

cultures of the same series of experiments. It is uncertain whether 

there was a significant difference in susceptibility to alteration 

between the clones of KD cells. 

The morphologically altered cells appeared in the sub­ cultures derived from only a few of the 10 initially treated 

cultures. If it is assumed that there was no segregation of the 

transformable cells, the transformation frequency is calculated to be 

approximately 3.3 x 10~7 per treated cell. It was impossible to test 

the possibility that further cell generations after 4NQO- treatment 

may result in a higher transformation frequency because the 

morphologically unaltered cells began to degenerate at 8 - 10 passages 

after treatment in these experiments. 

Although it may be dangerous to simply compare the different 

transformation systems, the transformation frequency obtained in this 

92 
experiment is of quite low magnitude compared to those obtained in 

rodent cells. The maximum frequency of chemical transformation per 

surviving cell is reported to be 6 x 10"1 - 10"3 in Syrian hamster 

embryonic cells, 10~2 - 10~3 in the subclone of BALB/3T3, (3,9), and 

10""^ in mouse prostate cell lines (1), varying between different cell 

sources, laboratories, criteria of the transformed phenotypes, and 

procedures, etc. The magnitude of the difference in the chemical 

transformation frequency between human diploid cells and rodent cells 

may be related to the difference in the stability of the ploidy in 

cultures as well as the difference in the cell sources and 

experimental conditions. It is known that the karyotype of the 

cultured rodent cells are generally unstable while human diploid cells 

are stable. It is possible to speculate about variations in the DNA 

repair activity (12) and variations in the presence and expression of 

the oncogene between the cells of the differnt species. It is 

unlikely that this difference in transformation frequency is due to 

the difference in the permeability across cell membrane of ^INQO or in 

the total amount of 4NQO bound to DNA. Thirty percent survival dose 

of iJNQO gave 3 x 10^ fold transformation frequency in A31-714 cells 

derived from BALB/3T3 (9). We have found that the amount of 

4NQO-adduct to guanine or adenine base was similar between human 

diploid fibroblast and A31-714 cells (8). 

BIOLOGICAL PROPERTIES OF THE TRANFORMED CELLS IN CULTURE 

Saturation density and the ability to grow in soft agar were 

examined in the morphologically altered lines derived from each focus 

and the untransformed cells of chemical-treated cultures. These cells 

were compared with the untreated and untransformed KD cells (table 2). 

Saturation density was determined after inoculating 5 x 10^ 

cells into 60 mm plastic dishes containing 5 ml regular culture 

medium. The medium was changed every 3 days, and the number of cells 

was counted daily. The saturation density was expressed as the cell 

number per cm 2 when counts on 3 successive days did not change 

significantly. Saturation density was increased in all the 

morphologically altered cells but not in the treated, morphologically 

unaltered cells. 

Growth of the suspended cells in soft agar was examined by 

the procedure as described previously (9) unless otherwise specified. The ability to grow in soft agar was also increased in the 

morphologically altered cells, although the increase was slight in the 

lines derived from cloned KD. The increase in the saturation density 

was not quantitatively parallel to the increase in the ability to grow 

in soft agar. 

93 
All the morphologically altered cells picked up from foci
 

grew exponentially and have not shown any sign of a limit to their
 

life span. Preliminary examination .indicated that all the
 

morphologically altered cells showed aneuploidy.
 

The results indicate that the morphologically altered cells
 

obtained possess the properties which are usually correlated with
 

neoplastic transformation of cells in culture.
 

TRANSPLANTABILITY INTO NUDE MICE
 

It has been reported that human tumor transplants or cells
 

freshly cultured from human malignant tumors give rise to tumors in
 

Nude mice (6,14,15). Cellular tumorigencity in nude mice has been
 

reported to be correlated with cell growth in semi-solid medium (4).
 Nude mice are thought to be one of the best available systems for
 

testing the malignancy of human cells.
 

To examine the.transplantability of the cells, the cells
 

were suspended in 0.2 ml Eagle's MEM serum-free medium and then
 

injected subcutaneously into 5 to 16-week old female nude (athymic)
 

mice, which were maintained under conditions of strict sanitation and
 

segregation from other strains of rodents. Groups of 10 mice with the
 

same distribution of ages in each group were designated. The mice
 

were checked for the development of tumors weekly. Biopsy samples
 

were removed surgically from one-half of the tumor-bearing mice in
 

each experimental group. Each biopsy sample was divided into two
 

portions, one portion being fixed for histological examination and the
 

other portion prepared for subsequent growth in vitro. Autopsies were
 

performed on each mouse that died or was sacrificed in moribund
 

conditions during the course of the study. Six months post-injection,
 all surviving mice were sacrificed and autopsies were performed.
 

Two concentrations each of 7 different morphologically
 

altered and unaltered human cells were injected into 14 groups of 10
 

nude mice each. All the morphologically altered cells grew as tumors
 

at the site of injection (table 3). The latent period for tumor
 

appearance varied from 2 to 21 weeks (average 6 weeks) depending on
 

the cell lines and the number of cells inoculated. The tumors tended
 

to grow progressively. Spontaneous regression of tumor was observed
 

only in 2 cases. No tumor was produced by injection of
 

morphologically unaltered cells. Examination of chromosome
 

preparations of some of the tissue culture transplants showed that the
 

tumor cells were of human origin. Taken together with the results of
 

the biological properties in culture, it is likely that the
 

morphologically altered cells are malignantly transformed cells.
 

94 
ACKNOWLEDGEMENTS
 

I thank Drs. Harry V. Gelboin and C. Wesley Dingman for
 

their support of this study. I am greatly indebted to Drs. Russell M.
 Madison and Robert M. Nim (Microbiological Associates, Bethesda, Md.)
 

for their contribution to the transplantation experiments and to Dr.
 Rufus S. Day for providing the KD cells and encouragement in pursuing
 

this project. I gratefully acknowledge the help of Ms. Janet D. Crow
 

and Ms. Barbara Heifetz.
 

95
 
Table 1. Saturation density and colony forming ability in soft agar
 

Cells
 

Untreated
 

Solvent-


treated
 

4NQ.NT1
 

4NQ.T-12
 

4NQ.T-22
 

NG.T-33
 

4NQ.TR-1^
 

4NQ.RT-2'4
 

4NQ.RT-34
 

Days in culture
 

after treatment
 

with chemical
 

4
 

95
 

95
 

95
 

95
 

105
 

97
 

97
 

97
 

Saturation
 

density
 

(xlO1* cells/cm2)
 

3.2
 

3.0
 

2.6
 

2.7
 

7.2
 

5.4
 

9.4
 

12.7
 

11.7
 

10.7
 

Colony forming
 

efficiency in
 

soft agar
 

< 5 x 10-7
 

-6
 < 1 x 10
 -6
 < 1 x 10
 

6


< 1 x 10	 ­

-4
 9 x 10


-3
 1 x 10
 

-3
 2 x 10
 

2 x 10-5
 

1 x 10-5
 

-6
 1 x 10


1

The cells of the current cultures which were treated with 4NQO and did
 

not show any morphological changes.
 2

The cell lines isolated from each focus area in the subcultures at 79
 

days after the treatment of KD cells with 4NQO.
 

-%he cell line isolated from a focus area in the subculture at 90 days
 

after the treatment of KD cells with MNNG.
 

^The cell lines isolated from each focus area in the subcultures at 83
 

days after the treatment of KD-1 clone with 4NQO.
 

96 
Table 2. Transplantability of the cultured
 

cells into nude mice
 

^The cells of the cultures which were treated with 4NQO and did not 

show any morphological changes. The cell lines isolated from each focus area in the subcultures at 

73 days after the treatment of KD cells with 4NQO. ^The cell line isolated from a focus area in the subculture at 90 

days after the treatment of KD cells with MNNG. ''The cell lines isolated from each focus area in the subcultures at 

83 days after the treatment of KD-1 clone with 4NQO.
 

97 
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1.	 Chen, T.T., and C. Heidelberger. "Quantitative Studies on the
 

Malignant Transformation of Mouse Prostate Cells by Carcinogenic
 

Hydrocarbons In Vitro." Int. J. Cancer, 4 (1969), 166.
 

2.	 Day, R.S. "Studies on Repair of Adenovirus 2 by Human
 

Fibroblasts Using Normal, Xeroderma Pigmentosum, and Xeroderma
 

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3.	 DiPaolo, J.A., K. Takano, and N.C. Popescu. "Quantitation of
 

Chemically Induced Neoplastic Transformation of BALB/3T3 Cloned
 

Cell Lines." Cancer Res., 32 (1972), 2686.
 

4.	 Freedman, V.H., and S. Shin. "Cellular Tumorigenicity in Nude
 

Mice: Correlation with Cell Growth in Semi-Solid Medium." Cell,
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5.	 Giard, D.J., S.A. Aaronson, G.J. Todaro, P. Arnstein,
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of Solid Tumors." J. Natl. Cancer Inst.. 51 (1973), 1417.
 

6.	 Giovanella, B.C., J.S. Stehlin, and L.J. Williams, Jr.
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Thymusless Mice. II. Malignant Tumors Induced by Injection of
 

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7.	 Igel, H.J., A.E. Freeman, J.E. Spiewak, and K.L. Kleinfeld.
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8.	 Ikenaga, M., Y. Ishii, M. Tada, T. Kakunaga, H. Takebe, and S.
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Responsible for Killing, Mutation, and Cancer." In: Molecular
 

Mechanism for the Repair of DNA. P. Hanawalt and R.B. Stelow,
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9.	 Kakunaga, T. "A Quantitative System for Assay of Malignant
 

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BALB/3T3." Int. J. Cancer, 12 (1973), 463.
 

10.	 Kakunaga, T. "Requirement for Cell Replication in the Fixation
 

and Expression of the Transformed State in Mouse Cells Treated
 

with 4-Nitroguinoline-1-Oxide." Int. J. Cancer. 14 (1974), 736.
 

98
 
11.	 Kakunaga, T., and J. Kamahora. "Analytical Studies on the
 

Process of Malignant Transformation of Hamster Embryonic Cells in
 

Cultures with 4-Nitroguinoline-1-Oxide." Symposia Cell. Chem.,
 20 (1969), 135.
 

12.	 Kondo, S. "DNA Repair and Evolutionary Considerations." Advan.
 in Biophysics, University of Tokyo Press, Tokyo; University Park,
 Baltimore, 7 (1975), 91-162.
 

13-	 Rhim, J.S., D.K. Park, P. Arnstein, R.J. Huebner,
 E.K. Weisburger, and W.A. Nelson-Rees. "Transformation of Human
 

Cells in Culture by N-Methyl-N'Nitro-N-Nitrosoguanidine." Nature,
 256 (1975), 751.
 

14.	 Provlsen, C.O., P.J. Fialkow, E. Kelin, G. Klein, J. Rygaard, and
 

F. Weiner. "Growth and Antigenic Properties of Biopsy-Derived
 

Burkitts's Lymphoma in Thymusless (Nude) Mice." Int. J. Cancer,
 11 (1973), 30.
 

15.	 Rygaard, J., and C.O. Povlsen. "Heterotransplantation of Human
 

Malignant Tumor to Nude Mice." Acta Path. Microbiol. Scand., 77
 

(1960), 758.
 

99
 
2.7.2	 HETEROPLOID CONVERSION OF HUMAN SKIN CELLS BY METHYLCHOLANTHRENE: POSSIBLE ROLE OF HYDROCARBON METABOLIZING EPITHELIAL CELLS
 

AARON E. FREEMAN1 , ROBERT S. LAKE, HOWARD J. IGEL, LISA GERNAND,
 

MARY ROSE	 PEZZUTTI, JAMES M. MALONE, COREY MARK2, AND WILLIAM F.
 

BENEDICT2
 

Department of Pathology, Children's Hospital of Akron,
 Akron, OH
 

A.	 QUESTIONS AND ANSWERS THAT SERVED AS A BASIS FOR THE RATIONALE IN
 

DEVELOPING HUMAN CELL MODELS OF TRANSFORMATION3
 

Q - Why is it important to develop in vitro assays for chemical
 

carcinogens?
 

A - There are two major reasons. First, we have the problem of
 

identifying and eliminating environmental carcinogens that may be
 

the direct cause of much human cancer. This project must include
 

many chemical agents already widely disseminated as well as all
 

of the new agents developed by industry each year. The cost of
 

such a study in laboratory animals is staggering. In vitro
 

assays, hopefully, would be quicker, more inexpensive, better
 

controlled, and more reliable than the equivalent test animals.
 Secondly, if we can induce cell transformation predictably and
 

under controlled conditions, we may also be able to determine the
 

molecular basis by which a cell becomes malignant and this, in
 

turn, may lead to better methods of cancer cure and prevention.
 

Q - But why mammalian cell transformation? Aren't bacterial
 

mutagenesis assays quicker, cheaper, and better?
 

A - Whether or not a potential carcinogen induces a cell to become
 

malignant depends on at least two genetic factors. First, does
 

the cell possess the necessary metabolic pathways for converting
 

distal carcinogens to an ultimate form and secondly, once this
 

ultimate carcinogen is produced, is the cell susceptible, i.e.,
 is it the specific target cell predisposed to becoming malignant?
 

1 - Present address: Torrey Pines Research Center, Microbiological
 

Associates, LaJolla, CA.
 

2 - From the Children's Hospital of Los Angeles, Los Angeles, CA.
 

3 - This section was provided as a personal discussion by A.E. Freeman.
 

100
 
Now let us take these two factors, metabolism and susceptibility,
 and ask whether or not all cells within a single individual are
 

equally prone to develop cancer. How about differences between
 

individuals within a species, between species, between genera,
 between phyla? Obviously, it is very difficult to be sure that
 

mutagenesis in bacteria has relevance to cancer in man. By
 

extending the data base to include in vitro transformation of
 

several species of mammalian cells, we hope to discover which
 

tests are relevant and which are not. If all tests should be
 

equally effective and relevant, we could utilize the cheapest and
 

quickest tests with confidence.
 

Q - Would carcinogen testing in animals be eliminated?
 

A - Certainly not at the present time because comparative in vitro-
 in vivo tests are needed to confirm the relevance of the in vitro
 

tests. The presence of a functional liver and many hormonal
 

modulations are just two factors that make the living animal an
 

ideal test system for carcinogenesis. Of course, if different
 

chemicals have varying carcinogenic potentials in different
 

animals, they may also have different potentials in man—and man
 

cannot be used as a test animal.
 

Q - Can a data base designed to correlate in vitro and in vivo tests
 

among the species be extended to include man?
 

A - The plan would be to correlate in vitro and in vivo tests in
 

nonhuman mammalian cells and then to compare in vitro
 

transformation of nonhuman mammalian cells with transformation of
 

human cells. When the data base is sufficiently complete, we
 

would have to assume that in vitro tests with human cells were
 

predictive of carcinogenic activity in man. Some verification of
 

this could be and indeed has been made with epidemiological
 

studies.
 

Q - What are the results of in vitro carcinogenesis studies with
 

human cells?
 

A - It has been frequently observed and is generally known that human
 

cells are resistant to the transforming effects of agents known
 

to be carcinogenic for rodents and for rodent cells in culture,
 however, few papers have documented this phenomenon
 

(3,5,6,12,14,18). The comprehensive studies by Igel, et al. (14)
 

subjected over 50 diploid human fibroblasts cell strains, derived
 

from normal tissues and from benign and malignant tumors, to a
 

number of carcinogens including methylcholanthrene,
 N-methy1-N'-nitro-N'nitrosoguanidine, and urethane. No evidence
 

of cell transformation was noted with the following exceptions.
 

101
 
Morphologically altered foci were seen in urethane treated
 

cultures derived from two siblings with von Recklinghausen's
 

disease. Many of these foci were isolated, but only two
 

developed into continuous cell lines. Each culture was
 

heteroploid and contained unique marker chromosomes indicating
 

they were derived from separate transforming events occurring in
 

two original parent cells (15). In addition to indefinite life
 

span in culture and heteroploidy, the cultures grew in soft agar,
 had increased fibrinolytic activity, and produced nodules, but
 

not progressively growing tumors in newborn mice (14,15). More
 

recently, Rhim, et al., have induced further changes in an
 

aneuploid cell culture derived from a human osteosarcoma (21).
 In this case, the transformed cells attained an important
 

characteristic of malignant cells, i.e., they produced malignant
 

tumors in nude mice. Both of these studies (14,21) are subject
 

to the same valid criticism. Since the cultures were derived
 

from human tumors, it is possible that treatment with chemicals
 

selected out a pre-existing population of malignant tumor cells.
 An attractive alternative interpretation is that each cell
 

culture was predisposed to transformation. Von Recklinghausen's
 

disease is transmitted by an autosomal dominate gene and is
 

characterized by formation of neurofibromas which often transform
 

in vivo to neurofibrosarcomas. Thus, diploid cells derived from
 

the neurofibroma could also be genetically predisposed to
 

transformation in vitro. In the case of the osteosarcoma cell,
 it should be noted that they are derived from tumors and are
 

aneuploid and thus could represent a tumor cell culture with
 

incomplete expression of full malignant potential, thus perhaps,
 could become fully transformed by a single mutation induced by a
 

carcinogen. In any event, it is evident that chemically induced
 

transformation of human cells is a rare event and has never been
 

reported with diploid human cells derived from normal tissues.
 

Q - Why are human cells so resistant to chemically-induced
 

transformation?
 

A - If a cell is to become transformed as a result of chemical
 

treatment, a number of events have to occur. The chemical must
 

reach the cell and enter it. Distal carcinogens have to be
 

metabolically activated to ultimate carcinogenic forms. Once the
 

ultimate carcinogen is formed, correct quantities of it must
 

reach the target sites where a "transformation potential" lesion
 

is produced. The target cell must be capable of transcribing and
 

translating the "transformation potential" lesion into phenotypic
 

expression. Obviously, these events occur readily in rodent
 

cells. We can speculate that certain human cells may be
 

impermeable to certain chemicals. If permeable, human cells may
 

not metabolize certain chemicals and, if so, may produce only
 

102
 
nontoxic noncarcinogens rather than carcinogenic products. If an
 

ultimate carcinogen is formed, it may be inactivated before
 

reaching the target site. The DNA of certain species may contain
 

fewer of the nucleotide sequences susceptible to a specific
 

carcinogen and thus may be more resistant to formation of a
 

lesion. Should a lesion be formed, DNA repair mechanisms may
 

correct it; and/or the cell may contain enzymes that destroy
 

defective messenger RNA. Further, certain virologists would
 

point out that mammalian cell species that are easily transformed
 

by chemicals are also the ones that possess readily demonstrable
 

C-type RNA viral genomes whereas this type of viral information
 

is extremely difficult to demonstrate in human cells. Although
 

this observation proves nothing, it leaves open the suggestion
 

that viral information may play a role in chemical carcinogenesis
 

and that human cells may be resistant to transformation due to
 

powerful cell regulation over viral expression.
 

Q - Is there evidence to support any of these proposed mechanisms?
 

A - In 1971, Deitz and Flaxman (5) wrote, "It is tempting .. . to
 

consider the possibility that susceptibility (by cells) to the
 

toxic effects (of chemicals) might indicate the potential for
 

achieving transformation because evidence suggests that both
 

processes may involve metabolism of the hydrocarbons by an
 

inducible aryl hydroxylase enzyme system (4,10,11)." In that
 

same report (5), 1 yg per ml of benzo(a)pyrene or
 

methylcholanthrene induced giant cell formation and a disorderly
 

pattern of growth in human skin epithelial cells, but not
 

fibroblasts, however, no evidence of malignant transformation was
 

noted. Diamond (3) related cytotoxic effects with metabolism of
 

polycyclic hydrocarbons in mammalian cell cultures. Of
 

particular interest was her finding that most of the human cell
 

strains studied had low levels of hydrocarbon metabolism and this
 

was reduced further upon subculture. In a study that effectively
 

combined and confirmed work of Dietz and Flaxman (5) and Diamond
 

(3), Huberman and Sachs (12) studied the hydrocarbon metabolizing
 

activity of epithelial cells derived from human embryos. They
 

found that epithelial cells metabolized 3-25 times more
 

benzo(a)pyrene than did fibroblasts from the same embryo. This
 

observation suggested to them that "the higher activity of
 

epithelial cells might be related to the higher incidence of
 

carcinoma rather than sarcoma in humans." This work has been
 

extended and confirmed in human skin by Fox, et al. (18).
 

Somewhat more recently Kouri, et al. (17) found a wide diversity
 

in hydrocarbon metabolizing activity in human fibroblast strains
 

derived from human embryos or adult human skin. All human
 

strains, however, had low hydrocarbon metabolizing activity
 

relative to Fu5-5 rat hepatoma cells.
 

103
 
All of this work taken together, leads to the following
 

conclusions. Human cell cultures have low levels of hydrocarbon
 

metabolizing activity relative to rodent cell cultures; human
 

fibroblasts have low levels of hydrocarbon metabolizing activity
 

relative to human epithelial cells. This specific increased
 

metabolic activity of epithelial cells may account for the
 

predominance of carcinoma rather than sarcoma in man. Human
 

epithelial cells with high hydrocarbon metabolizing activity may
 

be the targets of choice for cell transformation studies.
 

Q - It there evidence that certain viruses might play a role in
 

chemical carcinogenesis?
 

A - There is no evidence for this in human systems. Transformation
 

of rat cells, however, is greatly enhanced by murine C-type RNA
 

leukemia viruses. That is not a topic for discussion here.
 Those seeking more information are referred to a recent
 

publication (8).
 

Q - Do you have any new information regarding chemically-induced
 

transformation in human cells?
 

A - Yes, and it is presented and discussed in this paper.
 

B. MATERIALS AND METHODS
 

ESTABLISHMENT OF PRIMARY FORESKIN EXPLANT CULTURES
 

Portions of circumcised foreskin tissue were trimmed to remove
 

fat and connective tissue and minced with scissors in a small volume
 

of growth medium (Eagle's Minimum Essential Medium containing 1mM
 

sodium pyruvate, 0.1mM nonessential amino acids, 2mM glutamine, 100 U
 

penicillin per ml, 100 ug streptomycin per ml, 50 ug kanamycin per ml,
 and 10/t heat-inactivated fetal calf serum). The skin mince was washed
 

at least three times in growth medium to remove blood. For most
 

experiments, ten to twenty skin mince pieces (1-8mm^) were placed on
 

the medium-rinsed surface of 90mm plastic petri dishes and incubated
 

at 37°C, 95% humidity, 5% C02 in air. After 2 hours, taking care not
 

to dislodge the adhering skin pieces, the cultures were fed 3 ml of
 

growth medium and reincubated until about 1 cm of cell outgrowth was
 

obtained, usually by the tenth to fourteenth day after planting. Most
 

colonies, presumably derived from epidermis, consisted predominantly
 

of epithelial cells but fibroblasts were seen between colonies. Some
 

colonies, presumably derived from bits of dermis, seemed to consist
 

entirely of fibroblasts. For two other experiments, a 0.014mm split
 

thickness skin biopsy and full thickness foreskin tissue were minced
 

as described and planted on the dermal bed of dead sterile pigskin
 

104 
(7,9,13). A 1-2 cm diameter clearing of the pigskin substrate,
 corresponding to the diameter of virtually pure epithelial cell
 

outgrowth, occurred around the skin explants within 17 days after
 

planting.
 

COLONIAL ASSAY OF HYDROCARBON METABOLIZING ACTIVITY
 

Fifteen skin mince pieces (1-8nmP) were planted per 75 cm2
 

Falcon plastic flask by identical methods described above for petri
 

dishes. When individual colonies of fibroblasts or epithelial cells
 

attained a diameter of 16mm, plastic cylinders >(l6mm diameter) were
 

cemented over each island with sterile silicone grease. In order to
 

do this, sections of the flask opposite the cells were cut out with a
 

hot scalpel blade. An aluminum foil cover was then used to maintain
 

sterility. Each cylinder was filled with 2 mis of medium freshly
 

supplemented with ^H-Benzo(a)pyrene (Amersham Searle generally
 

labelled, 20 Ci per mmole diluted to 1 g per ml, 2 yCi per ml).
 After a 3-day incubation period at 37°C, water-soluble metabolites in
 

the medium were extracted as described by Kouri, et al. (17). The
 

cells growing in each cylinder were removed with a mixture of 0.25%
 

trypsin and 0.02$ disodium ethylenediaminetetraacetic acid in
 

calcium-magnesium free Hanks' Balanced Salt Solution, diluted in 0.4/f
 

trypan blue, and counted in hemocytometer. Background aqueous-soluble
 

3H-benzo(a)pyrene was determined by extraction of medium inoculated
 

without cells for 3 days. This background, representing 1-3? of total
 

input counts, was subtracted to determine net counts. The hydrocarbon
 

metabolizing activity (HMA) was expressed as pinoles of benzo(a)pyrene
 

converted from an organic-soluble to water-soluble form per 106 cells
 

per number of days. Generation of water-soluble benzo(a)pyrene
 

metabolites was linear over a 3-day incubation period.
 

TREATMENT WITH MCA
 

Thirteen to 17 days after planting cultures were treated for
 

7 days (3 medium changes) with growth medium containing the
 

appropriate concentration of MCA, or other chemical, dissolved in
 

acetone. Control cultures were exposed to the equivalent
 

concentration of acetone (0.1$) diluted in growth medium. All changes
 

of media and incubation were carried out in semi-darkness during the
 

week of treatment. After seven days, dishes and pigskins were rinsed
 

three times in growth medium to remove unbound MCA. Then growth
 

medium was replaced, and the cultures were incubated until confluency
 

was reached.
 

ESTABLISHMENT OF SECONDARY CULTURES
 

When grown on plastic, confluent primary cultures,
 consisting of 1-2 cm diameter epithelial islands surrounded by
 

105
 
fibroblasts, were removed with 0.25% trypsin in Hanks' Balanced Salt
 

Solution and subdivided 1:2 and/or 1:4 into Falcon plastic 75 cm2
 

flasks. When grown on pigskin, areas containing epithelial cell
 

outgrowth (as evidenced by clearing of the pigskin due to digestion of
 

the connective tissue matrix) were excised, minced with scissors in a
 

small volume of growth medium, and transferred to plastic flasks.
 Further subdivisions were adjusted to 1:2, 1:4, or 1:8 split ratios so
 

that physical subculture could be carried out weekly.
 

In our terminology, D refers to the approximate number of
 

population doublings based on the split ratios. Primary cultures are
 

those that have never been physically subdivided. Since skin
 

epithelial cells continuously maturate to form keratin, it is obvious
 

that the true number of population doublings always exceeds D. In
 

spite of this error, we consider the use of D representing the approx­ imate number population doublings to be preferable to the use of P
 

representing the number of physical subpassage.
 

SELECTION OF MORPHOLOGICALLY ALTERED FOCI
 

In some experiments, morphologically distinct colonies were
 

noted in MCA treated cultures. These were scraped from the
 

surrounding cell sheet with a sterile wire, removed with a capillary
 

pipette, and planted in a test tube in 1 ml of growth medium. Cell
 

lines derived from these colonies bear the number of the parent cell
 

line, followed by C (for colony) and a numerical designation to
 

identify the individual colony. For example, A566C-1 is colony 1
 

derived from A566.
 

OTHER METHODS
 

Growth in soft agar was done by the method of MacPherson and
 

Montagneir (19). Chromosome preparations at The Children's Hospital
 

of Akron were made by the method of Kajii (16). Chromosome
 

preparations at The Children's Hospital of Los Angeles were made by
 

the method of Paul (20). Banding of human chromosomes was done by the
 

technique of Seabright (23). Plasminogen activation was monitored as
 

described by Jones (15). To determine absolute plating efficiencies
 

the cell cultures were trypsinized and •suspended in growth medium
 

containing the lowest concentration of serum included in the test.
 One hundred cells were added to replicate plates containing medium
 

with fetal calf serum at the concentrations listed in table 8. The
 

cultures were inoculated without change of medium for 7-10 days.
 Colonies were fixed in 10% buffered formalin, stained with Giemsa, and
 

counted.
 

106
 
C. RESULTS
 

TABLE 1. Skin epithelial cell and fibroblast colonies derived from 13
 

different donors were tested for HMA after 21 to 50 days in culture.
 

A continuous spectrum of activity ranging from 1250 to 20,087 units
 

(pico moles BP/IO^ cells per 3 days) was found in epithelial
 

colonies. Epithelial colonies always had more activity than
 

fibroblasts from the same donor. In 9 of 13 donors, the relative
 

levels of HMA in epithelial cells and fibroblast correlated very well
 

but in the other 4 cases, the fibroblasts had low or undetectable
 

activity. Perhaps HMA of epithelial cells and fibroblasts are controlled by different genes, or perhaps the low levels of HMA in
 

certain fibroblaet colonies are artifactual, caused by, for example,
 changes in culture conditions or culture age. Based on the limited
 

number of donors tested, sex, race, or age of donor do not seem to be
 

factors in HMA expression.
 

TABLE 2. To see if HMA of epithelial cells was affected by culture
 

age, some cultures were maintained (with weekly medium changes) for
 

periods up to 55 days. Periodic monitoring of representative
 

epithelial colonies demonstrated that HMA was independent of culture
 

age. It should be emphasized that epithelial cells constantly maturate through cell division and thus, an "old colony" may be physiologically equal to a "young colony." Note that viable cell count
 

increased over the 55-day period of the experiment, but specific HMA
 

remained fairly constant.
 

TABLE 3. Although age of the epithelial cultures does not affect HMA,
 

upon subdivision both the epithelial cell population and the HMA
 

decreased precipitously.
 

TABLE 4. Epithelial colonies of rabbit, hamster, and mouse cells have
 

more HMA than fibroblastic colonies from the same donors. However,
 due to the limited number of individual animals tested, HMA levels
 

between species cannot be compared because of the possibility that
 

there arevariations between individuals (even in these inbred strains)
 

that are similar to that seen for humans.
 

NIH Swiss mouse and guinea pig cells did not form epithelial colonies
 

on plastic but did grow on pigskin where their HMA was measured.
 

Usually, however, cultures grown on pigskin had less HMA than
 

epithelial colonies grown on plastic. This may be due to physical
 

separation of carcinogen and cells by the dense layer of keratin that
 

forms when epithelium is grown on pigskin.
 

TABLE 5. Petri dishes containing individual colonies of rabbit or
 

human epithelial cells and fibroblasts were exposed to 12 chemicals
 

for 4 days. After an additional 10 days, the cultures were screened
 

107
 
microscopically for evidence of cytotoxicity. Certain chemicals were
 

selectively toxic to epithelial cells. These were BAP, DMBA, DBA, and
 

MCA—all polycyclic hydrocarbons. Thus the relative level of HMA in
 

epithelial and fibroblastic cells correspond with relative toxicity.
 On the other hand MNNG was selectively toxic to fibroblasts. Those
 

chemicals that were toxic at a dose of 6.25 yg or less are carcino­ gens. DBA, which was toxic at 25 mg per ml is a weak carcinogen. The
 

other chemicals tested are noncarcinogenic and are not toxic.
 

TABLE 6. This table is a summary of five experiments in which we
 

tried to transform human skin cultures with methylcholanthrene. In
 

the beginning we planned to look for morphological alteration as the
 

primary indication of transformation. We also planned monitoring of
 

chromosomes and fibrinolytic activity, growth in soft agar, and
 

plating efficiency. However, only the morphological examinations and
 

karyotypic analyses were made routinely. In none of the experiments
 

did we notice any striking morphological changes that were definite
 

and consistent from experiment to experiment or culture to culture.
 None of the small morphological changes that we did observe could be
 

identified when we read the cultures as coded cultures. However, when
 

karyotypic analyses were made we found that heteroploid conversion
 

occurred in each experiment but only in those cultures which had been
 

treated with methylcholanthrene. In the first experiment 2 of 2 cul­ tures treated with 1 yg MCA per ml became heteroploid. Experiment 2—
 the single culture treated with .5 yg MCA per ml became heteroploid.
 Experiment 3—heteroploid conversion was noted only in 1 of 4 cultures
 

treated with 1 yg MCA per ml. Experiment 4—we tested MCA at doses of
 

0 and 1 yg per ml. None of 6 control cultures, but 2 of 6 MCA treated
 

cultures became heteroploid. In the last experiment, which is still
 

in progress, we treated a number of primary cultures of epithelial and
 

fibroblast mixtures with varying doses of MCA and phenanthrene and
 

compared the degree of heterploid conversion in these culture to
 

similarly treated D3 fibroblasts which were free of epithelial cells.
 At present, we have 2 cultures which have become heteroploid, and both
 

of them had been treated with MCA. Note that the HMA level of the
 

primary cultures was over 9,000 U for the epithelial cells and over
 

2,000 U for the fibroblastic. D3 fibroblasts, however, had only 574 U.
 

TABLE 7. This table presents the actual ranges of chromosome counts
 

of a number of selected cultures listed in Table 6. No heteroploid
 

conversion was detected in any culture not included in Table 7.
 

In experiment 1, heteroploid conversion occurred in both cultures
 

treated with 1 yg MCA per ml but not in the 2 control cultures. Two
 

cell colonies, which were physically removed from the parent culture
 

and grown into mass cultures again, were also heteroploid.
 

108
 
In experiment 2, heteroploid conversion occurred in the culture
 

treated with 0.5 yg MCA per ml. This culture and its control were
 

sent to Dr. William Benedict for detailed karyotypic analysis by
 

chromosomes banding techniques. Marker chromosomes unique to this
 

cell culture were found in a number of metaphases at D40 and again at
 

D47.
 

In experiment 3, unique marker chromosomes were found at D18 and again
 

at D38. Other marker chromosomes were found in MCA induced hetero­ ploid cultures in experiment 4. Thus, in each of these experiments,
 unique marker chromosomes were found in MCA induced heteroploid
 

cultures. This means that there were proliferating aneuploid cells
 

and that heteroploidy was not due to cell senescence. More hetero­ ploid cultures were produced by MCA in experiment 5, but Dr. Benedict
 

has not yet completed his karyotypic analyses of these cultures.
 

TABLE 8. Some cultures have been tested for other characteristics
 

typical of transformed cells. The cell lines presented in this table
 

were derived from experiment 3 (see Table 6). The MCA treated
 

heteroploid culture had a higher plating efficiency than the control
 

culture at all levels of serum tested from 0.5 to 10/6. In 0.5$ serum,
 colonies were formed only by the heteroploid culture. We do not yet
 

know the plating efficiency in low serum of cultures derived from
 

these colonies.
 

109
 
TABLE 1 

METABOLIC CONVERSION OF BENZO(a)PYRENE 

TO A WATER SOLUBLE FORM BY HUMAN SKIN 

EPITHELIAL CELLS AND FIBROBLASTS FROM THE SAME DONOR 

HYDROCARBON METABOLIZING ACTIVITY 

(pmoles BP/10 cells/3 days 

SOURCE OF SKIN Plastic Substrate , 

Age of Culture Epithelial3' Fibroblasta> 

Sex Race Donor (year) Age (days) Colonies Colonies 

M NC 15 32 20,087+3,155 7,618+4,503 

M C NB 23 18,336 + 2,878 531 ± 350 

M C .05 32 17,329+9,425 1,665+2,840 

M C NB 24 17,212 + 2,560 2,287 + 684 

M C 19 30 14,581 + 4,334 Oc 

F C 11 24 11,751+5,750 2,778+ 754 

M C .5 50 10,118+4,346 2,540+1,946 

M NC 9 25 9,127 + 3,717 2,142 + 819 

M C .8 24 7,491 + 2,800 0° 

M NC 15 42 3,874 + 1,497 0° 

F C .12 29 2,810 + 678 699 + 400 

M C NB 28 2,100 + 583 436 + 150 

M C NB 21 1,250 + 400 219 + 89 

Q
 

Colony type is identified morphologically on plastic. Upon transplantation
 

back to pigskin, epithelial colonies form epithelium, fibroblast colonies
 

do not.
 

Average and standard deviation from 3-5 colonies derived from the same
 

culture flask. 

°Below background ABBREVIATIONS: NB-newborn; C-Caucasian; 

NC-Noncaucasian (Negro) 

110 
111

 

TABLE 2
 

EFFECT OF CULTURE AGE ON THE
 

BENZO(a)PYRENE METABOLISM OF HUMAN SKIN EPITHELIAL CELLS3
 

Primary Average Viable Cell Hydrocarbon Metabolizing Activity
 

Culture Description Count/60 mm dish pmoles BP/10 cells/ 1 day
 

Age (Days) (X10°) (N=3 plates)
 

6 .04 3,970 + 788 

13 Subconfluent 

(Epithelial) 

.07 2,940 + 980 

34 .78 2,389 ± 475 

41 2.1 2,244 + 838 

4? Confluent 12.8 2,855 + 296
 

(75-90$ Epithelial)
 

55 (10-25$ Fibroblasts) 19.3 2,786 + 894
 

Cultures derived from foreskin of donor 1.25 years old.
 
112 

TABLE 3 

EFFECT OF SUBPASSAGE ON EPITHELIAL CELL 

SURVIVAL AND BENZO(a)PYRENE METABOLISM OF HUMAN SKIN CULTURES

 

Culture Culture Age Percent Hydrocarbon Metabolizing Activity
 

Subdivision at Time of Epithelial pmoles BP/106

 cells/1 day
 

Level* Assay (Days) Cells/Dish (N=3 plates)
 

D 40 98 2,877 ± 701
 o
 

D 3 21 485 + 120
 1
 

D 3 0 555 + 180
 2
 

3 0 448 + 48
 °3
 

D

4 3 0 586 + 24
 

D 3 0 736 + 10
 5
 

"Cultures derived from foreskin of donor 0.75 years old, grown to confluence
 

(40 days), split 1:2 weekly thereafter with assay of triplicate dishes 3 days
 

after last split.
 
113

 

DONOR 

Rabbit, Strain III
 

Eabbit, Strain WH
 

Rabbit, N.Z. White
 

Hamster, Syrian
 

Mouse, C57 Bl/6
 

Mouse, NIH Swiss
 

Guinea Pig, Hartly
 

albino
 

TABLE 4 

METABOLIC CONVERSION OF BENZO(a)PYRENE 

BY MA^f^ALIAN SKIN CELLS OF VARIOUS SPECIES 

Specific Metabolic Rate
 

pmoles BP per 10 cells per 3 days 

Age of Culture/days Type of Colony on Plastic Cultures on Pigskin 

Plastic Pigskin Epithelial Fibroblast 

19 24 4,826 1,976 706 

19 24 9,045 2,415 287 

35 38 12,872 6,410 4,446 

30 14 1,391 162 1,844 

30 30 52,305 1,420 6,910 

30 30 No colonies 754 6,405 

30 30 No colonies 3,901 2,686 
- - -

_ 

_ 

-

_ 

_ 

-

_ 

_ 

-

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

_ 

114

 

TABLE 5
 

TOXICITY OF CHEMICALS TO RABBIT OR HUMAN SKIN
 

EPITHELIAL CELLS AND FIBROBLASTS GROWN ON PLASTIC
 

Skin
 

Carcin- Skin Input Concentration in 4 ml of medium - ug per ml
 

Chemical Abbrev. ogen Species 0.97 .195 .39 .78 1.56 3.125 6.25 12.5 25 50 100
 

_
 _
 _
 

anthracene A rabbit
 

human - - - - TEF
 

_
 _
 

benzo(e) pyrene BeP rabbit
 

human - TEF
 

benzo( a) pyrene BaP + rabbit	 TE TE TE TE
 •'-human	 TE TE TE TE TE
 

_
 _
 _
 

dibenz (a, h) anthracene DBA + rabbit
 

human - - - TE
 _
 7, 12-dimethylbenz(a) DMBA + ^rabbit TE TE TE TE
 anthracene •'•human - - TE TE TE TE
 _
 _
 

dimethylnitrosamine DMNA rabbit
 _
 

•'•human	 -
 TE
 

_
 diphenylnitrosamine DPNA rabbit - TEF
 human - TEF
 _
 _
 

methylazoxymethanol MAMA rabbit
 acetate human ~ ~
 

3-methylcholanthrene MCA + rabbit TE TE TE
 1-human - TE TE TE TE TEF
 _
 _
 

N-methyl-N-nitro-N- MNNG + rabbit TF TF
 nitrosoguanidine 2human TF TFE TFE
 

_
 pyrene PY rabbit
 

human - - TE
 

_
 phenanthrene PH rabbit TEF
 

human - - TE
 

_
 

^non toxic*; T toxic, E epithelial; F fibroblasts Selectively toxic to epithelial cells Selectively toxic to fibroblasts
 

Rabbit, New Zealand white; human, foreskin from 14 year old *Toxicity—gross cell alteration leading to death by 14 days
 after treatment
 
TABLE 6
 

HETEROPLOID CONVERSION OF HUMAN SKIN
 

CELL CULTURES BY METHYLCHOLANTHRENE
 

Number of Number
 

115 

Experiment 

Number Treatment 

1 Acetone 

MCA ­ 1.0 yg per ml 

2 Acetone 

MCA - 0. 5 yg per ml 

3 Acetone 

MCA - 0.1 yg per ml 

MCA ­ 0.5 yg per ml 

MCA ­ 1.0 yg per ml 

4 Acetone 

MCA - 1.0 yg per ml 

5 Acetone 

MCA - 1.0 yg per ml 

MCA - 3.0 yg per ml 

MCA ­ 5.0 yg per ml 

MCA -10.0 yg per ml 

Phenanthrene ­ 1 yg 

Phenanthrene ­ 10 yg 

Phenanthrene - 50 yg 

Phenanthrene -100 yg 

Acetone 

MCA - 1.0 yg per ml 

MCA - 3.0 yg per ml 

MCA - 5.0 yg per ml 

MCA -10.0 yg per ml 

Cultures
 

Per Test
 

2*
 

2*
 

1
 

1
 

4
 

4
 

4
 

4
 

6
 

6
 

2
 

2
 

2
 

2
 

2
 

per ml 2
 

per ml 2
 

per ml 2
 

per ml 2
 

2
 

2
 

2
 

2
 

2
 

Converted to
 

Heteroploidy
 

0
 

2*
 

0
 

1
 

0
 

0
 

0
 

1
 

0
 

2
 

0
 

0
 

1
 

0
 

1
 

0
 

0
 

0
 

0
 

0
 

0
 

0
 

0
 

0
 

Comments
 

From thighs of adult male
 

Foreskin
 

Foreskin
 

Foreskin
 

Primary Foreskin
 

HMA (pmoles/106 cells/3 days)
 

- epithelials cells 9,127 +
 

3,717
 

- fibroblasts 2,142+ 819
 

D3 fibroblasts 6
 

HMA (574 pmoles/10 cells/
 

3 days)
 

*0ne culture was also treated with 10 yg BdU per ml 
TABLE 7
 

CHROMOSOMAL CHANGES IN SKIN CELLS TREATED WITH MCA3
 

116 

Experiment I Culture Treatment Metaphases with Indicated Chromosome Count 

(Conditions) Code Dt <46 46 47-49 50-59 60-69 70-79 80-89 90-91 92 >92 

Expt. *1 A564 15 Acetone 2 8 (Primary on Plastic) A565 15 MCA - 1.0 ug 3 3 1 1 1 1 

A566 17 6 1 1 3 

A566C-1 +11 MCA ­ 1.0 ug +Bdu -10.0 ug 3 1 2 1 3 

A566C-2 +11 4 2 4 

Expt. 12 A802 40* Acetone 3 18 2 (Primary on Pigskin) 

43-50 

53* 20 5 14 3 1 54 5 5 

A804 40* 47-57* MCA - 0.5 ug 2 

6 17 6 2 llb 

lc 3C lc 7b lc 

Expt. J3 A1040 6 Acetone 4 16 (Primary on 

Plastic) 

18* 21-31 35-38* 3 

9 

3 

19 

30 

22 1 

A1047 6

18* 

 MCA - 1.0 ug 13 

2 

6 

5 1 2d 3d

 14d 2d 

21-35 3 22 2 2 

38-39* 4 le

 3S 5e 22 3e 

Expt. |4 A1113­ 3 Acetone 11 45 1 3 (Primary on Plastic) A1118 17 

29* 3 

1 59 28 1 7 4 

A1121­ 3

17 

29* 

 MCA - 1.0 ug 1 

1 

1 

9 

7 

11 1 2 1 

lf 2 lf 1 1 2 3 1 f 4f 

A1124 3

17 

 MCA ­ 1.0 ug 1 8 

3 1 1 1 1 

4 

Expt. 15 (Primary on 

Plastic) 

A1171, A1172 

A1175 

27

27

 Acetone 

 MCA - 3.0 ug 

5 14 

6 1 1 3 3 3 3 

A1179 27 MCA -10.0 ug 4 1 1 3 1 

A1188 26 Phenanthrene ­ 9 1 100.0 ug 

Footnotes on next page
 
FOOTNOTES TO TABLE 7 

Q

No heteroploid conversion was noted in culture lines listed in 

table 4 but omitted from this table, 

A total of 12 metaphases contained at least one similar 

dicentric marker chromosome. 

°Metaphases contain the same dicentric marker chromosome seen at 

D40. 

A total of 6 metaphases contained a common dicentric marker 

chromosome not seen in the A804 cell culture. 

p

At least a total of 5 metaphases contained the same dicentric 

marker chromosome seen at D1B. 

f

A total of 4 metaphases contain an abnormal marker chromosome 

not seen in the A804 or A1047 cell culture. 

*Coded culture analyzed by W.B. using a blind protocol. 

117 
TABLE 8
 

PLATING EFFICIENCIES OF
 

CELL CULTURES A1040 and A1047
 

Line # Treatment Passage Serum Concentration
 

10% 5% 1* 0.5?
 

A1040 Acetone 28 13.5 16 12.5 N.D.
 

32
 N.D.
 15
 7.0
 

36 N.D. 12 7.0 0
 

A1047 MCA - 1.0 yg per ml 28 58 56 57.5 N.D. 

32 N.D. 55 13.5 N.D. 

36 N.D. 57 43.0 14 

Q
 

Number of colonies per 60 mm petri dish, 100 cells planted, duplicate
 

dishes at passages 28 and 32, triplicate dishes at passage 36.
 

N.D. - Not done
 

Platin

g Efficiency 

N.D.
 

118
 
D. DISCUSSION 

Human fibroblasts may become aneuploid during their senescent 

phase (22). This, however, is not the basis for heteroploidy in our 

experiments because we have observed the proliferation of cells with 

unique marker chromosomes over a number of population doublings. In 

view of the well documented karyotypic stability of presenescent human 

cells in general and of our control cultures in particular, we 

consider it to be highly significant that heteroploid conversion 

occurred after MCA treatment in 8 separate actively growing cultures 

derived from 5 different human donors. 

Further study is needed to determine whether there is a relationship between in vitro heteroploid conversion of human cells and 

malignant transformation. There is, however, considerable evidence in 

other mammalian cell transformation systems that such is the case 

(1,2,24). In our experiments, there is some evidence that heteroploid 

conversion of human cells correlated with the acquisition of other 

characteristics of cell transformation. In experiment 2, the MCA 

treated heteroploid cell culture produced colonies in soft agar; in 

experiment 3, the MCA treated heteroploid cell culture had increased 

plating efficiency. In no case, however, has a continuously growing 

cell line been established. It should be noted that aneuploid human 

tumor cells usually have a short in vitro life span. In this sense, 

the reluctance of human aneuploid cells to proliferate under in vitro 

conditions is similar whether the cells are derived from tumors or 

from MCA treated cultures. 

We do not know how universal the tendency toward heteroploid 

conversion is in human diploid skin cells treated with carcinogens of 

various classes versus noncarcinogenic agents with similar chemical 

structures. However, in 5 experiments we have shown that MCA induces 

heteroploidy and in 1 experiment we have shown that a noncarcinogenic 

polycylic hydrocarbon (phenanthrene) does not have this effect even at 

a hundred fold increase in dose. 

The high frequency with which MCA induced heteroploid conversion 

in mixed skin fibroblast and epithelial cell cultures is in sharp 

contrast to the total lack of heteroploid conversion in MCA treated 

skin fibroblasts. Indeed, we have previously reported that MCA and 

other carcinogens only rarely induced any signs of cell 

transformation, including heteroploid conversion, in extensive 

attempts to transform a number of human fibroblasts derived from 

normal and malignant tumors (14). It seems likely that the epithelial 

cells either become heteroploid themselves and dedifferentiate to 

fibroblastic forms or that the epithelial cells, through their higher 

level of HMA, convert MCA to an ultimate carcinogenic form that causes 

119 
heteroploidy in cocultured fibroblasts. Either interpretation is 

consistent with the data, but we prefer the latter. If this is 

correct, MCA will induce heteroploidy in pure cultures of skin 

fibroblasts cocultivated with irradiated feeder layers of epithelial 

cells. 

REFERENCES
 

1. Benedict, W.F. "Early Changes in Chromosomal Number and 

Structure after Treatment of Fetal Hamster Cultures with 

Transforming Doses of Polycyclic Hydrocarbons." J. Natl. Cancer 

Inst.. 49 (1972), 585-590. 

2. Benedict, W.F. , N. Rucker, C. Mark, and R.E. Kouri. "Correlation 

Between Balance of Specific Chromosomes and Expression of 

Malignancy in Hamster Cells." J. Natl. Cancer Inst., 54 (1975), 157-162.

 

3. Diamond, L. "Metabolism of Polycyclic Hydrocarbons in Mammalian 

Cell Cultures." Int. J. Cancer, 8 (1971), 451-462. 

4. Diamond, L. "The Effect of Carcinogenic Hydrocarbons on Rodent 

and Primate Cells In Vitro." J. Cellular Physiol., 66 (1965), 183-197.

 

5. Dietz, M.H. , and B.A. Flaxman. "Toxicity of Aromatic 

Hydrocarbons on Normal Hyman Epidermal Cells In Vitro." Cancer 

Res., 31 (1971), 1206-1209. 

6. Fox, C.H. , J.S. Selkirk, F.M. Price, R.G. Croy, K.K . Sanford, and 

M. Cottier-Fox. "Metabolism of Benzo(a)Pyrene by Human 

Epithelial Cells in Vitro." Cancer Res.. 35 (1975), 3551-3557. 

7. Freeman, A.E. , H.J. Igel, B.J. Herman, and K.L. Kleinfeld. "Growth and Characterization of Human Skin Epithelial Cell

 

Cultures." In Vitro (in press), 1976.

 

8. Freeman, A.E., H.J. Igel, and P.J. Price. "Carcinogenesis In 

Vitro. I. In Vitro Transformation of Rat Embryo Cells: 

Correlations with the Known Tumorigenic Activities of Chemicals 

in Rodents." In Vitro, 11 (1975), 107-116. 

9. Freeman, A.E., H.J. Igel, N.L. Waldman, and A.M. Losikoff. "A 

New Method for Covering Large Surface Area Wounds with 

Autografts. I. In Vitro Multiplication of Rabbit-Skin 

Epithelial Cells." Arch. Surg., 108 (1974), 721-723. 

120 
10.

 Gelboin, H.V. , E. Huberman, and L. Sachs. "Enzymatic 

Hydroxylation of Benzopyrene and Its Relationship to 

Cytotoxicity." Proc. Natl. Acad. Soi., U.S., 64, (1968), 

1188-1194. 

11. Gelboin, H.W. , F. Wiebel, and L. Diamond. "Dimethyl- Benzathracene Tumorigenesis and Aryl Hydrocarbon Hydroxylase in 

Mouse Skin: Inhibition by 7,8-Benzoflavon." Publ. Am. Assoc. Adv. Soi.. 170 (1970), 169-171. 

12. Huberman, E., and L. Sachs. "Metabolism of the Carcinogenic 

Hydrocarbon Benzo(a)Pyrene in Human Fibroblast and Epithelial 

Cells." Int. J. Cancer. 11 (1973), 412-418. 

13. Igel, H.J., A.E. Freeman, C.R. Boeckman, and K.L. Kleinfeld. "A 

New Method for Covering Large Surface Area Wounds With 

Autografts. II. Surgical Application of Tissue Culture Expanded 

Rabbit-Skin Autografts." Arch. Surg.. 108 (1974), 724-729­

14. Igel, H.J., A.E. Freeman, J.E. Spiewak, and K.L. Kleinfeld. "Carcinogenesis In Vitro. II. Chemical Transformation of 

Diploid Human Cell Cultures: A Rare Event." In Vitro, 11 

(1975), 117-129. 

15. Jones, P.A. , W.E . Laug, and W.F. Benedict. "Fibrinolytic 

Activity in Human Fibrosarcoma Cell Line and Evidence for the 

Induction of Plasminogen Activator Secretion During Tumor 

Formation." Cell. 6 (1975), 245-252. 

16. Kajii, T., R.L. Neu, and L.I. Gardner. "Fibroblast Chromosome 

Preparations." Lancet, 1 (1967), 897. 

17. Kouri, R.E. , R. Keifer, and E.M. Zimmerman. "Hydrocarbon- Metabolizing Activity of Various Mammalian Cells in Culture." In 

Vitro. 11 (1974), 18-25. 

18. Leith, R.S., and L. Hayflick. "Efforts to Transform Cultured 

Normal Human Cells with Polycyclic Aromatic Hydrocarbons." Proc. Am. Cancer Res., 15 (1974), 86. 

19. MacPherson, I., and L. Montagnier. "Agar Suspension Culture for 

the Selective Assay of Cells Transformed by Polyoma Virus." 

Virology, 23 (1964), 291-294. 

20. Paul, B., and I.H. Porter. "Giemsa Banding in an Established 

Line of Human Malignant Meningioma." Humangenetik, 18 (1973), 

185-187. 

121 
21.

 Rhim, J.S., D.K . Park, P. Arnstein, R.J. Heubner, E.K. Weis burger, and W.A . Nelson-Reese. "Transformation of Human 

Cells in Culture by N-methyl-N'-nitro-N-nitrosoguanidine." 

Nature. 256 (1975), 751-753­

22. Saksela, E., and P. Moorhead. "Aneuploidy in the Degenerative 

Phase of Serial Cultivation of Human Cell Strains." Proc. Natl. Acad. Sol.. 50 (1963), 390-395. 

23. Seabright, M. "A Rapid Banding Technique for Human 

Chromosomes." Lancet, 2 (1971), 971-972. 

24. Yamamoto, R. , Z. Rabinowitz, and L. Sachs. "Identification of 

the Chromosomes that Control Malignancy." Nature New Biol. , 243 

(1973), 247-250. 

122 
3.

 CARCINOGENESIS STUDIES IN ORGAN CULTURE 

SYSTEMS 

3.1 ANIMAL TISSUES 

3.1.1 IN VITRO CARCINOGENESIS IN TRACHEAL ORGAN CULTURE 

BERNARD P.	 LANE
 

Department of Pathology, State University of New York at
 

Stony Brook, Stony Brook, NY
 

Fragments of tracheal epithelium and associated connective
 

tissues have been maintained in culture for studies of tissue function
 

(2,39,56,69), infectious processes (1,5-9,11-13,16,25-28,31-37,49,52,
 53,60-67,72-76), toxicity of various agents (20,22,23,54,51,70) and
 

carcinogenesis (17-20,24,40,42,44,46-48,54,58). For these investi­ gations a number of species have been employed including rat (17,18,
 

24,40-45,50,51,58,59), rabbit (22,23,56), mouse (49,59), cow (12,13,
 61-63), hamster (14,15,19,21,26,38,55,66,68,76), chicken (2,5-8,
 

11,67), pig (60), ferret (27) and human (4,9,10,16,20,25,27,29-36
 

46-48,53,57,71-75). Fetal (2,6,8,9,11,12,16,25), young (17,48,60) and
 

adult tissues (16,55,56) have been found to be suitable.
 

In most instances, lengths of airway have been cut
 

transversely by hand with a scalpel into ring-shaped segments lined by
 

mucosa. For some studies whole or split tracheas (14,17,48), squares
 

of mucosa and cartilage (4,16,25), mucosa alone (45) and strips
 

prepared by longitudinal sectioning of airway (75) have been used.
 Mechanical devices for rapid production of large numbers of uniform
 

ring cultures have been developed (43).
 

The tissue fragments have been incubated in many different
 

media, including Eagle's Minimal essential medium (8), Basal medium of
 

Eagle (8,11), Parker's Medium 199 (13,49), Leibovitz's L-15 (4,5,56),
 CMRL 1066 (4,21), McCoy's 5a (modified) (43) and Waymouth's MB752
 

(50,54). Comparison of several of these has demonstrated that complex
 

media sustains cellular proliferation more effectively than minimal
 

essential media and its modifications (55). The buffer for these
 

media has usually been sodium bicarbonate or HEPES. HEPES is more
 

convenient because it does not have to be equilibrated with a
 

particular gaseous environment, but it has been shown to alter
 

glycoprotein synthesis by the cultures (21). Most incubating media
 

contained serum or serum albumen although a number of studies have
 

been done with serumless media (55) or without adding serum to media
 

123
 
to which it is normally added (43). When serum has been deleted,
 

various additives including insulin and hydrocortisone have been used
 

(4,14). Maintenance of normal epithelial histology and cellular
 

differentiation is enhanced by addition of retinoids to the culture
 

medium (2,51). Retinoids also reverse squamous metaplasia (14,68).
 

Conditions of incubation have included free floating (59)i
 

steel mesh supported (48,59), clot supported (19) and partially
 

immersed rocker culture (14). The range of temperatures has been
 

35°-37°C. While high 02 levels in the environment have commonly been
 

used (20,58) 5% C02 in air has been shown to be effective for cultures
 

which can be sustained for periods of months (42,55).
 

Cultures have been evaluated by autoradiographic technique
 

for cellular proliferation (55,57), protein synthesis (51) and glyco­ protein synthesis (21). Measures of mitochondrial function (26,51)
 

have been made. There have been extensive studies of the histo­ chemistry and ultrastructure of cultures (4,71). Secretory activity
 

has been assayed (21,69) and the retention of ciliary function has
 

been used in chemical cytotoxicity studies (51) and infection assays
 

(6,12,36,60).
 

When injured mechanically (41) or chemically (42), the
 

epithelium or organ cultures responds with mitotic activity. The
 

response is limited however and eventually the cultures degenerate.
 Continuous exposure to carcinogens therefore results in atrophy or
 

death of cultures if the concentration of carcinogen is sufficient to
 

induce hyperplasia (42). Short term exposure to carcinogen dissolved
 

in the medium produces a sustained hyperplasia (18,58) and this effect
 

occurs more rapidly and at lower concentrations of carcinogen if serum
 

is deleted from the medium (44). Focal hyperplasia can also be
 

induced by local application of carcinogen using fibers coated with
 

the agent (54). Carcinogen has been shown to bind to epithelial cell
 

nuclei exposed in this way (38) and ultrastructural features of
 

epithelium exposed to carcinogen in vitro (7,44) include nuclear and
 

cell periphery alterations seen in vivo.
 

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124
 
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126
 
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28.	 Harrnett, G.B., and W.L. Hooper. "Test-Tube Organ Cultures of
 

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127
 
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Otolaryngol (Suppl), 188 (1964), 138-144.
 

35.	 Hoorn, B. "Organ Cultures of Ciliated Epithelium for The Study
 

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36.	 Hoorn, B., and D.A.J. Tyrrell. "Organ Cultures in Virology."
 

Prog. Med. Virol., 11 (1969), 408-450.
 

37.	 Howard, C.J., and L.H. Thomas. "Inhibition By Mycoplasma Dispar
 

of Ciliary Activity in Tracheal Organ Cultures." Infec. Imm., 10
 

(1974), 405-408.
 

38.	 Kaufman, D.G., V.M. Genta, and C.C. Harris. "Studies on
 

Carcinogen Binding in Vitro in Isolated Hamster Tracheas." In:
 

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39.	 Kent, P.W., P.P. Daniel, and J.T. Gallagher. "Metabolism of
 

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40.	 Lane, B.P., and S. Miller. "Benzo(a)pyrene-Induced Changes in
 

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825.
 

41.	 Lane, B.P., and S.L. Miller. "Response of Rat Respiratory
 

Epithelium in Vitro to Acute Mechanical Injury." Amer. J.
 

Pathol., 70 (1973), 35a.
 

42.	 Lane, B.P., and S.L. Miller. "Dose Dependence of Carcinogen-
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Tracheal Organ Cultures for Long Term Studies. I. Effects of
 

Serum on Establishment in Culture." In Vitro, 12 (1976), 147-154.
 

44.	 Lane, B.P., and S.L. Miller. "Carcinogen-Induced Changes in
 

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46.	 Lasnitzki, I. "The Effect of 3-4-benzpyrene on Human Foetal Lung
 

Grown In Vitro." Br. J. Cancer, 10 (1956), 510-516.
 

47.	 Lasnitzki, I. "Observations on Human Foetal Lung In Vitro."
 

Br. J. Cancer, 12 (1958), 547-554.
 

48.	 Lasnitzki, I. "The Effect of Hydrocarbon-enriched Fraction of
 

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49.	 Maase, E., and J. Van Der Veen. "Growth of Mouse Hepatitis and
 

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50.	 Marchok, A.C., M.V. Cone, and P. Nettesheim. "Induction of
 

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451-460.
 

51.	 Mass, M.J., and B.P. Lane. "Effects of Chromates on Ciliated
 

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52.	 Matsuyama, T. "Point Inoculation of Cultivated Tracheal Mucous
 

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53.	 Mclntosh, K., J.H. Dees, W.B. Becker, et al. "Recovery in
 

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57.	 O'Donnell, T.V., T.T. Crocker, and L.L. Nunes. "Maintenance of
 

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130
 
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131
 
3.1.2

 IN VIVO - IN VITRO STUDIES WITH RESPIRATORY TRACT 

EPITHELIUM EXPOSED TO CARCINOGEN 

ANN C. MARCHOK AND MARGARET TERZAGHI 

Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 

I. METHODS OF CARCINOGEN EXPOSURE AND ATTEMPTS TO DEFINE NEOPLASTIC 

PROGRESSION IN VITRO 

In Vivo Exposure to Carcinogens 

In vivo-in vitro experimental systems are particularly 

useful for investigating neoplastic progression in a target tissue: 

(a) because exposing the intact animal to the carcinogen allows for 

any necessary activation of the carcinogen, (b) the tissue of interest 

can be sampled at various selected times after injection or exposure 

to the carcinogen. With respect to (a), the carcinogen has been 

delivered transplacentally to fetal hamster cells in a host mediated 

in vivo-in vitro quantitative assay for chemical carcinogens (11). Long term cell cultures were derived from the brain cells of rat 

fetuses after a transplacental pulse of the nervous-system-specific 

carcinogen, N-ethyl-N-nitrosourea (19). The progression toward 

neoplasia in these ceTl lines was defined in five stages. The first 

four stages were phenotypic changes and the last stage was identified 

by growth in soft agar and the ability to form tumors. Organ 

cultures, made from the kidney of mouse embryos exposed transplacentally to several carcinogens, showed longer survival in culture 

than unexposed controls, as well as hyperplasia and the formation of 

cystadenomas in vitro (28). Kidney cells isolated as early as 20 

hours after injection of dimethylnitrosamine had a prolonged growth 

span in vitro, and exhibited a morphological transformation during 

subculture (4). The resulting cell lines acquired the properties of 

colony formation in methycellulose gels and Concanavalin A 

agglutination during subculture. 

In Vivo Exposure of the Respiratory Tract to Carcinogens 

A major limitation to in vivo chemical carcinogenesis
 

studies is the lack of knowing the actual amount of carcinogen
 

delivered and the areas of the target tissue exposed. This is also
 

true of the respiratory system. After a carcinogen is administered
 

intratracheally or by inhalation exposure, it is distributed unevenly
 

in the tracheo-bronchial tree and alveolar parenchyma (see 10,25 for
 

reviews). Subsequently, a redistribution of the material occurs as it
 

is cleared by various mechanisms. Several investigators have tried to
 

132
 
deal with these problems (16,18). Two new localized tumor-induction
 

systems recently developed in our laboratory have overcome many of
 

these difficulties (14,17,27). In one of these systems (27), the
 

carcinogen is topically delivered to a circumscribed region of the
 

trachea by a means of a special catheter. The second system is a
 

tracheal transplant model (14,17), which is particularly well suited
 

for quantitative studies, and is the system used for the in vivo-in
 

vitro studies to be described in detail here. With the tracheal
 

transplant system, the target tissue is well-defined, and uniform
 

exposure of the entire tracheal epithelium at predetermined dose rates
 

for various periods of time can be carried out (15).
 

Neoplastic Progression in the Respiratory Tract 

The difficulty in distinguishing, morphologically preneo­ plastic lesions in the respiratory tract from lesions caused by other
 

toxic effects of various substances has been discussed (24).
 Biological characteristics other than morphology are needed for
 

identifying preneoplastic lesions, as well as detecting progressive
 

changes in the epithelial cells during the evolution to neoplasia. One
 

approach to this problem has been to collect biopsy specimens from
 

human respiratory tract, and to grow the lesions in vitro as organ
 

cultures to detect differences in growth behavior (8,26). The highly
 

replicative state of dysplasias was maintained for 2 weeks, but
 

squamous metaplasias were poorly sustained. The development of
 

improved culture conditions for maintaining respiratory tissue in
 

organ culture for long periods of time as reviewed in earlier pre­ sentations and also developed by us (20) should make this approach
 

rewarding. A more experimental approach has been taken by Shabad, et
 

al. (29). In their experiments, benzo(a)pyrene was delivered trans­ placentally to mice and the lungs of the fetuses maintained in organ
 

culture. Hyperplastic preadenomatous and adenomatous type alterations
 

of the lung epithelium appeared in culture. Administration of the
 

noncarcinogen pyrene did not produce these effects.
 

We are studying the progression of neoplasia in the respira­ tory system by attempting to detect preneoplastic changes and indica­ tors of neoplastic transformation by identifying alterations in the in
 

vitro growth behavior of epithelial cells derived from respiratory
 

tissue exposed to carcinogens in vivo (21). We use the tracheal
 

transplant system described earlier (14,17) to uniformly expose the
 

trachea to carcinogen. In the current studies two approaches are
 

followed: (a) After exposure to carcinogen for brief periods of time
 

explants are grown as organ cultures to determine the fate of the
 

lesions in different nutritional environments; (b) In the second
 

approach, the explants are maintained under culture conditions which
 

stimulate outgrowth of epithelial cells. The cultures are monitored
 

for the number of explants that produce outgrowths, the rates of
 

133
 
epithelial cell outgrowths and changes in epithelial cell morphology.
 

This is correlated with the number of outgrowths that can be main­ tained as primary cell cultures under media conditions suboptimal for
 

that required to maintain unexposed tracheal epithelial cells in
 

primary culture for long periods of time (22). Early epithelial
 

changes are manifested by an increased survival potential. Further
 

indicators of changes in biological behavior due to carcinogen
 

exposures are sought after in the cell lines established from such
 

cultures.
 

II.	 METHODS FOR DEFINING ALTERED BIOLOGICAL BEHAVIOR OF EPITHELIAL
 

CELL POPULATIONS
 

Introduction 

The study of oncogenesis in epithelial cell systems
 

introduces some variables different from those encountered in
 

fibroblast models. Amongst these are (a) the possible involvement of
 

differentiation and de-differentiation in epithelial cell trans­ formation (3), (b) differences in the proliferation kinetics of
 

epithelial cell populations (5), and differences in the phenotypic
 

"markers" which can be used as indicators of transformation in
 

epithelial cells (33). Such factors need to be taken into
 

consideration when attempting to describe altered behavior of
 

epithelial cells in vivo or in vitro.
 

There are several ways of approaching the problem of
 

defining those characteristics of epithelial cell populations which
 

are specifically associated with the oncogenic process. Cell
 

cultures, amenable to detailed characterization in vitro, can be
 

established from in vivo tumor populations. This approach yields
 

useful information about the final stage or stages of oncogenesis, but
 

yields little information about the processes involved. Information
 

about earlier stages must be gained through a more detailed
 

examination of changes occurring prior to the appearance of a frank
 

carcinoma. Towards this end, cell populations can be exposed to a
 

carcinogen in vivo and in vitro and then observed at regular intervals
 

for carcinogen induced alterations in behavior in vitro and in vivo.
 The above general approaches to defining the altered behavior of
 

epithelial cell populations will be discussed and referenced below.
 

Characterization of Tumor Derived Cells in Culture 

Information obtained through this approach is in general as
 

follows: (a) proliferation kinetics and plating efficiency under
 

varying culture conditions (7,30,31,32), (b) morphology (light
 

microscopy and EM) (2,30,32), (c) TD50 (tumor dose-50*) as a measure
 

134
 
of oncogenicity in vivo, (d) attempts to correlate the in vivo
 

behavior of altered epithelial cell populations (1,3*0 or adjacent
 

fibroblast populations with the behavior of same in culture (6). The
 

above mentioned parameters have been described over variable lengths
 

of time passed in vivo and in vitro. This allows some investigation
 

into the effects of proliferation in vivo or in vitro on the
 

phenotypic make-up of a cell population.
 

Characterization of Epithelial Cell Populations During the Development of the Transformed 

Phenotype 

This approach to the characterization of transformed
 

epithelial cells allows for the identification of "early" markers.
 Cell populations can be exposed to a carcinogen in vivo or in vitro
 

and then described at intervals thereafter in vitro (12,13,23,35).
 Tumorigenicity of the cell population can be used as the final marker
 

of oncogenic transformation (12,13,23,35). Criteria described are (a)
 

increased capacity for continued proliferation in vitro relative to
 

normal non-exposed cell populations (12), (b) changes in morphology
 

(EM and light microscopy) (12,13,23,35,36), (c) growth in agar (36),
 and (d) tumorigenicity (12,13,23,35).
 

Other points are of particular note. Colburn (9) observes
 

changes in the differentiated state of epithelial cells during the
 

transformation process. Yamaguchi and Weinstein (36) have described
 

details of the transition from wild-type to transformed-type behavior
 

in a temperature sensitive mutant epithelial cell line.
 

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135
 
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138
 
3.1.3 INDUCTION AND SUPPRESSION OF DYSPLASTIC GROWTH IN ORGANCULTURED URINARY BLADDER EPITHELIUM 

DAVID H. REESE AND ROSALIND D. FRIEDMAN 

Lung Cancer Branch, Carcinogenesis Program, Divsion of 

Cancer Cause and Prevention, National Cancer Institute, NIH, 

Bethesda, MD 

In view of the general lack of improvement in survival rates 

for various forms of epithelial cancer over the last 25 years, it is 

apparent that the traditional approaches for treating epithelial 

cancer - chemotherapy, surgery, or radiation intervention after the 

onset of invasion - have not been satisfactory. A new approach, in 

which incipient disease is detected and treated by novel means well 

before invasion, is clearly needed. (For a detailed discussion of the 

rationales for this approach see ref. 25.) It will be necessary to 

rely heavily on experimental animal models for this approach. Before 

work of this nature can be pursued, however, it is essential that we 

have a better understanding of the early (preneoplastic) phase of 

cancer development in experimental animals. 

Although there is some disagreement on the interpretation of 

data (10), a considerable body of evidence (5,7,9,16,18,21-2H) has 

accumulated on the preneoplastic phase of human bladder cancer which 

suggests to most investigators that the disease is a multifocal 

process of long duration which begins as hyperplasia and progresses 

through increasing degrees of dysplastic growth and cytological atypia 

before invading. (These characteristics are not exclusively those of 

bladder epithelial cancer but are probably features common to many, if 

not all, types of epithelial cancer, human and experimental (8)). There is general agreement that many cases of human bladder cancer, 

like human cervical cancer, are preceded by a carcinoma in situ stage 

of sometimes considerable duration. (For a discussion of the significance and implications of bladder carcinoma in situ and earlier 

lesions see ref. 11.) A well-defined carcinoma in situ stage in the 

bladder epithelium has important diagnostic significance because it 

signifies a readily identifiable stage in cancer development which, by 

definition, is non-invasive but which shows a high probability of 

progressing to invasive cancer if intervening measures are not taken. 

Histologically, human bladder carcinoma in situ is characterized by 

dysplastic growth and cytological atypia (9,17). Unfortunately, a 

well-defined bladder carcinoma in situ stage has not, as yet, been 

identified in experimental animals. Precancerous changes have, 

however, been identified. In rats fed the carcinogenic nitrofuran, 

N-U(5-Nitro-2-furyl)-2-thiazolyl] formamide (FANFT), hyperplastic 

foci of endophytic and exophytic growth are seen by 8 weeks of FANFT 

139 
feeding (27). Recent evidence suggests that these lesions which exist 

at 8 weeks may be irreversible precancerous lesions (4,15). Were it 

not for the absence of severe cytological atypia, these lesions would 

thus fit the criteria for carcinomas in situ. In mice, to which the 

carcinogenic N-nitrosamime, butyl-(4-hydroxybutyl)-nitrosamime was 

administered continuously in the drinking water, endophytic growths 

which eventually became isolated as nests of Brunn were observed 4-6 

weeks prior to the appearance of identifiable cancer and were 

considered by the authors to represent the last precancerous stage 

prior to the appearance of overt cancer (2). In humans, endophytic 

growth with the formation of Brunn's nests is frequently observed in 

association with bladder carcinoma in situ (7>9) , however it is not 

clear whether these abnormalities constitute precursor lesions or 

whether they are merely manifestations of an unstable epithelium 

(17). Some investigators do conclude that endophytic growth "probably 

indicates aggressiveness even though actual infiltration has not 

begun" (12). 

It is not known at this point if the dysplasias which are 

characteristic of the precancerous changes observed in the experimental bladder cancer systems described above are in fact precursor 

lesions. It is clear, however, that dysplastic hyperplasia in the 

transitional epithelium is cause for concern because it is a salient 

feature of precancerous bladder epithelium and because there is a 

direct correlation between dysplastic growth and the degree of 

malignancy in human (1) and experimentally-induced (13) tumors. We 

think, therefore, that an analysis of the process by which dysplastic 

growth arises and how it can be suppressed in the transitional 

epithelium can provide valuable information for understanding and 

devising means for controlling both the preneoplastic phase of cancer 

development and the malignant progression of tumors. A recentlydeveloped organ culture system for bladder is being used to facilitate 

this analysis. 

We have demonstrated (19) that urinary bladders from the rat 

can be maintained in organ culture for prolonged periods of time in 

chemically-defined protein-free, culture media. It was further shown 

that while two culture media, Eagle's MEM (6) and Waymouth's MB752/1 

(26) allowed maintenance with only a small degree of non-dysplastic 

growth, Ham's F12 medium (14) produced extensive dysplastic 

hyperplasia which included the loss of normal transitional structure 

of the epithelium and endophytic growth culminating in Brunn's nests. 

These epithelial abnormalities mimic very closely the precancerous 

changes which are seen in the experimental bladder cancer systems 

discussed above (2,27). Recently, it has been found that F12-induced 

hyperplasia is suppressed by greater than 70% when hydrocortisone is 

added to the culture medium at a concentration of 2.1yM (20). Dysplasia 

140 
was also suppressed under these conditions. Nuclear-cytoplasmic 

ratios, which are elevated in Fl2-cultured epithelialcells (as well as 

in malignant cells), are also maintained at nearly normal levels by 

2.1 yM hydrocortisone (26). 

Although hyperplasia is often a prerequisite to dysplastic 

growth, we do not think that excessive hyperplasia is the sole 

explanation for dysplasia. Increased levels of hyperplasia can be 

induced in Waymouth's MB752/1 medium by elevating the concentrations 

of all media components without causing dysplastic growth (Reese, 

unpublished). Dysplasia may be caused, in large part, by an 

aberration in the normal maturation process of the transitional 

epithelium. This is suggested by recent pulse-chase experiments 

with 3H-thymidine on F12-and MB752/1-cultured epithelia (Reese, 

unpublished) which indicate that the rate of cell loss from 

F12-cultured epithelia is only 20% of that observed for MB752/1­

cultured epithelia. This suggests an abnormal accumulation of cells 

in F12-cultured epithelia which could result in the displacement of 

cells from their normal territories. Cells out of their normal 

territories may no longer be subject to the controls which determine 

ordered cell growth. This could thus explain dysplastic growth in F12 

media. It may also explain the dysplastic characteristics of 

malignant growth (3). 

REFERENCES 

1. Bergkvist, A. , A. Ljungquist, and G. Moberger. "Classification 

of Bladder Tumors Based on the Cellular Pattern." Acta Chir. 

Scand., 130 (1965), 371-378. 

2. Bertram, J.S., and A.W. Craig. "Specific Induction of Bladder 

Cancer in Mice By Butyl-(4-hydroxybutyl)-nitrosamime and the 

Effects of Hormonal Modifications on the Sex Difference in 

Response." Eur. J. Cancer, 8 (1972), 587-594. 

3. Cairns, J. "The Cancer Problem." Sci. Amer., 233 (1975), 64-78. 

4. Cohen, S.M., J.B. Jacobs, and M. Arai, et al." "Early lesions in 

Experimental Bladder Cancer: Experimental Design and Light 

Microscopic Findings." Cancer Res., 36 (1976), 2508-2511. 

5. Cooper, P.H. , J. Waisman, and W.H. Johnston, et al. "Severe 

Atypia of Transitional Epithelium and Carcinoma of the Urinary 

Bladder." Cancer, 31 (1973), 1055-1060. 

6. Eagle, H. "Amino Acid Metabolism in Manmalian Cell Cultures." 

Sci., 130 (1959), 432-437. 

141 
7.

 Eisenberg, R.B., R.B. Roth, and M.H. Schweinsberg. "Bladder 

Tumors and Associated Proliferative Lesions." J. Urol., 84 

(1960), 544-550. 

8. Farber, E. "Putative Precursor Lesions: Summary and Some 

Analytical Considerations." Cancer Res., 36 (1976), 2703-2705. 

9. Farrow, G.M. , D.C. Utz, and C.C. Rife. "Morphological and 

Clinical Observations of Patients with Early Bladder Cancer 

Treated with Total Cystectomy." Cancer Res., 36 (1976), 2495-2501. 

10. Foulds, L. "Neoplastic Development." Vol. 2, London, Academic 

Press, 1975, 161-204. 

11. Friedell, G.H. "Carcinoma, Carcinoma In_ Situ, and "Early 

Lesions" of the Uterine Cervix and the Urinary Bladder: 

Introduction and Definitions." Cancer Res., 36 (1976), 2482-2484. 

12. Friedman, N.B. , and J.E. Ash. "Tumors of the Urinary Bladder." 

In: Atlas of Tumor Pathology, Sect. 8, Fasc. 31a, Washington, Armed Forces Institute of Pathology, 1959. 

13- Fukushima, S., M. Hirose, and H. Tsuda, et al. "Histological 

Classification of Urinary Bladder Cancers in Rats Induced by 

N-butyl-N-(4-hydroxybutyl)-nitrosamine." Gann, 67 (1976), 81-90. 

14. Ham, R.G. "Clonal Growth of Mammalian Cells in a Chemically 

Defined, Synthetic Medium." Proc. Nat. Acad. Sci., 53 (1965), 

288-293. 

15. Jacobs, J.B., M. Arai, and S.M. Cohen, et al. "Early Lesions in 

Experimental Bladder Cancer: Scanning Electron Microscopy of 

Cell Surface Markers." Cancer Res., 36 (1976), 2512-2517. 

16. Koss, L.G., P.M. Tiamson, and M.A. Robbins. "Mapping Cancerous 

and Precancerous Bladder Changes: A Study of the Urothelium in 

Ten Surgically Removed Bladders." J. Am. Med. Assoc., 227 

(1974), 281-286. 

17. Koss, L.G. "Tumors of the Urinary Bladder." In: Atlas of Tumor 

Pathology. Second Series, Fasc. 11, Washington, Armed Forces 

Institute of Pathology, 1975. 

18. Melicow, M.M. "Histological Study of Vesical Urothelium 

Intervening Between Gross Neoplasms in Total Cystectomy." J_._ Urol., 68 (1952), 261-279. 

142 
19.

 Reese, D.H. , R.D. Friedman, and J.M. Smith, et al. "Organ 

Culture of Normal and Carcinogen-treated Rat Bladder." Cancer 

Res.. 36 (1976), 2525-2527. 

20. Reese, D.H. , R.D. Friedman, and M.B. Sporn. "Induction of Hyper­ plasia and its Suppression by Hydrocartisone in Organ-Cultured 

Rat Urinary Bladder." Cancer Res., 37 (1977), 1421-1427. 

21. Schade, R.O.K. , and J. Swinney. "Pre-cancerous Changes in 

Bladder Epithelium." Lancet, 2 (1968), 943-946. 

22. Schade, R.O.K. , and J. Swinney. "The Association of Urothelial 

Atypism with Neoplasia: Its Importance in Treatment and 

Prognosis." J. Urol., 109 (1973), 619-622. 

23. Simon, W. , J.J. Cordonnier, and W.T. Snodgrass. "The 

Pathogenesis of Bladder Carcinoma." J. Urol. , 88 (1962), 797-802. 

24. Skinner, D.G. , J.P. Richie, and P.H. Cooper, et al. "The 

Clinical Significance of Carcinoma In Situ of the Bladder and Its 

Association with Overt Cancer." J. .Urol., 112 (1974), 68-71. 

25. Sporn, M.B. "Approaches to Prevention of Epithelial Cancer 

During the Preneoplastic period." Cancer Res., 36 (1976), 2699-2702. 

26. Stinson, S.F., J.C. Lilga, D.H. Reese, R.D. Friedman, and M.B. Sporn. "Quantitation with an Automated Image Analyzer of 

Decreased Nuclear-Cytoplasmic Ratios Induced by Hydrocortisone in 

Organ-Cultured Rat Urinary Bladder Epithelial Cells." Cancer 

Res., 37 (1977), 1428-1431. 

27. Tiltman, A.J., and G.H. Friedell. "The Histogenesis of 

Experimental Bladder Cancer." Invest. Urol. , 9 (197D, 218-226. 

28. Waymouth, C. "Rapid Proliferation of Sublines of NCTC Clone 929 

(Strain L) Mouse Cells in a Simple Chemically Defined Medium 

(MB752/1)." J. Nat. Cancer Inst., 22 (1959), 1003-1017. 

143 
3.2	 HUMAN TISSUES
 

3.2.1	 EXPLANT CULTURE AND XENOTRANSPLANTATION OF HUMAN
 

BRONCHI
 

CURTIS C. HARRIS1, HERMAN AUTRUP1, GARY D. STONERr, MARION VALERIO2,
 

ELLIOTT FINEMAN2, PAUL W. SCHAFER3, LUCY A. BARRETT4, ELIZABETH M.
 

McDOWELL4, AND BENJAMIN F. TRUMP4.
 

Experimental Pathology Branch, Carcinogenesis Program,
 Division of Cancer Cause and Prevention, National Cancer
 

Institute, NIH, Bethesda, MD (1); Litton-Bionetics,
 Kensington, MD (2); Department of Surgery, VA Hospital,
 Washington, DC (3); and Department of Pathology University
 

of Maryland, School of Medicine, Baltimore, MD (4).
 

Epidemiologic studies of migrating groups, religious sects,
 

occupational populations and tobacco smokers as well as numerous
 

experimental investigations identifying carcinogenic chemicals found
 

in man's environment have strongly suggested human cancer is caused by
 

environmental agents, including chemical carcinogens (17). The
 

epithelia of the respiratory and gastrointestinal tracts are the most
 

important sites of these environmentally-related cancers. One of the
 

major problems with these studies is that they provide important, but
 

retrospective, information, i.e., "after the fact." Therefore,
 

experimental model systems have been developed in attempts to study
 

chemical carcinogenesis in target tissues. For example animal models
 

of human lung (27), colon (6,33) and pancreatic (22) carcinoma have
 

been developed during the last decade. While these animal models
 

successfully mimic many of the features of the human disease, the
 

extrapolation of carcinogenesis data from experimental animals to man
 

is a difficult problem (9,32). The extrapolation of data from both in
 

vitro carcinogenesis and mutagenesis studies is an even more complex
 

problem. Model systems for studying carcinogenesis in human tissues
 

have been developed (10). These systems provide an opportunity to
 

investigate chemical carcinogenesis in the primary target tissues for
 

human cancer. These model systems have three major facets: 1)
 

obtainment of viable human tissues which involves all of the medical,
 legal and ethical aspects of human experimentation; 2) maintenance of
 

these tissues in vitro; and finally 3) xenotransplantation of human
 

tissues into immunodeficient animals.
 

This guide to the literature will focus on the methodology
 

required to culture explants of human bronchus, how these explants can
 

be used to investigate the metabolic activation of chemical
 

carcinogens, and lastly, the xenotransplantation of both normal and
 

neoplastic tissues into the athymic nude mice.
 

1W 
A.	 EXPLANT CULTURE OF HUMAN BRONCHUS
 

The in vitro maintenance of explants of human bronchi under
 

controlled experimental conditions is an essential aspect of our
 

attempts to develop model systems using human target tissues; the
 

advantages of organ and explant culture are listed in Table 1.
 

Lasnitzki (19) is credited with being the first to maintain
 

human respiratory epithelium in culture. Since then other
 

investigators (2,4,20,21,28) have cultured lung tissue, including
 

human bronchus, for periods up to 3 weeks. These investigators used
 

culture medium containing serum, and/or the bronchial explants were
 

maintained on plasma clots. Based on studies using organ cultures of
 

hamster tracheas (3), a chemically defined medium for culturing human
 

bronchial explants has been developed (1,15,29; Table II). Human
 

bronchial specimens can be successfully cultured in this medium for
 

periods of 1 month, and for 6 months in medium supplemented with 5%
 

fetal bovine serum. Other conditions important for the culture of
 

human bronchi are also listed in Table II.
 

B. METABOLIC ACTIVATION OF CHEMICAL CARCINOGENS BY CULTURED HUMAN BRONCHI 

Tobacco smoke contains several chemical classes of known 

carcinogens (10,3*0- Of these, polynuclear aromatic hydrocarbons 

(PAH) were chosen for our initial studies. Previous reports by 

Lasnitzki (19) and Crocker et al., (4) indicated that both tobacco 

smoke condensates and PAH cause morphologic lesions, e.g., cytotoxi­ toxicity, hyperplasia and squamous metaplasia, in cultured respiratory 

epithelium. Using the chemically-defined medium described above to 

eliminate the complicating factor of serum, we reported that human 

bronchial explants bind carcinogenic PAH (7, 12-dimethylbenz(a)­ anthracene, 3-methylcholanthracene, benzo(a)pyrene and dibenz(a,h)­ anthracene) to cellular macromolecules (T4). The most potent 

carcinogen in experimental animals of the four tested, 7,12-dimen­ thylbenz(a)anthracene, had the highest level of binding to DNA. Recently we have investigated the interaction of benzo(a)pyrene (BP) 

with bronchial explants (10,12,13,15,31). Binding of BP" to cellular 

macromolecules is dependent on concentration of and duration of 

exposure to benzo(a)pyrene and to the temperature of incubation. Preliminary studies (8) suggest that the adducts formed between 

benzo(^)pyrene and DNA in cultured human bronchus have similar 

chromatographic properties as those adducts formed in both mouse skin 

and cultured hamster embryo cells. Autoradiographic analysis showed 

that bronchial epithelial cells bind approximately four times more BP 

than fibroblasts (15). While the intraindividual variation due to 

methodology is small (15), the variation of BP binding levels to DNA 

among individuals is quite marked (12); this 75-fold interindividual 

145 
TABLE I 

ADVANTAGES OF ORGAN AND EXPLANT CULTURE 

1. Maintains intercellular relationships between 

a. epithelial cells 

b. epithelium-mesenchyme 

2. Maintains differing state of differentiated state in 

epithelial cells 

3. Maintenance of tissue architecture allows 

a. morphological studies of tumorigenesis including 

possible identification of preneoplastic lesions 

b. correlation to histological lesions found in vitro to 

those observed in vivo 

Table II 

EXPLANT CULTURE OF HUMAN BRONCHUS 

A. Culture Medium 

1. CMRL 1066, insulin (1yg/ml), hydrocortisone (0.1 y g/ml),

 3-retinyl acetate (0.1yg/ml),

 penicillin G (100 units/ml),

 Streptomycin (100y g/ml),

 and amphotericin B (0.25yg/ml)

 

2.. with or without heat-inactivated 

fetal bovine serum 

B. Atmosphere and Incubation Temperature 

1. 50% 0_, 45$ N5 and 5% CO. 2.

 36.5°C 2 2 

C. Other Conditions 

1. submerge 50% of the time 

2. rock at 10 cycles per min 

146 
variation in the metabolism of BP is not surprising and may be due to 

genetic factors. Man is genetically a heterogeneous population, and 

pharmacogenetic studies in mono- and dizygotic twins have shown that 

the metabolism of drugs is controlled more by genetic factors than by 

exogenous agents (30). Since most environmental carcinogens, 

including PAH, require metabolic activation, one can hypothesize that 

genetic differences in the metabolic activation of chemical 

procarcinogens may be very important in determining an individual's 

risk of developing cancer. While indirect data supporting this 

hypothesis linking metabolic activation and individual susceptibility 

has accumulated in studies using cells in culture and experimental 

animals (16,32) it has not been adequately tested in man. 

A number of naturally-occurring and synthetic compounds both 

alter the metabolism of PAH and to have an anticarcinogenic action in 

experimental animals by decreasing tumor incidence and/or by 

increasing the latency period of induced tumors (reviewed in ref. 

16). Several of these chemicals have been tested for their ability to 

alter the level of BP binding to DNA in cultured human bronchial 

explants (13,15). Binding of BP was significantly inhibited by 

7,8-benzoflavone and to a lesser extent by disulfiram, a nonphenolic 

antioxidant containing sulfur, and vitamin E. Co-incubation with 

either nicotine or $-retinyl acetate did not alter the level of 

binding under the experimental conditions tested. These studies 

indicate the possibility to both quantify and modify the interaction 

of chemical carcinogens with macromolecules in human target tissues. 

In addition to polynuclear aromatic hydrocarbons, the 

metabolic activation of other chemical classes of carcinogens are 

being investigated. Ongoing studies (Harris et al., unpublished 

results) have shown that dimenthylhydrazine are metabolized in 

cultured human bronchi. While the metabolism of 1,2-dimethylhydrazine 

had not been previously studied in human tissue, slices of cultured 

human lung have been shown to metabolize dimethylnitrosamine (5). 

Matos et al., (24) have described hyperplastic and squamous metaplastic lesions in human fetal bronchial epithelium exposed to 

diethylnitrosamine. The morphological effects of dimethylnitrosamine 

and of 1,2-dimethylhydrazine on cultured human bronchus are as yet 

unkown. 

One objective of these studies is to determine if the 

metabolic pathways of carcinogen activation are the same in human 

target tissues as it is in animal models; if so, the extrapolation of 

carcinogenesis data among species is more likely to be valid than if 

the pathways of metabolic activation are different. While such 

comparative studies have only recently begun, the metabolic activity 

pathway of BP in cultured human bronchus appears to be similar to that 

found in several experimental animals. Other classes of chemical 

carcinogens are currently being studied. 

117 
C.

 XENOTRANSPLANTATION OF HUMAN BRONCHIAL SPECIMENS IN 

ATHYMIC NUDE MOUSE 

The ability of cells exposed to carcinogens in vitro to
 

produce tumors when transplanted into appropriate hosts is considered
 

to be the ultimate criterion for the assessment of malignant trans­ formation. While human cells transformed to malignant cells in vitro
 

cannot be transplanted into humans to test their tumorigenic proper­ ties, they can be xenotransplanted into immunodeficient animals; of
 

the several immunodeficient recipients tested, the athymic nude mouse
 

seems the most useful. A monograph (25) has recently been published
 

which describes the genetics, husbandry and experimental utility of
 

these animals. Adult and fetal normal human tissues have been
 

successfully maintained as xenotransplants (23,26). In addition,
 human tumors grow and occasionally metastasize in athymic nude mice.
 Surgical specimens of cancers do not grow as frequently in athymic
 

nude mice as cells from human cancers that have been initially
 

cultured as monolayers prior to xenotransplantation (7). This
 

observation may be due to ischemia in the solid tumor.
 

We have found that adult human bronchial specimens can be
 

maintained as subcutaneous xenotransplants in athymic nude mice for
 

periods in excess of 5 months (Valerio, M. _et al., unpublished
 

results). After an ischemic crisis and revascularization, the
 

bronchial epithelium regenerates and attains a normal morphology by
 

approximately 3 weeks. Using the approach described by Kendrick et
 

al., (18) for exposing heterografts to carcinogens containing beeswax
 

pellets, we have found that pellets containing 7,12 dimethyl­ benz(a)anthracene, inserted into the lumen of subsegmental human
 

bronchi, cause hyperplastic and squamous metaplastic lesions in the
 

bronchial epithleium (Valerio, M., Harris, C., et al., unpublished
 

results). Therefore, xenotransplantation is a useful technique both
 

for assays of the tumorigenicity of human cells exposured to carcin­ ogens in vitro and for exposing human xenografts to carcinogens in
 

vivo.
 

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2.	 Cailleau, R., T.T. Crocker, and D. Wood. "Attempted Long Term
 

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148 
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5. den Engelse, L., M. Gebbink, and P. Emmelot. "Studies on Lung 

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151 

4.1

4. MECHANISMS, METABOLISM, CO-FACTORS 

AND MARKERS 

 METABOLIC ACTIVATION OF CHEMICAL CARCINOGENS 

PETER N. MAGEE 

Pels Research Institute, Temple University School of 

Medicine, Philadelphia, PA 

The number of chemicals known to induce cancer in animals is 

large and it is constantly increasing as more carcinogens are dis­ covered, often as a result of new observations relating certain 

cancers with occupational or other exposure of human populations to 

synthetic industrial chemicals. A relatively small proportion of the 

known carcinogens has been studied in any detail. These compounds 

include various different chemical classes including certain 

polycyclic aromatic hydrocarbons, aromatic amines, aromatic azo 

compounds, biological alkylating agents, N-nitroso compounds, aliphatic azoxy compounds including cycasin, the naturally occurring 

glucoside of methylazoxymethanol, aflatoxins, pyrrolizidine alkaloids, urethane, 4-nitroquinoline-N-oxide, various chlorinated hydrocarbons 

and ethionine, the ethyl analogue of methionine. The chemical 

structures of these different types and classes of carcinogen are very 

different making it virtually impossible to predict potential 

carcinogenic activity from chemical structure except where there is 

close structural resemblence with known carcinogens. 

Chemical carcinogens can be divided into two main groups 

based on their capacity or lack of capacity to induce cancer at the 

site of injection or when applied to the skin. Those compounds that 

are active at the site of application are described as direct, or 

locally acting carcinogens, while those that are active in organs or 

tissues remote from the injection site are described as indirect or 

remotely acting carcinogens. These contrasting patterns of activity 

can be readily illustrated by reference to the biological behaviour of 

the chemically simplest carcinogenic N-nitroso compounds, dimethyl­ nitrosamine and N-methyl-N-nitrosourea. Dimethylnitrosamine which is 

chemically stable under physiological conditions, does not induce 

subcutaneous sarcomas at the injection site or tumors of the skin 

153 
after repeated topical applications. In contrast N-methylnitrosourea 

is unstable under physiological conditions and is a potent locally 

acting carcinogen. 

After administration to rats, dimethylnitrosamine is rapidly 

and virtually uniformly distributed throughout the body but it induces 

tumors only in the liver and kidney, with occasional lung tumors. 

These observations are most readily explained by assuming that the 

parent nitrosamine is not itself carcinogenic but that it is converted 

by metabolism in the body to yield a carcinogenically active 

metabolite or metabolites. It is now generally recognized, largely 

from the work of James and Elizabeth Miller of the University of 

Wisconsin, that most chemical carcinogens require similar metabolic 

activation. The Millers introduced the terms precarcinogen, proximate 

carcinogen and ultimate carcinogen to describe, respectively, the 

original, relatively or completely inactive molecule, various 

metabolites of the original compound with greater carcinogenic 

activity and the active molecular species that is responsible for 

conversion of normal cells into tumor cells. All the ultimate 

carcinogens so far suggested are highly reactive electrophiles that 

can react with cellular macromolecules under physiological conditions 

in vitro and are mutagenic in suitable test systems. Examples of 

extensively studied precarcinogens are the polycyclic aromatic 

hydrocarbon 7,12-dimethylbenz(a)anthracene, the aromatic amine 

2-naphthylamine, the aromatic azo dye p-dimethylaminoazobenzene (DAB) 

and the nitrosamine N-nitrosodimethylamine or dimethylnitrosoamine. 

Most precarcinogens are activated to form ultimate carcinogens by the 

enzyme systems known as microsomal mixed-function oxidases; 

occasionally activation is affected by other tissue enzymes; for 

example, M-nitroquinoline-N-oxide by diaphorase. Another distinct 

mechanism of activation is mediated by microsomal enzymes of the 

intestinal flora. The best understood example of this mechanism is 

activation of the naturally occurring glycoside of methylazoxymethanol 

or cycasin, found in cycad plants. 

Although the liver had long been recognized as the main 

organ for metabolism of foreign compounds, it was not until 1955 that 

Brodie and his colleagues at the National Institutes of Health 

demonstrated that the enzyme system concerned was located in the 

microsome fraction of liver homogenates. Subsequent work revealed 

that the enzyme activity was concentrated in the smooth membranes of 

the endoplasmic reticulum and there was the unusual requirement for 

reduced pyridine nucleotide (NADPH) and oxygen for activity. 

A variety of reactions with foreign compounds are catalysed 

including aromatic hydroxylation, oxidation of alkyl side chains, 

0-dealkylation, N-dealkylation, N-oxidation, sulphoxidation and 

replacement of S by 0. The basic mechanism of several of these 

154 
reactions is hydroxylation and the enzyme systems are, therefore, 

widely described as microsomal hydroxylases. Studies with 

isotopically labelled oxygen as the gas phase in hydroxylation 

reactions has indicated that one atom of oxygen from the atmosphere is 

incorporated into the hydroxyl group added to the foreign compound 

leading to the naming by Mason of the enzymes as mixed-function 

oxidases. It is well established that this microsomal enzyme system 

is involved in hydroxylation and other reactions that modify steroids 

and fatty acids as well as foreign compounds. 

The mixed-function oxidases are membrane bound 

multicomponent enzyme systems containing cytochrome P-450, cytochrome 

be, NADPH- and NADH cytochrome reductases and various lipid and other 

components. The system is inhibited by carbon monoxide, which binds 

to cytochrome P-^50 and by a number of other compounds of which 

SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride) is 

one of the most extensively used. Substrates of the microsomal mixed 

function oxidases cause spectral changes in cytochrome P-450 of two 

types. Type I spectra, exemplified by ethylmorphine, hexobarbitone 

and most foreign compounds show peaks at 385 my and troughs at *I20 

mu. Type II spectra, produced by the addition of aniline and some 

other bases, show a reverse type of change, with a trough at 390 my 

and a peak at ^30 my. 

Microsomal mixed-function oxidases are induced by treatment 

of animals with a variety of chemicals. There appear to be at least 

three different mechanisms of induction including that produced by 

phenobarbitone and most other drugs, that produced by polycyclic 

aromatic hydrocarbons and that by anabolic steroids. Most 

experimental work on mechanisms of stimulation has been done with 

phenobarbitone and either 3-methylcholanthrene or 3,1

<-benzo(a)pyrene. 

Recently, various polychlorinated biphenyls (PCBS) such as Aroclor 

125*1 have been found to be potent inducers and the most powerful agent 

known is a dioxin originally discovered as an impurity of PCBS. 

Induction of microsomal mixed function oxidases is accompanied by 

morphological changes in the liver characterized by marked increase in 

the smooth endoplasmic reticulum. The enzyme systems can be induced 

in cells in culture. Reactions catalyzed by the mixed-function 

oxidases that are important for chemical carcinogenesis include 

epoxidation, N-hydroxylation and N-dealkylation. 

Epoxidation is now thought to be an important metabolic 

pathway in the activation of polycyclic aromatic hydrocarbons, 

aflatoxins and some chlorinated hydrocarbons. Epoxidation of the 

polycyclics was first shown to occur at the so-called K-region of the 

molecule. The K-region is a bond having double bond character, like 

the 9,10 bond of phenanthrene, rather than aromatic character, the 

name being derived from Krebs, the German word for cancer. 

155 
Chemically, the K-region is a bond which takes part in addition 

reactions and it has been claimed to be essential for carcinogenic 

activity on theoretical quantum chemical grounds by A. and B. Pullman. 

Extensive studies on metabolism of benz(a)anthracene and 

other polycyclics by Sims and Grover in the United Kingdom and by 

Heidelberger and his colleagues in the United States have shown that 

epoxidation occurs at the K-region and at other bonds in the molecule 

and that this is followed by various competing reactions including 

conversion to the dihydrodiol by the microsomal enzyme epoxide 

hydrase, non-enzymic rearrangement and conjugation with glutathione by 

cytoplasmic glutathione-S-epoxide transferase, all of which lead to 

deactivation, and reactions with cellular constituents, thought to be 

responsible for the induction of cancer and other biological actions 

of the compound. More recent work suggests that epoxidation at the 

8,9 bond with formation of 8,9-dihydro-8-9-dihydroxybenz(a)anthracene 

may be more closely related to the carcinogenic action of 

benz(a)anthracene. The activating enzymes, usually called polycyclic 

aromatic hydrocarbon (PAH) hydroxylases, can be induced in vivo and in 

vitro by various inducers and can be inhibited by 7,8-benzoflavone. 

There is evidence for a similar type of activation by epoxidation for 

aflatoxin B. with the formation of the 2,3-epoxide, and for certain 

chlorinated hydrocarbons, including vinyl chloride, with formation of 

the corresponding epoxide or oxirane. 

Another important metabolic pathway for the activation of 

chemical carcinogens is N-hydroxylation, again discovered in the 

laboratory of the Millers in Wisconsin. This pathway is followed by 

the aromatic amine and azo carcinogens. Metabolic hydroxylation 

occurs on the carbon atoms of the aromatic rings (C-hydroxylation) and 

on the nitrogen of the amine groups (N-hydroxylation). 

C-hydroxylation is thought to represent deactivation and 

N-hydroxylation activation of the carcinogens. The details of these 

pathways were largely worked out with the aromatic amine, 

N-acetylaminofluorene (N-2-fluorenylacetamide; AAF). The hydroxylation product, N-hydroxyacetylaminofluorene (N-hydroxy AAF) is a 

good example of a proximate carcinogen, being more active than the 

parent molecule but not mutagenic or capable of reaction with 

macromolecules. Esterification of the hydroxyl group produces more 

reactive and mutagenic products. N-acetoxyacetylaminofluorene 

(N-acetoxy-AAF) is mutagenic and will transform cells in culture 

without further activation. 

Carbon tetrachloride, a relatively weak carcinogen, is 

activated by the microsomal enzymes, and the trichloromethyl radical 

has been suggested as a possible active species. Free radical 

formation may also be involved in the activation of other carcinogens. 

156 
N-dealkylation with formation of alkylating intermediates 

has been suggested as the mechanism of metabolic activation of the 

carcinogenic nitrosamines, dialkylhydrazines, alkyl azo- and azoxy compounds and the dialkylaryltriazenes. Dimethylnitrosamine is 

thought to undergo hydroxylation of one methyl group followed by 

release of formaldehyde, rearrangement to the diazohydroxide followed 

by decomposition to the methyldiazonium cation and then the 

methylcarbonium ion with release of molecular nitrogen and methylation 

of cellular macro-molecules and other components, 1,2-dimethyl hydrazine 

and azoxymethane appear to be metabolized in a similar manner. The 

carcinogenic nitrosamides such as N-methylnitrosourea, mentioned 

above, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) do not require 

enzymic activation for biological activity. MNNG has been widely used 

for in vitro cell transformation. 

Cycasin, the naturally occurring glucoside of 

methylazoxymethanol (MAM) is activated in quite another manner, i.e., by glucosidases of the microbial flora of the gastrointestinal tract. There is evidence that the further metabolic steps after release of 

the glucose moiety become identical with those for dimethylnitrosamine 

and N-methylnitrosourea. It is clearly improbable that cycasin would 

be active in transformation in vitro but the synthetic compound, methylazoxymethanol acetate (MAM acetate) is activated by tissue 

esterases and does have effects on cells in culture. 

From the above considerations, it is clear that in vitro 

transformation will not occur if the cells exposed to precarcinogens 

do not contain appropriate activating enzymes. An obvious way to 

circumvent this difficulty is to use agents such as MNNG, that do not 

require metabolic activation, or to prepare proximate carcinogens 

synthetically, e.g., epoxides of polycyclic hydrocarbons or N-acetoxy 

AAF. Another method, exploited by Heidelberger and his colleagues, is 

to use a "feeder" layer of irradiated cells, capable of activating the 

precarcinogen, upon which the target cells for transformation are 

grown. 

Activation may also be achieved by the so-called in vivo/in 

vitro method, developed by DiPaolo and his colleagues, in which 

pregnant animals are treated with the precarcinogen and cultures 

obtained from the progeny. Tissues for culture may also be taken from 

the treated animals themselves shortly after exposure to the 

precarcinogen. 

\^\ 
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 Levin, W., A.M. Conney, A.P. Alvares, I. Merkatz, and A. Kappas. 

"Induction of Benzo(a)pyrene Hydroxylase in Hunan Skin." 

Science, 176 (1972), 419-429. 

23. Nebert, D.W. , and J.S. Pel ton. "Evidence for the Activation of 

3-methylcholanthrene as a Carcinogen in vivo and as a Mutagen in 

vitro by Pl-450 from Inbred Strains of Mice." In: Cytochromes 

P-450 and be, D.Y. Cooper, 0. Rosenthal, R. Snyder, and C. Witmer 

(Eds.), Plenum Press, New York and London, (1975), 127-149. 

24. Nebert, D.W. , and J.S. Felton. "Importance of Genetic Factors 

Influencing the Metabolism of Foreign Compounds." Fed. Proc., 35 

(1976), 1133-1141. 

25. Nebert, D.W. , and H.V. Gelboin. "The in vivo and in vitro 

Induction of Aryl Hydrocarbon Hydroxylase in Mammalian Cells of 

Different Species Tissues Strains and Developmental and Hormonal 

Status." Arch. Biochem. Biophys., 134 (1969), 76-89. 

26. Oesch, F. "Mammalian Epoxide Hydrases: Inducible Enzymes 

Catalyzing the Inactivation of Carcinogenic and Cytotoxic 

Metabolites Derived from Aromatic and Olefinic Compounds." 

Xenobiotica, 3 (1972), 305-340. 

27. Selkirk, J.K., E. Huberman, and C. Heidelberger. "An Epoxide is 

an Intermediate in the Microsomal Metabolism of the Chemical 

"Carcinogen Dibenz(a,h)anthracene." Biochem. Biophys. Res. 

Commun., 43 (1971), 1010-1016. 

28. Sims, P., and P.L. Grover. "Epoxides in Polycylic Aromatic 

Hydrocarbon Metabolism and Carcinogenesis." Advanc. Cancer Res., 20 (1974), 166-274. 

29. Sims, P., P.L. Grover, A. Swaisland, K. Pal, and A. Hewer. "Metabolic Activation of Benzo(a)pyrene Proceeds by a 

Diol-epoxide." Nature (London), 252 (1974), 326-328. 

30. Swaisland, A.J., A. Hewer, K. Pal, G.R. Keysell, J. Booth, P.L. 

Grover, and P. Sims. "Polycyclic Hydrocarbon Epoxides: The 

Involvement of 8,9-dihydro-8,9-dihydroxybenz(a)anthracene-10,lloxide in Reactions with DNA of Benz(a)anthracene Treated Hamster 

Embryo Cells." FEES Letters, 47 (1974), 34-38. 

31. Wattenberg, L.W. "Dietary Modification of Intestinal and 

Pulmonary Aryl Hydrocarbon Hydroxylase Activity." Toxicol. Appl. Pharmacol., 23 (1972), 741-748. 

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 Wattenberg, L.W., and J.L. Leong. "Histochemical Demonstration 

of Reduced Pyridine Nucleotide Dependent Polycyclic Hydrocarbon 

Metabolizing Systems." J. Histochem. Cytochem., 10 (1962), 412-420. 

33. Wattenberg, L.W., and J.L. Leong. "Inhibition of the 

Carcinogenic Action of Benzo(a)pyrene by Flavones." Cancer Res., 

30 (1970), 1922-1925. 

34. Wattenberg, L.W., J.L. Leong, and P.J. Strand. "Benzpyrene 

Hydroxylase Activity in the Gastrointestinal Tract." Cancer 

Res., 22 (1962), 1120-1125. 

35. Whitlock, J.P., H.L. Cooper, and H.V. Gelboin. "Aryl Hydrocarbon 

(Benzopyrene) Hydroxylase is Stimulated in Human Lymphocytes by 

Mitogens and Benz(a)anthracene." Science, 177 (1972), 618-619­

36. Wiebel, F.J., and H.V. Gelboin. "Arylhydrocarbon (benzo(a)pyrene) 

hydroxylases in Liver from Rats of Different Age, Sex and 

Nutritional Status." Biochem. Pharmacol., 24 (1975), 1511-1515. 

37. Wiest, W.G. , and C. Heidelberger. "The Interaction of 

Carcinogenic Hydrocarbons with Tissue Constituents. II. 

l,2,5,6-dibenzanthracene-9,lO-C11* in Skin." Cancer Res., 13 

(1953), 250-254. 

AFLATOXINS 

1. Garner, R.C. "Microsome-dependent Binding of Aflatoxin Bl to 

DNA, RNA, Polyribonucleotides and Protein in vitro." Chem.-Biol. 

Interactions, 6 (1973), 125-129. 

2. Garner, R.C. "Chemical Evidence for the Formation of a Reactive 

Aflatoxin B-| Metabolite by Hamster Liver Microsomes." FEES 

Letters, 36 (1973), 261-264. 

3. Garner, R.C. "Reduction in Binding of C Aflatoxin B^ to Rat 

Liver Macromolecules by Phenobarbitone Pretreatment." Bioohem. 

Pharmacol., 24 (1975), 1553-1556. 

4. Garner, R.C., E.G. Miller, and J.A. Miller. "Liver Microsomal 

Metabolism of Aflatoxin B to a Reactive Derivative Toxic to 

Salmonella Typhimurium TA1530." Cancer Res., 32 (1972), 

2058-2066. 

5. Garner, R.C., E.G. Miller, J.A. Miller, J.V. Garner, and R.S. 

Hanson. "Formation of a Factor Lethal for S. typhimurium 

TA1530and TA1531 on Incubation of Aflatoxin B-| with Rat Liver 

Microsones." Bioohem. Biophys. Res. Commun., 45 (1971), 774-780. 

166 
6.	 McLean, A.E.M., and A. Marshall. "Reduced Carcinogenic Effects
 

of Aflatoxin in Rats Given Phenobarbitone." Brit. J. Exp. Path.,
 

52 (1971), 322-329.
 

7.	 Patterson, D.S.P. "Metabolism as a Factor in Determining the
 

Toxic Action of the Aflatoxins in Different Animal Species." Fd.
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8.	 Swenson, D.H., J.A. Miller, and E.G. Miller. "2,3-dihydro-2,3­ dihydroxyaflatoxin Bl: An Acid Hydrolysis Product of an
 

RNA-aflatoxin Bl Adduct Formed by Hamster and Rat Liver
 

Microsomes in vitro." Biochem. Biophys. Res. Commun., 53 (1973),
 

1260-1267.
 

9.	 Wogan, G.N. "Chemical Nature and Biological Effects of
 

Aflatoxins." Bacteriol. Rev., 30 (1966), 460-470.
 

10.	 Wogan, G.N. "Metabolism and Biochemical Effects of Aflatoxins."
 

In: Aflatoxin, L. A. Goldblatt (Ed.), Academic Press, New York
 

and London, 1969, 151-186.
 

AROMATIC AMINES, AROMATIC AZO COMPOUNDS
 

1.	 Cramer, J.W., J.A. Miller, and E.G. Miller. "N-hydroxylation: A
 

New Metabolic Reaction Observed in the Rat with the Carcinogen
 

2-acetyl-aminofluorene." J. Biol. Chem., 235 (I960), 885-888.
 

2.	 DeBaun, J.R., E.G. Miller, and J.A. Miller. "N-hydroxy-2­ acetylaminofluorene sulfotransferase: Its Probable Role in
 

Carcinogenesis and in Protein(methion-S-yl) Binding in Rat
 

Liver." Cancer Res., 30 (1970), 577-595.
 

3.	 Litwack, G., B. Ketterer, and I.M. Arias. "Ligandin: A Hepatic
 

Protein Which Binds Steroids, Bilirubin Carcinogens and a Number
 

of Exogenous Organic Anions." Nature, London, 234 (1971),
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4.	 Miller, E.G., and J. A. Miller. "Studies on the Mechanism of
 

Activation of Aromatic Amines and Amide Carcinogens to Ultimate
 

Carcinogenic Electrophilic Reactants." Ann. N.Y. Acad. Sci., 163
 

(1969), 731-750.
 

5.	 Miller, J.A., and E.G. Miller. "The Metabolic Activation of
 

Carcinogenic Aromatic Amines and Amides." Progr. Exp. . Tumor
 

Res.. 11 (1969), 273-301.
 

6.	 Weisburger, J.H., and E.K. Weisburger. "Biochemical Formation
 

and Pharmacological Toxicological and Pathological Properties of
 

Hydroxylamines and Hydroxamic Acids." Pharmacol. Revs., 25
 

(1973), 1-66.
 

167
 
N-NITROSO COMPOUNDS 

1. Blattman, L., N. Joswig, and R. Preussmann. "Struktur von 

Metaboliten des Carcinogenen Methyl-n-butylnitrosamines in 

Rattenurin." Z. Krebsforsch, 81 (1974), 71-73. 

2. Blattman, L., and R. Preussmann. "Struktur von Metaboliten 

Carcinogenen Dialkylnitrosamine in Rattenurin." Z. Krebsforsch, 79 (1973), 3-5. 

3. Blattman, L., and R. Preussmann. "Biotransformation von 

Carcinogenen Dialkylnitrosaminen. Weitere Urinmetaboliten von 

Di-n-butyl und Di-n-pentylnitrosamin." Z. Krebsforsch, 81 

(1974), 75-78. 

4. Brusick, D., and H. Andrews. "Comparison of the Genetic Activity 

of Dimethylnitrosamine, Ethyl Methanesulphonate, 2-acetylamino­ fluorene and ICR-170 in Saccharomyces cerevisiae Strains D^ Djj 

and Dg Using in vitro Assays With and Without Metabolic 

Activation." Mutation Res., 26 (1974), 491-500. 

5. Czygan, P., H. Greim, A.J. Garro, F. Hutterer, J. Rudick, F. Schaffner, and H. Popper. "Cytochrome P-450 Content and the 

Ability of Liver Microsomes from Patients Undergoing Abdominal 

Surgery to Alter the Mutagenicity of a Primary and a Secondary 

Carcinogen." J. Natl. Cancer Inst., 51 (1973), 1761-1764. 

6. Czygan, P., H. Greim, A.J. Garro, F. Hutterer, F. Schaffner, H. Popper, 0. Rosenthal, and D.Y. Cooper. "Microsomal Metabolism of 

Dimethylnitrosamine and the Cytochrome P-450 Dependency of Its 

Activation to a Mutagen." Cancer Res., 33 (1973), 2983-2986. 

7. Czygan, P., H. Greim, A. Garro, F. Schaffner, and H. Popper. "The Effect of Dietary Protein Deficiency on the Ability of 

Isolated Hepatic Microsomes to Alter the Mutagenicity of a 

Primary and a Secondary Carcinogen." Cancer Res., 34 (1974), 119-123. 

8. Druckrey, H., R. Preussmann, S. Ivankovic, and D. Schmahl. "Organotrope Carcinogene. Wirkungen bei 65 Verschiedenen 

N-nitroso-verbindungen an BD-Ratten." 1. Krebsforsch, 69 (1967), 103-201. 

9. Gletten, F., U. Weekes, and D. Brusick. "In vitro Metabolic 

Activation of Chemical Mutagens. I. Development of an in vitro 

Mutagenicity Assay Using Liver Enzymes for Activation of 

Dimethylnitrosamines." Mutation Res., 28 (1975), 113-122. 

168 
10.

 Gomez, R.F. , M. Johnson, and A.J. Sinskey. "Activation of 

Nitrosomorpholine and Nitrosopyrolidine to Bacterial Mutagens." 

Mutation Res., 24 (197*0, 5-7. 

11. Haga, J.J., B.R. Russell, and J.F. Chapel. "The Kinetics of 

Decomposition of N-alkyl Derivatives of Nitrosoguanidine." 

Cancer Res., 32 (1972), 2085. 

12. Heath, D.F. "The Decomposition and Toxicity of 

Dialkylnitrosamines in Rats." Biochem. J., 85 (1962), 72-81. 

13. Heath, D.F., and A. Dutton. "The Detection of Metabolic Products 

from Dimethylnitrosamine in Rats and Mice." Biochem. J., 70 

(1958), 619-626. 

14. Jose, P.J., and T.F. Slater. "The Stimulation of Lipid 

Peroxidation Produced by Dimethylnitrosamine in Rat Liver 

Microsomes in vitro; The Effects of Promethazine and Inhibitors 

of Drug Metabolism and a Comparison with Previous Studies Using 

Carbon Tetrachloride." Xenobiotica, 3 (1973), 357-366. 

15. Kato, R. , H. Shoji, and A. Takanaka. "Metabolism of Carcinogenic 

Compounds. I. Effect of Phenobarbital and Methylcholanthrene on 

the Activities of N-demethylation of Carcinogenic Compounds by 

Liver Microsomes of Male and Female Rats." Gann, 58 (1967), 

467-469. 

16. Kawachi, T., K. Kogure, Y. Kami jo, and T. Sugimura. "The 

Metabolism of N-methyl-N'-nitro-N-nitrosoguanidine in Rats." 

Biochem. Biophys. Acta, 222 (1970), 409-415. 

17. Kr'uger, F.W. "New Aspects in Metabolism of Carcinogenic 

Nitrosamines." In: Topics in Chemical Carcinogenesis, W. Nakahara, S. Takayama, T. Sugimura, and S. Odashima (Eds.), University of Tokyo Press, 1972, 213-235. 

18. McLean, A.E.M. , and P. Day. "The Effect of Diet and Inducers of 

Microsomal Enzyme Synthesis on Cytochrome P-450 in Liver 

Homogenates and on Metabolism of Dimethylnitrosamine." Biochem. Pharmacol., 23 (1974), 1173-1180. 

19. Magee, P.N. , and J.M. Barnes. "Carcinogenic Nitroso Compounds.M 

Advanc. Cancer Res., 10 (1967), 163-246. 

20. Magee, P.N. , R. Montesano, and R. Preussmann. "N-nitroso 

Compounds and Related Carcinogens." In: Chemical Carcinogens, C.E. Searle (Ed.), American Chemical Society, New York (1976), 

491-625, 

169 
21.

 Magee, P.N., and R. Schoental. "Carcinogenesis by Nitroso 

Compounds." Brit. Med. Bull., 20 (1964), 102-106. 

22. Magee, P.N., and P.P. Swann. "Nitroso Compounds." Brit. Med. Bull., 25 (1969), 240-2*11*. 

23. Mailing, H. V. "Dimethylnitrosamine: Formation of Mutagenic 

Compounds by Interaction with Mouse Liver Microsomes." Mutation 

Res., 13 (1971), 425-429. 

24. Mailing, H.V. "Mutagenic Activation of Dimethylnitrosamine and 

Diethylnitrosamine in the Host-mediated Assay and the Microsomal 

System." Mutation Res., 26 (1974), 465-472. 

25. Mayer, V.W. "Mutagenicity of Dimethylnitrosamine and 

Diethylnitrosamine for Saccharomyces in an in vitro Hydroxylation 

System." Molec. Gen. Genetics, 112 (1971), 289-294. 

26. Mayer, V.W. "Induction of Mitotic Crossing Over in Saccharomyces 

cerevisiae by Breakdown Products of Dimethylnitrosamine 

1-naphthylamine and 2-naphthylamine Formed by an in vitro 

Hydroxylation System." Genetics, 74 (1973), 433-442. 

27. Montesano, R. , A.J. Ingram, and P.N. Magee. "Metabolism of 

Dimethylnitrosamine (DMN) by Amphibians and Fish in vitro." 

Experientia, 29 (1973), 599-601. 

28. Montesano, R. , and P.N. Magee. "Comparative Metabolism in vitro 

of Nitrosamines in Various Animal Species Including Man." In: 

Chemical Carcinogenesis Essays, R. Montesano, and L. Tomatis 

(Eds), International Agency for Research on Cancer, Lyon, 1974, 39-56. 

29- Nakajima, D., A. Tanaka, and K. Tojyo. "The Effect of Metabolic 

Activation with Rat Liver Preparations on the Mutagenicity of 

Several Nitrosamines on a Streptomycin-dependent Strain of 

Escherichia coli." Mutation Res., 26 (1974), 261-366. 

30. Okada, M., and E. Suzuki. "Metabolism of Butyl (4-hydroxybutyl) 

Nitrosamine in Rats." Gann, 63 (1972), 391-392. 

31. Okada, M. , E. Suzuki, J. Aoki, M. liyoshi, and Y. Hashimoto. "Metabolism and Carcinogenicity of N-butyl-N-(4-hydroxybutyl) 

nitrosamine and Related Compounds, with Special Reference to 

Induction of Urinary Bladder Tumors." Gann Monograph on Cancer 

Research, 17 (1975), 161-176. 

170 
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 Popper, H., P. Czygan, H. Greim, F. Schaffner, and A.J. Garro. 

"Mutagenicity of Primary and Secondary Carcinogens Altered by 

Normal and Induced Hepatic Microsomes." Proc. Soc. Exp. Biol. 

Med., 142 (1973), 727-729. 

33. Rayman, M.P., B.C. Challis, P.J. Cox, and M. Jarman. "Oxidation 

of N-nitrosopiperidine in the Udenfriend Model System and Its 

Metabolism by Rat-liver Microsomes." Biochem. Pharmacol., 24 

(1975), 621-626. 

34. Schulz, U. , and D.R. McCalla. "Reactions of Cysteine with 

N-methyl-N-nitroso-p-toluenesulfonamide and N-methyl-N'-nitro-N­ nitrosoguaiiidine." Can J. Chem., 47 (1969), 2021-2027. 

35. Wheeler, G.P., and B.J. Bowdon. "Comparison of the Effects of 

Cysteine Upon the Decomposition of Nitrosoureas and 

l-methyl-3-nitro-l-nitrosoguanidine." Biochem. Pharmacol., 21 

(1972), 265-267. 

DIALKYLHYDRAZINES, ALKYL AZO- AND AZOXY COMPOUNDS, DIALKYLARYLTRIAZENES 

1. Dost, F.W. , D.J. Reed, and C.M. Wang. "The Metabolic Fate of 

Monomethylhydrazine and Unsymmetrical Dimethylhydrazine." 

Biochem. Pharmacol., 15 (1966), 1325-1332. 

2. Druckrey, H. "Production of Colonic Carcinomas by 

1,2-dialkylhydrazines and Azoxyalkanes." In: Carcinoma of the 

Colon and Antecedent Epithelium, W. J. Burdette (Ed.), Charles 

C. Thomas, Springfield, 1970, 267-279. 

3. Preussmann, R., H. Druckrey, S. Ivankovic, and A.V. Hodenberg. "Chemical Structure and Carcinogenicity of Aliphatic Hydrazo, Azo 

and Azoxy Compounds and of Triazenes, Potential in vivo 

Alkylating Agents." Ann. N.Y. Acad. Sci., 163 (1969), 697-716. 

4. Preussmann, R. and A. vonHodenberg. "Mechanism of Carcinogenesis 

With l-aryl-3,3-dialkyltriazenes. II. In vitro Alkylation of 

Guanosine RNA and DNA With Aryl-monoalkyltriazenes to Form 

7-alkylguanine." Biochem. Pharmacol., 19 (1970), 1505-1508. 

5. Preussmann, R. , A. vonHodenberg, and H. Hengy. "Mechanism of 

Carcinogenesis with l-aryl-3,3-dialkyltriazenes. Enzymatic 

Dealkylation by Rat Liver Microsomal Fraction in vitro." 

Biochem. Pharmacol., 18 (1969), 1-13­

6. Prough, R.A. , J.A. Wittkop, and D.J. Reed. "Evidence for the 

Hepatic Metabolism of Some Monoalkylhydrazines." Arch. Biochem. 

Biophys., 131 (1969), 369-373. 

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 Prough, R.A. , J.A. Wittkop, and D.J. Reed. "Further Evidence on 

the Nature of Microsomal Metabolism of Procarbazine and Related 

Alkylhydrazines." Arch. Biochem., 140 (1970), 450-458. 

8. Reddy, B.S., T. Narisawa, P. Wright, D. Vukusich, J.H. 

Weisburger, and E.L. Wynder. "Colon Carcinogenesis with 

Azoxymethane and Dimethylhydrazine in Germ-free Rats." Cancer 

Res., 35 (1975), 287-290. 

9. Weisburger, J.H. "Colon Carcinogens: Their Metabolism and Mode 

of Action." Cancer, 28 (1971), 60-70. 

10. Wittkop, J.A., R.A. Prough, and D.J. Reed. "Oxidative 

Demethylation of N-methylhydrazines by Rat Liver Microsomes." 

Arch. Biochem. Biophys., 134 (1969), 308-315. 

CYCASIN (METHYLAZOXYMETHANOL GLUCOSIDE), METHYLAZOXYMETHANOL ACETATE 

(MAM ACETATE) 

1. Kobayashi, A., and H. Matsumoto. "Studies on Methylazoxymethanol, the Aglycone of Cycasin. Isolation, Biological 

and Chemical Properties." Arch. Biochem., 110 (1965), 373-380. 

2. Laqueur, G.L. "Carcinogenic Effects of Cycad Meal and Cycasin, 

Methylozaxymethanol Glycoside in Rats and Effects of Cycasin in 

Germ Free Rats." Fed. Proc., 23 (1964), 1386-1387. 

3. Laqueur, G.L. "Contribution of Intestinal Macroflora and 

Microflora to Carcinogenesis." In: Carcinoma of the Colon and 

Antecedent Epithelium, W. J. Burdette (Ed.), Charles C. Thomas, 

Springfield, 1970, 305-313­

4. Laqueur, G.L., E.G. McDaniel, and H. Matsumoto. "Tumor Induction 

in Germ Free Rats with Methylazoxymethanol (MAM) and Synthetic 

MAM Acetate." J. Natl. Cancer Inst., 39 (1967), 355-371. 

5. Laqueur, G.L. , 0. Mickelsen, M.G. Whiting, and L.T. Kurland. "Carcinogenic Properties of Nuts from Cycas Circinalis L 

Indigenous to Guam." J. Natl. Cancer Inst., 31 (1963), 919-951. 

6. Laqueur, G.L., and M. Spatz. "Toxicology of Cycasin." Cancer 

Res., 28 (1968), 2262-2267. 

7. Matsumoto, H., and H.H. Higa. "Studies on Methylazoxymethanol, the Aglycone of Cycasin: Methylation of Nucleic Acids in 

vitro." Biochem. J., 98 (1966), 20c-22c. 

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8.

 Matsumoto, H., Y. Nagata, E.T. Nishimura, R. Bristol, and M. 

Haber. "B-Glucosidase Modulation in Preweaning Rats and Its 

Association with Timor Induction by Cycasin." J. Natl. Cancer 

Inst., 49 (1972), 423-434. 

9. Matsumoto, H., and P.M. Strong. "The Occurrence of 

Methylazoxymethanol in Cycas Circinalis L." Arch. Biochem., 101 

(1963), 299-310. 

10. Miller, J.A. "Comments on Chemistry of Cycads." Fed. Proc., 23 

(1964), 1361-1362. 

11. Nagata, Y. , and H. Matsumoto. "Studies on Methylazoxymethanol: 

Methylation of Nucleic Acids in the Fetal Rat Brain." Proc. Soc. Exp. Biol. Med., 132 (1969), 383-385. 

12. Narisawa, T., and H. Nakano. "Carcinoma of the Large Intestine 

of Rats Induced by Rectal Infusion of Methylazoxymethanol." 

Gann, 64 (1973), 93-95. 

13. Riggs, N.V. "Decomposition and Carcinogenic Activity of 

Azoxyglycosides." Nature, London, 207 (1965), 632. 

14. Shinohara, K., and H. Matsudaira. "Evidence for Caffeine- sensitive Damage in Methylazoxymethanol Acetate-treated L5178Y 

cells." Chem.-Biol. Interactions, 12 (1976), 53-63. 

15. Smith, D.W.E. "Mutagenicity of Cycasin Aglycone 

(methylazoxymethanol), a Naturally Occurring Carcinogen." 

Science, 152 (1966), 1273-1274. 

16. Spatz, M. "Toxic and Carcinogenic Alkylating Agents from 

Cycads." Ann. N.Y. Acad. Sci., 163 (1969), 848-859­

17. Spatz, M., and G.L. Laqueur. "Transplacental Induction of Tumors 

in Sprague-Dawley Rats with Crude Cycad Material." J. Nat. Cancer Inst., 38 (1967), 233-245. 

18. Spatz, M. , E.G. McDaniel, and G.L. Laqueur. "Cycasin Excretion 

in Conventional and Germ Free Rats." Proc. Soc. Exptl. Biol. Med., 121 (1966), 417-422. 

19. Spatz, M. , D.W.E. Smith, E.G. McDaniel, and G.L. Laqueur. "Role 

of Intestinal Microorganisms in Determining Cycasin Toxicity." 

Proc. Soc. Exptl. Biol. Med., 124 (1967), 691-697. 

20. Teas, H.J., and J.G. Dyson. "Mutation in Drosophilia by 

Methylazoxymethanol, the Aglycone of Cycasin." Proc. Soo. Exp. 

Biol. Med., 125 (1967), 988-990. 

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Cycad Research." Fed. Proc., 31 (1972), 1543-1546. 

22. Zedeck, M.S., S.S. Sternberg, J. McGowan, and R.W. Poynter. 

"Methylazoxymethanol Acetate: Induction of Tumors and Early 

Effects on RNA Synthesis." Fed. Proc., 31 (1972), 1485-1492. 

23. Zedeck, M.S., S.S. Sternberg, R.W. Poynter, and J, McGowan. 

"Biochemical and Pathological Effects on Methylazoxymethanol 

Acetate, A Potent Carcinogen." Cancer Res., 30 (1970), 801-812. 

ALKYLATING AGENTS 

1. Abell, C.W., H.L. Falk, M.B. Shimkin, E.K. Weisburger, J.H. 

Weisburger, and N. Gubareff. "Uracil Mustard: A Potent Inducer 

of Lung Tumors in Mice." Science, 147 (1965), 1443-1444. 

2. Brookes, P., and P.D. Lawley. "Alkylating Agents." Brit. Med. Bull., 20 (1964), 91-95.

 

3. Clapp, N.K. "Carcinogenic! ty of Nitrosarnines and 

Methanesulfonate Esters Given Intraperitoneally in RF Mice." 

Internat. J. Cancer, 12 (1973), 728-733­

4. Clapp, N.K. , A.W. Craig, and R.E. Toya. "Oncogenicity by Methyl 

Methanesulfonate in Male RF Mice." Science, 161 (1968), 913-914. 

5. Colburn, N.H. , and R.K. Boutwell. "The Binding of 

3-propiolactone and Some Related Alkylating Agents to DNA, RNA 

and Protein of Mouse Skin; Relation Between Tumor-initiating 

Power of Alkylating Agents and Their Binding to DNA." Cancer 

Res., 28 (1968), 653-660. 

6. Dickens, F. "Carcinogenic Lactones and Related Substances." 

Brit. Med. Bull., 20 (1964), 96-101. 

7. Hrushesky, W., D. Sampson, and G.P. Murphy. "Carcinogenicity of 

Ethylmethanesulfonate." J. Nat. Cancer Inst., 49 (1972), 

1077-1083. 

8. Jones, A.R. "The Metabolism of Biological Alkylating Agents." 

In: Drug Metabolism Reviews, F. J. DiCarlo (Ed.), Marcel Dekker, New York, 1974, Vol. 2, 71-100.

 

9. Laskin, S., M. Kuschner, R.T. Drew, P. Cappiello, and N. Nelson. "Tumors of the Respiratory Tract Induced by Inhalation of

 

Bis(chloromethyl) Ether." Arch. Environ. Hlth., 23 (1971), 

135-136. 

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Butterworths, London, 1966, 270 pp.
 

11.	 Preussraann, R. "Direct Alkylating Agents as Carcinogens." Fd.
 

Cosmet. Toxicol., 6 (1968), 576-577.
 

12.	 Price, C.C., G.M. Gaucher, P. Koneru, R. Shibakawa, J.R. Sowa,
 and M. Yamaguchi. "Mechanism of Action of Alkylating Agents."
 

Ann. N.Y. Acad. Sci., 163 (1969), 593-600.
 

1 3- Ross, W.C.J. Biological Alkylating Agents. Butterworths,
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14.	 Van Duuren, B.L. (Ed.) "Biological Effects of Alkylating
 

Agents." Ann. N.Y. Acad. Sci., 163 (1969), 589-1029.
 

15.	 Van Duuren, B.L., B.M. Goldschmidt, C. Katz, L. Langseth, G.
 

Mercado, and A. Sivak. "Alpha Haloethers: A New Type of
 

Alkylating Carcinogen." Arch. Environ. Health, 10 (1968),
 

472-476.
 

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Oncogenic Transformation Induced by N-methyl-N'-nitro-N­ nitrosoguanidine in Culture." Cancer Res., 34 (1974), 526-537.
 

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Adult Liver Epithelial Cells into Differentiated Hepatoma-like
 

Cells Under Conditions of Nutritional Stress." Proc. Nat. Acad.
 

Sci., USA, 69 (1972), 965-969.
 

3.	 Borenfreund, E., P.J. Higgins, M. Steinglass, and A. Bendich.
 

"Properties and Malignant Transformation of Established Rat Liver
 

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Cells from the Kidneys of Rats Treated with a 'single'
 

Carcinogenic Dose of Dimethylnitrosamine (DMN) Detected by
 

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175
 
5.

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Res., 33 (1973), 2627-2636. 

6. DiMayorca, G., M. Greenblatt, T. Trauthen, A. Seller, and R. Giordano. "Malignant Transformation of BHK 21 Clone 13 Cells in 

vitro by Nitrosamines. Conditional State." Proc. Nat. Acad. Sci., USA, 70 (1973), 46-49. 

7. DiPaolo, J.A., P. Donovan, and R. Nelson. "Quantitative Studies 

of in vitro Transformation by Chemical Carcinogens." J. Nat. Cancer Inst., 42 (1969), 867-876. 

8. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "Transformation of 

Hamster Cells in vitro by Polycyclic Hydrocarbons Without 

Cytotoxicity." Proc. Nat. Acad. Sci.. USA, 68 (1971), 2958-2961. 

9. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "In vitro 

transformation of Hamster Cells by Polycyclic Hydrocarbons: 

Factors Influencing the Number of Cells Transformed." Nature New 

Biol., 230 (1971), 240-242. 

10. DiPaolo, J.A., R.L. Nelson, and P.J. Donovan. "In vitro 

Transformation of Syrian Hamster Embryo Cells by Diverse Chemical 

Carcinogens." Nature (London), 235 (1972), 278-280. 

11. DiPaolo, J.A., R.L. Nelson, and P.J. Donovan. "Host-mediated in 

vivo - in vitro Assay for Chemical Carcinogenesis." Arch. Pathol., 95 (1973), 380-385. 

12. DiPaolo, J.A., K. Takano, and N.C. Popescu. "Quantitation of 

Chemically Induced Neoplastic Transformation of BALB/3T3 Cloned 

Cell Lines." Cancer Res., 32 (1972), 2686-2695. 

13. Freeman, A.E. , P.J. Price, H.J. Igel, J.C. Young, J.M. Maryak, and R.J. Huebner. "Morphological Transformation of Rat Embryo 

Cells Induced by .Diethylnitrosamine and Murine Leukemia 

Viruses." J. Nat. Cancer Inst., 44 (1970), 65-78. 

14. Freeman, A.E. , E.K. Weisburger, J.H. Weisburger, R.G. Wolford, J.M. Maryak, and R.J. Huebner. "Transformation of Cell Cultures 

as an Indication of the Carcinogenic Potential of Chemicals." J^ 

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15. Frei, J.V., and J. Oliver. "Influence of Methylnitrosourea on 

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176 
16.

 Frei, J.V., and J. Oliver. "Early Enhancement of Plating 

Efficiency of Primary Mouse Embryo Cells by the Carcinogen 

Methylnitrosourea." Cancer Res., 32 (1972), 2747-2752. 

17. Hard, G.C. , R. Borland, and W.H . Butler. "Altered Morphology and 

Behaviour of Kidney Fibroblasts in vitro Following in vivo Treat­ ment of Rats with a Carcinogenic Dose of Dimethylnitrosamine." 

Experientia, 27 (1971), 1208-1209. 

18. Hashimoto, Y. , and H.S. Kitagawa. "In vitro Neoplastic 

Transformation of Epithelial Cells of Rat Urinary Bladder by 

Nitrosamines." Nature (London), 252 (1974), 497-499. 

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Carcinogenic Hydrocarbon Activation." In: Topics in Chemical 

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20. Heidelberger, C. "Chemical Oncogenesis in Culture." Advanc. Cancer Res., 18 (1973), 317-366. 

21. Henderson, E.E. , A.J. Norin, and B.S. Strauss. "Induction of 

Permanently Proliferating Human Lymphoblastoid Lines by 

N-methyl-N'-nitro-N1-nitroso-quanidine." Cancer Res., 35 (1975), 358-363. 

22. Huberman, E., P.J. Donovan, and J.A. DiPaolo. "Mutation and 

Transformation of Cultured Mammalian Cells by 

N-acetoxy-N,2-fluorenylacetamide." J. Nat. Cancer Inst., 48 

(1972), 837-840. 

23. Huberman, E., T. Kuroki, H. Marquardt, J.K. Selkirk, C. Heidelberger, P.L. Grover, and P. Sims. "Transformation of 

Hamster Embryo Cells by Epoxides and Other Derivatives of 

Polycyclic Hydrocarbons." Cancer Res., 32 (1972), 1391-1396. 

24. Huberman, E., S. Salzberg, and L. Sachs. "The in vitro Induction 

of an Increase in Cell Multiplication and Cellular Life Span by 

the Water-soluble Carcinogen Dimethylnitrosamine." Proc. Nat. Acad. Sci. USA, 59 (1968), 77-82. 

25. Huberman, E., M. Traut, and L. Sachs. "Cell Susceptibility to 

the Cytotoxic Effect of the Carcinogens Dimethylnitrosamine and 

N-nitrosomethylurea." J. Nat. Cancer Inst., 44 (1970), 395-402. 

26. Kuroki, T., J. Ishizawa, and H. Sato. "Studies on Early Events 

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177 
27.

 Laerum, O.D. , and M.F. Rajewsky. "Neoplastic Transformation of 

Fetal Rat Brain Cells in Culture After Exposure to 

Ethylnitrosourea in vivo." J. Nat. Cancer Inst., 55 (1975), 1177-1187. 

28. Marquardt, H., and C. Heidelberger. "Influence of 'feeder cells' 

and Inducers and Inhibitors of Microsomal Mixed-function Oxidases 

on Hydrocarbon-induced Malignant Transformation of Cells Derived 

from C3H Mouse Prostate." Cancer Res., 32 (1972), 721-725. 

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With N-nitrosomethylurea." Nature (London), 220 (1968), W8-453. 

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178 
4.2	 CERTAIN ASPECTS OF CHEMICAL CARCINOGENESIS
 

PAUL O.P. TS'O, J. CARL BARRETT, STEPHEN LESKO, AND ROBERT MOYZIS
 

Division of Biophysics, Johns Hopkins University, School of
 

Hygiene and Public Health, Baltimore, MD
 

I.	 THE POSSIBLE INVOLVEMENT OF FREE RADICALS IN BENZO(a)PYRENE
 

CARCINOGENESIS
 

INTRODUCTION
 

A substantial amount of evidence has been accumulated in support
 

of the notion that metabolic activation is often a prerequisite for
 

the action of chemical carcinogens, particularly the polycyclic
 

hydrocarbons. One hypothesis postulates that metabolic activation of
 

carcinogens produces electrophilic intermediates which react with
 

nucleophilic sites of vital macromolecules in cells, thereby
 

initiating neoplastic transformation. This notion has often been
 

interpreted to mean that the crucial, causative, chemical event is a
 

two-electron reaction resulting in a stable, covalent bond between the
 

carcinogen and the suspected target molecule, DNA.
 

There has been other evidence, however, which indicates that
 

one-electron, or free radical, mechanisms may also be related to the
 

action of chemical carcinogens (27) as well as perhaps being
 

responsible for radiation damage to tissue. Free radicals as a group
 

are generally considered to be electrophilic reagents. They are
 

capable of altering important macromolecules in cells, not only by
 

covalently binding with the macromolecules, but also by cleavage of
 

chemical bonds leading to their degradation. The propagation
 

reactions between free radicals, produced either by carcinogens or
 

radiation, and cellular macromolecules are complex. One principal
 

mode, which has been documented for radiation-induced DNA damage (7)
 

is hydrogen atom abstraction. This process produces a reactive free
 

radical site in the macromolecule which can undergo many further
 

reactions leading eventually to degradation or strand scission. In
 

this section, the radical species produced by metabolism of
 

benzo(a)pyrene and the reactive reduced oxygen species produced in
 

one-electron autoxidations will be described. These molecules can 

cause demonstrable breakage in nucleic acids and inflict cytotoxic 

effects on treated cells. 

ENZYMIC FORMATION OF THE 6-OXO B(a)P RADICAL
 

Benzo(a)pyrene is metabolized by microsomal mixed function
 

oxidases to a number of products. The sixth position of B(a)P is
 

chemically more reactive than the other sites on the hydrocarbon (6),
 

179
 
but 6-OH-B(a)P often is not reported as a metabolite although its 

formation has been implied by the detection of the 6-OH-B(a)P 

glucuronide in vivo (9). Negata et al. (21) demonstrated that 

6-OH-B(a)P can be detected in the metabolism of B(a)P in rat liver 

homogenate, as well as in mouse and rat skin homogenates, via its 

phenoxy radical, 6-oxo-B(a)P. This observation has been confirmed and 

extended by our laboratory (16). 

Upon incubation of B(a)P in uninduced rat liver homogenates 

fortified with a NADPH-generating system at 37°, a metabolite was 

formed which gave rise spontaneously to an EPR signal. The metabolite 

and the radical were extracted quantitatively into benezene in which 

they were relatively stable. The metabolite also could be 

quantitatively converted to the radical by shaking with aqueous 

solutions of 2,6-dichloroindophenol or K_Fe(CN)g. The EPR signal 

obtained with B(a)P was identical to that observed after incubating 

6-OH-B(a)P in fortified rat liver homogenates and has been identified 

as the 6-oxo-B(a)P radical by its characteristic hyperfine structure 

(18,11). No EPR signal was seen when B(a)P or cofactors were 

eliminated from the incubation mixture or when the homogenate was 

heated at 65° for 10 minutes. These observations indicate that the 

oxidation of B(a)P to 6-Cfl-B(a)P was enzymic in nature. 

The decay of added 6-OH-B(a)P in rat liver homogenates showed 

first order kinetics with a rate constant of 0.29 minutes . The 

half-life of added 6-OH-B(a)P in this system, thus, was about 2.4 

minutes. The rate of degradation of 6-OH-B(a)P was not decreased by 

either elimination of the NADPH-generating system or by heating the 

homogenate at 65° for 10 minutes, indicating that this reaction was 

nonenzymic in nature. 

The products formed by incubating 6-OH-B(a)P in rat liver 

homogenates were extracted into benzene and isolated by aluminum oxide 

chromatography (18). The data show that 6-OH-B(a)P was oxidized in 

this system and the following were the only products detected after 40 

minutes of incubation: 6,12-B(a)P dione (15$), l,6-B(a)P dione (41$) 

and 3,6-B(a)P dione (44$). 

We have estimated the percentage of B(a)P metabolism proceeding 

through the 6-OH-B(a)P pathway by comparing the initial rate of 

6-OH-B(a)P formation (measured via the 6-oxo-B(a)P radical) with the 

initial rate of total B(a)P metabolism. The comparison indicated the 

percentage of metabolism proceeding through 6-OH-B(a)P was about 18$ 

and 20$ for the liver homogenates of female Sprague-Dawley and female 

ACI rats respectively. The reaction rates were measured at relatively 

high substrate concentration to ensure zero-order kinetics. This 

value of about 20$ of B(a)P metabolism proceeding through the 

180 
6-OH-B(a)P pathway agrees reasonably well with the proportion of B(a)P 

diones produced from B(a)P metabolism (1*0. Apparently, the bulk, if 

not all, of B(a)P diones formed during B(a)P metabolism have 

6-OH-B(a)P as a precursor. 

AUTOXIDATION OF 6-OH-B(a)P 

We have synthesized 6-OH-B(a)P from B(a) P using modifications of 

the procedure of Fieser and Hersberg (1939). It is a labile 

compound. In aqueous buff er-ethanol (1:1) solutions, synthetic 

6-OH-B(a)P is autoxidized to a mixture of three stable B(a)P diones, 

6,12-; 1,6-; and 3,6-, plus a small amount of an unidentified 

paramagnetic, violet-colored material (18). In this reaction 

molecular oxygen is consumed. Some of this oxygen is reduced by 

electron transfer which is evident by the apearance of H O during the 

autoxidation as determined by enzyme assay. H O is the most stable 

reduced oxygen species (besides HO ) and the easiest to determine. 

But the paramagnetic nature of ground state molecular oxygen virtually 

ensures that autoxidations proceed by one-electron steps, a notion 

supported in this specific case by the formation of 6-oxo-B(a)P 

radical in the autoxidation process. Therefore, it is virtually 

certain that other very reactive reduced oxygen species, such as the 

superoxide and hydroxyl radicals, are formed transiently in this 

autoxidation as well. The free radical derived from 6-OH-B(a)P by 

one-electron oxidation, the 6-oxo-B(a)P radical, is an obligatory 

intermediate in the autoxidation as indicated by its kinetics for 

formation and decay (18). 

The 6-oxo-B(a)P radical could be produced quantitatively in 

benzene by oxidation with aqueous K^FetCN^ and isolated in the 

absence of oxygen. Like 6-OH-B(a)P, this isolated free radical was 

autoxidized in aqueous buffer-ethanol solutions and yielded identical 

products. In addition to this data, recent experiments reveal the 

incorporation of ^80 into B(a)P dione products from 6-OH-B(a)P 

autoxidation with 02. All of this suggests a reaction mechanism for 

autoxidation of 6-OH-B(a)P in which molecular oxygen couples directly 

with the 6-oxo-B(a)P radical at the positions of high spin density 

(11), producing ketoperoxyl radicals. Several subsequent pathways 

from the ketoperoxyl radicals to B(a)P diones are conceivable. The 

most likely would involve the formation of hydroperoxides or 

disubstituted peroxides, followed by the decomposition of these 

peroxides to produce B(a)P diones. 

ACTION OF B(a)P DIONES OF DNA AND CELLS IN CULTURE 

As noted earlier, the products of 6-OH-B(a)P autoxidation are the 

6,12-; 1,6- and 3,6- B(a)P diones. These quinones are routinely 

identified products of B(a)P metabolism in various tissues and 

181 
generally account for about 20$ or more of observed products. 

Although easily isolated, they are not totally unreactive products of 

B(a)P metabolism. Incubation of B(a)P diones with T? DNA produces DNA 

strand breakage as measured by alkaline sedimentation. This ability 

to damage DNA is inhibited by removing oxygen and by the presence of 

either superoxide dismutase or EDTA. On the other hand, it is 

increased by the presence of a cellular reducing agent, NADPH, or a 

transition metal ion, Cu++. The observations suggest that the DNA 

strand scission is caused by reactive, reduced oxygen species produced 

in an one-electron redox reaction facilitated by B(a)P diones. 

The B(a)P diones are quite toxic to cells in culture. This is 

indicated by the inhibition of uptake of {3

H} thymidine into the DNA 

of Syrian hamster embryonic (SHE) fibroblasts in the presence of B(a)P 

diones. This inhibition is greater by 1,6 and 3,6 B(a)P diones than 

6,12 B(a)P dione. Cloning efficiency of SHE cells is also reduced by 

B(a)P diones; the most cytotoxic is 1,6 B(a)P dione, but all three 

diones are considerably more cytotoxic than B(a)P. 

PROPERTIES OF THE B(a)P DIONE/B(a)P HYDROQUINONE REDOX COUPLES (19) 

Quinones in general, including B(a)P diones, can undergo a wide 

variety of chemical reactions, but are best characterized by their 

ability to participate in reversible, one-electron, redox reactions 

involving the hydroquinone and semiquinone radical forms. B(a)P 

diones can be easily reduced to semi-quinones and hydroquinones as 

monitored by ESR and absorption spectroscopy. We have observed that 

even such cellular reducing agents as NADH, NADPH, cysteamine, and 

glutathione can reduce, wholly or partially, B(a)P quinones to 

hydroquinones in the absence of oxygen as shown below: 

Reducing agents: NaBH^, Na^O^ (dithionite), NAD(P)H, 

cysteamine, gluthathione 

This reduction of the B(a)P diones is readily reversible. Thus, B(a)P 

hydroquinones prepared from diones under anaerobic conditions are 

autoxidized very rapidly back to B(a)P diones by air in aqueousalcohol solutions. This cycle of quinone+hydroquinone-Kjuinone can be 

repeated many times as shown above. As a result of each autoxidation 

of B(a)P hydroquinone to B(a)P dicne, hydrogen peroxide is produced in 

near stoichionetrie amounts. In parallel with other autoxidations, 

superoxide and hydroxyl radicals are likely to be produced as well. 

The semiquinone radicals are reaction intermediates in this cycle. 

182 
There is considerable evidence in the literature to suggest that 

quinones of widely varying structure may act as electron acceptor 

substrates for many respiratory enzymes in cells (20). In the case of 

B(a)P diones, this enzyme reduction to B(a)P hydroquinones would be 

immediately followed by autoxidation to produce the dione and reduced, 

reactive oxygen species (F^C^, $2* -OH) . Using a commercially 

available diaphorase {NADH dehydrogenase from Cl. Kluyveri (1.6.99-)} 

we have demonstrated the operation of this cyclic process. 

THE METABOLISM OF BENZO(a) PYRENE BY PURIFIED RAT LIVER NUCLEI AND SYRIAN 

HAMSTER EMBRYO HOMOGENATES 

In view of the possible significance of metabolic activation of 

benzo(a)pyrene within nuclei to the problem of chemical carcinogenesis 

and the extensive use of Syrian hamster embryos as the experimental 

system for in vitro carcinogenesis, we have studied the biochemistry 

of B(a)P metabolism in purified rat liver nuclei and nuclear membranes 

as well as the homogenates of the Syrian hamster embryos. The main 

approach adopted in this study is to investigate whether the nuclei 

preparations and the microsomal preparations have independent and 

different metabolic pathways for B(a)P. The ideal situation would be 

to investigate the metabolic profiles of these two preparations and to 

compare them. 

Various reports have appeared in the literature which indicate 

the activation of B(a)P by nuclei preparations. However, it was 

difficult to rule out the possibility that nuclear activity resulted 

from contamination by microsomes (1,13,22,24,30,31). In fact, in a 

recent article (1) it was claimed that BP activation by these two 

fractions is indeed very similar. In our approach, in the first step 

of investigating this problem, i.e., to establish the possibility of 

differences between nuclear AHH versus microsomal AHH, we emphasize 

the metabolism at the sixth carbon of B(a)P versus other carbon 

positions of this hydrocarbon. This approach can be supported by two 

types of experimental systems: 

(1) By measurement of_tritium released from generally labelled 

and selectively labelled 6- H-B(a)P in the presence or absence of a 

modulator of AHH activity. 

(2) By electron paramagnetic resonance measurements of the 

radicals generated from phenolic metabolites of B(a)P after chemical 

oxidations by a series of selection one-electron oxidants. The ratio 

of the amount of radicals produced by these different oxidants 

corresponds to the proportions of phenols in the metabolic products. 

The nuclear preparation was first made (3,28) and then the 

nuclear membrane preparation was made from the purified nuclei. The 

183 
removal of DNA and other nuclei contents makes the preparation of the 

nuclear membrane much more manageable and far less contaminated (2). The data from the tritium release studies clearly indicate that the 

metabolic pathway at the 6 position is twice as extensive in the 

nuclear membrane preparation as opposed to that of the microsomal 

preparation. In addition, the inhibitory effect of 7,8-benzoflavone 

is very large in the microsomal preparation but is minimal in the 

nuclear membrane preparation. Preliminary results based on esr 

measurements of the 6-oxo radicals also substantiate this finding that 

there exists a higher percentage of metabolism at the 6 position of 

B(a)P, compared to all of the other positions of B(a)P, in nuclear 

membrane preparations than in microsomes. 

Research has indicated the appearance of the 6-oxo B(a)P radical 

upon the metabolism of B(a)P in Syrian hamster embryo homogenates. In 

addition, the early results suggest that the metabolic pathway at the 

6 position as compared to all other positions of B(a)P is two-three 

times higher in the .embryo preparations than in the liver 

preparations. This result implies that the metabolism at the 6 

position is a predominant pathway in the embryo preparation. 

Thus, research results on the metabolism of B(a)P in the nuclear 

membrane preparation and the Syrian hamster embryo homogenates are in 

accord with each other: nuclear preparations could have a pathway 

which is different and independent from that of the microsomes; 

biological materials which are rich in microsomal activities such as 

liver would therefore be expected to have a different profile of 

metabolism of B(a)P from that of biological material enriched in 

nuclei such as embryonic tissue. The nuclei preparation and the 

homogenates from biological material enriched with nuclei all show 

that the metabolic pathway at the 6 position of B(a)P could be a 

predominant pathway. This finding could have important implications 

for carcinogenesis research. 

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25.	 Schechtman, L., S. Lesko, R. Lorentzen, and P. Ts'o. "A Cellular
 

System for the Study of the Chemical Reactivity and Transforming
 

Ability of Benzo(a)Pyrene and its Derivatives." Proc. Am. Assoc.
 

Cancer Res., 15 (197*0, 66.
 

186
 
26.

 Shamberger, R., F. Baughman, S. Kalchert, et al. 

"Carcinogen-Induced Chromosomal Breakage Decreased by 

Antioxidants." Proc. Natl. Acad. Sol. USA, 70 (1973), 1461-1463. 

27. Slater, T.F. Free Radical Mechanisms in Tissue Injury. Pion 

Limited, London, 1972. P. Ts'o, J. C. Barrett, and W. J. 

Caspary, et al. "Involvement of Radicals in Carcinogenesis." 

In: Aging, Carcinogenesis, and Radiation Biology. K. Smith 

(Ed.), Plenum Press, New York, (1976), 373. 

28. Spelsbert, T., J. Knowler, and H. Moses. "Specific Methods for 

the Isolation of Nuclei from Chick Oviduct." Methods in 

Enzymology, 31 (1974), 263-279. 

29. Szent-Gyorgyi, A. , I. Isenberg, and S. Baird. "On the Electron 

Donating Properties of Carcinogens." Proc. Natl. Acad. Sci. USA, 

46 (1960), 1444-1449. 

30. Vaught, J., and E. Bresnick. "Binding of Polycyclic Hydrocarbons 

to Nuclear Components In Vitro." Biochem. Biophys. Res. Commun., 

69 (1976), 587-591. 

31. Watanabe, M., F. Ariji, K. Takusagawa, and K. Konno. "Aryl 

Hydrocarbon Hydroxylase in Liver Nuclei of C3H/He and DBA/2 

Mice." Gann, 66 (1975), 399-409. 

32. Wattenberg, L. "Inhibition of Chemical Carcinogen-Induced 

Pulmonary Neoplasia by Butylated Hydroxyanisole." J. Natl. 

Cancer Inst., 50 (1973), 1541-1544. 

II. THE RELATIONSHIP BETWEEN NEOPLASTIC TRANSFORMATION AND SOMATIC MUTATION 

To study the role of somatic mutation in Carcinogenesis, a system 

of normal, diploid Syrian hamster embryo (SHE) cells has been 

developed in which both processes can be investigated concomitantly. 

For somatic mutation, 8-azaguanine resistance (8AGr), 6-thioguanine 

(6TGr) (1,2,8,9,11,19,21,24,29,31-36,39-41) and ouabain resistance 

(ouar) (3,10,20,25,26) markers have been characterized quantitatively 

with SHE cells and a subdiploid transformed cell line (BP6T) derived 

from SHE cells. The mutation frequencies of 8AGr, 6TGr and ouar of 

the normal SHE and BP6T cells are within a 4-10 fold range. Isolated 

mutant clones of both cells are stable and have similar cloning 

efficiencies in normal and appropriate selective media. Isolated 8AGr 

and 6TGr clones have very low levels of HGPRT activity (33) and low 

reversion frequencies as measured by their growth in HAT medium 

(35,36). The uptake of potassium or rubidium by the Na+/K+ ATPase 

187 
system of the cells is inhibited by ouabain (20,26). The uptake of 

°°Rb by oua clones is less inhibited by ouabain than wild type cells, a finding consistent with the notion that the ouabain resistance trait 

is due to a mutation in the Na+/K+ ATPase enzyme. Transfonnation of 

SHE cells (6,7,12-18,22) has been quantitated by a morphological assay 

and isolated transformed clones are tumorigenic. The concomitant 

investigation of morphological transformation and somatic mutation of 

SHE cells revealed that the morphological transformation frequency is 

25-540 fold greater than the mutation frequency. 

It has been postulated by Foulds (18) and others that oncogenesis 

in vivo characteristically occurs as a progressive event, exhibiting 

numerous qualitatively different stages. It is our aim to determine 

whether neoplastic transformation of the Syrian hamster embryo system 

in vitro proceeds through such a series of progressive changes. The 

experimental design involves the examination of the temporal 

acquisition of several phenotypes of the transformed state following 

exposure of the cells to benzo(a)pyrene. 

It was found (4,5) that somatic mutation and morphological 

transformation can be obtained within 3-7 days after carcinogen 

treatment. However, attainment of other transformation properties, such as fibrinolytic activity (23,27,28,30,37,38), growth in soft agar 

and tumorigenicity requires additional growth of many weeks in culture. 

These results suggest that transformation in vitro of Syrian 

hamster embryo cells occurs in a progressive manner, therefore 

correlating the process of in vitro transformation with in vivo 

transformation. Thus, neoplastic transformation is a continuous, multistep, progressive process, while single-gene somatic mutation is 

a single-step process. Neoplastic transformation may be initiated by 

a mutational change, but cannot be adequately described by this 

mutational event. 

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Mutants of Cultured Human Fibroblasts." Science, 169 (1972), 

482-M85. 

2. Arlett, C.F., P. Turnbill, and S.A. Harcourt, et al. "A 

Comparison of the 8-Azaguanine and Quabain-Resistance Systems for 

the Selection of Induced Mutant Chinese Hamster Cells." Mutation 

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3.

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4. Barrett, J.C., D.L. Grady, and P. Jones. "A.A.C.R. Abstracts." 

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5. Barrett, J.C., B.D. Crawford, and P.O. P. Ts'o. "Abstract, First 

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6. Berwald, Y., and L. Sachs. "In Vitro Transformation of Normal 

Cells to Tumor Cells by Carcinogenic Hydrocarbons." J. Natl. Cancer Inst., 35 (1965), 6*11-657.

 

7. Casto, B.C., W.J. Pieczynski, and J.A. DiPaolo. "Enhancement of 

Adenovirus Transformation by Pretreatment of Hamster Cells with 

Carcinogenic Polycyclic Hydrocarbons." Cancer Res., 33 (1973), 819-824. 

8. Chu, E.H.T. , P. Brimer, K.B. Jacobson, and E.V. Merriman. "Mammalian Cell Genetics I. Selection and Characterization of 

Mutations Auxotrophic for L-Glutamine or Resistant to 

8-Azaguanine in Chinese Hamster Cells In Vitro." Genetics, 62 

(1969), 359-377. 

9. Chu, E.H.Y. , and H.V. Mailing. "Mammalian Cell Genetics II. Chemical Induction of Specific Locus Mutations in Chinese 

Hamsters." Genetics, 61 (1968), 1306-1312. 

10. Corsaro, C.M. , and B.R. Migeon. "Effect of Ouabain Resistance on 

Human Diploid Fibroblasts Carrying other Genetic Variants." Exp. Cell Res., 95 (1974), 47-53. 

11. R. DeMars. "Resistance of Cultured Human Fibroblasts and Other 

Cells to Purine and Pyrimidine Analogues in Relation to 

Mutagenesis Detection." Mutation Res., 24 (1974), 335-364. 

12. Di Paolo, J.A. , and P.J. Donovan. "Properties of Syrian Hamster 

Cells Transformed in the Presence of Carcinogenic Hydrocarbons." 

Exp. Cell Res., 48 (1967), 361-377. 

13. DiPaolo, J.A., P. Donovan, and R. Nelson. "Quantitative Studies 

of In Vitro Transformation by Chemical Carcinogens." J. Natl. Cancer Inst., 42 (1969), 867-874. 

14. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "In Vitro 

Transformation of Hamster Cells by Polycyclic Hydrocarbons: 

189 
Factors Influencing the Number of Cells Transformed." Nature New 

Biology, 230 (1971), 240-242. 

15. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "Transformation of 

Hamster Cells In Vitro by Polycyclic Hydrocarbons without 

Cytotoxicity. (cloning efficiency/carcinogen/protective agents/ 

frequency of transformation)." Proc. Natl. Acad. Sci., 68 

(1971), 2958-2961. 

16. DiPaolo, J.A., P.J. Donovan, and R.L. Nelson. "X-Irradiation 

Enhancement of Transformation by Benzo(a)pyrene in Hamster Embryo 

Cells." Proc. Natl. Acad. Sci., 68 (1971), 1734-1737. 

17. DiPaolo, J.A., R.L. Nelson, and P.J. Donovan. "In Vitro 

Transformation of Syrian Hamster Embryo Cells by Diverse Chemical 

Carcinogens." Nature, 235 (1972), 278-280. 

18. Foulds, L. Neoplastic Development. Academic Press, (1969). 

19. Gillin, F.D., D.J. Roufa, A.L. Beaudet, and C.T. Caskey. "8-Azaguanine Resistance in Mammalian Cells. I. Hypoxanthine- Guanine Phosphoribosyltransferase." Genetics, 72 (1972), 203-214. 

20. Glynn, I.M. "The Action of Cardiac Glycosides on Ion 

Movements." Pharmacol. Revs., 16 (1964), 381-407. 

21. Harris, M. "Mutation Rates in Cells at Different Ploidy 

Levels." J. Cell Physiolog., 78 (1971), 177-184; Harris, M. "Anomalous Patterns of Mutation in Cultured Mammalian Cells." 

Genetics Supplement, 73 (1973), 181-185. 

22. Huberman, E., P.J. Donovan, and J.A. DiPaolo. "Mutation and 

Transformation of Cultured Mammalian Cells by 

N-Acetoxy-N-2-Fluorenylacetamide." J. Natl. Cancer Inst., 48 

(1972), 837-840. 

23. P. Jones, W. Benedict, S. Strickland, and E. Reich. "Fibrin 

Overlay Methods for the Detection of Single Transformed Cells and 

Colonies of Transformed Cells." Cell, 5 (1975), 323-329­

24. Kao, F.T, and T.T. Puck. "Genetics of Somatic Mammalian Cells: 

Linkage Studies with Human-Chinese Hamster Cell Hybrids." 

Nature, 228 (1970), 329-332. 

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27. Ossowaski, L., J. Unkeless, A. Tobia, et al. "An Enzymatic 

Function Associated with Transformation of Fibroblasts by 

Oncogenic Viruses. II. Mammalian Fibroblast Cultures 

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28. Ossowski, L., J. Quigley, and E. Reich. "Fibrinolysis Associates 

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29. Peterson, A.R. , H. Peterson, and C. Heidelberger. "The Influence 

of Serum Components on the Growth and Mutation of Chinese Hamster 

Cells in Medium Containing Aminopterin." Mutation Res., 24 

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30. Quigley, J., L. Ossowski, and E. Reich. "Plasminogen, the Serum 

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31. Sato, K. , R.S. Slesinski, and J.W. Littlefield. "Chemical 

Mutagenesis at the Phorsphoribosyltransferase Locus in Cultured 

Human Lymphoblasts." Proc. Natl. Acad. Sci., 69 (1972), 1244-1248. 

32. Shapiro, N.I., A.E. Khalizev, E.V. Luss, et al. "Mutagenesis in 

Cultured Mammalian Cells I. Spontaneous Gene Mutations in Human 

and Chinese Hamster Cells." Mutation Res., 15 (1972), 203-214. 

33. Sharp, J.D. "Altered Enzymes in Drug-Resistant Variants of 

Mammalian Tissue Culture Cells." PNAS, 70 (1973), 3144-3149. 

34. Shin, S., R. Caneva, C.L. Schildraut, et al. "Cells with 

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38. Unkeless, J., K. Dano, G. Kellerman, and E. Rich. "Fibrinolysis 

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41. Wild, D. "Serum Effect on the Yield of Chemically Induced 

8-Azaguanine Resistant Mutants in Chinese Hamster Cell 

Cultures." Mutation Res., 25 (1974), 229-234. 

III. CHANGES IN DNA SEQUENCE ORGANIZATION AND GENE EXPRESSION IN NEOPLASTIC 

TRANSFORMATION 

In order to determine the role of nucleic acid changes in 

neoplastic transformation, we are investigating alterations in DNA 

sequence organization and the extent of new transcription in B(a)P 

transformed Syrian hamster embryo (SHE) cells (83,84). Ultimately, we 

hope to answer two fundamental questions. First, is DNA sequence loss 

or DNA sequence organizational change, either specific or nonspecific, 

causally related to neoplastic transformation? In other words, is it 

necessary to change the DNA, in some gross manner, for transformation 

to occur? Furthermore, even if DNA organization changes are not 

important for transformation, it seems obvious that transformation, 

whether virally or chemically induced, must involve the qualitative or 

quantitative change in the expression of at least one gene. Our 

second immediate objective, therefore, was to determine the extent of 

new transcription in transformed cells. Are ten genes "turned on" ­

or 10,000? 

We have begun our analysis by comparing a newly transformed subdiploid cell line (18 CL 10), transformed in vitro by B(a)P, and its 

192 
highly malignant tumor cell derivative (BP6T) to normal diploid SHE
 

cells. As few as ten BP6T cells produce tumors in 100$ of injected
 

newborn hamsters. Karyotypic studies indicate that both 18 CL 10 and
 

BP6T have a modal chromosome distribution of 40 in comparison to the
 

normal 44 chromosomes of cultured diploid SHE cells (37-39,92). This
 

loss in chromosome number appears to be the result of actual
 

chromosome loss and not due to Robertsonian fusions and
 

translocations. Losses of B and D group chromosomes are apparent, and
 

further studies utilizing various chromosome banding techniques (72)
 

are in progress to determine if this line exhibits the "balance" of
 

specific chromosomes found associated with malignancy by various
 

investigators (4,14,61,113).
 

These karyotypic studies are being extended to the molecular
 

level by analysis of DNA-DNA reassociation kinetics
 

(19-22,40,70,93,104,109,110). If one isolates DNA from normal SHE
 

cells, denatures it, and allows the sheared DNA to reassociate, at
 

least three kinetic components are apparent (34,36,45,56,74,98,115).
 Using T7 and E. coli as standards, approximately 5% of the genome
 

consists of highly repetitive or fold-back DNA sequences
 

(27,43,90,99,103) with a CotJ <io~3; 35% behaves as moderately
 

repetitive sequences (15,23,24,26,28,34,77,107) with an average CotJ
 

value of 2.5. These sequences consist of between 250 and 2,000
 

families repeated 500 to 5,000 times per genome; and 60% behaves like
 

nonrepetitive sequences present only once per genome (34). These
 

various kinetic components can be isolated in pure form. While the
 

reassociation kinetics of tumor cell DNA are not yet complete,
 

preliminary data indicate a 10$ decrease in DNA sequence complexity in
 

BP6T. This loss is in both repetitive and single-copy DNA sequences.
 Flow-microfluorometric analyses of normal and tumor cells indicate a
 

decrease of 5-10$ in DNA content in BP6T (32,71). Other probes of
 

specific DNA sequences such as the rRNA cistrons (12,42,47,52,
 64,87,112) and poly(A) tracts (9,100) are also being exploited. The
 

relevance of these intriguing DNA changes to neoplastic transformation
 

is currently under investigation.
 

The extent of new transcription in transformed cells is currently
 

being investigated by examining polysome-associated poly(A) mRNA, a
 

cellular component whose role in protein synthesis is well defined
 

(1,2,18,33,41,58,65,66,75,76,82,86,88,89,95). The sizes of poly(A)
 

mRNAs in SHE cells, 18 Cl 10, and BP6T appear similar, having a
 

heterogeneous distribution with a maximum at approximately 2,000
 

nucleotides. The gross turnover rate, measured by the approach to
 

steady-state labeling with ^H-Uridine in logarithmically growing cells
 

(16,17,57,67,91,100,102), indicates a similar turnover rate in all
 

three cell types, with a half-life approximately equal to the doubling
 

time of the cells.
 

193
 
There is also no apparent gross qualitative change in the types 

of mRNAs produced in these three cell types. Trace amounts of 

steady-state ^-labeled polysomal poly(A) nRNA were hybridized to a 

vast excess of hamster organ DNA, and hybridization was assayed with 

RNase (5,6,8,25,35,51,59,62,69,81,105). Under these conditions, the 

probability of forming a hybrid is related only to the concentration 

of the complementary sequence in the DNA. Repetitive sequence 

transcripts will hybridize at lower Cot values than will single-copy 

sequence transcripts. Fourteen to fifteen percent of the mRNA in all 

three cell types is complementary to mid-repetitive DNA sequences, while the remainder of the hybridizable mRNA appears to be 

complementary to single-copy DNA sequences. No significant 

differences are apparent between normal and transformed cells. 

Early investigations of gene transcription changes during 

transformation (30,31,73,85,107) were limited to changes in repetitive 

sequence transcripts (13,50,68,80,93,106,108). Recent advances in 

hybridization technology, however, have made reactions with 

single-copy transcripts possible ( 3, 7,10,11, W, 46,48,49, 53-55, 60, 63, 96,97,111,114). Under conditions of mRNA excess, the sequences 

complementary to the mRNA (mDNA) can be isolated from the remaining 

sequences (nmDNA) by hydroxyapatite chromatography. The hybridization 

conditions used are sensitive enough to detect down to one mRNA per 

cell. Normal, transformed, and tumor cell mRNAs saturate 0.64/f of the 

^H-normal cell single-copy DNA probe, indicating a complexity of 2.5 x 

10' nucleotides or approximately 12,500 genes of average size. Total 

poly(A) mRNA from 13 day hamster embryos saturates at least twice this 

amount of DNA. If one mixes normal and transformed cell poly(A) 

mRNAs, the percent saturation is a measure of the difference in 

sequence complexity of the two cell types. If all the mRNAs were 

different, the saturation value would be additive. If all the mRNAs 

are the same, no increase would be observed. When SHE and BP6T cell 

mRNAs are mixed, no increase is observed. Taking into consideration 

the accuracy of the technique, no more than M/t of 12,500 mRNAs are 

different between normal and transformed cells. 

Thus, the maintenance of malignant transformation can be 

accompanied by relatively small changes in the number of polysome­ associated poly(A) mRNA sequences; massive gene activation need not be 

invoked to account for the transformed state. Recycling experiments, using isolated mDNA and nmDNA, are in progress to detect, isolate, and 

identify any small amounts of new mRNA existing in transformed cells 

which have escaped notice by the simple addition experiments. 

194 
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204 
4.3 A STRATAGEM FOR EXPERIMENTS ON ONCOGENESIS IN VITRO
 

ANDREW R. PETERSON
 

Los Angeles County - University of Southern California
 

Comprehensive Cancer Center, Los Angeles, CA
 

Studies of oncogenesis in vitro were made possible by the
 

development of techniques for culturing oncogenically transformable
 

fibroblasts (U). Using such cells, an ideal approach to experimental
 

oncogenesis would be to take them to pieces, to modify the pieces
 

under carefully controlled conditions, to reassemble the cells in
 

stages, and so to trace, step by step, the process of oncogenesis.
 

Experiments with nuclear transplantation (18) are moves in this
 

direction, but the methods are still at an early stage of development.
 

An alternative to the above ideal approach is to look, in
 

cultured cells, for correlations between the class of phenomena known
 

as oncogenic transformation and other classes that are better
 

understood at the molecular level. At best, this strategem will
 

produce uniform associations under a limited number of conditions. At
 

least, such associations will provide a basis for deciding whether
 

oncogenic lesions belong in the same set as those that lead, by known
 

processes, to other phenomena.
 

A precondition for the use of the latter stratagem is a detailed
 

investigation of the techniques employed to establish the associa­ tions. Wide usage of a procedure is not a sufficient guarantee that
 

such scrutiny has been applied. For example, the method of measuring
 

mutagenesis in eukaryote cells using 8-azaguanine (AG) resistance as a
 

marker has been widely used, but it is only recently that the
 

importance of replating monolayer cells before selection, of using
 

high concentrations of AG, and of using dialyzed serum has been
 

pointed out by a number of people (cited in 17). Procedures that
 

incorporate these details yield results quite different from those of
 

earlier studies (see below).
 

Alkaline sucrose sedimentation analysis of DNA molecular weight
 

is another technique that can lead to anomalous results. We
 

previously used this method to study the DNA of transformable mouse
 

fibroblasts (C3H/10TJ cells) treated with N-methyl-N'-nitro-N­ nitrosoguanidine (MNNG). On that occasion we concluded that the
 

single strand breaks that we found were produced not by enzymes, but
 

by the action of alkali on methylation lesions (1U). In this we were
 

probably correct, as others have provided evidence of a molecular
 

basis for the phenomenon (6,8,21,23). Nevertheless, our measurements,
 which suggested that the lesions were apurinic sites, suffered from an
 

error due to an anomaly of sedimentation of large DNA molecules at
 

high speed (11,14,24,25).
 

205
 
Lately, we have applied the techniques of alkaline sucrose
 

sedimentation analysis, and of measurement of mutagenesis using AG
 

resistance as a marker, to look for correlations between DNA damage,
 mutagenesis, and oncogenic transformation produced by methylating
 

agents in cultured mammalian cells. We have not been able to study
 

mutagenesis in 10T1/2 cells, so we have used Chinese hamster
 

fibroblasts (V79 cells) for those studies. We have been able to
 

measure the production of alkali labile lesions and their repair in
 

both cell lines.
 

For the mutagenesis studies, monolayers of V79 cells were treated
 

for 2 hours with a range of doses (LD5-LD80) of MNNG or methyl methane
 

sulfonate (MMS). The treated cells were allowed to divide at least
 

four times, and were then trypsinized and replated at low density (10^
 

cells per 100 mm dish), and exposed to 40 ug of AG per ml in medium
 

containing 10$ dialyzed, heat-inactivated, fetal calf serum. Colonies
 

of AG resistant cells were fixed, stained, and scored after 9 to 10
 

days.
 

For the alkaline sucrose sedimentation studies, monolayers of V79
 

or 10T1/2 cells were labeled for 24 hours with 1i*C-thymidine,
 incubated in ^C-thymidine-free medium for 90 minutes treated for 2
 

hours with a range of doses (LD5-LD80) of MNNG or MMS, labeled for 4
 

hours with ^H-thymidine at various times after treatment, and lysed
 

for 30 minutes to 1 hour on the surface of alkaline sucrose gradients
 

(5-25$ sucrose, 1.7 M sodium ions, 1 mM EDTA, 0.06 M j>-amino
 

salicylate, pH 13). The gradients were centrifuged at 13,000 rpm for
 

7 to 12 hours, fractionated, and the radioactivity in each fraction
 

was determined. The number of alkali labile lesions in the parental
 

DNA was determined from the molecular weight of the 1^C-DNA, the
 

effect of these and other lesions on DNA synthesis was determined from
 

the molecular weight of the ^H-DNA.
 

We and others have shown that the above mutagenesis procedure
 

measures mutations at loci that control the activity of hypoxanthine:
 

guanine phosphoribosyl transferase (HGPRT) in the V79 cells (cited in
 

17,22). Furthermore, dose response curves for a variety of agents are
 

linear over large ranges of dose (2,5,17,22). We found that MNNG is
 

about 700 times more mutagenic than MMS on a molar basis, and about 35
 

times more mutagenic than MMS per lethal hit (Table 1). These
 

findings suggest a relationship between the mutagenic potencies of
 

MNNG and MMS that is similar to that observed in bacteria (9), but
 

quite different from that suggested by experiments on V79 cells on a
 

previous occasion (19).
 

In our system, the DNA sediments as single strands with a weight-
 average molecular weight of 7x10^ daltons, and is uncontaminated with
 

proteins or lipids. The DNA does not sediment anomalously at the low
 

206
 
speed (13,000 rpm) that we now employ. The criteria by which we have
 

determined the molecular weight, the extents of contamination and
 

anomalous sedimentation have been discussed by ourselves and others
 

(cited in 13,14). We found in V79 cells that single-strand break
 

production by MNNG is about 700 times greater, on a molar basis, than
 

that with MMS. Furthermore, MNNG produced about 35 times more breaks
 

per lethal hit than did MMS (Table 1). These data are not consistent
 

with a mechanism of single-strand breakage at alkali labile apurinic
 

sites, because the methylating agents produce similar numbers of such
 

sites per lethal hit in V79 cells (18). The results are consistent
 

with a mechanism of breakage at alkali labile phosphotriesters
 

(1,6,10,21,23).
 

Our latest results are summarized in table 1. It can be seen in
 

V79 cells, that the relationships between the mutagenic potencies of
 

MNNG and MMS on one hand, and single-strand breakage by these agents
 

on the other, are very similar. These findings suggest that the
 

lesions responsible for both phenomena belong in the same subset of
 

methylation lesions in the DNA. That this subset is the set of all
 

0-methylations is suggested by studies of the mechanisms by which
 

these agents methylate DNA (6,7,12). The relationship between
 

single-strand break production by MMS and MNNG in 10T1/2 cells appears
 

to be virtually identical with the associations found in V79 cells.
 

Therefore, careful measurements of the extent of oncogenic
 

transformation produced by MNNG and MMS in 10T1/2 cells will indicate
 

whether lesions in the set of ()-methylations of DNA are responsible
 

for that transformation. Experiments aimed at obtaining those
 

measurements are in progress.
 

The present data suggest several avenues of approach for future
 

experiments. One consists of determining whether the lesions
 

responsible for the single strand breaks are identical with those
 

responsible for mutagenesis. Another approach is to look for
 

parameters of DNA structure and function that change, on treatment
 

with MNNG and MMS, in a manner related to single strand break
 

production, rnutagenesis and oncogenic transformation. A third line of
 

attack is to look for the above relationships in cells treated with
 

chemical carcinogens that are not methylating agents.
 

We have begun to examine two new parameters of DNA structure and
 

function, namely, the number and size of newly synthesized DNA
 

molecules made in cells treated with MNNG and MMS. We find that the
 

size of newly synthesized DNA molecules is reduced to a greater extent
 

in MNNG treated cells than in MMS treated cells (16). This is con­ sistent with results obtained by others (20). By contrast, the number
 

of newly synthesized DNA molecules is reduced by the same extent in
 

cells treated with MMS and in cells treated with MNNG. The inter­ pretation of these data in relation to oncogenic transformation and
 

mutagenesis must await further scrutiny of the procedures that we used.
 

207 
TABLE 1. Associations among DNA damage, mutagenesis, and cytotoxicity
 

produced by methylating agents in V79 Chinese hamster cells and in
 

10T1/2 transformable mouse fibroblasts. Data are the values of slopes
 

from straight line curves fitted by linear regression analysis.
 Standard errors are determined from the standard errors of the
 

estimates as described in reference 25.
 

a) The units for mutation frequency are AG.resistant colonies x 10~
 survivors x control mutation frequency" . See reference 24 for
 

details of the calculation.
 

b) The units for single strand breaks are breaks x 10—8 daltons. See
 

reference 4 for details of the calculation.
 

c) Mutagenesis data from 10T1/2 cells are not available at this time.
 

d) At least 2 measurements were taken with at least 3 doses, ranging
 

from LD5 to LD80, of MNNG or MMS.
 

DNA damage, mutation, and cytotoxicity: 

CELLS AGENT CYTOTOXICITY MUTATION FREQUENCY3 SINGLE STRAND BREAKSb 

D (yM) per yM per DQ per pM per DQ 

V79 MNNG 7.99 +1.04 1.10 +0.20 8.76+1.81 1.03 +0.29 8.25+2.56 

779 MMS 154+26.2 0.0015+0.0006 0.23+0.09 0.0015+0.008 0.23+1.30 

10T1/2 MNNG 8.36+1.56 ?C 1.01 +0.25 8.48+2.54 

10T1/2 MMS 210 +25.2 ?c 0.0029+0.0013 0.61+0.29 

Number of measurements : 

V79 MNNG 29 28 9 

V79 MMS 10 30 9 

 8 10T1/2 MNNG 0 2 9 

10T1/2 MMS 8 0 14 

208
 
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15. Peterson, A.R. , H. Peterson, and C. Heidelberger. "Reversion of 

the 8-Azaguanine Resistant Phenotype of Variant Chinese Hamster 

Cells Treated with Alkylating Agents and 5-Bromo-2'­ deoxyuridine." Mutat. Res., 29 (1975), 127-137. 

16. Peterson, A.R. , M. Mulkins, and C. Heidelberger. "Alkaline 

Sucrose Sedimentation Studies of DNA Lessions that Impede DNA 

Synthesis in Transformable Mouse Fibroblasts Treated with 

Methylating Agents." Proc. Am. Assoc. Cancer Res. , 17 (1976), 122. 

17. Peterson, A.R. , D.F. Krahn, H. Peterson, C. Heidelberger, B.K. Bhuyan, and L.H. Li. "The Influence of Serum Components on the 

Growth and Mutation of Chinese Hamster Cells in Medium Containing 

8-Azaguanine." Mutat. Res., 36 (1976), 345-356. 

18. Prescott, D.M. , and J.B. Kirkpatrick. "Mass Enucleation of 

Cultured Animal Cells." Methods in Cell Biology, D.M. Presscott 

(Ed.), Academic Press, New York, 7 (1973), 189-203. 

19. Roberts, J.S., J.E. Sturrock, and K.N . Ward. "DNA Repair and 

Alkylation-induced Toxic, Mutagenic and Cytotogical Effects in 

Mammalian Cells." In: Chemical Carcinogenesis, Part A. P.O.P. T'so and J.A. Dipaolo (Eds.), Dekker, New York, (1974), 401-423­

20. Scudiero, D., and B. Strauss. "Increased Repair in DNA Growing 

Point Regions After Treatment of Human Lymphoma Cells with 

N-Methyl-N'-Nitro-N-Nitrosoguanine." Mutat. Res., 35 (1976), 311-324. 

210 
21.

 Shooter, K.V. , and R.K . Merrifield. "An Assay for Phospho­ triester Formation in the Reaction of Alklating Agents with 

Deoxyribosenucleic Acid In Vitro and In Vivo." Chem. Interact., 13 (1976), 223-226. 

22. Van Zeeland, A.A. , and J.W.I.M . Simons. "Linear Dose-response 

Relationships after Prolonged Expression Times in V-79 Chinese 

Hamster Cells." Mutat. Res., 35 (1976), 129-138. 

23- Walker, I.G. , and D.G. Ewart. "The Nature of Single-strand 

Breaks in DNA Following Treatment of L-cells with Methylating 

Agents." Mutat. Res., 19 (1973), 331-341. 

24. Walker, I.G., A.R. Peterson, J.S. Bertram, and C. Heidelberger. "Correspondence." Cancer Res., 35 (1975), 267-268. 

25. Zimm, B.M. "Anomalies in Sedimentation. IV Decrease in 

Sedimentation Coefficients of Chains at High Fields." Biophys. Chem., 1 (1974), 274-291. 

211 
4.4

 THE USE OF DIPLOID HUMAN FIBROBLASTS TO MEASURE THE 

EFFECT OF DNA REPAIR ON THE CYTOTOXIC AND MUTAGENIC 

ACTION OF CARCINOGENS 

VERONICA M. MAHER AND J. JUSTIN McCORMICK 

Carcinogenesis Laboratory, College of Osteopathic Medicine, 

Michigan State University, East Lansing, MI 

Although it has not yet been possible to prove that 

chemical and physical carcinogens cause cancer by producing mutational 

changes in cells, this theory, first proposed by Boveri in 1914 (5), 

has become widely accepted in the last eight years since the mutagenic 

action of "active forms" of carcinogens was reported (10,33,35,36). 

This is because a wide range of data point to mutations as the common 

denominator in the etiology of cancer caused by chemicals of widely 

different structure and by physical agents such as ionizing radiation 

and ultraviolet radiation. In this paper, we will review the use of 

cytotoxicity and mutagenicity experiments with diploid human cells in 

culture as a tool to dissect various aspects of this problem. It is 

obvious that such procedures can be used in conjunction with 

biochemical, biophysical, or genetic techniques to yield additional 

information on the mode of action of carcinogens in cells and many of 

the cited papers, in fact, do this. 

Normal human fibroblasts are now routinely grown in a large 

number of laboratories. Usually they are obtained from skin biopsies 

or from foreskin tissue removed at circumcision, but may also be 

obtained from other body tissues or from cell banks such as The 

American Type Culture Collection (Rockville, Md.) . The methods for 

growing and handling such cells have been described in detail 

(17-19). With a little care, such fibroblasts routinely give cloning 

efficiencies of 20 to 60%. Normal human cells are not immortal but 

die off after 50 to 70 passages in culture as has been established by 

Hayflick (24). However, this is generally not a limitation since a 

single culture produces enough cells for a large number of experiments 

of the type described below. Normal human fibroblasts can be made 

into "immortal" lines by transformation with SV40 virus (53) if one 

wishes to preserve cells with particular characteristics, although 

this may prove objectionable in some cases because the cells are no 

longer "normal." 

One special advantage of normal human fibroblasts is that 

one can obtain a wide variety of cells defective in various biochemical pathways including DNA repair pathways. These latter are of 

particular value in examining questions such as the relationship 

between DNA repair, cytotoxicity, and mutagenesis. In animal cell 

systems only rarely has a DNA repair-defective cell been reported 

(20,21). 

212 
One reason for using biological parameters such as 

cytotoxicity or mutagenicity as end points, rather than biochemical or 

biophysical, is that they are generally much more sensitive indicators 

of carcinogen interaction with cells. For example, ultraviolet 

irradiation survival curves for normal cells show that killing begins 

at about 12 ergs/mm2 and one reaches 10% survival at about 60 ergs/mm2 

(32). Repair-defective human cells show cytotoxicity at even 

1 erg/mm2 (32). Biochemical experiments designed to measure excision 

repair following ultraviolet irradiation (including our own) generally 

use doses of 75 to 150 ergs/mm2 because of the insensitivity of the 

assay (6,7). Chemical carcinogen effects on human fibroblasts can 

also be determined at lower doses by biological parameters than by 

biochemical or biophysical parameters. 

ARYL HYDROCARBON HYDROXYLATING (AHH) ACTIVITY OF HUMAN FIBROBLASTS 

Diploid human fibroblasts in culture seem to have little or 

no AHH activity which is necessary to activate polycyclic hydrocarbon 

carcinogens (McCormick, unpublished data). Foreskin tissue of newborns, as well as some early cell passages of cells derived from 

foreskin, contain measurable levels of the enzyme (28) . After 

foreskin derived cells are in culture for 2 or 3 passages, they are, 

with rare exception, negative for this enzymatic-activity. It is not 

clear whether any of the enzymatic activity found in foreskin or early 

passage cells from foreskin cultures is contributed by fibroblasts 

since homogenates from whole skin are used for the assays of foreskin 

and early passage of cells contain epithelial cells (which are later 

lost) as well as fibroblasts and epithelial cells generally possess 

AHH activity. Practically, this means that human fibroblasts in 

culture can be utilized to test for active derivatives of compounds 

such as polycyclic hydrocarbons. They show no cytotoxicity from the 

parent polycyclic hydrocarbons (59). Therefore, in our studies we 

have utilized chemically synthesized active forms (32,31*). It is also 

possible to generate active forms of such polycyclic hydrocarbons by 

adding a rat liver microsomal fraction prepared according to the 

method of Frantz arid Mailing (22), to the human cells along with the 

parent hydrocarbon molecule (Maher, et al., unpublished data). It is 

probably also possible to use irradiated feeder layers of cells which 

are able to metabolize carcinogens such as has been done by Huberman, 

et al., (25), but we know of no examples of such experiments with 

normal human fibroblasts. 

EFFECT OF DNA REPAIR ON THE CYTOTOXICITY OF DIPLOID HUMAN FIBROBLASTS BY CARCINOGENS 

Cytotoxicity experiments are usually the first method of 

measuring a biological response in cells. This can be done by cloning 

213 
experiments or by following cell numbers (by hemocytometer or 

electronic counter) after treatment of cells with an appropriate 

agent. Such experiments give base-line data on the sensitivity of 

cells to various agents. They are quick and provide reliable data on 

the sensitivity of cells to the toxic action of agents and when they 

are coupled with the use of cells with different characteristics, etc. 

(see below), they can provide evidence as to the mechanism(s) by which 

an agent kills cells. Such experiments have been carried out with 

ionizing radiation (3,54,55) with ultraviolet radiation 

(31,32,34,37,53), and with a variety of mutagenic, carcinogenic, or 

otherwise toxic compounds (23,31,32,34,39,46,47,52,61). Cytotoxicity 

studies can be especially useful when one wishes to examine the 

interaction of two agents. We and others have examined the 

synergistic effect of caffeine on the cytotoxic effect of ultraviolet 

radiation (4,32,39). 

If cells are available with a defect in a pathway which 

affects the uptake or the metabolism of a cytotoxic compound or the 

repair of lesions caused by an agent, such defects will generally 

result in a change in survival. When such defective cells are 

compared with normal cells, for cytotoxicity, one can learn something 

of the biological effect. One of the clearest examples of this 

situation is from studies on cells from xeroderma pigmentosa (XP) 

patients (32). Such patients develop multiple carcinomas of the skin 

when exposed to sunlight (45). Cleaver found that fibroblasts from 

such patients were unable to excise pyrimidine dimers from DNA (7). 

More recently, it has been found that such excision-repair defective 

XP cells (classical) fall into 5 complementation groups based on their 

ability to restore excision repair capacity when heterokaryons are 

formed by fussion of XP fibroblasts (26,45). In addition, there 

exists one small group of XP patients which are clinically 

indistinguishable from the classical XP patients, but whose cells do 

not have a defect in the rate of excision repair. These patients have 

been called "XP variants" by Cleaver (8). Recently, Lehmann, et al., 

(27) have shown that such variant XP cells have a defect in some 

post-replication repair process so that such cells after ultraviolet 

irradiation, take abnormally long to convert low molecular weight DNA 

into high molecular weight DNA. 

Figure 1 shows a series of survival curves demonstrating the loss 

of cloning ability by normal cells, classical XP cells from various 

complimentation groups, and variant XP cells following treatment with 

ultraviolet radiation. It is clear from the data that XP cells are 

more sensitive than normal cells. Furthermore, the survival of 

various classical XP cells correlates with their excision repair 

capacity, i.e., those with the least repair capacity have the greatest 

toxicity, etc. From this type of experiment along with biochemical 

measurements of repair capacity, one can determine the effect of DNA 

214 
215

 

Figure 1. Survival Curve for Cloning Ability Versus Dosage
 
repair processes on cell survival. In our studies, we have found that 

similar survival curves are found when cells are treated with active 

derivatives of some of the carcinogenic polycyclic hydrocarbons and 

aromatic amides (31,32,3^,40), but not with carcinogenic chemical such 

as N-methyl-N'-nitro-N-nitrosoguandine (MNNG) (Maher, et al., unpublished results). These data indicate that DNA excision and post- replication repair pathways are utilized for DNA damage resulting from 

these polycyclic hydrocarbons and aromatic amides as well as ultra­ violet radiation, but not that from methylating carcinogens such as 

MNNG. Similar conclusions have been reached by Stich et al., (47) who 

compared the cloning ability and DNA repair capacity of normal cells 

with one strain of XP cells following treatment with 4-nitroquinoline 

1-oxide (4NQO), some of its analogs, and MNNG. The effect of 4NQO on 

the survival of one of the XP strains has also been studied by Takebe 

et al., (52). Regan and Setlow, who used a biochemical approach, arrived at similar conclusions regarding repair in XP cells (44) . Research on XP cells has recently been reviewed by Cleaver and Bootsma 

(9). 

OTHER DNA REPAIR-DEFICIENT CELLS 

Patients with the autosomal, recessive defect, ataxia 

telangiectasia (AT) have an increased incidence of cancer of various 

types (49). Fibroblasts from such patients are abnormally sensitive 

to ionizing radiation as seen in cytotoxicity curves. Some of the AT 

cell lines tested lack the ability to excise lesions in the DNA 

produced by the ionizing radiation (42). However, cells from other AT 

patients show a similar increase in cytotoxicity when exposed to 

ionizing radiation but as yet no defect in DNA repair has been 

identified. 

Cells from patients with Fanconi's anemia (FA), a disease 

characterized by a predisposition to leukemia, have recently been 

shown to be about 6 times more sensitive to Mitomycin C treatment than 

normal cells. These cells seem to have a failure in removing the 

cross-link in DNA caused by this agent (23). 

HOST CELL REACTIVATION OF VIRUSES 

Host Cell reactivation of plaque-forming DNA viruses which 

are treated with carcinogenic agents is a method of determining the 

ability of cells to repair damage caused by such agents without 

treating cells directly. It yields data that are similar to survival 

curves. Various viruses have been used by different workers to study 

reactivation by normal and XP cells as well as caffeine effects 

(1,12-15,29,30,43,53). In general, the work of these workers agrees 

with the survival data for the cells discussed above. 

216 
CONCLUSION REGARDING CYTOTOXICITY ASSAYS IN REPAIR-DEFICIENT 

CELLS 

From the data we have reviewed, it is apparent that in 

human cells there are various DNA repair enzyme pathways that are 

responsible for the repair of DNA photoproducts as well as DNA 

carcinogen adducts. On the basis of the present data, one would 

assume that there are at least three partially independent pathways. 

It seems likely that other repair pathways remain to be identified. 

One extremely important area that as yet has received little attention 

is "post-replication repair." Most generally, this term refers to the 

ability of cells to replicate DNA containing a lesion by some sort of 

by-pass mechanism. The XP variant cells described above are the only 

known human cells with a defect in this mechanism (63). Such 

mechanisms are especially important since our data on XP cells 

demonstrate that excision repair in such cells is essentially 

error-free (32) (see below). Thus, it appears that mutations are 

caused when cells replicate DNA containing lesions, i.e., carry out 

post-replication repair. 

EFFECT OF DNA REPAIR ON THE FREQUENCY OF MUTATIONS INDUCED IN 

DIPLOID HUMAN FIBROBLASTS BY CARCINOGENIC AGENTS 

It is now possible to carry out mutagenicity studies with 

diploid human fibroblasts (16,56). Although such studies are much 

more laborious because of the numbers of dishes involved (see below) 

than the cytotoxicity studies described above, they are capable of 

providing a quantitative determination of spontaneous mutation rates 

(17) and of the ability of chemical (40,41,59-61) or physical 

carcinogens to induce mutations (3,11,37,38,62). Two types of 

selection systems have been successfully used for such studies: 

(1) resistance to purine analogs such as 8-azaguanine 

(SAG) (3,37,38,40,59-61) and thioguanine (TG) (11,62), 

etc. and 

(2) resistance to the membrane active drug, ouabain (41). 

The advantages and disadvantages of the first system have been 

outlined in considerable detail in recent reviews (16,56). Briefly, 

resistance to SAG or related analogs can be obtained in a "single 

step" selection (50,51). It is usually, but not always (16), 

accompanied by a reduction in the level of activity of the enzyme 

hypoxanthine (guanine) phosphor!bosyl transferase (HGPRT, B.C. 

2.4.2.8). In addition, a "single step" back selection system is 

potentially available for assaying the frequency of revertants. This 

is because when endogenous purine synthesis pathways are blocked by 

the presence of a folio acid antagonist, such as aminopterin, cells 

217 
lacking HPRTase activity should not be able to grow if the only 

source of purines is exogenous hypoxanthine even when exogenous 

thymidine is supplied (HAT medium) (16). This situation can be 

complicated, however, by the acquisition of resistance to the blocking 

agent itself. (M. Fox personal commun.) Use of SAG resistance as a 

genetic marker has the advantage that the HGPRT locus is located on 

the X-chromosome and, therefore, is essentially present as a single 

gene (monosomic). This allows the selection of a single mutation to a 

recessive trait without the need for two independent mutational events 

or the requirement of starting with cells which are heterozygous for 

the gene in question. (A related system developed and described by 

DeMars (16) involves loss of the enzyme, adenine phosphoribosyl 

transferase (APRT, E.G. 2.4.2.?).) The gene for this latter trait is 

on somatic chromosome #16 which restricts the use of this marker to 

cells heterozygous for this trait. 

Because of the phenomenon known as "metabolic cooperation" 

(48,57) in which non-mutant cells transfer material to SAG-resistant 

mutant cells and thus make the latter susceptible to killing by SAG, 

it is not possible to select for mutants if the cell density in the 

dishes is too high (3,38,40,57). Reconstruction experiments have 

demonstrated the need for correction factors to take into account this 

phenomenon as well as the effect of the different cloning efficiencies 

of the cells used (57,58). 

The second system, i.e., resistance to ouabain, has been 

described in detail by Thompson and Baker (56). Ouabain is a specific 

inhibitor of the Na+-K+-activated ATPase of the plasma membrane, the 

enzyme responsible for the active transport of K+ into the cell and 

the extrusion of Na . The many advantages of this system have been 

outlined (56). It also gives stable mutants in a "single step" 

selection. However, there is no back selection system for assaying 

revertants. The trait is co-dominant and, therefore, it is possible 

to select ouabain resistant mutants at reasonable frequencies. It has 

been used by Mankowitz, et al., (41) to quantitate the frequency of 

mutations induced in diploid human fibroblasts by the carcinogen, 

ethylmethanesulf onate. 

Resistance to purine analogs, such as SAG or TG, has to 

date been the genetic marker used most widely for studies on the 

induction of mutations in diploid human cells in culture 

(3,37,38,40). With the noted exception (41), we know of no published 

reports concerning the induction of mutations in such fibroblasts 

using any other marker. This selection system, developed by Szybalski 

and his co-workers (50,51) was successfully adapted and perfected to 

quantitate the induction of mutations to SAG resistance in normal 

diploid human fibroblasts by Albertini and DeMars (2,3) using X-rays 

as the mutagenic agent. We further adapted their techniques in order 

218 
to quantitate the induction of mutations by the chemical carcinogen 

N-acetoxy-2-acetylaminofluorene (40). 

More recently, we have revised our own techniques and 

investigated the effect of DNA excision repair capacity of human 

fibroblasts on the frequency of mutations induced by UV-radiation by 

comparing normal cells with several strains of classical XP cells 

(32,34,37,38,40). Our results indicate that the excision repair 

process itself is essentially "error free" since the frequency of 

UV-induced mutations are much higher in XP cells which lack excision 

repair than in normally repairing cells. What is still more 

significant, at equicytoxic doses in the normal and classical XP 

strains, the induced mutation frequencies are equal. 

We have investigated this question using "K-region" 

epoxides of carcinogenic polycyclic hydrocarbons and obtained 

essentially identical results. The frequency of mutations is higher 

in the excision repair defective cells than in the normal (59). 

It has also been possible to use such mutagenicity studies 

to investigate the effect of a defective post-replication repair 

process which has been identified in XP variants (27) . We find that 

these cells, like classical XP's, have higher UV-induced mutation 

frequencies than normal. Since such XP variant

as subject to sunlight-induced carcinoma of the

patients, such results add strength to the link

and carcinogenicity. 

 patients

 skin as

 between

 are equally 

 classical XP 

 mutagenicity 

CONCLUSION 

This brief review demonstrates that the use of diploid 

human fibroblasts has become an established method for examining the 

cytotoxicity and mutagenicity of carcinogenic agents. The unique 

availability of a series of DNA repair-defective cells (XP) has 

contributed greatly to our knowledge of how various carcinogenic 

agents affect cells. It is to be hoped that the recent discovery that 

AT and FA cells have DNA repair deficiencies (different from XP) will 

lead to further insights into the relationship between DNA repair, and 

the cytotoxic and mutagenic action of carcinogens. 

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Ultraviolet Irradiation and of Hydrocarbon Epoxides in Strains of 

Xeroderma Pigmentosum." Nature, 258 (1975), 760-763. 

40. Maher, V.M. , and J.E. Wessel. "Mutations to Azaguanine 

Resistance Induced in Cultured Diploid Human Fibroblasts by the 

Carcinogen, N-acetoxy-2-acetylaminofluorene." Mutation Res., 28 

(1975), 277-284. 

41. Mankovitz, R. , M. Buchwald, and R.M . Baker. "Isolation of 

Ouabain-resistant Human Diploid Fibroblasts." Cell, 3 (1974), 221-226. 

42. Paterson, M.C. , B. P. Smith, P.H. Lonman, et al. "Defective 

Excision Repair of X-ray-Damaged DNA in Human (Ataxia 

Telangiectasia) Fibroblasts." Nature, 260 (1976), 444-446. 

43. Rabson, A.S., S.A. Tyrrel, and G.Y. Leggallais. "Growth of 

Ultraviolet-Damaged Herpes Virus in Xeroderma Pigmentosum 

Cells." Proc. Soc. Exptl. Biol. Med., 132 (1969), 802-806. 

44. Regan, J.D., and R.B. Setlow. "Two Forms of Repair in the DNA of 

Human Cells Damaged by Chemical Carcinogens and Mutagens." 

Cancer Res., 34 (1974), 3318-3325. 

45. Robbins, J.H., K.H . Kraemer, M.A. Lutzner, et al. "Xeroderma 

Pigmentosum—An Inherited Disease with Sun Sensitivity, Multiple 

223 
Cutaneous Neoplasms, and Abnormal DNA Repair." Ann. Intern. 

Med., 80 (1974), 221-248. 

46. Stich, H.F., and R.H. San. "DNA Repair Synthesis and Survival of 

Repair Deficient Human Cells Exposed to the K-region Epoxide of 

Benz(a)anthracene." Proc. Soc. Exp. Biol. Med., 142 (1973), 115-158. 

47. Stich, H.F., R.H. San, and Y. Kawazoe. "Increased Sensitivity of 

Xeroderaa Pigmentosum Cells to Some Chemical Carcinogens and 

Mutagens." Mutation Res., 17 (1973), 127-137. 

48. Subak-Sharpe, H., R.R. Burk, and J.D. Pitts. "Metabolic 

Cooperation Between Biochemically Marked Mammalian Cells in 

Tissue Culture." J. Cell. Sci., 4 (1969), 353-367. 

49. Swift, M., L. Sholman, M. Perry, and C. Chase. "Malignant 

Neoplasms in the Families of Patients with 

Ataxia-telangiectasia." Cancer Res., 36 (1976), 209-215. 

50. Szybalski, W., andM.J. Smith. "Genetics of Human Cell Lines 

1.8-azaguanine Resistance, A Selective 'single step' Marker." 

Proc. Soc. Exp. Biol. Med., 101 (1959), 662-666. 

51. Szybalski, W. , E.H. Szybalska, and G. Ragni. "Genetic Studies 

with Human Cell Lines Analytic Cell Culture." Natl. Cancer Inst. 

Monogr., 7 (1962), 75-89. 

52. Takabe, H., J. Furuyama, Y. Miki, and S. Kondo. "High 

Sensitivity of Xeroderma Pigmentosum Cells to the Carcinogen, 

4-nitroquinoline-1-oxide." Mutation Res., 15 (1972), 98-100. 

53. Takebe, H. , S. Nil, M.I. Ishii, and H. Utsuml. "Comparative 

Studies of Host-cell Reactivation, Colony Forming Ability and 

Excision Repair After UV Irradiation of Xeroderma Pigmentosum, 

Normal Human and Some Other Mammalian Cells." Mutation Res., 25 

(1974), 383-390. 

54. Taylor, A.M.R. , D.G. Harnden, C.F. Arlett, et al. "Ataxia 

Telangiectasia: A Human Mutation with Abnormal Radiation 

Sensitivity." Nature, 258 (1975), 427-429. 

55. Thacker, J., and R. Cox. "Mutation Induction and Inactivation in 

Mammalian Cells Exposed to Ionizing Radiation." Nature, 258 

(1975), 429-431. 

224 
56.

 Thompson, L.H. , and R.M . Baker. "Isolation of Mutants of 

Cultured Mammalian Cells in DM Prescott." Methods in Cell Biol., 6, Academic Press, New York, (1973), 210-277. 

57. van Zeeland, A.A. , M.C.E . van Diggelen, and J.W.I.M. Simons. "The Role of Metabolic Cooperation in Selection of HGPRT

 

Deficient Mutants From Diploid Mammalian Cell Strains." Mutation 

Res., 14 (1972), 355-363. 

58. van Zeeland, A.A. , and J.W.I.M . Simons. "The Use of Correction 

Factors in the Determination of Mutant Frequencies in Populations 

of Human Diploid Skin Fibroblasts." Mutation Res., 3^ (1976), 149-158.

 

ADDITIONAL REFERENCES 

59. Maher, V.M. , J.J. McCormick, P.L. Grover, and P. Sims. "Effect 

of DNA Repair on the Cytotoxicity and Mutagenicity of Polycyclic 

Hydrocarbon Derivatives in Normal and Xeroderma Pigmentosum Human 

Fibroblasts." Mutation Res., 43 (1977), 117-138. 

60. Maher, V.M. , R.D. Curren, L.M. Ouellette, and J.J. McCormick. "Role of DNA Repair in the Cytotoxic and Mutagenic Action of 

Physical and Chemical Carcinogens." In: In Vitro Metabolic 

Activation in Mutagenesis Testing. F.J. de Serres, J.R. Fouts, J.R. Bend, and R.M . Philpot, (Eds), North Holland Biomedical 

Press, Amsterdam, (1976), 313-336. 

61. Stark, R.M. , and J.W. Littlefield. "Mutagenic Effect of BUdR in 

Diploid Human Fibroblasts." Mutation Res., 22 (1974), 281-286. 

62. Cox, R., and W.K. Masson. "X-ray Induced Mutation to 

6-Thioguanine Resistance in Cultured Human Diploid Fibroblasts." 

Mutation Res., 37 (1976), 125-136. 

63. Lehmann, A.R. , S. Kirk-Bell, C.F. Arlett, S.A. Harcourt, E.A. deWeerd-Kastelein, W. Keijzer, and P. Hall-Smith. "Repair of 

Ultraviolet Light Damage in a Variety of Human Fibroblast 

Strains." Cancer Res., 37 (1977), 904-910. 

225 
4.5 ENHANCEMENT OF DNA VIRAL TRANSFORMATION BY CHEMICAL 

CARCINOGENS AND MUTAGENS1 

BRUCEC. CASTO 

BioLabs, Inc., Northbrook, IL 

Enhancement of viral carcinogenesis by chemical carcinogens 

in vivo was first described by Rous and Kidd (20), who demonstrated an 

increase in virus-related papillomas following treatment of rabbits 

with Shope papilloma virus and the horizontal retort tar of the 

Oster-Gasfabrik of Amsterdam. Subsequently, Rous and Friederwald (19) 

and Rogers and Rous (18) observed the development of malignant 

carcinomas in sites jointly treated with Shope papilloma virus and 

chemical carcinogens that were not observed when virus or chemical 

were applied independently. 

Tumors induced by fibroma virus in animals pretreated with 

tar or benzo(a)pyrene (B(a)P) were larger and regressed at a slower 

rate in contrast to tumors induced by virus alone (1,2). In addition, 

some of the tumors demonstrated neoplastic characteristics, including 

invasiveness. The enhancement of tumor formation did not occur if 

chemicals were applied several months before virus infection nor if 

chemical treatment was begun 10 days after virus innoculation (2). In 

the preceding studies, the increase in viral-induced tumors was 

restricted to the site of chemical application. This latter observation, and the temporal relationship between virus and chemical 

addition suggested that the chemical carcinogen acted directly upon 

the eventual target cells and thereby increased the susceptibility of 

a population of individual cells to virus transformation. 

Subsequently, treatment of cells in vitro with x-irradiation 

(13,17,21) BCdR, BUdR (3,23) or lUdR (3,13,16), 4NQO (14,22), and with 

UV-irradiation (3) was shown to increase the frequency of transformation induced by the carcinogenic DNA viruses SV40, polyoma, human 

adenovirus types 12 and 31, and the simian adenovirus, SAY. 

In a series of experiments utilizing hamster embryo cells 

(HEC) and SAY, it was shown that cells pretreated with chemical 

carcinogens followed by infection with SAY demonstrated an increased 

sensitivity to SAY transformation. The polycyclic hydrocarbons B(a)P, 

DB(a,h)A, DB(a,c)A, DMBA and MCA enhanced SAY transformation but the 

non-carcinogens phenathrene, pyrene and perylene did not (3,4,5,6,8). 

1. The work upon which this publication is based was performed 

pursuant to Contract No. NCI-N01-CP-45615 with the National 

Cancer Institute, Department of Health, Education and Welfare. 

226 
Similarly, HEC treated with Ac-AAF, AFB.,, ara-C, B-PL, caffeine, MNNG, 

MMS, and MAM-Ac enhanced SA? transformation as evidenced by an 

absolute increase in the number of SA? foci per plate and a relative 

increase in the transformation frequency (4,5,6,9,10). Recently, the 

inorganic metal salts BeSOj,, CdCl-,, CaCrO^, K2Cr04, NiSO^, NaAs02, PbAc2, PbO, and PbCrO|, were shown to increase the sensitivity of HEC 

to SA? transformation following 18 hours exposure of the cells to the 

above metals; treatment with Ti02 was ineffective (11) . 

Although chemical exposure for 1 to 2 hours prior to virus 

addition was sufficient to increase SA? transformation by AcAAF, B-PL, EMS, MNNG, MMS, and PS (9,10 ) longer exposures were necessary for 

B(a)P, B(e)P, DB(a,b)A , DB(a,c)A , DMBA, MAM-Ac, and MCA (4,8,9 ) 

presumably because the latter compounds require more extensive 

metabolic activation than the former. Not only was the time of 

incubation with chemical found to be critical, but also the sequence 

of addition of chemical and virus. B(a)P, DMBA and AFB., were shown to 

enhance when added prior to SA? inoculation and cell transfer (5,6,8), but inhibited transformation up to 20-fold when added after cell 

transfer (5,6,8,10). In contrast, ara-C, 6-Ac-B(a)P and caffeine 

enhanced SA? transformation more efficiently when added after virus 

inoculation and cell transfer (6,10). A third group of chemicals 

including Ac-AAF, B-PL, EMS, MMS, MNNG, and PS enhanced equally well 

regardless of time of addition with respect to virus inoculation 

(5,9,10). 

Simultaneous treatment of HEC with B(a)P and 

benz(a)anthracene (BA) or 7,8-benzoflavone (ANF) 24 hours prior to SA? 

inoculation inhibited the lethal effects associated with B(a)P 

exposure and resulted in an increase in the absolute number of SA? 

foci. However, because of the significant increase in cell survival, the frequency of SA? transformation decreased relative to that 

observed in cells treated only with B(a)P (6). Similar results were 

observed when cells were treated with DMBA and BA or ANF, but were not 

as pronounced when MCA was substituted for B(a) P or DMBA (6). Pretreatment with BA or ANF 24 or more hours prior to B(a)P , DMBA or 

MCA treatment had little or no effect on enhancement of SA? 

transformation by the latter 3 chemicals (6, unpublished data). 

The enhancement of viral transformation by chemicals is 

probably not due to an effect on virus or viral DNA, as the cells are 

treated 2 to 24 hours prior to virus addition. Further, an effect on 

viral adsorption or penetration is unlikely since many of the chem­ icals enhance when added 5 to 8 hours after virus. It is also 

improbable that the chemicals act by selecting cells sensitive to 

viral transformation. If the latter were true, one would expect only 

a relative increase in the proportion of transformed foci among 

surviving cells. However, with the majority of chemicals tested, there 

22? 
has been an absolute increase of 2- to 5-fold in the number of SA7 

foci per plate where either negligible or extensive cell death occurs 

following chemical treatment. It has been suggested that irradiation 

or chemical treatment of cells enhance viral transformation by 

creating additional attachment sites for virus in cell DNA 

(3,5,9,13,17). These sites could be expressed during scheduled DNA 

synthesis opposite regions of unrepaired damage in parental DNA (12) 

or as a result of the excision-repair system of the cell (6,10). In 

either case, host cell repair of these damaged areas prior to the 

availability of viral DNA, should abrogate the expected increase in 

viral transformati on. 

Hirai et al. (15) have shown that treatment of Chinese 

hamster cells with 4NQO increased both the frequency of SV40 

transformation and the integration of SV40 DNA into cell DNA. Similarly, Casto and Miyagi (7) found increased amounts of SA7 DNA in 

Syrian HEC DNA following 2 hour treatment of the cells with MMS. With 

both 4NQO or MMS treatment, the period of enhancement was directly 

correlated to the time of repair (B.C. Casto and H.F. Stich, unpublished data; 4). Additionally, Coggin (13) showed that a delay 

of 10 hours in adding SV40 to x-irradiated hamster cells reduced 

enhancement by 10%. Alternatively, chemicals that are slowly removed 

by excision-repair, such as B(a)P or DMBA (10) and chemicals that are 

not removed such as BCdR (5) enhance transformation up to 72 hours 

after treatment. In the case of DMBA, enhancement declined progres­ sively over 72 hours (unpublished data) with the highest level of 

enhancement occurring when virus was added immediately after treat­ ment. With BCdR, enhancement was undiminished when SA7 was added 24, 48, or 72 hours after treatment (5). All chemicals that cause DNA 

repair increase SA7 transformation of HEC (6,10); however, 51% of all 

chemicals tested that enhance transformation do not induce demonstra­ ble DNA repair synthesis. It is suspected that these chemicals 

enhance by being bound to cell DNA and creating gaps for attachment of 

viral DNA during scheduled DNA synthesis as suggested by Cleaver (12). 

The enhancement of viral transformation provides a mammalian-cell 

assay that not only detects chemical carcinogens and mutagens, but 

also provides a system for recognition of those compounds that act as 

co-factors for carcinogenic viruses and chemicals that may not be 

detected by microbial systems. 

REFERENCES 

1. Ahlstrom, C.G. , and C. H. Andrewes. "Fibroma Virus Infection in 

Tarred Rabbits." J. Pathol. Bacteriol., 47 (1938), 65-86. 

228 
2.

 Andrewes, C.H. , C.G. Ahlstrom, L. Foulds, and W.E. Gye. "Reaction of Tarred Rabbits to the Infectious Fibroma Virus." 

Lancet, 2 (1937), 893-895. 

3- Casto, B.C. "Enhancement of Adenovirus Transformation by 

Treatment of Hamster Cells with Ultraviolet-irradiation, DNA Base 

Analogs, and Dibenz(a,h)anthracene." Cancer Res., 33 (1973), 402-407. 

4. Casto, B.C. "Enhancement of Viral Oncogenesis by Chemical 

Carcinogens." In: Chemical Carcinogenesis, Part B, The 

Biochemistry of Disease, Vol. 4, P.O. P. Ts'o and J. A. DiPaolo 

(Eds.), Marcel Dekker, N.Y. , (1974), 607-618. 

5. Casto, B.C., and J.A. DiPaolo. "Virus, Chemicals, and Cancer." 

Prog. Med. Virol., 16 (1973), 1-47. 

6. Casto, B.C., and J.A. DiPaolo. "Combined Chemical and Viral 

Carcinogenesis." Excerpta Medica Internatl. Cong. Series No. 350, Vol. 2, Chemical and Viral Oncogenesis. Excerpta Medica 

Amsterdam, 1974, 122-127. 

7. Casto, B.C., and M. Miyagi. "Studies on the Nature of the 

Enhancement of Viral Transformation by Chemical Carcinogens," 

Abstracts XI International Cancer Congress, 2 (1974), 76. 

8. Casto, B.C., W.J. Pieczynski, and J.A. DiPaolo. "Enhancement of 

Adenovirus Transformation by Pretreatment of Hamster Cells With 

Carcinogenic Polycyclic Hydrocarbons." Cancer Res., 33 (1973), 819-824. 

9. Casto, B.C., W.J. Pieczynski, and J.A. DiPaolo. "Enhancement of 

Adenovirus Transformation by Treatment of Hamster Embryo Cells 

with Diverse Chemical Carcinogens." Cancer Res., 34 (1974), 72-78. 

10. Casto, B.C., W.J. Pieczynski, N. Janosko, and J.A. DiPaolo. "Significance of treatment interval and DNA Repair in the 

Enhancement of Viral Transformation by Chemical Carinogens and 

Mutagens." Chem.-Biol. Interactions, 13 (1976), 105-125. 

11. Casto, B.C., W.J. Pieczynski, R.L. Nelson, and J.A. DiPaolo. "In 

Vitro Transformation and Enhancement of Viral Transformation With 

Metals." Proc. Am. Assn. Cancer Res., 17:12 (1976). 

12. Cleaver, J.E. "Repair of Damaged DNA in Human and Other 

Eukaryotic Cells." In: Nucleic Acid-Protein Interations 

229 
Nucleic Acid Synthesis in Viral Infections, D.W. Ribbons, J.F. Woessner, and J. Schultz (Eds.), North Holland, Amsterdam, 1971, 87-112. 

13. Coggin, J.H., Jr. "Enhanced Virus Transformation of Hamster 

Embryo Cells In Vitro." J. Virol., 3 (1969), 458-462. 

14. Diamond, L., R. Knorr, and Y. Shimizu. "Enhancement of Simian 

Virus 40-Induced Transformation of Chinese Hamster Embryo Cells 

by 4-nitroquinoline-l-oxide." Cancer Res., 34 (1974), 2599-2604. 

15. Hirai, K. , V. Defendi, and L. Diamond. "Enhancement of Simian 

Virus 40 Transformation and Integration by 4-nitroquinoline-l­ oxide." Cancer Res., 34 (1974), 3497-3500. 

16. Ledinko, N. , and M. Evans. "Enhancement of Adenovirus 

Transformation of Hamster Cells by N-methyl-N'-nitro-N­ nitroso-guanidine, Caffeine and Hydroxylamine." Cancer Res., 33 

(1973), 2936-2938. 

17. Pollack, E.J., and G.J. Todaro. "Radiation Enhancement of SV40 

Transformation in 3T3 and Human Cells." Nature, 219 (1968), 520-521. 

18. Rogers, S., and P. Rous. "Joint Action of a Chemical Carcinogen 

and a Neoplastic Virus to Induce Cancer in Rabbits; Results of 

Exposing Epidermal Cells to a Carcinogenic Hydrocarbon at Time of 

Infection With the Shope Papillcma Virus." J. Exptl. Med., 93 

(1951), 459-488. 

19. Rous, P., and W.F. Friedewald. "The Effect of Chemical 

Carcinogens on Virus-Induced Rabbit Papillomas." J. Exptl. Med. , 79 (1944), 511-539­

20. Rous, P., and J.G. Kidd. "The Carcinogenic Effect of a Papillcma 

Virus on the Tarred Skin of Rabbits. I. Description of the 

Phenomenon." J. Exptl. Med., 67 (1938), 399-428. 

21. Stoker, M. "Effect of X Irradiation on Susceptibility of Cells 

to Transformation by Polyoma Virus." Nature, 200 (1963), 756. 

22. Stich, H.F., 0. Hammerberg, and B.C. Casto. "The Combined Effect 

of Chemical Mutagen and Virus on DNA Repair, Chromosome 

Aberrations and Neoplastic Transformation." Canad. J. Genet. Cytol., 14 (1972), 911-917. 

23. Todaro, G.J., and H. Green. "Enhancement by Thymidine Analogs of 

Susceptibility of Cells to Transformation by SV40." Virology, 24 

(1964), 393-400. 

230 
4.6

 MORPHOLOGICAL TRANSFORMATION, CHROMOSOMAL CHANGES 

AND INCREASES IN PLASMINOGEN ACTIVATOR IN IN VITRO 

CARCINOGENESIS 

WILLIAM F. BENEDICT
 

Department of Medicine, Children's Hospital of Los Angeles,
 University of Southern California School of Medicine
 

Los Angeles, CA
 

A.	 MORPHOLOGICAL TRANSFORMATION BY CANCER CHEMOTHERAPEUTIC
 

AGENTS IN THE C3H/1(rr/2CL8 CELL LINE
 

We have chosen to study whether or not various cancer chemotherapeutic agents produce morphological transformation in the
 

C3H/10TsCL8 mouse cell line developed in the laboratory of Dr. Charles
 

Heidelberger (21) for several reasons. First, the molecular
 

mechanism(s) of action of many of these agents have been studied
 

extensively. Secondly, there is considerable data on the in vivo
 

carcinogenicity of most of these chemotherapeutic agents and finally,
 

the majority of these drugs are presently being used for the treatment
 

of malignant and often nonmalignant conditions in man. Therefore,
 

these studies should have relevancy not only to help investigate the
 

actual mechanism(s) of oncogenic transformation in vitro and to
 

compare results obtained in cell culture with in vivo carcinogenicity
 

data, but also would be important in our attempt to determine the
 

significance of short-term assays as potential screens for hazards to
 

man. Since there are many long-term survivors for various types of
 

malignancy following treatment with chemotherapeutic agents, we should
 

be able in the future to determine whether those same agents which
 

cause morphological transformation in cell culture also produce
 

primary or secondary malignancies in man. Thus, cancer chemothera­ peutic agents may be a unique group of compounds, particularly since
 

the actual plasma level of the majority of these agents is already
 

known. In general, the concentrations used in cell culture for the
 

transformation studies are actually achieved in the plasma level in
 

man (5).
 

Various subclasses of chemotherapeutic agents have been studied
 

including alkylating agents, antitumor antibiotics, antimetabolites,
 

and miscellaneous synthetics. Those agents which have been shown to
 

be carcinogenic in vivo also produced morphological transformation,
 whereas those agents which thus far have not been shown to be
 

carcinogenic in vivo did not cause transformation in vitro (5 and
 

Table 1). Thus, there appears to be a good correlation between known
 

carcinogenesis in vivo and transformation in vitro utilizing these
 

agents. Consequently, it would seem that this system as well as
 

others hold promise in screening for potential carcinogens, especially
 

231
 
TAPLE 1
 

SUMMARY OF CHROMOSOMAL DAMAGE, TRANSFORMATION AND
 

CARCINOGENICITY PRODUCED BY CANCER CHEMOTHERAPEUTIC AGENTS2
 

Chemotherapeutic
 

Agent
 

Alkylating Agents
 

Melphalan
 

Thiotepa
 

Antitumor Antibiotics and
 

Natural Products
 

Actinomycin-D
 

Bleomycin
 

Vincristine
 

Antimetabolites
 

Methotrexate
 

6-Mercaptopurine
 

Miscellaneous Synthetics
 

Hycanthone
 

5-aza-C
 

Tilorone
 

Chromosomal
 

Damage In
 

A(T1)Cl-3 Cells
 

+
 

+
 

+
 

+
 

+
 

+
 

+
 

Morphological
 

Transformation In
 

C3H/10T 1/2CL8 Cells
 

+
 

+
 

+
 

+
 

+
 

+
 

+
 

Known
 

Carcinogenic!ty
 

In Animals
 

+
 

+
 

+
 

+
 

±*
 

_c
 

+
 

+
 

232 

o
 

These findings are presented in detail in Reference 5.
 

Unknown at high dose exposure.
 

Produces lymphomas only, possibly secondary to immunosuppression.
 
when methods for metabolic activation of all types of chemicals can be 

incorporated into the transformation system such as those now being 

used for mutagenicity studies (1,13). 

B. CHROMOSOMAL CHANGES AND THE EXPRESSION OF MALIGNANCY 

Our present feeling about the importance of chromosomal changes 

in relationship to the expression of malignancy has recently been 

reviewed (3). We originally showed that aromatic polycyclic hydrocarbons produced aneuploidy in cells within 24 to 72 hours after 

exposure (4) and suggested at that time that the rapid occurrence of 

chromosomal changes might be important in cell transformation. 

Subsequently, various chromosomal banding techniques became available 

(9,22 ) which allowed individual chromosomes to be identified with 

certainty. Thus, it became possible to study the relationship between 

specific chromosomal changes and malignant expression. 

The first evidence for specific chromosomal imbalances being 

related to the expression of malignancy came from the laboratory of 

Dr. Leo Sachs (25). It was shown in chemically transformed hamster 

cells that the number 5y and 51f) chromosomes appeared to have genes 

for the expression of transformation and/or malignancy and the number 

?2 and 7o chromosomes were thought to have genes for the suppression 

of these same properties. Likewise, in polyoma virus transformed 

hamster cells, it was reported that genes for the expression of 

malignancy were located on the 5/- chromosome and genes for the 

suppression of malignancy were located on the 5_ and 75 chromosomes 

(24).

 6 * 

At this time, we were also studying in the same hamster embryo 

system the relationship between specific chromosomal changes and the 

expression of malignancy (7). In cells transformed by ara-C, 

tumorigenicity was associated with an increased number of 5 

chromosomes compared to the number of 73 chromosomes, i.e., 

tumorigenicity increased when the ratio of 5y to 7o chromosomes was 

greater than one. Additional independent studies also published on a 

hamster embryo cell line transformed with dimethylnitrosamine again 

suggested that genes for the expression of malignancy and transformation were located on the 5j chromosome and to a lesser extent on the 

5-|2 chromosome (8). The 73 chromosome was also felt to contain genes 

for the suppression of malignancy in these cells. Thus, although 

there appears to be variation at times in the specificity of chromosomes with genes for the expression or suppression of malignancy, the 

implication from these studies is that chromosomes which contain 

genetic information involved in oncogenesis are far from random. 

We have also found a high correlation between rapid chromosomal 

damage produced in the A(T..)C1-3 hamster cell line by various cancer 

233 
chemotherapeutic agents and morphological transformation in vitro or 

carcinogenicity in vivo. This is summarized in Table 1. The chromosomal damage was produced within 24 hours after treatment with various 

concentrations of the drugs. The A(T^)C1- 3 hamster cell line is being 

utilized by us as a potential screen for human mutagens and/or 

carcinogens, since it has a pseudodiploid chromosomal complement (7) , 

rapid growth in cell culture (6), and also has a unique property for 

fibroblasts in that the cells have highly inducible aryl hydrocarbon 

hydroxylase activity (unpublished data). This enzyme system is 

important in the activation of many carcinogens to their ultimate 

carcinogenic form(s), particularly in the case of aromatic polycyclic 

hydrocarbons. Thus the cell line may be excellent for testing many 

types of potential mutagens and/or carcinogens, because the parent 

compound can be metabolized to its active form(s) when most classes of 

compounds are exposed to the cells. However, a microsomal activation 

system or some other activation system may have to be incorporated 

into this cell line for certain classes of compounds to be evaluated 

adequately. 

Specific chromosomes involved in the suppression of malignancy 

have also been reported in mouse cells (10). Malignancy appeared to 

be related to whether or not one or two of the same marker chromosome 

was present in the cell. When mixed populations containing cells with 

either one or two of this particular marker chromosome were injected, 

only cells containing one complement of the specific marker chromosome 

were found in the tumors. This marker was formed by a translocation 

of the number 7 chromosome onto the number 19 chromosome, and it was 

concluded that either the 7 or 19 chromosome contained genetic 

information for the suppression of malignancy

the addition of an extra marker chromosome

expression of tumorigenicity. 

 in

 seemed

 these

 to

 cells,

 inhibit

 since 

 the 

Finally, chromosome changes have been found in human diploid 

cells which have been transformed either with chemicals or viruses 

(11,17). Abnormal chromosomal patterns have also been a consistent 

finding following treatment of human diploid epithelial cells with 

methylcholanthrene (12). Therefore, possible specific chromosomal 

imbalances related to the expression of malignancy can now be studied 

in human diploid cells after in vitro transformation. We have, in 

fact, outlined a general hypothesis on how specific chromosomal 

changes can be related to malignant expression following transformation with all carcinogens including DNA oncogenic viruses, RNA 

oncogenic viruses, and chemical or physical agents (3). 

234 
C.

 INCREASES IN PLASMINOGEN ACTIVATOR FOLLOWING IN VITRO TRANSFORMATION 

Many in vitro characteristics of transformed cells, including a 

crisscross morphological pattern and growth in semi-solid medium, have 

been shown to be at least partially related to the presence of a 

serine protease which is usually produced by malignant cells at a 

higher level than their normal counterparts (19). This protease, 

named plasminogen activator (20,23), has also been shown to be 

increased in many chemically transformed mammalian cells (1ti). 

Presently, fibrin overlay methods have been developed to detect single 

transformed cells or colonies of transformed cells (14). Since the 

transformed cells can not only be identified using these techniques, 

but also can be isolated (15), early transformed cells hopefully could 

be cloned for further investigations regarding the nature of the 

transformation event(s). An increase in plasminogen activator may, in 

addition, be useful to identify chemical transformants prior to 

detection by more standard techniques. 

In general, it would appear that an increase in plasminogen 

activator is an early event following chemical transformation in 

several systems, including secondary hamster embryo cells (2) , and the 

C3H/10T4CL8 cell line (16) which are now in wide use as model systems 

for evaluating chemical carcinogens. 

REFERENCES
 

1. Ames, B.N. , W.E. Durston, E. Yamasaki, and F.D. Lee. "Carcinogens are Mutagens: A Simple Test System Combining Liver 

Homogenates for Activation and Bacteria for Detection." Proc. Natl. Acad. Sci. USA, 70 (1973), 2281-2285. 

2. Barrett, J.C., B.D. Crawford, D.L. Grady, L.D. Hester, P.A. Jones, W.F. Benedict, and P.O. P. Ts'o. "The Temporal Acquisition 

of Enhanced Fibrinolytic Activity by Syrian Hamster Embryo Cells 

Following Treatment with Benzo(a)pyrene (B(a)P). " Cancer Res. (submitted). 

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Expression of Malignancy." In: The Molecular Biology of the 

Mammalian Genetic Apparatus, Part B. Relationship to Somatic 

Genetics, Cancer and Aging. P.O.P. Ts'o (Ed), Elsevier North 

Holland, Inc. Vol. 2 (1977), 215-226. 

4. Benedict, W.F . "Early Changes in Chromosomal Number and 

Structure After Treatment of Fetal Hamster Cultures with 

Transforming Doses of Polycyclic Hydrocarbons." J. Natl. Cancer 

Inst., 49 (1972), 585-590. 

235 
5.

 Benedict, W.F. , A. Banerjee, A. Gardner, and P.A. Jones. "Induction of Morphological Transformation in Mouse C3H/10TJC1-8 

Cells and Chromosomal Damage in Hamster A(T-|)C1-3 Cells by Cancer 

Chemotherapeutic Agents." Cancer Res., 37 (1977), 2203-2209. 

6. Benedict, W.F. and T.A. Khwaja. "A New Hamster Fibrosarcoma 

Model for _In Vitro/In Vivo Evaluation of Cancer Chemotherapeutic 

Agents." Medical and Pediatric Oncology, 2 (1976), 271-278. 

7. Benedict, W.F. , N. Rucker, C. Mark, and R. Kouri. "Correlation 

Between the Balance of Specific Chromosomes and the Expression of 

Malignancy in Hamster Cells." J. Natl. Cancer Inst., 54 (1975), 157-162. 

8. Bloch-Shtacher, N., and L. Sachs. "Chromosome Balance and the 

Control of Malignancy." J. Cell Physiol.. 87 (1976), 89-100. 

9. Caspersson, T., C. Johanson, and E. Modest. "Identification of 

Human Chromosomes by DNA Binding Fluorescent Agents." 

Chromosoma, 30 (1970), 215-227. 

10. Codish, S.D., and B. Paul; "Reversible Appearance of a Specific 

Chromosome Which Suppresses Malignancy." Nature, 252 (1974), 610-612. 

11. Croce, C.M. , D. Aden, and H. Koprowski. "Somatic Cell Hybrids 

Between Mouse Peritoneal Macrophages and Simian-Virus-40­ Transformed Human Cells. II. Presence of Human Chromosome 7 

Carrying Simian Virus 40 Genome in Cells of Tumors Induced by 

Hybrid Cells." Proc. Natl. Acad. Sol. USA, 72 (1975), 1397-1400. 

12. Freeman, A.E. , R.S. Lake, H.J. Igel, L. Gernand, M.R. Pezzutti, J.M. Malone, C. Mark, and W.F. Benedict. "Euploid to Heteroploid 

Conversion of Human Skin Cells by Methylcholanthrene." Proc. Natl. Acad. Sci. (in press). 

13. Huberman, E., and L. Sachs. "Cell-Mediated Mutagenesis of 

Mammalian Cells with Chemical Carcinogens." Int. J. Cancer, 13 

(1974), 326-333. 

14. Jones, P.A., W.F. Benedict, S. Strickland, and E. Reich. "Fibrin 

Overlay Methods for the Detection of Single Transformed Cells and 

Colonies of Transformed Cells." Cell, 5 (1975), 323-329­

15. Jones, P.A. , W.E. Laug, and W.F . Benedict. "Clonal Variation of 

Fibrinolytic Activity in a Human Fibrosarcoma Cell Line and 

236 
Evidence for the Induction of Plasminogen Activator Secretion 

During Tumor Formation." Cell, 6 (1975), 245-252. 

16. Jones, P.A. , W.E. Laug, A. Gardner, C.A. Nye, L.M. Fink and W.F. Benedict. "In Vitro Correlates of Transformation in C3H/10J 

Clone 8 Mouse Cells." Cancer Res., 36 (1976), 2863-2867. 

17. Kakunaga, T. "Transformation of Human Diploid Cells by Chemical 

Carcinogens." Proc. Nat. Acad. Sci. (submitted). 

18. Laug, W.E. , P.A. Jones, and W.F. Benedict. "Studies on the 

Relationship Between Fibrinolysis of Cultured Cells and 

Malignancy." J. Natl. Cancer Inst., 54 (1975), 173-179. 

19. Ossowski, L., J.P. Quigley, G.M. Kellerman, and E. Reich. "Fibrinolysis Associated With Oncogenic Transformation. Requirement of a Plasminogen for Correlated Changes in Cellular 

Morphology, Colony Formation in Agar and Cell Migration." 

J. Exp. Med., 138 (1973), 1056-1064. 

20. Ossowski, L., J.C. Unkeless, A. Tobia, J.P. Quigley, D.B. Rifkin, and E. Reich. "An Enzymatic Function Associated with 

Transformation of Fibroblasts by Oncogenic Viruses. II. Mammalian Fibroblast Cultures Transformed by DNA and RNA Tumor 

Viruses." J. Exp. Med., 137 (1973), 112-126. 

21. Reznikoff, C.A. , D.W. Brankow, and C. Heidelberger. "Establishment and Characterization of a Cloned Line of C3H Mouse 

Embryo Cells Sensitive to Postconfluence Inhibition of 

Division." Cancer Res., 33 (1973), 3231-3238. 

22. Seabright, M. "A Rapid Banding Technique for Human 

Chromosomes." Lancet, 2 (197D, 971-972. 

23- Unkeless, J.C., A. Tobia, L. Ossowski, J.P. Quigley, D.B. Rifkin, and E. Reich. "An Enzymatic Function Associated With 

Transformation of Fibroblasts by Oncogenic Viruses. I. Chick 

Embryo Fibroblast Cultures Transformed by Avian RNA Tumor 

Viruses." J. Exp. Med., 137 (1973), 85-111. 

24. Yamamoto, T., M. Hayashi, Z. Rabinowitz, and L. Sachs. "Chromosomal Control of Malignancy in Tumors from Cells 

Transformed by Polyoma Virus." Int. J. Cancer, 11 (1973), 555-556. 

25. Yamamoto, T., Z. Rabinowitz, and L. Sachs. "Identification of 

the Chromosomes that Control Malignancy." Nature (New Biol.), 243 (1973), 247-250. 

237 
4.7 CELL SURFACE ANTIGENS AS MARKERS FOR THE TRANSFORMED
 

PHENOTYPE'
 

LOUIS M. FINK2, ROBERT M. EVANS2, J. CARL BARRETT3, SARA M.
 

CLARKE2
 

Department of Pathology, University of Colorado Medical
 

Center, Denver, CO (2); and Department of Biophysics,
 

Johns Hopkins Medical School, Baltimore, MD (3)
 

CELL SURFACE ANTIGENS AS MARKERS FOR THE TRANSFORMED PHENOTYPE
 

One. potential approach for understanding neoplastic
 

transformation is the study of the plasma membrane of cells.
 Alterations in this cellular structure may result in many of the
 

physiological properties which are associated with neoplastic
 

transformation. Probably the major impetus in stimulating studies on
 

the cell surface was the report that some plant lectins agglutinate
 

transformed cells better than untransformed cells (1,17). Later it
 

was found that many tumor cells contain altered transplantation
 

antigens (2) or embryonic antigens (6). Each of these discoveries
 

suggests that the surface membranes of tumor cells are different from
 

normal cells. However, even after extensive study, the mechanism by
 

which membrane changes are related to other phenotypic characteristics
 

of transformed cells or tumorigenicity is not known.
 

Limitations in our present technology have made the study of
 

plasma membrane difficult. Methods developed for isolation of plasma
 

membranes are largely unsatisfactory because of contamination by
 

endoplasmic reticulum and other cellular components. However, this
 

may be because these cellular components are intimately associated
 

with the membrane. Another difficulty is that the membrane must be
 

subjected to drastic procedures before the specific activity of
 

certain membrane associated enzymes appears to rise. These procedures
 

often allow the loss of certain membrane components. The most
 

effective reagents for the dissociation of membranes are detergents
 

such as sodium dodecylsulfate (SDS). This frequently used detergent
 

1This work was supported in part by grants CA-15823, CA-13419 and
 

CA-15109 from the National Institutes of Health and grant BC-189 from
 

the American Cancer Society. Dr. Fink is recipient of Research Career
 

Development Award CA-00050 from the National Institutes of
 

Health. Sara Clarke is recipient of training grant GM-977.
 

238
 
has proven useful for analytical procedures where the molecular size 

is to be analyzed, but may ruin the functional properties of the 

proteins. The use of nonionic detergents may allow preservation of 

enzyme or antigenic activity, but frequently the membrane is not 

sufficiently dissociated to allow biochemical analysis of many of the 

components. 

One technique, which has aided greatly in membrane study, is the 

surface labeling of viable cells in culture (4,10,21). These 

reactions are catalyzed by enzymes that are too large to enter the 

cell. Using these techniques, interesting differences between normal 

and neoplastic cells have been found (3,4,5,7-15,18,20,22,24). 

This paper will describe some of the methods we have used to 

study the cell surfaces of neoplastic cells and discuss some of the 

potential uses of similar techniques in studying genesis and evolution 

of neoplastic cells. 

CELL SURFACE LABELING 

Lactoperoxidase catalyzed iodination of cells primarily results 

in labeling of the available tryosine moieties of proteins (21). It 

has been reported that this reaction may also label cell membrane 

lipids (polar and neutral) (16). The method can be restricted to 

labeling the cell surface proteins when the cells are exposed for a 

short period of time, when there are no extensive intracellular 

peroxidases, and when the cells are intact. 

We use a ten-minute incubation at 22 C in this procedure and 

believe the cell surface proteins are selectively labeled for the 

following reasons: (1) There is a marked selectivity of labeling. 

That is, certain proteins show a very high or low specific activity. 

(2) Pretreatment of the cells with trypsin (1.0 ug/ml for ten minutes) 

removes most of the proteins which are usually labeled. (3) 

Postiodination trypsin treatment markedly alters the proteins which 

are labeled. Glucose oxidase is used to generate H

20? directly 

because we feel that cells are more viable during this treatment. 

In the studies to be described, the cells are iodinated when they 

are confluent and washed three times with PBS prior to iodination. 

After the iodination, the cells are scraped, washed, and lysed in the 

presence of phenylmethylsulfonylfluoride (PMSF) , a protease inhibitor. The nuclei and DNA are removed by centrifugation. The samples 

are boiled in the presence of B-mercaptoethanol and SDS, are count 

matched, and electrophoresed on a 1.5% polyacrylamide gel (PAG) with a 

4.2J stacking gel. E. coli and T4 phage proteins are used as 

molecular weight markers. The 1^5 autoradiograph and Coomassie 

239 
blue stained protein profiles are compared to those obtained when the 

cells are metabolically labeled with C1** L-amino acids. 

Fig. 1 A and C show the gels of normal tertiary Syrian hamster 

cells. The most intensely labeled iodine band is approximately 

220,000 daltons (Fig. 1C). This protein has been termed LETS (Large 

External Transformation Sensitive) by Hynes (13). Other names which 

have been given to this protein are CSP (cell surface protein) (25), SF antigen (fibroblast surface antigen) (23), galactoprotein a (4), Zeta (22), and band 1 (10). Comparing the labeling pattern of 

transformed hamster cells (Fig. 1D) (a gift from W. Benedict, Los Angeles Children's Hospital) to the untransformed cells 

demonstrates the lack of this protein on the transformed cell 

surface. However, when metabolic C^ labeling is used, both 

transformed and untransformed cells are labeled in the 220,000 MW 

region (Fig. 1 A and B) . Analyses of several other chemically 

transformed Syrian hamster cell lines (obtained from J.C. Barrett, Johns Hopkins School of Medicine) were made and the results are shown 

I

125 in Fig. 2. The A-6, T-6, and U-19 lines show loss of the

labeling in the LETS region. A-12 shows a reduced amount of label as 

compared to the normals (U-18, E-11, B-14). Dr. Barrett, earlier in 

this volume, has described the biological characteristics of these 

lines. A-6, T-6, and U-19 are tumorigenic, grow in soft agar, and 

produce extensive zones of fibrinolysis in fibrin overlay 

experiments. A-12 is less tumorigenic, grows poorly in agar, and has 

decreased fibrinolytic activities. Table 1 illustrates that for the 

lines analyzed there appears to be a correlation between the amount of 

labeling of LETS and the other markers of transformation in this 

study. In support of this, iodination of SV^Q transformed human WI-38 

cells and untransf ormed WI-38 cells reveals that the transformed cells 

lose the 220,000 MW iodinatable protein (unpublished observations). However, studies on the transformation of mouse C3H101/2 cells (15) 

did not reveal any correlation between the amount of LETS protein and 

the other characteristics of transformation (Fig. 3). 

ANTIBODY PREPARATION AGAINST 220,000 MW CELL SURFACE PROTEIN 

Because we felt that assays had to be developed which could 

detect phenotypic characteristics of single cells, we decided to 

prepare an antiserum against specific cell proteins and test whether 

this antiserum could be used to detect altered cells in populations. 

Gels containing lysates of 1.5 x 10

o 

 cells were run and stained 

with Coomassie blue. The region which corresponded to the I'^S 

220,000 MW band was cut out, emulsified in PBS, diluted in complete 

Freund's adjuvant (FA) and injected subcutaneously into rabbits. This 

was repeated at day 14 with incomplete FA. Lysates of cells 

containing the 220,000 dalton protein were used to boost the animals 

240 
 I12^ C1i Fig. 1 SDS polyacrylamide gel autoradiograms of and *

labeled normal and transformed Syrian hamster embryo 

fibroblasts. 

14 A.

 Normal fibroblasts labeled with C L-amino acids 

B. Transformed fibroblasts [A(T )C13]labeled with 

C1^L-amino acids 

C. Normal fibroblasts lactoperoxidase I labeled 

D. Transformed fibroblasts lactoperoxidase I12^ labeled 

The arrow denotes the 220,000 MW protein. 

241 
Fig. 2 SDS polyaorylamide gel autoradiogram of lactoperoxidase 

1125 labeled normal and transformed Syrian hamster embryo 

fibroblasts. 

A. U-19 — spontaneously transformed 

B. U-18 — late passage fibroblasts 

C. T-6 — tumor cell lines 

D. E-11 — early passage fibroblasts 

E. B-14 — late passage fibroblasts 

F. A-12 — transformed after treatment with benz(a)pyrene 

G. A-6 — transformed after treatment with benz(a)pyrene 

The arrow denotes the I ipc J 220,000 MW protein. 

242 
Table 1
 

CHARACTERISTICS OF TRANSFORMED AND UNTRANSFORMED
 

SYRIAN HAMSTER EMBRYO (SHE) CELLS
 

CELLS ORIGIN LETS PROTEIN
 

E-ll Early passage, embryo 4+
 

culture from inbred
 

animals
 

A-12 B(a)P transformed cell 14­

line
 

A-6 B(a)P transformed cell
 

line
 

T-6 Tumor cell line
 

B-14 Late passage SHE cells 4+
 

U-18 Late passage SHE cells 3+
 

U-19 Spontaneously transformed ­

cell line
 

a. Colony Growth in Soft Agar
 

b. Plasminogen Activator Activity - 0-4+ scale
 

c. Not Tested
 

TUMORIGENICITY


Negative, with 10


cells inj .
 

50% pos. at 104


cells inj .
 

75% pos. at 102


cells inj .
 

100% pos. at 101


cells inj .
 

Neg. at 106 cells


Neg. at 107 cells


100% pos. at 107


cells
 

SAa PAb
 

 Neg. 1+
 

 3% 2+
 

 51% 4+
 

 96% 3-4+
 

 0% 2+
 

 0% 2+
 

N.T.C N.T.
 

243 
125 Fig. 3 SDS polyaorylamide gel patterns of lactoperoxidase I 

labeled mouse cell lines. Densitometer tracings are given 

for Coomassie blue staining ( ) and I ^ 

autoradiographs ( ) of the gels. 

A. 13H — transformed after treatment with 5-fluorodeoxyuridine 

B. J2BB — transformed after treatment with 5-fluorodeoxyuridine 

C. B-b-Cll — transformed after treatment with cigarette 

smoke condensate 

D. MCA-TC115 — transformed after treatment with 3 

methylcholanthrene 

E. C3H/10TiCL8 — untransformed parental cell line 

125 Arrow indicates the position of the I -labeled protein 

(MW 250,000). 
on day 21, 22, and 23. The animals were exsanguinated on day 30 which 

is prior to a time at which they could respond to new antigens in the 

crude lysate. This antiserum contains an antibody directed against an 

iodinatable 220,000 MW cell surface protein as determined by 

immunochemical methods. 

IMMUNOFLUORESCENT STAINING OF CELLS WITH ANTI-220,000 DALTON ANTISERUM 

In these experiments, tertiary cultures of Syrian hamster fibro­ blasts and transformed cells were stained with the antiserum and 

fluorescein-labeled goat anti-rabbit IgG to detect the antigen- antibody complexes. In all studies, non-immunized or pre-immunized 

rabbit serum was used as a control. Fig. 4 shows the staining pattern 

of unfixed, untransformed Syrian hamster fibroblasts and their 

corresponding phase microscopy. The staining seems to be at the 

surface of these cells. Unfixed transformed Syrian hamster cells 

(A(T..)C13), prepared in an identical manner, have little to no 

staining. Fig. 5 demonstrates the staining of fixed transformed 

cells. Most of the cells show a diffuse staining pattern. We believe 

that acetone or ethand fixation allows penetration of the antibody and 

the staining suggests that there is an intracellular form of the 

antigen in transformed cells. This may be true in some transformed 

cells and not in others. The gels show 220,000 MW C1^-amino acid 

labeled or Coomassie blue stained protein in some transformed lines 

but not others. From studies on several transformed lines it appears 

that the decrease or loss of I12^-220,000 cell surface protein may 

occur by more than one process, i.e., decreased synthesis, failure to 

be retained on the cell, or by allowing a cryptogenic form to occur. 

Other experiments on the staining of cells with the antibody 

suggest that the antigen is not actin, myosin, actin-binding protein 

or cold insoluble globulin. Adsorption of the antiserum with intact 

normal cells removes the antibody with resultant loss of its staining abilities. When a transformed cell line was used to adsorb the 

antiserum, only a slight reduction in the staining intensity was 

observed. 

CYTOTOXICITY AS A METHOD OF SCORING A PARAMETER OF THE TRANSORMATION 

PHENOTYPE 

It has previously been shown that Chinese hamster cells are 

killed by treatment with rabbit antiserum containing antibodies 

against cell surface proteins of Chinese hamster cells in the presence 

of complement (19). Since Syrian hamster cells are either devoid or 

contain a markedly reduced amount of the 1^^-220,000 MW cell surface 

protein, we tested the capacity of our antibody in the presence of 

complement to kill normal cells. Table 2 demonstrates that a 1:800 

dilution of antiserum is cytotoxic to untransformed cells in the 

presence of *\% complement. 

245 
Fig. 4 Indirect jmmunofluorescent staining of Syrian hamster

(unfixed) with anti-220,000 MW antiserum and

anti-rabbit fluorescein conjugate. 

 cells 

 goat 

A. Epifluorescence (420X) 

B. Phase contrast (420X) 

246 
Fig. 5 Indirect immunofluorescent staining of transformed Syrian
 

hamster cells treated with formalin and acetone (420X).
 

2*17
 
Table 2 

Effect of Anti 220,000 Dalton Cell Protein 

on Tertiary Syrian Hamster Cells 

Number of 

Treatment of Cells Surviving Colonies 

Media alone 19 

Rabbit complement 25 

Antiserum 23 

Complement and normal rabbit serum 23 

Complement and antiserum 1 

a

Colonies macroscopically scored after 7 days incubation. Initial 

plating was 2,000 cells per 35 mm plate. Average of 12 separate 

experiments performed in duplicate. 

248 
The controls (media alone, rabbit complement, antiserum, and 1:800 

dilution of normal rabbit serum plus complement) show no effect on the 

cells. Table 3 shows that the antiserum has much less toxicity on 

transformed cells. Table 4 compares the cytotoxic effects of the 

antiserum on three transformed Syrian hamster cell lines with the 

untransformed hamster fibroblasts. The differences in the killing 

observed between untransf ormed and transformed

each type of cell shows a variability either in

the availability of this protein on its surface. 

 cells

 the sy

 indicate

nthesis

 that 

and/or 

SUMMARY 

Cell surface labeling techniques have allowed the examination of 

changes in the distribution of a 220,000 MW protein in normal and 

transformed cell populations. Antiserum produced against this protein 

has made possible the study of the distribution in individual cells by 

indirect immunof lucres cence and the scoring of the transformed 

phenotype by selective killing in the presence of complement. 

The problems we are now attempting to study are: 

1. What are the properties of the clones which allow different 

percentage killing, e.g., is there heterogeneity within 

clones? 

2. Are the survivors different in other phenotypic properties 

(other than resistance to the antibody); i.e., what is their 

(a) growth in agar, (b) lectin agglutinability, and (c) 

turnorigenicity? 

3. If there is a true heterogeneity, is it reestablished after 

the selective pressure of the antibody? What modulates the 

amount and regeneration of heterogeneity? 

4. Can the selective killing of untransformed cells be used in 

assays of chemical car ci no genesis to allow quantitation by 

counting surviving clones? Is this an early or late even in 

the progression of events which seems to occur after 

exposure to carcinogens? 

249 
Table 3 

Effect of Anti 220,000 Dalton Cell Protein 

on Transformed Syrian Hamster Cells3 

Number of . Treatment of Cells Surviving Colonies 

Media alone 208 

Rabbit complement 228 

Antiserum 222 

Complement and normal rabbit serum 231 

Complement and antiserum 172 

Hamster cells were transformed after treatment with 1-B-D-arabino­ furanysylcytosine (Ara-C). 

Colonies macros co pi call y scored after 5 days incubation. Initial 

plating was 1,000 cells per 35 mm plate. Average of 7 separate 

experiments performed in duplicate. 

250 
Table 4
 

Comparison of the Cytotoxicity of Anti 220,000
 

Dalton Cell Protein on Untransformed and
 

Transformed Syrian Hamster Cells
 

Cells treated Percent of Killing3
 

Untransf ormed 96
 

ACT^CIS 25
 

P-MCA-75 17
 

A-6 59
 

Number of surviving colonies in
 

the presence of antiserum and
 

complement
 

Percent of killing = 1 - x 100
 

Number of surviving colonies in
 

the presence of normal rabbit
 

serum and complement
 

251
 
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253 
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254 
4.8 BIOPHYSICAL CYTOLOGICAL

CARCINOGENESIS 

 APPROACHES TO IN VITRO 

MORTIMER L. MENDELSOHN 

Biomedical and Environmental Research Program 

Lawrence Livermore Laboratory, Livermore, CA 

Biophysical cytology deals with the manipulation, 

measurement, identification, and sorting of cells and subcellular 

components by physical proceses (77,80). Differential centrifugation, 

optical scanning, and flow cytometry are the principal approaches of 

relevance to in vitro carcinogenesis. General sources of information 

are found in the literature on automated cytological diagnosis, 

automated cytogenetics, computer image processing, and biophysics 

(40,64,65,68,69,77,80,83,94,100-102). 

The measurement and interpretation of cells and chromosomes 

by optical scanning is now a highly developed and commercialized 

capability. Cells can be recognized on the basis of content and 

distribution of stainable constituents, as well as size and shape of 

cytoplasm or nucleus. Both fluorescent and absorption stains are used 

(15,47,69,83,94,103). Recently developed but yet to be published 

approaches promise major increases in processing speeds, approaching 

those of flow cytometry. 

In flow cytometry, objects in monodisperse suspension flow 

past optical or electrical detectors. Initially this technique was 

used to count cells and estimate cell volume by the change in 

electrical resistance in a minute aperture. The main emphasis today 

is on fluorescence measurement of cytochemically deposited 

fluorochromes. Precise measurements of the distribution of properties 

can be assembled automatically and objects are processed at rates of 

about 1000 per second. In flow sorting, the objects are separated on 

the basis of their cytometric values. Several commercial instruments 

are now available for both processes (12,27,45,46,51,63,68,90,91, 

95-97,99). Light scatter signals can be generated simultaneously 

with fluorescence and are useful as a rough indicator of size 

(14,46,63,70,96). 

The key to both scanning and flow approaches is the availability 

of a rich assortment of relevant cytochemical probes. In addition to 

the large classical histochemical literature (which is not referenced) 

and the specific techniques referred to below, there are several 

general methods and sources of information (12,15,18,28,29,33,40,48, 

53, 64, 65, 67, 68, 74, 77, 78, 80, 98,100,101). 

Differential centrif ugation and gravity separation are 

widely practiced at the cellular and subcellular level. Several 

255 
techniques are directly relevant to bulk separation of specific types 

of cells or chromosomes (30,34,62,104). 

The application of biophysical cytology to in vitro carcinogenesis can take several forms: 1) the definitive assay of the 

transformed cell; 2) enrichment of transformed cells; 3) separation of 

transformed cells for biochemical, biological, or visual evaluation; 

4) detection of transformed cells in vivo in blood or other accessible 

sites. These are potential, not yet achieved capabilities and their 

fruition will depend on the discovery of suitable markers for the 

transformation state. 

Perhaps the best studied candidate markers are those involving DNA and the genetic apparatus. DNA content is the most quantitative cytochemical property of mammalian cells; it has been extensively 

measured and is known to be frequently abnormal in cancer cells 

(1,5,8,12,15,18,25,28,37,39,47,56,58,67,71,85,86,92,99,103,107). Qualitative differences in DNA have also been described in abnormal, 

malignant and transformed cells (2,8,11,13,19,26,29,35,41,54,61,67, 

71,85,86). Also, it is generally recognized that malignancy and 

perhaps transformation are highly correlated with chromosomal changes 

(4,9,37,56,60,69,75,79,81,82,92,95,104,105). Biophysical cytology is 

able to address these properties with currently available methods. 

The identification and quantitation of enzymes is a richly 

developed cytochemical capability which is just beginning to be 

adapted to flow systems. Many enzymes have been linked to cancer and 

a few to transformation; it should soon be possible to measure one or 

more rapidly and accurately in single cells (24,31,33,42,44,48,50,51, 

53,55,59,72,76,78,88,89). 

Fluorescence depolarization measures the rotational mobility 

of molecular probes in solution or in situ in living cells. Recently, 

the technique has been extended to flow cytometry allowing further 

testing of the evidence that transformed cells may have altered 

fluidity of membranes or other cell components (3,16,87). 

Immunofluorescence has been successfully applied to flow 

cytometry. With further development of specific probes, it should be 

possible to study the membrane, chromosomal, and intracellular 

antigen-antibody reactions that mark the transformed cell 

(17,21,23,26,32,35,48,49,57). 

In addition to the potential for direct identification of 

the transformed cell by biophysical cytology, there are several 

indirect applications worth listing briefly. Flow systems are 

excellent tools for cytokinetic analysis. Thus presently available 

methods can be used to study the early and late kinetic changes of the 

256 
evolving transformation (6,7,20,22,36,38,40,43,58,61,73,86,93,106);
 

viable cells can be identified and sorted (45,66); and with impending
 

developments such as vital stains for DNA content, there is the
 

potential to synchronize cells by sorting prior to carcinogen
 

treatment (3,7,8,10,34,38,43,54,86). Also, as markers for cell
 

differentiation are developed, there is the exciting possibility of
 

using sorting to enrich for the very cells that are most vulnerable to
 

transformation (3,8,15,21,23,29,30,32-34,42,44,48,50,52,53,55,59,74,
 84,89).
 

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266 
4.9 AUTOMATIC TECHNIQUES IN CELL BIOLOGY: POSSIBLE APPLICATIONS 

TO IN VITRO CARCINOGENESIS 

DONALD A. GLASER 

Department of Molecular Biology 

University of California 

Berkeley, CA 

I. INTRODUCTION 

Rapid progress in many biological and medical research fields 

depends on observing growth of colonies of microorganisms or animal 

cells on solid surfaces. Very often the pace and even the ultimate 

success of the work is limited by the number of colonies that can be 

observed under well-defined conditions in order to find a rare mutant, 

measure the rate of mutation induced by seme chemical, or determine 

the probability that a virus causes a neoplastic transformation in a 

growing animal cell. In addition to the need for examining very large 

numbers of colonies for these purposes, it is extremely useful to be 

able to make accurate quantitative measurements of the response of 

these colonies to a variety of environmental factors. Our methods are 

designed to apply modern automation methods including materials 

handling and computer aided pattern recognition to make possible large 

experiments and quantitative observations on microorganisms and animal 

cells in tissue culture. A wide variety of studies in fundamental 

biology and applications to practical biological and medical problems 

should be aided by these methods. The work in progress ranges from 

the study of the genetics and physiology of E.coli bacteria and other 

microorganisms to studies of mammalian cell genetics and the response 

of such cells to a variety of hormones, mutagens, and potential 

carcinogens and teratogens. It is expected that these various 

projects will provide large numbers of mutants of various cell lines 

very difficult to obtain by conventional hand methods for detailed 

study in a number of laboratories. They will aid in the mapping of 

genetic markers in a variety of microorganisms and animal cells which 

will be important in understanding the biochemical genetics and 

regulatory systems of these organisms. Feasibility studies for 

screening programs testing the effectiveness of putative 

chemotherapeutic agents and examining suspected environmental 

mutagens, carcinogens and teratogens can be carried out. These 

methods are applicable to a wide range of difficult medical and 

biological problems now being attacked in many places but they also 

make possible an attack on problems usually considered intractable by 

conventional methods. 

26? 
II.

 ADVANTAGES OF A LARGE-SCALE AUTOMATED SYSTEM FOR THESE APPLICATIONS 

What does this kind of large-scale automation have to contribute 

to biomedical science? In general, many experiments profit from the 

economy of scale and the need for precise control of environmental 

conditions for the possibility of accurate quantitative measurements. 

In particular: 

(a) Accurate Control of Biological Conditions. With the use of 

suitable sensors and control circuitry cheaply and reliably 

monitored with small computers, growth conditions can be 

controlled very accurately over a large biological sample 

for extended periods of time and can be changed on a 

precisely repeatable schedule so that all portions of the 

biological sample will have experienced exactly the same 

conditions of growth. 

(b) Accurate quantitative measurements of growth and other 

biological responses to the environment are possible which 

cannot be done at all by manual methods in many cases. 

(c) Accurately timed measurements of biological response can be 

made on a schedule optimized for the experimental result 

desired without regard to personal fatigue and convenience 

of the experimenters. 

(d) Advantages of Large Scale 

1. Rare events for which no biological selection can be 

devised can be discovered for study. 

2. Extensive statistics can be collected for known or for 

commonly occurring classes of events. 

3. Many similar experiments can be done at the same time in 

parallel so certain kinds of experimental results can be 

achieved more rapidly than when serial experimentation 

is required. 

4. The cost of developing sophisticated technology can be 

spread over many projects which can share the technology 

even when no single project can justify the necessary 

investment of time and funds. 

5. Mutants and special strains can be provided for many 

small groups which would otherwise be unable to attack 

268 
problems requiring highly specialized starting

of cells. 

 strains 

III. TECHNIQUES OF OBSERVATIONS 

Optical methods of observation were chosen in order to make rapid 

and accurate measurements on very large numbers of colonies. Time- lapse photographs of growing colonies are taken with 35 mm black and 

white film using five different colors of illuminating light in 

sequence. Between successive photographs, the colonies can be exposed 

to a wide variety of temperature, gaseous environment and radiation 

regimes in addition to treatment with drugs, nutrients, enzyme sub­ strates that show a color change if the enzyme is present, and various 

other types of vital stains. Quantitative measurements of these 

photographs are carried out by a flying-spot scanner (similar in 

operation to a very high resolution television camera) connected 

directly with a sizeable computer. These measurements can produce 

curves describing rate of growth, colorimetric response, changes in 

appearance and other optically detectable consequences of the known 

treatment and allow by this means the quantitation of the response and 

the selection of individual colonies that respond in a particularly 

specified way. 

IV. TECHNIQUES OF COLONY INCUBATION AND MANIPULATION 

Colonies are grown in conventional 100 mm petri dishes or on 

large glass sheets (about 40 x 80 cm) covered with agar or other 

growth substrate. The dishes or trays are handled manually in a 

presently operating prototype and will be handled automatically in a 

large machine now under construction. Temperatures are held constant 

and uniform to better than 0.1°C and other biologically important 

parameters are similarly controlled. Inoculation, colony picking, restreaking, replica plating, administration of drugs, nutrients and 

other liquids are all carried out by automated equipment under 

computer control. The capacity of the final system will be about 10° 

colonies per batch. 

V. RESULTS 

The system has been used successfully for isolating large numbers 

of E.coli mutants for particular studies. For example, 1.4 million 

colonies were examined to isolate 2266 heat sensitive mutants which 

included 110 mutants unable to synthesize DNA at 41°C but apparently 

normal at 30°C. These have been mapped and shown to include 11 genes 

located at different sites on the E.coli map. A number of other 

nutritional and regulatory mutants of E.coli and Salmonella have been 

isolated. Present experiments include extension of this work to cold 

269 
sensitive mutants of E.coli, to heat and cold sensitive mutants of 

yeast and to a variety of other bacterial projects. 

By analysis of the appearance of colonies growing on agar, it has 

been possible to identify bacterial pathogens that cause human urinary 

infections so that an error rate of less than about 1 in 1000 is 

experienced in identifying such pathogens from a panel of about 12 

unknowns. 

Chinese hamster ovary cells grown on agar in this system reveal 

subtle differences in colony morphology not detected when the same 

cells are grown in the conventional way on plastic under liquid 

nutrients. These subtle differences breed true in many cases but the 

colony morphology and growth rate is found to depend very sensitively 

on small amounts of insulin, testosterone, and cyclic AMP. This 

combination of stability of clonal appearance with sensitivity to 

hormonal (and also mutagenic) agents will be the basis for a large 

scale mutant hunt in which we will try to find large numbers of heat 

sensitive Chinese hamster ovary cells including those defective in DNA 

synthesis and its control. Other animal cell experiments designed to 

furnish the basis for a test method for carcinogens, teratogens and 

mutagens in mammalian cells are being developed. 

REFERENCES 

1. Couch, J., J. Berk, D.A. Glaser, J. Raymond, and T. Wehr. "Automated Recognition of Bacterial Strains by Analysis of Colony 

Morphology." (Proc. 13th Internat. Congress of Genetics, Berkeley, Calif., August 1973). Genetics (June 1973 suppl.), 54. 

2. Glaser, D.A. , "Automation of Colony Identification and Mutant 

Selection." In: New Approaches to the Identification of Micro­ organisms, C.G. Heden and T. Illeni, (Eds.), John Wiley and Sons, Inc., N.Y. , (1975), 5-12. 

3. Glaser, D.A. , and C.B. Ward. "Computer Identification of 

Bacteria by Colony Morphology." In: Frontiers of Pattern 

Recognition, Acad. Press, N.Y. , (1972), 139-162. 

4. Glaser, D.A. , and W.H . Wattenburg. "An Automatic System for the 

Growth and Analysis of Large Numbers of Bacterial Colonies Using 

an Environmental Chamber and a Computer-Controlled Flying-Spot 

Scanner." Ann. N.Y. Acad. Sci., 139 (1966), 243-257. 

5. Raymond J., J. Couch, D.A . Glaser, and C.T. Wehr. "Automatic 

Selection of Conditionally Defective Mutants of Microorganisms." 

270 
(Proc. 13th Internat. Congress of Genetics, Berkeley, Calif., August 1973). Gentics (June 1973 suppl.), 226. 

6. Wehr, C.T. , L. Waskell, and D.A. Glaser. "Isolation and 

Characterization of Cold-Sensitive DNA Mutants of Escherichia 

Coli K12." (Proc. 13th Internat. Congress of Genetics, Berkeley, Calif., August 1973) Genetics (June 1973 suppl.), 293. 

ADDTIONAL REFERENCES: 

7. M.W . Konrad, B.Storrie, and D.A. Glaser, "Clonal variation in 

colony morphology and growth of Chinese hamster ovary cells 

cultured on agar", Cell, 10 (1977), 305-312. 

8. C.G. Sevastopoulos, C.T. Wehr, and D.A . Glaser, "Large-scale 

automated isolation of Escherichia coli mutants with 

thermosensitive DNA replication," submitted to Proc. Natl. Acad. Sci. 

9. C.G. Sevastopoulos and D.A. Glaser, "Mutator action by 

Escherichia coli strains carrying dnaE mutations," submitted to 

Proc. Natl. Acad. Sci. 

271 

5.1

5. WORKSHOP ON LABORATORY PROCEDURES 

 THE USE OF EARLY PASSAGE HAMSTER EMBRYO CELLS FOR THE
 

IDENTIFICATION OF CHEMICAL CARCINOGENS
 

ROMAN J. PIENTA
 

Chemical Carcinogenesis Program,
 NCI Frederick Cancer Research Center,
 Frederick, MD
 

METHODOLOGY
 

Culture Medium. Dulbecco's modified Eagle medium (DMEM)
 

containing sodium pyruvate and a low glucose content (Grand Island
 

Biological Company, Grand Island, N.Y.), supplemented with 2mM
 

L-glutamine and 20% sterile fetal bovine serum (FBS) (Rehatuin, F.S.,
 Reheis Chemical Company, Phoenix, Arizona), heat-inactivated at 56°C
 

for 30 minutes was used in all bioassay experiments. DMEM was
 

prepared either from powdered stock with deionized double-distilled
 

water and sterilized by pressure filtration through 0.22y Millipore
 

filter, or purchased as sterile liquid medium. No antibiotics were
 

used.
 

Quality Control. The reproducibility of the bioassay
 

employing early passage hamster embryo cells, requires stringent
 

control of the quality of serum, culture medium, and plastic culture
 

vessels. Only those lots of FBS specified by the supplier to be free
 

of adventitious microbial or viral agents were considered for use. To
 

confirm the absence of mycoplasma or bacteriophage, aliquot samples
 

were retested by independent laboratories. Aliquot samples of trypsin
 

solution were similarly monitored for contamination by mycoplasma.
 

All lots of DMEM, FBS, as well as plastic vessels (Falcon
 

Plastics, Oxnard, Ca.) were pretested for their ability to support
 

optimal growth of standard pools early passage hamster cells for two
 

subcultures when seeded to form individual colonies or monolayers.
 

Hamsters. Embryos from timed-pregnant Golden Syrian
 

hamsters of the outbred, systematically randomized LVG/LAK strain,
 purchased from Lakeview Hamster Colony, Newfield, N.J., served as the
 

source of primary embryo cells subsequently used as target or feeder
 

cells in all experiments.
 

273
 
Target Cells. Pregnant hamsters were sacrificed by carbon 

dioxide asphyxiation, usually on the 13th day of gestation. The uteri 

were removed aseptically and the embryos were transferred to a sterile 

petri dish containing warm calcium-, magnesium-free tissue culture 

diluent (TCD) prepared from 20X stock containing: I60g NaCl, 8g KC1, 

0.71g Na2HP04 and 2 ml 1% phenol red in 1 liter of deionized 

double-distilled water. For use, the concentrate was diluted to IX 

with deionized double-distilled water, supplemented with 0.1% glucose 

and adjusted to pH 7.4 with 1.5% NaHCOSThe embryos were decapitated, eviscerated, and the remaining 

tissue was minced finely with scissors or apposing scalpels, and then 

washed with TCD to remove extraneous red blood cells and debris. 

After a short preliminary wash with 0.25% trypsin solution to further 

remove any remaining red blood cells, the tissue fragments were 

disaggregated by treating the sample with trypsin at least twice for 

20 minutes at 37°C and pooling the supernatant cell suspensions. The 

cells were washed once by centrifugation at 200 x g for 10 minutes, 

resuspended in a minimum measured amount of TCD and counted. 

Approximately 1 X 107 cells were planted per 75 cm2 flask in DMEM 

and incubated at 37°C in a humidified atmosphere containing 10$ C02 

and air. When approximately 80-90/f confluent (usually 2-3 days) the 

cells were trypsinized, washed and dispensed into glass ampoules at 

2.5 or 5 X lo6 cells/ml in DMEM containing a final concentration of 

1.5% dimethylsulfoxide (DMSO) (Crown-Zellerbach Corp., Camas, Wa.) and 

20% FBS. If necessary, the cell suspension was adjusted to pH 7.4 by 

gassing with pure CC>2 immediately prior to flame-sealing the 

ampoules. The ampoules were placed into a mechanical freezer (Revco) 

at -85°C for 1 1/2-2 hours and immediately transferred to a liquid 

nitrogen freezer and stored in the liquid phase at -195°C until needed. 

Cells from aliquot samples were tested for their ability to 

undergo morphological transformation by the reference carcinogen, 3-methylcholanthrene (3MC). Suitable responsive pools of cells were 

then used in bioassay experiments. Nonresponding cultures were 

discarded. 

For use in the bioassay, an ampoule of frozen target cells 

was rapidly thawed by immersion in a 37°c water bath and seeded into a 

75 cm2 flask containing 20 ml DMEM and incubated at 37°C. The 

medium was changed daily until the culture was approximately 80-90/t 

confluent when the cells were trypsinized and seeded at 500 cells per 

dish in 2 ml of DMEM into 50mm plastic petri dishes previously seeded 

with x-irradiated feeder cells. 

Feeder Layer Cells. Primary cultures of cells subsequently 

used as feeder cells were prepared in a manner similar to that used 

for target cells. 

274 
Chemicals. Chemicals were stored over silica gel in a 

dessicator at either -20 C or ^ C, as required to maintain 

stability. Bioassays were performed in rooms illuminated by yellow 

lamps (Sylvania F40GO) to minimize photodecomposition of chemicals. 

All test chemicals were coded prior to bioassay to eliminate 

bias when stained dishes were monitored for transformation. Stock 

solutions were always prepared immediately prior to use. 

Water-soluble chemicals were dissolved in DMEM, while water-insoluble 

chemicals were initially dissolved or suspended in DMSO at 1-10 

mg/ml. These were further diluted with DMEM to desired concentrations. 

Safety Precautions. To minimize exposure of personnel to 

potentially biohazardous materials, all test chemicals were weighed 

out in an approved chemical fume hood and stock solutions were 

prepared in a vented laminar flow safety cabinet. Respirator masks, 

disposable gloves, and protective outer garments were worn. All cell 

culture manipulations were performed in laminar flow cabinets (Baker, 

Sanford, Me., or Contamination Control Inc., Kulpsville, Pa.). 

Chemically contaminated spent culture media and wash solutions were 

collected by vacuum aspiration onto absorbant material and 

subsequently incinerated. To avoid contamination from recycled 

glassware, only disposable labware was used and this was incinerated 

after use. Stained dishes were handled with gloves to avoid 

contamination by residual carcinogens. 

Bioassay. The procedure previously described (Berwald and 

Sachs, Nature, 200, 1182, 1963; DiPaolo, et. al., J.N.C.I. 42, 867, 

1969, Nature 235, 278, 1972) was modified to incorporate cryopreserved 

primary cell cultures as sources of target and feeder cells. Usually 

four days before use as a feeder culture, an ampoule of cryopreserved 

primary cells was rapidly thawed by immersion in a 37°C water 

bath. The cells were seeded into a 75 cm2 flask containing 20 ml 

DMEM and were refed daily. The day before x-irradiation, the cells 

were trypsinized and reseeded into 75 cm2 flasks at a concentration 

appropriate to obtain 75-80/6 confluence on the next day. The cells 

were then irradiated with 5000R by means of a Picker Portable 

Industrial x-ray machine fitted with 0.55 mm copper and 0.25 mm 

aluminum filters and with the cell monolayer 30 cm from the x-ray 

source. The cells were immediately washed with TCD, trypsinized and 

seeded at 6 X 10^ cells per 50 mm dish in 2 ml DMEM in the number of 

dishes required for the bioassay. 

For use as target cells in the standard bioassay an ampoule 

of cryopreserved cells from a pretested culture was rapidly thawed and 

seeded into a 75 cm2 flask containing 20 ml DMEM and incubated at 

37°C. The medium was changed daily and when the culture was approximately 80-90/& confluent it was trypsinized. Approximately 500 

275 
cells contained in 2 ml DMEM were seeded into 50 mm plastic dishes 

when had been seeded 24 hours previously with x-irradiated feeder 

cells. On the next day, graded doses of test chemicals, prepared 

double-strength in DMEM, were added to the cultures in 4 ml amounts to 

obtain the desired final concentrations of the chemicals in the 

culture medium. Control cultures were similarly treated with DMEM 

alone, or DMEM containing a final concentration of 0.2% DMSO. 

The cultures were incubated for 8 days without disturbing or 

refeeding. They were then washed twice with TCD to remove residual 

unbound chemical remaining on the dishes, fixed with methanol, and 

stained with Giemsa. After air-drying, the stained dishes were 

monitored by means of an Olympus Model JM zoom stereomicroscope (7-40X 

magnification) to identify normal and transformed colonies. 

Criteria for In Vitro Transformation. Although a 

heterogeneous population of colonies is observed in this system, the 

endpoint used for determining carcinogenic activity is the presence of 

fibroblast-like colonies morphologically altered beyond that observed 

in normal cultures. Cells in a transformed colony exhibit three- dimensional growth with extensive random-oriented growth and 

crossing-over at the periphery of the colony (DiPaolo, et. al., Cancer 

Res. 31, 1118, 1971, Pienta, et. al., Proc. 3rd. Int. Symp. on Cancer 

Detect. Prevention, 1976 in press). These cells usually have an 

increased ratio of nucleus to cytoplasm, are more basophilic, and are 

variable in size (Sanford, J.N.C.I. 53, 1481, 1974). The center of 

transformed colonies usually exhibits dense piling up of cells. These 

criteria are summarized in the chart. 

SOME CHITERIA FOR M DRPHOLOGIC TR ANSFORMATION OF IIAMSTER EMBRYO CELLS 

Colonia 1 Morphology 

Example Center Periphery Evaluation 

A P+ X+ Transformed (Contiguous Colony) 

B P+ X- Normal 

C P- X- Normal 

D P+ X+ Transformed (Semi dispersed 

Colony) 

P- = No piling up of cells 

P+ = Dense piling up of cells 

P = Moderate piling up of cells 

X- = Little or no crossing over of cells 

X+ = Extensive crossing over of cells 

276 
STANDARD HAMSTER EMBRYO CELL
 

IN VITRO CARCINOGENESIS BIOASSAY
 

Day 0
 

Reconstitute frozen standard feeder cells
 

from liquid nitrogen
 

Day 1
 

Refeed feeder cells
 

Day 3
 

Seed feeder cells onto 50 mm dishes at 10
 

cells/dish; reconstitute frozen standard target
 

cells
 

Day U
 

X-irradiate feeder cells with 5000.R, then 

refeed onto 50 mm dishes at 6 X 10 cells/ 

dish; refeed target cells 

Day 5 

Seed target cells onto feeder layer cells
 

at 500 cells/dish
 

Day 6
 

Add chemicals and incubate dishes without
 

disturbing or refeeding
 

Day 13
 

Fix with methanol, stain with Giemsa and monitor
 

dishes for transformation
 

277
 
5.2

 QUANTITATIVE FOCUS ASSAY FOR TRANSFORMATION OF SYRIAN 

HAMSTER EMBRYO CELLS BY CHEMICAL CARCINOGENS 

BRUCE C. CASTO 

BioLabs, Inc. Northbrook, IL

 

Cell Cultures. Primary hamster cell cultures (HEC) were 

prepared by trypsinization of eviscerated and decapitated embryos 

after 13-14 days of gestation. Cells were resuspended in Dulbecco's 

MEM medium or BioLabs Modified Dulbecco's medium (Casto, 1973) herein 

referred to as BMD, supplemented with 5% heat-inactivated Reheis fetal 

bovine serum (FBS), and 0.22g% NaHC03. Approximately 1.0 X 107 cells 

in 20 ml of medium were plated into 100-nm LUX plastic Petri dishes 

and incubated in a 5$ CC>2 atmosphere at 37°C. Total cell counts after 

3 days usually ranged from 1.0 to 1.2 X 107 cells per plate. Secondary HEC were prepared by transferring 5 X 106

 cells into 100-mm 

dishes in 20 ml of the above medium, but with 10% FBS. 

Chemical Transformation. Stock solutions of chemicals were 

prepared at concentrations of 1 or 10 mg/ml in acetone. Appropriate 

dilutions were made in complete medium to give the desired final 

concentrations. 

Secondary HEC were plated at 5 X 104 cells/60-mm dish in 

4 ml of BMD containing 10? FBS and O.llgJ NaHC03. After 24 hours, 4 ml of chemical dilution (as 2X concentrations) were added to each of 

5 to 10 plates per dilution and incubated for 3 days at 37°C in 5% 

C02. The culture fluid was then replaced with fresh medium (0.22g$ 

NaHC03) containing chemical dilutions at IX concentrations for an 

additional three days. After a total of 6 days exposure, the chemical 

containing medium was removed and the cultures fed with 6 ml of BMD, 10* FBS, and 0.22g* NaHC03 at 3 to 4 day intervals. After 21 to 25 

days from the time of chemical addition, the cells were formalin­ fixed, Giemsa stained, and scored for transformed foci. Verification 

of the transformed morphology was made with a stereomicroscope at 10X 

to 30X magnification. 

Cell lethality due to chemical was determined on plates 

seeded with 1000 cells and treated as above. The surviving colonies 

were formalin fixed and stained with 0.02% crystal violet 

approximately 8 days after seeding. The number of surviving colonies 

among cells treated with chemical was divided by the number of 

colonies in solvent treated cells to give the surviving fraction of 

chemically treated cells. 

278 
Figure 1. Transformed cell focus in hamster embryo cells following 6
 

days of exposure to 250 ug/ml of phthalazinone. Cells were
 

fixed and stained 21 days after removal of the chemical. x^O
 

279
 
Figure 2. Transformed cell focus in hamster embryo cells following


days of exposure to 2 ug/ml of 3-methylcholanthrene. Cells
 

were fixed and stained 21 days after removal of the
 

chemical. x80
 

280
 

 6 
To obtain cell lines from the transformed foci, selected
 

Petri dishes, after 17-20 days incubation, were overlayed with medium
 

containing 1% methylcellulose (Methocel). Five to 7 days later,
 individual foci were transferred with a Pasteur pipette and each
 

placed into a 60-mm dish in 3 ml of medium. The cells were fed every
 

3 to 4 days until a sufficient number was present for passage into
 

fresh dishes. The cultures were first passaged from one dish into 5
 

dishes and after establishment in culture were passaged weekly at 1:10
 

split ratios.
 

REFERENCES
 

1.	 Casto, B.C. "Enhancement of Adenovirus Transformation by
 

Treatment of Hamster Cells With Ultraviolet Irradiation, DNA Base
 

Analogs, and Dibenz(a,h)anthracene." Cancer Res., 33 (1973),
 402-407.
 

2.	 Casto, B.C., N. Janosko, and J.A. DiPaolo. "Development of a
 

Focus Assay Model for Transformation of Hamster Cells In Vitro by
 

Chemical Carcinogens." (submitted)
 

3.	 Evans, C.H., N. Janosko, B.C. Casto, and J.A. DiPaolo.
 "Development of a Quantitative Focus Assay for Transformation of
 

Hamster Cells by Chemical Carcinogens." Proc. Am. Assn. Cancer
 

Res., 17 (1976), 193.
 

281
 

5.3 IN VITRO CULTURE OF DIPLOID HUMAN SKIN EPITHELIAL CELLS 

FOR CHEMICAL TRANSFORMATION STUDIES 

ROBERT S. LAKE, AARON E. FREEMAN, HOWARD J. IGEL, MARY ROSE 

PEZZUTTI, AND MELVIN L. KROPKO 

Department of Pathology, Children's Hospital of Akron, 

Akron, OH 

Three problems are common to the culture of primary 

epithelial cells from a variety of epithelial tissues. These are: 

(1) Nutritional and cultural conditions for growth and 

maintenance of epithelial elements 

(2) Physical or selective removal of fibroblasts 

(3) Conditions for subculture with long-term retention 

of epithelial character. 

No one culture method yet exists which solves all of these 

problems. In this demonstration we show three culture methods 

currently being developed for in vitro carcinogenesis studies with 

human skin epithelium. Each of these methods for the in vitro culture 

of adult human skin has distinct advantages or disadvantages as 

outlined in Table 1. 

The first method is the growth of skin explants on a 

substrate of sterile dead pigskin (1). In this system, skin mince 

pieces are planted on pigskin which rests dermal side up on an organ 

culture type grid (Fig. 1A). Medium is added to the level of the 

grid such that the mince is nourished through the pigskin. After a 

period of 5-9 days, epithelial cells are seen to migrate from the 

mince onto the pigskin dermis and have commenced replication (Fig. 

IB). A continuous process of replication and differentiation ensues 

over the following weeks as clearing of the pigskin through digestion 

of the dermal substrate occurs. These clear areas represent digested 

pigskin dermis covered with the four layers of skin found in vivo — 

basal layer (germinative layer), squamous layer, granular layer, and 

horny layer (keratin) (Fig. 1C). Expansion of the epithelium 

continues until the pigskin surface is confluent with new epithelium. 

Several subcultures to new pigskin substrate can be achieved by either 

scraping new growth from the digested pigskin, mincing and replanting 

onto new pigskin, or by cutting out cleared areas and inverting on new 

pigskin. 

283 
284 
285 

Figure 1 (A) Progressive clearing of pigskin associated with human
 

skin epithelial cell growth. Each clear zone is
 

derived from one mince piece.*
 

(B)	 Histological section of a 14-day outgrowth of human
 

skin epithelial cells (NE) on pigskin. Pigskin
 

collagen fibers (PC) under the epithelium have been
 

partially digested while the pigskin epithelium (PE)
 

remains intact. (120X) Hematoxylin-Eosin stain.*
 

(C)	 Histological section of a 21-day outgrowth of human
 

skin epithelium on pigskin. Skin cells organize into
 

the basal (B), squamous (S), granular (G), and horny
 

(H) layers characteristic of in vivo skin. (240X)
 

Hematoxylin-Eosin stain.*
 

(D)	 Biopsy of an in vitro grown human epithelial skin
 

graft three months after surgery. Note reformation of
 

true epithelium with intercellular bridges and a
 

basement membrane (arrow) separating the dermal and
 

epidermal layers (6UOX) Hematoxylin-Eosin stain.
 

*	 Figure A, B, C from Freeman et al. (1) with permission
 

from In Vitro.
 
Grafting of new epithelium to an isogeneic host is achieved
 

by inverting the pigskin onto a prepared graft site so that the in 

vitro grown human cells are in direct contact with the wound bed and 

the pigskin serves as a protective dressing. After several days, new 

characteristic epithelium is found over the wound (Fig. ID). 

For in vitro carcinogenesis studies the pigskin system has 

the disadvantage that biochemical analysis is difficult because of 

interference of pigskin protein and dead dermal elements. In 

addition, periodic microscopic examination of the epithelium in a
 

horizontal plane is impossible; however, vertical sectioning of fixed
 

pigskin yields histological sections with excellent definition.
 

The second method involves growth and differentiation of
 

skin explants or trypsin-collagenase digests of skin on a layer of
 

6000R-irradiated mouse 3T3 fibroblast feeders (2). In this system,
 

(Fig. 2) the feeders serve to accelerate the growth of epithelial
 

cells (keratinocytes) while simultaneously retarding the growth of
 

dermal fibroblasts which invariably contaminate primary skin
 

cultures. Subculture is achieved by EDTA removal of feeders and a
 

small number of fibroblasts followed by complete dissociation of
 

epithelium with trypsin-EDTA. Upon replanting on new mouse 3T3
 

feeders, new epithelial islands arise with approximately 1% plating
 

efficiency. Two to four subcultures are feasible. The disadvantages
 

of this system are the possible contamination of human cells with
 

mouse endogenous viruses or other mouse genetic material and
 

interference of mouse cells with biochemical analysis of the donor
 

epithelial elements. Due to the low plating efficiency it is
 

difficult to increase the total population of epithelial cells on
 

subculture.
 

The third method is conventional explant growth on a tissue
 

culture grade plastic. Superior growth and longevity of primary
 

cultures is obtained in a closed or low gas exchange vessel (flask
 

T-75 or T-25). Open dishes for unknown reasons yield slower growth
 

and premature keratinization. Foreskin or biopsy skin mince pieces
 

are placed on a dry vessel surface and allowed to adhere for 0.5-3
 

hours without medium (dried on). Dulbecco's Modified Eagle's Medium
 

(DMEM) containing 5%-lO% fetal bovine serum (FBS) is then added.
 After 20-50 days of incubation at 36.5°C in a humidified incubator at
 

IQ% C02 certain mince pieces yield exclusively fibroblast colonies and
 

others yield true keratinizing epithelial colonies (Fig. 3). If the
 

original skin sample is cleaned exhaustively by scraping away most of
 

the dermis and underlying connective tissue, 70 to 90/6 of the mince
 

pieces will yield epithelial colonies free of fibroblasts. Each donor
 

sample will vary with respect to the relative growth of epithelial
 

versus fibroblastic elements; this is dependent on the amount of
 

dermis in each mince piece and the number of mince pieces per unit
 

area of the culture vessel.
 

286
 
Figure 2 (A) Thirty days of human skin epithelial cell growth on a
 

feeder layer of 5 X 105 irradiated 3T3 mouse fibroblasts. Foreskin
 

was digested overnight in 0.25$ trypsin, 0.125$ collagenase, and
 

seeded at 1 X 105 viable cells per 25 cm2 Falcon flask. Rhodanile
 

blue stain.
 

(B) Microscopic view of an epithelial cell island in the
 

above flask. (125X) Most islands emanate from clusters of input
 

cells as seen in the center of this island. Surrounding cells are 3T3
 

mouse fibroblast feeder cells.
 

28?
 
Figure 3. (Sheet 1 of 2) 

288 
Figure 3 (A) Forty-three days of epithelial cell growth from skin
 

explants grown in plastic flasks. Giemsa stain.
 (B) Microscopic view of an epithelial cell island in the
 

above flask. (125X) Note the fibroblasts apparent at the periphery
 

of this island.
 (C) Twenty-five days of growth from skin explants on
 

plastic. Two islands are keratinizing epithelium (right); the other
 

is dermal fibroblasts (left). Origin mince pieces to the far left
 

have no cell outgrowth; these are usually pieces of fat. Giemsa stain.
 

Figure 3. (Sheet 2 of 2)
 

289
 
The obvious advantage of this method is that direct phase 

microscopic examination is feasible, and individual colonies can be 

examined for some biochemical property such as macromolecule synthesis 

or carcinogen metabolism free of interference from feeder cells and 

pigskin proteins. A major disadvantage of this system of skin growth 

is that subculture by conventional trypsinization and replanting is 

uniformly a failure since only fibroblasts remain after 1 or 2 

subcultures. 

For in vitro chemical carcinogenesis studies, we hope to 

combine these methods to characterize critical variables and develop a 

transformation assay in some animal model. With increased survival of 

epithelial cell types on a feeder system there will be the potential 

of retaining more target cells with the requisite number of cell 

divisions during and after treatment with chemicals. Using plastic 

grown mixed epithelial and fibroblast cultures we have demonstrated 

that skin epithelial cells have greater carcinogen metabolism than 

dermal fibroblasts from the same donar (3). With the advantage of 

being able to graft epithelium from pigskin to an isogeneic host with 

reformation of true skin epithelium it may be possible to demonstrate 

carcinogenicity if the target cells are of epithelial origin. 

TABLE 1. SKIN EPITHELIAL CELL CULTURE METHODS
 

METHOD
 

Pigskin Substrate
 

Plastic with Feeders
 

Plastic
 

ADVANTAGES
 

Grafting 

Slow Fibroblast Take Over 

Histopathology 

Subculture 

Growth From Dispersed Cells 

Survival- of More Epithelial 

Cell Types 

Subculture 

Slow Fibroblasts Take Over 

Microscopic Examination 

Explant Colonies of Epithelial or Fibroblast Origin 

Biochemical Analysis 

Microscopic Examination 

DISADVANTAGES 

Biochemical 

Analysis 

Microscopic 

Examination 

Agents From 

Feeder Cell 

Biochemical 

Analysis 

Low Plating 

Efficiency 

Subculture 

Rapid Fibroblast 

Take Over 

290 
REFERENCES
 

1.	 Freeman, Aaron E., Howard J. Igel, Brenda J. Herrman, and Karen
 

L. Kleinfeld. "Growth and Characterization of Human Skin
 

Epithelial Cell Cultures." In Vitro 12 (1976), 352-362.
 

2.	 Freeman, Aaron E., Robert S. Lake, Howard J. Igel, Lisa Gernand,
 Mary Rose Pezzutti, James M. Malone, Corey Mark, and William F.
 Benedict. "Euploid to Heteroploid Conversion of Human Skin Cells
 

by Methylcholanthrene." Proceedings Natl, Acad. Sci. (Submitted).
 

3.	 Rheinwald, James G., and Howard Green. "Serial Cultivation of
 

Strains of Human Epidermal Keratinocytes: The Formation of
 

Keratinizing Colonies from Single Cells." Cell 6 (1975), 331-344.
 

291
 

PROCEDURES 

Cell Cultures 

Stocks of normal fibroblasts derived from foreskins were
 

established, cultured, and stored until use in liquid nitrogen as
 

described previously (2,3). Excision repair defective fibroblasts,
 derived from skin biopsy specimens of XP patients as well as
 

Lesch-Nyhan patients were obtained from the American Type

Collection (Rockville, Md.) and similarly cultured. Cells

these experiments were from stocks in passages 6-18. 

 Culture 

 used in 

Culture Medium 

Stocks were cultured in 250 ml plastic flasks in a humid 

atmosphere of 5% CO2 and air at 37°c in Eagle's medium with Earle's 

salts supplemented with 15% fetal bovine serum (GIBCO, Grand Is., N.Y.) and gentamycin (50 yg/ml) (Scherring Corp., Union, N.J.). Hams 

F10 lacking hypoxanthine (GIBCO) supplemented with 15% fetal bovine 

serum and streptomycin (100 ug/ml) and penicillin (100 units/ml) was 

the medium used for experiments unless otherwise stated. 

Selective Medium 

The medium for selecting and culturing 8-azaguanine 

(AG)-resistant cells was Ham's F10 lacking hypoxanthine and 

supplemented with 15$ calf serum (GIBCO, Grand Is, N.Y.) and SAG at 

2 X IO-SM (Sigma, St. Louis, Mo.). The conditions were standardized 

by choosing lots of serum which not only supported high cloning 

efficiencies in XP strains, but also did not interfere with 

selection. The concentration of SAG used for the selection (viz., 2 X 

10~5M) was demonstrated to be high enough to insure that surviving 

colonies would represent SAG-resistant cells in the population (see 

Fig. 1). Although SAG and thioguanine give very similar dose response 

curves, 8-azaguanine was preferred to thioguanine as a selective agent 

in the present studies because, under our conditions, it was 

demonstrated that SAG prevented a logarithmically growing cell 

population from undergoing even a partial doubling, whereas 

293 
291

 

Figure 1. Cytotoxicity of 8-azaguanine in human fibroblasts (from Ref. 7) 
thioguanine allowed one or more population doublings before growth
 

cessation (Maher, et al, unpublished data). This phenomenon would
 

lead to complications caused by high cell density (see below,
 "metabolic cooperation").
 

Cytotoxicity Assay (Survival of Cloning Capacity) (See Fig. 2A) 

The techniques for determining the % survival of the cloning 

capacity of the cells following exposure to these compounds have been 

described (3,4). Briefly, appropriate numbers of cells (250-3000) 

were plated in 60 mm dishes and allowed time (ca. 10-12 hours) for 

attachment. The culture medium was replaced with serum-free medium 

and the compound to be tested was introduced by micropipette into the 

individual dishes. After 3 hours the medium was removed and the cells 

were refed with culture medium. Since proteins possess strong 

nucleophilic centers, the latter medium containing 15% fetal bovine 

serum should inactivate any remaining traces of electrophilic 

reactants. All operations with the compounds were carried out in 

subdued light and cells were protected from light until they had 

undergone several cell divisions. Cells were refed 3 times weekly 

until clones developed to macroscopic size (ca. 12-14 days), (see Fig. 2). Cloning efficiencies for the untreated cells ranged from 20-35$ 

for normal fibroblasts, 10-25$ for XP2BE and XP4BE, and 10-15$ for 

XP12BE. The cloning efficiency of the treated cells, divided by the 

cloning efficiency of the control cells which received solvent only 

determined the cytotoxicity of the compound and is expressed as a 

percent. The final concentration of acetone in the medium (0.45$) was 

not cytotoxic to the cells. 

For ultraviolet irradiation (254 nm) of cells, the protocol 

was as described above with the following modifications. When the 

medium was removed after 10-12 hours, the cells were rinsed in 0.9$ 

saline, exposed to a known dose of ultraviolet irradiation, and medium 

containing 15$ serum was added to each dish. Cells were refed 3 times 

weekly until clones developed to a macroscopic size. Ultraviolet 

irradiation was carried out with a UVS-12 or UVS-54 ultraviolet lamp 

(Ultraviolet Products, San Gabriel, CA). The dose was determined with 

a J-225 meter from the same manufacturer. 

Mutagenicity Assay (See Fig. 2B) 

For each dose, a population of 1.0-2.4 X 106 cells was 

plated in a total of 120 60 mm dishes. After a 12-14 hour cell 

attachment period, the medium was replaced with serum-free medium and 

the cells exposed to the carcinogen as described above. A 

cytotoxicity experiment was carried out in parallel with each 

mutagenicity experiment to determine the number of survivors. After 3 

295 
A. CYTOTOXICITY
 

B. MUTAGENESIS 

0.7 - 1.8 x 1C 

cells/dish 

100-250 dishes 

per dose 

Rinse, 

Irradiate, 

Give Medium 

Expression Time 

Permit 3-4 

Tell Doublings 

8-Azaguanine 

selection 

medium 

Fresh 8-Azaguanine medium 

every 2 days 

Fix, stain 

and count 

dishes with 

clones 

Figure 2. Experimental protocol for UV exposure 

of human fibroblasts (from Ref. 6) 

Figure 3. Left: normal skin fibroblast colony
 

Right: azaguanine resistant colony (from Ref. 5)
 

296
 
hours the medium was removed and the cells refed culture medium
 

containing 15$ fetal bovine serum. Each population was allowed
 

sufficient time for the surviving cells to undergo 3 doublings which
 

were required to overcome phenotypic lag of cellular expression of
 

azaguanine resistance before 8-azaguanine selection was begun.
 Because azaguanine has been shown to be unstable in the presence of
 

serum (10), the cells were refed selection medium 3 times weekly for
 

ca. 21 days until macroscopic resistant colonies could be detected by
 

examining the petri dishes in a darkened room with a lamp which gave
 

off a focused beam of light. Dishes in which a macroscopic resistant
 

colony (500 or more cells) was detected were maintained; the rest were
 

rinsed, fixed, stained, and examined for undetected resistant colonies
 

composed of at least 100 cells. The frequent refeeding of the petri
 

dishes might be expected to lead to the formation of satellite
 

colonies which would result in an overestimation of the number of
 

mutants. That this is not the case is demonstrated by the fact that
 

the number of 8AG-resistant colonies formed follows a Poisson
 

distribution (see example below). Furthermore, the P(o) method used
 

to determine the number of mutants (see below) prevents one from
 

making such an overestimation. A control population of 1-1.5 X 106
 

cells was included with every experiment to determine the background
 

mutation frequency. The frequencies of spontaneous or preexisting
 

mutants in each of the experimental populations used in this study
 

were closely matched so that a valid comparison between different
 

strains could be made.
 

Reconstruction Experiments to Determine Efficiency of Recovery of Mutants 

Since overcrowding of cells in the dishes can result in an 

apparent reduction in mutation frequency caused by "metabolic 

cooperation" (9) , in every experiment a known number of 8AG-resistant 

Lesch-Nyhan cells was seeded into ten of the control and ten of each 

set of experimental dishes as well as into a set of dishes lacking a 

monolayer of cells to provide an estimate of the efficiency of the 

recovery of mutants under the conditions of each experiment. The 

average number of colonies of Lesch-Nyhan cells in each series of 

dishes containing a non-resistant monolayer divided by the average 

number of colonies of Lesch-Nyhan cells found in the dishes which 

lacked such non-resistant cells yielded the efficiency of recovery and 

was expressed as percent. The relationship between the efficiency of 

recovery of azaguanine resistant cells 'and the total number of cells 

viable or non-viable) attached to the surface of the 60 mm dishes at 

the time selection was begun has been determined from a large series 

of such reconstruction experiments with human fibroblasts and is shown 

in Fig. 4. This empirically-generated data was used to pre-determine 

the number of cells to be exposed to the various doses of the 

hydrocarbon to epoxides to insure that the surviving population could 

297 
Figure 4. Relationship between efficiency of recovery of 8-AG resistant colonies and the density 

of non-resistant cells in the dish at the beginning of selection (from Ref. 7) 

298 
undergo three doublings without the total number of cells in the dish
 

at the beginning of selection (6 days later) exceeding that which
 

would permit an efficiency of recovery of at least 30-UOyL That
 

Lesch-Nyhan cells can validly be used to estimate the efficiency of
 

recovery of AG-resistant human cells has been recently demonstrated by
 

Van Zeeland and Simons (9). They published a similar curve based on
 

the density of untreated cells.
 

Determining the Frequency of Induced Mutations 

The method used to quantitate the frequency of mutations has
 

been described in detail by Albertini and DeMars (1) and by us (3,
 7). In summary, this consists of determining the probability of a
 

mutational event occurring per experimental culture dish from the
 

number of culture dishes which contain no clones: P (0) = exp (-x)
 

where x is the probability of a mutant event per dish. For each
 

experiment this value is corrected first, for the efficiency of
 

recovery of mutants and secondly, for the small contribution of the
 

background mutation frequency. From the probability of mutational
 

event occurring per dish, the number of viable-cells per dish, and the
 

cloning efficiency of the cells in each particular experiment, the
 

frequency of induced mutational events per 10^ survivors is
 

determined. The need for correcting for cloning efficiency has been
 

demonstrated (10). (See example in Table I.)
 

Analysis ofAzaguanine Resistant Colonies 

At the end of 21 days of selection, resistant colonies were
 

detected as described above. A representative proportion of such
 

colonies were isolated and cultured in selection medium containing 8AG
 

until they reached sufficient size for the cells to be tested for
 

levels of hypoxanthine guanine phosphoribosyltransferase (HPRT, E.G.
 2.4.8.2.) enzyme activity as described by Albertini and DeMars (1). A
 

second group of resistant colonies was isolated, and cultured in
 

non-selective medium for 11-13 generations. At this time they were
 

replated at low number and tested for ability to form colonies in
 

normal culture medium, in azaguanine selection medium, and in medium
 

containing hypoxanthine, aminopterin, and thymidine (1) which selects
 

against cells with little or no HPRT enzyme activity.
 

The research reported from the authors' laboratory was supported
 

by Grants CA-21247 and CA-21253 awarded by the National Cancer
 

Institute, D.H.E.W.
 

299
 
TABLE I. EXAMPLE OF CALCULATING THE FREQUENCY OF

 

INDUCED MUTATIONS FROM THE DISTRIBUTION OF 

RESISTANT CLONES IN THE POPULATION [|(0) METHOD] 

Cloning efficiency of the cells = 0.2 Control Dose 1 Dose 2 Dose 3 

Number of cells plated into dish 10,000 12,000 15,000 18,000 

Percent cell survival 100 60 35 18 

Viable cells per dish 10,000 7,200 5,250 3,240 

Dishes with 0 clones 91 87 80 82 

Dishes with 1 clone 3 5 5 4 

Dishes with 2 clones 1 1 1 1 

Dishes with 3 clones 0_ 1 1 1_ 

Total mutant clones 5 10 10 9 

Total dishes scored 95 94 87 88 

p/ 0 \ a _ (Dishes with 0 clones) 

(Total dishes scored) 0.958 0.925 0.920 0.932 

x (chance of mutant event per dish) 0.043 0.077 0.084 0.071 

Efficiency of recovery (data from reconstr.) 50% 60% 50% 50% 

Corrected xc 0.086 0.129 0.167 0.141 

Chance of a spontaneous or pre-existing mutant per dish 0.086 0.062 0.045 0.028 

Chance of an induced mutant per dish — 0.067 0.122 0.113 

Frequency of induced mutants per 10 viable cells — 0.93 2.32 3.49 

Frequency of induced mutants per 10

corrected for 20% cloning efficiency 

 viable cells — 4.6 11.6 17.4 

Probability of a dish containing no clones. 

Calculated from P(0)=ex; Poisson Distribution Function. 

°Chance of a mutant event per dish corrected for efficiency of recovery (see text). 

Obtained from control population dishes and calculated for each population. 

e

Calculated by subtracting the chance of a spontaneous mutant from total chance, x c . 

Calculated from chance of induced mutant event per dish divided by number of viable cells. 

300 
REFERENCES
 

1.	 Albertini, R.J., and R. DeMars. "Somatic Cell Mutation Detection
 

and Quantification of X-Ray-induced Mutation in Cultured, Diploid
 

Human Fibroblasts." Mutation Res., 18 (1973), 199-224.
 

2.	 DeMars, R., and W.E. Nance. "Electrophoretic Variants of
 

Glucose-6-phosphate Dehydrogenase and the Single-active-X in
 

Cultivated Human Cells." In: Retention of Functional
 

Differentiation in Cultured Cells. V. Defendi (Ed.), Wistar
 

Press, Philadelphia, (1964), 35-46.
 

3.	 Maher, V.M., and J.E. Wessel. "Mutations to Azaguanine
 

Resistance Induced in Cultured Diploid Human Fibroblasts by the
 

Carcinogen, N-acetoxy-2-acetylaminofluorence." Mutation Res., 28
 

(1975), 177-284.
 

4.	 Maher, V.M., N. Birch, J.R. Otto, and J.J. McCormick.
 "Cytotoxicity of Carcinogenic Aromatic Amides in Normal and
 

Xeroderma Pigmentosum Fibroblasts with Different DNA Repair
 

Capabilities." J. Natl. Cancer Inst., 54 (1975), 1287-1294.
 

5.	 Maher, V.M., R.D. Curren, L.M. Ouellette, and J.J. McCormick.
 "Effect of DNA Repair on the Frequency of Mutations Induced in
 

Human Cells by Ultraviolet Irradiation and by Chemical Carcin­ ogens." In: Fundamentals in Cancer Prevention, P.N. Magee,
 S. Takayama, T. Sugimura, and T. Matsushima (Eds.), University of
 

Tokyo Press, Tokyo (1976), 363-382.
 

6.	 Maher, V.M., R.D. Curren, L.M. Ouellette, and J.J. McCormick.
 "Role of DNA Repair in the Cytotoxic and Mutagenic Action of
 

Physical and Chemical Carcinogens." In: In Vitro Metabolic
 

Activation in Mutagenesis Testing. F.J. de Serres, J.R. Fouts,
 J.R. Bend, and R.M. Philpot, (Eds.), North Holland Biomedical
 

Press, Amsterdam (1976) 313-336.
 

7.	 Maher, V.M., J.J. McCormick, P.L. Grover, and P. Sims. "Effect
 

of DNA Repair on the Cytotoxicity and Mutagenicity of Polycyclic
 

Hydrocarbon Derivatives in Normal and Xeroderma Pigmentosum Human
 

Fibroblasts." Mutation Res., 43 (1977), 117-138.
 

8.	 Maher, V.M., L.M. Ouellette, R.D. Curren, and J.J. McCormick.
 "The Frequency of Ultraviolet Light-induced Mutations is Higher
 

in Xeroderma Pigmentosum Variants than in Normal Human Cells."
 

Nature, 261 (1976), 593-595.
 

301
 
9.	 va Zeeland n , A.A., and J.W.I.M. Simons. "The Use of Correction
 

Factors in the Determination of Mutant Frequencies in Populations
 

of Human Diploid Skin Fibroblasts." Mutation Res., 34 (1976),
 149-158.
 

10.	 van Zeeland, A.A., M.C.E. van Diggelen, and J.W.I.M. Simons.
 "The Role of Metabolic Cooperation in Selection of HGPRT
 

Deficient Mutants From Diploid Mammalian Cell Strains." Mutation
 

Res., 14 (1972), 355-363.
 

302
 
APPENDIX A:
 

Survival Curves
 

1.	 Compute the average number of clones per dish.
 

2. Chart the number	 of cells plated originally and the dose of agent.
 

3.	 Compute cloning efficiency as follows:
 

Cloning efficiency	 = Average clones per dish
 

Number of cells plated
 

4.	 Compute percent survival as follows:
 

Percent survival = Cloning efficiency (experimental)
 

Cloning efficiency (control)
 

5.	 On semilogarithmic paper plot percent survival (log axis) versus
 

dose.
 

303
 
APPENDIX B: 

Procedure for Staining Dishes 

1. Pour medium off into sterilizing bucket. 

2. Put bottom of dish containing cells into perforated bucket 

3. Plates in buckets go into: 

a) 0.ti? - 0.9? saline for quick rinse (app. 30 seconds) 

b) 95% methanol for fixing app. 5 minutes 

c) 2% methyline blue 10 minutes 

d) running tap water in sink for few minutes 

4. Each plate is then individually rinsed in tap water for several 

minutes. 

5. Plates then turned upside down on large trays and placed in an 

incubator at 37°C to dry, taking care to keep sets separated and 

identifiable. 

304 
APPENDIX C: 

Standard Procedure for Starting Primary Cultures (Mincing) 

1. Wash tissues 2 times with 8-10 ml F-10 containing 500 units/ml 

Pen-Strep in 60 mm dish. 

a) discard medium after each wash 

2. Haras F-10 liquid medium is added back to the dish, but not so as 

to float the tissue. 

3. Tissue is then minced up using aseptic technique; the tissue to be 

minced refers to pieces ca. 1 cm square which are cut from the 

whole foreskin. These pieces are then minced into ca. 1 mm pieces. 

4. The minced tissue is then transferred to a dry 60 mm dish where: 

a) it is held firmly in place by pressing down lightly with a 

small amount of sterile silicone grease on one corner 

b) this dish then receives app. 5.0 ml of medium (Ham's F-10, 15% 

fetal calf serum, and pen strep) 

5. After two days this medium is discarded and new growth medium is 

added. 

a) the culture should then be refed every day 

6. Epithelial cells will be seen in @ 3-5 days. 

7. Fibroblasts will be seen in @ 3-5 days. 

8. When good colonies of fibroblasts are seen, cells may then be 

transferred. Epithelial cells are lost within the first or second 

transfer of these cells. 

305 
APPENDIX D: 

Procedure for Transfer of Cells 

1. Have receiving vessels (dishes, flasks) prelabeled and containing 

proper amount of growth medium (20 ml/250 ml flask, 5 ml/60 mm 

dishes). 

2. Trypsin and serum free medium for rinsing should be warmed to 

37 C for best results. Trypsin, 0.25? (Gibco). 

3. Have canulas attached to syringe in hood set-up and ready to use. 

4. Drain off growth medium by aspiration. 

5. Cover cells with normal amount of warm 37 C serum free medium 

(20 ml/250ml flask, 5 ml/60 mm dish) and allow to stand for a few 

minutes, discard. 

6. Replace warm 37°C serum free media with trypsin (3 ml P-60, 7-8 

ml T-75). 

a) observe culture under scope 

b) when 80-90$ of the cells have rounded up, remove trypsin 

c) rinse culture with minimal amount of serum-free media to 

dilute trypsin 

7. Replace trypsin with 6.0 ml regular growth medium (3.0 ml for 60 

mm dishes) and force cells off bottom using canula with syringe 

and gently pipetting them. 

a) bottom of dish may be seen to be free of cells by reflecting 

in light at proper angle 

8. Transfer suspended cells to prelabeled culture flasks. 

306 
APPENDIX E: 

Procedure for Freezing Cells 

1. See cell transfer steps 2-6. 

7. Replace rinsing medium with enough growth medium containing DMSO 

Freezing Medium=DMSO growth medium = F-10 + PCS 15$ + DMSO 5% to 

fill the respective freezing vials - but only in one culture 

vessel [eg. 4 T-75's to freeze each freezing vial-1.5 ml 1.5 x 4 = 

6.0 ml - this 6.0 ml is put in only 1 of the T-75's and this is 

used to force off the cells from the bottom of the culture 

vessel]. This medium containing cells is then transferred to the 

next flask and so on until all 4 flasks have been completed. 

8. This 6.0 ml of cells is then placed in freezing vials. 

Note: This technique is for a 1:1 ratio regardless of the no. of 

cells of T-75 freezing vial, but may be easily adjusted if a 

different ratio is wanted. 

307 
5.5 FIBRINOLYTIC ACTIVITIES AND QUANTITATIVE SOFT AGAR ASSAYS 

J. CARL BARRETT 

Division of Biophysics 

Johns Hopkins University, School of Hygiene and Public Health 

Baltimore, MD 

PREPARATION OF FIBRINOGEN 

References: (1,4) 

IMPORTANT: Use no glass! 

1. Dissolve 10 grams fibrinogen (60-70$ clottable) in 500 ml of 

0.1 M phosphate buffer (pH 6.41) in a plastic beaker. 

2. Dilute solution with 500 ml double distilled H20 and allow to 

stand overnight in the refrigerator. 

3. Filter off formed precipitate and obtain clear supernatant 

(fibrinogen I) 

4. To supernatant, add slowly (stirring at room temperature) 330 ml 

of saturated ammonium sulfate, then stir approximately 30 minutes. 

5. Centrifuge down the flocculent white precipitate. 

6. Dissolve pellet in ~100 ml 0.6 M NaC1 and 0.003 M EDTA. May 

take several hours. Pellet should be dissolved before moving to 

cold room. 

7. Dialyze overnight against 0.6 M NaCl and 0.003 M EDTA in the cold. 

8. Dialyze against PBS (Ca+, Mg+ free) in cold. 

9. Filter sterilize through 0.22'jj Nalgene sterile filter. May need 

to prefilter 1-3 times through fluted filter paper and may need 

to dilute and warm (37°C) before filter sterilizing. 

10. Determine protein concentration and freeze at 10 mg/ml (16 CD 

280/ml). 

308 
PREPARATION OF PETRI DISHES COATED WITH 125I-FIBRIIM 

1.

12"5 I-Fibrinogen prepared by the method of Helmkamp, et al. (3). 

2. Pipette radioactive fibrinogen solution into a petri dish 

(10yg/cm at 2000-5000 cpm/yg of fibrinogen). 

3. Bake at 45 °C overnight. 

4. Convert the fibrinogen to fibrin by incubating the plates with 

2 ml E MEM with 5% fetal calf serum 2 hours at 37°C. 

5. Wash 2 times with PBS. 

6. Add 2 ml assay buffer (Tris HC1 - 0.1M pH 8.0). 

PREPARATION OF HARVEST FLUID 

1. Wash the cultures 2 times with PBS. 

2. Add fresh medium without serum (E MEM, or E MEM with 5% acid 

treated fetal calf serum). 

3. Incubate for 18 hours at 37°C. 

4. Collect the medium and freeze (-20°C). 

PREPARATION OF INTRACELLULAR PLASMINOGEN ACTIVATOR 

1. Wash the cultures 2 times with PBS. 

2. Scrape the cells into buffer with rubber policeman. 

3. Centrifuge and wash 2 times (1000 xg 5 minutes). 

4. Add ~ 10 vol. of 0.5% Triton in Tris-HCl 0.1M pH 8.0. 

5. Aspirate with Pasteur pipette. 

6. Centrifuge to pellet the nuclei (1000 xg, 10 minutes). 

7. Freeze the supernatant (-20 C). 

309 
PREPARATION OF PLASMINOGEN AND PLASMINOGEN FREE SERUM 

Reference: (2). 

1. Couple cyanogen bromide activated sepharose-4B with L-lysine 

(1 gm of lysine per 5 ml of activated, moist sepharose). 

2. Prepare a column (1 ml of bed volume/5 ml of serum), slowly pass 

the serum through and then elute with 0.3 M phosphate buffer. 

3) Elute the plasminogen with 0.1M EACA in phosphate buffer. 

4) Repeat and dialyze the plasminogen extensively against PBS. 

PROTOCOL FOR FIBRIN OVERLAY ASSAY 

1) Mix 2x EMEM (or 2 MEM) with dog serum (4:1). 

2) Tube out 2x EMEM with 20% dog serum in 2 ml aliquots, incubate at 

42°C. (One tube/plate). 

3) To each tube add 2 ml of 2.5% agarose at 42°C. 

4) Suction off medium from plate and wash with medium without serum. 

5) To one tube, add 0.5 ml Fibrinogen II (10mg/ml and mix. Replace 

in water bath. 

6) Add 20-50 pi thrombin to tube, mix rapidly, and quickly pour on 

plate. 

7) Let solidify 5-10' at room temperature, then put into 37° CO^ 

incubator. 

8) Read at 3 hr and 5 hr and 24 hr for lysis. 

PROTOCOL FOR GROWTH IN SOFT AGAR 

1) Prepare 1.2% agar, 0.4* bacto-peptone. Melt in boiling water 

bath, equilibrate to 45 C. 

2) Prepare 2x medium with 20% serum. Equilibrate to 45 C. 

3) Mix the above 1:1 to get O.b% agar, 0.2% peptone in Ix medium and 

10? serum at 45°. 

4) Plate 4ml of this mixture onto 60mm culture dishes for base 

layer. Let firm at room temperature for 15 minutes. 

310 
5) For testing 10.5 cells, to the mixture prepared in step (3), add 

an equal volume of cell suspension at SxlO2* cells/ml in Ix medium 

(90$)/fetal calf serum (10J). This gives a suspension of 2.5 x 

10^ cells/ml at 0.3% agar and 0.1% peptone. 

6) Immediately place 4 ml of this suspension over base layers to 

complete agar plates at 10.5 cells. Let firm in 24° incubator 

for 1 hour, then switch to 37°c incubator for 2 weeks when they 

are examined for macroscopic and microscopic colony formation. 

7) If additional incubation time to allow further enlargement of 

clones is desired, incubation at 37° is continued with weekly 

additions of 1 ml growth medium to preclude dehydration of the 

agar. 

DEFERENCES 

1. Barrett, J.C., B.D Crawford, and P.O.P. T'so. "Quantitation of 

Fibrinolytic Activity of Syrian Hamster Fibroblasts Using 

^H-labeled Fibrinogen Prepared by Reductive Alkylation." 

Cancer Res.. 37 (1977), 1182-1185. 

2. Deutsch, D.G. , and E.T. Mertz. "Plasminogen: Purification from 

Human Plasma by Affinity Chromatography." Science. 170 (1970), 1095-1096. 

3. Helmkamp, R.W. , R.L. Goodland, W.F. Bale, I.L. Spar, and L.E. Mutschler. "High Specific Activity lonation of Y-Globulin with 

I

131

 Monochloride." Cancer Res.. 20 (1960), 19^5-1950. 

4. Laki, K. "Polymerization of Proteins: The Action of Thrombin on 

Fibrinogen." Arch. Biochem. Biophys.. 32 (1951), 317-324. 

311 
5.6

 ASSAYS FOR SOMATIC MUTATION STUDIES ON SYRIAN HAMSTER EMBRYONIC CELLS 

J. CARL BARRETT 

Division of Biophysics 

Johns Hopkins University School of Hygiene and Public Health 

Baltimore, Md. 

A. PROTOCOL FOR THE INDIRECT POPULATION ASSAY FOR SOMATIC MUTATION STUDIES 

OF SYRIAN HAMSTER EMBRYONIC CELLS 

1. IxlO6 Syrian embryonic fibroblasts of passage 2 to passage 3 

(same cells used for in vitro neoplastic transformation) are 

seeded in a 75 cm2 flask. 

2. After 15 hours, the cells are treated with MNNG at a concen­ tration of IxlO6

 or 5x10-6 M for 2 hours, or with B(a)P at a 

concentration of 1 yg/ml or 10 yg/ml for 24 hours. 

3. After the exposure time, the flasks treated with MNNG are washed 

thoroughly with phosphate buffer-saline (PBS) and the flasks 

treated with B(a)P are washed five times by the complete medium 

(containing 5% serum), the same washing procedure used in the 

neoplastic transformation assay. 

4. The flasks are then allowed to reach confluency in 4-7 days. 

5. The flasks at confluency are subcultured at a split ratio of 1:10 

and allowed to grow to confluency. Confluency is normally obtained in 7 days with untreated cells but 5xlO~6

 M MNNG or 10 

yg/ml B(a)P treated cells require 2-3 weeks to reach confluency. 

6. At each passage, 105 cells are seeded in a 100 mm petri dish. After 15 hours, 8-azaguanine containing medium is added to the 

petri dish to a final concentration of 40 

312 
B.

 PROTOCOL FOR THE DIRECT CLONAL ASSAY FOR SOMATIC MUTATION STUDIES ON 

SYRIAN HAMSTER EMBRYONIC CELLS 

1. 105 Syrian hamster embryonic fibroblasts are seeded in a 100 mm 

petri dish. 

2. After 15 hours, these cells in the petri dishes are treated with 

MNNG and B(a)P as described earlier in the indirect population 

assay. Parallel experiments with 2 x 104 cells/100 mm petri 

dishes are conducted to determine toxicity as measured by the 

decrease in cloning efficiency. 

3. These cells are then washed, refed with complete medium for a 

recovery period of 1-3 days. 

4. After the recovery period, 40 yg/ml of 8-azaguanine is added to 

the medium and the cells are grown in this selective medium for 

3-4 weeks with refluiding before fixed, stained, and scoring for 

mutated colonies. 

313 
5.7

 ANALYSIS OF DMA STRAND BREAKS BY ALKALINE SUCROSE 

GRADIENTS 

ANDREW R. PETERSON 

Los Angeles County—University of Southern California 

Comprehensive Cancer Center, Los Angeles, CA. 

Five to 30% sucrose gradients were prepared by 4 cycles of 

freeze-thawing (1) of tubes containing 18% sucrose in 0.1M EDTA which 

was adjusted to pH 12.6 with 10M NaOH. Primary hamster cell cultures, after 1 days' incubation, were passaged to 60-ram plastic dishes using 

4 x 105 cells per dish. Immediately, or after 24 hours, the cells 

were pulsed with 0.5 pCi/ml of H-labeled thymidine (3H-TdR) for 24 

hours. After H-TdR labeling, all cultures were changed to GERM with 

0.5% FBS and 0.22 gm$ NaHCOS, and held for an additional 24 hours. At 

this time each dish contained approxi- mately 1O6 cells. The test 

chemicals were usually prepared as 1-10 rag/ml stocks in acetone, acetone-water, or DMSO and added to prewarmed medium to give the 

desired final concentration. The HEC were treated for 2 hours or for 

18 hours, washed Ix, and removed from the dish with EDTA. Following 

centrifugation, the cells were resuspended in EDTA to give 10 cells 

per 0.2 ml. Two-tenths ml of the cell suspension was then added to 

the top of a sucrose gradient tube layered with 0.3 nil of lysing 

solution C\% sarkosyl in 0.0.5? EDTA). The cells were then lysed at 

room temperature for 1 hr, placed in an SW-50 rotor, and centrifuged 

for 1 hr at 30,000 rpm in a Model L-2 ultracentrifuge at 20°c. Three 

drop fractions were collected directly into scintillation vials 

following bottom puncture, neutralized with 1.0 ml of 0.2N HC1 and 

prepared for counting by addition 5 ml of Bray's scintillation fluid 

to each vial. Counts were made in a Packard Tri-Carb scintillation 

spectrometer, 10 minutes per sample. Data were plotted as percent of 

the highest count in each gradient. A shift in the peak of 

radioactivity greater than 3 fractions (more than 6 mm) from the 

control peak was considered positive evidence for chemical-induced DNA 

strand breaks. 

REFERENCES
 

1. K.L. Baxter-Gabbard. "A simple method for the large-scale 

preparation of sucrose gradients." FEES Letters, 20 (1972), 

117-119. 

2. Casto, B.C. , W.J. Pieczynski, N. Janosko, and J.A. DiPaolo. "Significance of Treatment Interval and DNA Repair in the 

Enhancement of Viral Transformation by Chimical Carcinogens and 

Mutagens." Chem-Biol. Interactions, 13 (1976), 105-125. 

314 
5.8 ANALYSIS OF DNA REPAIR SYNTHESIS BY AUTORADIOGRAPHY 

ANDREW R. PETERSON 

Los Angeles County—University of Southern California 

Comprehensive Cancer Center, Los Angexes, CA. 

Primary HEC were passaged to 60-mm petri dishes, containing 

a 22-mm square glass coverslip, at 500,000 cells per dish. After 24 

hr incubation, the cultures were changed to medium without arginine, 

containing 5-0% dialyzed FBS, to inhibit scheduled DNA synthesis 

(Freed and Schatz, 1969). After incubation in the arginine-free 

medium (AFM) for 48 hr, the medium was withdrawn an appropriate 

concentrations of the test chemicals in AFM were added to each of 4 

plates of cells. Depending upon the particular experiment and the 

chemical being tested, the exposure period was either for 2 or 18 hr. 

After treatment, the cells were rinsed once in AFM and 2.5 ml of AFM 

with 1.0/J FBS was added to each plate3 To each of 2 plates per 

chemical concentration was then added H-TDR at a concentration of 

10 uCi/ml. The coverslip cultures were pulsed for 6 hr (0-6 hr), 

rinsed 3x, fixed 15 min in Carnoy's, rinsed 2x with 20% ethanol and 2x 

with 10$ ethanol, (10 min per rinse) and air-dried. The remaining 2 

plates were labeled as above with H-TdR beginning 6 hr after 

treatment and continuing through 24 hr (6-24 hr). The coverslips were 

fixed to slides (cell side up) with mounting medium (Boyd, 1955), 

dipped in subbing solution (Boyd, 1955) and air-dried. The coverslips 

were then coated with Kodak Nuclear Track Emulsion (NTB2). Exposure 

was accomplished by either of two methods: (1) placed in a foil 

covered slide box and held at 4C for 14 days, or (2) impregnation of 

the partially dried film with scintillator (Durie and Salmon, 1975) 

and incubated overnight at -20°c. After developing, the cells were 

stained for 5 min with 0.1$ crystal violet in 0.1M citric acid. The 

number of grains per nucleus was counted on 100 cells per slide for 

each of the controls nd chemical concentrations. 

REFERENCES
 

1.	 Boyd, G.A., "Autoradiography in biology and medicine." Acedemic
 

Press, Inc., New York, NY, 1955.
 

2.	 Casto B.C., W.J. Pieczynski, N. Janosko, and J.A. DiPaolo.
 "Significance of Treatment Interrval and DNA repair for the
 

enchancement of viral transformation by chemical carcinogens and
 

mutagens." Chem.-Biol. Interactions., 13 (1976), 105-125.
 

315 
3.

 Durie, B.G.M. , and S.A. Salmon. "High-speed scintillation 

autoradiography." Science, 190 (1975), 1093-1095. 

4. Freed, J.J., and S.A. Schatz. "Chromosome aberrations in cultured 

cells deprived of a single essential amino acid." Exptl. Cell 

Bes.. 55 (1969), 393-409. 

316 OU.S. GOVERNMENT PRINTING OFFICE: 1978 720-339/8271 1-3 

DHEW Publication No. (NIH) 78-844