Screening systems

Transformed cell lines containing a reporter gene operatively linked to a genetic control element that is responsive to growth factor-stimulated cell proliferation and/or oncogene-mediated neoplastic transformation are provided. Also provided are methods for using such transformed cell lines to screen for growth factor antagonists and/or antineoplastic agents.

TECHNICAL FIELD 
This invention relates to recombinant vectors comprising genetic control 
elements that are sensitive to the stimulation of cell division by growth 
factors and/or to oncogene-mediated neoplastic transformation. This 
invention further relates to recombinant vectors comprising such genetic 
control elements operatively linked to reporter genes, cells stably 
transformed with such vectors, and methods for using such transformed 
cells to identify antagonists of growth factors and/or oncogene-mediated 
neoplastic transformation. 
BACKGROUND OF THE INVENTION 
Most screening systems currently used to identify potential antineoplastic 
drugs evaluate the ability of compounds to kill rapidly-growing cells in 
culture. Drugs identified in such systems are thus generally not specific 
for tumor cells but are also toxic to rapidly-growing normal cells in the 
body. Out of more than 400,000 compounds that have been evaluated in such 
systems, fewer than twenty have shown an acceptably low level of toxicity, 
and even these compounds show toxic effects in most cancer patients. 
More effective cancer chemotherapy will require the identification of new 
drugs that act to specifically kill cancer cells or to suppress the 
transformed phenotype, while exhibiting low toxicity to normal cells. To 
find these new drugs, new screening systems will be required. 
Our understanding of the molecular basis of cancer has been revolutionized 
by the identification of a relatively small set of normal cellular genes 
called protooncogenes which, when altered, can produce neoplastic change 
Bishop, Ann. Rev. Biochem. 52:301 (1983); Varmus, Ann. Rev. Genetics 
18:553 (1984)!. Alterations in protooncogene expression can occur for a 
variety of reasons, including mutations, nucleotide substitutions, 
chromosomal translocations, gene amplifications, and the insertion of 
mobile genetic elements. As a result of such changes, the expression of 
protooncogenes may be altered or they may be mutated to encode altered 
protein products. 
The proteins encoded by protooncogenes play an important role in governing 
many aspects of cell growth and development. Mutant or activated 
protooncogenes are believed to make specific contributions to the 
phenotypes of tumor cells and hence are called oncogenes. 
One of the remarkable features of cellular protooncogenes is that they have 
shown extraordinary conservation during evolution. Several of these genes 
have been identified in organisms as diverse as yeast, mammals, birds, 
fish and insects. This evolutionary conservation suggests that the 
proteins encoded by the protooncogenes must have important functions in 
normal cell growth and development, with each directing a particular event 
in the complex system of signals that regulates the proliferation and 
differentiation of cells. Changes in any one or more of these genes can 
lead to cancer (Bishop, supra; Varmus, supra). 
The proteins encoded by protooncogenes fall into several groups. Some are 
growth factors--polypeptides that signal cells to divide. Others are 
receptors for growth factors, molecules that are embedded in the cell 
membranes and respond to growth factors. Another group is known as the 
group of G proteins, which transmit signals from receptors to other 
components of the signal-transduction pathway. Others are protein kinases 
which phosphorylate other proteins. Still others are nuclear proteins that 
are involved in DNA transcription Weinberg, Science 230:770 (1985)!. A 
schematic representation of the pathway by which signals generated outside 
a cell can transmit information to the nucleus to produce cell division is 
shown in FIG. 1. 
More recent research has focused on the part played by negative regulators 
of cell growth in the development of cancer. These negative regulators are 
known as tumor suppressor genes (also known as recessive oncogenes or 
anti-oncogenes). Unlike oncogenes of viral and cellular origin, which 
appear to act in a dominant manner to confer transformed characteristics, 
loss of both copies of these recessive oncogenes is required for 
neoplastic change Stanbridge, Bioessays 3:252 (1985)!. 
A protein called the Rb protein which is encoded by one such anti-oncogene, 
the retinoblastoma anti-oncogene, is presumed to act in the control of the 
cell cycle. Oncogenes carried by DNA tumor viruses such as SV-40 large T 
antigen and adenovirus EIA function by complexing with and inactivating 
the Rb protein Whyte, et al., Nature 334:124 (1988)!. 
Although oncogenes have been linked to tumor growth, the signalling 
pathways controlled by oncogene proteins are not limited to growth control 
alone. Oncogene-encoded proteins probably regulate other biological 
activities such as transmission of nerve impulses, phototransduction, 
chemotaxis, differentiation, etc. Alterations in pathways controlling such 
activities may play an important role in other diseases such as 
atherosclerosis and Alzheimer's disease. Hence, specific drugs designed to 
inhibit the activities regulated by mutant oncogene proteins may prove 
useful in the treatment not only of cancer, but of many other diseases as 
well. 
When rat embryo fibroblasts undergo neoplastic transformation, 
microfilaments containing actins and myosins are reorganized from a bundle 
state into a randomly interwoven meshwork Pollack et al., Proc. Natl. 
Acad. Sci. USA 72:994 (1975)!. This phenomenon, known as actin cable 
network diffusion, has been found to be a common characteristic of many 
such transformed cells. Studies indicate that changes in different 
cytoskeletal components are not an indirect consequence of transformation 
but are specific to the oncogenes that cause transformation Franza et 
al., Cancer Cells 1:137 (1984); Leavit, J., in Human Fibroblast 
Transformation (Ed., G. Milo), CRC Press Inc., 1989, pp. 1-28!. 
Changes in the arrangement of cytoskeletal components have been associated 
with alterations in cell growth rate, attachment, saturation density and 
the expression of the differentiated phenotype. Such changes may favor 
neoplastic growth and play an important role in tumor initiation or 
progression. Although detailed understanding of the molecular mechanisms 
involved in these cytoskeletal changes is lacking, it is clear that some 
genes which are silent in normal cells are turned on in transformed cells, 
and that certain others that are expressed in normal cells are turned off 
following transformation. 
Studies by Leavitt et al. Nature 316:840 (1985)! and Garrels et al. 
Cancer Cells 1:137 (1984)! have shown that smooth muscle .alpha.-actin 
isoform is expressed in both Rat2 and REF52 cells and is repressed 
following neoplastic transformation of the cells by several RNA and DNA 
tumor viruses. 
Investigations on the human smooth muscle myosin light chain-2 (MLC-2) 
isoform have shown that the MLC-2 gene also is specifically repressed when 
fibroblasts undergo neoplastic transformation Kumar et al., Biochemistry 
28:4027 (1989); Kumar et al., in Cytoskeletal Proteins in Tumor Diagnosis, 
1989, Weber et al., Eds., Cold Spring Harbor Press, p. 91!. Revertants of 
such transformed cells show normal levels of MLC-2 gene expression. 
In view of the diverse roles played by oncogenes in cellular regulation and 
the relationship of oncogene activity to diseases such as cancer, it would 
be desirable to identify agents that can specifically alter 
oncogene-mediated biological processes, thereby reversing or suppressing 
the disease state. There is thus a need for specific in vitro screening 
systems for that purpose. 
The proliferation and differentiation of mammalian cells are controlled by 
a family of polypeptide growth factors Holley, Nature 258:487 (1975)!. 
All polypeptide growth factors act by binding to specific cell surface 
receptors that, upon activation, transduce a broad range of signals 
leading to cell growth and differentiation James et al., Ann. Rev. 
Biochem. 53:259 (1984)!. A number of growth factors and their receptors 
have been characterized in recent years, including, e.g., epidermal growth 
factor (EGF), fibroblast growth factors (FGFs), platelet-derived growth 
factor (PDGF; a dimeric protein consisting of two "A" chains, two "B" 
chains or one "A" chain and one "B" chain), insulin-like growth factors 
(IGFs) and Bombesin. Many of the growth factor receptors have an intrinsic 
tyrosine kinase activity and contain very closely related structural 
elements. 
Each growth factor may have a specificity for certain cells or tissue 
types. In many cases, however, they can also induce a response in other 
cell types. For example, EGF, the major target of which is epithelial 
cells, can also elicit a response from fibroblast cells. Fibroblast growth 
factor (FGF) is a potent stimulator of vascular endothelium and thus may 
be important in angiogenesis. At the same time FGF can stimulate other 
cell types such as fibroblasts and smooth muscle cells. PDGF is a key 
mitogen for smooth muscle cells and fibroblasts but has no direct effect 
on vascular endothelium or epithelium. 
It has long been known that transformed cells in culture are generally able 
to grow in much lower concentrations of serum than are nontransformed 
cells. Serum is the normal source of growth factors for cultured cells. It 
was later discovered that fibroblasts transformed by certain retroviruses 
secrete factors which transiently induce normal cells to express a 
transformed phenotype Todaro et al., in Genes and Proteins in 
Oncogenesis, 1983, Weinstein and Vogel, Eds. Academic Press., New York, 
N.Y., pp. 165-181; DeLarco et al., Proc. Natl. Acad. Sci. USA 75:4001 
(1978); Todaro et al., Cancer Res. 38:4147 (1978)!. 
These factors, known as transforming growth factors (TGFs), consist of two 
functionally and structurally distinct groups of factors called 
TGF-.alpha. and TGF-.beta. Sporn et al., Nature 313:745 (1985)!. 
TGF-.beta. acts as a growth inhibitor for certain cell types, and as a 
mitogen for other cell types. The discovery of these TGFs led to the 
suggestion that one of the ways by which cells become transformed is by 
endogenous production of growth factors for which they have receptors 
Sporn et al., N. Eng. J. Med. 303:878 (1980)!. This internal production 
of growth factors is believed to serve as a constant stimulus for 
continued cell division, releasing the cells from their normal endogenous 
physiological controls. 
The binding of growth factors to cellular receptors stimulates an array of 
biochemical responses, including changes in ion fluxes, activation of a 
number of protein kinases and alternation of transcriptional rates of 
several genes. These events culminate hours later in DNA replication and 
cell division. Recent studies have led to the delineation of pathways by 
which signals, generated at the membrane by the binding of a growth factor 
to its receptor, are transduced to the nucleus Ullrich et al., Cell 
61:203 (1990); Williams, Science 24:1564 (1989)!. Increased expression of 
genes encoding transcription factors is an important element of the signal 
transduction mechanism which assures long term transcriptional response of 
cells to growth factors. 
Smooth muscle .alpha.-actin isoform is expressed in both vascular smooth 
muscle and fibroblast cells Vandekerckhove et al., Differentiation 14:123 
(1979); Leavitt et al., Nature 316:840 (1985)!. Actively proliferating 
aortic smooth muscle cells are known to contain relatively low levels of 
.alpha.-actin protein, whereas post-confluent cells show a nearly 
three-fold increase Owens et al., J. Cell Biol. 102:343 (1988); Corjay et 
al., J. Biol. Chem. 264:10501 (1989)!. Addition of PDGF to quiescent 
aortic smooth muscle cells results in a decrease in the steady state level 
of .alpha.-actin mRNA (Corjay et al., supra). 
Abnormal cell proliferation due to the action of various growth factors is 
associated with a number of diseases such as neoplasia, atherosclerosis 
and myelofibrosis. To alleviate these conditions, it would be desirable to 
identify agents that can antagonize the actions of the responsible growth 
factors. 
One of the most direct approaches to the identification of growth factor 
antagonists has entailed the use of assays based upon the binding of 
radiolabeled ligands to cellular receptors. Such assay systems are quite 
laborious and time consuming, however, and determination of the 
specificity of a given antagonist requires the use of a number of 
different radiolabeled growth factors and membrane receptor preparations. 
An even more serious drawback to such assays is that they can detect only 
antagonists which act at the receptor level and interfere with growth 
factor binding. As noted above, however, a complex sequence of events 
occurs after a growth factor binds to its receptor. Intervention at 
multiple points by appropriate antagonists may thus be possible, but 
antagonists acting at points other than at the receptor cannot be 
identified by radioligand/receptor assays. 
There is therefore a need for a more broadly-based growth factor antagonist 
screen that could identify a much wider range of antagonists, regardless 
of their locus of action. 
SUMMARY OF THE INVENTION 
The present invention fills the above-mentioned needs by providing 
materials and methods for such screening. 
More particularly, this invention provides methods for identifying 
antineoplastic agents comprising: 
(a) providing a mammalian cell line containing: 
(i) a recombinant vector comprising a reporter gene operatively linked to a 
genetic control element responsive to oncogene-mediated neoplastic 
transformation, the rate of expression of which reporter gene is 
measurably altered when the cell line undergoes such neoplastic 
transformation, and 
(ii) an oncogene, the expression of which renders the cell line 
neoplastically transformed; 
(b) contacting the neoplastically-transformed cell line of step (a) with a 
sample suspected to contain an antineoplastic agent; and 
(c) measuring the level of expression of the reporter gene, 
whereby an antineoplastic agent in the sample is identified by measurement 
of a level of expression of the reporter gene substantially similar to 
that of cells of the same cell line incubated in parallel which have been 
transformed by the vector of step (a)(i) but lack such oncogene and have 
not been exposed to the sample. 
In some embodiments of the invention, the level of expression of the 
reporter gene is suppressed following neoplastic transformation. 
Antineoplastic agents reverse such suppression, causing an increased level 
of reporter gene expression. In other embodiments, the level of expression 
of the reporter gene is increased following neoplastic transformation. 
Antineoplastic agents cause a decreased level of reporter gene expression 
in such embodiments. In all of the embodiments, expression at the lower 
level is preferably negligible. 
This invention further provides methods for identifying growth factor 
antagonists comprising: 
(a) providing a mammalian cell line containing a recombinant vector 
comprising a reporter gene operatively linked to a genetic control element 
responsive to proliferation of the cell line, the rate of expression of 
which reporter gene is measurably decreased when the cell line is 
stimulated to proliferate by a growth factor; 
(b) contacting the cell line of step (a) with a quantity of a growth factor 
sufficient to stimulate proliferation of the cell line and with a sample 
suspected to contain an antagonist of the growth factor; and 
(c) measuring the level of expression of the reporter gene, 
whereby an antagonist of the growth factor in the sample is identified by 
measurement of a substantially increased level of expression of the 
reporter gene, compared to the level measured in cells of the same cell 
line incubated in parallel with the growth factor but without the sample. 
Preferably, the genetic control element used is a human MCL-2 isoform gene 
promoter or a human smooth muscle .alpha.-actin promoter, with the latter 
promoter being most preferred. 
This invention still further provides recombinant vectors and host cells 
transformed by such vectors, for use in the methods of the invention.

DESCRIPTION OF THE INVENTION 
All references cited herein are hereby incorporated in their entirety by 
reference. All nucleic acid sequences disclosed follow the normal 5' to 3' 
convention, as read from left to right. Standard single-letter 
abbreviations are used for the nucleotide bases in the sequences (37 
C.F.R. .sctn. 1.822). 
As used herein, the term "reporter gene" is defined as either a DNA 
molecule isolated from genomic DNA, which may or may not contain introns, 
or a complementary DNA (cDNA) prepared using messenger RNA as a template. 
In either case, such DNA encodes an expression product that is readily 
measurable, e.g., by biological activity assay, enzyme-linked 
immunosorbent assay (ELISA) or radioimmunoassay (RIA). 
The term "genetic control element" has two meanings herein. It means a DNA 
sequence (molecule) which, when operatively linked to a reporter gene in a 
host cell, is capable of responding to oncogene-mediated neoplastic 
transformation of the cell by either stimulating or suppressing expression 
of the linked reporter gene. It also means a DNA sequence which, when 
operatively linked to a reporter gene, causes a down regulation (i.e., 
reduction) in the level of expression of the reporter gene when a cell 
harboring the element is stimulated to multiply by a growth factor. 
The term "oncogene-mediated" refers to a process by which cells are 
neoplastically transformed by an oncogene, either directly or indirectly. 
Such transformation may involve unidentified intermediaries which are 
involved in the complex control of cell division (see FIG. 1) and may be 
initiated directly by expression of an oncogene within a cell, or 
indirectly in a process by which another neoplastic agent such as a 
chemical carcinogen or radiation causes expression of an otherwise latent 
oncogene within a cell. 
As used in this invention, the term "neoplastic transformation" is defined 
as a process by which a normal cell obtains an altered phenotype 
characterized by: 
(a) morphological change (normally flat cells become rounded and actin 
cable network diffusion occurs); 
(b) loss of contact inhibition (cells pile up and become multi-layered in 
tissue culture); 
(c) anchorage independence (cells can grow in soft agar without substrate 
contact); and 
(d) tumorigenicity (cells injected into immune-deficient animals produce 
tumors). 
An "antineoplastic agent" is defined as a chemical compound that can 
substantially reverse or suppress oncogene-mediated neoplastic 
transformation as defined herein, thereby restoring the normal cell 
phenotype. This restoration of the normal cell phenotype is accompanied by 
a concomitant alteration in the rate of reporter gene expression measured 
in the methods of this invention. 
A "normal" cell in the context of this invention is a cell that does not 
manifest the above-mentioned phenotypic characteristics but which may or 
may not exhibit an altered karyotype and an indefinite lifespan in tissue 
culture (i.e., normal cells may be cells of an established cell line). 
The term ".alpha.-actin promoter" means a particular genetic control 
element which has a nucleotide sequence corresponding to the sequence of 
bases of a region of the human smooth muscle .alpha.-actin gene. Parts of 
the human smooth muscle .alpha.-actin gene relevant to the present 
invention have been disclosed by Reddy et al. J. Biol. Chem. 265:1683 
(1990)! and by Ueyama et al. Mol. Cell. Biol. 4:1073 (1984)!. 
Cells which have been "stably transformed" have recombinant DNA 
incorporated into their genomic DNA. Such stably incorporated DNA is 
retained by the transformed cells because it is introduced into the cells 
with a selection marker which forces retention when the cells are grown in 
a selection medium. The present invention preferably employs mammalian 
cell lines that have been stably transformed. 
In some embodiments of the invention, which can be used to screen for 
antineoplastic agents, the vectors contain reporter genes operatively 
linked to genetic control elements that are responsive to 
oncogene-mediated neoplastic transformation. Genetic control elements that 
can be used in these embodiments include all DNA sequences which, when 
operatively linked to a reporter gene in a host cell, are capable of 
responding to oncogene-mediated neoplastic transformation of the cell by 
either stimulating or suppressing expression of the linked reporter gene. 
Especially preferred are promoters and enhancers meeting such functional 
requirements. 
One genetic control element that can be used to screen for antineoplastic 
agents is a murine VL30 transcriptional element. Owen et al. Mol. Cell. 
Biol. 10:1 (1990)! have shown in transient expression assays that 
expression of a firefly luciferase gene operatively linked to a VL30 
long-terminal repeat/mouse major .beta.-globin promoter construct is 
increased in mouse NIH 3T3 cells harboring the construct, following 
neoplastic transformation of the cells by the human Ha-c-ras EJ bladder 
carcinoma gene. Another element that can be used which responds to 
oncogene-medicated neoplastic transformation by increasing expression of a 
linked gene is the ras-responsive human .beta.-polymerase promoter 
described by Kedar et al. Mol. Cell. Biol. 10:3852 (1990)!. 
Other genetic control elements that can be used to screen for 
antineoplastic agents, in contrast to the foregoing elements, respond to 
oncogene-mediated neoplastic transformation by suppressing expression of 
genes to which they are operatively linked. Such elements, which include, 
e.g., the promoter regions of the rat thyroglobulin gene Avvedimento et 
al., Proc. Natl. Acad. Sci. USA 85:1744 (1988)!, the major 
histocompatibility (MCH) class I gene Ackrill et al., Oncogene 3:483 
(1988)!, the human smooth muscle .alpha.-actin isoform gene Leavitt et 
al., Nature 316:840 (1985); Garrels et al. Cancer Cells 1:137 (1984)! and 
the human smooth muscle myosin light chain-2 (MLC-2) isoform gene Kumar 
et al., Biochemistry 28:4027 (1989); Kumar et al., in Cytoskeletal 
Proteins in Tumor Diagnosis, 1989, Weber et al., Eds., Cold Spring Harbor 
Press, p. 91!. 
Although the above-mentioned elements are promoter sequences, 
transcriptional enhancer sequences can be used as well. Enhancers are 
genetic elements that can influence the level of expression of genes with 
which they are associated. Unlike promoters, which must be positioned 
upstream (i.e., in the 5' direction) of the genes they control, enhancers 
may be either upstream or downstream. 
One enhancer that can be used in this invention is the polyoma virus 
enhancer described by Imler et al. Nature 332:275 (1988)!, which mediates 
Ha-ras activation in mouse myeloma and fibroblast cells. Another has been 
located within promoter sequences of the MHC class I gene. Lenardo et al. 
EMBO J. 8:3351 (1989)! have demonstrated that N-myc oncogene expression 
in a rat neuroblastoma cell line leads to reduced binding of a 
transcription factor that activates this enhancer. The result is 
suppression of MCH class I gene expression. 
Genetic control elements preferred for use in screening for antineoplastic 
agents produce suppression of reporter gene expression following 
oncogene-mediated neoplastic transformation. Such elements are preferred 
because they are unlikely to yield false positive results with agents that 
are merely cytotoxic, instead of specific for oncogene-mediated processes. 
Toxic substances will cause general metabolic damage in the host cells and 
will not produce an elevation of gene expression. Especially preferred are 
the promoter regions of the human MLC-2 isoform gene and the human smooth 
muscle .alpha.-actin isoform gene, the latter of which is used to 
illustrate the invention in the Example below. 
In other embodiments of the invention, which can be used to screen for 
growth factor antagonists, the vectors contain reporter genes operatively 
linked to genetic control elements that are responsive to stimulation of 
proliferation of the cell lines by growth factors. Genetic control 
elements that can be used for this purpose include any DNA sequence which, 
when operatively linked to a reporter gene in a host cell, are capable of 
responding to growth factor-stimulated proliferation of the cells by down 
regulation of the level of expression of the linked reporter gene. 
Especially preferred are promoters meeting such functional requirements. 
Surprisingly, it has been found that the promoters of the human smooth 
muscle MLC-2 isoform gene and the human smooth muscle .alpha.-actin gene 
can be used to screen for both antineoplastic agents and growth factor 
antagonists. For that reason, these promoters are preferred for use in 
this invention. 
Especially preferred is the region of the human smooth muscle .alpha.-actin 
gene which begins at the 5' (upstream) end with nucleotide residue -896 
and extends in the 3' (downstream) direction to encompass the remainder of 
the 5' flanking region, exon 1, intron 1, and the 5' noncoding sequences 
from exon 2. Residue -896 is numbered relative to the first base of the 
transcription initiation site of the gene, with that base being designated 
+1. 
The sequence of much of the involved region of the gene is defined in the 
Sequence Listing by SEQ ID NO:1, wherein bases 1 to 1127 correspond to 
bases -896 to +232 of the sequence of FIG. 3 of Reddy et al., supra. The 
guanosine residue at position 896 of SEQ ID NO:1 corresponds to the first 
base of the transcription initiation point of the human smooth muscle 
.alpha.-actin gene (i.e., +1). Bases 1-6 and 902-910 of the sequence 
defined by SEQ ID NO:1 define an EcoRI and DraIII restriction site, 
respectively, which are used below to make an illustrative embodiment of 
the invention. 
Other relevant regions of the human smooth muscle .alpha.-actin gene that 
can be employed in this invention are disclosed by Ueyama et al., supra, 
which provides in FIG. 2 a restriction map covering, inter alia, exons 1 
and 2 and intron 1 of the human smooth muscle .alpha.-actin gene. 
All present embodiments of the genetic control elements based upon the 
human smooth muscle .alpha.-actin gene comprise DNA having a nucleotide 
sequence corresponding to the sequence of about base 1 to about base 910 
of the sequence defined by SEQ ID NO:1. Other embodiments are longer in 
the 3' direction, containing one or more additional bases having a 
sequence corresponding to the sequence of bases 911 to 1127 of the 
sequence defined by SEQ ID NO:1. Still other embodiments contain 
additional bases comprising all or part of intron 1 and/or exon 2, up to 
but not including the translational start signal of exon 2. 
Those skilled in the art can readily make such embodiments using the 
sequence information in SEQ ID NO:1 and the known restriction map of the 
human smooth muscle .alpha.-actin gene (Ueyama et al., supra). By the 
application of standard sequencing methods, the complete nucleotide 
sequence of intron 1 can also be determined, thereby permitting the 
construction of embodiments not terminated by a known (or determinable) 
restriction cleavage point, if desired. 
In particluarly preferred exemplary embodiments described in the Example 
below, the genetic control element of one plasmid, p.alpha.AP126, has a 
nucleotide sequence corresponding to the sequence of bases 1 to 910 of SEQ 
ID NO:1. The genetic control element of another plasmid, p.alpha.API127, 
has a nucleotide sequence corresponding to all of the sequence defined by 
SEQ ID NO:1 and, in addition, contains intron 1 (.about.1.5 kb) and the 
first (5') 13 base pairs of exon 2 of the human smooth muscle 
.alpha.-actin gene. Deletion of part of intron 1 was accomplished by 
restriction endonuclease cleavage and ligation of a 54 bp double-stranded 
oligodeoxyribonucleotide linker, the sequence of which is defined by SEQ 
ID NO: 2. In both of these exemplary plasmids, the genetic control 
elements are operatively linked to E. coli LacZ coding sequences (encoding 
.beta.-galactosidase). 
Plasmids p.alpha.AP126 and p.alpha.API127 can be used both to screen 
antineoplastic agents and to screen growth factor antagonists. 
The genetic control elements of the invention can be prepared by standard 
methods based upon the known sequences of the genes. For example, they can 
be chemically synthesized using the phosphoramidite solid support method 
of Matteucci et al. J. Am. Chem. Soc. 103:3185 (1981)!, the method of Yoo 
et al. J. Biol. Chem. 764:17078 (1989)!, or other well known methods. 
Alternatively, since the sequences of the elements and the site 
specificities of the many available restriction endonucleases are known, 
one skilled in the art can readily identify and isolate the elements from 
genomic DNA and cleave the DNA to obtain a desired sequence. The PCR 
method Saiki et al., Science 239:487 (1988)! can also be used to obtain 
the same result. Primers used for PCR can also be designed to introduce 
appropriate new restriction sites, to facilitate incorporation into a 
given vector. 
Isolation of the human smooth muscle .alpha.-actin promoter from a cosmid 
library constructed using human placenta DNA has been described in detail 
by Reddy et al J. Biol. Chem. 265:1683 (1990)!. The same approach taken 
by Reddy et al. can be used to isolate other promoter sequences. 
In the Example below, the human smooth muscle .alpha.-actin promoter used 
in the construction of exemplary plasmid p.alpha.AP126 was obtained by 
restriction endonuclease cleavage of plasmid p.alpha. (Reddy et al., 
supra). The promoter could as easily have been obtained, however, by the 
use of the polymerase chain reaction (PCR) method Saiki et al., Science 
239:487 (1988)!. 
To generate DNA containing the human smooth muscle .alpha.-actin promoter 
as present in plasmid p.alpha.AP126, two oligodeoxynucleotides having 
nucleic acid sequences defined by SEQ ID NO:3 and SEQ ID NO:4 are 
synthesized and used as primers for PCR using human genomic DNA (Clontech 
Laboratories, Inc., Palo Alto, Calif.) as template. The resulting DNA 
fragment is blunt-ended by enzymatically filling in the overhangs, and the 
fragment is ligated to prepared plasmid pCH126 as described below to 
produce plasmid p.alpha.AP126. 
Insertion of a genetic control element into a vector is easily accomplished 
when the termini of both the element and the vector comprise the same 
restriction site. If this is not the case, it may be necessary to modify 
the termini of the element and/or vector by digesting back single-stranded 
DNA overhangs generated by restriction endonuclease cleavage to produce 
blunt ends, or to achieve the same result by filling in the 
single-stranded termini with an appropriate DNA polymerase. Alternatively, 
any site desired may be produced by ligating nucleotide sequences 
(linkers) onto the termini. Such linkers may comprise specific 
oligonucleotide sequences that define desired restriction sites. The 
cleaved vector and the control elements may also be modified if required 
by homopolymeric tailing. 
Any of the well-known reporter genes can be operatively linked to one of 
the foregoing elements. Examples of suitable reporter genes include but 
are not limited to E. coli .beta.-galactosidase An et al., Mol. Cell. 
Biol. 2:1628 (1982)!, xanthine-guanine phosphoribosyl transferase Chu et 
al., Nucleic Acids Res. 13:2921 (1985)!, galactokinase Shumperli et al., 
Proc. Natl. Acad. Sci. USA 79:257 (1982)!, interleukin-2 Cullen, Cell 
46:973 (1986)!, thymidine kinase Searle et al., Mol. Cell. Biol. 5:1480 
(1985)!, firefly luciferase De Wet et al., Mol. Cell. Biol. 7:725 
(1987)!, alkaline phosphatase Henthorn et al., Proc. Natl. Acad. Sci. USA 
85:6342 (1988)!, secreted placental alkaline phosphatase Berger et al., 
Gene 66:1 (1988)! and chloramphenicol acetyltransferase (CAT) Gorman et 
al., Mol. Cell. Biol. 2:1044 (1982); Tsang et al., Proc. Natl. Acad. Sci. 
USA 85:8598 (1988)!. Many of these and other useful reporter genes are 
available from commercial sources. 
Expression products of the reporter genes can be measured using standard 
methods. For example, bioassays can be carried out for biologically active 
proteins such as interleukin-2. Enzyme assays can be performed when the 
reporter gene product is an enzyme such as alkaline phosphatase or 
.beta.-galactosidase. Alternatively, various types of immunoassays such as 
competitive immunoassays, direct immunoassays and indirect immunoassays 
may be used. 
Such immunoassays involve the formation of immune complexes containing the 
reporter gene product and a measurable label. As used herein, the term 
"label" includes moieties that can be detected directly, such as 
fluorochromes and radiolabels, and moieties such as enzymes that must be 
reacted or derivatized to be detected. 
In competitive immunoassays, samples from induced cultures (following cell 
disruption if the reporter gene product is not secreted) are incubated 
with an antibody against the reporter gene product and a known amount of 
labeled reporter gene product. Any unlabeled product produced by the cells 
competes with the labeled material for binding to the antibody. The 
resulting immune complexes are separated and the amount of labeled complex 
is determined. The reporter gene product produced by the cells can be 
quantified by comparing observed measurements to results obtained from 
standard curves. 
Direct immunoassays involve incubating culture samples with a labeled 
antibody against the reporter gene product and separating any immune 
complexes that form. The amount of label in the complexes is determined 
and can be quantified by comparison to standard curves. 
Enzyme-linked immunosorbant assays (ELISAs) can also be carried out by well 
known methods, e.g., as described in U.S. Pat. No. 4,665,018 to Vold. 
The particular label used will depend upon the type of immunoassay used. 
Examples of labels that can be used include, e.g., radiolabels such as 
.sup.32 P, .sup.125 I, .sup.3 H and .sup.14 C; fluorescent labels such as 
fluorescein and its derivatives, rhodamine and its derivatives, dansyl and 
umbelliferone; chemiluminescers such as the various luciferin compounds; 
and enzymes such as horseradish peroxidase, alkaline phosphatase, lysozyme 
and glucose-6-phosphate dehydrogenase. 
The antibody or reporter gene product, as the case may be, can be tagged 
with such labels by known methods. For example, coupling agents such as 
aldehydes, carbodiimides, dimaleimide, imidates, succinimides, 
bisdiazotized benzadine and the like may be used to tag the antibodies 
with fluorescent, chemiluminescent or enzyme labels. 
The genetic control elements used in this invention can be inserted into 
many reporter gene-containing vectors, including but not limited to 
plasmids pSV2Apap, pMAMneo-CAT, pMAMneo-LUC, pSVOCAT, pBCO, pBLCAT2, 
pBLCAT3, pON1, pCH110, pCH126 and various plasmids described by De Wet et 
al., supra. Where a desired vector contains a different promoter, the 
promoter can be excised using standard methods and replaced by a promoter 
that is responsive to oncogene-mediated neoplastic transformation. In the 
Example below, the SV-40 promoter in plasmid pCH110 was excised and 
replaced with the human smooth muscle .alpha.-actin promoter. 
As used herein, the term "recombinant vector" includes both recombinant 
plasmids such as those mentioned above and recombinant retroviral vectors, 
which can also be engineered as described by Geller et al. Proc. Natl. 
Acad. Sci. USA 87:1149 (1990)! to contain a genetic control element 
operatively linked to a reporter gene. 
The foregoing recombinant vectors can be used to transform any cell that is 
normal but capable of becoming neoplastically transformed, as herein 
defined. Cells from fish, amphibian or avian sources could be used as long 
as they meet the foregoing requirements, but mammalian cells are 
preferred. Although cells from fresh tissue explants (primary cells) could 
in principle be used, the use of established cell lines is preferred. Many 
such cell lines are available including, e.g., the Rat-2 (ATCC CRL 1764), 
Rat-6, NIH 3T3 mouse (ATCC CRL 1658), FRTL Fischer rat thyroid (ATCC CRL 
1468) and L-M (TK-) mouse (ATCC CCL 1.3) cell lines. 
The choice of a cell or cell line for use in screening antineoplastic 
agents will be dictated by the known or determinable specificities of the 
genetic control element and oncogene used. For example, the MHC class I 
promoter can be used in conjunction with the Ad 12 E1A oncogene in primary 
baby rat kidney cells Ackrill et al., Oncogene 3:483 (1988)!. The polyoma 
virus transcriptional enhancer/ras oncogene can be used in mouse L-M (TK-) 
cells. The human smooth muscle .alpha.-actin promoter/ras oncogene can be 
used in Rat-2, Rat-6 or other cells which normally express .alpha.-actin. 
The rat thyroglobulin promoter/TL src or TL mos oncogenes can be used in 
FRTL rat thyroid cells. NIH 3T3 cells can be used with the human 
.beta.-polymerase promoter, in conjunction with the ras oncogene. 
Although cells for use in the present invention could be transiently 
transformed, the use of stably-transformed cells is preferred. Stable 
transformation of a mammalian cell line can be accomplished by using 
standard methods to co-transfect the cells with one of the above-mentioned 
recombinant vectors and with a second vector which confers resistance to a 
selection agent such as an antibiotic. Alternatively, transformation can 
be carried out with a single vector containing both the genetic control 
element/reporter gene component and the selection marker gene. Recombinant 
retroviral vectors can also contain a selection marker gene to produce 
stable transformation. In the Example below, co-transfection was carried 
out using plasmids pIBW and pMAMneo, which provide a dominant selectable 
marker for resistance to antibiotic G418 (neomycin) in mammalian cells. 
Other well known plasmids such as pSV2neo can be used for the same 
purpose. 
Neoplastic transformation of cells which have been stably transformed by a 
genetic control element/reporter gene construct is most conveniently 
carried out by further transformation of the cells with an expression 
vector containing a desired oncogene such as the src, neu, sis, raf, abl, 
N-ras, Ki-ras or Ha-ras oncogene. These oncogenes are well known in the 
art and in use in laboratories around the world. Most are also available 
for purchase from commercial sources or from the American Type Culture 
Collection NIH Repository of Human and Mouse DNA Probes and Libraries. 
They can be obtained already incorporated into a vector suitable for cell 
transformation or they can be excised from the vectors in which they are 
provided and inserted into another vector, using standard methods. 
Insertion of oncogenes into cells via recombinant vectors is most 
convenient, although expression of latent oncogens can be induced in the 
host cells instead by the use of an agent such as radiation (preferably 
X-radiation) or a chemical carcinogen. For example, Rhim et al 
Carcinogenesis 8:1165 (1987)! have shown that a latent H-ras oncogene is 
activated following transformation of certain human fibroblasts by 
3-methylcholanthrene. 
Oncogenes can also be introduced into the cells using viral vectors 
carrying the genes. Examples of such viral vectors include, e.g., 
retroviral vectors described by Dotto et al. Nature 318:472 (1988)!. 
In screening antineoplastic agents using the methods of the invention, the 
level of reporter gene expression is first preferably measured in cells 
which have been transformed with a genetic control element/reporter gene 
construct but are not neoplastically transformed. The cells are then 
neoplastically transformed, contacted with serial dilutions of a sample 
suspected to contain an antineoplastic agent (e.g., a solution in which a 
compound has been dissolved or a fraction or pool from a chromatography 
column or another purification method) or control buffer, and following a 
period of incubation to allow such agent to affect expression of the 
reporter gene, the level of expression of the reporter gene is measured. 
An antineoplastic agent in the sample is identified by measurement of a 
level of reporter gene expression substantially similar to that of the 
cell line prior to neoplastic transformation. 
In cases where the genetic control element used causes an increased level 
of reporter gene expression following neoplastic transformation (e.g., the 
ras-responsive murine VL30 transcriptional and human .beta.-polymerase 
promoter elements), controls are preferably run in parallel using the same 
type of cells transformed with the same reporter gene operatively linked 
to a promoter that is not responsive to neoplastic transformation. 
Antineoplastic agents that are specific antagonists of oncogene-mediated 
neoplastic transformation and are not merely generally cytotoxic will 
cause reduced reporter gene expression in cells harboring the 
oncogene-responsive element only. 
The choice of cells or cell lines for use in screening growth factor 
antagonists will be dictated by the known or determinable specificities of 
the genetic control element and growth factor(s) used. For example, the 
human smooth muscle .alpha.-actin promoter can be used to screen for 
antagonists of a variety of growth factors in Rat-2, Rat-6 or other cells 
which normally express .alpha.-actin. Growth factors that can be used in 
this system include but are not limited to TGF-.alpha. and -.beta.; 
PDGF-AA, -BB or -AB; EGF (epidermal growth factor); bFGF (basic fibroblast 
growth factor), insulin-like growth factors and Bombesin. Preferably, the 
cells will be stably transformed as described above. 
In screening growth factor antagonists using the methods of the invention, 
cells are provided which are transformed with one of the recombinant 
vectors of the invention. The cells are plated in a culture medium 
appropriate to the kind of cells used. Because mammalian or avian cells 
are typically passaged and plated in medium containing serum, the cultures 
are preferably incubated for a period of at least several days prior to 
beginning the assay, to permit the cells to deplete the medium of serum 
growth factors and to thereby become quiescent. 
The cells are then stimulated to proliferate by addition to the culture 
medium of none (control) or varying quantities of a growth factor(s) to 
which the cells are responsive and for which an antagonist is sought. 
Parallel cultures containing the varying growth factor quantities are also 
treated with samples suspected to contain antagonists of the growth 
factors. These samples can be aqueous or water-miscible solutions in which 
isolated compounds have been dissolved, or individual or pooled fractions 
from purification steps such as chromatographic or electrophoretic 
fractions. 
If desired, the growth factors can be dissolved in a physiologically 
compatible solvent such as dimethyl sulfoxide (DMSO) prior to aliquoting 
in the culture medium. Carrier proteins such as bovine or human serum 
albumin may be added to the medium to prevent adsorptive loss of low-level 
quantities of growth factors on test tubes used to make dilutions, 
pipettes and/or culture vessels. 
All of the cultures are then incubated together under conditions in which 
the growth factors, in the absence of an antagonist, will stimulate 
proliferation of the cells. Typical incubations are carried out at 
37.degree. C. in a humidified CO.sub.2 incubator, although the choice of 
conditions will be apparent to those skilled in the art and will depend, 
e.g., upon the nature of the cells, the medium used and the type of 
culture container. 
Incubation is continued for a period of time to permit development of a 
strong proliferative response, at which time the level of expression of 
the reporter gene is measured by an appropriate assay. The optimal time 
for making the measurement after growth factor addition is determined by 
routine experimentation but will typically be in the range of about 24 to 
72 hours for mammalian or avian cells, preferably 48 hours. 
The highest levels of reporter gene expression will be measured in the 
control (growth factor-free) cultures. Where a culture contains a growth 
factor alone, a reduction in the level of reporter gene expression will be 
measured, the degree of which will be a direct function of the quantity of 
growth factor added to the medium. Growth factor antagonists present in 
the samples added to some of the cultures will be identified by measuring 
a substantially increased level of reporter gene expression, compared to 
the level measured in the parallel cultures containing growth factor 
alone. 
A substantially increased level of reporter gene expression is defined as 
an increase of at least about 5%, preferably about 50% and more preferably 
about 90-100% of the level measured in the complete absence of growth 
factor. Of course, the degree of increase may be influenced by the 
quantity of antagonist present in the sample compared to the quantity of 
growth factor used and the efficiency of the antagonist. 
EXAMPLE 
The present invention can be illustrated by the following, non-limiting 
Example. Unless otherwise specified, percentages given below for solids in 
solid mixtures, liquids in liquids, and solids in liquids are on a wt/wt, 
vol/vol and wt/vol basis, respectively. Sterile conditions were maintained 
during cell culture. 
General Methods and Reagents 
Unless otherwise noted, standard recombinant DNA methods were carried out 
essentially as described by Maniatis et al., Molecular Cloning: A 
Laboratory Manual, 1982, Cold Spring Harbor Laboratory. 
Small scale isolation of plasmid DNA from saturated overnight cultures was 
carried out according to the procedure of Birnboim et al. Nuc. Acids Res. 
7:1513 (1979)!. This procedure allows the isolation of a small quantity of 
DNA from a bacterial culture for analytical purposes. Unless otherwise 
indicated, larger quantities of plasmid DNA were prepared as described by 
Clewell et al. J. Bacteriol. 110:1135 (1972)!. 
Specific restriction enzyme fragments derived by the cleavage of plasmid 
DNA were isolated by preparative electrophoresis in agarose followed by 
electroelution (Maniatis et al., supra, p. 164). Gels measuring 9.times.5 
1/2 cm were run at 50 mA for 1 hour in Tris-Acetate buffer (Maniatis et 
al., supra, p. 454) and then stained with 1 mg/ml ethidium bromide to 
visualize the DNA. Appropriate gel sections were excised and melted at 
65.degree. C. for 10 minutes and then diluted with 5 ml of a low salt 
buffer containing 0.2M NaCl, 20 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The 
DNA was then concentrated using a Elutip-D column (Schleicher and Schuell 
Inc., Keene, N.H.) following the manufacturer's instructions and 
precipitated at -20.degree. C. with ethanol in the presence of 10 mg of 
yeast tRNA carrier (Bethesda Research Laboratories, Bethesda, Md.). 
The restriction enzymes, DNA polymerase I (Klenow fragment) and T4 DNA 
ligase were products of New England Biolabs, Beverly, Mass., and the 
methods and conditions for the use of these enzymes were essentially those 
of the manufacturer. T4 DNA ligation was carried out for 16 hours at 
4.degree. C. in a buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM 
MgCl.sub.2, 20 mM dithiothreitol, 1 mM ATP and 50 mg/ml bovine serum 
albumin. Klenow blunt-ending of single-stranded DNA ends was carried out 
in restriction enzyme buffer which had been adjusted to contain 1 mM dGTP, 
dATP, dCTP and TTP. 
Plasmids pIBW and pCH110 are available from Pharmacia LKB Biotechnology, 
Inc., Piscataway, N.J. Plasmid pMAMneo was obtained from Clontech 
Laboratories, Inc., Palo Alto, Calif. Plasmid pH06T1 Spandidos et al., 
Nature 310:469 (1984)! comprising the Harvey ras (Ha-ras) oncogene was 
used to neoplastically transform Rat-2 and Rat-6 cells, both of which are 
normal rat cell lines deficient in nuclear thymidine kinase. 
Plasmid pH06T1 is functionally equivalent to plasmid pHB-11 (ATCC 41013), 
which also contains the Ha-ras oncogene and can be used with equal 
efficacy to neoplastically transform such cells. One of the known viral 
vectors carrying the ras oncogene could also be used instead of pH06T1 to 
carry the gene into the cells. 
Growth factors were purchased from Collaborative Research (Beverly, Mass.). 
Chlorophenol-red-.beta.-galacto-pyrannoside (CPRG) was purchased from 
Boeringer-Mannheim Chemicals (Indianapolis, Ind.), while 
5-Bromo-6-chloro-3-indolyl-.beta.-galactopyrannoside (X-gal) was obtained 
from Sigma Chemicals (St. Louis, Mo.). Goat antibodies against human PDGF 
and goat anti-human IgG are available commercially. 
Both Rat-2 and Rat-6 cells are sublines of the Rat-1 line described by Topp 
Virology 113:408 (1981)!. Both are functionally equivalent and have 
produced similar data using the constructs and methods described below. 
Rat-2 cells were obtained from the American Type Culture Collection, 
Rockville, Md. (accession No. ATCC CRL 1764). 
Synthetic oligonucleotide primers having nucleic acid sequences as defined 
in the Sequence Listing by SEQ ID NO:3 and SEQ ID NO:4 can be synthesized 
by standard methods. 
Cell Culture 
Rat-2 and Rat-6 cells were grown in Dulbecco's Modified Eagle's Medium 
(DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM 
glutamine and 100 gg/ml gentamycin. Both cell lines were maintained in a 
humidified incubator with 5% CO.sub.2 at 37.degree. C. The media and cell 
culture reagents were purchased from Hazleton Biologics, Inc., Lenexa, 
Kans. Stock cultures containing about 5.times.10.sup.6 cells per 100 mm 
dish were routinely split 1:5 or 1:6 by trypsinization and replating every 
3 or 4 days. 
Transfection 
Plasmids were transfected into 5.times.10.sup.5 Rat-2 or Rat-6 cells in 100 
mm culture dishes (Becton Dickinson & Co., Lincoln Park, N.J.), 
essentially as described by Graham et al. Virology 52:456 (1973)!. 
Co-transformation with pIBW or pMAMneo and p.alpha.AP126 or 
p.alpha.API127was carried out at a ratio of 1:10 (pIBW or 
pMAMneo:p.alpha.AP126 or p.alpha.API127). Stably-transformed cells were 
selected in a medium consisting of the above-mentioned medium supplemented 
with 200 .mu.g/ml G418 (Sigma Chemical Co., St. Louis, Mo.). After two 
weeks of incubation in selection medium (replaced every 3 days), 
individual G418-resistant (neomycin-resistant) colonies were picked by the 
agar-overlay method (Reid, L. C., in Methods in Enzymology, Vol. LVIII, 
1979, Jakoby et al., Eds., Academic Press, New York, N.Y.) and expanded 
into mass culture. 
.beta.-Galactosidase Assays 
.beta.-Galactosidase activity in cultured clones was detected by the X-gal 
method, essentially as described by An et al. Mol. Cell. Biol. 2:1628 
(1982)!. Briefly, duplicate sets of stably-transformed Rat-2 or Rat-6 
colonies containing about 10.sup.5 cells/well were incubated in 24-well 
tissue culture plates (Becton Dickinson) in 1-ml volumes of DMEM for 4 
days at 37.degree. C. The cells in each well were then fixed for 15 
minutes in 1-ml volumes of a solution containing 1% glutaraldehyde, 0.1M 
sodium phosphate buffer (pH 7.0) and 1 mM MgCl.sub.2 and then incubated 
for 4 hours at 37.degree. C. with a solution containing 0.2% X-gal 
(5-bromo-4-chloro-3-indolyl-.beta.-galactopyranoside; Sigma Chemical Co., 
St. Louis, Mo.), 10 mM sodium phosphate (pH 7.0), 150 mM NaCl, 1 mM 
MgCl.sub.2, 3.3 mM K.sub.4 Fe(CN).sub.6.3H.sub.2 O and 3.3 mM K.sub.3 
Fe(CN).sub.6. Following the incubation, the X-gal solution was removed and 
10% glycerol was added to the wells. Positive colonies showed blue color 
which was stable in 10% glycerol solution. 
Quantitative .beta.-galactosidase assays were carried out on Rat-2 or Rat-6 
transformants essentially as described by Miller Experiments in Molecular 
Genetics, 1972, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 
N.Y.!, except that although o-Nitrophenyl .beta.-D-galactoside (ONPG) was 
used as the substrate for antineoplastic agent screening, 
chlorophenol-red-.beta.-galactopyrannoside (CPRG) was used instead for 
growth factor antagonist screening. Briefly, about 1.times.10.sup.4 cells 
were seeded in 0.1 ml of DMEM into the wells of a 96-well microtiter plate 
(Becton Dickinson) and incubated at 37.degree. C. At various times 
thereafter, the medium was removed and the cells were rinsed with 
phosphate buffered saline. 
The cells were lysed for 20 minutes at 4.degree. C. with 0.05-ml aliquots 
of a 50 mM sodium phosphate solution (pH 7.0) containing 5 mM 
.beta.-mercaptoethanol and 0.5% Nonidet P-40 (octylphenol-ethylene oxide) 
detergent. After lysing the cells, 50 .mu.l of LacZ reaction buffer 
containing 800 .mu.g/ml ONPG in 50 mM sodium phosphate buffer (pH 7.0), 10 
mM potassium chloride, 1 mM MgSO.sub.4 and 50 mM .beta.-mercaptoethanol 
were added to each well. The plates were incubated for 4 hours at 
37.degree. C., after which the reaction was terminated by the addition of 
30 .mu.l of a freshly-prepared solution of 1M Na.sub.2 CO.sub.3. The 
absorbance of the solutions in the wells was measured at 420 (ONPG) or 590 
(CPRG) nm. 
Construction of Plasmid ph.alpha.AP126 
To produce an exemplary genetic control element/reporter gene construct, 
plasmid pCH110 containing the E. coil lacZ gene under the control of the 
SV-40 early promoter) was prepared by excising the SV-40 promoter by 
digesting the plasmid with PvuII/HindIII. The cleaved plasmid was then 
blunt-ended by filling the HindIII overhangs with T4 DNA polymerase and 
ligated to form plasmid pCH126, which contained a unique HindIII site 5' 
of the lacZ gene. Plasmid pCH126 was then cleaved at the HindIII site. 
The human smooth muscle .alpha.-actin promoter was excised from plasmid 
p.alpha. Reddy at al., J. Biol. Chem. 265:1683 (1990)! by cleaving the 
plasmid with EcoRI/DraIII. Following gel purification, an about 840 bp DNA 
fragment containing the promoter was blunt-ended by enzymatic overhang 
filling and then ligated to the HindIII-digested plasmid pCH126 at 
15.degree. C. for 22 hours in a ligation mixture consisting of 50 mM 
Tris-HCl (pH7.5), 10 mM MgCl.sub.2, 1 mM ATP and 10 units of T4 DNA 
ligase. 
The ligation mixture was introduced into competent E. Coli strain 
DH5.alpha. cells (Bethesda Research Labs, Gaithersburg, Md.) using the 
CaCl.sub.2 transformation procedure (Maniatis et al., supra, page 250). 
Using nucleotide sequence information disclosed by Reddy et al., supra, 
transformant clones bearing the human smooth muscle .alpha.-actin promoter 
in the correct orientation were identified by restriction digestion of 
partially-purified plasmid DNA with EcoRI and Pstl. This digestion 
produced five restriction fragments of 206, 752, 995, 2300 and 3400 bp 
from plasmids having the correct promoter orientation. Further analytical 
confirmation of the correct plasmid was obtained by digestion with Apal 
and Pstl, which produced expected restriction fragments of 270, 958, 2182 
and 4232 bp. The plasmid thus identified was designated p.alpha.AP126 
(FIG. 2). 
Construction of Plasmid p.alpha.API127 
A 1.8 kb BamHI/EcoRI fragment containing the second exon of the human 
smooth muscle .alpha.-actin gene was isolated from a cosmid library 
constructed in C2BX using human placental DNA (Reddy et al., supra). This 
fragment was then cloned into plasmid pAT153 (Maniatis et al., supra, page 
6) which had been prepared by cleavage with the same enzymes to produce a 
first intermediate construct. This intermediate was digested with PvuII 
and EcoRI, gel purified and ligated with a 54 bp double-stranded 
oligodeoxyribonucleotide having a 5' to 3' nucleotide sequence defined by 
SEQ ID NO:2. The 3' to 5' strand of this double-stranded 
oligodeoxyribonucleotide was complementary to the 5' to 3' strand except 
for a four-base (TTAA) extension at the 5' end, which created an EcoRI 
site when the strands were annealed together. Both strands were chemically 
synthesized using standard methods. 
Ligation of the double-stranded oligodeoxyribonucleotide produced a second 
intermediate construct containing intron 1 sequences and exon 2 sequences 
immediately upstream of the translation initiation ATG codon. The second 
intermediate plasmid was digested with BamHI and then ligated with an 
EcoRI/BamHI fragment containing the promoter and first exon of the human 
smooth muscle .alpha.-actin gene, to produce a plasmid designated 
pAI-AT153. 
Plasmid pAI-AT153 was digested with EcoRI to produce a 3.5 kb restriction 
fragment that was isolated, blunt ended and then ligated into 
HindIII-cleaved, blunt-ended plasmid pCH126. The result was plasmid 
p.alpha.API127, which is shown schematically in FIG. 2. The plasmid 
containing the promoter in the correct orientation was identified by 
restriction cleavage and gel analysis. Upon digestion with BamHI, the 
correct plasmid yielded two bands 4.5 and 7.0 kb in size. 
Antineoplastic Agent Screen 
Neoplastic Transformation 
Prior to assaying for the effect of ras oncogene expression on 
.beta.-galactosidase production, 5.times.10.sup.5 of the 
stably-transformed Rat-2 or Rat-6 cells were neoplastically transformed by 
transfecting the cells with 10 .mu.g of plasmid pH06T1 DNA, as described 
above. Two days after transfection, the cells from one plate were split 
into 5 plates and grown for about 3 weeks. The medium was replaced with 
fresh medium every 3 days during this period, and the cultures were 
observed for the presence of multilayered cell foci. Such foci were then 
picked, cloned, expanded and assayed for .beta.-galactosidase activity. 
The resulting cells are referred to as neoplastically transformed (or 
ras-transformed) below. 
Effect of ras Transformation on .beta.-Galactosidase Expression 
Two-tenths-ml aliquots of DMEM with 10% fetal calf serum containing 
1.times.10.sup.4 Rat-6 cells which had been co-transfected with plasmids 
p.alpha.AP126 and pMAMneo (with and without further ras transformation) as 
described above were plated into the wells of 96-well microtiter plates 
and incubated at 37.degree. C. in a humidified 5% CO.sub.2 incubator. At 
various times after plating, the cells were analyzed for 
.beta.-galactosidase activity, with the results shown in FIG. 3. 
As shown in FIG. 3, Rat-6 cells stably transformed with plasmid 
p.alpha.AP126 but not further neoplastically transformed (Untransformed) 
showed a relatively high level of .beta.-galactosidase activity, which 
increased over time as the cells multiplied. In contrast, 
.beta.-galactosidase activity was initially suppressed in the cells which 
had also been neoplastically transformed (ras-transformed) and remained 
suppressed, despite continuing cell proliferation. 
To determine whether the suppression of lacZ gene expression observed in 
the ras-transformed cells was due to an oncogene-mediated phenomenon, 
revertants of the neoplastically transformed cells were prepared 
essentially as described by Yanagihara et al. Oncogene 5:1179 (1990)! and 
assayed for .beta.-galactosidase activity. 
Such revertants were prepared by treating 20 100-mm culture dishes, each of 
which contained 10.sup.6 Rat-6 cells which had been stably transformed 
with both plasmid p.alpha.AP126 and plasmid pH06T1, with 10 ml volumes of 
DMEM containing 5 .mu.g/ml 5-azacytidine and 10% FCS for 24 hours at 
37.degree. C. Following this incubation, the medium was replaced with 
azacytidine-free DMEM with 10% FCS for a 24-hour recovery period, and then 
with the same medium containing 200 .mu.g/ml cis-4-hydroxyproline. 
This medium was replaced with fresh medium twice weekly for 3 to 4 weeks, 
after which colonies comprising apparently flat cells were marked and 
isolated using the agar overlay method. Such colonies were expanded in 
DMEM with 10% FCS and subcloned in microtiter plates. The cells in one 
revertant clone designated D-3 showed consistently flat morphology over 
several subpassages. Another clone designated D-3A was obtained by further 
subcloning of clone D-3. 
Revertant clones D-3 and D-3A were plated into microtiter plates, together 
with Rat-6 cells that had been stably transformed with plasmid 
p.alpha.AP126 and further neoplastically transformed with plasmid pH06T1 
(ras-transformed) or not (Untransformed), and analyzed as described above 
at various times following plating for .beta.-galactosidase activity. The 
results are shown in FIG. 4. 
There, it can be seen that while the activity in the ras-transformed cells 
was suppressed as before, the activity in the cells from both revertant 
clones was similar to that of the untransformed cells. Suppression of lacZ 
gene expression was therefore dependent upon oncogene-mediated neoplastic 
transformation. Mutational revertants which no longer displayed the 
neoplastic phenotype showed release of the suppression at the human smooth 
muscle .alpha.-actin promoter. 
That this release of suppression was specifically related to the reversal 
of oncogene-mediated neoplastic transformation has been shown by results 
obtained screening a substantial number of potential antineoplastic agents 
with the .alpha.-actin promoter/.beta.-galactosidase/ras system. Agents 
shown by other tests to be generally cytotoxic which do not restore the 
normal cellular phenotype do not cause increased lacZ gene expression. 
Growth Factor Antagonist Screen 
Plasmid p.alpha.AP126 or p.alpha.API127 was co-transfected with plasmid 
pIBW into Rat-2 cells to produce stable transformant clones. 
G418-resistant colonies were isolated and expanded. A duplicate set of 
colonies was stained with X-gal substrate to identify colonies expressing 
.beta.-galactosidase. In this way, clones stably incorporating plasmids 
p.alpha.AP126 and p.alpha.API127, designated Y2 and Z2, respectively, were 
isolated. A third clone designated SR.alpha.LacZ was similarly prepared 
using plasmid pSR.alpha.LacZ, a recombinant vector containing the lacZ 
gene of E coli operatively linked to an SR.alpha. promoter Takebe et al., 
Mol. Cell. Biol. 3:280 (1983)!. Clone SR.alpha.LacZ was prepared as a 
control for nonspecific effects, because the SR.alpha. promoter is not 
responsive to growth factor-stimulated cell proliferation. 
Growth factor assays were carried out by growing the above-mentioned stably 
transformed Rat-2 clones in DMEM containing 10% FBS and 200 .mu.g/ml G418 
antibiotic. The cells were seeded in 0.1 ml volumes of the medium into the 
wells of a 96-well microtiter plate (Becton Dickinson) at a density of 
5.times.10.sup.4 cells/well and incubated at 37.degree. C. Twelve days 
after plating, various growth factors dissolved in DMEM containing 1 mg/ml 
bovine serum albumin (BSA) as a carrier were added. Samples containing 
various concentrations of potential growth factor antagonists dissolved in 
water or DMSO were added to some of the cultures, together with the growth 
factors. 
Forty-eight hours after addition of the growth factors, the cells were 
lysed for 20 minutes at 4.degree. C. with 0.05-ml aliquots of a 50 mM 
sodium phosphate solution (pH 7.0) containing 5 mM .beta.-mercaptoethanol 
and 0.5% Nonidet P-40 (octylphenolethylene oxide) detergent. After lysing 
the cells, 50 .mu.l of LacZ reaction buffer containing 800 .mu.g/ml CPRG 
in 50 mM sodium phosphate buffer (pH 7.0), 10 mM potassium chloride, 1 mM 
MgSO.sub.4 and 50 mM .beta.-mercaptoethanol were added to each well. The 
plates were incubated for 4 hours at 37.degree. C., after which the 
reaction was terminated by the addition of 30 .mu.l of a freshly-prepared 
solution of 1M Na.sub.2 CO.sub.3. The absorbance of the solutions in the 
wells was measured at 590 nm. 
To determine whether clone Z2 could respond to growth factor stimulation 
with a decreased level of .beta.-galactosidase production, cells from the 
clone were plated, stimulated with various growth factors and then assayed 
for .beta.-galactosidase activity. The growth factors tested included 
TGF-.alpha. and -.beta., PDGF-AA, EGF and bFGF, all over a concentration 
range of from 0 to 10 ng/ml of culture medium. The results are shown in 
FIG. 5. 
The data of FIG. 5 show that all of the growth factors tested caused a 
marked decrease in the level of .beta.-galactosidase production, compared 
to the level observed in the control (carrier BSA only) cultures. Similar 
results were obtained using the Y2 clone transformed with plasmid 
p.alpha.AP126. In contrast, exposure of clone SR.alpha.LacZ to the same 
growth factors produced essentially no change in the level of lacZ 
expression. 
As is also shown in FIG. 5, the median effective concentration for the 
various growth factors was about 1 ng/ml. This concentration is also known 
to be effective in inducing mitogenesis of fibroblast cells Corjay et 
al., J. Biol. Chem. 264:10501 (1989); LaRocca et al., Cancer Cells 2:106 
(1990); Battegay et al., Cell 63:515 (1990)!. 
To demonstrate use of the foregoing system to detect a known growth factor 
antagonist, clone Z2 cells were plated and subjected to 
.beta.-galactosidase assay as described above in the presence of 
TGF-.alpha., TGF-.beta. or PDGF-AA homodimer (all at a final concentration 
of 10 ng/ml) with or without neutralizing antibodies against PDGF 
(anti-PDGF) or control goat anti-human IgG antibodies (anti-IgG), both at 
a final concentration of 3 .mu.g/ml. Controls were also run containing BSA 
carrier alone with or without one of the antibodies. The results are shown 
in Table 1. 
TABLE 1 
______________________________________ 
Antibody Antagonist Assay 
O.D. 
Growth Factor Antibody (590 nm) 
______________________________________ 
-- -- 0.183 
-- Anti-PDGF 0.180 
-- Anti-IgG 0.180 
TGF-.alpha. -- 0.022 
TGF-.alpha. Anti-PDGF 0.022 
TGF-.alpha. Anti-IgG 0.025 
TGF-.beta. -- 0.022 
TGF-.beta. Anti-PDGF 0.171 
TGF-.beta. Anti-IgG 0.025 
PDGF-AA -- 0.022 
PDGF-AA Anti-PDGF 0.182 
PDGF-AA Anti-IgG 0.022 
______________________________________ 
The data of Table 1 show that, as before, stimulation of the cells to 
proliferate by TGF-.alpha., TGF-.beta. and PDGF alone caused a marked 
reduction in .beta.-galactosidase production. In the presence of 
neutralizing antibodies against PDGF, however, the levels of 
.beta.-galactosidase activity produced by PDGF and TGF-.beta. approximated 
control levels. The control anti-IgG antibodies had no effect. 
The effect of the neutralizing anti-PDGF antibodies on the PDGF was 
expected; presumably the antibodies bound to the growth factor, thereby 
preventing its binding to the cellular receptors. The effect of these 
antibodies on TGF-.beta. was not surprising, because it is believed that 
TGF-.beta.-induced mitogenesis is an indirect effect which is mediated by 
PDGF Moses et al., Cell 63:245 (1990)!. Moreover, TGF-.beta. has been 
shown to induce both the PDGF A chain gene and c-sis, which encodes the 
PDGF B chain Coffrey et al., J. Cell. Physiol. 132:143 (1987); Leof et 
al., Proc. Natl. Acad. Sci. USA 83:2453 (1986)!. The proliferative effects 
of TGF-.alpha., on the other hand, appear to be direct, and not the result 
of intermediary PDGF activity. 
As a further test of the utility of the methods of the invention to 
identify growth factor antagonists, the foregoing assay system was used 
with the growth factor antagonist, Suramin. Suramin is an organic compound 
that is capable of blocking growth factor-receptor interactions La Rocca 
et al., Cancer Cells 2:106 (1990)!. 
This test was carried out by plating and subjecting clone Z2 cells to 
.beta.-galactosidase assay as described above in the presence of EGF, 
TGF-.alpha., TGF-.beta., bFGF and the PDGF-AA and PDGF-BB homodimers (all 
at a final concentration of 10 ng/ml) with or without 30 .mu.M Suramin. 
Controls were also run containing BSA carrier alone with or without 
Suramin. The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Antagonistic Effects of Suramin 
O.D. 
Growth Factor Suramin (590 nm) 
______________________________________ 
-- - 0.180 
-- + 0.178 
TGF-.alpha. - 0.020 
TGF-.alpha. + 0.170 
TGF-.beta. - 0.020 
TGF-.beta. + 0.177 
PDGF-AA - 0.022 
PDGF-AA + 0.168 
PDGF-BB - 0.022 
PDGF-BB + 0.175 
EGF - 0.024 
EGF + 0.172 
bFGF - 0.024 
bFGF + 0.178 
______________________________________ 
As shown in Table 2, Suramin strongly antagonized the effects of all of the 
growth factors tested. 
Plasmid Deposit 
E. coil strain DH5.alpha. harboring plasmid p.alpha.API127 was deposited 
Jul. 10, 1991 with the American Type Culture Collection (ATCC), Rockville, 
Md., and assigned Accession No. ATCC 68645. This deposit was made under 
the provisions of the Budapest Treaty on the International Recognition of 
the Deposit of Microorganisms for the Purposes of Patent Procedures. 
Many modifications and variations of this invention can be made without 
departing from its spirit and scope, as will become apparent to those 
skilled in the art. The specific embodiments described herein are offered 
by way of example only, and the invention is to be limited only by the 
terms of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1127 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
GAATTCGAGACGAGATTTGGGTGGGGACGTAGAACCAAACCATATCACCTGGTCTCTCTA60 
CTTCCTGTCAAGGAGGTTAGTGGGCAGAGAGGAGGGCTACAGAGGCTTCCTTTGAACAAT120 
CTCCTTTCTTTTCCAAACTACTTCTTTGACAGGCTGCTGGGTAGACTCTCTGGTCAAAGG180 
ATGGTCCCTACTTATGCTGCTAAATTGCTCGGTGACAAATTAGTAGACAAAGCTAATGCA240 
CCAAAAAAATGAATGTAGTTATAGTAATGCTAACATCCAAATTCCTCTTTGTAAGACATA300 
GGCCTGTCAACCTTGTCTCCATACTTCAATTCCTATTTCCACTCACCTCCCTCAAGAACT360 
TGATTTATAAACAGTGTGCCTACCATAAAATCATCACTCCCTCTATGTATTTATAGACGA420 
CTGAAGGAATATCTTTCTTCTTTGCATGCTACCGTGGTAGAAGGGTTTTAAAAGTCCGTG480 
CTAGGCAGAGGCAGCCCTTTCTGCCCCTTTCTGTTCTCAGTTTATTAGGAAATGGCCTGA540 
AATTCCAGCATGATAGCAAGCTGGCATCCTCTGTGGAATGTGCAAACCATGCCTGCATCT600 
GCCCATTACCCTAGCTCAGTGTCTCTGGGCATTTCTGCAGTTGTTCTGAAGGCTTGGCGT660 
GTTTATCTCCCACAGGCGGCTGAACCGCCTCCCGTTTCATGAGCAGACCAGTGGAATGCA720 
GTGGAAGAGACCCAGGCCTCCGGCCACCCAGATTAGAGAGTTTTGTGCTGAGGTCCCTAT780 
ATGGTTGTGTTAGACTGAACGACAGGCTCAAGTCTGTCTTTGCTCCTTGTTTGGGAAGCA840 
AGTGGGAGGAGAGCAGGCCAAGGGGCTATATAACCCTTCAGCTTTCAGCTTCCCTGAACA900 
CCACCCAGTGTGGAGCAGCCCAGCCAAGCACTGTCAGGGTAAGTGGCGCCAGGCCAAGGA960 
TGTGACTTATAGATTCCAGTGGCTCTTTTAATTACCCGGTATAATAAGACATCATCTGCA1020 
GGGATTTGGCTGGGTTCATGCACTGATATTTCTGAATGAAGATTGTACTACTAAAATGAT1080 
TGTAGCTTTTGGCTTTAATGATCTAACGTTAAAGACAGGGCTAATAT1127 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
CTGAGGCTGCTTCCTCCCTGTTTTCTATAGAATCCTGTGAAGCAGCTCCAGCTG54 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
GAATTCGAGACGAATTT17 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
CACACTGGGTGGTGTTC17 
__________________________________________________________________________