SOURCES AND EFFECTS
OF IONIZING RADIATION
United Nations Scientific Committee on the Effectsof Atomic Radiation
UNSCEAR 1993 Report to the General Assembly,
with Scientific Annexes

UNITED NATIONSNew York, 1993

NOTE
The report of the Committee without its annexes appears as Official Records of theGeneral Assembly, Forty-eighth Session, Supplement No. 46 (N48/46).
ll1e designation employed and the presentation of material in this publication do not imply
tl1e expression of any opinion whatsoever on the part of tlie Secretariat of the United Nations
concerning the legal status of any country, territory, city or area, or of its authorities, or concerning
tl1e delimitation of its frontiers or boundaries.
The country names used in this document are, in most cases, those tliat were in use at thetime the data were collected or the text prepared. In other cases, however, the names have been
updated, where tliis was possible and appropriate, to reflect political changes.

UNITED NATIONS PUBLICATION
Sales No. E.94.IX.2
ISBN 92-1-142200-0

ANNEX E
Mechanisms of radiation oncogenesis

CONTENTS

Page..........................................552
I. STRUCTURE AND FUNCTION OF MAMMALIAN CELLS ........ 553
A. CELLS AND TISSUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
B. CHROMOSOMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553

INTRODUCTION

I1. PRINCIPAL THEORIES OF ONCOGENESIS . . . . . . . . . . . . . . . . . . . 554
A . INDUCTIVE AND DEVELOPMENTAL PROCESSES .......... 555
1. DNA as a principal target for radiation action ............. 555
2. The single-cell origin of neoplasia . . . . . . . . . . . . . . . . . . . . . 555
3. Genetic changes in oncogenesis ....................... 556
4. Multistage cellular development in oncoge~~esis. . . . . . . . . . . . 563
5 Viral involvement
570
B. HUMAN SUSCEPTIBILITY TO RADIOGENIC CANCER
571
1. Homozygous deficiencies inDNA repair, cell inactivationand chromosome breakage
571
2. Heterozygous carriers of genetic traits . . . . . . . . . . . . . . . . . . 573
3 Systemic factors .................................. 574
4 Immunodeficiency and cell surveillance mechanisms ........ 576.

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III EXPERIMENTAL INVESTIGATIONS OF CELLULAR
AND MOLECULAR MECHANISMS
OF RADIATION ONCOGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
A . EPIDEMIOLOGICAL STUDIES .........................578
B MOLECULAR STUDIES OF MUTAGENESIS ANDREPAIR: POSSIBLE IMPLICATIONS FORNEOPLASTIC INITIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
580
C IN VITRO STUDIES WITH CELLULAR SYSTEMS
1. Convcr~tionalsystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
2 Novel systcri~s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
D. IN V N O STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
1. General experimental strategies . . . . . . . . . . . . . . . . . . . . . . . 582
2. Molecular studies of induced animal neoplasms . . . . . . . . . . . . 583.

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U N S C M R 1993 REPORT

552

Pagc

1V. COMPARATIVE ASPECTS OF ONCOGENESIS BYRADIATION AND CHEMICALS . . . . . . . . . . . . . . . . . . . . . . . . . . . .585
V.

FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .588
A.

B.

IMPLICATIONS OF ADVANCESIN RELATED RESEARCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . .588
METHODOLOGICAL AND CONCEPTUAL ASPECTS . . . . . . . . 589

CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .593

Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Figures
600
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603.................................................

INTRODUCTION
1. Basic information on cancer induction(oncogenesis) by radiation conles from epidemiological studies of exposed human populations. These
give evidence of the relatively wide range of neoplasms involved and of the latency periods and thedose levels at which they are observed. It may be thatthe limits of resolution of such data are being reached,
and for absorbed doses of less than 0.1-0.5 Gy of low
linear energy transfer (LET) radiation, there may be
increasing dependence on extrapolation procedures.
Since most human exposures to radiation occur atdoses substantially below 0.5 Gy, the validity of such
extrapolations is a crucial factor in risk estimation.

2. The extrapolation of radiation response to lowlevels of dose is made with dose-effect models derived
from physical and biophysical studies of the action ofradiation on biological systems [Cl,GI, U2]. The
principal appeal of these nlodels is that they attempt todescribe dose-effect with simple linear or linearquadratic equations. While in recent years considerable
efforts have been made to validate such models ofradiation action with respect to fundamental biologicalfactors, such as the macron~olecular structure ofcellular targets and h e influence of post-irradiation
repair processes [a,G2], the unqualified applicationof simple biophysical equations to a complex multifactorial biological process such as cancer induction is
problematic. That is not to say that simple extrapolations and approximations from animal and cellulardose-effect data will not be possible, but ralher that
there is as yet insufficient knowledge to make themwith confidence.
3.
So that future extrapolation of high-dose
epidemiological data may be made with confidence, il

is crucial that a much more detailed picture be gainedof the cellular and rtlolecular processes that mediate
oncogenic change in mammalian cells. Much of themechanistic knowledge of radiation action is derived
from studies on cell inactivation, chromosonial change,
mutagenesis and the repair of deoxyribo~~ucleic
acid(DNA). These data will all continue to contributetowards a broad solution to the problem, but until they
can be placed in the correct context through theidentification of specific neoplasia-associated cellular
events, their direct relevance to oncogenesis willremain uncertain.
There is strong evidence that oncogenesis is a4.
multi-step process involving the accumulation of aseries of genetic and epigenetic changes in a clonal
population of cells, that different steps characterizedifferent neoplasms and that the whole process isstrongly influenced by genetic, physiological andenvironmental factors. The characterization of mechanisms of radiation oncogenesis is therefore a daunting
challenge. Its importance to future estimates of cancer
risk is, however, sufficiently great that the challengemust be accepted.

It is also important to stress that in addition to its
5.
obvious relevance to radiological protection, thefurther understanding of the mechanisms of radiation
or~cogenesiswould contribute substantially to fundamental cancer research, a very active and rapidly
developing field in which breakthroughs are beingsought. This Aluiex outlines some of the principal
problems, broad theories and experimental strategies
regarding the mechanisms of radiation oncogenesis
and attempts to anticipate future studies and theirpotential application in radiation risk estimation.

ANNEX E: MECHANISMS OF RADIATION ONCOGENISISI.

STRUCTURE AND FUNCTION OF MAMMALLAN CELLS

6 . A brief description of the ccll, its structure andfunctioning in the mammalinn organism will serve as
background i~~formation
for Ute discussio~~
to follow.
Only those 1110rc basic aspects of the ccll and of~nolecularbiology that arc relevant to this Annex are
given here. More detailed inforniation can be found in
the references (c.g. [Al, C7, W1,W2,W51).

A.

553

CELLS AND TISSUES

7.
The cell is the basic unit of all organisnis. Eachmammalian cell is bounded by a complex, semi-pcrmcable lipid and protein membrane, which actively
mediates the two-way flow of metabolites and presents
protein receptor sites to allow the cell to interact with
its environment. Contained within the membrane is ahighly organized aqueous milieu, the cytoplasm, whichcontains numerous organelles, principally the nucleus
containing the genetic material of the cell (see below),
mitochondria (for the generation of biochemicalenergy), the endoplasmic reticulum (providing surfaces
for biochemical reactions) and protein-nucleic acidcomplexes termed ribosomes (for protcin synthesis).
Proteins synthesized on ribosomes niay act structurally, as catalysts of biochcrnical reactions (enzymes),or play a coordinating role in cell physiology (intracellular regulators, growth factors, cytokincs andhormones).

At certain points in their life history cells are
8.
required to reproduce themselves; this is achieved bytransit through the ccll cycle. Most cells established inculture have cell cycle transit times of 18-24 h; in r h ocell cycle times range up to a week, these longer
periods reflecting varying periods of quiescence in one
phase of the cycle. In this cycle the genetic material of
the cell is replicated and shared equally betwecn thetwo identical daughter cells that result from the ccll
division that terminates the cycle.

9. Even at the single-cell level, the complexity ofthe biochemical and biophysical reactions that are
required for maintenance of biological function isimmense. In multicellular organisms, however, cells
are required to act in concert in order to provide
specialized tissue functions. Beyond that, tissue function itself is required to be interactive, s o that thewhole physiology of the organism is coordinated andresponses to appropriate environmental conditions areavailable.
10. Although grossly simplistic, the above outline issufficient to demonstrate ltic need for a highly
complex network of information transfer, which musthave its origins in single-ccll function arid response.
The source of this lies in the ccll nucleus, specificallyin the nucleoprotcin complexes that make up themic~oscopicbodies ternted chroniosomcs.

11. Chromosomes are basic components in cellular
reproduction. Before every somatic ccll division,
chromosomes are duplicated, and cach daughter ccll
norn~allyreceives an idelltical set of chrornosonics.
Each mammalian species is characterized by aparticular and constant chromosome number, size andmorphology.

12. Each chromosome contains a single nucleic acidacid (DNA), which ispolymer, deoxyribo~~ucleic
complexed along its length by proteins such as thoseof the histone family and others with roles in theregulation of chroniosomc structure and DNA
metabolism. The DNA fomis the crucial structural
entity, but the whole nucleoprotcin complex has aseries of orders of structure progressing throughsecondary solenoid-like bodies (nucleosomes), whichstack together to form tertiary chromatin fibres. The
individual chromosome, the quaternary structure, iscomposed of a cornplcx arrangement of these fibres
rnaintaincd by rnatrix and scaffold proteins.

13. Although the maintenance of overallchromosome structure is crucial for DNA metabolic
processes such as condensation, transcription,
replication, reconibination and repair, it is the D N A
polymer itself that is the source of cellular information
and, thereby, physiological control.

14. The information is encoded in a linear sequenceof alternating aromatic (nuclcic acid) base pairs. The
pairing is achieved by a double-stranded helicalarrangement, where the four bases, adenine (A),thymine (T), guanine (G) and cytosine (C), on one
strand are covalenlly linked to a sugar-phosphatebackbone and specifically pair (A with T, G with C)
with the bases on the opposite strand throughhydrogen bonding. The relatively weak hydrogenbonding between base pairs allows unwi~tdingof the
DNA duplex, which is necessary for some aspects of
DNA processing. In particular, DNA replication
through DNA polymerase enzyme activity has to occur
with high fidelity cach time the cell is preparing todivide. This fidelity is achieved by urtwirtding the
DNA and replicating the base seque~tccon the two
strands.

15. The base pair code in DNA is arranged insubunits of three bases, termed codons. Amino acids,
the building blocks of protcin, are specified by codons,
and a string of codor~s is able to determine the554

UNSCEAR 1993 REPORT

structure and thereby the function of a single protein(polypeptide chain). Such a functional string of DNA
codons represents the basic unit of cellular informationand hereditary, the gene. Three specific codons act as"stop" signals, thereby indicating the point at which
the gene code is tenninated. W i l e all others specify
an amino acid, the code is degenerate in that there isrnore than one codon for rnost amino acids, i.e. 61
coding units determine the 20 natural amino acids.
Other non-coding DNA sequences associated with agiven gene act to regulate its activity. In mammaliancells it may be estimated that there are approxin~ately
100,000 genes, each of which depends for its correctfunction on maintaining a constant DNA basesequence. Changes in these sequences by base pair
substitution, loss or addition can cha~rgegene function;such changes are ternled genetic mutations. However,damage to the DNA does not always result inmutations, since cellular DNA repair enzymes areoften able to restore both damaged DNA base anddamaged sugar phosphate backbone.
16. Most cellular DNA does not code for proteins,and even allowing for base sequences that act to control gene expression, there appears to be much functional redundancy in the mammalian genome. Thisfeature implies that DNA damage in certain chromosome regions will have little or no consequence for thecell, while in other regions damage may change theactivity of key genes, leading to changes in cellular
properties. Gene expression occurs when a singlestranded nucleic acid, ribonucleic acid (RNA), is
synthesized (transcribed) from the gene. In mammaliangenes only part of the linear base sequence actuallycodes. The coding segments (exons) are separated by
non-coding segments (introns), which often constitutethe major portion of the gene. Consequently, theprimary RNA transcribed from the whole gene has tobe appropriately cut and spliced before it is fully functional. This messenger RNA (mRNA) contains a stringof codons complementary to those of the gene, andthese are used by cellular ribosomes to construct the
specified protein. Transcriptional processes and hence
gene product availability are closely coordinated incells by a series of interacting feedback loops.

17. In a given mammalian species, all cells containthe salnc gcnes; these cells may, however, differ in therelative activity of those genes. Developmental processes involve the selective functional activation andinactivation of rrrany genes. While a core function is
maintailred by so-called housekeeping genes, specialized functions in tissues require the expression of
appropriate sets of (luxury) genes in certain cellli~reages.This reprogramming of the genome has to bestably maintained, since such specialized functions
have to operate throughout the subsequent life-span of
the cell. It may therefore be seen that this coordinatedprogranlrning underlies cellular and tissue differeatiation and is hence a central feature of nornlal organogenesis and tissue maintenance. Gene programming,
which must be initiated early in ernbryogenesis, isbelieved to have its origins in gene activation/
inactivation through secondary biochemical modification to DNA, such as DNA base methylation, changes
in chromatin structure and/or stable protein binding[H16, M44].Growth factors, hormones and cytokines
also play a role in the mediation of differentiation in
many tissues. Such stable changes in gene expression
and cellular properties (phenotypes) in the absence ofDNA base pair changes are essentially non-genetic andare often referred to as being epigenetic. The specificrearrangernent of immunoglobulin germ-line DNA
sequences is known to play a key role in the generation of irnmunocompetence in white blood cells; DNA
rearrangement is not, however, believed to be a majormechanism of cell differentiation.
18. While fundamental knowledge of gene function,
control and mutation may be traced back to pioneer
work in micro-organisms [HI, Jl], higher organisms
have progressively more complex biological problems,
including cancer developn~ent (oncogenesis). 111 a
broad histopathological sense, cancer in mammaliantissues preselrts itself as a caricature of rronnal celland tissue development. In sorne way, intrinsic biological control at both the cellular and tissue level has
been subverted. Chapter I1 outlines some of thetheories relating to oncogenesis, particularly those thatare relevant to its induction by the exposure ofmarnrnalian cells to ionizing radiation.

11. PRINCIPAL THEORIES OF ONCOGENESIS
19. During the last decade there has been aremarkable increase in knowledge of the roles of chromosomal change, of specific cellular genes and of inherited nlutatiorrs in human oncogenesis. However,huge gaps remain in the understanding of the problem.
In the context of radiation oncogenesis, great uncer-

tainty exists in the nature and consequences of thegenetic/epigenetic events that mediate the inductive,
promotional and progressior~processes, in the mechanisrns and corltributior~of human genetic susceptibility
and in differences in oncogenic mechanisms following
exposure to radiation or chernical carcinogens.

ANNEX E: MECIIANISMS OF RADlAnON ONCOGENEISA.

INDUCTIVE AND DEVELOI'RI ENTAI,
I'KOCESSES
1. DNA us n principnl turget
for mdintion llction

20. The genetic material (DNA) of the cell is ~ I ~ O W I I
to be daniaged following exposure to ionizing
radiation. Many induced DNA base damages havebeen identified (e-g. [F13]), alld the illduction of DNA
strand breakage in cells has recently been reviewed[W12]. There is conipelling evidence from a range ofin virro cellular studies that the main detrimental
effects of radiation derive from its ability to da~nagc
cellular DNA. First, at the cellular level it has beenshown through selective radioisotope irradiation Illat
the cell nucleus contains the principal targets
determining radiosensitivity (see, e.g. [H2, H3)).
Secondly, radiation-induced chromosomal damage may
be quantitatively correlated wilh cell inactivation (see,e.g. [J4,Ll]) and, to a lesser extent, mutation [C3].
Thirdly, in highly radiosensitive mutant strains of both
micro-organisms and cultured nlammalia~icells, there
is a good correlation between radiosensitivity andgenetic deficiency in the cellular processes that act on
DNA damage P I ] . It has also been shown that theinduction of DNA double-strand breaks (dsb) in cellsby the introduction of restriction endonuclease
enzymes mirnics the chromosomal darnage resulti~~gfrom radiation exposure [ N l ] and that there is aquantitative correlation between DNA dsb induction1
repair and cellular radiosensitivity [Rl]. Althoughnone of these observations exclude the irivolvemer~tof
non-DNA targets in some aspects of cellular response,
they provide strong evidence for induced DNA
damage as the principal mechanism of radiation action.
Most of these data relate, however, to radiationinduced chromosonie damage and cell inactivation.
While the evidence is less extensive, in vitro celltransformation has been shown to be induced by DNA
breaks produced in cells by restriction endonucleases[B9]: other evidence, outlined in paragraphs 162-165,
provides additional support for a DNA target for invitro cell transfonnation.
21. Microbial genetic techniques have alsoestablished a convincing correlation between thecapacity of physical and chemical agents to induce
somatic niutations in DNA and their activity as
carcinogens (see, e.g. [A2]). This basic somatic
mutation theory of cancer induction, although defining
the principal macroniolecular target for radiation
oncogenesis as DNA, leaves unanswered imporla111
questions regarding the number of cells that need to be~iiutatedin order to initiate the process, the number ofgene mutations that are required, whether specific geneniutations in target cells are needed and whether thesemutations are dominant or recessive in tlieir action. In

555

recent years, the application of modeni methods ofcell and ~noleculargenetics to the characterization of~ieoplasmshas led to a significant increase in theunderstandi~~gof Inany of these questions.
2.

'I'l~esingle-cell origin of neoplnsio

2 2 In order to understand the niolecular mechanismsof oricogcncsis and the role of genetic and epigcrietic
cl,al,ge, it is n,osl imponalll lo firstthecellular derivation of disease. Evidence for the
monoclo~~alorigin of neoplasia comes from a numberof sources.
23. In fc~nalemammals, all of whom are heterozygous for X-chromosome-linked genes, X-chromosome inactivation leads to a mosaic of somatic cells intissues with cells of different clonal origin expressing
different forms of the same gene product (i.e. isoenzymes), depending on which X-chromosome
remains active. Neoplasms in female mammals have,in the vast n~ajority.ofcases, been shown to have onlya single form of the gene product (i-e. a single
phenotype), implying that the neoplasm was of monoclonal origin [Fl]. Studies utilizing restriction enzyme
polymorphisms of characterized and anonyrnous DNA
sequences, chromosomal markers and oncogene andimmunoglobulin gene rearrangements have all confirmed the original X-chron~osomebiochcrnical studies[TI, W3]. Convincing evidence for the monoclonal
origin of human and murine colorectal cancer has been
provided by cytogenetic and nlolecular studies ofbenign arid malignant manifestations of this disease[F8, P7]. Most, if not all, malignant human colorectal
carcino~nasarise from pre-existing benign adenomas[S17]. Thus, although normal colonic epithelium ispolyclonal, adenomas arise within single pockets ofepitheliurti, indicating that they were initiated within amonoclonal population of stem cells [FS, F9]. The
characteristic cytogenetic and molecular features ofsuch adenomas fully supports this contention. In
addition, early prc-neoplastic haemopoietic cells
carrying characteristic chro~nosomal rearrangements
have been shown to convert in a mor~oclonalfashion
to overt ~iialignancy(paragraph 72); in animal studies,
the monoclonal origin of chemically induced tumours
is further supported by the finding of characteristic
point mutations in ras proto-oncogenes of rodent skinpapillomas/carcinomas and breast tumours (paragraphs
188 and 189). Studies on the p53 gene in humantumours associated wilh chemical or sunlight exposure
also support the monoclonal origin of neoplasia(paragraph 194).
24. Overall, it may be concluded that neoplasms withdouble phenotypes are rare and that neoplasia developsi~~frequently
from a mixed population of normal cells.
Double pl~enotypes have been recorded in some

556

UNSCEAR 1993 REPORTi ~ ~ d u c eani~ualdneoplasms, usually where high dosesof a potent carcir~ogerrwere employed. This probably
rellects Ulc coalescence of adjacent primary neoplasms, but there is some cvidcl~cetlial some doublepl~cnotypcpolyploid neoplasms niay originate throughthe fusion of two single-phe~rotypecells [TI].

25. Despite the co~iipelling c v i d e ~ ~ cthatesingleneoplasms arise from single ancestral progenitor cells,
solnc caution needs to be exercised with regard to theimplications for oncogenic nicchanisn~s.Since manyrounds of cell reproduction and clonal evolution/
selectio~roccur between neoplastic initiation and final
malignancy, the observations summarized above do
not exclude initiation of a number of target cells,
polyclonal pre-neoplasia and subsequent predominanceof a single successful cancer clone. In these contexts,
models of radiation carcinoge~~esis
requiring induced
damage in more than onc cell have been proposed[MI], and it has bee11 argued that radiation-induced
nlurinc acute rnyeloid leukaernia may not develop, asco~lverltior~allyvicwed, through a series of clonal progressions v 2 ] . It is inherently difficult to study clonal
contribu~ionsto pre-neoplasia ill most irradiated tissues
and the problem is not one that will be easily resolved.
26. However, these qualifying remarks do not in themain detract from the conclusion that the majority ofneoplasn~soriginate from darnage to single cells. In
principle, therefore, the traversal of a single target cellby one ionizing track from radiation has a finite probability, albeit low, of initiating ~~eoplasticchange. On
this basis, although only a proportion of induced preneoplastic cells may convert to malignancy, the initiation process may be expected to show a non-thresholdresponse. This may be contrasted with a hypotheticalprocess whereby neoplasia is initiated through daniage
accumulation in an adjacent set of target cells, producing, for example, a "field effect" in the tissue,
resulting in aberrant differentiation. The predictions
arising from this are (a) that a significant proportionof benign and malignant neoplasms will be polyclonal;(b) that the requirement for multi-cell damage willtend to produce a dose-effect relationship having aclear low-dose threshold. The current state of knowledge provides little general support for such hypotheses, and in the case of human bladder cancer,
where field effects have been specifically postulated,recent molecular studies show multiple site tulnours to
be monoclo~~alin origin [S47]. Against this, sonicbenign neurofibronias have, however, been shown to
be of polyclonal origin [W6], and threshold-like
responses have been reported for the induction ofsome ani~naltunlours by radiation (see, e.g. [05]).
Related to this are problems regarding the role ofrnultiple events ("hits"), genomic inslability andcellular defense rnechanisrns in neoplastic development. These receive comIne~rtlater in the Annex.

3.

Cerietic changes in oncoge~~esis

27. During tl~elast decade, investigation of the genes
directly i~lvolved ill neoplasia has been one of the~rrost rapid growth areas ill cellular and niolecular
generics. Conscquci~tly,only a very brief sulrlnlary is
possiblc (see [ B l , B6, B18, H4, H46, S18, S63, W14,
W20] for reviews). AlUlough the association between
chro~nosomalchange and neoplasia was established in
the early years of cancer research, the contribu~ionofs ~ ~ e c i fgcnctic
ic
changes to the neoplastic phenotype
had to await the advent of recombinant DNA
techniques, wliich allow the identification, isolation(cloning) and characterization of relatively small DNA
seque~~cesfrom whole genoniic con~plexes.
(a)Proto-oncogene activation

28. In this area of research, a major breakthroughwas acfiieved when it was shown that oncogenic DNA
sequences (v-one) in an avian virus had related protooncogene sequences (c-one) in normal avian DNA[Sl]. This approach initially identified c-src but was
subsequently extended to other proto-oncogenes, e.g.
c-ras (v-ms of rat sarcoma virus) and c-a61 (v-abl ofAbelsor~ murine leukaemia virus). Using v-onc
molecular probes, it has been possible to isolate andsubseque~~tlycharacterize cellular DNA sequences that
ellcode a range of oncogenes [Bl]. These and othercellular oncogenes have been characterized by otherexperimental approaches:
(a)

(b)

(c)

(d)

by transferring DNA purified or cloned from aneoplasm into suitable host mammalian cells andeslablishi~~ga correlation between the genomic
integration of a specific donor DNA sequenceand the acquisition of a quasi-neoplastic cellular
phenotype (see, e.g. [C4, G31);
by identifying host DNA sequences that areconsiste~rtly and specifically activated by theadjacent integration of oncogenic viruses (see,e.g. [M3, N21);and characterizing DNA sequencesby isolati~~gthat are located in the breakpoint region ofchromosorual
rearrangements cor~sistently
associated with specific neoplasms (see, e.g.
[ D l , H4, S631);
by using molecular hybridization techniques to
idcntify new or~coger~esthat have conservedc e r t a i ~DNA~sequences and are therefore part ofa large gene family, some niembers of which are
already known.

29. The characterization of oncogenes in human andanimal neoplasms and in in virro cellular systems
initially leads to the followi~rgbroad conclusions:(a)altered expression of oncogenes may occur
through p o i ~ mutation~t[B6, B18],chromosomal

@)
(c)

(d)

(e)

translocation and juxtaposition with anotherDNA sequence [H4,S631, insertion of relatively
short and specific viral or genomic sequences[N2] or through gene anlplification (generationof multiple oncogene copies) (see, e.g. [N7]);
there is also evidence that epigcnetic
mechanisms, such as changes in the extent of
DNA methylation, may also play a role in theexpression of genes involved in neoplasticchanges [FD,J5, S191;
these mechanisms are often characteristic ofparticular oncogenes but are by no means
mutually exclusive;
the activation of particular oncogenes may
characterize histopathologically related
neoplasms, but equally, the same oncogene may
play different temporal roles in different
neoplasms [B6];a given neoplasm will often contain structural
andlor activational changes in a number ofknown oncogenes, and there is evidence foroncogene cooperation in the developn~entof
neoplastic phenotypes [H12]; thus, long latency
periods might be explained by the need toaccumulate such changes;
oncogene activation often generates a dominantphenotypic trait.

There is an ever-growing list of proto-oncogenes,
many of which have known chronlosomal locations
and functions within the cell (see e.g. Table 1); someof these are referred to at various points in this Annex.

neoplasms requires the sequential mutationfloss ofboth copies of a specific suppressor gene. According
to these proposals, the same mutant gene is involvedin both filmilia1 and sporadic forms of the neoplasm.
In the for~nercase, the first nlutation occurs prezygolically and is thus present in all sonlatic cells of
the offspring. This is then followed by n~utationof theseco~ldgene in target sornatic cells of the affectedorgan. I n the latter case (sporadic neoplasms). both
mulatio~~soccur post-zygotically in the same target
sonlatic cell. In general, tumour suppressor genes may
be viewed as providing negative cellular effects, forthe purposes of nlaintaini~~gcells in a normal
proliferative state andlor regulating normal cellulardifferentiation programmes.
3 2 The positive phenotypic role of oncogenes
provides strong selection in experimental systems(paragraph 28) and was a major factor in their early
identification and characterization. In contrast, suchselection is far more difficult to achieve for suppressor
functions, and as a consequence, research in this area
did not make rapid progress until recently.
Nevertheless, there is now persuasive evidence thatsuppressor genes play a normal role in ccll cycle
control, cell senescence, signal transduction anddifferentiation and that their loss can contribute to thedevelopruent of a broad spectrum of neoplasms.

clear relevance to oncogenesis, dominantly acting
oncogenes probably reflect only one aspect of thewhole phenotype, determined in part by early scientificmethods. In recent years, this view has received strong
support from cytogenetic and molecular studies, whichhave revealed consistent loss of chromosomal regions
and genes in a range of human neoplasms (M4, P I ,S18, S19, S341. Such specific losses during oncogenic
development are suggestive of a major role fornegative regulatory genes and recessive effects inoncogenesis.

33. The function of tunlour suppressor genes was
first experimentally demonstrated in somatic cellhybrids produced from the in vitro fusion of normal
and tunlour cells (NT hybrids) [H29, S18]. In thesestudies, it was shown that genetic contributions fromnormal cells suppressed the neoplastic potential of thetumour cells, i.e. the malignant state was recessive andsubject to negative regulation. As NT hybrids
proliferated, they often lost chromosomes, and the lossof certain chromosomes was accompanied by areversion to a neoplastic phenotype. Thus, tumour
suppression was seen to be specific to certain
chromosonles and, by implication, to specific genes.
Recent technical advances allow the introduction of
single nor~nalchromosomes into tumour cells, and thismicrocell transfer technique has been used to nlap
putative suppressor genes to specific humanchron~osomes(see, e.g. [S34, T31).

31. The loss of negative regulatory signals fromgenes has been suspected for some years to be an
important aspect of neoplastic development. Suchgenes, often collectively referred to as turnour
suppressor genes, were originally proposed by
Knudson to be specifically involved in the hereditary
predisposition to certain autosomally deternlined
childhood cancers, in particular retinoblastoma [ K l ,PI21 of which 30%40% are of the heritable type.
Knudson's two hit hypothesis for the induction of such

34. The genetic pedigee analysis of cancer-pronefamilies has also proved to be a powerful tool in theidentification of tumour suppressor genes and theirlinkage on the human gcnon~e.Here it is important lo
recognize that for autosorilal "loss of function" genetic
traits associated with tumour suppression, theappearance of tumours in ofkpring is manifested as adominant trait Although the genes function in arecessive fashion at the cellular level, their dominancewithin family pedigrees rellects h e high probability(b) Turnour suppressor gene inactivation

30. It may be argued, however, that despite their

55 8

UNSCWR 1993 REPORT

that, in heterozygous orfspring, the normal gene will
be lost from a target somatic cell, thus initiating thespecific tuniour. These losses may, in some tumours,
be evidenced by specific chromosome deletion events,
and the identification of 13q deletions in retinoblastonia and I l p deletions in Wilms' tumour (seeIS18, S191) provided important early clues as to thelocatiori of the respective tumour suppressor genes.
This approach has also been extended to familial
adenomatous polyposis (FAP) [S20] and multiple
endocrine ncoplasia type 2 [L8], but here allelic losses
do not always occur in the cliromosonial region that is
associated with the cancer predisposition locus.

35. Molecular approaches to suppressor gene
identification in human tumours have centred on theanalysis of the loss of specific chromosome regions
where parental genetic contributions may be identified
by the presence of DNA restriction fragment length
polymorphisms (RFLPs); that is, where the two autosoma1 chromosome regions may be genetically distinguished in normal cells of the patient, it becomes
possible to use molecular analysis to distinguishspecific DNA losses through the loss of heterozygosity
in tumours [M4, S191. Thus, consistent DNA losses
for a set of linked genes in a specific chromosome
region may be taken as preli~riinaryevidence for thepresence of a tumour-specific suppressor gene in theregion. This has proved to be an extremely powerful
technique for resolving subchromosomal losses andhas provided important information on the positionand linkage of putative suppressor genes for a range ofhunian tumour types [S18]. It is becoming increasingly
clear that many different tumour types are characterized by specific losses, that the same losses are often
apparent in different tumour types and that multiple
losses are not uncommon. Parental effects on geneloss have also been observed in some tumours (paragraphs 58-60), and overall, it is now obvious that theloss of suppressor gene function plays a very important role in oncogenesis at both the initiation andprogression levels [S18, S 191.
36. From these crucial observations on thechromosomal linkage of tumour suppressor genes it
has proved possible to obtain molecular clones
representing the RB,WT,p53 and K-REV I genes (see[S18, S191) and, more recently, candidate genes forhuman FAP characterized by colorectal carcinoma
associated with chromosome Sq DNA sequence losses[F8,K15,N161. A putative suppressor gene (NF-1) for
neurofibroniatosis has also been isolated [W6].
Table 2 provides a summary of the suppressor geneloci implicated in human tumorigenesis. For some ofthese it is now possible to suggest specific cellularfunctions and link these to oncogcnic processes.
37. For RB and p53 genes, a principal role of theprotein products appears to be in the control of thecell cycle IC10, N23] (see also paragraphs 43-52). In
the case of FAP, tlie closely linked and structurally
related APC and MCC genes on Sq were candidate
determiaants of colorectal carcinogenesis, and it has
been suggested that they may act in concert to control
the proliferation of colonic epithelium. Both genes
were fouud to be soniatically mutated in sporadicallyarising tumours, and APC was found to carry pointt nut at ions in the germ line of FAP patients [KlS,
N161. Moreover, the APC gene has been implicated in
gastric and pancreatic tumours in man [N17] and, inthe mouse, is also a gcnn line detenninant of gastrointestinal tract cancer [S48]. APC somatic gene
inactivation is principally associated with point
mutation or deletion, but in one case of human coloncancer, L1 transposon insertion was detected [M46].
38. While RB gene function has provided a relatively
straightforward example of recessive suppressor genefunction, not orlly in eye tumours but also in othertumour types (see, e.g. [L21]), other tumour
suppressor genes may not operate in such a siniple
manner, and multiple locus interactions may be a morecommon feature of such genes. There is evidence thatpredisposition to and development of Wilms' tunlour
is genetically complex [D12, F101, and recent studies,
noted above, suggest that the phenotypic variation
between FAP kindreds might be explained by theinteraction between APC and MCC genes. In addition,some uricertainty still surrounds the genetics of the
p53 gene. This gene has been shown to have theproperties of a suppressor gene in colorectal carcinoma[B19], and its germ line function in Li-Fraumeni
patients (paragraph 127) supports this contention.
However, p53 in certain forms has also been shown tocooperate with ras in transforming primary rodent
cells [H17], where it appears to act in a quasidominant fashion (see also [DlS]).
39. In this context, the interaction between sequential
gene losses and gene activation has been most clearly
demonstrated in human colorectal cancer. Heremutations in at least four or five genes appear to berequired for full malignant conversion, with fewer
events being required for benign changes [F8, K15,
N161. There is also evidence that while there may besome degree of preference in the sequence of somatic
cell genetic events in colorectal carcinogenesis, theoverall accuniulation of such changes may be more
important [Fa, F l l ] ; these changes are illustrated in
Figure 1. Chromosome 5 genes may be APC and/or
MCC chromosorne 18 losses may involve the DCC
gene and chromosorne 17 losses may involve thep53
gene.
40. The escape from cellular senescence and theacquisition of cellular i~nmortalityis believed to be an
important feature of the oncogenic process. Recent

ANNEX E: MECllANlSMS OF IUDIATION ONCOGENE5IS
studies suggest that cellular scncsccncc is a process ofquasi-differentiation, which results from recessivechangcs in growth-inhibiting genes [G8].The p.53
gene has been i~i~plicatedin the immortalization ofsome murine cells in culture [H39, R151, and a rolefor normal suppressor gene function i n maintaining thesenescence process now seerns likely; thus, a loss ofsuch genes could be viewed as an integral part of ncoplastic developnient.
41. The immortalization, or life-span extcnsion, ofcells in vino may be achieved by the introduction ofviral or cellular oncogenes, and this may be linkcd
with the function of these genes in controlling cytoplasmic signal transduction and/or cell proliferation(paragraphs 43-52). 111 vivo, many oncogenic viruses,
while eliciting a chronic proliferative response intissues, do not induce one-step oncogenesis, arid
further cellular mutations are needed. In the case ofviral hepatocellular carcinoma, p53 mutation appears
to at least partially nieet this need (paragraph 117).
Similar conclusions may be formed from the observation that many immortalized human cell lines do notform tumours when transplanted into immunodeficient
mice (see, e.g. [C17]). Thus while immortalization, or
life-span extcnsion, may well involve tumour suppressor gene changes and be an important step in theevolution of most tumours, it is not itself an unambiguous marker of tumorigenic potential.

559

and non-mutational but stable changcs to gcncs, such
as DNA methylation.

(c)Cellulnr proliferativetn~nscription

controlandgene

43. Cellular growth and proliferation are controlled
U~roughthe co~istraintsof the cell cycle [M27, N121.
Quiescent cells in the Go phase require a mitogenic
stimulus to enter an active phase (GI), where geneactivity is greatly increased. At the end of the G I
phase, DNA replication is initiated; this replication
phase is teniicd S. Chromosome condensation,
initiated at the end of the next (G2)phase andproceeding through Uie prophase, is followed byassembly of the replicated chromosonie on the mitotic
apparatus; this M phase, and indeed the whole cycle,
is cornpletcd when the niitotic cell divides, providing
the two daughter cells with an equal shaie ofcytoplas~n and chromosomes. If mitotic stimulation
continues, the daughter cells enter a second cycle via
GI: if not, they may fall back into a quiescent state.
44. The cell cycle is a highly conserved and ordered
process (see [H50, M47, N23, S62]), which like mostcomplcx biological events is subject to both positive
and negative regulation. At the single-cell level,
mitogenesis and DNA replication/condensationneed tooccur at opti~nalpoints in cell development and becompleted "on time", such that chromosomes segregate
equally to the two daughter cells. Mistimirlg of cell
cycle events would obviously have adverse physiological consequences for the dividing cell or would
create genetic abnontialitics in its clonal progeny.
Equally, the rate of initiation and progression of
lleighbouring cells through the cycle must also be
closely controlled s o that tissue maintenance anddevelopment proceed without inappropriate clonal
expansion.

4 2 In conclusion, it may be seen that both positive
regulatory signals from activated oncogenes and theloss of negative signals from suppressor genes contribute, in an interactive fashion;to the development of
neoplasia. As may be anticipated, oncogenes andtumour suppressor genes have a normal role to play incell physiology, and it is through lack of or inappropriate expression, relatively minor structuralchange and combined effects that they enable the cellto evade the normal constraints of proliferation,
migration and terminal differentiation and enter a
45. It may be argued, therefore, that the expression
phase of neoplastic evolution. The specific role ofof cell cycle defects may be related to some of thesome oncogene and suppressor gene products is
illherent characteristics of oncogenic transformation.
known. These include growth factor, growth factor
While this relationship has been suspected for many
receptor, transmernbrane signalling protein,years, it is only recenUy that insights have been gained
cytoplasmic message transducer, DNA binding protein
into the molecular and biochemical control of the celland a range of regulatory proteins including
cycle and, particularly, the crucial roles played bytranscriptional factors influencing all these functions
some tumour-associated genes (see [B40, N18, R21I).
[A13, B1, B6,B18, H4, H46, S18, S63, W141. It may
46. Studies with yeasts [M27, N12] initially provided
be envisaged, therefore, that in combination oncogene
activation events, the loss of regulatory gene functio~~s evidence that the activity of two classes of proteins,
cdc-like protein kinases and cyclins, was central to celland cpigenetic changes affecting gene activity cancycle control. Acting in concert as complexes, theseproduce a cascade of inappropriate or defective geneproteins catalyse essential steps of cell cycle
expression, thus generating a ~netas~able
and grossly~~rogressiondirectly via phosphorylation of otherabnonnal cellular phenotype. These cl~arigesmay be
nuclear proteins or indirectly via activation ofbrought about by a variety of different changes tosecondary regulators. In general, these controls alsoDNA, point mutation, chromosome translocation/
apply in the case of animal cells p 2 3 ] .
insertion, intragenic deletion, chromosomal deletion

A N N M I:: MECttANISMS OF RADIATION ONCOGliNLSlS

chemical pathways associated with the ccll cyclc is amajor factor in oncogcnic transformation. In view of
the tuniorigcnic conscqucllces of RE and p53 germl i ~ l cn ~ u t a t i o ~in~ sman, it IIiily also be argucd that, insome cases, the loss of ccll cyclc control is intrinsic totuniour initiation. Collcrc~~tn~olcculara ~ l dbiocl~cniicalri~odclsarc ~ i o we~ncrgiugU~atexplain how the proteir~products of so~iickcy gcncs of ~ h ctulnour suppressor
and proto-oncoger~etypcs i~~tcract
wit11 the other components of the ccll cyclc in ordcr to effect fine control.
Current models include tumour supprcssor proteins
with cycliris and transcription factors as the primarycontrol switches, with proto-oncogenes fulfilling asecondary function. If, for the sake of argument, theloss of cell cycle control al~crcdproliferative response,
together with defective damage response, is regarded
as a consistently early event in tumorigenesis, thenirrespective of the physical or cben~icalnature of the
carcinogenic initiator, it nlay be that rnutatiorial loss orinactivation of suppressor genes, such as RB or p53,
is the most effective route for tuniour initiation. Whilestill uncertain, molecular studies on p53 gene mutations in a variety of hunian solid tumours (see, e.g.
m30]) provide some support for this, as do the datathat linkp53 tumour mutations with environmental exposure to carcinogeru (paragraphs 194 and 223-226).

(d)

Gene dosage, dominant negative effects a n dgenoniic imprinting

54. It may be argued that the sequcr~tialmutation oftarget somatic cells is the most important feature ofoncogenesis. These changes must occur within an
evolving clone of cells, and in ordcr to arise with areasonable probability at the early phases of thedisease, it may be necessary for that clone to be
expanding at an abnormally high rate. It may be
expected, therefore, that many neoplasia-initiating
events will, in appropriate in vivo circumstances,provide target cells with some degree of proliferative
or selective advantage. For dominantly acting
oncogenes, such as those of the RAS family, a specificpoint mutation may satisfy this requirement [B6, BlS].
Similarly, the activation of MYC and ABL protooncogenes through, for example, chromoson~c-specifictranslocation [H4, N3, N9] could similarly provide a
one-step growth stirnulus to appropriate target cells.
For autosomal recessive gcries (tumour suppressor
genes), the loss of function of one gene copy will, in
principle, only reduce gene product availability to50%. Such a modcrate reduction of g c ~ i cproductavailability as a consequence of this change in genenunibcr (usually ternicd gene dosage) would not
obviously provide the ;~ppropriate proliferative
stimulus to the cell. In familial neoplasnis such as
rctinoblasto~na,the loss of the first RB suppressor
gene occurs pre-zygotically, thus obviating the need

561

o~i
31), but sincefor initial clonal c x p a ~ ~ s i (paragraphRB gene loss also characterizes non-familial
rctinoblasto~na and ostcosarco~na,tl~crercniains thecorisiderablc problc~ii of c x p l a i ~ ~ i ~apparently~grecessive "loss of functionn genes, which at thecellular level may havc s o ~ n cdcgrcc of do~ninancc.

55. The problem of si~lglcautosori~algcne loss andphenotypic effect has persisted for sorne years, andalthough it is still to be fully resolved, there are anunibcr of mecllanisn~sthat would explain i t
56. For some autosonial loci involved intumorigencsis, gene dosage (one versus two copies of
the gene) may be critical, such that loss of even 50%of the gene product in a mutated cell will give rise toa phenotypic change associated with a degree ofproliferative/selective growth advantage F8]. In someindividuals this effect might be emphasized byothewise minor structural differences between alleles,
i.e. genn-line mutations of very low penetrance(paragraph 127). Alternatively, particular forms ofstructural gene mutations may produce so-calleddominant negative effects, whereby an abnormal
protein detenni~iedby the mutated gcne interferes withthe function of the normal protein specified by its nonmutant honiologuc [H18]. It has bccri suggested thatsuch dominant negative effects underlie the oncogenic
functions o f p 5 3 suppressor gcne niutations (see [DlS,
W141). Although gene transfer studies indicated thatin some tumorigcnic cclls wild type p53 is dominant
to mutatcdp53 [C18], more recent i~lvcstigationsshowthat oncogenic fonns ofp53 inhibitp53-regulated gene
expression, thus providing a basis for the selection of
p53 mutant cells during tuniorigcnesis [K17]. It may
be secn, therefore, that gene dosage or dominantnegative effects can underlie the action of sometumour suppressor gene mutations, but considerable
uncertai~itystill surrounds the implications of single
allelic mutation for tumour initiation.
57. A further possible solution to the problem hasrecently emerged from molecular studies on sporadic
tumours thought to be initiated through suppressor
gene loss. For sporadic tumours associated with RB
and other putative tumour suppressor genes (retinoblastonia, osteosarcoma, rhabdoniyosarcoma, neuroblaston~aand Wilrns' turnour) it has been observedthat major chromosome segrncnt loss events occur
preferentially fro111 the rnatcrnally i~lheritcdchromosome and that mutant paternal loci arc usually retainedin the neoplasnl (see IF23, H16, R7, R81). These
results run countcr to the nonnal expectation that insporadically arising neoplasn~s there should be anequal probability that somatic ccll gene loss will occur
from maternal and paternal chromosomes. A processtermed gcnomic iniprinting [H19, MI41 has been
i~lvokedto cxplain these fisdings.

562

UNSCEAR 1993 REPORT

58. Genomic imprinting is a poorly understood
epigenetic process by which differential expression ofcertain autosonial chromosomal rcgions is imposed insomatic cells of the offspring following some fonn ofdifferential chromosonlal niodification of male andfemale gametes. In such situations the successful
enibryonic/neonatal developlnent of tile offspring is
Uiought to be dependent upon the inheritalice of equal
parental genetic contributions for that chron~osonlal
region. The molecular mechanisms of such imprinting
effects are not well understood, but current proposals[B41,H16, R8] include the differential methylation of
DNA in imprinted regions, such that the activity of thehypomethylated genes inherited from one parent
significantly exceeds that of the hypermethylated
genes inherited from the other. Thus, somedevelopmentally associated autosomal recessive genes
do not make equal phenotypic contributions to the cell.
59. On the basis of this hypothesis and for theexamples cited in paragraph 57 it may be seen that aninactivating mutation in the more active paternallyderived suppressor gene of the target somatic cell
could reduce gene product availability to a level thatderegulated cellular proliferation/differentiation andresulted in the excessive clonal expansion of preneoplastic cells. This expanding clonal population
canying the first suppressor mutation may thencomplete sufficient cell divisions to allow for the
probability of spontaneous loss of the second, lessactive suppressor gene copy of maternal origin.
According to the hypothesis [R7,R8], this second
mutational event greatly increases the probability thatthe cell clone will progress towards malignancy.
60. Although an imprinting effect on gene mutability
has also been suggested for the RB gene in theinitiation of osteosarcoma [TS], the above hypothesis
does account for Ihe molecular findings in sporadic
tumours, and if it is a more general phenomenon, there
may be important implications for radiation
oncogenesis. Specifically, an increasing numbcr ofpotentially imprinted chromosomal regions and genes
involved in differentiation and development are being
identified in man and the mouse [B41,C19, H19,
H52].The involvement of negative regulatory genes inliormal cell differentiation and development may meanthat some potential tumour suppressor genes are
imprinted in specific tissucs and, therefore, the loss of
one copy may be sufficient to initiate neoplasticchange. This, together with the finding of a DNA
deletion mechanism for radiation niutagenesis(paragraph 154), implies that radiation may be an
efficient "single eventn initiating agent for certain
neoplasms associated with the loss of tumour
suppressor gene function. However, the whole processof imprinting remains poorly understood. The recent
observation of imprinting-like effects on the 1(9:22)

translocation in lluriial~ chronic n~yelogenous
leukacmia and N-MYC anipliSication in neuroblastoma
indicates that such clfects may also extend to certain
proto-oncogene activation events [F23, H40, R161.

(e)

Cl~romosomulfragile sites

61. Clirolnoso~iialrearrangement and deletion is amajor feature of oncogenic development, and there isan ongoing debate as to the significance of specificsites of chromosomal instability (fragile sites, c-pa)
lH2.5, H26, L9, M5, S21]. These sites, classified
according to their frequency in the human populationand their inducibility by different chemical treatmentsof cells, have been suggested as possible predisposing
factors in oncogenesis. Some Gagile sites are also
believed to be preferential targets for the clastogenic
action of DNA-damaging agents, including ionizing
radiation [Yl]. Despite statistical analyses showing nooverall association between common fragile sites andcancer-associated chromosome breakpoints Ff48,
S211, an association with rare and distamycin A
inducible fragile sites rernains, particularly withrespect to certain leukaemias [H20, L9, S21]. Overall,
it appears that the expression of DNA damage is nonuniformly distributed within the mammalian genome,that certain highly recombinogenic sequences are
preferentially involved in induced chromosomalchanges and that certain classes of these may
contribute towards oncogenesis.
62. The lnolecular structures of fragile sites are notknown, although it seems likely that they represent
certain reiterated (repeat) DNA sequences that may
recombine at a high frequency. In this context, it has
been suggested that interstitial teloniere-like repeat
sequences may be highly recombinogenic [H21].
These DNA repeat sequences, in their nom~alterminal
position on chroniosomes, buffer against chromosomal
erosion and instability during DNA replication [MIS,
M29, S221. However, in some lower organisms,
teloniere-like repeat (TLR) sequences are highly
recornbinogenic; it is this feature, together with limited
chromosonle aberration and mapping data, that initially
prompted tile speculation that, when located ininterstitial sites, they may represent a subclass ofmammalian c-fra [H21]. Further support for telomere
sequence instability has come from both microbial[K18] and in viao n~an~maliancell studies p19], butother investigators [I21 failed to find an association
between a specific human c-fro, FRA2B, and a TLR
sequence array located in the same chromosomal
region.
63. Cytogenetic evidence of telomere-associatedchron~osomalinstability has been obtained in studies
with some human leukaeniias (see, e.g. [S35]), andANNEX E: MI:Ct+lNlSMS OF RhDlATION ONCOGENEIS

chromosome break Ilealing mechanisms involving thede novo addition of telomeric sequences have beendeveloped from studies with lower eukaryotes (see,e.g. [H31]). Under certain circumstances it appears
that these DNA repeat sequences are subject toconsiderable modification in somatic cells, perhaps
reflecting their structure and/or the lack of telonierase
cnzylnes, which can act to extend tlie repeat arrays[B20]. This apparent instability is not, however,restricted to somatic cells, since studies in the mouseclearly show the arrays to be hypervariable withinmouse strains, a feature that might be explained bytheir capacity to initiate DNA recombination and DNA
replication slippage or as a consequence of the repeat
sequence modification by telomerase, which isbelieved to occur during gametogenesis [S36, S371. A
possible factor in teloniere-like sequence instability is
the unusual secondary structure that these sequences
adopt in vitro [B20, B42, K19]. Such secondary
structures, if they were to occur in vivo, might also
contribute to the fragile site properties suggested
above. The contention that such sites may beparticularly prone to radiation-induced breakage andrearrangements and that some such events are involved
in radiation oncogenesis is supported by recent studies
on the nature of the chromosome 2 deletions andrearrangements that are believed to initiate murine
myeloid leukaemogenesis (paragraph 183). Alphaparticle-induced chromosomal instability has recently
been demonstrated in murine haemopoietic cells[K25],but this has yet to be specifically linked withfragile site expression or neoplastic change.
64. Telomeric DNA sequences are also believed toplay a role in cell senescence [G8], and it has beenshown that the ageing of human fibroblasts isaccompanied by telomeric shortening [H22]. This may
be viewed as increasing inherent chromosomal
instability and could be a factor in age-related
carcinogenesis. There is no doubt that the current
intense interest in the involvement of telomeric andother genomic repeat sequences (see, e.g. [BIO, B42,
K20, S491) 1
chromoso~nal fragility andrearrangement will yield data of direct relevance tomolecular mechanisms of chromosomal instability andtheir role in oncogenesis.

4.

hlultistnge cellulnr development inoncogenesis

65. The concept of multistage oncogenesis, originallyproposed by Berenblum and Schubik in 1948, hasbeen a most valuable and durable concept. In modem
developments of this theory, oncogenesis is divided,
albeit imprecisely, into three phases: initiation,
promotion and progression.

(n)

563

Tunlour initiution nnd other enrly events in
oncagenesis

66. The initiation of oncogenesis may be most
simply viewed as one or more stable cellular events
arising spontaneously or induced by exposure to a
carcinogen, which predisposes carrier cells to
subsequent neoplastic conversion. In the case ofneoplasms induced in man or experimental animals bysingle acute doses of a carcinogen, the agent isassumed to act as an initiator by damaging a specificcellular target in a stable and irreversible fashion [B6,
B18, Y2]. Throughout this Annex it is argued, on thebasis of animal and human data, that specific somatic
mutations in target genes are initiating events forneoplasia.
67. In the preceding Section, cytogenetic andmolecular findings on suppressor gene mutations andproto-oncogene activation were discussed in respect of
their possible roles in oncogenesis, including initiation.
In subsequent Sections, data from experimental studies
will be discussed, with an emphasis on the molecularnature of induced initiating events. Here it is sufficient
to emphasize that at low doses of ionizing radiation,
it is knowledge of the induction of early initiating
events that may b e most important to the understanding of radiation effects. That is not to say
however that radiation plays no part in the other stagesof oncogenesis.
68. In principle, it appears that neoplasia may beinitiated either through proto-oncogene activation or
suppressor gene inactivation. Thus, a multiplicity of
induced somatic mutations may contribute towards
human radiation oncogenesis, and even for a single
histopathological form of neoplasia, there may be anumber of possible initiating events, albeit withdifferent probabilities of contributing towards overt
malignancy. The necessity for a single clone toaccumulate further somatic mutations before malignant
conversion must be rate-limiting and implies that only
a minority of initiated cells progress beyond the preneoplastic phase (paragraph 219).
69. At this early phase, aberrant pre-neoplastic
clones may be lost through, for example, metabolic insufficiency, non-specific cell selection, the suppressive
effects of neighbouring cells, terminal differentiation,
cell senescence, programmed cell death (apoptosis) orcell surveillance mechanisms. Thus, the capacity of an
initiated cell clone to expand in a relatively undifferentiated state may be viewed as a crucial aspect of theearly phase of the disease (paragraph 54). In thisrespect, the manifestation of benign tissue dysplasias(e.g. papillomas, adenomas and hacmopoietic hyperplasia) may be viewed as clonal expansion, limited
perhaps by a con~binationof the factors noted above.

564

UNSCEAR 1993 REPORT

70. Specific ~ ~ i i ~ l iofp 11111iour-initiati~~gl~smutationsassociated wilh RAS, RB, 1V, p53, NF-1 a ~ APC~ dgenes arc discusscd clscwhcrc in this AJIIICX,
and thecvidc~icethat mutation o l tllcsc genes in appropriate
norliial target cells prcdisposcs to malignant convcrsion is rclativcly strollg. For other tunlour mutations it
is not yet possiblc to conclude t11;it they i~ritiatcdthe
tumour, only th;~tthey arc presclll at an carly prcneoplastic phasc, before full cli~~ical
progression of
disease is n~aoifcstcd.
71. In h u r n a ~ncoplasia,~
particularly lcukaemias andlymphomas, carly phases of neoplastic dcvelopn~cnt
are frequently associated with consistent chromosonial
rearrangements, such as the 9:22 translocation inchronic n~ycloidleukacrnia (CML), the 5q-deletion in
myclodysplastic syndrome and the 14:18 translocation
in follicular lymphoma [ H l l , S63]. In such cases the
progression of early iridolcrit disease is usually accompanied by the selective clonal expansion of neoplastic
subpopulations containing further chromosomal abnornialitics. In general, in both haemopoietic and solid
tumours, the more advanccd and aggressive the disease the greater the degree of chromosomal change.
Tables 3 and 4 illustrate consistent chroniosonial changes recorded in hunian Icukacmias/lymphomas andsolid tumours, respectively (see [S45]). In the case ofh e 9:22 translocation in CML it has been establishedthat a BCR-ABL fused gene is produced at the translocation brcakpoint; this has tyrosine kinase activity,
which produces a stablc mitogcrlic stimulus to tile cell.
Of particular note arc observations of different formsof BCR-ABL fusion in acutc and chronic myelogcnous
leukaemia and a likely correlation between mitogcnicity of the BCR-ABL gene product and the relative
aggression of the two mycloid ricoplasrns (see [G24,N91). For the 14:18 trarislocatior~ in follicularlymphoma, molecular studies show that a ncwly
recognized proto-oncogene BCL-2 is uprcgulated byjuxtaposition with an active immunoglobulin hcavy
chain gene (see [Ng]). It has recently been establishedthat such overexprcssio~iof BCL-2, rather than being
niitogenic, serves the purpose of blocking progranmed
cell death [H23] and extending the in vh*olife-span of
B-cells (see also [El I]). Thus, ccrtain gcne activation
events in tumorigencsis do not simply increase proliferation rates but instead block or prolong the normal
differentiation pathway. Olhcr examples of chromosomal translocations in acutc B-cell and niyeloid
ncoplas~nsare thc t(15; 17) in sonic acutc myclogenous
lcukacniia (i~~volvingthe PML end rctinoic acidreceptor genes), the t(1:19) in pre-B ALL (involving
the PBX and E2A gcncs) and the t(17;19) in B-ccll
ALL (involving the HFL aud E2A gc~lcs)(scc [ElO]).
111 addition, the I lq brcakpoint gene thought to be
involvcd in t(4;lI) and t(9:ll) acute leukae~iiiashassignificant hornology to thc trx transcription factor
gcne of Drosoplrila ID131.

72. Characteristic cl~romoso~nal
rcarrangcnicnts arc
also seen ia I I U I I I ~ I I T-cell 1c11kacmi;is;many of Ulcse
i ~ ~ v o l vspecificesites on cl~romoso~~ics
7 and 14. In
peripheral ly~iipl~ocytcs
of at;ixia-lclaagicctasia, c l o ~ ~ a lrcarraagcn~entso l this type 11;ivc hccri obscrved in a~rurnbcr of ~)aticr~ts
s l l o w i ~ ~110
g otllcr cvidc~iccof
nialignaacy. Alll~ougl~
olic~lprcscnl at rcl:itivcly highfrequencies, tllcsc cloncs tlo 1\01 usually convert tofrank lcukaeniia: in a few cases, however, nialignant
progression of thcsc clorics Ilas bccn observed (see[T6]). Molecular studics have now cstablishcd thatmany of these rcarrangc~ncnts in T-ccll neoplasms
involve vcry spccific recombination between T-cell
receptor (TCR) gcncs and othcr chroniosonial regions
encoding transcription factors such as HOX 11 (see[H32,R31). The activation of these factors U~rougb
TCR translocatio~iwill tc~idto alter the developnicntal
programmcs of T-ccll precursors and could explain theearly clonal expansion observed in some ataxiatclangicctasia patients. A more co~iipletediscussion ofmany of the chromosomal events noted here isprovided by Solon~onet al. [S63].
of the nicchanism of low
73. In any co~~sidcration
doses of radiation to i11iti;itc oncogcncsis it isimportant to attanpt to establish the relative
probability with which a s i ~ ~ g lionizingetrack willi~~tersecta given DNA targct, causing a tuniourinitiating mutation. This is co~rsidercdin the following
and loss-of-function ~iiutations.
for gain-of-functio~~
74. Activation of proto-oncogenes through gain-offunction nuta at ions appears to occur via two principal
rneclia~iisms.For p r o t o - o ~ ~ c o g c ~such~ c s as RAS, theDNA base-pair (bp) changes required for activation
are very restricted, and hence the molecular targct (fordirect effects) will bc small, pcrhaps o~lly-10 bp [B6,B18]. For t l ~ c gene-specific chromoson~altranslocalions ilivolvi~igthe juxtaposition of protoo ~ ~ c o g e ~such~ c s as ABL, BCL-2 and HOX 11 withothcr rclativcly specific activating encs, the nioleculartargct may be larger, pcrhaps 10--lo4 bp [H4,H32,
R3, S63). However, for these events it may inprinciple be necessary to daniagc DNA at two specificsites rather than one, thus effectively reducing ovcralltarget size. Relative to thc size of the whole genome,-lo9 bp, such gain-of-function, gene-activating
mutations would sccln thcrcforc to present \'cry srnali
targets for si~lglc-trackradiation a c t i o ~ Nevertheless,~.
OII a targct-size hasis it h;is bccn proposcd that the
primary e v c ~ ~int human CML call bc a radiationinduced BCR-ABL tr;ir~sloaitio~~[H41].

ri

75. This s i t u a t i o ~Illiiy~ be r o ~ ~ t m s t ewith
d the loss-offunction ~uutations cl~nraclcristic of the tuniour
suppressor roles of gc~lcssuch as RB, IVT, APC andp53. Loss-of-functioa of lulllour suppressor genes may
small intragcnic dcletionoccur tlirough point 111utatio11,

ANNIX li: MECllANIShfS OF RADIATION ONCOGENFSIS

or larger deletions spanning whole cl~romoson~csegments (-lo7 bp) IH30, S18, W141. I l e priacipal
liniit to the size of the DNA deletions associated withsulqwcssor gene rnutatio~~
will be the dcgrce to which
the ccll can susli~in viability followi~~gsubstantial
genetic losses. Sincc this will vary with d ~ locationcofh e targct gene in relation to csscr~tialDNA sequences
icof
and, correspondingly, with t l ~ cg c ~ ~ c tbackgroundthe cell itself, it is impossible to prcdict more
precisely the targct sizcs for tl~csedeletions. It should
also bc noted that in the case of d o r n i ~ ~ negativea~~teffects, such as those postulatcd for p53, deletion of
the whole gcne may not be effective (paragraph 56).

76. In spite of thcse uncertainties and qualifications
made in paragaphs 158 and 159, this simple biophysical argument would predict that radiation-induced
loss-of-function mutations may dominate the spectrumof potential initiating events for carcinogenesis. On thebasis of the figures given above, the probabilities
between "lossn and "gain" events may differ byperhaps two orders of magnitude. It remains to be
seen how realistic thcse estimates might be, but some
support for this argument has conlc from molecularstudies on radiation-induced somatic cell mutation(paragraph 154).

77. It may be concluded that turnour-initiating
niutations probably vary in fornl, but on the basis ofrelative target sizes, it secnls likely that tumour
suppressor gene mutations nlay be the predominant
form in radiation oncogcnesis. Alone, such initiating
events in normal targct cells will not be sufficient lo
produce a malignant phenotype, which would require
both clonal expansion of initiated cells (promotion)and the accumulation of further epigcnctic and genetic
changes (promotion plus progression). The point ofmalignant commitment in a developing neoplasm istherefore difficult to specify; indeed, there may not be
an "all or nothingn transition but rather an increasing
probability that pre-neoplastic cells will bypass normal
cellular constraints and convert to frank neoplasia.
Consequently, while oncogenesis may bc operationallysubdivided into initiation, pron~otionand progression,these definitions grossly simplify a geneticallycomplex process of ccll development U~atwill vary
between neoplasms.
(b) Turnaur promotion
78. In expcrimc~~talanimal systems, promoters are
identified as agents that, a l o ~ ~ havee , low oncogenic
potential but that arc able to greatly enhance h e yieldof neoplasms induced by prior exposure to a subcarcinogcnic dose of an initiator [ C l l , S2, Y2].
Agents that strongly promote oncogenesis generally do
so at low concentrations, but in contrast to initiators,

565

repeated or chronic exposure is usually necessary. A
third distinguishing feature is that, unlike initiation,
pro~rlotional effects arc usually reversible. In thcse
respects prornolion has the properties of a11e p i g c ~ ~ c t i cprocess, involving ~~lctastablecl~angcs in gcne
expression and ccll ular/tissuc responses that havedramatic consequences for the initiated ccll and its
clonal progeny. In the rllajority of cxpcrimcr~talanimal
systems, initiation and promotion procedures produce
an increase in prc-neoplastic lesions or benign
neoplasms, and for nlost promoters there appear to be
no dramatic effects on oncogenic progression. The
whole question of carcinogcnic interaction between
radiation and chemicals, il~cludinginitiation/promotion,
has been discussed in dcph by Strcffcr el al. [S2] andTrosko et al. [T12].
79. Neoplastic proniotion following ionizing
radiation has been studied in both experimental animalsystcms and in vitro transformation systcms w6, K10,
W,S2, T121. However, much of the detailed knowledge of nlechanisms of promotion has come fromchemical initiation/proniotion studies with rodent skincarcinomas [ C l l , Y2], coupled with detailed biochemical investigations on the cellular consequencesof promoter exposure [N5, N14, T11, T12, W4].

80. The nature of promoter action has, until recently,
been obscure. The term promoter is an operational
definition encompassing a diversity of chernicalentities ranging from the classical phorbol esters
through phenobarbital and bile acids to growth factors,
hormones and ill-defined dietary components. However, tissue wounding and stress IS231 should also be
included in any broad definition. Clearly, not all thesefactors will operate in all tissues, and it is likely thata range of biochcrtlical pathways can be involved. It
is through studies with the phorbol esters, such as
TPA(12-o-tetradeconylphorbol-13-acetate),
ditcrpenes,
indole alkaloid and polyacetate promoters, that anunderstanding of certain aspects of promotional
mechanisms has emerged. High affinity receptors forthese promoters have been identified in mammaliancells [N5, W4]. In the case of TPA, that receptor, orpart of it, is the calciunl- and lipid-dependent enzyme
protein kinase C.
81. Protein kinase C, through its ability to phosphorylate and activate a range of cellular proteins andinduce the expression of cellular genes, plays a crucialrole both in signal transduction across ccll membranes
into the cytoplasm and ill subscqucnt cellular responses. In this rcspcct, protein kinase C is ar tbe
crossroads of a nu~nbcrof biochemical pathways thatare known to mediate cellular proliferative response tohormones, growth factors and cytokines. Promotermediated activation of protein kinase C will tend loenhance these pathways through a cascade of protein

pl~osphorylationand gene expression events, a principal outcome of which will be a disturbance in tissue
homeostasis. Although diverse in their structure, mosttun~ourpromoters share the basic property of inducing
a degree of tissue hypcrplasia and inflammation; thishistopathological feature probably slenls from thedisturbance of tissue homeostasis.
8 2 Endogenous promotior1 is likely to play a far
greater role in human oncogenesis than extrinsic
chemical factors, and in this respect it is important torecognize that protein kinase C is normally stimulated
by increasing cellular diacyl glycerol (DAG) levels
through lipid tunlover [NS]. Since DAG levels havebeen shown to be increased following the action of avariety of cellular growth factors and cytokines (see,e.g. [M16, M17]), it follows that these factors
probably play a major role in oncogenic promotion.
This view is also consistent with observations
indicating that the promotional wounding response inRous sarcoma virus tumorigenesis is mediated bytransforming growth factor-I3 [S23].

83. However, it has become increasingly clear thattunlour promotional pathways are not solely mediatedthrough the biochemical pathways involving protein
kinase C and that changes in cellular com~nunicationand oxidative metabolism [C21, T11, T12] may also
occur in response to exposure to many tunlour
promoters.
84. The maintenance of tissue homeostasis, which
requires cells to establish a critical balance between
proliferation and differentiation, is known to involve
not only responses to high molecular weight systenlicfactors (growth factors and hormones) but also lower
molecular weight ( ~ 1 , 0 0 0daltons) ions and metabolites that are exchanged between neighbouring cells
linked by so-called gap junctions at cell n~embranes[L14]. In facilitating cellular commurlicatior~ andbiochemical coupling, these junctions are believed toplay an important role in the local coordination of cell
pro1iferation.
85. The establishment of gap junctions is detem~inedby a family of conserved mammalian genes, theactivity of which appears to be controlled by both
systemic factors and intracellular processes mediatedthrough signal transduction pathways. A critical link
between gap junction cornrnunication and oncogenesis
was established by the observation of the stable loss ofcoupling in many tunlour cells (K13,T12j. However,such losses in tulnour cells may often be selective, in
Ihat tumour cells remain coupled to each other but
lose the ability to communicate with normal cells
IN13, Y7]. 'Illus, it became possible to postulate thatthe signals exchanged between normal cells(hornologous coupling) adequately regulate certain

proliferation/ differentiation responses but that the
selective loss of heterologous coupling allows tumour
cells to become more autonornous and less receptive
to tissue regulation.
86. Tlie relevance of cellular communication in
tunlour promotion became obvious when it was shownthat a wide range of turnour promoters had the capacity to induce transient dysfunction in gap-junctionmediated processes [M30, N14, Y7]. Additional
studies show such inhibition of coupling to be part ofthe ~ ~ o r ~ proliferativeual
response in tissues, in that
inhibition occurs when cells are drawn from a quiescent phase in order to complete tissue growth or
repopulation [PlO]. It seems likely, therefore, that insome tissues gap junction fom~ationand loss is asecondary compoacnt of the complex cellular machinery that drives the cell cycle (paragraphs 48-50).
87. Thus, albeit in a transient fashion, the inhibitionof gap junction communication by promotional stimulation nlay be viewed as a means whereby one layerof cellular proliferative control is removed; this would
tend to elicit a mitogenic response in all undifferentiated stem-like cells in tissue. The magnitude ofthat response may not, however, be uniform, in thatcells carrying tu~nour-initiating mutations in genes
central to cell cycle control, such as RB, might be
expecled to respond most strongly. The involvementof tumour-associated mutatior~saffecti~rgcell adhesion
proteins, e.g, DCC (deleted in colon cancer) [ F l l ] , ingap-junction mediated processes is also possible.
88. The potential complexity of tumour promotionn~echanisnisis further increased by the finding that
some, but not all, promoting agents induce bursts ofoxidative nletabolism in exposed cells, which lead tothe generation of short-lived, free chemical radicals[C21]. Such radicals are highly reactive within the celland are able to induce damage in a range ofn~acromolecularstructures, including cell membranes
and chroruosomes.
89. 111 the case of TPA exposure, it has been
established that induced radicals such as superoxides,
peroxides and arachidonic acid metabolites are able toattack cellular DNA, inducing a range of cytogenetic
abnor~nalilies[C21, D11, P l l ] . On the basis of theseobservations i t has been proposed that TPA-generated
chemical radicals act at an early stage of prornotion(stage 1) and convert carcinogen-initiated cells to astate in which they are more sensitive to proliferative
stimulatio~~(stage 2 promotion) [F15].

90. I s the mouse skin papilloma/carcinoma system,
TPA has bee11 shown to be a "complete" promoter inthat it fulfils both stage 1 and stage 2 promotional
requireme~~ts;in colrtrast, the related phorbol ester

ANNEX E: MECIWISMS OF RADIATION ONCOCiENESIS

RPA fulfils o~nlysL~gc2 rcquircnic~nts,i.e. it is not
clastogcnic. The innporta~~ccof c h r o ~ ~ l o s o ~daniagc
ticin the stage 1 convcrsio~nphase was fuflhcr cstablishcd
by studics showing that at low doscs ~ h calkylatirng
clastogcrnic agent, mclhyl ~nctlnancsulphonatc actcd
synergistically with RPA to cffcct coni~)lcteprornnotion[F16]. Since UIC convcrsio~n s h g c of pro~tnotiorn
involvcs chromosonnal damagc, it may ccntrc on
charngcs in gene activity or chroniosomnl instability;
the specificity of such cvc~ntshas not, I~owcver,been
establ ishcd.

91. The cytogenctic observations notcd above requircthat a much broadcr vicw be taken of the involvementof genetic changes in the promotional phases of oncogenesis. The boundary bctwccn initiation and promotion is becoming increasingly artificial, defined onlyby the tumour systcm under consideration, thephysico-chemical nature of the insult applied and thecellular niechanisrii bcing sought. Hcrc it is sufficicnt
to say that, mechanistically, both initiation and prornotion probably involve a combination of genetic andepigenetic cellular cvcnts and together thcy drive aclonal population of cclls through a prc-neoplastic
phase to a point whcrc malignant co~nversion isassured. Perhaps they differ most clearly in thc
temporal requircnnc~ntsfor phcnotypic changc and, on
current belief, the larger co~~tributionthat stable
mutagenic changc makes to the i~nitiatio~nprocess.

92. Ionizing radiation is a powerful clastogcn andwould certainly induce ninny of the forms of cytogenetic damage currently associated with the conversion stage of tumour pronnotion. What may bequestioned is the cxtcnt to which radiation might
induce the epigcnctic cha~ngesassociated with stage 2
promotion. As notcd below, it sccnis that, in general,
radiation acts as only a weak promotor of neoplasticchange.
93. Through its ability to damage tissues and inducea mitogenic response in rcpopulating cclls, radiation
could be regarded as a stage 2 promoting agent. Sucheffects will, howevcr, only be significant at relatively
high doses, where substantial ccll inactivation has
occurred. Thus, it may be argued that for radiologicalprotection considerations, this form of promotional
activity is of minor irnportancc. This view may betempered, howcvcr, by rcccnt cvidcr~ccof upregulationof protein kinasc C and proto-o~ncogencgene products
by relatively low doscs of radiation [S50, W7, W21],
and it will be i~nportant to establish whether suchbiochemical rcsponscs extend to other promotional
processes, particularly, perl~i~ps,
those deterniirni~ng
intercellular communication.
Recent expcriniental studies usi~ng the niouse
skin papilloma/carcinoma system havc highlighted
94.

567

sonic of thc difricultics laccd ie i~pproaching thequestion of ilntcractioo bctwccn radiation and othercarci~nogcnicagcnls. Hcrc it l ~ a sbeen shown that beta
irradiation induces rcsistarlce to chc~~nicallyinduccd
These
papillomi~but not carci~nonlaforniatiorn [M31],
indn~ccsa DNA
obscrvatio~nsmay i~nply(hilt rijdiatio~~rcpair process that acts diffcrcntii~llyo ~ tllc
r cl~c~nicallyand i~tduccd lcsior~sdriving pro~notion-dcpc~~dcrnt
indcpc~ndcntskin carcinogcncsis. Howcvcr, given thc
complexity of t h ~p r o ~ ~ i o t i o ~processes~i~ldescribed
hcrc, oUier explanatior~sshould not be excluded. In thc
same cxpcrimer~tal systcm, chro~nicbeta irradiation
lacked action as a complctc or stage 2 tumour
proniotcr but did show wcak but positive action instage 1 promotion [M37].

95. The suppression of cellular transformational
processes by so-called anticarcinogc~uor antipromoters has been studied using in vuro transformation
systems. Protease inhibitors, such as antipain andchymostatin, appcar to strongly inhibit radiationtransformation in an irreversible fashion, suggestingthat thcy may act at thc level of DNA damage modification [KlO].However, since such inhibitors also
suppress the promotional activity of TPA, there may
in ccll surface receptor-promoter
also be i~~volvemcrnts
biridi~igprocesses or the subsequent protein-kinasc-cmediated lnessage tralnsduction process. Other chemical factors, such as retinoids, ascorbic acid, lymphotoxin and vitamin E, havc also been shown to havc
anticarcinogcnic and/or antipromotional activities (see[Cll,K10,T20,YS]).Some of these factors may also
act in v h ~ o[Cll,C15, C16, L15j. In the case of
d-limoncne it has bccn suggcstcd that i~iducedchanges
in the intracellular location of GTP-binding proteinssuch as p21ra rniight underlie the anticarcinogcnic
action [C27].

96. While no single molecular mechanism of antipromotion may exist, at the cellular level, promotional
processes are most consistently characterized by thetransient loss of intercellular communication. Relevantto this is recent evidence showing that strongly
antipromoting agents such as retinoids markedly
increase intcrccllular cornniu~nication[H48, M451. In
virro antipromotion by ascorbic acid may, however,involve oxygen radical re~riovalfrom cells [YlO].

97. In conclusion, the promoter cnhanccnicnt ofappropriate biochcniical pathways in carcinogcninitiated cclls call be viewed as interacting with thestable biochcrnical sequelae of the initiating cvcnl ina target cell in a llnallrlcr that elicits a supranonilal
prolilcrativc response. Such a mechanism, dependingon the strength of t l ~ eprornotcr and the duration of
cxposure, would tend to establish i~nitiatcdclones ill
their host tissucs rapidly and efficiently, thusincreasing the frequency and proliferative capaci~yof

UNSCMR 1993 REPORT

568

pre-neoplastic lesions. Until these transient changes arestabilized through perhaps further clonal mutations,they are, in principle, reversible. Assuming gap junction dysfunction to be a pivotal feature of promoti011
IT121, then upregulation of junction formation could
be regarded as an important mechanism of antipromotion [H48].On the basis of current experimental
knowledge, this may be achieved using exogenous
agents such as retinoids but, in vivo, e~idogenous
processes involving the regulation of tumour suppressor genes and other cell cycle control factors may
be expected to be of greater importance.

(c)

Malignant progression

98. During the life history of a neoplasm there isoften a progressive tendency towards increased
malignancy. This is most frequently seen as a stepwisechange in both tumour histopathology arid aggression[F3, N3]. In the case of solid tumours, an extended
blood supply niay be recruited; subsequently, nietastatic properlies emerge, allowing the neoplasm tospread to distant sites. Phenotypically, neoplastic
progression appears to be the most complex of thethree phases, and considerable histopathological,
cellular, cytogenetic and molecular variation may beseen, even within a single progressing neoplastic
clone. Importantly, progression through clinicallydefined phases is generally, in the absence of clinicalintenlention, irreversible. This, together with otherfeatures discussed later, is suggestive of sequential
somatic mutation and consequent selective clonal
proliferation.
99. Although the histopathological manifestations ofneoplastic progression have been well-documented[F3], it is only recently that some understanding has
been gained of the underlying cellular and molecularmechanisms. The apparently stepwise transitions thatcharacterize the progression from benign preneoplastic lesions to aggressively metastatic neoplasms
are thought to represent clonal evolution and selection
processes driven by genetic and epigenetic cellularchanges [K6,N3, N9, S19].
100. At the cytogenetic level these changes are often
evidenced by secondary chromosomal translocations,deletions or duplications. Some of these, particularly
those appearing at low clonal frequency, probably
represent "cytogenetic noise" and may be regarded as
neutral. Others appear more consistently in dominantclones and are likely to involve the activation,
amplification or loss of specilic genes. These may beviewed as positively contributing towards clonal
selection, dominance and, thereby, neoplastic
progression. Examples of consistent secondary
cytogenetic changes in huntan neoplasms are the 8:14

translocation iu some cases of acute lyniphocytic
Ieukaemia (hWC oncogene activation), the trisomy ofchromosome 7 in advanced melanoma (ERB B genedosage?) and the appearance of homologously stainingr e g i o ~ ~ands double-minute chromosomes in neuroblastoma (N-MYC a~nplificatiol~)[HI1, N3]. Secondary
mutations potcetially releva~ilto r~eoplasticprogression
change, and in
do not always involve cl~ro~nosomal
RAS mutations are believed to occur
some ~~eoplasnis,
during progression and contribute towards tumour
aggressiol~(see, e.g. [ R l l , V2)). Such studies havealso highlighted the genetic polyrnorphisrns in tumours
associated with genoniic instability [Rll]. The polyclonal evolution of tuniours may also be studied using
experinie~~tal
molecular markers; for example, using
plasrnid-transfected inouse fibrosarcoma cells, it has
been possible to identify individual clones that haveacquired properties associated with preferential
metastasis [ES].

101. Secondary chrornosonial changes and oncogene
activation events have also been characterized in a
number of animal neoplasms induced by ionizing
radiation or chemical carcinogens and have received
comnient elsewhere in this Annex. Animal models ofneoplastic induction are also beginning to demonstrate
the possible roles of induced DNA da~nagein oncogenic progression. Neoplastic progression in chemically initiated murille skin has been shown to beenhanced by treatment with radiation [Bll]. Thisobservation may be consistent with the specific losses
from chro~nosorne7 that have been shown to contribute to the progression of rnurine skin carcinomas[B12, B29].
102. Studies of this type should extend the knowledgeof differences ill the genetic events that mediate
neoplastic initiation and progression and shouldii~dicate the potential co~itribution of physical andchemical agents in the two phases. In this respect, it isimportant to recognize that in the case of protracted or
fractionated exposures, ionizing-radiation-induced
cellular damage may, in principle, contribute to both
neoplastic initiation and progression. Parallel but more
complex co~isiderationsapply in the case of combined
exposure lo radiation and chemical carcinogens [S2].
103. Tl~erc is growing evidence that neoplastic
progrcssio~~
may be greatly i~lfluencedby the acquisition, at a relatively early stage in h e process, of
intrillsic g e ~ i o ~ n iinstability.
c
Benign lesions usuallycontain few cells with mitotic abnormalities, but thesecells usually increase in frequency as the neoplasm
progresses. This is often accompanied by increases inploidy, chroriiosornal breakage, non-disjunction andsister chroniatid exchange [S13], i.e. all the features ofthe dcvelop~iientof abnormalities in DNA metabolism.

104. Wlrile the mcclranis~risu~rdc.rlyi~~gthese 11utativc
defects arc still obscure, the conscqurnccs are crucial
to the understanding of rrcoplastic progression. I~rtrinsic chron~osomaliastability will greatly increase thefrcqucncy of spontaneous and induced genetic and cpigenetic change witlri~rthe cvolvirig neoplastic cloac.
This provides the dynanlic hcterogcncity at the cellularlevel that is the hallmark of clonal neoplastic progression. Loss of ccll cycle control, established during
earlier pliascs of the neoplastic proccss (paragraphs
43-53), may play a role in the ge~icticinstability thatcharacterizes tumour progression. I t is also possiblethat mechanisms of gcnomic instability involving thecxprcssion of recombinogenic sites and telomerc-likc
repeat scqucnccs in DNA may contribute to this process: for some human Icukacmias thcrc is somc cvidence for telomere sequence instability [S35] (paragraphs 61 and 64), and one fonn of heritable coloncancer is characterized by widcsprcad instability of
dinuclcotide repeat sequences (see paragraph 122).

105. The principal phenotypic characteristic of themalignant progressio~lof many tunlours is the ability
to spread (metastasize) from the primary tumour massand to establish secondary growth foci (metastases) atother sites. Figure I1 provides a schematic reprcscntation of the steps involved in the spread of tumours.
Such tumour dissemination requires primary tuluour
cells to acquire a range of new properties, particularly
those that determine the relationship between thetumour and its host tissues (see [A4, D5, D6, F3, F5,
H33, H42, K2, I(5,L5,M,N6, S14, V3, V4)). Mctastasizirrg cells are first required to invade normal
tissues and penetrate blood and lymphatic systems.
Subsequently, penetrating cells need to be able to survive passage in these circulatory systems, exit the systerns and then establish themselves in surrounding normal tissuc. To what extent have experimental approaches succeeded in resolving the complex cellular andmolecular processes involved in ~nctastaticgrowth?
106. The molecular strategies so successful in
identifying proto-oncogene and turnour suppressor
gene activities in earlier phases of the neoplastic
process have not proved to be wholly satisfactorywhen applied to metastatic mechanisms. While transferof activated ras oncogenes to cell lines such as
C3HlOT'A and 3T3 has apparently resulted in one-stepmetastasis [E6]. the conlplcxity of the process in vivo
makes it highly unlikely that invasive properties could
simply emerge through single gene mutations. It may
be, therefore, that dircct approaches to the idcntification of metastasis genes using such atypical rodent
cell lines will tend to give a misleading irnprcssion of
the process.
107. Indirect approaches have compared ge~ie
activities in primary and metastatic tumours of thesalnc origin or type. Using these strategies someevidence for M S gene activation during metastatic~)rogrcssion has cnlcrged (see, e.g. [V2]), but for
tunlours in sure it lras proved difficult to obtain clear
corrclatio~lsbetween levels of RAS gene activity andthe i~lvasivecapacity of the turnour. Indeed in somestudies with human colonic tumours, directly conflicting results have bcen obtained [H33]. Similar conflictis evident in rcspect to FOS gene activity in somc
cxpcrilncntal nlamnlary tumours. Oversll, with the
cxccption of corrsistcnt MYC gene amplification incertain solid tu~riours(small cell lung carcinoma andncuroblastorna) arid HER-UNEUgene amplification in
many rnammary turnours, here is a marked lack ofcorrelatiori betwccri known oricogerie activation eventsand clinical staging criteria [H33]. There is somc
evidence that the activity of the protein kinase C gene
[ G l l ] is one determinant of the metastatic proccss,but, again, the available data do not allow sirnple correlations to be established. However, although general
correlations between metastasis and tumour geneactivity have yet to be established, some comment ispossible on specific aspects of the problem.
108. The initial stcp in metastasis is the attachment of
the primary tunlour cell to the extracellular stromal
tissue matrices and basement membranes [LlO].
Cadherin proteins are believed to play a role in celladhesion, and iin inverse correlatio~~has beensuggested betweell E (epithelial) cadherin expression
in tumour cells and the loss of cellular differentiation
associated with increased nictastatic properties [H42,S511. In addition, so-called integrin glycoproteins arealso believed to act as ccll surface receptors forcellular attachnlcnt, and it has been shown that manymeta-stasizing tumours strongly express thesemolecules at cell surfaces [C22, W1.51; there may also
be a relationship betwccn cancer development and thesynthesis of certain extracellular matrix proteins suchas tenasci~r[K27]. Following such attachment, tumour
cells then use a variety of proteolytic enzymes todigest the matrix in order to penetrate normal tissues;of particular current interest is Ihe involvement ofmetalloproteinases (MP) in this process. The activityof these enzymes is regulated by a specific tissue
inhibitor, TIMP, which has bcen shown to have antimetastatic activity [S38], and an importarrt role for
TIMP in suppressing malignant phenotypes is now
suspected [H33, H421. A specific protease encoded bytlre transirr geric is also known to be expressed during
the skin papillo~nato carcirlonla progression in thenou use [ B l l ] . The metastatic behaviour of someturnours has bec~rlinked to the expression of variant
CD44 glycoprotei~r(see [H42]). The finding that CD44
serves to activate both B and T lymphocytes has led
to the suggestion that ~ninlicry of lymphocyte
behaviour may be an important aspect of the metastatic proccss [A8].

570

UNSCMR109. More recently, the invasive characteristics ofhuman breast carcinoma have been direclly linked toh e expression of a n~et;~lloproteinasc
ternled stronielysin-3 [B21]. Of particular iniportance is the observation thnt this matrix-digesting enzyme is expressedin stronlal rather than tuniour cells, with strornelysin-3
gene expression being confined to tissue surrounding
only invasive mammary tumours. Thus, the acquisitionof metastatic properties appears to involve the specificlocal sti~nulation of normal breast tissue throughsignals received from the tumour; these data provide
one of the clearest examples of the intinlate relationship between norrnal arid tumour cells during tunlour
progression.
110. Such interactions are also a crucial aspect oftumour blood supply recruitment. Once a solid tumour
has expanded beyond a few millimetres diameter, it
becomes necessary for the tumour mass to be servedby new blood vessels. Blood supply recruil~iientfrom
normal tissues (angiogenesis) is known to be an active
process mediated by the secretion of angiogenic
factors such as fibroblast growth factor, epidennai
growth factor, transforming growth factor arid angiogenin from the tumour [F17]. In some cases, othercytokines produced by infiltrating leucocytes are also
believed to play a role.
111. Cell to cell colnrnunication through theestablishment of gap junctions has been shown to be
selectively lost in many progressing tumours, and theexpression of connexins have been analysed in a rat
liver tumour system [S65]. Communication between
normal cells is believed to be part of the regulatory
mechanism for cell proliferation, and its loss may
result in the relaxation of the conuols that restrictinvasive growth. Since a number of oncogenes havebeen shown to downregulate gap junctiorr forniation[Tll], the appearance of activated forms of thesegenes may enhance tutnour progression via the loss ofcellular communication.

112. Other phenotypic changes, such as increased cell
mobility, may also contribute towards the metastatic
spread of tumours. The molecular signals that increase
turnour cell mobility and thereby promote invasivenessare poorly understood. Secretion of autocrine mobility
stimulating factor has, however, been shown to correlate with h e invasive properties of human bladdercarcinoma [G12], and a so-called scatter factor
involved in epithelial cell motility has been shown tobe identical with hepatocyte growth factor, thereceptor of which is coded by the MET protooncogene (see IH421). Some genes involved in the
ccllular control of metastasis have been isolated by
cDNA procedures based on the over- or underexpressiorr of certain mRNA species in metastasizing
tunrours. Of particular note is the chromosome 17

encoded g e ~ NM23,~ewhich appears to function as anietastasis suppressor [L22, S39]; the loss of NM23
exprcssio~rlras been correlated with poor survival inbreast caricer [L23].
113. In co~rclusion, from a clinical viewpoint, theacquisition of ~nctastaticproperties is perhaps the mostcritical aspect of the neoplastic process. Since much of
the lethality of human malignancy derives fromsecondary metastatic growth, tunlour progression is amajor dctcr~iiiningfactor in the judgement of tissue
weighting in radiological protection. For example, skintumours, most of which only rarely metastasize to
distant sites, have low lethality, and this is reflected ina mucl~ lower tissue weighting than that given tobreast or lung where, largely as a consequence of
secondary growths, the lethal fraction is very muchhigher. Clearly, an understanding of the cellular andmolecular mecha~iisrnof the metastatic process will beof long-term value in making illformed judgenients onsuch weighting factors.
114. In spite of the difficulties experienced inresolvi~rg the complex mechanisms involved, somespecific aspects of the metastatic process arc now
becoming clearer. DNA and gene transfer studies have
yet to provide broad guidance on tumour gene
involven~ent,but the overall approach remains valid:some studies (e.g. [R13]) do show promise; cellhybridization [C23] and cDNA screening procedures
are also proving to be valuable [S39]. However,
perhaps the most critical aspect of the metastaticcascade is the complex interaction between invasive
tunlour cells and the surrounding stromal structuresand cells. In this area significant progress is being
made in understanding the underlying mechanisms; it
is to be hoped that this will also contribute to thedesign of more effective therapeutic procedures.

5, Viral involvement
115. Viruses are believed to irrfluence the appearanceof neoplasms in experimental animals and man by anumber of different mechanisms [N2, 01, P13, W16,
231 and, worldwide, may account for around 15% ofcancer incidence in man [Z3]. Viral oncogenesis iscurrently believed to proceed via the following routes:
(a)
(b)

(c)

through suppression of host systems for the
eli~nil~atio~rof tumour cells (e.g. avian
reticuloe~rdotheliosisand feline leukaemia virus);by sti~nulating cell proliferation through thespecific ioteraction of viral and cellular proteins,
either transiently or in a persistent manner (e.g.
human papilloma virus and cytomegalovirus);through the transduction of acquired andactivated viral oncogenes and growth-regulating
genes to host cells (e.g. Rous sarcoma andEpstei~r-Barrviruses);ANNEX ,YE: MECtIANISMS 01:RADIATION ONCOGENISIS(d)through site-specific intcgration into the genomeof host cclls, resulti~rg in the activation or
inactivatio~l(inscrtioaal mutagcnesis) of criticalhost gelies (e.g. avian Ieukaeniia i111d hepatitis B
viruscs).

The molecul;~rmcchanisms of some of tllcse processcs
have been studied in detail, but while both DNA andRNA viruscs have bccn iniplicated in the actiology ofa number of human arrd animal neoplasms (c.g. anogenital cancer, skin cancer, livcr cancer, leukaemias
and lymphonias), the overall extent of viral involvement in human oncogenesis remains uncerlain. Also,in many instances it is clcar that the viral componentof disease reprcscnts only part of a more complex
picture involving interaction with other risk factors(Table 5).
116. In recent years a close association between
certain human viruses and specific host genes has
become apparent, and the following cxamples serve toillustrate some of the mcchanisms currently believedto operate in human viral oncogenesis.
117. First, a tra~lsfonning protein (E7) of humanpapilloma virus 16 (HPV 16, associated with anal andcervical papillomas) has becn shown to bind the Rb
suppressor protein; a second HPV 16 protein (E6)complexes with the p53 suppressor protein in the cell.
Thus, the transforming potential of this virus may be
mediated by dual inhibition of tumour suppressor
activity [W9]. Secondly, human cyton~egalovirus(HCMV) infection, possibly associated with Kaposi's
sarcoma and cervical cancer, has been shown to lead
to upregulation of cellular FOS,JUN arrd MYC protooncogenes. This occurs prior to the onset of viralprotein synthesis and may be mediated by a hit andrun process, whereby the interaction of viral particles
with cell surfaces triggers cellular proliferation [B13].
Thirdly, the Epstein-Barr virus (EBV) gene BCRFl
has been shown to be hon~ologousto the cytokine
synthesis inhibitory factor gene IL-10, suggesting that
this oncogenic herpes virus uses a captured cytokine
gene to enhance its survival in the host, thus potentiating its oncogenic propertics in respect to Burkitt's
lymphoma [M18]. Fourthly, human T-lymphotrophic
virus (HTLV) has been shown to be associated with aunique form of adult T-cell leukaeniiaflymphoma(ATL), with the two main foci of infection being inJapan and the Caribbean (see IB351). The mechanisms
o f T-cell oncogerlesis following HTLV infection are
uncertain but may involve a combination of immunosuppressive effects [J6, K23) and T-cell proliferative
dysregulation elicited by virally encoded proteins suchas the products of the tax and rer genes 1231. Finally,
hepatitis virus and aflatoxin B1 (a mutagenic foodcontaminant) appear to be strongly interactive factors
in the induction of human hepatocellular carcinoma(HCC), which is prcvalcnt in southern Africa and571

eastern Asia [B22]. The insertional mulagenesis ofhost genes by hepatitis B virus has bccn demonstrated,
and recent evidc~rcestrongly suggcsts that the atlatoxin c o ~ ~ ~ p o nof
c nriskt derives frorii the i~rductionof
target livcr cell mutations that inactivate the p53
tumour suppressor gene [B23, H34]. The still uncertain role of gerni-linc retroviral clenlents in humantumorigenesis has bccn reviewed reccnly [L24].
118. Thcse observations provide evidence that the
oncogenic potential of many human viruses derives
from their capacity to provide a chronic growth
stimulus to cells, often by the interaction of viral
oncoproteins with cell cycle control proteins [N28],
but that direct mutational damage may underlie theaction of other viruses. The apparent synergy between
hepatitis B virus and a specific environmental DNA
damaging agent (aflatoxin BI), noted above, together
with data on retroviral involvement in some radiationinduced animal neoplasms (see paragraph 180) and theinducibility of virus-like elements in mice by radiation[P14], suggests that viral processes may well synergistically influence a component of human radiation
oncogenesis; there is, however, only preliminary
evidence for this in rcspcct to laryngeal carcinoma
associatcd with papilloma virus infection (see [W16]).

B. HUMAN SUSCEPTIIIILITYT O RADIOGENIC CANCER

1. Homozygous deficiencies in DNA repair,cell inactivntion and chroniosome breakage

119. There is increasing awarcness of the strong
influence of gem-line gene mutations in human oncogenesis (paragraphs 31-34). In addition to the familial
traits associated wilb specific DNA sequence loss andorgan-specific neoplasms (e.g. retinoblastoma, Wilmsaniridia, multiple endocrine neoplasia and familial
adenomatous polyposis), there is, in the context of thisAnnex, the most important problem of cancer susceptibility through deficiencies in DNA metabolism [C12,F1, H5]. The clearest example of this is found in thegenetically complex autosomal recessive trait xeroderma pigmentosum (XP). In this genetic disorder,
defects in the repair of DNA photoproducts appear to
be causally linked with a high incidence of skinneoplasia in thc sun-exposed regions of affectedpatients [B14, C12]. Hurrian and rodent genes involvedin the rcpair of UV-induced damage have been
isolated and characterized (see, e.g. [T13, V7]), andthere is much new information on the cellular andmolecular mechanisn~sof UV carcir~ogenesis(see, e.g.
[A6, B361). A detailed description of thesemechanisms is, however, outside the scope of thisAnnex.

572

UNSCWR 1

120. For ionizing radiation, direct links between theepidemiological, genetic and nicchanistic aspects ofoncogenesis are less well cstablislled. However,potentially rclcvant DNA repair genes arc now being
idcatified arid isolated (see, e.g. [ K l l , TlS]), andstudies on the autosoma1 rccessivc human genetic
disorder ataxia-tclangicctasia continue to provide
potentially important infor~tiation on the association
between cellular radioscnsitivity, DNA repair
deficiency and cancer proneness (G7, G13]; acandidate gene for ataxia-telangiectasia group D has
rcccntly bccn isolated [K29].
121. Cellt~lur rudiosensXvity - cell irzuctivu(ion.
Although largely restricted to the inactivation ofcultured skin fibroblasts, in virro clonogcnic
techniques have highlighted the rclativcly wide rangeof low-LET cellular radiosensitivity in the normal
h u n i a ~population~[A3, A12, C13, L l l ] . These data,for both acute and chronic exposures, show that only
cells from ataxia-telangieclasia and Nijmegen break
syndrome (NBS) hon~ozygotcsclearly fall outside thenornlal range of radiosensitivity arid that patients witha variety of other putative DNA repair deficiencies arecont.~ined within the relatively broad normalis, however, only a crude
distribution. Cell inactivatio~~
surrogate for overall cellular radiosensitivity andobviously fails to rcllect the gc~icticcomplexity of
oncogenic processes. The lack of correlation between
intrinsic radiosensitivity in fibroblast andT-lymphocyte cell s t r a i ~ ~alsos casts some doubts on
the predictive value of studies on cell inactivation[G15]. Consequently, the data cited can only be used
to provide comment on a single aspect (DNA damage
and repair) of the problem; they should not be usedalone as an indicator of the distribution of ncoplasia
susceptibility in a radiation-exposed human population.
In spite of these reservations, the overall approach has
yielded much useful information (see [A12, G91).
122. Chrornosornal rudimensifivity and jragile sites.
The quantitative estimation of radiation-ieduced
chromosomal abnonnalities also identifies ataxiatelangiectasia and NBS homozygous genetic disorders
radiosensitive [T6, T7]. Whileas being abnon~~allythese data are somewhat more relevant to oncogenesis
than cell inactivation, here again it may be argued that
Uley are of limited value to radiological protection.
This view may, however, be tc~npered by recent
observations suggesting that a range of hu~nandisorders predisposi~~g
to cancer are characterized bycell-cycle-dependent chron~osonial radiosensitivity[S24]. This is a potelltially important observation lhat
demands furtller study. The potential relevance tooncogenesis of' specific chromosomal sites of fragility
and enhanced recombination is discussed in paragraphs
61-64. For both ionizing radiation and chemical agent

exposure of cells, tl~crcis growing evidence for the
expression of cliro~noso~ualsites of preferential
induced breakage; in some studies an association withfragile sites and cancer-specific cl~roniosonie
breakpoiats lias also bccn suggested [D7, M19, S25,
S26, Y 11. Of particular note arc the proposed mutagen
sensitive sites (MSS) of hu111a11chSq, which at thecytogenctic levcl, appcar to correspond to the 5q
breakpoiaLs that characterize myelodysplastic
syndromes (MDS) [M20, Yl].Since these myeloid
disorders and l~coplasn~sare believed to besignificantly radiogenic [M21, R9, V5], confirmationof 59 breakpoint co~~cordance
for MSS and MDS
would bc of sonic importance. Losses from the 5q
region in MDS are now believed to centre on theinterferon regulatory factor-1 gene [W24]. Recent
studies on site-specific chromosomal brcakpoiuts inradiation-induced n i u r i ~ ~acuteeniyeloid leukaemias(paragraphs 182 and 183) add support to thecontention that heritable predisposition to breakage atfragile sites on certain chromosomes may influence
susceptibility to radiation leukaemogenesis. The moststriking example of geno~nicinstability associated withhuman cancer predisposition is, however, provided byhereditary non-polyposis colorcctal cancer (HNPCC),
which is deter~ili~~edby a 2plS-16 locus [P16]. This isbelieved to account for 4%-13% of all colorectal
carlccrs in industrial natior~sand is therefore morecommon than FAP, which accounts for around 1%ofsuch cancers. Contrary to nornlal expectations basedon the HNPCC locus being a tumour suppressor gene,
HNPCC tumours do not show characteristic DNA
losses in the 21115-16 region [A16]. Instead, suchtumours exhibit widespread alterations in shortdinucleotide (CA), repeat sequences suggestive ofgenornic instability mediated through a dorninant
defect in a DNA replication factor. Such instability
can be viewed as a means whcrcby mutation rates for(CA),-associated genes are elevated, thus enhancing
tumour dcvclop~nent. The finding of an excess ofother tulnour types in HNPCC kindreds i~~dicates
thatsuch mechanisms may not be restricted to colonicncoplasrtis.
123. Although not associated with oncogenic
processes, the recent characterization of the X-linked
fragile site (FRA X ) mutation associated with heritablemental retardation in man allows some comment on
the g e ~ ~ e t i cof
s cllro~~~osorne
fragility. The FRA X
mutation confers not only X-chromosome fragility insomatic cells but also appears to be unstable in germ
cells and subject to structural modification that is
depcndc~~ton its parental route of i~lheritance[ 0 2 ,Y8].This feature appears to contribute to the bizarre
ge~teticsof FRA X i1111eritancearid expression [R17,S53] and, if it were to apply to cancer-related fragile
sites, would tend to disguise the underlying genetic
basis of familial predisposition. These FRA X data also

ANNEX I:

MECtlANISMS OF

directly 1i11k cellular repair observations with thehighlight the potential i~nportancc of DNA rcl)ciit
of riidiogenic skin carcinoma.
c l ~ r o m o s o ~ ~instability~:~lfor c c r t i ~ i ~ ~ cli~lical~na~lifcstatior~disease stales [R171, a theme that is ecllocd clscwhcrc
126. The frcquer~cy of known highly penctratingin tl~isAnnex.
llu~llan~nonogcnicdiseases that are possibly associatedwith clcvatcd susceptibility to radiation oncogcnesis islow. Examplcs of UIC estimated frcquencics of
2. Heterozygous crrri-iers of genetic traits
occurrence of sonlc of these canccr-prone humanr~iutatiolisarc listed in Table 6. Conscquendy, in t e m ~ s
124. Whilc the overall picture of hunlan g c ~ ~ c t i c of the \vhole population, the highly pc~lctrating~~lutatiorisdo not appcilr to present a significant
influc~lceson canccr susccptibility is bcco~ilingclcarcr,
problem for risk estimation. Howcvcr, more frequent
the information available is based largcly on studies
mutations, such as ataxia-telangiectasia heterozygotes,
with highly penctrating dominant nuta at ions arid
(estimated frequency of -I%), who show illcreased
ho~~iozygousmutations at autoso~ai~lloci. The
incidences of breast cancer, wilh a relative risk of 6.8
frequency of such clearly recognizable disorders in theclainlcd in one study [PZ, S4, S661, together with apopulation is low, and while thcre are obvious
possible but still contentious increase in radioimplications for affccted individuals and their families,
the ovcrall contribution to canccrs in thc populalio~~ sc~lsitivity[S64], could make significant contributions
to population risks.
may be relatively small. In contrast, for autosonla1
recessive traits, the frequency of carrier heterozygotes
127. However, it is most important to recognize thatwill be much higher. In the case of the ataxiacancer
is esserltially a nlultifactorial genetic diseasetclangiectasia disorder, epidcrniological studies point
andthatgenes determining cancer susceptibility will
towards increased risk of spontaneous neoplasms,
differmarkedlyin the probability of expression
particularly breast cancer [P2, S4, U2], and it is(pcnetrance),asmeasuredby the appearance of one orpossible that such carriers of the ataxia-telar~giectasia
moretumoursinmembers
of families carrying themutation represent a human subpopulatio~~at increased
appropriate
nlutations.
The
often-cited
retinoblastoma
risk of radiation-induced neoplasia.
(RE) and Wilms' tumour (147) gene mutations arehighly penetrating and express as bilateral childhood
125. Similar considerations may apply to the farnilial
tumours in a high proportion of carriers. Mutations oftraits associated with organ-specific neoplasia.
lower penetrance have been suspected for many years.
Cytogenetic and nlolecular studies wilh these imply
However, since they tend to express, perhaps as single
that cancer proneness is often associatcd with the loss
adult tumours, in many fewer carriers, they also wouldof one germ-line copy of a tunlour suppressor gene.
be much more difficult to detect in the population,
The spontaneous loss of the other copy in a targct
even if they occurred. overall, at a higher frequency.
somatic cell would then explain the elevated Gequcllcy
This view has received some support from the findingof organ-specific neoplasia and the apparentof relatively low penetrance p53 germ-line lnutations
phenotypic dominance of these traits. An explanationin rare cancer-prone Li-Fraumeni syndrome (LFS)
that demands the reduction of target gene nur~ibcrin
patients [S41] and mutations associated withcells from two to one also implies that certain tissues
predisposition to breast and ovarian cancer [H35, K21,
of affccted individuals would be at a considerably
S521. One of thcse breast canccr genes, BRCAI,
elevated risk of radiatiou-induced ncoplasia. Sonle
encoded on chromosome 17, is strongly associatedaspects of therapy-related neoplasia in familial cancer
with early onset disease and appears to act as apatients have been discussed [SS, U3]. Although thetumour suppressor. Such heritable forms of breast
paucity of data precludes detailed analysis, there iscancer may account for around 5 % of the total inevidence of an i~icreased yield of thcrapy-relatedindustrialized countries. Since one in ten women
second tumours in familial retinoblastonla patientsdevelops breast cancer in her lifetime, perhaps as[E12], an obsenlation that is consistent with the known
many as or~ein 200 carry genes that predispose to thisinvolvcn~cnt of the RB gene in the iriitiatiot~ ofneoplasm [K21]. Howcvcr, with Uic exception of
tunlours other than rctieoblastonla, including
BRCAl most of these mutations are probably of low
osteosarcoma [T5]. With respect to radiogenic
pcnetrance. In the case of p53, cancer predisposing
neoplasms in dominant fa~nilial traits, intriguing
germ-line mutations arc by no means restricted to LFS
observations have also been made with basal ccll
fanlilies [M38, TI71 and include one family showing
nevus syndro~ne(BCNS), where there is unambiguous
abnormal expression of wild-type p53 protein [B30].
evidence of radiotherapy-induccd nlultipie skin128. Recent observations on the effects of gcnomic
neoplasms [SS]. Although there is evidence for postimprinting on the expression of tunlour suppressor
irradiation rcpair/recovery defects in BCNS cells (seegenes (paragraphs 57-60) are also relevant to patterns[A3, L11, N21 I), the disorder may be hcterogcnous.
of heritable tulllour susceptibility. Gcnomic imprinting
and at this stage of knowledge it is not possible to

S C ~ U C I I C C S and19P3 UEPORT

may be considered to he a process whereby the dominance of mutant g c ~ cxprcssio~~~c
is ~nodificdaccordi~~gto whrll~crit is inl~critcdfro111the mother or UIC father(H19, M43, S27]. 111 tllc case of imprinted cancer-susceptibility gcncs, UIC expected M c ~ l d e l i apatterns~~oftumour incidc~~cc
in :~ffcctcdfan~ilieswill tend to be
distorted, dcpcnding critically on parental routes ofinllcritancc. An illustratio~~of the inl~eri~?nce
patternof a human disease wilh imprinting effects is shown in
Figure 111. The term nlatcr~~al
imprinting is used to
imply that there will be no phc~lotypicexpression of
the abnormal allele whcn transn~ittcdfro111the mother.
and paternal imprinting is used to imply that there will
be no phenotypic expression when transniitted fromthe father. Because there will be a phenotypic effectonly whcn the gene or the chromosome segment inquestion is transmitted from one or Ibe other parent,
there are a nuniber of unaffected carriers. There are
equal numbers of affected rnales and affected females
or of unaffected male and unaffected female carriersin each generation [HI91 (see also Annex G,"Hereditary effects of radiation").
129. In addition to the imprinting-like effects seen for
the IZB- and WT-related gencs in man, it may be that
this process influcnccs the expression of other tunlour
suppressor and growth-factor gc~lcswith known orsuspected involvement in oncogencsis [C23, F23,
H191. The inherita~~cepattern for familial glomus
tumours in man has been shown to be consistent withinlprinting [V6]; clinical manifestations, including
neoplasia, in h e human Bcckwith-Wicdennann syndrome are also believed to involve imprinting effects
mediated by paternal inheritar~ceof bolh copies of aregion from the short arnl of chronioson~e11 (paternal
disomy/niaternal deficiency) (see [L16]). It may bereasonably predicted that other less-well-defined"cancer families" will, in detailed studies, show
pcdigree distributions indicative of imprinting-like
effects. The observation of parent-of-origin effects on
leukaemogenic translocations Itlay also have
implications for Ihe genetics of haemopoietic
neoplasms [ H a ] .
130. In conclusion, based on the frequency of the
known highly penetrating mutations, it might beconcluded that genetic susceptibility to cancer is 1101 amajor factor in the forrnulation of radiation risk
estimates. However, the increasing appreciation ofpartial effects in hetcrozygotes, the variable penetranceof mutations, and cpigenctic modifying factors shouldsound a note of caution on this conclusion.
131. An overall genetic contribution to cancer risk intile human population of around 20% has been suggested (see [B24]). Although considerable uncertainty
exis& this suggested value implies that as knowledge
accrues it should be possible to begin to consider

isdividui~l risk. For future epidcn~iologicalof ~)clssiblcgenetic effects on radiationi~~vcstigation
carci~~ogcnesisi t will he i a ~ p o r t a ~to~ t selectively
I~istoryof ncol)lasia along will1 otherconsider fan~ili;~lrelevant factors, s11c11;IS age of onset in relation to age
at exposure. 111 this spccific context, although othercxplaaatio~rsare ~)ossiblc,tile recent arid unexpected
findings of an elevated relative risk of early o ~ ~ s c tbreast cancer anlong t l ~ csurvivors of Ibe atomic
1nig11taccord with the appabombings in Japan [WO]
rently high genetic component of this disease (seeparagraph 127). I t sllould be emphasized, however,that [he full establ ish~ncr~tof such relationships
d e m a ~ ~ dextensives
investigations of fattiilial cancer
incidences and, wl~erepossible, ~nolecularanalysis of
relevant g e m - line DNA sequences in order to ascertain the carrier status of the affected individual. If
progress in this most important area of human genetics
can be maintained, it nlay be necessary to modify
current views on the expected distribution of induced
cancers in human populations.

3.

Systenlic factors

132. Although Ulcrc is, at present, a paucity of
infonnativc data, the ide~~tificationof initiating events
for the principal radiation-iaduced neoplasms is offor the further development ofcritical ilnporta~~cc
mechanistic models of oncogcnesis and the validationof dose-effect relationships. Armed with knowledge ofthe target cell and llle initiating event, it may bepossible to rnake infonned judgements on the effects
of post-irradiation repair processes, on dose rate andon radiation quality effects. However, since oncogenic
processes involve far more than initiation, it is of
considerable importance to gain a further understanding of the factors that influence the expression
and development of neoplastic change, i.e. age, sex,
dietary and honnonal factors. In this context, animalmodels of radiatio~~
oncogencsis can be of great value
in gaining a broad understanding of these factors (see,e.g. [F2]), and there is also valuable information to beobtained from human population studies.
133. Hontioaal status is known to be a major factor in
the appearance oftumours ie breast, ovarian, testicularand prostate tissues in rnan and may also be implicated in the progression of other hor~nonc-sensitiveor
horn~onc-producingtumours, such as those arising inadrenal a ~ thyroid~ dtissues (L17, M32]. Since, however, honnol~csand other cytokincs play a ubiquitousand ~ ~ ~ a i n t c r ~ofa nallc e tissues,role in the developme~~ttheir action should not be considered to be restricted
to the tunlour types noted above. On current knowledge and with few cxccptio~~s,
Lhe principal oncogenicrole of such systen~icfactors centres on their action as
tumour pro~notersor co-carcinogens [M32].

A N N M li: MECIIANISMS 01: R/\DIATION ONCOGENESIS

134. The influence of systcmic endocri~lcfactors inhuman radiation oncogcncsis is most strongly cvidericed by age- and sex-related cffccts on brcast
canccr incidence [Ul]. Thcsc observatio~~s
imply that
hormonal status is a major deternii~~antof thc
devclopnient of tliis neopl;~smarid that initiated cclls
may remain dormant in breast tissues for long periods.
Ccllular and molecular studics oudincd elscwherc(paragraph 189) support this contention and are alsobroadly consistcnt with currcllt vicws on the mcchanisms of neoplastic promotion and progression (paragraphs 78-114) that are believed to mediate oncogenesis. Cellular interactions involving hornlones andgrowth factors are also known to influcricc neoplastic
yicld in experimental systcnis through promotional andselection processes; there can bc IitUe doubt that such
effects are crucial to neoplastic yiclds in man and may
be determined by physiological changes deriving fromboth genetic and environmerital influences.
135. Diet is thought to be a significant factor in carcinogcnesis, with fat intake perhaps the niost important
determinant of cancer in different human populations[C15]. Promotional mechanisms centred on dietary
lipid effects on the activity of endocrine systems, on
prostaglandin synthesis, on immune functions and on
bile acid production have been proposed for some neoplasms and may be supported by epidemiological andanimal studies [C15]. Althougli cellular and molecular
data relating directly to thcse proposals have yet to bepresented, it may be relevant that the promotional
pathways involving protein kinase C are known to
involve cellular lipid tunlover (paragraph 82), and it ispossible that their activity is influenced by lipid or
lipid metabolite availability in tissues. Non-genotoxic
chemicals present in the diet or in the cnvironmcnt
have also been proposed as factors in hurnan carcinogenesis [C16]. Through their capacity to elicit achronic proliferative stimulus to cells, it is possiblethat many of thcse may act as relatively non-specifictumour-promoting agents: the role of i ~ ~ d u c emitod
genesis and endogenously induced DNA damage in
the initiation of oncogenesis has also bccn considcred(see, e.g. [AlO, A1 1, C161). Although somewhat outside the scope of this Anncx, the rate at which cells
are believed to sustain and repair e r ~ d o g e ~ ~ oDNA
usdamage, in relation to that small amount of additional
damage induced by a low dosc of radiation, hasreceivcd detailed comment [B43, B44, L27, S66,
W27] and is of considerable importance to therelationship between DNA repair and radiatiori
carci~logcnesis(sec also paragraphs 154-159).
136. The possible cellular mechanisnis of antitumorigenic agents, such as retinoic acid, have bee11
outlined in paragraphs 95-97. Animal studies, particularly with the mouse skin papilIoaia/carci~io~lia
system, clearly demonstrate the anti-tuniorigenic action575

of rctirioic acid in vi~vo [R14], and recently, ther c t i ~ ~ oacid
i c trcalmcnt of tumour-sensitive traosgc~lic
mice carrying an active germ-line copy of a v-ras
ollcogcne has bcen shown to dramatically delay oreven conipletcly inhibit the appearance of promoterinduced papillonias k 1 5 ] .
137. Honnorie pro~~iotion(paragraph SO), incrcasingg c ~ ~ o m iillstabilityc(paragraph 104) and intrinsic
progressional processes (paragraphs 98-114) have bccn
rncntioncd as possiblc factors in age-rclated
carcinogcncsis. Also, for familial tumours such as
rctinoblastoma and somc brcast and colon tumours,
early onset of induced malignant discase would, forgenetic reasons, be anticipated. For the early onset(childhood) acute lymphocytic leukacmia, howcver, adifferent mechanism may opcrate; it has been
proposcd that this diseasc rcsults from two sequential
mutations in haemopoietic target cells [G6]. The firstof these is postulated to occur in ulero, whcrc rapid
ccll proliferation is required for haemopoietic
development, and U I ~second during the neonatal
period, when a clonal population of thcse cells is
expanding in response to a primary antigenic stimulus.
138. Anillla1 niodels provide the basis for quantitativein vivo studies on radiation oncogenesis and doseeffect relationships (see, e.g. [E2, M I , M2]), andspontaneouslinduced ratios for malignancy strongly
influence views on the choice of radiological risk
models (i.e. relative vs. absolute risk) (see, e.g. [S12]).
M e r e therc is a paucity of human cpidcmiological
data, as in the case of leukaemogcncsis by boneseeking alpha-emitters, animal studies can provide thepreliminary data. For exanlple, the filldin of induced
acute mycloid leukacmia in low dose z'R~ exposed
mice at a higher frequericy than that of osteosarcoma
may have important implications for human radiation
risk estimates (H9, HlO]. The inhomogeneity of dose,
which is inherent in bone-seeking and otheriritcrnalizcd radioisotopes, highlights a major systemic
uncertainty in radiation oncogenesis, i.e. the identity
and in vivo distribution of target cells for oncogenesis.
The pattcrri of isotopc distribution and decay inrelation to organ specific target cclls is probably thc
major dctcrminant of oncogenesis by these isotopes,
yct very little is known about target ccll identity atideven less about distribution.
139. In conclusion, the highly interactive multi-stepnature of oncogenesis demands that systemic factors
will greatly ir~fluencethe probability that a carci~iogeninitiated cell in tissue will complctc all steps and givc
rise to an overt malignancy. Positive factors such as
factor-mediated promodietary and hor~~ional/growthtio~ial mechanisms may tend to drive the process
forward, while tcnninal cellular diffcrcntiation, procellular
grammed ccll death, cellular commu~~ication,
surveillance and dietary/cndogenous anti-oncogcnic

compounds niay restrict or ever1 abrogate tu~nourdevelopnient. Any specific judgement on the probability that a single tumour-initiated stem cell will
progress to nialignancy requires a greater knowledgeof the complex ii~terplaybetween these factors t11a11is
currently available. In general, however, it seems
certain that tl~e~~cgalive
factors greatly outweigh thepositive and that, although only crudely estimated,
perhaps less than one in a n~illion initiated cells
con~plete the full transition to overt n~alignancy(paragraph 21 8).

4. Immunodeficiency a n dcell surveillance mechanisms
140. Some human genetic imniunodeficiency diseases,
such as the Wiskott-Aldrich syndrome [P3], arecharacterized by susceptibility to certain neoplasms.
However, the role of host immune-response in o n c e
genesis is a problem that has yet to be satisfactorily
resolved (see, e.g. (S431). In the context of oncogenic
mechanisnis, some specific coniment is necessary.

~eby endo143. 111 t l ~ ecase of o r ~ c o g c ~activation
genous proccsscs, i.c. cl~romosometranslocation, gene
amplification or point n~ubtion, either normal or
minimally modified p r o t c i ~ ~havesbeen seen to be
expressed (paragrapl~s28 aud 29). Cl~aenicricfusion
protcins (paragrap11 71 and Table 1) arc a possible
excel)tio~~to this. Conscquer~tly,such mechanisn~swould aot, in genrrill, provide slrongly antigenic geneproducts to act as high-affinity "non-sell" targets for
B- and T-cell mediated irr~rriune mechanisms. Thisconclusion accords with the low immunogenicity seen
in most spontaneous neoplasms [K2]. Oncogenic processes associated with gene losses would not obviously have any direct consequences for conventional
inlmunc response.

141. In the case of oncogenic DNA viruses, such as
Epstein Ban virus (EBV) that carry transforming
genes, there is evidence that immune func~ionsefficiently suppress oncogenesis [P4] and that EBVcarrying lympho-proliferative disorders normally only
develop where there is evidence of host immune
deficiency. It has been suggested [K2] that the rather
unusual spectrum of human neoplasms that develop in
the case of inherited or acquired immuno-deficiency
reflects the involvement of oncogenic DNA viruses intumour actiology. In such cases the expression of"foreignn viral genes in neoplastic cells could be seen
as providing specific targets for host inlnlune
functions. Viral oncogenesis often depends on thepersistent proliferation of the virus in host tissues(paragraphs 115-118), and this is obviously influenced
by the degree to which the host is able to mount aneffective imniune response against the highly specific"non-self" viral proteins.

144. T-cell mediated immune recognition depends on
the precise presentation of antigens on target cellsurfaces tlirough Ule action of proteins, collectively
termed the major histocompatibility complex (MHC),
and there is evidence that during the course of theirdevclop~nentmany tu~noursdevelop antigenic determinants that can elicit irn~nunereactions. Tumour
antigens have been characterized in a range of humanarid aniriial tumours [G14], but the contention that they
are capable of sti~ilulating an effcctive immune
reaction may be questioned. Melanoma probably represents the best antigenically characterized humantunlour, i111daround 40 different tumour antigens havebeen identified using monoclonal antibody techniques.
These include antigens of MHC, pigment, growth
factor receptor arid cell menibranelmatrix origin p 3 6 ] .
A feature of these antigens that illustrates the central
probler~iof tumour inimunology is that none are truly
tumour-specific; all are expressed, albeit in some cases
weakly, by other normal tissues. The lack of specificity of the vast majority of tumour antigens may wellunderlie the apparent failure of T- and Bcell mediatedimnlune systems to mount an effective response toneopli~sticcells arising in tissues. As noted above, thevirally-associated human turnours represent a clear andwholly understandable exception to this.

142. For oncogcnesis mediated by the insertion of aviral sequence close to cellular oncogenes the situationis less clear. The target gene nlay be activated by theinsertion of only a short, non-coding viral sequence(the viral long terminal rcpcat or enhancer) [J2]. This
would not tend to generate imniur~ogenicityin h e cell.
If, however, the whole viral genome were inserted, theexpression of virally encoded proteins could elicit an
immune response to eliminate the carrier cell [K2].
The activation of normally silent transposons has been
demonstrated in human testicular tumours [B34], andia one case of colon cancer h e target APC gene had
been inactivated by transpositional insertion (M461.
The implication of such activational and insertional
events for imrnune response is however uncertain.

145. Thus, while T- and B-cell mediated immune
systc~nsmay under some circumstances modify thedevelop~nentof non-viral human tumours, clear evidcnce that they have the capacity to eliminate earlypre-neoplastic cells in tissues is lacking. Indeed, if
such efficient syslenls were to be available, it would
be necessary to seek explanations as to why such cells~ t tissues for long periods prior to
can rcalain d o r r n a ~ inpromotional stin~ulatioaby systemic factors such as
honnoacs (paragraphs 78-97). Also, from an experimental point of view, an explanation is needed for thefact that very large studies with mice, genetically
deficient in T-cell immunity, have failed to provide
any evidence of elcvatcd tunlour susceptibility (see[B25, S421).

ANNEX E: MECI-IANISMS 01.'
RADIATION ONCOGENBIS
146. Immunodeficiency is a characteristic feature of
the ataxia-tclangiectasia human genetic disorder [RlO].
Wlile the inlnlune defect in the disease may contribute to the increased frequency of lymphoreticularneoplasia in ataxia-tciangicclasia, chromosomal
rearrangements that are characteristic of the predominant T-ccll leukaen~iassuggest that the underlyi~~gdelect in DNA metabolism resulting in misreconibination of T-cell receptor genes [R3] may be a more
important determinant of neoplasia incidence.

147. There is also evidence that the skin plays asignificant role in the body's immune systcnl and thathngerhans' cells in skin originate in bone marrow
and are irnmunocompetent [S28]. With regard toimmune effects in carcinogencsis, it has been shownpatients show an excess ofthat in~n~unosuppressedsquamous cell carcinomas [K7], and clinical observations have led to suggestions that a depression of thecell-mediated immune function in skin, as a consequence of UV-sensitivity, may contribute towards theincidence of skin neoplasia in XP patients [B14].

577

latory role for lymphoid cells in selectively preventing
the self-rcnewal and accumulation of early neoplastic
cells has also bee11 proposed [G4].
149. The overall extent of NK or NK-like ccll
function in the elimination of tumour cells and thedevelopment of malig~lancyin man remains uncertain.
However, in animal systenls it has been shown that thetransplantation of cloned NK cells to NK-dcficicnt
host mice leads to resistance to radiation-induced
thymic ly~tlpholtiaand the inhibition of lung nodule
developr~lent following implantation of nlelanoma
tumour cells [W17]. The selective activation of NK
cells has also been shown experimentally to inhibit
tumour ~iletastasis[H37j.The implications of theseobservations for radiation carcinogenesis in man
re~naiilto be established, but they do provide evidence
for the existence of cell-mediated processes thatdcfcnd against tumour growth and metastasis [H38,
Y3]. In spite of this, their apparent lack of specificity
poses questions on the overall efficiency of theirscavenging capacity.

148. While there is relatively good understanding of150. In conclusion, it has been argued that with thethe nature and extent of T- and B-cell mediatedexception of a possible viral component, T- and B-cell
immune reactions, the same is not true of the potential
mediated immune response may not play a major role
defence mechanism provided by natural killer (NK)in moderating human radiation oncogenesis. However,cells; these appear to recognize abnormal cells without
specialized immune functions in certain organs and the
MHC involvement and are far less discriminating inexistence of non-immunogenic cell surveillance
their action than T-cells [B2]. Recognizing certain cellmechanisms imply that a proportion of early presurface receptors that are not necessarily antigenic,
neoplastic cells may be eliminated before they become
NK cells bind and then secrete membrane-perforating
established. Other nlechanisms defending against
proteins that lead to target cell lysis [Y3]. Thus cells
tumour induction or development, including DNA
misplaced in tissues, cells over-expressing certain
membrane proteins and/or cells with co~~for~natior~alrepair, programmed cell death, terminal differentiationand phenotypic suppression, are noted in othermembrane changes may be eliminated. The accidental
Sections of this Annex. Together, these will reduce theelimination of normal cells by NK cells is known toprobability that a specifically damaged target cell will
occur [Y3],and this relatively low specificity of action
progress to hank malignancy. An estimate of thismay provide NK cells with a capacity to eliminate
probability, while of considerable importance topre-neoplastic cells showing characteristic changes atradiological protection, is extremely difficult to make.
membrane surfaces. In this general context, NK cells
Nevertheless, in paragraph 219 a first approximation
have been shown to exert selective inhibitory effects
calculation is illustrated.
during post-irradiation hacmopoicsis [P8], and a regu-

111. EXPERIMENTAL INVESTIGATIONS OF CELLULAR
AND MOLECULAR MECHANISMS OF RADIATION ONCOGENESIS
151. Given the complexity of the cellular genetic
events involved in oncogenesis, how should the principal questions regarding the nlecha~~ismsof radiation
oncogenesis be framed? With a view lo exploring
experimenlal strategies, these questions may begrouped as follows:(a)what is the nature of radiation-induced initiating
events? Is DNA tile principal cellular target, and

(b)

(c)

if so, what is the effect of post-irradiation DNA
repair on the fate of these potentially initiating
lesions?
what is the identity, distribution and radiosensitivity of target cells for the major induced
neoplasms?
what are the consequences of an initiating eventto a given target cell, and how does this event

(d)

(c)

ir~teractwith or determine promotional or progressional events in order to give rise to theovertly neoplastic cellular phenotype?
in what ways do genetic, hormonal and/or environmental factors affect the expression of initial
oncogenic damage and the subsequent progression towards malignancy?
how closely can experin~entallyderived data on
oncogcnic change be related to other biological/
biophysical effects of radiation and, crucially, toin vivo human oncogcncsis.

152. Unanlbiguous allswers to most of the above
questions are not yet possible. The questions serve
here only as a franiework for discussion. Nevertheless,some specific comment is possible froni both in vitro
and in vivo approaches to the problem.

A. EPIDEMIOLOGICAL STUDIES
153. Epiderniological studies of human groups
exposed to low-LET radiation show that a range ofneoplasms are represented in excess and, broadly, thatthese do not differ markedly from those arisingspontaneously in the population (see, e.g. [Ul]). That
is not to say that different tissue sensitivities orcharacteristic mechanisms do not occur for radiation
oncogencsis but rather that no unique neoplastic signature of human radiation exposure is, as yet, apparent(paragraphs 223-227). This may be contrasted with theorgan-specific neoplasms that characterize exposure tocertain chemical agents, e.g. asbestos and mesothelioma, vinyl chloride monomer and hepatic angiosarcoma, benzene and leukaemia and aflatoxin andhepatocellular carcinoma (B22, TI61 (paragraphs 9193). The basis of these observations, although
uncertain, may be associated with the evidence that,through energy deposition and chemical radical
interaction in DNA, radiation is able to induce adiversity of genoniic lesions, ranging from damage tosingle bases to gross DNA deletions and rearrangements. Again, this may be contrasted with s o n ~ echemical agents, which have characteristic chemical
specificities in their interaction with DNA and also
target certain organs (paragraphs 188-203). Thus, theepidemiological characteristics of radiation oncogenesis would be explained, if the spectrum anddistribution of induced cellular initiating evenb was
not grossly different from that arising sponlaneously
and if low-LET radiation sirnply i~lcreased thefrequency of the commonly occurring neoplasms,
albeit with different levels of excess in different target
organs. Although attractive in its simplicity, h i s hypothesis may need to be modified in the light ofmolecular information on i~iducedso~naticcell nlutalion. As noled later in Chapter IV, the physiologicalg uptake,and bioche~nical processes g o v e r ~ ~ i n the

distributio~~and cxcretioil of radioactive isotopes canlead to dose-inlioniogcncity and, subsequently, organspecific neoplasia. There is lirnitcd information onsuch carcinogenic effects in man [ U l , U2); andAnnex F, "111fluenccof dose and dose ratc on stochastic effects of radiation" summarizes some of thereleva111animal clata.

IJ. MOLECULAR STUDIESOF MU'I'AGENESIS AND REPATR:
POSSIBLE IMPLICATIONS FORNEOPLASTIC INITIATION
154. If, as iniplied in previous paragraphs, themajority of neoplasms are initiated through gene- andcell-specific somatic niutalion, then the molecularcharacteristics of radiation-induced gene mutation may
be informative. Detailed studies of radiation-induced
mutatio~~at the HPRT, APRT, TK and DHFR loci of~ n a m ~ n a l icells
a r ~ show that the principal mechanism ofradiation-induced mutation is through gross genctic
change, usually DNA deletions [El, H7, M6, M33,
N20, S29, T2, T8, U l l ] . These data in no way
excludc radiation niutagenesis through point mutation,
which has been convincingly demonstrated at the
APRT locus [G5,M33], but rather suggest that ionizing radiation is a relatively weak point mutagen. The
molecular analysis of spontaneously arising somatic
cell mutanls suggests that point mutation may be thepredominant spo~~taneous
mutagenic event, allhoughspontaneous deletion mutants have also been characterized [M33, S29, T2].

155. It is, however, important to recognize that mostof these data relate to mutation induction in a limited
selection of genes and, generally, at relatively highdoses and dose rates (see [T8]). In recent studies, it
has been show11 that fk mutations in a murine(L5178Y)cell line are induced in a dose-rate-dependent fashion but, apparently, with an increase in the
proportion of tnultilocus mutations correlating withdecreased dose ratc [E4]. In this study, niutations at
the lrprt locus, which are thought to be single-locus
events, showed no such dose-rate dependence. In contrast, olher studies (see IF22, M221) show that induced
lrprr rnutation rates decrease with dose rate or dose
fractionation. It has also been demonstrated [M22] thatthe specific fraction of full lrprt gene deletion in
Chinese hamster ovary cells is not altered by postirradiation rcpair processes, suggesting that DNA
repair acts with equal fidelity on the majority ofpote~~tiallyniutagct~ic lesio~u (see also h i e x F,
"I~~fluenceof dose and dose rate on stochastic effectsof radiation"). This latter study also provided preliniinary e v i d e ~ ~ cfore Iyrt breakpoint hot spots in the 3'
segment of the gene. Since oxygen concentraliori is
k11ow11 lo aSfect Lhe spectrum of radiation-induced

ANNM E: MECHANISMS OF RADIATION ONCOGENIiSISDNA lesions, it may be an additional and importantvariable, particularly for DNA base damage that might
give rise to point mutations.

156. There is growing evidence that spontaneous andradiation-induced gene deletions in cultured
mammalian cells may involve preferential DNA
brcakage at certain short repeat sequences [M33, M39,
M491. The mechanism for the induction of such
deletions remair~s uncertain but could include theinduction of DNA double-strand breaks, followed byrecombination and/or replication slippage pastsecondary DNA structures produced by the pairing of
the repeats [M33]. The potential iniportance of thismechanisnl for oncogenesis is evidenced by thefinding of similar short, direct repeats at the
breakpoints of deletions in the RB gene in humanretinoblastoma, and it also appears that the repeat
sequences involved in gene deletions vary according
to the locus that is being studied [C25]. The catalytic
properties of such repeat sequences, while still to be
fully resolved, could therefore be a significant factor
in the relative radiosensitivity of different tunlour
genes and, through this, could be a determinant of theinducibility of the neoplasms with which they areassociated. At present the information available islargely restricted to small intragenic deletions, and it
is of some importance to determine the relevance of
the DNA deletion n~echanisn~
noted above for thegross chron~oson~allosses that characterize someradiogenic neoplasms [B42].
157. A similar picture with respect to gross genetic
damage also emerges from nlolecular studies on thenature of Ihe DNA-repair defect in radiosensitive
ataxia-telangiectasia human cells. These data imply
lhat misrepair of DNA double-strand scissions may be
the major determinant of ataxia-telangiectasia
radiosensitivity and that the misrepair takes the formof DNA deletion and/or rearrangement around the siteof the scission [C6, D2] (see also Annex F, "lnfluenceof dose and dose rate on stochastic effects ofradiation"). Some of the uncertainties about therelevance of this misrepair mechanism for the ataxiatelangiectasia phenotype have been resolved by the useof a DNA repair assay utilizing cell-free extracts[G16,N151. Here it has been shown that, whereas
nuclear extracts of normal and ataxia-tclangiectasia
cell lines do not differ markedly in the efficiency withwhich they rejoin enzyme-induced double-strand
breaks in plasmid DNA, they do differ in the fidelity
with which certain fonns of breaks are repaired; fromthe data presented it appears likely that the nature of
the DNA sequence at the breakpoint may influence
repair fidelity. Such in virro approaches may haveconsiderable potential for resolving the molecularmechanisms of oncogenic initiation and could, forexample, be used as model systems to explore the579

importance of double-strand breaks and DNA repcat
s c q u e ~ ~ c ine s the induction of DNA deletions in cloned
turnour suppressor genes such as RB, APC and p53.
Honiologous recombinatio~lis thought to be involved
in sonle cases of tumour suppressor gene niutagenesis,
and model in virro approacl~esto this mechanism andits induction by radiation have been described [B31].

158. DNA ruisrcpair niay also result in the appearanceof DNA trarlslocations as well as deletions, and it isrelevant chat the predoniinant, spontaneously arising
T-cell neoplasms in ataxia-telangiectasia patients often
involve specific chron~osomal rearrangenlent [T6]
centred on incorrect recombination of DNA sequences
associated with the assembly of mature T-cell receptor
genes [MI. Owing to the apparently small DNA target
involved, the efficient induction of such precisetranslocations by radiation may be questioned(paragraphs 74 and 75). It may be, however, that as aconsequence of specific recombination affinity,
radiation-induced molecular damage outside the target
sequence may catalyse site-specific misrecombination[B42]. Again, this problem could, in principle, b e
approached using in vitro molecular strategies.
159. Overall, the above molecular studies may be
used to argue that radiation-induced somatic cell
mutations associated with the initiation of oncogcnesis
are, perhaps, more likely to be specific DNA deletions
and/or rearrangements than point mutations. However,there is insufficieat evidence to be dogmatic about this
inlportant aspect of or~coger~ic
initiation, and it may b elhat the genomic domain within which a target gene islocated is a major determinant of the predominantmolecular mechanism of induced genetic change. It
may also be oversimplistic to view mutagenic mechanisms as the principal deterniinant of oncogenesis. A
significant factor in the emergence of an establishedneoplasm will be the degree of proliferative advantage
and/or selection associated with early pre-neoplastic
events. Thus, while ionizing radiation may be a weak
point mutagen for a given gene, the selective advantage conferred to a target cell by a specific point
mutation in that gene may in some circumsta~~cesoutweigh the relatively low overall frequency of suchmutations induced within the target cell population. In
conclusion, recent attempts to relate specific mutations
in radiation-associated human tumours to then~utagenicaction of ior~izi~lg
radiation [VlO] highlightthe pressing need for more detailed infonnation on then~cchanismsof radiation mulagenesis and repair. The
induction of cellular repair by low doses of ionizhg
radiation has been demonstrated in certain experi~ n e ~ ~ sysicms,
tal
but the irnplications of this foroncogenif processes remain obscure (see A m e x F,
"lnnuence of dose and dose rate on stocl~asticeffeclsof radiation").

UNSCEAR 1993 REPORT

580

C. IN VITHO STUDIESWITH CEI,I,UIAR S YSTEhIS
1. Conventionill systems
160. The early work of Rcznikoff ct al. [R2] provided
strategies and techniques for the d c v c l o p ~ n e ~of~ tin
11iiro oncogenic cell transformation systems. These
clonogcnic systeas, such ;IS that based on C3HlOTE
cells, seek to characterize and quantify radiationillduccd changes in quasi-nornial cells that ~nimicorarc associated with neoplastic cellular phenotypes, e.g.
loss of contact inhibition, growth in semi-solid'ledium IH61.
The positive and negativearguments
the relevance of these in vitro techniques to oncoge"es" in "'0 ha''been di'c"'~'I['tand it issufficient here to make only a few tFncra1 points
aboutin Iheof oncogenic
mechanisms.
161. First, irrespective of the detailed cell biology ofthe systen~s,in vitro studies that relate radiation exposure to the induction of a clonogenic, neoplasia-associated cellular phenotype should be viewed as being
potentially informative. Secondly, the characteristics ofthe established rodent cell systems allow rapid assayof the transfomlcd phenotype and ready manipulation,
for the purpose of addressing questions of dose effect,
dose rate, radiation quality, post-irradiation repair andtumour promotion. Also of great potential benefit is
the possibility that such techniques could be applied to
cultured human cells.
162. For the induction of the in vifro transfornied
cellular phenotype there is compelling evidence thatthe principal target is genomic DNA. The gross morphological cellular changes that signal transformation
may be produced by DNA-mediated oncogene transfer,and there is a good correlation between the ability of
the given agent to induce ccll transionnation and its
capacity to induce DNA-damage (see also paragraphs
20 and 21). In addition, there is evidence that in virrotransformation progresses by at least a two-stepprocess and that many agents and conditions thatil~fluencein vivo oncogcnesis have parallel effects onin vitro cell transformation [H6, K10, W].

163. There remain, however, rnany uncertainties
regarding the true relevance to in vivo or~cogenesisof
quantitative and qualitative studies with many existingin vifro systems. The pri~~cipal
in vitro cellular systems are based on immortalized rodent ccll lines, andquantitative, radiation-induced transforniation of normal human diploid cells has yet to be convi~icingly
demonstrated. In the established rodent ccll systems
there has been considerable controversy on the shapeof dose-effect curves [L2], on the i~ifluellceof radiation quality and, reccatly, 011 the existence of a reverse

dose-rate effect for high-LET radiations (see [C14]).
Overall, UIC relative si~nplicityof Ulcsc systenis is
a l ~ j ) e a l i ~but
~ g , their direct application to quantitative
aspects of 11u1n:lnradiological risk is perhaps prcmalure. Some of these aspects are discussed in Annex F,"Influence of dosc and dosc rate on stochastic effectsof radiatio~~".
164. Mechanistic studies of in virro cell transfonnat s in some instanlion illdieate that initiating e v c ~ ~may,ces, be i ~ ~ d u c eatd a surprisi~~glyhigh frequency in an
irradiated population of C3H10T44 cells [K3]. Evidcncc has also been presented that, at low doses, 1 2 5 ~incorporated into DNA is extremely effective at
illducillgthe trallsformationo[ BALB/3=3 cells [L18].
Indeed, atlevels producing only -30 decays percell, ale higll blefonnation frequency obselved (104
per survivillg
implies that only asmallllulllberof ullspecified DNA sites need to be damaged
to convert the cell to a neoplastic phenotype. Similarc o ~ ~ c l u s i oare
~ ~ alsosemerging from in vivolin vitro
studies wilh rat mamniary and thyroid clonogcns [CS];such high-frequency induction is, however, difficult toreconcile wilh the overall picture of specific genetargets for neoplastic initiation that is emerging fromstudies with human tumours. The molecular mechanisms underlying this process remain obscure, butestablishing the generality of such high frequency
induced events is of obvious importance. The molecular aspects of the radiation-induced transformed
phenotype have been studied using DNA-mediatedgene trausfer techniques, a ~ ~althoughddoniinantly
acting tra~isformir~g
DNA sequences have been shown
to be carried by overtly transformed C3HlOT44 cells,lheir identity has yet to be fully established [B3, K22].
The presence of unidentified transforming sequences
has been demonstrated in post-irradiation masscultures of C3HIOT'h cells [L2], and recently, DNAtransfer techniques have been successfully employedin Ihc niolecular cloning of a transforniing gene froma radiation-induced C3HlOT'h transformant F20]; itrcniairs to be seen l~owcverwhether this gcnc represents the principal target for radiation-induced
initiation of C3HlOTE transformation. In addition,
myc o ~ l c o g c rcarrangernellt~~chas been characterized ina radiation transfornicd C3HIOTX clone [S6] and myc
and r f gcnc cxpressio~~has been shown to be
increased in anolher such bansfomled clone [L25].
165. M i l e it is possible that the identification ofchariictcristic gene activation events in in virro transformed cells may allow com~rienton the broad mechanisn~sof radiation oncogencsis, i t is more likely thatsuch ide~~lificationwill be of greatest value in theunderstantli~~gthe systems themselves. With conventional rodent it1 vi~rotransforrnation systems, doubt islikely to remein about the true significance of single
interactive
cell it? virro response to the more co~l~plex
processes that mediate in vivo oncogenesis.

ANNEX E: MIXI IANISMS OF RADIATION ONCOGENESIS

2.

Novel systenis

166. While quantitative and qualitative data fro111
conveotional ccll transfornlation systems will continue
to be useful, more attention is now being focused on
Uic potentially n ~ o r e rclevant systems that utilize
human cells, cclls of epithelial origin and cells frorii
hacnlopoietic tissues (see IC91). It is alrc;~dyapparent
that the conventiorral criteria for in vitro celltransforniation, i.e. loss of contact inhibitions/focus
formation, growth in semi-solid media, i~nnlortalizalion and relatively rapid progression to an overtly
oncogenic phenotype, do not apply generally incellular systcms. Cellular and molecular studies ofhuman neoplasms strongly suggest that different
trarisfonnational processes may operate in different
ccll lineages, and this should be reflected in the invitro systems that are employed. The nlost significant
progress that could be anticipated would be the designof in vitro cellular systems that were linked, bycytogenetic and rnolecular studies, to specific in vivo
neoplastic and prc-neoplastic conditions. It cannot be
expected that such systems will be sirnple or quantitatively precise [H13], but they should reveal more
about oncogenic mechanis~ns than the currently
favoured systems based on rodent fibroblast cell lines.
167. New developments in it1 vitro cell transformation
have recently been reviewed [C9], and it is sufficienthere to highlight a few of the nlost important findings.
Studies with human diploid fibroblasts have been
discussed by McCorniick et al. [M9], with particularemphasis on the lack of clear evidence for i ~ ~ d u c e dmalignant transformation in these cells. The
immortalization of human fibroblasts, unlike that ofcultured rodent cells, appears to be a very rare eventand probably represents a rate-limiting step in thepathway towards malignancy. It has bcen shown,however, that a rare, radiation-induced, immortalized
human fibroblast strain may be transformed lo a fully
malignant pbenotype by the introduction of anactivated H-ras oncogene [N8, N l l ] . I n a series ofsubsequent experiments [M40] it has been shown thatintermittent in vitro exposure (total dose 28 Gy) togamma rays over 50 passages rcsultcd in theimmortalization of hurilan fibroblasts but that final
malignant conversion could not be demonstrated until
the 547th passage (2.800 d in culture). These studies
provide a clear dcmo~lstrationof the multi-step natureof in vuro malignant transformatio~r in humanfibroblasts and, un~ortunately,the difficulties that are
faced in obtaining quantitative estimates of induced
transformation with these cells.
168. A human hybrid cell system has also beendeveloped in order to quantify radiation effects on aputative tumour suppressor gene encodcd on chromosome 11. The quantitative, cellular and rnolecular581

aspects of this system are currently under ir~vestigatio~i[R4], and good progress niay be anticipated. Although
based on inimortalized strailrs of human cells and used
so far for characterization and chcn~icalcarcinogcncris
studies, niarrirnary epithelial [S54], kidney epithelial[T18], ski11 kcratinocyte [B7, F6, R18, R19], colonicc~~itlielialIW25] and urinary epithelial cell [R5]systc~ns also show irrtercsti~ig potential for bolhmechanistic and quantitative studics.
169. Cytogcnctic and molecular studies of chen~icallytransfomied variants of the SV-HUC human uroepithelial cell line havc been particularly rewarding[K15]. It lras been shown Uiat chromosome 3 p deletions are characteristic of the high-grade carcinomas
produced in transplanted nude niice following in vilro
chenlical transformation. What is particularly noteworthy is that loss of heterozygosity for 3p encodcd
DNA sequcoces have been reported in a variety ofhuman carcinomas, thus for the first time providing aputative link between in vitro i ~ ~ d u c ecellularddamageand carci~rogenesis in vivo. An extension of thisapproach to radiation-induced transformation should be
encouraged, but it should be recognized that, atprcsent, the assay system as developed does not lend
itself to detailed quantitative studies and that the viralinlniortalization process niay influence the transformational response.
170. Novel cell trarisfonnation systclns that include invivo and in vitro phases have been developed for
mammary [C5], thyroid [M23] and tracheal [T9]tissues of rodents. In the mamniary system, transplantation of dissociated mamniary tissue to subcutaneous fat pads has allowed the quantification ofdifferentiatirig mammary clonogens and, through this,esti~natesof radiosensitivity. A tissue-dependent postirradiatiolr repair process has been characterized, as
has the induction of dysplastic (pre-neoplastic) lesions
resulting from radiation damage to clonogens. The
proniotional effects of elevated prolactin and glucocorticoid deficiency are evidcrit in thcse studies, and
it is also apparent that neoplastic initiation is arelatively common cellular event [C5].
171. A fiit pad transplantation technique also underlies
the in vivo in vitro studies on thyroid carcinogenesis.
The radiation induction of prc-neoplastic lesions wasshown to be dependent on hormonal status (thyroidstimulating honnone), and again, initiating events wereobserved at a surprisingly high frequency [CS]. Cellcell interactions havc been shown to influence strongly
Ihe cxprcssio~lof oncogenesis in both mammary andUiyroid systems. Sinlilar findings have been madeusing an in vivo in vitro rnodcl of trachcal epithelialtransforlnation in the rat [TlO].
Here, direct cell-cell
contact in tissues and a diffusible factor from cultured
cells have bcen shown to suppress the expression ofearly oncogenic changes. In the case of in vitro

UNSCFAR 1993 REPORT

582

suppression tlie factor is believed to be transforming
growth factor D (TGF D), which niay exert its effectthrough the induction of terminal differenti;~tion[TlO].
Thus, the emergence of initiated cells and theirsubsequent proliferative advantage niay depend on
their ability to escape Croni T G F B mediated terniinal
differentiation.

172. Despite the increasing knowledge of tlie complex
interactions between growth promoting and inhibitory
factors in the regulation of haeniopoicsis andassociated cell lineage di fferentiation p 8 , M241,
transformation systems for cells of haemopoietic origin
are at a relatively early stage of development. However, the availability of growth factor-dependent
haeniopoietic cell lines p 9 , G10, S30] and their
oncogenic transformation by specific haeniopoiesisrelated genes [B15, L12] are providing powerful tools
with which it may be possible to relate transforliiing
events in vuro to those that characterize haemopoietic
neoplasms in vivo. In the case of osteosarcomagenesis,
an in vkro system for murinc mandibular condyles isbeing developed to study the cellular and molecular
evcnts that mediate skeletoblast differentiation andoncogenic transformation [S15]. In this system it has
been shown that fos expression precedes the in virro
osteogenic differentiation of cartilage cells [C28].

D. IN VIVO STUDIES'

1. Geneml e x p e r i n ~ e t ~ tstrategiesnl173. It has been argued in previous paragraphs thatthe most plausible molecular form of neoplastic initiation is specific gene mutation that predisposes appropriate target cells to subsequent malignant conversion;
studies with familial human neoplasms have also
shown that, under some circumstances, it is possible togain detailed information on the identity of tumourinitiating events in man. However, the central problem
for cellular and molecular studies using inducing
agents and experimental animal systems is that during
the post-irradiation latent period, a number ofgenetic/epigenetic changes will have accumulated
within the evolving neoplastic cell population, andmany of them will be represented in the final
malignant clone that is available for study. Whatcriteria can be used to deterniine which of thesechanges was induccd by the carcinogen and istherefore the initiating event?
174. Two main experimental strategies have evolved
to approach this crucial question:(a)to use molecular techniques to search forconsistent molecular change in specific genes of
the induced neoplasm and attempt to relate any
characteristic DNA changes to the known DNA-@)dalriaging properties of the iriducirig agent;
to ide~ilifycarididate initiating events as consistcllt ~nolecularand/or chromosomal changes intllc overt induced neoplasnl and subsequently
trace the induction of these evelits back to theimmediate post-treatment proliferation of cells in
the target organ.

175. The first strategy has been effectively employed
in b e putative identification of m s proto-oncogenes as
principal targets for early evcnts in chemically induced
mammary, liepatic and skin carcinonlas in rodents(paragraphs 188 and 189). In general, the success of
this approach to clicn~icaloncogenesis depends on the
induced initiating events being base-pair specificchanges in a restricted set of codons of a known gene(see, e.g. [B37]). In this case the chemical specificitiesof the carcinogen will be immediately apparent andinfornlative. The relative lack of DNA damage specificity following exposure to ionizing radiation niay
mean that this strategy is less appropriate to radiation
oncogenesis. Consistency of molecular genetic change
in a given neoplasm infers a major contribution of thatchange to the success of the malignant clone but neednot be informative on the stage in clonal evolution atwhich the change took place. Point mutation within alimited set of ras gene codo~lshas been demonstrated
in radiation-induced murine lymphomas [D3,D4,
N191, but alone, these data arc insufficient to identify
ras activation as an initiating event for this neoplasm.
More recently, however, it was shown that K-ras
codon mutation spectra in tumours induced by gamma
rays are different from those in tumours induced byneutrons [S31]. These data were used to argue thatdifferences in physical properties between the tworadiation qualities might influence mutational spectraand hence that K-ms activation might represent an
initiating event for radiation lymphomagenesis. Thereis, however, some uncertainty in the biophysicalinterpretation of these data, and it remains possiblethat ras mutations play a greater role in thedevelopment of lymphoma than in its initiation. Even
greater problems of interpretation surround otherobservations of structural and functional changes inoncogenes of radiation-induced animal neoplasms(paragraphs 179-187). In no case is it possible to be
confide~~t
about the identity of the radiation-induced
inilialing event (e.g. [F4, S71). In this area, however,
the implementation of polymerase chain reaction techniques (PCR) for rapid gene mutation analysis [E8,R l l ] during early post-irradiation cell proliferation
periods may be expected to have a major impact on
future research.
176. The second experirnental strategy has beensomewhat more successful, particularly in providing
comment on the status of characteristic chromosomalchanges in radiation-induced haemopoietic neoplasms.

ANNEX C: MITI IANISMS OF KN>IA'IlON ONCOGENESIS

177. Trisonly of chromoso~ne(ch) 15 is a characteristic cytogcnctic fca~ureof radiation-induccd murille
Ulynlic lymphoma. Cytogerietic studics of pre-neoplastic animals strongly suggest that the appcarancc of
the extra ch15 copy is associated with tllc later stagesof neoplastic developme~~t.In Co~ItriiSt, the early
appearance of a ch 1:s translocation i n a significant
proportion of prc-neoplastic animals suggests that thisis a better candidate for a lymphoma-initiating cvent[M7]. There is, as yet, no information regarding the
involvement of specific genes in either of thesechron~osomalchanges.
178. The deletion and rearrangement of ch2 is a consistent feature of radiation-induced acute niyeloid
leukaemia in the mouse [B4, B16, H81. Using an invilro irradiation and transplar~tationtechnique, similar
ch2 rearrangements were characterized in rapidly
proliferating haemopoietic cell clones within five daysof irradiation, and it was coricluded that these nlay
represent candidate initiating events for murine acuteniyeloid leukaemia [B16, S8]. More recently, a strong
statistical concordance has been established between
radiation-induced ch2 breakpoints in irradiated muririe
haemopoietic cells and those in acute niyeloid leukaemia [B16]. In addition to strengthening the link
between ch2 delctionfrearrangement and lcukaemic
initiation, these data also suggest that induced
breakage may occur at specific radiation-sensitive sites
on this chromosome. Stable chromosori~al changes
have also been recorded in irradiated and repopulating
haernopoietic cells in the rat [M35].

the spccific role of these and their derivation fromgemi-line ecotropic provirus rcliiains unccrtain (J2,
N41. Some evidence for proviral illduction by radiationand integration into coninion gcnomic domains inlynipho~nasand derivative ccll lines has recently beer1
obtai~icd,but the genes within these domains are not~ I I O W I I132, S551. The weight of evidence now tends tofavour a rolc for provims in lymphoma dcvelopn~cnt
rathcr than initiation, and there are studies that iniplythat radiation lcukacmogeriesis in NFS mice does not
involve C- and B-type rctrovimses [03]. Indeed,
recent evidence suggests that infection by activatedviruses is not necessary to explain the indirect
inductio~iof radiogenic lymphomas [S56, S57]. It has
been proposed that thc dcpletion of T-cell precursors
in the bone marrow, together with the depletion ofthymic lymphocytes, is sufficient for regenerating
thymus cells to undergo pre-neoplastic changes,
possibly due to an aberrant expression of genes
involved in the growth and differentiation of thynius
cells [M50, S56, S571.
181. It has been shown that the injection of syngeneic
bone marrow cells into split-dose-irradiated mice canprevent lymphoma development, possibly as aconscqucnce of the rapid repopulation of recipientthynius by donor lymphoid cells [K4, S9]. Overall, it
may be concluded that complex cellular processes
inediatc radiation lymphoriingenesis and that dircct
radiation damage to target cells may not be aprerequisite for the appearance of oncogenic events. In
this respect, four interactive factors nlay be invoked:(a)

2. Molecular studies
of induced ani~ilalneoplasms
179. The molecular aspects of radiation oncogenesis
in animal models of thymic lymphoma, myeloid leukaemia and osteosarcoma have recently bccn reviewed[J2] and are outlined here briefly, together with someobservations on rodent skin, mammary and lung carcinogenesis. This whole area has also been explored ina number of reccrlt syniposia (see [B28, E71).
180. Thymic lyrphorna. Studies of thyniic lyrtiphomagenesis in mice have provided evidence that anindirect mechanism is involved [K4]. It has been
repeatedly shown that thymic lymphomas may develop
from immature lyn~phoidcells present in non-irradiated thymuses that are subcutar~couslygrafted into splildosc-irradiated, thyniectomized recipients [K4, M8].
Since weakly leukaeniogcnic activity is often present
in cell-free extracts of primary lymphomas and its
potency increases with serial passage, a viral role inlymphomagenesis was suspected [L4]. Although
recombinant retroviral complexes have been reproducibly detected in radiation-induced lymphomas [J2],

583

@)
(c)
(d)

activation of potentially leukaemogenic virus
from germ-line provirus and subsequentreco~nbinationto yield more potent virus;
thymic involution and regeneration, giving a
population of virus-susceptible cells;
impairment of cell surveillance mechanisnis;
impairment of bone nlarrow and thymocyte cellfunction, leading to reduced thymic repopulation
and altered gene expression.

In addition, codon mutations in N- and K-ras
oncogenes, T-cell growth Pdctor changes together withspccific chromosomal rearrangements have been
detected in radiation-induced lymphomas, and some ofthese have been suggested as early events in lymphomagenesis (paragraph 175, IN191); in contrast, recent
data tend to argue against the involvement of p53
mutations in radiation-induccd and chemically-induced
lynlphonlas [B33]. Attempts to characterize and isolate
target cells for radiation ly~uphoriiagenesisarc also
undcr way (M261.
182. Acrrfe rnyeloid leukaernh. There is little
evidence to suggest that radiation-induced murine
acute myeloid leukaemia proceeds via an indirect(viral) nlcchanism. In contrast, the specific deletion/

584

UNSCEAK 1993 REPORT

rearrangement of chromosome 2 is a consistent cytogenetic feature of induced nlurine acute myeloid
leukaenlia [B4, B16, H8, H44], and evidence has beenobtained that ch2 rearrangenlent is iltduced directly by
x rays in multipotential haemopoietic cells, in somecases generating a cellular phenotype associated withpreferential haemopoietic recruitnlent and/or proliferative advantage [B16, S8]. It has also been shown thatch2 events may be observed in bone marrow cultures
cstablisbed from irradiated mice long before theanticipated appearance of acute myeloid leukacmia[T4] and that the induction of ch2 rearrangement is anearly step in leukaemogenesis but is not sufficient forthe development of nlyeloid malignancy [H27, H28).
Preliminary evidence for deletion of the homeobox
gene, hox 4.1, and activation of the cytokine gene,interleukin-1B (II-14, has been obtained in some ch2rearranged acute myeloid leukaemias [BS, S10, S l l ] .
However, the evidence presented for the involvementof these genes in leukaemic initiation was notcompelling, and more recently, detailed molecularanalyses of the 11-1 genomic region on ch2 failed toprovide evidence of the structural rearrangements
predicted in acute myeloid leukaemia [S32, S33]. In
these analyses it was found, however, that some acute
myeloid leukaemias show very similar methylation
pattern changes in the If-1 region, and it wassuggested that these may be associated with certain
forms of ch2 rearrangement and deletion. Other
studies have shown that the ch2-encoded his-1 locus
is involved in virally induced myeloid leukaemia inthe mouse and may represent another candidate genefor the initiation of acute myeloid leukaemia [A9].

183. Cytogenetic studies with radiation-induced
rnurine acute myeloid leukaemias and irradiatednormal haemopoietic cells have yielded evidence thatch2-encoded fragile sites are involved in leukaemogenic initiation [B16]. These investigations alsosuggest that the interstitial ch2 sites in question havea strong recombination affinity with the terminal(telomeric) ends of other chromoson~es,implying thatthey may indeed be teloniere-like repeat sequences(paragraphs 62 and 63): it was also suggested that theexpression of these sites might be influenced bygenomic imprinting. More recently, interstitial
telomere-like sequences of an inverted repeat form
have been cytogenetically mapped close to the relevant
eh2 breakpoints, and h e use of telomeric sequences asmolecular probes has provided some evidence thatgerm-line variation at certain telomere-like repeat
sequences may be associated with genetically determined leukaemogenic radiosensitivity in the mouse(S431. Following these studies, it has been suggested
that the telomere sequences at ch2 fragile sites may
promote the formation of recombinogenic secondary
DNA structures that are bigilly radiosensitive [B32].
Finally, while ras mutation is not a common feature ofmurine nlyeloid Ieukaemias, the activation of N-rtls
has been reported in radiation-induced myeloid
leukaemias i n the dog [G17].
184. Oslrosarcotna. Although weakly oncogenic
viruses have been isolated fro111 radiation-induced
murille osteosarcomas, there is no evidence for common gcnorr~icinsertion sites nor is there an obligate
requirement for such viruses in osteosarcomagencsis[J2, V8]. A non-essential role for such viruses inoncogenic progression appears, perhaps, to be more
likely. Molecular studies have revealed the amplification of a ch15 domain encoding c-myc and the Mlvi-1
proviral integration site in up to 30% of 224~a-induced
osteosarcomas, and evidence for altered expression of
K-ras, myc, sis, abl, bas and fos has been reported insonic tumours [J2, S59]. Structural and/or expressional
cilanges have also been reported in the p53 gene ofsome osteosarcornas, but to date, there is no evidence
for consistent structural changes in the retinoblastoma(rb) gene [J2, S581. Although specific comment on thestatus of these molecular changes in radiation osteosarcomagenesis is not yet possible, the humanevidence of RB and p53 gene involvement in the earlyphases of the human neoplasm [C29, T5] demands
further studies wilh murine osteosarcomas.
185. Skin carcinomas. Ionizing radiation i n d u e sskin carcinomas in both the mouse and the rat [J3,S7], but little is known of Ule molecular mechanisms
involved. Recently, it has been shown that theactivation of K-ras and myc oncogenes is frequently
observed in primary rat skin carcinomas induced byionizing radiation [S7]; the status of these changes in
the oncogenic process remains uncertain, but myc
seems most likely to function relatively late in skincarcinogenesis [G18]. In nlurine studies it has beenshown that the gene encoding the transin protease isoverexpressed in carcinomas but not in benign
papillomas. It was inferred from these observatio~~sthat transin gene expression might be involved inneoplastic progression by enhancing tumour invasiveness [ B l l ] .
186. Mammary carcinomas.
The induction of
mammary carcillomas in mice has been studied by isolating mammary epithelial cells at intervals following
irradiation in vivo and subsequently selecting clonalcell lines in vifro. After low in virro passage, isolated
cell lines produced normal ductal outgrowth in transplanted mice, but with increasing passage cells became
increasingly tumorigcnic and invasive. Chromosomal
abnormalities and myc (but not ras) gene expression,increased with passage and tumorigenic potential. On
the basis of studies on r b and p53 gene expression it
was suggested that alteration of expression of tumour
suppressor genes might be an early event in the
oncogenic transformation of mammary epithelial cells

ANNEX E: MECtlANlShlS OF MDIATION ONCOGENISIS[E7]. This in virro system l ~ a salso bee11 used toinvestigate the acquisition of a~igiogenicactivity inirradiated niarlilriary cells and its ter~iporalrelationshipwith tumorigenic potential [U12]. Although ras protooncogene involvcnient in clicr~iically induced
mamliiary carcino~lla in rodc~ttsis becoming ullderstood (see [G19]), the situation regarding ionizitig
radiation has not been resolved.
187. Lung carciriornas. Lung carci~iogenesis has
been studied in a number of animal species [M41],585

and in contrast to tile situation in l ~ u ~ i i ait~ lseemssatpresent unlikely U~ateither the rb or p53 gene plays aniajor role in induced lung neoplasia [G20, M411.
Studies on ras gene chaligcs in pluloniuni-induced rat
lung tunlours imply tl~atthe activation of this gcnc
may be a relatively early cvent in t l ~ egeneration ofpro1 iferative pulnionary lesions [S60], while in thedog, the expression of the epidernial growth factor
receptor gene was elevated in a significatit proportionof lung tumours and proliferative foci arising after
exposure to plutoniu~~i[G21].

IV. COMPARATIVE ASPECTS OF ONCOGENESIS BYRADIATION AND CHEMICALS
188. It has been suggested that ionizing radiation may
principally, but not exclusively, initiate oncogenesis
through niechariisms involving DNA deletion andlor
rearrangement. While many chemical agents also
induce gross (chromosomal) damage in mammaliancells, the mechanisms of induction are fundamer~tallydifferent from those of radiation [C3, E3]. In addition,
molecular studies of induced somatic mutation show
the majority of chemical agents to act principally as
point mutagens [S29, T2]. Although the mechanisnisof chromosome aberration induction by radiation andby the majority of chemical agenls differ in terms ofcell cycle dependence, the gcnomic distribution ofdamage and the DNA repair/reconibination processes
involved, there may not be niajor differences in the
final stable fornis of many induced chrornosomalchanges in surviving cells. It should be emphasized,
however, that the distribution of these changes within
the genorne may be expected to be differel~tfor radiation and chemical agenrs. In spite of these uncertaintics it appears that the major ~iicchanisticdifference at
the cellular level between radiation-induced andchemically induced oncogencsis is likely to centre on
their relative efficiencies for the induction ofneoplasms that are principally initiatcd through point
mutation and activation of proto-oncogenes. Chemicaland physical carcinogenesis has been reviewed
recently [H46], and the strongest cxperimenlal
evidence for the existcrlce of such an initiating
mechanism for chemical otlcogencsis comes fromstudies of ras proto-oncogene activation in somerodent neoplasms. In many chemically induced skin,mammary and hepatic turnours, codon-specific point
mutations responsible for ras activation correspond to
those predicted by the luiown base-pair-specificmutagenic action of the agent used as the initiator, i.e.
ras niutation appears to be the initiating event [B6].

189. Recent studies [Kg] have extended theseobservations by showing that followirig nitrosomethylurea exposure, ras activatilig events may be detectedin nornial rat manilnary glands only two weeks after
lreatnient, i.e. at least two months before the onset ofovert neoplasia. It appears, therefore, that these
specific chemically induced somatic mutations remain
dormant in tissue until promotional, hornlone-mediatedproliferalive responses mobilize ras activated cells andallow them to progress towards neoplasia. Broadly,these and other data, e.g. [B37, M42, S61], althoughcurrently limited to only a few neoplasms and chemical agents, identify carididate initiating events as point
mutations and further strengthen the link betwecn
mutagenic and oncogenic mechanisms. However, otherstudies indicate that ras activation is not the only
potential initiating event for rat mammary carcinomas
and that different initiating mechanisms may havedifferent carcinogen dose and promotional dependencies [ Z l , 221. Although still at an early stage ofdevelopment, molecular studies on in vifrotransformed
cells are also consistent with the existence of different
molecular mechanis~nsfor radiation and chemical carci~~ogenesis[C9]. However, for molecular events in agiven iicoplasm that follow initiating damage, i.e.
those contributing towards neoplastic progression,
radiation-induced and chcrnically induced neoplasms
may be expected to share some common genetic
changes [N7].
190. Two major issues emerge from the suggested
lr~echa~iistic
differences that rnay distinguish radiation
oncogcnesis. First, since different neoand chc~l~ical
plasrns may depend for their initiation on different
forms of n~oleculardamage to Ihe potentially oncogenic DNA sequence, e.g. point mutations rather thanDNA rearrangement or deletion, then differences inthe predominant induced neoplasias following human
exposures to radiation and chemical carcinogens may
be expected. Secondly, if radiation and chenlicalagents induce characteristically different spectra ofinitiating lesions, then different genetically deterniined
post-exposure cellular DNA-repair processes will tend
to operate to modify and/or remove that damage.
Since the efficiency arid fidelity of these repair
systems will be a significant determinant of induced
oncogenesis, the above differences in repair imply thathuman genetic susceptibility to induced neoplasia byradiation and chemicals will not necessarily corrcspond. Although this speculation is broadly supportedby the lack of cellular cross-sensitivity between radiation and chemicals in the majority of human genetic
diseases showing hypersensitivity to DNA damaging
agents [A2, F4, H5], the mutations in these disorders
will not be fully representative of those having effects
on ~nutagenesisand oncogenic initiation. This is an
important problem in human cancer susceptibility, of
which there is insufficient knowledge.
191. Tomatis [TI61 has reviewed those chemical
agents that are known to have carcinogenic activity inman, and among these are agents for which exposure
would occur occupationally (e.g. asbestos, aromatichydrocarbon derivatives and vinyl chloride monomer),
medically (e.g. cyclophosphaniide and phenacetin) or
environmentally (e.g. aflatoxins, nitrosamines, soots,tars and oils). The majority of these agents do not act
directly but require specific endogenous biochemical
modification in order to generate the carcinogenic
species (see [H46]).
192. Certain workers in the plastics industry arc
occupationally exposed to vinyl chloride monomer(VCM). The enzyme-mediated activation of VCM (theprocarcinogen) to carcinogenic metabolites such as
chloroelhylene oxide and 2-chloroactelaldehyde occurs
principally in the liver. Such activation produces
tissue-specific exposure to the direct carcinogen and in
the case of VCM produces a very clear excess of therare hepatic neoplasm, angiosarcoma, in exposed
workers [B8].
193. Occupational exposure to aromatic hydrocarbon
compounds, in particular benzene, has been associatedwith lhe development of haemopoietic neoplasms[T16]. Most polycyclic aromatic hydrocarobons, suchas bcnzo(a)pyrene (BP), are not directly carcinogenic;
however, through the action of cytochrome P450linked mixed function oxidases and cpoxide hydrases,
BP may be converted to active carcinogens, such as
BP 7,8-dial,-9,10 epoxide (see [W18]). Studies withthe mouse skin papilloma/carcinoma system suggestthat the carcinogenic action of this diol epoxide derivative centres on its capacity to form adducts on the
guanine (G) residues of DNA. Misrepair of theseadducts results in base-pair substitutions at G residues,
and these have been linked with the rus proto-oncogene activation events that are believed to initiatemouse ski11 carci~rogcncsis(see paragraphs 188-189).
194. As noted earlier, anatoxin B1 (AFB), present insome fungally co~~ta~ninatedfoods, is associated withthe prcvalericc of human liver cancer in parts ofsouthcni Africa and eastern Asia [B22]. AFB ismctabolizcd, principally it1 the liver, by the mixed
oxidase erlzyme system to produce several products,
including a highly reactive 2,3-epoxide derivative.
This reacts with the 7 position of guanine residues in
DNA and represents a major DNA adduct in the liverof AFB-exposed rats (see [W18]). The importance of
this reaction for human liver carcinogenesis has
recently been demonstrated by molecular studies ontumour DNA from hepatocellular carcinoma (HCC)
patients from regions where the disease is prevalent,probably as a consequence of AFB contamination.
These studies revealed that the G-T base substitutions at codon 249 of the p53 suppressor gene,which might be predicted from AFB epoxide action
dominated the p53 mutational spectrum observed[B23, H34]. As well as highlighting the importance of
p53 suppressor gene mutations for the initiation of
HCC, these studies provide the first clear links
between environmental carcinogen exposure, procarcinogen activational processes and gene-specifictumour mutations in man. An essentially similar
approach with thep53 gene has been used lo study the
involvement of specific UV photoproducts in sunlightmediated human skin carcinogenesis [B36]. In essence,the specific form of p53 mutation in HCC and skintumours is providing a signature of human AFB andUV exposure, respectively, [H46]; the potential implications of these data are discussed in paragraphs 221
and 222. However, it should be noted that in the caseof the mouse, liver carcinogcnesis following chemical
cxposure may not be associated with consistent p53
mutation [G20].
195. The p53 point mutations observed in HCC andother solid tumours [C31, H30, H46, L13] also serve
to illustrate the point that the initiation of oncogenesisby chemical agents is in no way restricted to protooncogene activation but may also occur via themutation of tumour suppressor genes. In such cases,
the expectation is that those mutations will tend tocluster in the regions of the gene specifying certain
active sites in the protein product. Detailed analyses ofp53 gene mutations in human tumours by polymerase
chain reaction techniques have provided ample
evidence of this [H30, L13] (Figure IV). In the
distribution illustrated in the Figure, the horizontal axis
represents the 393 codons of the human p53 genefrom N-terminal p H 2 ) to C-terminal (COOH) ends.
The vertical bars represent the relative proportion ofANNFX E: MEClbWISMS OF RADIATION ONCOGENLSIS

niutations that occur at eacli codon of ll1ep53gene, as
sequcrlced froni 94 different primary tu~nours,xcnografts or cell l i ~ ~derived
esfroni turnours (brain, breast,colon, oesophageal and 1u11gturnours and neurofibrosarcomas, ostcosiirconias, rl~adomyosarcomas andT-cell lymphomas). The higl~est percentage is at
position 273, which accounted for 13% of all mulalions [L13]. Tuniour types a11d p53 mutations havebeen tabulated [C31, H301, and further infomiation on
tumours of the thyroid and cervix is available [C30,11, K241. Some studies show that p53 mutation can,however, arise at the malignant conversion stage of
oncogenic development indicating that p53 mutational
spectra will not always be iriforniative on carcinogenic
initiation. Nevertheless, current p53 mutational
analyses have been most informative, and the same
approach will doubtless yield important irifonnation on
the mutational damage sustained by other tumour
suppressorgenes, such as APC [KlS, N16], thought tobe involved in the initiation of other tun~ours.
196. Since many cnvironn~entalchemical carcinogens
are subject to specific cellular activation andtor
degradative processes, human genetic heterogeneitywith respect to these processes may be an importantfactor in detcrniining the specific dose of the carcinogen to target cells [A7, W19]. These activation/
degradative processes will also affect the dose of the
carcinogen to different target tissues and thereby
influence the speckurn of induced neoplasms.
197. For external radiation, the induction ofpotentially oncogenic cellular lcsions may be viewed
as a more direct and genetically less cornplex process
influenced principally by Uie LET and the penetrative
properties of the radiation, by DNA metabolic functions in target cells and, particularly for high-LET
radiation, by biophysical factors such as the a o s s section of the cellular rlucleus of target cells and by
the packaging density of the DNA. If, as implied, asignificant genetic detern~inantof the radiation initiation of oncogenesis resides in the activity and fidelityof DNA repair processes, then it may be that there isa greater degree of human genetic variation in cancer
susceptibility following exposure to low-LET radiation
than there is following high-LET radiation. This
speculation derives fro111observations of the apparent
lack of (correct) cellular repair following high-LET
irradiation of cultured hunian cells. If high-LET
induced genomic lesions are inherently difficult torepair correctly [C8], then less genetic variation andof repair functions niay be aaticipatcd inless i~~flucnce
the hunian population. The ~ilainqualification to thisspeculation is that it is based on studies of radiationinduced potentially lethal l c s i o ~ and~ s may not apply inor oncogenic damage.
full to potentially ~nutage~iic
198. In contrast, for intenial radiation, the chemicaland biochemical properties of the isotope will deter-

587

nine in vivo nietiibolie routes for uptake, transport,
distribution and excretion (see, e.g. IPS]). All theseprocesses will have genetic dctcrminnnts, and in thisrespect, or~cogcnesisby isotopes taker1 into the bodylias some parallels wid1 cl~cmici~l
oncogenesis and niay
be subject to a greater degree of genetic heterogeneity
than predicted for cxtcn~alradiation.

199. For radon, a co~nbinationof physical, physicochemical and physiological factors will interact todetermine radon inhalation, its concentration in
respiratory compitrtments and the subsequent dose
distribution in tissues. The active carcinogenic species
following radon exposure is, however, likely to be thedaughter nuclides in the decay chain. Hence, theunderlying mechanism of radon carcinogeriesis will be
subject to a series of complex interactive factors
involving riot only initial radon uptake and distribution
but also the biokinctics and half-lives of the radon
daughters (see Annex A, "Exposures from natural
sources of radiation").

200. More straightforwardly, some alpba-emitting
actinides have a high affinity for specific glycoproteinspresent on bone surfaces and niay indeed be bound by
metal transport proteins. Consequently, the greatest
radionuclide deposition and, thereby, accumulation ofalpha-particle dose often occurs at active endostealbone surfaces. Metabolic factors therefore deterniinethat the principal somatic cells at risk are osteogenic
cells at bone surfaces and haetnopoietic cells in peripheral marrow close to bone surfaces. Clear evidence
for radium-induced osteosarcornas has been obtainedin both man and experimental animals. However,alpha particles from some actinides deposited in bone
appear to be only weakly leukacmogenic [H9, H10,
S161. It is possible that this latter observation reflects
the hypersensitivity of rilultipotential haemopoietic
cells to the lethal effects of track traversals by single
alpha particles, in which casc there will be a low
probability of survival for irradiated target cells;
equally, however, the spatial rclationsliip between sitesof deposition of radionuclides and large1 cells and thealpha-particle track LET may also be critical [B26,
S16]. In this latter context it is important to recognizethat alpha-ernifling actinides distribute differently
between bone volunle and bone surface, and it is,therefore, not appropriate to gericralizc on the in vivo
dose to different target cell populations.
201. In the casc of radionuclides taken up inparticulate form, tlie action of macrophages in thee n g u l h i e ~ ~dissolutiont,and nuclide redistribution intissues is of pri~iic importance. These factors willundoubtedly influence the relationships between dose
and target tissues IS46j. In human patients receiving
colloidal alpha-emitting thorolrast as a contrast
medium, distribution of dose to tissues will have been

UNSCEAR 1993 IEPOI1T

588

subject to cell-mediated effects. In these patients thereis evidence of both Ule accu~nulationof the isotope inliver and a dramatically increased risk of liver cancer[ U l , U21. Studics have been initiated to relate p53
niulations in thcse tumours to potential radiationinduced darnage [W26].
202. I t should, however, be stressed that riiany of theargumcnls presented l~ererelate to genctic and physiological effects on the i~litiationof oncogcnesis. Genetic
and physiological hctcrogcneity with respect to promotional and progressive processes in tumour developmerit must be of irriportance to the overall incidence of
induced neoplasia in the population and could indeed
be the dominant factors. The relative roles of radiationand chemical agents in these processes are noted inparagraphs 78-1 14 and discussed in detail elsewhere(see, e.g. [S2, T141). Here it is sufficient to suggest,that for a highly interactive multi-step disease such as
cancer, it is only possible to generalize on the relativeroles of radiation and chemical agents in the steps that
may currently be defined on the basis of incomplete
mechanistic knowledge. Better knowledge shouldmake the task somewhat easier, but it is important torecognize the current extent of ignorance.
203. In conclusion, the niechanisms of oncogenic
initiation by radiation and chemicals differ substantially and may be subject to different genetic andphysiological factors. Consequently, the determi~ratioliV.

of relative risk fro111radiation and chemicals will not
be an easy lask. A report o f t h e United States National
Council on Radiation Protection and Measurements[NlO] considers in detail the comparative aspects ofthe carci~iogenicily of io~~izingradiation andchemicals, and it is clear from Illat report and the outline prc-..e~~tedhere that the deterniination of relativerisk froni radiation and cheltiicals is not at all straightforward. For radiation and those chemical agents withknown mechanisms of action on DNA, it may bc
possible to make some assessments on the basis ofmolecular dosimetry. In such cases the abundance ofinitial mutagenic/carci~~o&enic
lesions in the DNA ofcells of the target organ following exposure to a
carcinogen may provide a crude experimental indicatorof potency. However, it should be recognized that forthis strategy to have a firm scientific base, it isnecessary to know the different spectra of mutagenic
lesions and their repair/misrcpair characteristics and,perhaps niosl importantly, to have a much better
understanding of the mccllanistic differences thatcharacterize the oncogenic processes that drive theinduction of different neoplasms. In such a future
experimental approach to comparative carcinogenicity
it will be essential to be able to relate animal and in
virro data to man. In this respect, it should be notedthat the current quantitative knowledge of human risk
from exposure to ionizing radiation greatly exceedsthat for any chemical carcinogen.

FUTURE PERSPECTIVESIN REIATED RESEARCH

discussed in other Sections. Of particular note is thea~iticipatcdavailability of molecular probes for genesthat deterniine human sensitivity to radiation and othergenotoxic agents (e.g. [ F l , G7, H45,K11, K28, T151).
When available, these will find use in the elucidationof hu~iiangenetic heterogeneity with regard to radiosensitivity, with the final prospect of identifying
subpopulations that may carry elevated risks of oncogenesis. In this context, the identification and molecular cloning of tumour genes associated with cancer
susceptibility (Table 6 ) is also of crucial importance.
Studies on the latter have largely focused on the dominantly expressing genes of high penetrance, such as
RE, WT and APC, but the prospect of identifying lessevident genes of low penetrance, which may be more
frequent in the population, is probably of grcater
iniportance. In addition to increasing the knowledge ofo~~cogcnic
mechanisms at the human population level,such infor~natio~imay have significant social andeconomic iniplications.

205. During the last few years much progress has
been made in elucidating mechanisms of DNA-repair
and niutagenesis; some of these advances have been

206. The characterization of genes involved inmarn~nalianDNA repair and radiosensitivity may also
provide the means of identifying (or of generating

204. It will have become obvious that although someunderstanding of the mechanisms of radiation oncogenesis is beginning to emerge, the complexity of thewhole process is such that rapid progress on a broad
front should not be anticipated. Nevertheless, it should
be noted that the main component of understanding
has come within the last decade and owes much lo theapplication of modern methods of cell and molecularbiology. It may be that the best prospects for the
future lie in the strategic implementation of thesemodern methods, with particular emphasis on the full
integration of radiation oncogenesis research into thecontinually expanding field of cancer biology. A few
of the many potential growth points and future needs
in research on radiation oncogenesis are briefly
discussed in the following Sections.

A.

1MI'1,ICATIONS OF ADVANCESANNIX E: MECIMNISMS OF RADIATION ONCOGENISIS

5x0

cytogenctirs [P6] and it1 silrr chromosome hybridizathrough gc~leticmanipulatio~r) radiosensitive, DNAtion to detect pre-lcukacnlic conditions in radiationrepair deficient mutants of the mouse. The availability
exposed individuals.
of such mutant strains would provide a very powerfultool for deL7iled whole animal studies on the rela209. Future molecular approaches to the detection oftionship between DNA-repair and oncogenesis. Recent
studies imply that severe co~nbinedin~mur~odeficie~~tprc-neoplasia may lot l~oweverbe restricted to theeasily accessible liaemopoietic and lyalphatic systems.
defect in DNA(scid) micc may have an i~~llerent
In circunlstances wllere a small number of exfoliatem e ~ b o l i s mthat confers cellular radiosensitivity [B27,cells can be obtai~lcdfrom tissues, polymerase chain
F12, MIO]; mouse mutants of this type could provide
reaction techniques have sufficient resolving power tovaluable models for radiation oncogenesis studies. In
detect specific tumour-associated gene mutations, anda similar way, genetically manipulated mice (transthis approach has been employed to identify, forg e n i c ~ )carrying germ line copies of activated oncoexample, p53 gene mutations in cells present in thegenes (see, c.g. [HIS, H24, L26, M11, R20, V91) may
urine of bladder cancer patients [S44], as well as thealso have an important future role to play in understanding the mechanisms of radiation oncogenesis. In
BCR-ABL gene fusions in pre-leukacnlias [M43]. The
this context, genetically engineered murine models ofuse of such new molecular and biochemical markersof neoplasia clearly have great potential for the early
retinoblastonla and o f p 5 3 deficiency in micc may bediagnosis of neoplasia, thereby allowi~lgfor early andparticularly valuable [D14, W l l ] . Other so-called
more effective clinical intervention (see [M34]).
turnour suppressor gene "knock-out" mice will doubtless be available in the near future.
210. However, although the power and speed ofpolymerase chain reaction techniques may, in prin207. Recenlly, important advances have been made by
ciple, be sufficier~tto col~lributeto the evaluation of
the successful long-term repopulation of the haernothe consequences of human radiation exposure, such
poietic systems of scid irnmunodeficier~t micc withas occurred in the immediate vicinity of the Chernobyl
engrafted human haemopoietic cells [KB,M12, M131.
accident, it may be argued that there is, as yet,
These scid-human chimaeras may provide the means
insufficient fundamental knowledge to effectively use
for studying haemopoietic and leukaemogenic responsethetechniques as a screening procedure (see parain an experimental animal system. This generalgraphs222 and 223).
approach, i.c. the use of mouse-human chimaeras, may
become an iri~portantfocus of future studies on human211. Similar considerations apply in the case of autoradiation oncogenesis, in that it may allow the study
mated cytogenetic screening for stable cytogenetic
of a range of transplanted human cells in murine host
events, although here there can be no doubt that, if
tissues.
they had been available at the time, they would havegreatly
facilitated cytogcnetic analyses in atomic bomb
208. On a more practical note, two relatively recently
survivors
in Japan (see, e.g. [AS, B171). Despite thesedeveloped techniques may have application in thereservations,with increasing technical innovation, thedirect assay of radiation-associated somatic cellcollection
andniaintenance of archival neoplasticchanges in radiation-exposed human groups. First, amaterialfromradiation-exposed
human populations
novel technique now allows small samples of humanshouldbegiven
seriousconsideration.
The potential
blood to be assayed for the frequency of certain
benefit to knowledge that would accrue from suchsomatic mutations; this technique has been employed
coordinated procedures should not be underestimated.
to examine residual HPRTand glycophorin gene mutations in atomic bomb survivors in Japan [AlS, H14,
H49, L7].The technique may have broader application
B. hlETHODOLOCICAL AND[U13], particularly if it becomes possible to identify
CONCEPTUAL ASPECTS
gene-specific protein variants associated with haemopoietic neoplasms and to use flow-sorting techniques
212. Over many years, a substantial number ofon blood samples in the same way as recently
experimental animal n~odelsof induced neoplasia havedescribed for glycophorin A locus variants in bomb
been developed. In the long term these models will be
survivors [K9]. Secondly, a very powerful molecularmost informative if quantitative studies on induced
technique, polymerase chain reaction (PCR) (see, e.g.
neoplasia
are coupled with cellular, cytogenetic and[E8, F7, R1 I]), now allows rapid and specific genemolecular
studies of the nlcchanisms of induction. It
analysis in samples comprising very few cells
may,therefore,
be argued that where possible, murinc(conventional methods require much larger samples).
modelsofneoplasia
should be favoured, since for the
If specific genomic changes associated with thecytogenetics are available, amouse
well-establishedinitiation of human haemopoietic neoplasms aredetailed
genetic
map,
i~~fornlation
on nou use-man geneidentified, this tech~~iquecould, in principle, betichomologies,
informationonthe cellular andemployed in conjunction with new autonlatcd

93 REPORT

molecular aspects of dcvelop~nentalbiology arid anever-increasing number of relevant reco~nbinantDNA
probes. It is not likely that an equivalent data resource
will become available for another mammalian species.
This having been said, the rat offers considerable
advantages for the sti~dyof so~ilcnlodel neoplasms,
e.g. those of the lung. L h e r c such benefits ouhveightechnical difficulties, it is obvious that the biologicalrelevance of the ariirnal systc~nshould take priority.
213, The degree of conimitmcnt that is required toresolve oncogenic mechanisms in whole animal
systems is so considerable that aninial neoplasms
having the greatest relevance to human radiogenic
neoplasms should be emphasized. Detailed histopathological comparisons of both neoplastic arid pre-neoplastic conditions are crucial to tlle choice of suchanimal models. At present mechanistic studies areemphasizing induced leukaelriias and osteosarcomas,
and it is in these areas that the most rapid progress
may be anticipated. However, with a view to representing the whole problem, it will be necessary toapproach the broader task of uridcrstanding inductive
mechanisms for a range of solid tumours, such as
those of the breast, liver, kidney, lung and colon.
Studies using in vivo in vilro transformation systems
are already making an important contribution in thisarea (paragraphs 170-172), and a new dominant mutation predisposing mice to gastro-intestinal tract
neoplasms [ M Z ] may be of great value. Since thispredisposing gene in the mouse is the homologue ofthe human APC gene (colorectal cancer susceptibility)(5481, there is also the prospect of exploring genetically determined susceptibility to radiation
oncogenesis using aninial models. Recent findings
regarding rodent susceptibility loci for mammary[G23],kidney [W23], liver [B39] and myeloid [S43]neoplasms tend to heighten expectations in lhis
important area of research. The further cytogenetic andmolecular study of therapy-related neoplasms in man(see, e.g. (B38, R9, V5,W221) also holds considerable
promise and should be encouraged, particularly since
the approach may not only provide direct information
on early events in radiation oncogenesis but couldalso, in principle, contribute towards an understalldingo f human cancer susceptibility following radiation.
214. Of great importance is the prospect of being able
to bridge experimentally between species. One strategy
providing for such bridging would be to develop
further the organ-specific in vivo in virro approaches
to rodent radiation oncogenesis. These experimenlal
systems currently incorporate an in virro phase toreflect the cellular mechanisnis and quantilative
radiation responses that underlie the in vivo inductionof relevant radiogenic respiratory, mammary, thyroid
and bone neoplasms. If it becorncs possible to extend
the in vitro approach to hu~nantissues, perhaps usingtranspla~rtationto irnrnune-suppressed mice (paragraph206), then a direct mechanistic and quantitative cornparison between rodent arid llun~anin vitro phases
coultl be achieved. Such studies, together with quantitative estir~iates of lumour induction in the wholeaaimal, would then provide for direct extrapolation tomechanisms and risk in man. Existi~igrodent in vivoin vitro transforination systems, as well as providing
a potential for tlle identification of initiating events forh e neoplasm in question, have already highlighted the
crucial role of hornlonal and growth factor effects in
the inductive process. The experimental elucidationand qua~itificationof parallel effects in human tissues
would be of substantial importance. Thus, while it
may not be possible to realize the full theoreticalpotential of this strategy, it does provide a major goal
for future studies and one that unites in vivo and in
virro approaches to the problem.
215. One important methodological implication ofrecerit advances in cancer research concerns the limitations of current in vitro and in vivo models of induced
neoplastic change for determining the loss of specific
DNA sequences in induced tumours. Most of theexisting rodent-based animal models of tumorigenesis
utilize inbred or closely related hybrid strains ofanimals; similarly, animal cell culture systems fromthe mouse, the rat and the hamster are usually of
inbred animal strain origin.
216. It has been shown that in outbred humanpopulations, the loss of specific genes from tumours ismost easily detennined by the molecular analysis ofloss of DNA heterozygosity [S19]. Highly inbred
animal systems do not allow for this, since for any
given autosonial gene the two copies are, by definition, identical, and distinguishing the complete deletionof one of them is not straightforward. Thus, while forthe purpose of providing reproducible quantitative
estiniates of radiation-induced neoplasia it is logical toemploy inbred animal strains, this choice can be seen
to impose important niethodological limitations to theresolution of the ~nolecular mechanisms of oncogenesis. The extension of a selected number of animal
and cellular niodels of radiation tumorigencsis tocross-bred animals, while not a trivial task, now seems
to be essential. This will allow the gene deletion
nicchanisnl strongly suspcctcd of being involved inradiation oncogericsis to be approached not only inrespect to specific candidate genes but also using themore ge~ierallyapplicable new method of detennini~lg
loss of heterozygosity by analysing highly dispersedand polymorphic DNA repeat sequences, such as polypurine-pyrimidine tracts [C25]. It is notable that thiswhole approach is already being successfully imple~nentedfor ani~nalstudies of che~ilicalcarcinogenesis(e.g. [B27]). The couplillg of these techniques with therapidly improving techniques of fluorescence in sitcc

ANNEX E: hlEClLWISMS OF MDIATION ONCOGENESIS
hybridizatio~~(FISH) for single gene location and"painting" of chroniosonies jF18, L19] should further
enhance the prospects for resolv-ing niccha~~isrnsofradiation ,oncogct~csis at both the nlo]ecu]ar alld
cytogenetic levels.
217. 111spite of the optimistic note sounded abovc, it
is most likely that an increased understanding of themolecular mechanisms of oncogenesis will derive fromdetailed studies of neoplastic phenotypes induced
experimentally at relatively high total doses. To what
extent will i t be possible to provide specific commenton the quantitative aspects of tumour induction at the
low doses that are of prime concern in radiologicalprotection?
218. The principal problem in providing any answer
to this question concerns the great unccrtai~~ties
involved in judging the probability with which a single
neoplasia-initiated cell in tissue gives rise to a malignancy. As noted elsewhere, the current lack of understanding of the interactive factors that detemline thisprobability is such that cvcn a first approximate..
calculation is highly dependenta series
necessary biological assumptions. N e v e r t h c l ~ ~ its , isuseful to at least illustrate such an estimation.
219. The assumptions made are as follows:
the human body contains a total of 1014 cells;
between one and ten cells per 10,000 have stemlike properties and, as such, are potential targets
for induced neoplasia, i.e. 1 0 ~ ~ - 1 target
0 ' ~ cells
per individual;
the typical ncoplasia-initiating event ccntrcs on
single gene inactivationfloss mutations in one often possible genes in target cells;
the average acute low-LET induced mutation rate
(per ccll) for these genes is similar to thatobserved. in v i m fir the HPRT gcac i.e.
-10.' G ~ - ' ;an acute low-LET exposure of 1 Gy to a population generates a 10% cxcess risk of n~alignancy,
i.e. one excess tumour for ten exposed
individuals;
1 Gy of low-LET radiation gellerates 1,000
eleclron tracks in each cell.

591

a target cell will ive rise to an excess malignalley is l0-'~-10*I§ (i.e. 1 0 - ~ ' - 1 0 -t~ 1,000).
~

1" rcsl)cct to low-LET background radiation of, say, 1
mGy per year, cacl~ccll will receive, on average, oneback i~~tcrscclionI)cr year. Thcrcforc:
the probability per car of malignancy will bearound 1 0 ' ~1i.c. 10' -10" (target cells) x 10-1410." (probability of malignant conversion)];for 5 0 years of exposure this gives a lifetime
risk of 5 x loe3 (50 x lo4), i.e. 0.5%;
assutning a natural cuniulativc frequency ofn~alignancyof 20%, then 1 in 40 (20% + 0.5%)
cancers in the population are due to low-LET
natural background radiation.

6

I t should be noted that the illustrative calculations
above take no account of dose and dose-rate effects
for low-LET radiation and that (c) above does notdepend on the assumptions made in respect to target
cell numbers or on the probability of malignant
conversion.

220. The realism of such calculations is, however,highlydepending as it does on a seriesof biological estimates of frequency, each of which
may be incorrect by an order of magnitude or more.
However, the exercise points out some questions thatneed answers from rescarch in cellular and molecularbiology if the problenis of estimating risk at low doses
arc to be solved. The most important questions centre
on (a) the number of target cells in different tissues;@) the number of relevant target genes in cells (thismay well be dependent on cell type and tissue); (c) the
mutagenic mechanism of initiation and the induced
mutation rate for different target genes: (d) thc
mechanisms governing the probability that a given
initiated cell will progress to full malignancy.

221. Further to this, epidemiological observations ofconstant relative cancer risk over time in the atomic
bomb survivors i n Japan F20], together with theexperimental data outlined in this Annex, imply thatradiation acts prirlcipally at an early phase inoncogenesis, probably at the initiation stage. Thus, forexcess cancer in an irradiated population, theefficiency with which the initiating- mutation is
induced [nay be a rnajor factor in the contributions
It may then be inferred that:specific neoplasms make to that excess. In this respect,(a) one excess malignancy occurs within 1 0 ~ ' - 1 0 ~ ~ Ihe absence of evidence for any measurable excess oftarget cells receiving 1 Gy (i-e. 10 x 1 0 ' ~ - 1 0 ~ ~ ) : chronic lyniphocytic leukaemia (CLL) might be
explained by a s t r i ~ ~ g erequirement
r~t
for CLL initiation(b) the rate of target gene inactivations with ten
bygene-specificchromosonietranslocatior~,
which haspossible target genes per cell is l o 4 G ~ - (i.e.
'i~~volved
in
the
early
development
been
shown
to
be
10 x
of some human T-cell neoplasms [R3].It has been(c) the number of initiating rnutations within theseargued in paragraphs 74 and 75 that such events
tar el cells g i v i ~ yrise to a single nialignancy ispresent very small r~lolecular targets for radiation
10 -lo8 (i.e. 10 r 1 0 ~ ~ - 1 0 " ) ;action,and, on this basis, the T-ccll lcukaen~ogenicof(d) the probability that a single track interscctio~~

g

592

UNSCEARpotcnti;~l of radialion migl~tbe expected to be low.
Expcrimeatal evidence for tbc i~iductionof BCR-ABL
gene fusion by high dose x-irradiation has, however,been prese~~tcd[13]. Otlicr cxpla~~ationsfor this
cpidcmiologicel observation are also possible. For
cxamplc, a high sensitivity of T-cell targets toradiation-induced apoptotic death might efficiently
rcrriovc i~~itiatcd
cells fro111 irradiated target T-cell
populations: an c x p l a ~ ~ a t iofo ~this~ general for111 hasrecently bccn offered in respect of t l ~ edifferential
cancer iricidcncc in the large and s~tiallbowel [P15].
It is also important to consider whclher cellular andniolecular techniques have tile po~cntialto distinguishradiation-induced neoplasms from those arising
sporrtaneously or from otlier fonns of carcinogen
exposure.
222. As noted in paragraphs 188 and 189, thespecificity of marry gcnotoxic chernical agents in theirinteraction with nuclcic acids may provide acharacteristic mutational signature in DNA, which
infers that cellular exposure to this agent has takenplace. In cascs where target genes for neoplasia
induction have been identified it thus beconles
possible to catalogue the DNA basc sequence of suchmutant genes in hurrian tun~ours,with a view to
identifying characteristic DNA base changes indicativeof the action of agents of known mutagenic andcarcinogenic potcnti;~l.

223. Inactivating niutations in the p.53 turnour
suppressor gcne are s t r o ~ ~ g l suspectedyof being
involved in the early development of a wide range ofhunian solid tumours, arid recent analyses o f p 5 3 genes t ~ c t u r e in hunian carcinonias are beginning toprovide evidence that turnours of divergent aetiologies
arising at various sites do riot exhibit the same spectraof mutational change to this gcne [C31, H30, H46]
(Figure IV). Characteristic p53 mutations are mostevident for hepatocellular carcinoma associated withenvironniental exposure to aflatoxin B1 [B23, M34,
0 4 1 and skin tumours associated with sunlight
exposure [B36], but within this rapidly developing
field of molecular epidemiology (see also [M34]),
parallel advances in rclatiorr to other che~nicalcarcinogens arid other target gcnes should be
anticipated.
224. Ionizing radiation docs not, however, show thesame degree of mutagenic specificity as chemical
agents and, on currcnt evidence, it may tend to act
principally through DNA dclction/ rearrangement
rather than basc pair change. Nevertheless, in a recent
study [VlO] rare p53 gene d e l e t i o ~ ~ands base pairchanges wcrc shown to characterize radon-associatedlung canccr in uranium miners; it may be that thisunusual mutatiosal spcctrum is associated with alphaparticle-induced damage to tl~ep.53 gene of target

respiratory cells. Mut:~tionsof t l ~ cpS3 gene have also
been cliaracterizcd in radiation-associated huniansarcotrias [B38], a ~ l dK-ras mutation has been shown
i n radiation-associated follicular
to be lliore frccluc~~tcarciaooias of t l ~ c thyroid lllan i n sponhncously
arising ~rcol)las~ns[W22]. In ncitbcr a s c , however, isit possible to be certain dl:~t thcsc ~nutntio~iswerespecifically i~rduccd by t l ~ cradiatioe. Even greater
uncertainty surrou~ldsthe iatcrprctation of the MOS
gene poly~norphisms in n o n ~ ~ itissue~lof radium
exposed i~~dividuals[H471.

225. The niolccular characterization of DNA base pairchanges in tumour-associated gcnes using polymerase
chain reaction (PCR) as employed in many of theabove studies is a flexible, rapid and reasonably
straightforward procedure [ES]. In contrast, themolecular characterization of DNA deletions,particularly tl~ose that extend outside the gcne ofinterest, is niorc difficult, because it demands adetailed knowledge of gene and gene-flanking
sequences. While small intragcnic DNA deletions willtend to ericonipass or disturb the reading frame ofthose sequences encoding fu~rctionalprotein sites, fordeletions resulting in loss of tllc whole gene there is
no apriori reason why the deletion breakpoints should
be characteristic of that gene or of the mutagenic
agent in question.
226. There is, however, limited evidence thatradiation-induced DNA deletions in mammalian cells
may involve specific breakpoinu. Whether such
deletion breakpoiuts rnight be characteristic ofradiation-induced damage remains an open question,
but the presence of DNA repeat sequence motifs atsonic DNA sequericc breakpoints in somatic cellmutants [M33] and, albeit less certainly, in someneoplasia-associated chromosomal changes [B16, B32]
does allow for this possibility. However, on current
knowledge, it seems highly u~llikelythat DNA eventsof this or other types will be unique to radiationinduced daniage (see, e.g. [A14, M331); if so, this willtend to limit the general utility of niolecular analyses
between radiationfor the purposes of discri~rii~~ating
induced, chcnrically irlduced and spontaneously arisingneoplas~us in 11umn11populations. Nevcrthclcss, therate of progress i n h i s wl~olearea is such that at
prcsc~lt it is prudent to rcscrve judgcnient on this, unusual p.53 mutation
crucial issue. I ~ ~ d c c dt11c
spectrum observed in Uic lung tumours of uranium
millers [VlO] hints at the utility of this approach inrespect to the p53 gene.
tl~eidentification of
227. From a predictive vicwpoi~~t,
initiating e v c ~ ~ tfor
s radiation orrcogcr~csis is not
limited to its ~)olcetial to discri~ninate between
tu~r~oursof i~lductiveand spontalleous origins. Even if
it only becomes possible to idc~itifyin a more general

ANNEX E: MECIIANISMS OF RADIATION ONCOGI:NL9IS

593

fashion the principal categories of i~lduced DNA
damage and target genes associated with radiation
oncogenesis, there still remain important inlplications
for future research.

possible to link the induction of the initial, potentially
oncogcnic damage with specific energy loss events in
DNA, U~us allowing a biologically more realisticn~icrodosin~etric
extrapolation of radiation effects.

228. First, the provision of such data may allow thedesign of quanti~ativclyreliable cellular systc~nsthat
mimic or reflect the relevant ~nolccular event...
Secondly, it would become possible to specifically
explore the post-irradiation repair of the relevant initial
DNA lesions (see [WlO]), particularly if these were to
involve some form of gene or DNA sequence specificity. Thirdly, using Monte Carlo simulations (see, e.g.
[N22]) of radiation track structure, it ~iliglltbecome

229. However, as noted irr paragraph 5, a resolutionof the mcclianis~ns of radiation oricogencsis would
also providc an essential input into the whole field ofcanccr research. It is only through such integration thatthe whole of the complex multi-stage oncogcnic
process will be better understood, aqowing thc
formulation of realistic cellular and molecular models
to replace, or at least complement, the existing
empirical approaches to wrlccr risk projection.

CONCLUSIONS
230. Ionizing radiation induces a broad spectrum ofneoplasms in both man and experimental animals. The
basic mechanisms of the induction, pron~otionand progression processes are not yet well understood; however, some points from considerations in this Annex
can be summarized.

231. Point mutation, chromosomal translocations anddeletions may all play roles in the initiation andprogression of neoplasia. Some of these changes areshared by different neoplasms. Others appear to be
restricted to certain tumour types.
232. Studies on hurnan susceptibility to neoplasia
associated with inherited defects in DNA nietabolism
or the loss of tumour suppressor gcnes are making
important contributions to the understanding of oncogenic mechanisms and the different modes of inhcritance of cancer-proneness in the human population.
233. Neoplastic initiation as a consequence of specificsomatic mutation is thought to provide target cellswith some degree of proliferative dysrcgulatio~~.
Thcsc
events may not be phenotypically expressed as prencoplastic conditions until a promotional proliferative
signal is received.

234. Point mutations or chromosonial translocations
that activate proto-oncogenes or mutations that lead toloss of function of turnour suppressor gcnes may bc
considered as potential initiating events for oncogenesis. Relative target sizes for the illduction of these
events by radiation would tend to favour tunlour
suppressor genes as the most radiosensitive targets.
235. Strong evidence for tumour suppressor genes astargets for oncogenic initiation has been obtained instudies of germ-line mutations that predispose to
canccr. Some of these genes appear to play a central
role in the control of the cell cycle.

236. Neoplastic promotion arises mainly as a consequence of induced changes in gene expression ininitiated cells. It may occur through the action of
specific chemicals, honnoncs and growth factors oncell surl'ace receptors, and the ensuing proliferative
responses may favour the establishment of pre-neoplastic clones in tissues. In some cases the enzyme
protein kinase C is thought to mediate promotional
processes; changes in intercellular communication may
be an important aspect of promotion, but the inductionof endogenous DNA damage is also a feature of somestrong promoting agents.

237. Neoplastic progression is a complex, multifaceted
process that appears to involve a series of subsequent
genetic changes within the evolving pre-neoplastic
clone of cells; these changes may include changes ingrowth rate, growth factor response, invasivcness andmetastatic potential. The development of intrinsic
gcnor~licinstability in neoplastic cells may providc thecellular heterogeneity in tumours that drives the
progressional process.
238. Both physical and biological factors influence
radiation oncogenesis, as shown in experimental aninial systcnis, but few specific details of cellularnicchi~nisn~shave eniergcd from quantitative studics.
l n virro cell transformation studics have highlighted
some aspects of cellular oncogenic mechanisms (frequency of potentially initiating events; effects of LET,
dose rate and repair, and neoplastic promotional
mechanisms). While in vitro studies on specific genei n v o l v c ~ ~ ~ einn tradiation-induced cell transforn~ation

594

UNSCEAR 1993 REPOR'T

have yet to yield detailed infomiation on the niolecularmechanisnis of oricogcnic initiation by radiation, rioveltransformation systenis, already making an impact inchemical carcinogenesis, show considerable potential.

239. Cell niutagcncsis and DNA repair data may be
used to argue that oncogcnic initiation following
ionizing radiation may occur more frequently throughDNA rea~~angcmentldeletion
than through point mutation, but this may well depend on the gene in question. Chromosonies or cellular oncogenes have beer1
shown to be changed in a number of radiation-induced
experimental neoplasms, but the temporal position androle of most of these changes are uncertain. In thecase of murine acute myelogenous leukaemia, theinduced initiating event is thought to be a spccificchromosomal rcarrangemcnt/dclction leading to theloss of critical genes. In contrast, other studies suggestthat point mutations in ras proto-oncogenes may beinitiating events for radiation-induced murine thymic
lymphomas. Some progress is also being made in themolecular characterization of radiation-induced osteosarcomas and turnours of the skin, breast and lung.
240. There are a number of mechanistic differences
between chemical and radiation oncogenesis, including
differences in DNA damage induction/repair and theactivational/degradative processes that influence the
carcinogenicity of many chemicals. An important difference in oncogenic niechanisms may derive fioni therelative efficiency with which chemical carcinogens
and radiation induce point mutations. It is suggested

Ihat radiation and chemical oncogenesis arc subject todiffering degrees of hunian genetic variation.

241. Molecular studies with certain hunian tumours
have drawn alterition to lllc possibility Illat spccificpoint niulntion in tuniour geries may serve as a signature of prior exposure to chenlical carcinogens. On
current knowledge, the same approach may not be
equally inforn~ativefor ionizing radiation exposures,
but tl~e possible preferential involvemcrit of DNA
repeat sequences in radiation-induced DNA deletionand of certain DNA bases in point niutations may
provide a focus for furthcr study.
242. The development of new methods of investigation promises further advanccs in understanding. Novelin virro cell transformation systems arid cellular/
niolecular studies with these and with neoplasms
induced ill outbred animals [nay be a productive area
for the study of oncogenic mechanisms. Recent
advanccs in the corislruction of transgenic andchimaeric mice, new cellular cytogenic and molecularapproaches to the assay of in vivo somatic cellchanges, and studies on radiation-associated humantumour cells appear to have great potential.
243. In order to take advantage of modem methods ofcell and n~olecularbiology and to anticipate further
technical advanccs, turnour material obtained fromradiation-exposed hunlar~ populations should be
systematically stored. This material may prove to bea very iniportant resource for future molecular studies
of oncogcnic mechanisnis.

A N N M E: MECHANISMS OF RADIATION O N C O G E N F S I S

595

Tahle 1T h e L ~ n c t i o na n d c h r o n ~ o s o r ~ i lto~cla t i o n o f s o m e h u t n a n o n c o g e n e s[A13, U 1, CI, M36, S63]

Clmifia~ion

C n m h factor

Chromatoma1 lororion' YS
f 1 s .1

22q 12-13
l l q 13l l q 13

PDGF P chain
FGF family
FGF family

7q 12-13
17q 21

EGF receptor
EGF r a p l a - l i k e
CSF-I reccptor
SCT recepta
Exprascd in nerve cells
Expressed in spleen ~ n braindExprascd in ganulocyt&mocyia
Bound to T c d l reccpor
Bound lo C D 4 m canplex
olaemcric protein (CML)

INT-2

Tyrohinc kinarc

FHB BIERB B2INEU
FhfS
NT
S3C

YES

FPYFESFYNLCKBCRIABL

Sainehhrecnine kinaw

GTP tinding p r d d n *Regulatory factas

OchersI'roduc~lfuncrion"

Oncogcnc% 34
4q 11-12
20q 12-13
18q 21
ISq 25-26
6q 21
Iq 32-35
2?qlyr)qN.UOS
RAFIMIL

4 11

II-RASRRAS
N-RAS

l l p 151
12p 12 1
Ip 13-22

Message tnnductim, muutcd in many tumarr t j p a

JUN
FOS
MYB
MYC
N-MYC
ERE4
PMLIRAm
HFLIEM
PRYIEM
H0.W Il p 31-32
14q 21-31
6q 22-24

2p 23-24
17q 11-21
15q 2U17q 11
17q 2U19p 13
1q 23/19p 13
10q 24

Binds Far (tranmiption faam)
Binds Jun ( ~ n n t a i p t i mfactor)
Erprused in uythmblasts
Tnnrcripdmhcpliatim factor
Amplified in ncuroblastorna
Thyroid hormone reccp~or
Chacmnic protdn (hnrcription factor)
Chaemeric protein (tranrcription b a a )Chaemeric protein (tranrcription factor)
Tmnsoipdon factor

BCL-2

18 q 21

3P 25

4 24

GTP: guanaine triphospbte; PDGF: platcladcrived govnh factor; FGE fibroMast go*
factor; SCF: stern cell facta; CML chronic mydogenour leukaemia.

Oocycc matuntim
Fuaccion downstram c€ RAS

Amplified in neurobladoma

G n v o l of apoptosis
f a a a ; EGF: epidermal g r w h facta; CSF: colony stimulating

UNSCMR 1943 REPORT

596

Table 2
Suppmsor genes In hun111ntunlours
lW141
Derccred by ccll hybZzarion or chromamme trms/rr
Tumour type

Chromaromal location

IP
3~
6
Y

11

Ncurdartoma
Renal carcinanr
Endometrial a r d n a n a
Endometrial u r c i n a n a
Ncuroblastornn; con'cal urcinann; Wilms' tumwr

Dcrrcrd through l n x of lulcrayp&y or direct mdeculnr probing
Chrommomal locarion

IP
Iq
3~

%
'h
1 9
1 1 ~
1lq
13q
17p
17q
18q
2-q

Tumwr ~ p eMelanoma; multiple endocrine acoplrsia type 2; newoMastana; medullary thyroid
carcinoma; pheodvcinocytanr: d u a l d l a r c i n a n a
Breast cardnonu
Small cell lung arcinoma; rdcno o r c i n a n a of lung: cervical carcinoma:
von flippel-Lindau disease. renal a l l caranomn
Familial a d m a n r t a u pdyposis; cdotwtal cardnana
Dladdcr carcinana
A s t r c q t o m ; multiple endocrine !m~pladatype 2
Wilms' tumour; rhabdomycaarcanr; h a carcinoma: heparoblastoma;transi~icnalcdl Madder a r c i n o m , lung carcinoma
Multiple endocrine neoplasia type 1
Rctinoblastoma; ortcorarcoma; m a l l cell lung mrcinann; ductal brcast carcinoma;
stomach carcinoma: Madda u r a n a n r ; d o n carcinoma
Small d l lung cardnoma: cdaecul c a r a m ; b l e s t a r d n a n a , ortcourcana;aslrocytonu quamolu cdl lung carcinoma
Neuroftbranntais type 1
C o l a e a s l carcinoma
New&~branatuistype 2; mcningioma; acoustic ncuromn: phmchromocytoma

ANNEX E: MECllANlSMS OF RADIATION ONCOGENEIS

597

Table 3
Consistent chron~oson~lllchanges in l c u k ~ i c r ~ ~undl u s lyrnphonl~is

IS451

I

Neoplarm

Chromarome aburwion

lri~kermim(hronic myelad leukntmia

1(%22)(q34:q I I)

Acute myeltid lcukacmia
MI
M2
M3
M4 uith ahonnal easinophils
M Sa
M1. M2 M4 with inncasui tssophilsMI, hQ M4, MS, M6t(9;22)(q34:ql I)1(8;21Xq22;q22)t(15; 17)(q22;q12)
inv(lb)(p13q22)1(9;1l)(p22;@3)
1(6;9)@U:qW)
hfmorany S/dd(Sq)Mmasony 7Md(7q)Msany 8Olronic l y q h c y i i c leukacrnia

1(ll:l4)(q13;q32)
Trisany 12

Acute lymphocylic lcukaernia

1(9;22)(q34;qll)((4; 1l)(q2 1;q23)

Aculc B e l l leukamia

1(~:14~~24;~32)
1(2;8)@12q24)t(8:22)(q24;q11)

Acute T a f l leukacmia

inv(lJ)(qllq32)t(1J: 14Xq l l;q32)(8: 14xq24;q 11)1(10;14)(q24;qll)t(11;14)(p13qll)-

Lymphomas
Burkilt's lymphomat(8: 14Xq24;q32)
1(28Xp12;q24)1(8;22)(q24;ql I)

Small non-dcavcd cell lymphoma, large d l immunoMastic lymphomat(8; 14)(q24;q32)

Follicular mall dcavcd cell lymphoma((14; 18)(q32;q21)

Small cdl lymphocflic lymphana

Trisany 12

Small cdl lymphocy(ic transTormed to diffuse luge cell lymphoma((11;14)(q13;q32)
Pdycylhermia v c n

Pdyqkacmia v a akI(209)

UNSCEAR 1993 REPORT

598

Tnble 4

Consistent c h r o n l o s o n i ~ r lc h n n g c s In s o l i d tumours
IS451
Ncoplarm

Chromnromc n k r t u i m

Alveolar rhabdomymarcornat(2;13)(q37;q14)

Bladdn carcinomn

Struaural changes or 1i(5p)Structural changes or 11

B r a r t carcinoma

Structural Aanges or 1
l/dd(lag)

Euing'r u r c u n * Akin's tumourheurapithdiana[(I 1;22)(q24:q12)

K h c y caranma

I/dd(3)(p11-21)t(5; 14)(q 13;qZ)

Luge bou;el cancer

Structural Aanges of 1
Triray 7
Structural changes of 17

lipomal(12Xq13-14)

Malipant rnchnana

Udd(l)(p 12-22)t(1;19)(q12;p13)t/dd(6p)h(6p)Trisany 7

Mcningianr

M m m a n y 22

Mixed mlivary gland adcnorna

1(3)(~21)~/dd(R)(q12)t/dd(12)(q13-15)

Myxad lipmarcomt(12;16)(q13-14;pll)

Neuroblastanadel(lHp31-32)

Ovarian a r a n a at(6; 14)(q21;q24)
Structural c h m g a of 1

P r c r ~ t i cm a n a n a

dmiq22)dcI(lO)(q24)

Rainoblutana

Structural changes of 1i(6p)dd(13)(q14)/-13

Small cdl lung aranomadeI(3Xp14p23)

Synovia Yleomat(X;18)(pll;qll)

Tcrticular tnatormlrcminomai(l2p)

Uterine cudnoma

Struaural and numerical changes of 1

Wilm' tumour

Structural changes or I
l/dd(ll)(plf)A N N E X E: MECIIANISMS O F RADIATION ONCOGENESIS

599

Tnble 5
V i r u l t u s o c l u t l o n s In l i u n t r l n oncogcnc-rls

IW161

v;ra (VP~)

Orher risk fi~rturs

Assarialad rumours

Tcell viruses (RNA)

Tccll IcubemiaAympham

Imtivirwcs (WNA)

Kapori'r sarcoma, lymphoma

Concurrcnl viral infections
Lnmunc deficiency

I3urkiti's lymphma
lmrnunoblas~iclymphNuopharynpal carcinoma

Kapori's sarcoma (7)G r v i d neoplasia (7)Malaria
Immuoc deficiency
Dietary compmentr
gendYpe
Papillana riruxs, l d x c c o
Immune dcfidcncy
1Il-A g a d Y F

uIlcpatitis B v i ~ s (DNA)Liver anccr

Analoxin. alcohd. tobacco

Papillana viruses (DNA)C u r i d and anal ntoplasia
laryngeal carcinoma
Skin carcinoma

T h c c q herpa vim and immunc ddiciacy
X ray& tobacco
Sunlight. pnctic fadors influencing skin pigrncnmtim

Ilcrpcs virw (DNA)
Eprt Jn-Barr virus

Herpu simplex
Cylanegdovirur

G r d d neqlasia (7)-Table 6
Es1inintc-r of t h e f r e q u e n c y of some c a n c e r - p r o n e h u m a n n i u l a t i o n s

Mutarion
Ataxia-telangiectasia(homozygotes)
Ataxia-telangicciasia(betero zygotes)
Retinoblastoma
Wilms-aniridia
Basal cell nevus syndrome
Neurolibromatosis
Familial adenomatous polyposis
Non polyposis colorcctal cancer
Familial brcast cancer
Li-Fraumeni syndrome*

Principal neoplasms

Phc~~otypicma~~+sfafio~~

Chromosomal
locatio~t

Frequency perlive births

Leukaemias lymphomas

Autosomal recessivelllal- 1 per 100.000

Mammary carcinomas

Autosomal recessive

1 l/al1 per 1 0 0 "Rc~inoblastoma.ostwsarcoma
Nephroblastoma
Skin carcinoma. medulloblastoma
Neurofibromas. C N S tumours
Colorcctal c a r c i n o m aColorectal carcinomas
hfammary and ovariancarcinomas
Wide range o f malignancies

Autosomal dominant
A u ~ o s o n ~ da ol m i n a n ~Autosomal dominantAutosomal dominantAutosomal dominantAutosomal dominantAu~osornaldominant

13q
I ~ P

Autosomal dominant

17p- I per 20.000- 1 per 30.000
50.000c 1 per9q- 1 per 5,000- 1 perb 10.000

17q

5q
ZP
17q

C

c 1 per 50.000-'Ihc number and location d ataxia-tclangicctada genes has ye! to be conclusively dacrrnincd; the numkr of gcnctic wmplcmcnlaion groups in rtadstclangiectoda is the maja delerminant d hcterozygde Irqucncy.
Accounts for around 14% of cdorcctal cancers in the populalim.
It has been a l i m a i d that sr many as 1 in 200 wanen carry one d a number of gcnu lhpl predirporc hear1 can= ( p r a p s p h 127).

APC andlor MCC
gene mutation

Meth I gmu
losses &om

DP~A

DCC gene

RAS genemutalionmutallon

Olherchromosome
loss&

pS3 genemutation

ANNEX E: MECHANISMS OF RADIATION ONCOGENrSIST'

U f N f W IMPORTANT METASTASIS
7

Figure 11.
Posslhle steps Involved In the metastauc spread of solid turnours.
IH421

ml

602

UNSCEAR 1993 REPORT

Unaffcclcd male
AlTec~ed male
Carrier male0@

Unaffecled fcmnlc
N f c c ~ dlemalc
Carrier femalc

Figure 111.
Idealized pedigrees Tor maternal and paternal gcnonlic imprinting.
r~191

Figure IV.
The dktrlbullon or pS3 tunlour cell gene mutations In man observed in a wide range of human neoplasms.
The hodzonhl axls represents the 393 condons or lhe p53 gene rron~ the NH2 l o Ule CO OH k m i n a l s .
P-131

ANNEX 1::

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