Recent evidence for oncogenesis by insertion
mutagenesis and gene activation

FAROLD E. VARMUS

Francisco, California 94 143

Depaflment of Microbiology and Immunology, University of California, San

I Introduction 309

11  Identifying potential cellular genetic contributions to oncogenic mechanisms  31 0

1 Cellular homologues of viral oncogenes 31 0
2 Cellular genes that transform cultured cells 31 2

In Oncogenesis by viruses lacking onc genes 313

Keywords: Retroviruses, Proviruses, Viral oncogenesis, Oncogenes, Inser-
tion mutagenesis, Transformation, Avian leukosis, Enhancement of
transcription

-

[Summary

ative cellular oncogenes have recently been identified by their homology
h known viral oncogenes and by their capacity to transform the behaviour
cultured cells. Retroviruses lacking oncogenes may induce tumobrs by

or activating such cellular oncogenes. Analysis of such tumours
novel mechanisms for modulating expression of eukaryotic genes,
es new candidate oncogenes, and suggests possible mechanisms for
nesis by non-viral agents.

Introduction

r many years, tumour virologists paid lip service to the possibility that the
  soma1 sites at which viral DNA integrates might have a determining
ce upon the neoplastic process. Until recently, this notion has had few
nts, for several good reasons. (i) Insertion of viral genomes into host
eemed most likely to affect host genes by disrupting them; it is difficult
envisage a cancer arising from the inactivation of one gene in a diploid
  . (ii) Although more promising consequences of viral insertions were
  e (e.g., gene activation, creation of a hybrid gene, or inactivation of a
  a locus for which the host was heterozygous), such speculations were

pported by experimental observations. (iii) The integrated (proviral)
ms of DNA and RNA tumour viruses appeared to be inserted randomly in
                         Cancer Surveys Vol. 1 No. 2 I982

310 Harold E. Varrnus

the host genome, reducing the likelihood of introducing viral DNA at the
presumably rare positions conducive to oncogenesis. (iv) Mounting evidence'
for transforming genes (oncogenes) carried by many DNA and RNA tumour
viruses seemed to obviate the need for more complicated and unsubstantiated
mechanisms of viral oncogenesis.
Events of the past couple of years have refocused attention upon her-
tional mechanisms for viral oncogenesis and have provided some specific
experimental reagents with which to pursue the idea that many types of
cancer of unknown cause-not only certain virus-induced cancers-might
have their roots in the disordered expression of host genes. My aim in this
brief review is to provide the nonspecialist with an understanding of the
experimental findings that have contributed to the current viewpoint.
Two overlapping areas of study are central to the ensuing discussion: (i)
efforts to define cellular genes whose altered expression might contribute to
tumour formation and (ii) efforts to decipher the mechanisms by which
tumours are caused by RNA tumour viruses (retroviruses) lacking their own
oncogenes.

II  Identifying potential cellular genetic contributions to oncogenic
  mechanisms

At least two classes of host genes can now be considered candidates for some
role in oncogenesis-a group of genes identified by their homology with
retroviral oncogenes and another group identified by their capacity to induce
transformation of a cultured mouse fibroblast cell line.

1

The first of these groups came into view during a search for the origins of the
genetically defined transforming gene (src) of Rous sarcoma virus (RSV).
Molecular hybridization reagents specific for src annealed to normal cellular
DNA from the natural host for RSV (chickens) and to DNA from all other
vertebrates, suggesting that the homologous cellular sequences had been rela-
tively well conserved throughout evolution (Stehelin et al., 1976; Spector et
aI., 1978a). Tests for RNA and protein products of the src-related sequences
demonstrated that they constituted a competent coding domain normally
expressed at a low level (Spector et al., 1978b; Collet et al., 1978; Opper-
mann et al., 1979). Additional tests for genetic linkages and polymorphisms,
and for the structure of the domain, indicated that the src-related domain was
a structurally conventional cellular gene (now called c-src), with multiple
introns, little or no variation among members of a species, and no apparent
linkage or resemblance to the endogenous proviruses that inhabit the germ
lines of many animals (Hughes et a1 ., 1979a, b; Parker et al., 1981; Shalloway
et al., 1981).
Subsequent studies of a large number of other RNA tumour viruses cap-
able of transforming cultured cells and inducing tumours rapidly in animals
have unearthed over fifteen distinct viral oncogenes, each of which is closely

Cellular homologues of viral oncogenes

Oncogenesis by insertion mutagenesis and gene activation

31 1

[elated to (and presumably derived from) a cellular gene (Bishop, 1981;
/Bishop and Varmus, 1982). For convenient communication, these genes have
n generically labelled `v-onc's', the viral oncogenes, and `c-onc's', their
ular counterparts (Coffin et al., 1981). Each member of these classes has
n assigned a trivial name (e.g., src, myc, mos; see Table 1) according to
cently published rules. We may be approaching the limit of the number of
c's that will ultimately be identified, since two related sets of oncogenes
andfps, bm and rus) have been isolated in viruses arising in different host
ecies (Shibuya et al., 1980; Andersen et al., 1981); moreover, some
  enes have appeared repeatedly in independent virus isolates within the
  pecies (Table 1). Nevertheless, there may be additional cellular genes
  lar potential that, for one reason or another, are not susceptible to
sduction by retroviruses. Other means will be required to identify those

  account of the functions and products of the several c-om's is beyond
   view of this short essay (see Bishop and Varmus, 1982). For our
  ses, it is sufficient only to outline the evidence supporting the simple
  nce that at least some cellular homologues of v-onc's do themselves
  ncogenic potential. Such potential could be achieved in two ways: by
ations that alter the nature of the gene products or by modulations of
  expression that raise the level above a threshold required for neoplastic
cts. For at least two c-onc's (c-mos and c-rus), the latter possibility has
  found sufficient by using a strong promoter of transcription to enhance
  ssion of the cellular genes (Oskarsson et al., 1980; Blair et al., 1981;

able 1. Cellular sequences identified as probable oncogenic sequences in the genomes of

nc Number of
quence virus isolates Example

Animal origin

Rous sarcoma virus (Prague strain)     Chicken
Fujinimi sarcoma virus Chicken
Y73 sarcoma virus Chicken
UR-2 virus Chicken
Avian myelocytomatosis virus-29 Chicken
Avian erythroblastosis virus Chicken

(v-onc's) have been found in the genomes
mologous to host sequences (c-om's) with
been assigned to the onc sequences (Coffin
virus isolates containing each onc sequence,
m which the onc sequences have apparently

312 Harold E. Varmus

DeFeo et al., 1981). These experiments required the molecular cloning and
subsequent joining in vitro of two components: a cellular oncogene and a
region of retroviral DNA, the long terminal repeat unit (LTR), Which
encodes a strong promoter. When reintroduced into cultured cells, the `acti-
vated' c-onc's are capable of transforming them to a neoplastic phenotype.
Earlier experiments indicated that c-src could contribute at least partially to
the production of an onhgenic protein, since competent transforming viruses
could be isolated from tumours induced in chickens with deletion mutants of
RSV lacking part of v-src (Hanafusa et ai., 1977). Biochemical studies of the
recovered viruses suggested they were generated by recombination between
c-src and the defective v-src (Wang et al., 1979; Vigne ef al., 1980; for a
dissenting view, see Lee et al., 1981).
It should be emphasized, however, that for most c-om's there is as yet no
direct evidence for their oncogenic potential; in these cases, the structural
differences between v-om's and c-om's may prove to be critical determinants
of function. Even in the provocative situations considered below, in which
c-onc's are activated in tumours induced by retroviruses that lack V-om's, it is
premature to conclude that the enhanced expression of a c-onc is directly
responsible for tumour growth.

2  Cellular genes that transform cultured cells

The second class of putative cellular tumour genes has been defined by using
mouse NIH/3T3 fibroblasts to assay the transforming potential of naked
DNA from a variety of sources, including chemically induced tumours,
tumours induced by viruses lacking om genes, spontaneous tumours, and
normal tissues (Shih et al., 1979, 1981; Cooper et al., 1980; Cooper and
Neiman, 1980; Lane et al., 1981). Although the efficiency of transformation
is quite low, DNA from the transformed cells can be used, in turn, to trans-
form fresh cells; the transforming elements can be classified with respect to
their susceptibility to restriction endonucleases or their linkage to highly
repeated cellular sequences (Shihet al., 1981; Murray et al., 1981; Perucho et
al., 1981); and, in a few cases, the transforming components have been
molecularly cloned in prokaryotic host-vector systems (Goldfarp et a1 ., 1982,
G. Cooper, R. Weinberg, personal communications). Although little is pres-
ently known about the structures, products, and normal functions of these
putative oncogenes, they are likely to be genetically altered in the tumour
cells from which they are derived, since DNA similarly prepared from normal
cells is inactive in the transformation assay. As in the case of c-om's and
v-om's, it is for the dost part uncertain whether the important differences
reside in structural or in regulatory domains. The observation that the shear-
ing of DNA from normal cells may confer transforming potential upon it
suggests that regulatory changes may be more important, since the shearing
might remove the restraining influence of cis-dominant controlling elements
and allow potential transforming genes to be inserted within highly active
transcriptional units (Cooper et al., 1980). However, the experiments with
normal cell DNA may be misleading: it is not known whether the transform-

OncoRenesis by insertion mutagenesis and gene activation

3 13

g activity of DNA from tumour cells is derived from the same set of genetic
ci as the activity in sheared DNA from normal cells.
It is possible that transforming genes defined by the NIH/3T3 cell assay are

-overlapping with c-om's; however, there is reason (described below) for
osing that c-om's and transforming genes may play different roles in at
certain types of neoplasia. The number of potential transforming genes

still elusive, but it is striking that DNA from certain classes of tumour cells
   mary tumours [Lane et al., 19811, neural tumours and carcinomas
   . , 19811, chemically transformed cell lines [Shilo and Weinberg
   or T cell leukaemias [Lane et al., 19821 and human lung and

r carcinomas [Perucho et al., 1981; R. Weinberg, personal communi-
]) exhibit class-specific patterns using restriction endonucleases.
ver, the transforming element from two human colon and two human
arcinomas appears to be the same (Perucho et al., 1981; Murray el al.,

Such observations suggest that a common gene is affected in each
type and that the number of potential transforming genes may be less

number of distinguishable types of cancers.

Oncogenesis by viruses lacking om genes

the late 1970s, amidst the proliferation of viral oncogenes and their pro-
s, increasing attention was also given to the previously elusive question of
some retroviruses can cause tumours despite the apparent absence of
forming genes from the viral genomes. There were several reasons for
rekindled interest. First, the long latency between infection and the
arance of tumours suggested that viruses lacking v-onc's might be more
vealing than v-one' viruses about the pathogenesis of human tumours
eichetal., 1982). Second, the application of restriction mapping to proviruses
tumours induced by v-om- viruses indicated that these tumours were clonal
r at least dominated by clones), so that the sites of proviral insertion could

onveniently studied (Steffen and Weinberg, 1978; Cohen et al., 1979).
the newly perceived structure of proviral DNA-with identical
ory domains (long terminal repeats or LTRs) present at both ends
oked hypotheses in which proviruses exerted regulatory influences
anking host DNA (Coffin, 1979; Quintrell et al., 1980; Robinson et al.,

most tumours induced by viruses lacking oncogenes, pathogenetic
  are still far from understood. However, in one case, the induction
  mphomas in the bursa of Fabricius by avian leukosis viruses
  re is strong evidence for insertion mutagenesis and gene activa-

    ogenic process. The experimental findings that dominate the
    ows: (i) all tumours are clonal and carry at least a portion of an
provirus (Neiman et al., 1980; Payne et al,, 1981; Neel et al., 1981;
g et al., 1981); (ii) in several instances the provirus is structurally defec-
and no viral genes are expressed, implying that viral gene products are
necessary to maintain the tumour state (Payne et al., 1981); (iii) virtually
umours bear proviruses within the same cellular genetic domain (Neel et

314 Harold E. Varmus

al., 1981; Payne et al., 198l), a region first identified by Hayward and his
colleagues (1981) as the c-onc called c-myc; (iv) all the tumours bearing
insertions in the c-myc region exhibit levels of expression of c-myc RNA 10-
to 100-fold above levels in normal B cells (Hayward et al., 1981; Payne et a[.,
1982); and (v) DNA from the tumours contains activated transforming genes,
as determined by assay in NIH/3T3 cells, with the transforming DNA free of
ALV or c-myc sequences (Cooper and Neiman, 1980, 1981).
How should these findings be viewed in relation to the pathogenesis of the
disease? It is likely that ALV spreads efficiently through the bursal cell poPu.
lation in a newly infected susceptible animal, inserting proviral DNA more or
less at random in the genomes of large numbers of cells. In the rare cell (about
1 in lo6) that acquires a provirus in the c-myc domain, the neoplastic process
can presumably be initiated by overproduction of c-myc RNA and protein.
(The c-myc protein has not been identified, and there is no proof that it is
inherently oncogenic; furthermore, the v-myc domain is usually expressed in
virally transformed cells as a hybrid protein also containing peptides encoded
by viral structural genes [Bister et al., 19771.) It is probable that additional
mutations or rearrangements of cellular genes occur subsequent to activation
of c-myc, including one change that activates a gene capable of transforming
NIH/3T3 cells. As the tumour nodule enlarges and the genetically evolving
cellular population competes for dominance of the tumour mass, advantage
belongs to tumour cells in which all proviruses, including the inciting provirus
in the c-myc domain, are inactivated (e.g., by partial deletions), since any
immune response against viral antigens will no longer operate against such
cells. However, retention of at least a single LTR near c-myc may be
necessary to maintain the tumour state, even though viral gene products are
not required (Payne et al. ,1981).
An immediate question raised by the study of chicken lymphomas is rele-
vant to understanding gene regulation in eukaryotes, as well as tumorigenesis:
how does an insertion of ALV proviral DNA amplify the expression of
c-myc? The initial premise, and the simplest, was that in each case an ALV
LTR would be positioned `upstream' from the genetic target, in an orienta-
tion that would permit it to act as a promoter for transcription (Nee1 et al.,
1981; Payne et al., 1981). Although the majority of tumours do contain this
arrangement of c-myc and an ALV LTR, with RNA transcripts that appear to
have been initiated within the LTR and extended into c-myc (Hayward et al.,
198l), there is also a substantial number of tumours in which other
arrangements are observed (Payne et al., 1982). In several tumours, the ALV
provirus is upstream from c-myc, but in the transcriptional orientation
opposite that of c-myc; in at least one tumour the provirus is downstream
from c-myc. With these two unexpected arrangements, the ALV LTR cannot
act simply as a promoter to enhance the expression of c-myc. The most
obvious possibility is that the ALV LTRs indirectly potentiate the
transcriptional activity of the chromosomal domain in which they have been
inserted, affecting the strength of promoters normally used or recruiting
promoters normally inactive.

Although the biochemical bases for such phenomena are not understood,

Oncogenesis by insertion mutagenesis and gene activntion

315

[potentially related findings in more malleable contexts suggest that the
[phenomena are experimentally accessible. Thus, regions near the origin of
keplication in SV40 DNA and the LTR of RSV (but not the LTR of the
Lon-tumorigenic chicken virus, RAV-0) markedly increase the frequency of
\ransformation of thymidine kinase-deficient (tk-) mouse cells to a tk+
bhenotype when present in a micro-injected plasmid containing the complete
herpes simplex tk gene in either orientation (Capecchi, 1980; P. Luciw and M.

pecchi, unpublished). Enhancing properties of SV40 DNA upon the
ression of linked promoters have been mapped within a 72 bp repeat
mally positioned upstream from the promoter for the early genes; this

equence has a strong influence upon transcription of the early genes (Gruss
&I al., 1981; Benoist and Chambon, 1981); it can stimulate expression of
linked heterologous genes using their natural promoters or adjacent
bromoter-like sequences (Banerji et a[., 1981; M. Fromn and P. Berg, per-
bnal communication; M. Botchan, personal communication); and it can be
eeplaced during SV40 replication by a functionally homologous domain of the
burine sarcoma virus LTR (Levinson et al., 1982).

The findings with ALV-induced lymphomas raise a host of additional ques-
ions the most approachable being whether similar events occur in tumours
   by other viruses without onc genes. Several pieces of evidence
   ge belief that they do. (i) In avian B cell lymphomas induced by a
etrovirus unrelated to ALV (the reticuloendotheliosis virus called chicken
    virus) and by a virus differing only modestly from ALV (the
   stosis-associated virus, MAV), proviral insertions near c-myc have
    tified, sometimes accompanied by deletions within and amplification
  interrupted domain (Noori-Daloii et al., 1981; D. Westaway and C.
  vici, unpublished). However, the level of c-myc expression has yet to
    rmined in these cases and tumour DNA has not been tested for trans-
ming activity in NIH/3T3 cells. (ii) Preliminary evidence implicates pro-
lira1 insertion near another cellular oncogene (c-erb), the progenitor of the
fransforming gene of avian erythroblastosis virus, in the rare induction of
&rythroblastosis by ALV (T. Fung and H. J. Kung, personal communication).
fiii) A significant proportion (about 50%) of mammary carcinomas induced
by the mouse mammary tumour virus (MMTV) in C3H mice contain pro-
viruses in the same 20 kb region of the host genome (R. Nusse, unpublished).
owever, this region has been defined only by the presence of proviral DNA:

' nit has not been found. The analogy with ALV-induced lymphomas is streng-
hened by the observation that DNA from MMTV-induced mammary
umours transforms NIH/3T3 cells; the pattern of inactivation of the trans-
orming principle by restriction endonucleases suggests that the same

ne may be involved in each of five cases and in a chemically induced
  mary tumour and a spontaneous human mammary carcinoma
., 1981). As in the case of ALV-induced lymphomas, the trans-

orming sequences seem to be unlinked to proviral DNA.
It would be premature to conclude from these provisional data that all
Retroviruses without oncogenes induce tumours in similar ways. On the one

P no known cellular oncogenes reside within it, and an activated transcriptional

316 Harold E. Varmus

hand, we are far from understanding how oncogenic transformation occurs in
the most successfully explored example, ALV-induced lymphoma; on the
other hand, some of the most important viruses in this category-including
murine, bovine, and feline leukaemia viruses, as well as the newly described
human isolates associated with T cell lymphomas and leukaemias (Poiesz et
al., 1980; Hinuma et al., 1981)-have yet to be adequately assessed from
these new perspectives.
Some insight into the significance and function of putative oncogenes
affected in various tumours may be provided by asking whether viruses, such
as ALV, that induce several kinds of neoplasm do so by activating the same or
different oncogenes in each target cell. Conversely, it will be of interest to
determine whether the same oncogene is affected by different viruses in a
single target cell, as may be the case for c-myc in B cell lymphomas (see
above). It is possible that the answers to these questions will depend upon the
normal functions of the various cellular oncogenes (e.g., whether they are
general regulators of growth or determinants of differentiation); in any case,
the answers may serve as guides for exploring the involvement of such genes
in spontaneous tumours and in tumours induced by non-viral agents.
A profound understanding of neoplastic mechanisms involving putative
cellular oncogenes will doubtless demand detailed descriptions of the pro-
ducts of these genes. There is reason to be optimistic about achieving these
immediate goals, judging from recent successes with viral oncogenes
(reviewed in Tooze, 1980 and Bishop and Varmus, 1982). It will be a more
difficult task to assign functional attributes to the genes and their products.
The demanding question that now dominates the study of viral oncogenes will
ultimately need to be addressed with cellular oncogenes as well: What
biochemical properties of their products are responsible for the various mani-
festations of the neoplastic phenotype?

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