Ann. Rev. Genet. 1984. 18553412
Copyright D 1984 by Annual Reviews Inc. All rights reserved

'THE MOLECULAR GENETICS

OF CELLULAR ONCOGENES J

Harold E. Vurmus

Department of Microbiology and Immunology, University of California, San Francisco,
California 94143

CONTENTS

INTRODUCTION ............................................... _.._.........

DEFINITIONS OF CELLULAR ONCOGENES ................................................
c-onc's: The Proto-Oncogenic Progenitors of Retroviral Oncogenes . . . . . . . . . . . . . . . . . . .
Identification of Cellular Oncogenes by Functional Tests for Dominant Mutations
Using Genetic Rearrangements to Identify Oncogenes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Proviral insertions can activate expression of cellular proto-oncogenes . . . . . . . . . . .
Oncogenes at translocation breakpoints.. . . , , . . . . , . . . . . . . . . . . . . . . . .
Oncogenes in amplified DNA . , , ... . . ..... , , ... .... , .. .. . .... . .. .. _.. . .._. .... . .. .. .. .. . ...

MOLECULAR PROFILES OF ACTIVATED ONCOGENES.. . . . . . . . .
Base Substitutions in Mutant ras Alleles ................................
Insertionally Mutated c-myc Loci.. . . . . . . . . . . . , . . , . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~

Structural and Functional Properties of Translocation Bre
    Oncogenes ...............................................

FROM c-onc TO v-onc: COLLUSION OF MUTATIONAL MECHANISMS

The Favored Model for Transduction of Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Functional Significance of Differences Between c-onc's and v-onc's . . . . . . . . . . . . . .
INACTIVATION OF INTEGRATED v-onc GENES: A SAMPLER OF MUTATIONAL
      MECHANISMS ..... ......................................

NEW DIRECTIONS TOWARD A MOLECULAR DEFINITION OF CANCER

.................................. ....

ble Hosts . . . . . . . . . . . . . . . . . . . . . . . . . .

Identifiing Genes Whose Products Might Interact with Oncogene Proteins

..... .... ........ .... ........... , .... .

A CONCLUDING PERSPECTIVE.. . . .

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554 VARMUS

INTRODUCTION

Eventually the techniques of nucleic acid chemistry should allow us to itemize all the
differences in nucleotide sequence and gene expression that distinguish a cancer cell from its
normal counterpart, and perhaps at that point the steps involved in carcinogenesis will cease
to be in doubt. (25)

Widespread interest in cellular oncogenes as potential substrates for the somatic
mutations believed to underlie neoplastic change has brought students of cancer
closer than could reasonably have been expected a mere four years ago to
meeting the challenge laid down by Cairns (25). Efforts to define cancerous
lesions at the nucleotide level have, moreover, been instructive on a broader
front, providing a panoramic view heretofore unavailable of the kinds of
mutations that afflict the somatic cells of higher eukaryotes.
My objectives in this review are to summarize the nature of the recently
defined mutations of cellular oncogenes and to evaluate the hypothesis that they
are giving us an accurate picture of both genetic change in somatic cells and the
mutational basis of cancer. This evaluation must take into consideration the
difficulties inherent in genetic attacks upon oncogenes: the poorly defined
complexity of neoplastic phenotypes; the co-existence of multiple genetic
changes in a single tumor cell; and the uncertain weight to be given to various
lesions that may influence gene expression, alter the amino acid sequence of an
oncogene product, or both. It is expressly not my intention to review the
structure, origin, and biochemical functions of oncogenes; this has been
accomplished in many other places in the recent past (1618, 39, 253, 254).
However, it seems appropriate to begin by describing the experimental
strategies used to identify normal and mutant cellular oncogenes (Table l), in
part because the extent to which these strategies depart from classical genetics
underlies current difficulties in correlating phenotypic changes in tumor cells
with individual mutations.

Table 1

Proto-oncogenes Active oncogenes

c-one's v-onc's'

Wild-type form of transforming genes
Wild-type form of rearranged genes

Categories of cellular oncogenes based on mode of discovery

Transforming genesb
Rearranged genes`

a Cell-derived sequences to which oncogenic activity has been ascribed in retroviral

Cellular genes competent to transform an appropriate recipient cell, e.g. NIHi3T3

Cellular genes altered by insertion mutations (Table 4). chromosomal transloca-

genomes (see Table 2).

cells (see Table 3).

tions (Table 5). amplification (Table 6). or other rearrangements.

CELLULAR ONCOGENES 555

DEFINITIONS OF CELLULAR ONCOGENES

Much of the recent work in molecular oncology is motivated by faith in the
proposal that a neoplastic cell develops from its normal progenitor as a con-
sequence of changes (probably multiple) in some members of a restricted set of
cellular genes. Such changes could be epigenetic, but the prevailing belief that
cancer has its origins in mutations (24, 25) and the persuasive power of
identified alterations in the structure of DNA (the subjects of this review) have
focused attention primarily upon genetic events. The mutant genes are known
as oncogenes, and corresponding wild-type alleles are called proto-oncogenes
(or normal cellular oncogenes).

c-onc's: The Proto-Oncogenic Progenitors of Retroviral
Oncogenes

Members of the largest group of proto-oncogenes have been identified ex-
perimentally by the homology of their nucleotide sequences to retroviral
oncogenes, those regions of certain retroviral genomes believed to be responsi-
ble for swift induction of tumors and neoplastic transformation of cultured
cells. The homologous cellular genes, known for convenience as c-om's (39,
are deemed proto-oncogenes because they have apparently served as targets for
genetic transduction by retroviruses and thus are the normal cellular progenitors
of viral oncogenes (v-onc's) (17, 18, 218) (Table 2).
Although the dividends of conferring the status of proto-oncogenes upon
these cellular genes have been considerable, it must be acknowledged that the
basis for doing so, the genetic definition of v-onc's, has not been uniformly
rigorous. At one extreme, numerous conditional and non-conditional mutants
of the src gene of Rous sarcoma virus (RSV) have convincingly demonstrated
that v-src is necessary for both the initiation and maintenance of the trans-
formed phenotype (125); other mutants of RSV, as well as manipulation of
cloned v-src DNA, have established that the v-src gene is also sufficient for
transformation (72, 122, 133a). At the other extreme, host-derived sequences
present in the genomes of highly oncogenic viruses have sometimes been
granted the provisional designation of v-onc's without supporting genetic
evidence, even when the genome is also inhabited by another v-om for which
there is genetic confirmation of neo-plastic activity. Belief that such sequences
have oncogenic potential of their own or the capacity to enhance the oncogenic-
ity of accompanying oncogenes (e.g. by broadening the range of cellular
targets for neoplastic effects) can be traced to at least three sources: (a)
transduction of each cellular gene is a rare event for which there is likely to be
some selective advantage for the virus (e.g. tumorigenic capacity); (b) in some
cases of retroviruses bearing components derived from two independent cellu-

Table 2 Retroviral oncogenes (v-onc's)"

<

G

Genetic basis

Species of Conditional Non-conditioflal In vitro Multiple

transductiond 5

Prototype virus(es)k v-om origin mutantsb mutantsb recombinantc

RSV

Yl3lESV

FuSVlPRCII

UR2

MC29

MH2

AMV

E26

AEV

SKV770

REV-T

Mo-MSV

Ab-MLV

MSV-3611
BALB-MSV

~

src
yes'
fpsf

ros

myc

myc

milg

mYb
mYb

ets

erbA
erbB

ski

re1

mos

ab1

raf
Ha-rash

chicken

chicken

chicken

chicken

chicken

chicken

+

chicken

chicken

chicken

chicken
turkey

mouse

mouse

mouse

mouse

+

+

+

+
+

+

+

+

+

+

+

+

+

+

+

Ha-MSV
RaSV

Ki-MSV

FBJ-MSV

FBR-MSV

ST-Fe SV, GA-FeSV
SM-Fe SV

GR-Fe SV

PI-FeSV. SSV

Ha-rash
Ra-rash

Ki-ras

fos

fos
fox
fes'

fms
fgr e

actin'

sis

rat

rat

rat

mouse

mouse

cat

cat

cat

cat, woolly
monkey

+

+

+

t

+

+

+

+

+

+

+

+

+

+

+

~~~

'Further description and bibliography can be found in (1618).
bDetails and references can be found in (17, 18, 125. 126).

+ means that the v-onc sequence has been found to have transforming activity when moved to a new context by in vitro manipulation of cloned DNA (see 19).
+ means that the onc sequence has been encountered in two or more viruses that appear to have independently transduced the same c-onc from one or more host species (see

17. 18).

* v-yes and v-fgr appear to be derived from the same cognate c-onc (144a).
'v-& and v-fes are derived from the same cognate c-onc (206).
sv-mil (also known as v-mht) and v-rufare derived from the same cognate c-onc (102, 225).
hHa-rus of BALB-MSV (also known as v-bus), Hams of Ha-MSV, and Ra-rus of RaSV are all derived from the same cognate c-onc. called c-Ha-rus (6, 181).
'Part of the mammalian p-actin sequence is found in the genome of GR-FeSV (144a).
jA mutant of MH2 that appears not to express v-mil has normal or augmented oncogenic activity (161).
'Abbreviations: RSV. Rous sarcoma virus; Y73, Yamaguchi 73 sarcoma virus; ESV, Esh sarcoma virus; FuSV, Fujinarni sarcomavirus; MC29, myelocytomatosis-29 virus; MH2.

Mill Hill-2 virus; AMV, avian myeloblastosis virus; AEV, avian erythroblastosis virus; REV-T, reticulo-endotheliosis virus, strain T; Mo-MSV, Moloney murine sarcoma virus;
Ab-MLV, Abelson murine leukemia virus; Ha-MSV, Harveymurinesarcoma virus; RaSV, Rasheed rat sarcoma virus: Ki-MSV. Kirsten murine sarcoma virus; ST-, GA-, SM-, GR-,
PI-FeSV, Snyder-Theilen, Gardner-Arnstein, Susan McDonough. Gardner-Rasheed. Parodi-lrgens feline sarcoma viruses; SSV, simian sarcoma virus.

558 VARMUS

tar genes (e.g. v-myc and v-mil in MH2 virus), both co~ponents have been
found singly in other transforming retroviruses (e.g. MC-29 and MSV-3611)
(102,225); and (c) in the case of avian e~throblastosis virus (AEV), mutants
constructed in vitro suggest that one v-onc sequence (v-erbA) can potentiate the
oncogenic action in the erythropoietic cell lineage of another, more critical
sequence (v-erbB) (70,203). For the majority of v-om's there is at least some
supportive genetic evidence for oncogenic potential, although usually no direct
evidence for a role in maintenance, since conditional mutations have only been
isolated in a few cases (Table 2). More commonly the evidence is restricted to
multiple isolations of the same oncogene in different highly oncogenic viruses
and the retention of transforming activity when the viral oncogene is placed in a
new context by the manipulation of cloned DNA.
For several years, the contention that the cellular progenitors of retroviral
oncogenes might be the instruments of oncogenic change rested upon supposi-
tion. Even before the structural differences and similarities between c-onc's
and vLonc's were recounted with the high resolution now possible, the two
versions of each gene were recognized to be sufficiently alike to propose that
partial recapitulation of events that produce active v-onc's from c-one's might
occur during tumorigenesis. Moreover, e-onc's are well-conserved during
metazoan evolution, commonly expressed in most or all tissues, and sometimes
regulated in temporal or lineage-specific fashion (1 6), implying that they might
play a central role in growth and development, processes that seem particularly
likely to be altered during neoplasia. [The latter idea has received dramatic
recent confirmation by the findings that c-sis appears to encode the platelet-
derived growth factor (59, 251) and c-erbB the epidermal growth factor
receptor (60).] However, new approaches were required to implicate c-UIZC'S
directly in neoplasia.

Identi~cat~on of Cellular Oncogenes by Functionat Tests for
Dominant Mutations
The second means to identify cellular oncogenes perceives active oncogenes

rather than proto-oncogenes. DNA transformation procedures, used for several
years to demonstrate, for example, the oncogenic activity of nuclear DNA from
cells containing integrated genomes of DNA and RNA tumor viruses, elicit
neoplastic transformation when DNA from a wide variety of tumor cells is
introduced into established lines of rodent fibroblasts, most commonly NIW
3T3 cells (39,43,207a, 253). Identified sources of active DNA by now include
primary human tumors and cell lines; spontaneous and virus-, chemical-, and
radiation-induced tumors of rodents and birds; and chemically-transformed cell
lines (Table 3). The interpretation of such findings-an interpretation for which
there is now considerable support (see below)-is that neoplastic wansfonna-
tion of the recipient cells is dependent upon the acquisition of a mutant allele.

CELLULAR ONCOGENES 559

The mutant allele is believed to act in a dominant manner in both recipient and
donor cells, and its effect upon the recipient cells is alleged to simulate its
biological activity in the donor cells. The presumption that a mutated gene is
present in transformation-competent DNA was based initially upon the failure
to induce the transformed phenotype in recipient cells with DNA similarly
prepared from normal cells. However, the existence of a mutant gene in
competent DNA requires more direct proof for several reasons: (a) sheared
DNA from normal sources can transform cells at low frequency (43), presum-
ably because under these circumstances strong promoters and proto-oncogenes
from either donor or recipient cells can recombine to generate an efficiently
expressed oncl )gene; (b) some wild-type c-onc proto-oncogenes (e.g. c-mos,
c-Ha-rusl, and c-fos) can transform cells in culture when placed under the
control of active transcriptional promoters (20, 31, 137, 159); and (c) rare
acquisition of multiple copies of proto-oncogenes during transfection could
presumably produce neoplastic change (akin to that proposed to occur as a
result of gene amplification or transcriptional activation) without mutation in
the donor cells.
Although the assay for oncogenes in NIW3T3 cells has stimulated remark-
able advances in our understanding of oncogenic mutations, it has come under
criticism for an obvious reason: it offers a functional test for genetic lesions in a
cultured mouse fibroblast that usually differs both in cell lineage and species
from the tumor cells whose genes are under scrutiny. This potential weakness
may well provide at least part of the e~p~a~a~on for two of the most striking
characteristics of collective experience with the NIH/3T3 assay: the failure to
observe transforming activity in DNA from 80-90% of spontaneous human
tumors (39, 112, 174, 253) and the high proportion of active oncogenes that
have proven to be mutant members of the rus gene family (see below). Recent
attempts have been made to exploit other types of cell lines (193) or different
assays for neoplastic effect (1 19; M. Wigler, personal communication) to seek
additional oncogenes by DNA transfection. Although these alterations in
methodology still have generally led to isolation of mutant ras genes, M.
Wigler and colleagues (personal communication) have tentatively identified
novel oncogenes in a human mammary carcinoma line, MCF-7, by treating
NIH13T3 cells with tumor cell DNA and a plasmid bearing a dominant genetic
marker, then observing for tumor growth after inoculation of mice with pooled,
co-transfected cells.
The NIH/3T3 cell assay also has been criticized on the grounds that the
recipient cells, from an established line, are already abnormai. Inspired by
requirements for two adeno- or polyoma virus genes to achieve full transforma-
tion of primary rodent embryo cells (62,182), Land et al(114) and Ruley (192)
have used embryo cells to show that two activated, molecularly cloned cellular
oncogenes coliaborate to produce a mo~holog~cally transfo~ed cell with

560 VARMUS

Table 3

Candidate oncogenes detected as transforming genes in the NIH/3T3 cell assay

Examples of cell types in which

Genes transforming activity is found"

c-Ha-ras 1

Bladder carcinoma cell line (human)

Urinary tract tumors (human)
Lung carcinoma cell line (human)
DMBA and NMU-induced mammary carcinomas

DMBAmPA-induced skin papillomas, benign and

Melanoma cell line (human)

(rat)

malignant (mice)

References

55, 83, 112, 164,

70a
260
220; see footnote c

8, 9

197

below

2

Mammary carcinosarcoma line (human)

MC, BP, DEN and MNNG-transformed primary

Myeloid tumor cell line (mouse)


Colon carcinomas and cell lines (human)

                                       See footnote d below
                                          223


                                          246a
c-Ki-ras2 -- Lung carcinomas and cell lines (human) 55, 174, 135
                                    55, 174, 135

cells (guinea pig)

Pancreatic carcinoma cell line (human)
Gall bladder carcinoma cell line (human)
Rhabdomyosarcoma cell line (human)
Ovarian carcinoma, primary (human)
Acute lymphocytic leukemia line (human)
hfC-induced fibrosarcomas and MC-transformed

y-irradiation induced thymoma (mouse)
MC-induced thymic lymphoma and macrophage

fibroblasts (mouse)

174
174
174
68
67
65, 165,208

88
246a

N-ras

tumor lines (mouse)

BP-induced fibrosarcoma (mouse) 246a
Neuroblastoma cell line (human) 210
Burkitt lymphoma line (human) 77

Fibrosarcoma and rhabdomyosarcoma cell lines

Promyelocytic leukemia line (human) 143
T cell leukemia line (human)                213a
Melanoma cell lines (human)             2; see footnote e

Teratocarcinoma line (human, late passage)       228a
Lung carcinoma line (human) 259a
Acute and chronic myeloblastic leukemia (human)   67, 77
Lung carcinoma cell line (mouse)             246a

89, 133

(human)

below

Carcinogen-induced thymoma (mouse) 88

c-mos       Plasmacytoma cell lines (mouse)           36, 78, 184
c-erbB-related   ENU-related neuroblastoma cell lines (rat)       168, 208, see foot-

note f below

CELLULAR ONCOGENES 56 1

Blym

Tlyrn


tx-lb

tX-Zb

tX-3b

tX-4b

Unnamed

Unnamed

ALV-induced bursal lymphomas (chickens)
Burkitt lymphoma lines (human)
Intermediate T cell lymphoma lines (human)
MLV-induced T cell lymphomas (mouse)
Mammary carcinoma cell line (human)
MMTV and DMBA-induced manima~ carcinomas

PfeB tumor cell lines (human)
AbMLV-induced pre B tumor cell lines (mouse)
Myeloma and plasmacytoma cell lines (human,

Mature T cell lymphoma lines (human, mouse)
Pre B cell leukemia cell line (human)
MNNG-t~ated osteosa~oma cell line

(mouse)

mouse)

41, 42, 85
56
117, 118
117, 118
116
116

I I7
115

117

117

160

38b

* 6bbreviations: MC, methylcholanthrene; DMBA, dimethylbenzanthracene; TPA, tetradecanoyl- 13-phorbol
acetate; NMU, nitrosomethylurea; MNNG, N-methyl-N-nitro-N-nitroguanidine BP. benzfafpyrene; DEN,
diethylnitrosamine; ENU, ethylnitrosourea.

bNomenclature suggested by M.A. Lane and G. Cooper (personal communication).
`M. Barbacid, personal communication.
dM. Kraus et al. personal communication.
`R. A. Padua, personal communication.
R. Weinberg, personal communication.

tumorigenic potential. These results are provisionally interpreted to mean that
one of the genes (an active form of myc, adenovirus ElA, or polyoma virus
large T antigen gene) provides a nuclear function akin to that presumed to be
active in established lines and required for prolonged growth, whereas the
second gene (an active member of the rus gene family, adenovirus ElB, or
polyoma middle T antigen gene) provides a cytoplasmic function responsible
for the properties conventionally associated with the transformed phenotype
(114). This sort of test appears to be particularly helpful for assessing the
functional properties of putative oncogenes cloned from tumor cells (rather
than for discovering new oncogenes) and for determining the contribution made
by each member of a collaborating pair to the oncogenic phenotype in the
recipient cell. However, it is uncertain whether two oncogenes are inevitably
required (and hence whether they can rightly be called complementary genes):
D. Spandidos & N. Wilkie (personal communic~tion) have shown that a mutant
c-Hams1 allele expressed under the control of a strong heterologous promoter
does not require co-transfection with a myc-like gene for full transformation of
primary rodent cells.

The l~keli~ood that multiple mut~tions conspire to produce a full-fledged

562 VARMUS

cancer cell raises the disconcerting possibility that some of the mutations have
phenotypic consequences that cannot be perceived in the absence of the others.
One way to deal with this dilemma may be to exploit the traditional strategem of
isolating phenotypic revertants of neoplastic cells (259). For example, partial
phenotypic revertants of the human fibrosarcoma line HT1080 can be returned
to a more advanced neoplastic state by transfection with the mutant N-ras gene
formerly isolated from HT1080 cells with the NIW3T3 cell assay (C. J.
Marshall, personal communication). In a modest variation on this theme,
non-tumorigenic cell lines established from human bladder cancers have been
rendered tumorigenic by introduction of a mutant c-Ha-rusl gene from the
human bladder cancer line, EJ (C. J. Marshall, personal communication).
Again, however, phenotypic revertants or tumor cells with partially trans-
formed phenotypes have not yet been used as target cells in DNA transfection
studies designed to isolate new oncogenes from unfractionated tumor DNA.
The accumulated attempts to identify active oncogenes by transformation of
cultured cells with tumor cell DNA have yielded a number of candidate
oncogenes (Table 3), but the majority of positive results can be traced to one of
three members of the ras gene family, a group of genes that encode closely
related proteins generally about 21 kd in size. Two of these genes, known in the
human genome as c-Ha-rasl and c-Ki-ras2 (see footnotes to Tables 2 and 3),
are members of the c-onc group; the third is called N-ras, a gene recognized as a
member of the rus group by immunological cross-reactions of its product with
antisera raised against products of other rus genes and, more definitively, by
comparison of the deduced amino acid sequence of N-rus protein with corre-
sponding sequences from other family members (210, 229).
Cooper and Lane and their colleagues have used the NIH/3T3 cell assay to
implicate several genes outside the rus family as oncogenes; each oncogene is
apparently specific for tumor cells within a cell lineage or at a certain stage in a
cell lineage, as often deduced from the lists of restriction endonucleases that do
and do not inactivate transformation competence (41,42, 115-1 17) (Table 3).
However, among these putative oncogenes only chicken and human Blym have
been molecularly cloned and sequenced, and for these genes only transforma-
tion-competent alleles are available (56, 85). Hence, in no instance outside of
the rus group has a mutation that renders a proto-oncogene competent to
transform NIH/3T3 cells been proven by delinition of the nucleotide sequence.

Using Genetic Rearrangements to Identify Oncogenes

Some circumstantial evidence is very strong, as when you find a trout in the milk. (Thoreau)

Identification of genes that are structurally altered in tumors has proven to be an
important if difficult means to register candidate oncogenes. Implicit in this
endeavor is the assumption that a gene found to be altered in most cells in a

CELLULAR ONCOGENES 563

tumor is likely to be selected for and therefore contributory to the neoplastic
phenotype. Clearly this is a hazardous simplification, particularly if, as sus-
pected from some experiments (e.g. 32), cancerous cells have an altered
mutation rate or if some extraneous mutations are highly favored to occur. To
achieve a credible identification of a putative oncogene through a purely
structural analysis, multiple sightings of the same gene in mutant form would
seem to be a minimal requirement, some effect upon the expression or com-
position of its product is expected, and a detectable phenotypic consequence of
introducing the rearranged allele into another cell is desirable, although infre-
quently available to date.
A structural approach to the discovery of oncogenes is, of course, further
circumscribed by the complexity of the eukaryotic genome. Even the limited
number of suspected targets for oncogenic mutations (e.g. the c-onc group of
proto-oncogenes) presents too large a challenge to propose to seek mutations
detectable only by nucleotide sequencing procedures. [There is a notable
exception: base substitutions that alter restriction endonuclease recognition
sites in certain ras genes occur with sufficient frequency that it is feasible to
survey tumor DNAs for these mutations (c.f. 195).] Efforts to find mutant
oncogenes, particularly novel ones, by a direct inspection of the genotype have
been restricted to lesions that alter the mutant gene or its environs sufficiently to
affect the mobility or abundance of its restriction fragments. Three kinds of
genetic rearrangement have been found to affect c-onc proto-oncogenes, and
each kind offers useful if laborious means to identify new oncogenes.

PROVIRAL INSERTIONS CAN ACTIVATE EXPiiESSION OF CELLULAR PROTO-

ONCOGENES  Most retroviruses lack v-onc genes and fail to transform cul-
tured cells but are nevertheless competent to induce tumors, generally after a
protracted latent period (233). There is now broad support for the idea that
many tumors that arise following infection by v-onc- retroviruses contain
mutant cellular oncogenes activated by proviral insertions (243). It is generally
held that such insertion mutations are primary events in tumorigenesis and that
their effect is to stimulate expression of the target gene by the provision of a
strong viral promoter or enhancer element within the viral long terminal repeat
(LTR) (48a, 74, 94, 145a, 155, 166a, 167, 170a, 184).

Insertion mutations caused by the proviruses of horizontally transmitted
retroviruses or of endogenous retrovirus-like elements have been found to
afflict several cellular genes, some previously known as c-onc's (c-myc,
c-erb B, c-myb, c-mos, and c-Ha-ras) (Table 4). In most of these situations, the
target gene for insertion mutation can be identified by examining restriction
digests of tumor DNA for rearrangements using molecular probes for c-onc
genes (74, 94, 147, 151a, 184, 217). Other candidate oncogenes (e.g. int-1,
int-2, pim) have come to view as repeated targets for insertion mutation only by

563 VARMUS

applying methods akin to what has been called transposon tagging by students
of mobile elements in Drosophila (14) (Table 4). To exploit this approach
optimally requires that a tumor contain only a single copy of newly acquired
proviral DNA. On the assumption that the single copy is acting as an insertional
mutagen for an adjacent cellular gene, the viral DNA is molecularly cloned
with some of the flanking DNA. Part of the retrieved cellular DNA is then used
as a molecular probe to ask whether the same region of the host genome is
occupied by insertions in other tumors (156). It has also been possible to isolate
cellular sequences that appear to be targets for oncogenic insertion mutations
from tumors bearing two or more proviruses (48a, 170, 236).
The conclusion that commonly interrupted cellular domains are sites of
functionally important insertion mutations rests, in part, upon the assumption
that retroviral proviruses integrate into the host genome without significant
regional specificity. Although the available evidence argues that many sites in
host chromosomes can accommodate proviral DNA (244a), it is premature to
claim that integration occurs randomly. Other kinds of evidence are therefore
used to sustain the view that the identification of candidate oncogenes by
transposon tagging reflects the selective advantage conferred upon rare cells in
which the insertion mutations occur, rather than integration at highly preferred
sites. For example, the vast majority of the multiple MMTV proviruses in
tumors with insertions in znt-1 or int-2 are located outside these domains, and
each tumor appears to carry an insertion in only one int-1 or one int-2 locus
(156, 170). Evidence for an adjacent coding domain that is expressed at
enhanced levels in the tumors and is evolutionarily conserved also fosters belief
that the common insertion site harbors an oncogene (155).
In some instances-x.g. B cell leukemias induced by bovine leukemia virus,
(86a, IOS), T cell leukemias associated with T cell leukemia virus (204a), and
nephroblastomas induced by myeloblastosis-associated virus (MAV) (157)-
persistent efforts have failed to identify genetic targets for insertion mutation by
probing for c-onc rearrangements or by transposon tagging, even though the
tumors appear clonally derived. Such negative findings could imply that
insertion mutations are not involved in these tumors, that several genetic targets
are used, or that the insertions are remarkably far from the activated gene.
Under certain circumstances, the existence of abundant transcripts from the
mutated gene can assist in the identification and molecular cloning of the gene,
e.g. when the transcripts contain viral sequences because the LTR serves as
promoter for adjacent cellular sequences. For example, cloning of cDNA
containing LTR sequences from a MAV-induced chicken nephroblastoma
permitted the identification of the target gene for proviral insertion mutation as
c-Ha-ras, despite the failure to perceive rearrangements by restriction mapping
(D. Westaway, unpublished data).

Although heightened expression of an oncogene is believed to be the primary

Table 4

Candidate oncogenes affected by insertion mutations

Gene Insertion mutagen"

c-myc ALV, CSV, RPV

csv
RPV
MAV
Mo-MLV, MCF-MLV

FeLV

                           Documented
                     effect upon
Tumor type Species expression

References

B cell lymphoma
B cell lymphoma
B cell lymphoma
B cell lymphoma
T cell lymphoma

T cell lymphomas

chicken
chicken
chicken
quail
rat, mouse

cat

71, 73, 94, 167
151a

see footnote c below

see footnote d below
43a, 217; see footnote e


147

below

c-erb B ALV erythroblastosis chicken -t 74

c-Ha-ras      MAV . nephroblastoma           chicken           + see footnote d below

c-mos       IAP plasmacytomas            mouse          + 36, 78, 184

c-myb

Mo-MLV

plasmacytoid mouse

lymphosarcomas

144; see footnote f

below

int-1 MMTV mammary carcinoma mouse + 156
inr-2 MMTV mammary carcinoma mouse + 170
Mlvi- 1 Mo-MLV T cell lymphoma rat 236
Mlvi-2 Mo-MLV T cell lymphoma rat 235
Mlvi-3 Mo-MLV
RMO-int-1 Mo-MLV

T cell lymphoma rat
T cell lymphoma rat

see footnote g below
120

pim-1 MCF-MLV, MO-MLV T cell lymphoma mouse + 48a

c-raf MLV-LTR~ transformed fibroblast mouse 14c

`Abbreviations: ALV, avian leukosis virus; CSV, chicken syncytial virus; RPV, ring-necked pheasant virus; MAV, myeloblastosis-associated virus; Mo-MLV, Moloney murine

bCloned DNA containing an MLV LTR was used as an insertion mutagen via transfection.

leukemia virus; FeLV, feline leukemia virus; IAP, intracisternal A particle; MMTV, mouse mammary tumor virus; MCF-MLV, mink cell focus-forming murine leukemia virus.

C. Simon, W. Hayward, personal communication.
D. Westaway, unpublished data.

eG. Selton, A. Berns, Y. Li. N. Hopkins, P. O'Donnell, E. Fleissner, personal communication.
`G. Shen-Ong et al, personal communication.
g P. Tsichlis, personal communication.

566 VARMUS

consequence of tumor-associated proviral insertion mutants, in some situations
listed in Table 4, expression of the disrupted cellular domain has yet to be
assessed. Furthermore, even in those instances (e.g. ALV insertion mutations
of c-myc or MMTV mutations of int-1) in which tumor cells contain relatively
high concentrations of RNA transcribed from the target gene, it is difficult to
know whether the appropriate normal cell has been chosen for comparison. The
insertions may also affect the efficiency of translation (e.g. by forming a
shortened hybrid leader region in mRNA) or truncate the gene product (e.g. in
ALV insertions within c-erbB). Lastly, it is now apparent that potentially
significant alterations other than the insertion mutation may occur within
affected loci; these will be described in greater detail below.

ONCOGENES AT TRANSLOCATION BREAKPOINTS  It has been recognized for
many years that chromosomal translocations a e common in tumor cells and
that certain combinations of chromosomes occur repeatedly in certain types of
tumors (189-191, 261). Proto-oncogenes are sufficiently numerous and dis-
persed to inhabit virtually all chromosomes, so it is not credible to implicate a
proto-oncogene in neoplasia merely because it is located somewhere within a
partner in a frequent translocation event. Emphasis has instead been placed
upon more stringent criteria: that a gene involved in the neoplastic process must
be sufficiently close to the recombination site to have undergone detectable
structural or functional alterations or must be within reach of the breakpoint as
gauged, say, by restriction mapping with probes for adjacent sequences. Given
the enormity of eukaryotic chromosomes, it is daunting to consider the routine
identification of novel oncogenes by direct isolation of breakpoint regions.
Sensibly, greater attention has been directed instead to the issue of whether
proto-oncogenes known to be residents of translocated chromosomes might be
positioned close to the breakpoints and whether their structure or expression is
deranged as a consequence. To date, two known proto-oncogenes, c-myc and
c-abl, have met the criteria that implicate them as oncogenic products of
translocations (Table 5). A large number of B cell tumor cell lines of both
human and murine origin harbor translocations that join the c-my gene on one
chromosome to one of the three immunoglobulin loci on another (107, 169,
191) (Table 5), in the manner originally predicted by Klein (106). The Phi-
ladelphia (Ph') chromosome in chronic myelogenous leukemia (CML) links
c-ab1 from one end of chromosome 9 to a restricted region of chromosome 22
(54, 87, 95). Novel putative oncogenes have been tentatively identified at
chromosomal breakpoints in B cell tumors because they, like c-myc, are joined
to immunoglobulin genes from the partner chromosome (Table 5).

The translocations of c-myc have been most closely studied and appear to

CELLULAR ONCOGENES 567

perturb expression of the oncogene by either transcriptional or translational
mechanisms, with ancillary mutations within c-myc perhaps further augment-
ing the oncogenicity of the translocated allele; these issues will be addressed in
a later section.

ONCOGENES IN AMPLIFIED DNA  The initial sightings of ampli~ed proto-
oncogenes in human tumor cell lines grew out of attempts to explain unex-
pectedly high levels of expression of c-onc genes or to test the possibility that
karyological markers of gene amplification in tumor cell lines-homogeneous-
ly staining regions (HSRs) or double minute chromosomes (DMs) (45Gmight
harbor c-onc's (3, 37, 51, 199). Although a search for amplified oncogenes is
inherently biased toward those proto-oncogenes already in hand, at least one
novel oncogene (known as N-myc) has been discovered as an amplified gene in
many neuroblastoma cell lines and tumors by virtue of limited homology with
the second exon of c-myc (111, 198). Since methods for enriching genomic
DNA for its amplified components are at hand4.g. rate zonal sedimentation
of DMs (80), gradient fractionation to prepare chromosomes enriched for
markers bearing HSRs (104), isolation of middle repetitive DNA based upon
reannealing kinetics (138), differential screening of cDNA libraries (1 1 I), and
reannealing of restriction fragments within agarose gels (188)--candidate
oncogenes may be advanced on the basis of their frequent amplification in
certain types of tumors, even without any overt relationship to known proto-
oncogenes. Other clues to amplified oncogenes have recently been exploited.
The human tumor line A43 1, long known to express high levels of epidermal
growth factor receptor (67a, 88a), was successfully tested for amplified num-
bers of c-erbB genes (124a, 136,237a) when c-erbB was proposed to encode
the receptor (60).

In all cases in which expression of amplified oncogenes has been examined,
augmented levels of RNA have been encountered, although not always in direct
proportion to the extent of amplification (201; F. Alt, personal communica-
tion). However, the effects of oncogene amplification may not represent a
simple gene dosage phenomenon: preceding or consequent mutations within
amplified genes, translocation to new chromosomal contexts, or co-
amplification of adjacent genes may have additional influences (see below).
Amplifications of cellular oncogenes are proposed to influence tumor pro-
gression to advanced stages of malignancy. Two recent findings support this
view: amplification of N-myc has been observed only in those neuroblastomas
clinically assigned to Stages III or IV (22), and amplification of c-myc is
regularly detected in those variants of small cell lung carcinoma cell lines with
highly malignant growth potential (127).

Table 5 Translocated cellular oncogenes

.-

Known or                                  Locus on
candidate                Partner partner

Tumor                    oncogenes       Chromosome       chromosome      chromosome        References

Mouse plasmacytoma cell lines      c-myc         15(D2/3)       12(F1)        IgH          I, 44, 46, 131,

Rat immunocytoma cell lines        c-my           7             6             ?             224

205

wl
cn
W

Human Burkitt lymphoma cell c-myc S(q24) Wq32) IgH
lines 2(p 13) IEK
                                    22(q11) w

Human chronic myelogenous       c-ab1        9(q34-+pter)     22(ql1) bcr


Human B cell leukemia and       "bcl- 1 "(?)"      1 m13)       14(q32)

leukemia cell lines


lymphoma

Murine plasmacytoma "TKNS-l"(?)" 10 6(C2) CK

I, 47, 49, 50, 52,

98, 231

87, 95

237a


168

Murine plasmacytomas "p\Jt"( ?)" 15 6(C2) CK see footnote b

below

"The oncogenic potential of these sequences is speculative.
bJ. Adam, personal communication.

Table 6  Amplification of cellular oncogenes

Oncogene Tumor

Degree of

Source amplification

~-

Reference

~ ~__ ~~~~

c-myc Promyelocytic leukemia cell line, HL60 and     Human      20 x a         51, 37, I53

c-myc APUDoma cell line, COL0320            Human      40 x .. +I+         3

primary tumoIg

c-myc Small cell lung carcinoma cell lines (variants)     Human      5-30X         ? 127
c-myc CSV-induced bursal Iymphoma            Chicken      5-1OX         ? 151a
N-myc Primary neuroblastomas (Stage 111 and IV)     Human      5- 1000 X       +/+ 22, Ill, 198, 201

N-myc Retinobiastoma cell line, Y79, and primary     Human 10-200 x       +I+ 111, 119a, 200

N-~Yc Small ceil lung carcinoma cell lines and     Human 50 X          t- i? See footnote c below

c-ab1 Chronic myeloid leukemia cell line, K562      Human 5-1ox        ? 38, 95


c-myb Acute myeloid leukemia               Human 5-IOX         ? 167a

c-Ki-ra~2~ Primary carcinomas of lung, colon, bladder Human 4-20 x ? See foo!:mte d below %
                                                                           0
                                                                           z

Colon carcinoma cell line, SW480 ? 135 n
                                                                          0

and neuroblastoma cell lines



tumors


tumor I

n

c-myb Colon carcinoma cell lines, COL0201/205 Human lox ? 4 rn

c-erbB Epidermoid carcinoma cell line, A431 Human 30 X ? 124a, 136, 237a $


                            @

and rectum

c-Ki-ras      Adrenocortical carcinoma cell line, Y1       Mouse 30-60x         +!f 199

N-ras       Mammary carcinoma line, MCF7         Human 5-1OX ,    ? see footnote e below

t:

PDMs were observed in early passages of HL60 cells and an abnormal banding pattern in 8q was later observed by Wowkll et ai (153); both appear to coincide with C-J~~C

ampli~cation.

some of these tumors, rearrangements of c-Ki-ms2, with and without amplification, were also observed (M. Barbacid, personal communication)

1. Minna, personal communication.
dM. Barbacid, personal communication.
e M. Wigler, personal communication.

570 VARMUS

MOLECULAR PROFILES OF ACTIVATED ONCOGENES

Strategies for the identification of mutant cellular oncogenes are currently
limited by their ability to detect only certain structural lesions (mainly rear-
rangements of various kinds) or lesions with certain functional consequences
(transformation of cultured fibroblasts). Nevertheless, as summarized in the
following sections, the observed genetic alterations seem to represent a diverse
sampling of the possibilities for somatic mutation in vertebrate cells.

Base Substitutions in Mutant ras Alleles

Nucleotide substitution and concomitant amino acid change in members of the
ras gene family is the genetic alteration that most commonly explains the
transforming activity of tumor cell DNA (Tables 3, 7). The findings have
particular force because it has been possible to use in vitro recombinants of wild
type and mutant ras genes to validate the functional significance of the identi-
fied mutations (e.g. 28, 227, 260) and because changes affecting the same
codons, 12 and 61, have been encountered in multiple mutant alleles of
c-Ha-rasl, c-Ki-ras2, and N-ras (Table 7).
The reiterative pattern of these lesions raises two immediate questions: (a)
what is the effect of amino acid changes at the observed positions on the
behavior of ras gene products? (b) does the finding of repeated lesions at the
same sites imply a hot spot for mutation or a selection for random mutations that
confer transforming activity upon a proto-oncogene?
The prevailing hypothesis holds that mutations may occur at various sites in
ras genes (and presumably throughout the genome), but that strong selective
pressures favor the outgrowth of cells that undergo changes in a restricted
number of sites within coding regions. It has been conjectured, for example,
that replacement of the glycine residue normally encoded by the twelfth codon
of c-Ha-rasl by amino acids with side chains will significantly alter the
conformation of p2 I~-~~-~~~~ or the nucleotide binding site predicted to reside in
this domain (17 1, 196,258). Support for the functional importance of positions
12, 59, and 61 in p21c~Ha~"" and for the likelihood that random mutations at
these sites are selectable has come from three genres of in vitro mutagenesis
experiments. First, A. Levinson & W. Colby (personal communication) have
used synthetic oligonucleotide primers to generate mutants of c-Ha-rasl encod-
ing 18 of the possible amino acids other than glycine at the twelfth position. All
of these, save pro-12, are capable of transforming NIH/3T3 cells, although the
phenotypes vary from highly transformed to relatively subtle ones. Second,
Fasano et a1 (67b) have used sodium bisulfite to produce C to T transitions at
many positions in c-Ha-rasl and then have tested cloned mutants for their
ability to transform NIH/3T3 cells. Oncogenic mutations occurred only at a
restricted number of sites, including codons 12, 13, 59, 61, and 63. Three of

CELLULAR ONCOGENES 57 1

these positions conform precisely to the positions of mutations encountered in
spontaneous tumors and in viral rus oncogenes (Table 7), and all are close to
such sites. The change observed at position 59, ala -+. thr, is particularly
interesting because it is one of the three differences in amino acid sequence
between c-Ha-rusl and v-Ha-rus, implying that the viral oncogene has ac-
quired two alterations independently sufficient to induce cellular transforma-
tion. This prediction has been confirmed by showing that a virus bearing a
v-Ha-rus gene with gly-12 and thr-59 is as oncogenic as wild-type Ha-MSV (P.
Tambourin, D. Lowy, personal communication). Third, K. C. Vousden andC.
Marshall (personal communication) have mutated c-Ha-rus1 in vitro with a diol
epoxide and found that five of twenty-one resultant transforming genes have
restriction site changes in the eleventh or twelfth codon.
The identification of single nucleotide substitutions, both transitions and
transversions, that account for the activation of rus proto-oncogenes in spon-
taneous human tumors (Table 7) is consonant with the widely disseminated
notion that environmental mutagens serve as carcinogens (5). Further support
for this idea has emerged from the isolation and structural analysis of mutant
ras alleles from tumors induced experimentally in rodents with a variety of
chemical carcinogens (Tables 3 and 7). For example, with strlking reproduci-
bility, both dimethylbenzathracene (DMBA) and nitrosomethylurea (NMU)
induce rat mammary tumors bearing activated c-Ha-rasl alleles, the lesions in
all examined instances being G to A transitions in the twelfth exon (22; M.
Barbacid, personal communication). In the classical two-stage model for
induction of skin carcinomas in the mouse with an initiator (e.g. DMBA) and
tumor promoter, Balmain & Pragnell(8) have shown that the c-Ha-rus gene is
activated, even at a relatively early stage (benign papillomas) (9). Similarly,
mouse cells transformed in culture with methylcholanthrene (MCA) and MCA-
induced murine fibrosarcomas carry activated c-Ki-ras genes (65, l65,206a,
246a).
Some central issues and assumptions about ras point mutations remain
uncertain, if not controversial:
1. Are ras mutations dominant? Although most evidence favors the idea that
these mutations act in a simple dominant fashion, it is not uncommon to
encounter results that undermine confidence in that assumption. Some tumor
lines appear to carry only the mutant allele and hence are homo- or hemizygous
for it, as judged by analysis of restriction sites that overlap the altered codon
(195); in one instance, the human cell line SW480, the same twelfth codon
mutation in c-Ki-rus2 is present in two alleles distinguished by an apparent
polymorphism in a non-coding region (29). Furthermore, mutant alleles are
sometimes amplified [e.g. a four- to eight-fold increase in c-Ki-ras2 in SW480
cells (135)], duplicated by tetraploidy [e.g. in the EJ bladder carcinoma line
(U. Franke, personal communication)], or expressed more frequently than the

Table 7 Mutations in transforming ras genes ul

-4
h)

                     Codons

rus allelc 12 I 59 61 References

<
P

Source of allele

Normal

Normal

Ha-MSV

RaSV

Bladder Ca lines

Lung Ca line

Mammary Ca

Normal

Ki-MSV

Lung Ca line

Colon Ca line

Human c-Ha-rusl

Rat c-Ha-rasl

v-Ha-ras

v-Ra-ras"

Human EJIT24-Ha-rust

Human HS242-Ha-rasl

Rat NMU-Ha-raJ I

Human c-Ki-rus2

v-Ki-rus

Human Calu-Ki-ras2

Human SW480-Ki-ms2

GGC

GCC

CAG

GGA
g'Y

GGT
gIY

ala


?


ACA

rzq

GCA
ala

GCC
ala

GCC

ala


?


GCA

-

-

-

ala

GCA
ala

GCA
ala

28

222

55a

181

227. 196, 186. 28. 230

260

222

13%

237

209, 29

29

Lung Ca

Lung Ca line

Bladder Ca line

Lung Ca line

Lung Ca line

Normal

Neuroblastoma line

Teratocarcinoma line, late passage

Fibrosarcoma line

Malignant melanoma line

Lung carcinoma line

Human LL- I 0-KI-ras2

Human A2182-Ki-rus2

Human A1698-Ki-rus2

Human PR371-Ki-ras2

Human PR3IO-Ki-ras2

Human N-rus

Human SK-N-rus

Human PA I -N-rus

Human HT1080-N-rus

Human Me1 N-ras

Human SW1271 N-rus

?

?

?

?


GCA
ala

GCT
ala

GCT
ala


?

-

?

- CCT
ala

- GCT
ala

?

?

?

?

his

CAA
gln

?

I95

I95

I95

145

see footnote b below

229

229

8

5

                  r
                  r

see footnote c below P

228a

;d

0
0

see footnote d below

259a %

'Amino acid positions .aligned with those of other ras proteins
bM. Perucho, personal communication.

dR. A. Padua. personal communication.

A. Hall, C. Marshall, personal communication.

ul
4
W

574 YARMUS

normal allele [e.g. in the CaLu tumor line (29)l. And when introduced into the
NIH13T3 cell containing normal ras alleles, it is usual to observe several copies
of the mutant gene. On the other hand, it has been possible to demonstrate
heterozygosity at the mutant locus in some instances [e.g. a primary human
lung carcinoma (195)l. On balance, it seems likely that the ras mutations are
dominant but dose-dependent, with consequent selection for cells in which the
mutant allele is augmented by chromosomal duplication, gene amplification,
gene conversion, or preferential expression. It is still uncertain how the find-
ings with ras alleles are to be reconciled with evidence that at least some aspects
of the oncogenic genotype behave as recessive properties in somatic cell
hybrids (e.g. 214).
2. When are ras mutations acquired? Although it is generally held that ras
mutations arise somatically during oncogenesis, only in rare instances has this
hypothesis been directly validated: e.g. in chemically induced tumors in inbred
mouse strains (222) and in a patient whose lung carcinoma contained a muta-
tion that-created a Sac1 site not present in multiple samples of normal tissue
(195). Greater debate, however, surrounds the question of whether ras muta-
tions are early or late events in tumor cell development, a question that can be
extended to consider the possibility that the mutations are merely products of
the exaggerated mutation rates observed in tumor cells 02). There are now
numerous instances of mutant ras alleles in primary tumors of man and animals
(Tables 3 and 7), countering earlier proposals that such mutations occurred
only in cultured cells. Moreover, mutations in c-Ha-ras appear to be early
events in chemically induced skin tumors, as is evident by transformation
assays when the tumors are still benign by traditional criteria (9). Based on
studies of human melanoma (2) and teratocarcinoma (228) lines, however, it
has been argued that ras mutations may be late events in some settings; most
tellingly, only one of five lines derived from a single melanoma patient
exhibited a mutant gene, implying either that the mutation arose in culture or
that the tumor was heterogeneous with respect to the lesion. At present, it
appears that ras mutations are somatic events that can occur at various stages of
the neoplastic process, but better functional tests will be required to establish
the contribution the lesions make to the cell types in which they are encoun-
tered.

Insertionally Mutated c-myc Loci

LOCATIONS OF PROVIRUSES  A large number of insertion mutations caused
by ALV, CSV, and MLV proviral DNA introduced into c-myc loci have now
been analyzed structurally in DNA from avian bursal and rodent T cell lympho-
mas. The c-myc genes of chickens, mice, and'man appear to consist of three
exons (239), the first of which is non-coding and presumed to have a regulatory
function (e.g. 119). Mapping with restriction endonucleases indicates that most

CELLULAR ONCOGENES 575

of the insertions in bursal lymphomas reside within the first intron of c-myc
between the non-coding 5' exon and the exon that contains the apparent
initiation site for translation of c-my; furthermore, most of the proviral
mutagens are oriented to allow their LTRs to serve as promoters for c-myc (7 1,
94, 167; R. Swift, H. Robinson, personal communication). Since there is little
reason to believe that retroviral integration occurs preferentially at certain sites
in host genomes or with preferred orientation, it seems likely that the common-
ly encountered configurations of viral DNA in c-myc loci imply a high prob-
ability of efficient expression of c-myc. Proviruses within the first intron appear
to be clustered in a fashion that conforms to the idea that insertions at some sites
are selectively advantageous over insertions at other sites (207; R. Swift, H. J.
Kung, personal communication). Nucleotide seqyencing of a few of the host-
proviral junctions (146,256; R. Swift, personal communication) shows that the
insertions are at different positions within the intron without apparent homolo-
gies between insertion sites or between host and viral sequences at the recom-
bination sites, findings consistent with studies of host-viral junctions from
proviruses not known to affect the behavior of adjacent cellular genes (244a).
The clustering of proviruses within the c-rnyc intron is particularly likely to be
affected by the presence of nearby sequences that can serve as splice donor sites
to join with the splice acceptor site at the 5' end of the second exon of c-myc.
Insertions have been found within the splice donor site from the first exon of
c-myc (146; C. Nottenburg; unpublished data), just upstream of a cryptic splice
donor site that was also used during the transduction of c-myc to generate the
v-myc gene in MC-29 virus (207,252,256), and on the 5 ' side of another intron
sequence that conforms to the splice donor consensus (146).
Other arrangements have been encountered within insertionally activated
c-myc loci in bursal lymphomas: proviral DNA in the first intron (or further
upstream) in the opposite transcriptional orientation [( 167); M. Linial, cited in
(90)], on the 3 ' side of the coding domain in the same transcriptional onenta-
tion (167); and on the 5' side of the first exon in the same orientation (162; H.
Robinson, personal communication). In the former two situations the LTRs
cannot serve as promoters for c-myc and activation of either a novel c-myc
promoter or the normal promoter must occur, presumably under the inflnence
of the enhancer element described in restriction fragments containing avian
virus LTRs (129). The initiation sites for transcription under these circum-
stances have not yet been mapped.
In MLV-induced T cell lymphomas of mice and rats, c-myc insertion
mutations that forbid the use of the MLV LTR as a promoter for c-myc seem to
be the norm. Almost all of these insertions are located within or on the 5  side of
the first c-myc exon and directed away from the gene (43a, 217; Y. Li, N.
Hopkins, G. Selton, A. Berns, personal communication), although a few are
positioned downstream of c-myc, in one case at least 24 kb beyond the third

576 VARMUS

exon (43a). Proviral insertions with a transcriptional orientation directed away
from the activated oncogenes are also commonly observed in MLV mutations
of pim (48a) and in MMTV mutations of inf-1 and int-2 (see below).

ADDITIONAL MUTATIONS IN PROVIRALLY MUTATED LOCI  In the several
instances in which host-viral junctions have been examined at the nucleotide
level in c-myc insertion mutations, it appears that the accepted rules of retrovi-
ral integration have been followed: two base pairs have been removed from the
ends of LTRs during insertion, and six bp at the host insertion site have been
duplicated (146, 256). However, as first recognized from restriction mapping
of the proviral mutagens (71, 73, 145a, 166a, 167), deletions and possibly
other secondary rearrangements have altered normal proviral structure. Most
commonly, these deletions remove regions of viral (and perhaps cellular DNA)
encompassing the 5' LTR in proviruses located on the 5' side of the c-rnyc
coding sequence in the same transcriptional orientation. There is experimental
support for the claim that one effect of such deletion mutations is more efficient
transcription of c-myc from the 3' LTR (48). However, other sorts of deletions
have also been observed: proviruses on the 5' side of c-myc in the same
transcriptional orientation have sustained a loss of their 3' ends, so that
transcription of c-myc proceeds from the 5 LTR through viral coding domains
(162) in the manner predicted to precede transduction of proto-oncogenes (see
below), and proviruses in the opposite transcriptional orientation have incurred
deletions that prevent expression of viral genes, e.g. by removing viral signals
required in cis for synthesis, processing, or translation of RNA (166a, 167).

Two proviral deletion mutants have been examined by nucleotide sequenc-
ing. One retains only a single copy of the ALV LTR that lacks two bp from both
ends and is flanked by a six-base duplication of cell DNA, indicating that it
arose by homologous recombination between the LTRs of an initially intact
provirus (256). Since the provirus in this case was inserted in the same
transcriptional orientation as c-myc, the residual LTR is positioned to serve as
promoter for a hybrid transcript that includes the coding exons of c-myc. The
other sequenced deletion mutation occurred in a provirus (from tumor LL3)
positioned in the opposite transcriptional orientation to c-myc (256). Although
the LTRs were spared, the approximately 1 kb deletion removed the tRNA
primer binding site, the RNA packaging site, the splice donor site, and the
translational initiation codon for all the viral proteins. Both deletion mutations
must have occurred during or after infection, since the proviruses lack se-
quences required in cis for transmission through the virus life cycle.
Nucleotide sequence alterations in c-myc, as well as deletion mutations in
proviruses, are present in some insertionally mutated loci. Comparison of a
partial sequence of the mutant c-rnyc locus from tumor LL3 with the homolo-
gous region of normal chicken c-myc (252) revealed three additional G residues

CELLULAR ONCOGENES 577

in the short portion of intron between the insertion site and the second exon (D.
Westaway, unpublished data) and ?hree nucleotide substitutions in the first 200
nucleotides of the second exon (256). One of these, a silent C -+ A transver-
sion, was later judged to be an inherited polymorphism because the same
change was observed in two other c-myc alleles from the same flock (D.
Westaway, C. Nottenburg, unpublished data). However, a second change that
does not alter the coding sequence, a G -+ A transition, must represent a
somatic mutation because it inactivates an SstI recognition site shown to exist in
both c-myc alleles in normal tissue from the bird carrying tumor LL3. A third
nearby change, a C 3 A transversion that converts a proline codon to a
threonine codon, appears to be a somatic mutation as well, since the change is
not found in alleles from the same flock that harbor the polymorphic marker.
A further genetic alteration of c-myc has been observed in bursal lymphomas
induced by CSV (15 la). Some insertionally mutated alleles are five- to ten-fold
more abundant than single copy genes and the unaffected alleles are not
detected. Apparent loss of normal c-myc alleles, presumably a result of loss of
one member of a chromosomal pair, also seems to occur in some ALV-induced
bursal lymphomas, perhaps accompanied by low level amplification of mutant
c-myc, since only the rearranged allele can be identified by the Southern
transfer procedure (71, 73, 94, 167).
The discovery of additional mutationsdeletions, base substitutions, and
amplification-at the site of proviral insertion mutations raises further ques-
tions. When do the additional mutations occur? Do they contribute to the
cellular phenotype, providing a selective advantage? Since cells bearing multi-
ple mutations within c-myc dominate the tumor mass, either the additional
mutations must be generated at the time of (or before) the proviral insertions or
they must produce a selectable change. Proviruses with deletions of the type
encountered in c-myc loci have not been observed among the many chosen
randomly for structural scrutiny (244a); however, under selective conditions
(see below), various deletion mutants, including those resulting from homolo-
gous recombination between LTRs, can be recovered at low frequency (245).
Thus, barring some marked stimulus to deletion formation in lymphoma cells,
a selective mechanism is likely to operate. Thehming of nucleotide changes in
c-myc seems less certain, since tl-ere is insufficient information about the effect
of proviral integration upon hc ' sequences surrounding those proviruses
chosen randomly for study. There i - rears to be concerted mutation, however,
since some of the changes do not affect the coding potential of c-myc and seem
unlikely to affect the efficiency of expression.
The selectable properties that might be conferred upon bursal cells by the
additional mutations have not yet been rigorously defined. Deletion mutations
within proviral DNA could mitigate the host's immune response against tumor
cells by preventing synthesis of virus-specific proteins that serve as antigens on

578 VARMUS

the cell surface. Proviral deletions might also have the more intriguing effect of
augmenting expression of c-myc (48). Amplification of insertionally mutated
alleles could provide selective advantage on quantitative grounds. Lastly,
nucleotide changes that alter the amino acid sequence of c-myc protein could
render the protein more effective as an oncogenic factor.
What can be done to discern the phenotypic effects (if any) of these multiple
mutations within c-myc alleles? Removal of the mutant alleles from the com-
plex environment of the tumor cells to a new context would seem to be
necessary to measure their effects, but to date the only convenient, direct assay
for activated myc alleles is the co-transformation assay in rat embryo cells
(1 14). In this assay, the crucial determinant of activity appears to be an efficient
transcriptional promoter upstream from c-myc coding sequence in the correct
orientation. Thus, normal c-rnyc exons transcribed from LTRs linked to c-myc
in vitro are competent in the co-transformation assay, whereas c-myc alleles
from tumors with proviral insertions in atypical arrangements are not, even
when the e-rnyc gene has undergone nucleotide substitutions (W. Lee et al,
unpublished data). More quantitative means to assess transformation efficien-
cies or the use of more appropriate cell types (e.g. B lymphocytes) may be
required to elicit the consequences of the mutational events recorded in the
c-myc alleles described above.

Characteristics of Other Proviral Insertion Mutations
in Proto-Oncogenes

The several other cellular loci found afflicted by insertion mutations (Table 4)
have been subjected to less scrutiny thus far than the c-myc alleles from bursal
lymphomas. Nevertheless, a number of interesting contrasts with the activated
c-myc alleles have come to light.
1. The insertion mutations in c-erbB described in ALV-induced erythro-
blastosis (74) seem likely to result from proviruses that are flanked on both
sides by parts of c-erbB. This inference is based on the idea that c-erbB encodes
the EGF receptor, a protein of approximately 138-175,000 Mr, whereas the
abundant c-erbB protein in erythroleukemia cells is similar in size (approx-
imately 62-75,000 Mr) and peptide composition to the pfoduct of v-erbB (H.
Beug, H. J. Kung, personal communication), apparently representing the
carboxy terminal half of the EGF receptor [(60) and references therein]. This
finding suggests that the oncogenic activation of the c-erbB gene may depend
on truncation of the protein product in both viral transductants and insertionally
mutated loci. A limited analysis of an LTR-c-erbB junction from one erythro-
blastic tumor shows that the LTR is in the same transcriptional orientation as
c-erbB, just upstream from an exon homologous to v-erbB, and that two bp are
missing from the LTR as anticipated in a normally integrated provirus (74).

CELLULAR ONCOGENES 579

2. MMTV proviruses are about equally distributed on the 3' and 5' sides of
the int-1 locus, but all save two are directed transcriptionally away from the
gene (155; R. Nusse, personal communication). A similar situation obtains in
MMTV-activated int-2 loci (56a, 170). Hence, the observed transcriptional
activation of these genes normally depends on the effect of a viral enhancer
rather than of a viral promoter. Two other features of int-1 and int-2 distinguish
them from some of the other targets of oncogenic insertion mutations: first, the
genes are not expressed at detectable levels in any of the normal tissues
examined to date, including mammary glands from pregnant and lactating
females (56a, 156; Y. K. T. Fung, unpublished data), implying that the genes
may be converted from transcriptionally inactive to active genes rather
than merely stimulated to higher levels of expression; second, these two genes
have not been encountered among retroviral oncogenes (i.e. they are not
c-onc's).
The int-1 gene is composed of four exons that contain the entire coding
domain, with a relatively long (approximately 800 bp) non-coding region at the
3' end of the final exon (A. van Ooyen, R. Nusse, personal communication; G.
Shackleford, T. K. T. Fung, unpublished data). Most of the MMTV insertions
on the 5' side of int-1 appear to reside upstream from the transcriptional
initiation site, with the exception of two insertions in the same transcriptional
orientation as int-1 (R. Nusse, personal communication). MMTV insertions on
the 3' side of int-1 are sometinics positioned within the final exon, between the
end of the coding sequence and the polyadenylation site (155); as a result, int-1
transcripts conclude with the U3 sequence copied from the MMTV LTR. Since
the U3 region is approximately 1200 bp in length (57), insertions of this type
produce a transcript longer than the usual 2.6 kb mRNA. The absence of the 2.6
kb int-1 RNA species in tumors containing the longer, allele-specific RNA
confirms that activation of int-1 functions in cis but not in trans.
Few lesions other than the MMTV insertions in mutant int4 and int-2 alleles
have been implicated in the activation process. Restriction mapping has not
revealed overtly mutated proviruses in most cases, although a mutant int-2
allele has been found to contain a solitary LTR, the presumptive product of
homologous recombination between MMTV LTRs (G. Peters, C. Dickson,
personal communication). The few host-viral junctions that have been se-
quenced show only the expected absence of two bp from the end of the LTR (R.
Nusse, personal communication). The mutant alleles do not appear to be
amplified in number, but structural or functional tests for additional mutations
within the int-1 locus have not been performed.
3. Insertions of intracisternal A particle (IAP) proviruses into the mouse
c-mos gene have been described in cell lines derived from two BALB/c
plasmacytomas, one tumor induced with mineral oil (MOPC 21) and one
induced with pristane (XRPL24). In the latter case, the 4.7 kb IAP insert is

580 VARMUS

positioned within the coding sequence of the only known exon of c-mas, 66
codons downstream from the start of homology with one of the v-mos alleles in
the transcriptional orientation opposite to that of c-mos (27, 184). The conse-
quences of the insertion include the transcriptional activation of an oncogenic
sequence that is normally silent (139), producing a 1.2 kb RNA species
presumably initiated from an occult promoter and generating a transforming
gene competent to cause neoplastic change in NIH/3T3 fibroblasts (184). In
cell lines derived from MOPC 21 (including a popular partner in the formation
of hybridomas, P3 X63 Ag8), the IAP DNA is located 58 codons closer to the
5' end of c-mos in the same transcriptional direction (36). In this arrangement,
c-mos RNA is at low or undetectable levels despite the presence of a 3' LTR
positioned to serve as a promoter for downstream transcription (36, 78); it is
not known whether the relatively poor expression of c-mos in derivatives of
MOPC 21 is still adequate to affect the phenotype of the plasmacytoma
cell.
A striking peculiarity of the IAP insertion in XRPC24 cells warrants further
comment. It is generally held that IAP proviruses are defective for production
of infectious extracellular virus particles, but the intracisternal particles are
thought to synthesize new IAP DNA molecules from IAP RNA and to direct its
integration by retroviral mechanisms. Analysis of IAP-host junctions supports
this view: two bp from the ends of LTRs appear to be missing at the junctions
and six bp of host sequence are duplicated to flank the IAP DNA (27, 113).
However, in the XRPC24 insertion mutation, the two IAP LTRs exhibit
considerable sequence divergence, a finding inconsistant with the generation of
the LTRs from sequences present only once in the RNA template for reverse
transcription (244a). The marked differences between the LTRs could be
explained by a high mutation rate following the insertion, by synthesis of the
insertional mutagen from an IAP with heterodimeric RNA, by recombination
between heterologous IAP DNAs prior to integration, or, most provocatively,
by transposition of IAP DNA without passage through an RNA intermediate.
More examples of newly integrated IAP DNAs or recovery of the progenitor of
the XRPC24 IAP mutagen will need examining to judge among these possibili-
ties.
4. The c-rnyb rearrangements in several plasmacytoid lymphosarcoma lines
derived from Ab-MLV-infected animals occur in the absence of Ab-MLV
proviruses and were originally proposed to result from insertion and deletion of
proviral DNA (144). However, cloning of the affected loci has revealed the
presence of helper MLV proviruses, with small internal deletion mutations, in
an intron upstream from known c-myb exons and in the same transcriptional
orientation as c-myb (G. Shen-Ong, D. Mushinski, M. Potter, personal com-
munication). It seems likely that these MLV insertional mutagens, rather than
Ab-MLV, were the primary determinants of tumorigenesis.

CELLULAR ONCOGENES 58 1

Structural and Functional Properties of Translocation
Breakpoints near Cellular Oncogenes

GENERAL FEATURES OF c-myc; IMMUNOGLOBULIN GENE TRANSLOCA-
TIONS  Efforts in several laboratories to characterize the translocations in
murine plasmacytoma (PC) cell lines and human Burkitt lymphoma (BL) lines
that join immunoglobulin (Ig) genes to c-myc have yielded a rich but complex
harvest [see reviews in (107, 119, 169, 191)]. Although each translocation
event appears to have its interesting peculiarities best viewed by descriptions of
individual cases (see below), common features have emerged from a census of
the translocations examined to date.

1. Whenever t(8;14) is observed cytogenetically in a BL line, c-myc with
distal portions of chromosome 8 is transferred next to the Ig heavy chain
constant region locus on 14qf, and when t(12;15) is observed in a PC line,
c-myc from chromosome 15 is joined to the heavy chain locus on chromosome
12. In both species, the heavy chain and c-myc genes are joined head-to-head in
divergent transcriptional orientations. The less frequent unions of human
chromosome 8 with a chromosome carrying a light chain gene [t(8;22) and
t(2;8)] have only been examined in a few BL lines, but again the patterns seem
constant; with t(2;8) or with t(8;22), the light chain constant regions, some-
timrs preceded by at least part of variable regions, appear on 8qt on the 3' side
of c-myc, with the joined loci in the same transcriptional orientation (47, 52,
62a, 63, 230b). The role of c-myc in the relatively rare t(6;15) in plasmacytoma
lines (96) has yet to be established at the molecular level (J. Adarns, personal
communication).
2. In BL lines with the less common translocations, there is concordance
between the type of light chain encoded at the site to which c-myc is translo-
cated and the type of light chain produced, even though the translocation
chromosome contains a non-functional (allelically excluded) locus (12 1).
There is, however, at least one exception to this rule, a BL line in which t(8;22)
is accompanied by production of kappa light chains (129a).
3. In the PC lines, most breakpoints within c-myc map to a region of 1-2 kb
that includes the first (non-coding) exon and part of the fist intron. Breakpoints
within the heavy chain locus have generally fallen in a switch (S) region. Most
commonly, the latter sites are within a domain containing an Sa region, usually
S, recombined with S, (implying that heavy class switching might have
occurred prior to the translocation) or within an Sa region not directly joined to

4. In the BL lines with t(8;14), the c-myc breakpoints appear to be scattered
on either side of the first (non-coding) exon, far enough from c-myc in several
instances to fail to produce altered c-myc restriction fragments. Most of the
heavy chain gene breakpoints in BL lines are within the Sp region, although
significant exceptions exist [e.g. the breakpoint in the Daudi line appears

SF (44).

582 VARMUS

to be within the VH3 region (64), and in the Raji line it is within an S, region
joined to S, (176)l.
5. Although breakpoints within the Ig and c-myc loci are sometimes clus-
tered within a few kbp, there is no evidence for homology between recombined
sequences or for strict sequence specificity. The Ig breakpoints within S
regions generally lie near consensus S sequences, implying that the enzymes
involved in heavy chain class switches participate in translocations. Fortuitous
S-like sequences may also influence the sites of recombination within c-myc in
some cases @Ob), and the sequence GAGG has been noted to lie 10-20 bp from
the breakpoint in c-myc in five PC lines (17Oc).
6. The reciprocal products of translocation events can be found by molecular
(if not by karyological) techniques, implying that reciprocal exchange is the
norm.
7. Reciprocal translocations are imprecise, with loss of a few to a few
hundred nucleotide pairs from one or both chromosomal partners. Unexpected
nucleotide pairs (1-5bp) are sometimes found at junctions in the recombina-
tional products, and in one case thus far a duplication was generated at a
breakpoint. These findings suggest a general model for translocation involving
staggered single-stranded cuts at the breakpoints, with polymerization or nu-
cleolytic activity prior to ligation (80b).
8. Alterations in the nucleotide sequence of c-myc are common accompani-
ments of translocations in BL lines. Generally these are confined to the first
exon and hence do not affect the amino acid sequence of the c-myc protein
(175a, 230b, 232), but in some cases changes are also found in the second exon
(176). Thus far, a limited search has not revealed significant nucleotide
sequence changes in translocated c-myc genes in PC lines (L. Stanton, C.
Croce, M. Cole, personal communication).
9. Some features of the rearranged chromosome carrying the coding region
of c-myc suggest that c-myc should not be expressed: recombination inevitably
occurs with the allelically excluded Ig locus, the c-myc gene is often deprived
of its normal promoter, and the Ig components are not suitably positioned to
provide a promoter for c-myc. Nevertheless, production of c-myc RNA occurs
from the translocation chromosome, with little or no expression of the c-myc
allele that remains on the normal chromosome, sometimes producing what
appear to be overtly high levels of c-myc RNA (7, 63, 131, 150) and at other
times more subtle differences [e.g. changes in differential usage of the two
transcriptional initiators in the first exon (119, 232).] In a few instances (e.g.
93) such effects may be mediated by cell-specific enhancer elements identified
within immunoglobulin loci (10, Sl), but normally the known enhancers are
relegated to the translocation product lacking c-myc coding exons (e.g. 176).
Even in the absence of known enhancers, the expression of translocated c-myc
genes seems dependent upon the cellular environment, since it is repressed in

CELLULAR ONCOGENES 583

hybrids between BL cells and mouse fibroblasts (149, 150). Effects of trans-
location upon the translation efficiency of c-myc mRNA have been proposed to
result from deletions or base changes affecting the first exon (175a, 232),
particularly since a portion of the first exon appears to be inversely repeated in
the second exon (194); formation of a stable duplex might normally impede
translation of c-myc mRNA, and an inability to form it could augment the yield
of c-myc protein. However, direct comparisons of amounts of c-myc protein in
BL or PC lines have yet to be published.
Many of the features of these translocations may reflect the specialized
properties of genetic partners that normally undergo programmed rearrange-
ments (the Ig genes) and of the type of cell (early or late B cells) in which the
events occur. The location of chromosomal breakpoints within S regions, the
loss of nucleotides at recombination sites, and the presence of somatic muta-
tions in c-myc (moved to the position normally assumed by the hypermutable
VH genes) are characteristics that mirror events in the ontogeny of Ig genes
(234). It remains to be determined whether translocations involving many sites
occur during the development of such tumors, with selection for those that
activate c-myc, or whether there is a strong predisposition to the kinds of
translocations observed. There is little reason to suppose that external initiating
factors in tumoI igenesis affect the translocation process: there is no evident
correlation with the agents used to induce PC tumors in mice (pristane or
mineral oil) or with the presence or absence of Epstein-Barr virus in BL lines.
Regrettably, translocations have yet to be described in PC or BL removed
directly from the hosts.

SPECIFIC EXAMPLES OF C-myC; IG TRANSLOCATIONS IN PC AND BL

LINES  Properties of the best studied translocation products illustrate some of
the general features of c-myc-Ig translocations.

1. The PC cell line X63 Ag8 (sometimes known as P3 and commonly used to
generate mouse hybridomas) was derived from the tumor MOPC 21 induced in
a BALB/c mouse by peritoneal injections of mineral oil. c-myc is joined to the
heavy chain locus at sites within the first exon of c-myc and within an S, region
directly joined to Sy2b in a manner that forbids distinction between a transloca-
tion that preceded and one that followed heavy chain switching. Comparison of
the sequences of both recombinational products with the sequences of germ line
c-myc and IgH genes reveals that sevenbp have been lost from chromosome 15
during the reciprocal translocation events; a single A:T nucleotide pair of
unknown origin is present at one junction point (61,80b, 148). In addition, 491
bp have been deleted by homologous recombination between short direct
repeats on the 3' side of the switch region, although it has not been unequivo-
cally demonstrated that this deletion occurred in the tumor cell rather than
during molecular cloning. Notably, X63 Ag8 and sibling lines contain three

584 VARMUS

other candidate oncogenes described elsewhere in this essay: a c-nios gene
activated by an IAP insertion mutation (36,78), a transcribed gene on chromo-
some 10 joined to a kappa light chain locus by translocation (168), and a
transforming gene unrelated to rus genes (1 17).
2. A second BALB/c-derived PC line, MPC 11, has undergone a transloca-
tion involving the first exon of c-myc and the Sy2b region; in this case, nuc-
leotides have been lost from both chromosomes: 11 bp from chromosome 15
and approximately 300 bp from chromosome 12 (216). Short inverted repeats
are present near the breakpoints in c-myc, but these have not been directly
implicated in the translocation mechanism.
3. The PC line 5558 has lost about 1 kb of c-myc on the 5 ' side of the site in
the first exon that has been joined to an S, sequence in a S, S, domain (44), and
the same or a second deletion has removed at least part of S, (80b). The
chromosome containing the c-myc coding region from the 3' side of the
breakpoint has an extra A:T base pair at the junction, as in X63 Ag8, where2,s
the reciprocal product contains an extraneous four bp sequence (AACC) at the
junction (80b). Sequencing of c-myc exons on this chromosome reveals only a
single non-coding change from the germ line c-myc sequence (L. Stanton,
personal communication).
4. The situation in the PC line W267 is similar to that in 5558. Twelve bp are
missing from the translocation breakpoint in the first intron of c-myc and about
400 bp from the breakpoint in S,. In addition, three extraneous bp (CTT) are
present at the fusion site in the reciprocal translocation product (80b).
5. In the PC line HOPC 11, 106 bp from the breakpoint region in the first
intron of c-myc are duplicated and present in both products of the translocation
event (80b). This finding provides the best support for the claim that the
translocation mechanism involves staggered cutting at the breakpoints. In this
cell line, 1.6 kb have been lost from the partner site in the S, region of
chromosome 12, and an extraneous 5 bp sequence (CCTAT) is present at the
joint on the reciprocal product of translocation (80b).
6. In the cell line BL22, a site about 1 kb on the 5 ' side of the first c-myc exon
is joined to S, (12). Although the second and third c-myc exons do not deviate
from the germ line sequence (12), the first exon displays small deletions and
several nucleotide substitutions (232). Similar changes confined to the first
exon-7% base changes and a 35 bp duplication-have been encountered in
the translocated c-myc allele from the Daudi BL line, t(8;14), with a breakpoint
at least 12 kb 5' of c-myc (175a). The amount of c-myc RNA is not appreciably
greater in BL22 than in lymphoblastoid cell lines, but the first of the two
transcriptional initiation sites is used 4-5 times as frequently as in lymphoblas-
toid cell lines (232).
7. The BL line, Raji, resembles BL22 in that a site about 2 kb on the 5' side
of c-myc is joined to S,, but the S, region is fused with S, and changes in the

CELLULAR ONCOGENES 585

c-myc sequence are not confined to the first exon (176). In addition to several
substitutions and a 10 bp deletion in the first exon, there are three short
deletions in the first intron (including one that removes the splice donor site),
25 nucleotide substitutions dictating 16 amino acid changes and a three'bp
insertion in the second exon, and 9 nucleotide substitutions in the second
intron. The third exon does not differ from published germ line sequence. In the
Raji line, unlike most BL lines, the normal (untranslocated) c-myc locus is also
transcribed (175a), prompting the suggestion that the product of the translo-
cated locus is defective in a trans-active repressor function (175a, 119). It is
possible that the striking changes in Raji c-myc are consequent to concerted
mutagenic activity during heavy gene class switching or di Iing translocation.
Although normal tissue is not available from the patient in which the tumor
arose, it is unlikely that these many differences are inherited polymorphisms.
8. In the BL line JBL2 with t(2;8), alterations in the structure and expression
of c-myc are associated with translocation breakpoints at least 20 kb down-
stream from c-myc and upstream of the kappa chain constant gene. A 2.5 kb
region that includes the first c-myc exon has been duplicated on the recombined
chromosome, and the duplicated exon contains several nucleotide substitutions
and a small internal duplication. The concentration of c-myc RNA is elevated
three-five-fold over levels in lymphoblastoid lines, with one RNA species
retaining sequences from the first intron and another appearing to be spliced
normally (230b).
9. Nucleotide substitutions in c-rnyc have also been observed in BLlines, LY
67 and MAKU, with t(8;22) (175a). The changes are most numerous in first
exon and intron, but cDNA cloned from the LY 67 line reveals  T-C
transition in the second exon that dictates a change of ser-62 to pro-62 in c-myc
protein (175a).

10. An 8;22 translocation in line BL37 has joined a site 5 kb upstream of Ck
to a site 4 kb downstream of c-myc (98). During recombination, 21 bp were lost
from chromosome 22. In this variant translocation, the joining sites bear no
relationship to S regions of heavy chain loci, Despite the distance between the
c-myc promoter and the Ig locus (> 10 kb), a marked effect on expression of
c-myc was observed; c-myc RNA is fifteen-fold more abundant than in lym-
phoblastoid lines and the first of the two initiation sites is preferred over the
normally favored second site.
11. The BL line, Manca, differs from most BL lines with t(8;14) in two
respects: the breakpoint in c-myc is within the first intron and, more exception-
al, the breakpoint in the Ig heavy chain locus is on the 5' side of Sp,, placing a
known enhancer element next to the coding exons of c-myc (93). Thus,
transcription of c-myc may be affected by an enhancer element and translation
may be augmented by the absence of sequences from the first exon in c-myc
mRNA (194).

586 VARMUS

12. In another BL line with t(8;14), ST486, the Ig heavy chain enhancer is
joined to the first exon of c-myc on the reciprocal translocation product, 8q-
(46a, 79); short transcripts containing sequences from the first exon of c-myc
are abundant, whereas the c-myc coding exons are expressed at near normal
(79) or augmented (46a) levels, presumably from 14q+. Differences in regula-
tory mechanisms are implied by findings that expression of the first exon on the
reciprocal product is suppressed in cell hybrids formed with human lympho-
blastoid cells, whereas expression of the coding c-myc exons is suppressed in
hybrids formed with mouse PC cells (46a). Both translocation products have
been isolated from this line: although a simple, non-homologous joining to S,
was found 280 bp downstream from the first c-myc exon, restriction mapping of
the reciprocal product suggested that additional undefined rearrangements had
occurred (79).

THE PH~ CHROMOSOME  The piece of clwomosome 9 transferred to chromo-
some 22 during the reciprocal translocation that generates the Ph' chromo-
some, a common marker in chronic myelogenous leukemia (CML) in man, is
karyologically invisible and hence estimated to be less than 5000 kb in size.
Detection of DNA from chromosome 9 on Ph' was initially accomplished by
using a probe"for the c-ab1 gene (54), and chromosomal walking later identified
the breakpoint on chromosome 9 in one CML cell line to lie within 14 kb
upstream of a region homologous to v-abl(95). The DNA specific for chromo-
some 22 from the 9-22 chimeric clone was subsequently used to identify a
region of about 5 kb, called bcr, that encompasses all of the chromosome 22
breakpoints in leukemic cells obtained from 17 of 19 CML patients (87). (The
two negative cases did not have karyological evidence of the 9;22 transloca-
tion.) The chromosome 22 breakpoints appear to be scattered throughout the
bcr region, though nucleotide sequencing of the junctions has not been re-
ported. The sites of recombination on chromosome 9 seem to be less restricted
and are usually at least 40 kb upstream from known exons of c-abl. These
findings focus attention on the possibility that the bcr region may have some
role in leukemogenesis; although bcr is transcribed in at least some CML lines
(J. Groffen, personal communication), there is no information about its coding
potential. Similarly, the effect of the translocation upon the behavior of c-ab1
has not been fully defined. c-ab1 mFWA of atypically large size and increased
abundance (27a, 38a) and a larger than normal form of the c-ab1 protein with an
active tyrosine kinase (0. Witte, personal communication) have been recently
observed in the CML line K562. Moreover, in the same line, the Ph' chromo-
some is accompanied by a modest amplification and an incompletely elucidated
rearrangement of c-abl, with co-amplification of a resident marker for chromo-
some 22, the h Ig gene (38, 95). Other Ph'-positive CML lines that do not
display amplification or local rearrangement of c-abl also contain augmented
levels of an enlarged c-ab1 RNA (38a, 27a). Neither the X Ig gene [which is at

CELLULAR ONCOGENES 587

least 45 kb distant from bcr and retained on Ph' (175)] nor c-sis [which is far
from bcr on chromosome 22 and transferred to 9q+ during formation of Ph'
(ll)] is considered likely to play a direct role in CML.

OTHER TRANSLOCATIONS AND REARRANGEMENTS IN E4 CELL TUMOR

LINES  Perlmutter et a1 (168) have described a 6;10 translocation in the NS-1
line, a derivative of MOPC21, that joins sequences about 1 kb on the 5 ' side of
the Ig kappa constant locus to an unidentified region of chromosome 10, callcd
TKNS-1. Although there is no sequence homology at the breakpoints, there is
an int~guing similarity between the involved region of the IgK locus and a
nearby region of the Igrc locus that is joined to the S, region from chromosome
12 in a bizarre t(6;12;15) rearrangement in the PC line 7183 (242). In the latter
line, the other end of the S, domain is joined to an unidenti~ed region of
chromosome 15 (Le. not c-myc). There are now some circumstantial reasons to
believe that the TKNS-1 region may be functionally significant: it is rearranged
in another mouse tumor cell line, the Abelson MLV-induced B cell line, 18-81;
modestly amplified in X63 Ag8, another line derived from MOPC 21; ex-
pressed in the NSl cells but not in several normal organs; and evolutionarily
conserved (R. Perlmutter, personal communication).

It is possible that the effects of translocations upon gene expression can be
mimicked by deletions or inversions. Weiner et a1 have described interstitial
deletions of band D of mouse chromosome 15 that appear to augment the
expression of e-myc (assigned to 15 D2/3), with or without rearrangements
detectable by restriction mapping of c-myc (255). These deletions are found in
Ab-MLV producing PC lines, but it is not known whether the rearrangements
are directly related to virus infection.

Structural Features of Amplified Oncogenes

The current picture of the molecular events that amplify proto-oncogenes is at
least as fuzzy as our view of amplifications affecting other genes. Because a
phenotype as simple and selectable as drug resistance cannot as yet be ascribed
to oncogenic amplifications, it has not been possible to define experimental
conditions to identify factors, such as inhibitors of DNA synthesis (23), that
might influence the frequency with which such amplifications occur. Nor has a
full structural analysis of any amplified units bearing oncogenes been
attempted.
Nevertheless, a few instructive features of amplified oncogenes have been
described.

1. Amplified oncogenes may exist as part of either DM chromosomes or
HSRs, even within sibling lines derived from a single tumor (3, 199).
2. The crudely approximated sizes of some amplified domains bearing

oncogenes-I00 to 1000 kb or more-are similar to the sizes of amplified

588 VARMUS

domains bearing other recognizable genes (197a). Accordingly, additional
presumably adjacent genes may be co-amplified with the implicated
oncogenes, at least one other in the mouse adrenocortical tumor with
amplified c-Ki-ras (80a) and several others in the IMR 32 line with ampli-
fied N-myc (1 11).

3. Oncogene-containing HSRs may be located in chromosomal contexts other
than those in which the oncogene is normally situated. For instance, N-myc,
a normal resident of chromosome 2p23-24, is amplified within HSRs found
on a different chromosome in each of several neuroblastoma cell lines (1 1 1,
201). In a subline of the COL0320 tumor line, amplified c-myc (derived
from 8q24) is present in an HSR situated on both sides of the centromere in a
marker chromosome that also contains part of the X chromosome (K.
Alitalo, C. C. Lin, unpublished data). It is possible that the identified sites
are predisposed to amplify transposed DNA (e.g. after integration of one or
more DM chromosomes) in a sense analogous to that observed for metaboli-
cally-selectable genes inserted at different sites in cells subjected to DNA
transformation (247).
4. Amplified oncogenes may exhibit other kinds of potentially oncogenic
mutations that presumably preceded an amplification step. For example, the
transformation-competent, point-mutated c-Ki-ras2 allele in the human
colon carcinoma line SW480 and the translocated c-ab1 locus on a Ph'
chromosome in the human CML line K562 are both modestly amplified (38,
95, 135). In a COL0320 subline bearing DM chromosomes, many of the
amplified copies of c-myc have lost the first (non-coding) exon as a result of
a rearrangement that joins a site in the first intron to DNA of unknown
provenance (3; M. Schwab, unpublished data); an atypical c-myc RNA
species presumed to represent the rearranged locus is at least as abundant as
RNA from the unrearranged gene. A rearrangement of c-erb-B also appears
to have occurred during its amplification in A43 1 cells (1 36). On the other
hand, no coding changes in codons 12,59, and 61 have been encountered in
c-Ki-ras cDNA clones from the mouse tumor line Y 1 (D. George, personal
communication).
5. Although oncogene amplification is assumed to be a somatic event, direct
evidence is generally lacking. In at least one patient with neuroblastoma, it
has been formally proven that N-myc amplification is a somatic event, since
a normal complement of N-myc genes is present in normal tissue (200).

FROM C-OIZC TO V-OIZC: COLLUSION OF

MUTATIONAL MECHANISMS

The most flagrant examples of mutant oncogenes are to be found among the
retroviral oncogenes, the transduced, multiply mutated, and highly tumorigen-

CELLULAR ONCOGENES 589

ic forms of cellular proto-oncogenes. What molecular events underwrite the
transduction mechanism? What changes in the oncogenes ensue? And which
changes are significant for the biological activity of the v-onc's? The complete
process of transduction of proto-oncogenes by retroviruses occurs too infre-
quently to studied under controlled conditions. Thus, the events must be
reconstructed instead from those artefacts presently available-the sequences
of v-onc's and c-onc's-and conjectures tested against further sequence data
and against attempts to recapitulate portions of the scheme under more favor-
able conditions than those existing naturally.
Several important differences between v-onc's and c-onc's have been en-
countered in the many comparisons now available (239).

1. v-onr's appear to be truncated versions of c-onc's, frequently lacking
coding sequence from both ends of the transduced domain. When missing
-the portion encoding the amino terminus of the c-onc product, the trans-
duced domain is generally expressed under viral translational signals as a
fusion protein encoded in part by viral replicative genes (gag or env).
Occasionally, v-om's retain the coding sequence from one or both ends of
the c-onc progenitor (e.g. in the cases of v-SYC, FBJ-fos, or v-Ha-rus), but
regulatory signals for synthesis and processing of RNA are invariably
provided by retroviral sequences (including the LTRs) present on both 5'
and 3` sides of oncogcnic sequences.
2. All of the introns that lie between transduced exons in c-onc's are absent
from v-onc's; but in some instances [e.g. v-src (226, 228), v-myb (108,
1 10) and MC29-myc (207, 252; C. Nottenburg, unpublished data)] there
remain short regions of intron-derived sequences from the 5' side of the first
transduced exon. These intronic sequences may be contiguous with the
exonic sequences in the host genome and thus include an intact splice
acceptor site that is used to generate both c-onc and v-onc mRNAs (as is true
for myb and src genes); or the intronic sequences may be non-contiguous
with exonic sequences, having been positioned upstream from a cryptic
splice donor site activated during the molding of the viral oncogene (e.g. in
the generation of MC29-myc).

3. Comparisons of the sequences of transduced genes and transducing retrovi-
ral genomes frequently display short (e.g. 4-6 bp) stretches of homology at
the recombination sites on either side of the onc region (239).
4. Several changes in the sequence of the transduced domain are present in
each v-onc. These commonly take the form of multiple base substitutions,
including silent, conservative, and non-conservative changes; in addition,
deletions of various sizes are often encountered [e.g. in the PRCII--s gene
(29a, 99)], and some of these shift the reading frame [e.g. in the FBJ-fos
gene (24)l.

590 VARMUS

Two central concerns arise from these comparisons: (a) what mechanisms
for transduction are compatible with observed differences between c-onc's and
v-onc's, and (b) which of the differences between c-om's and v-onc's are
significant factors in oncogenic potency?

The Favored Model for Transduction of Oncogenes

Any transduction mechanism must recombine retroviral sequences with both 5 '
and 3' sites in or near the oncogene, eliminate all introns between transduced
exons, and permit retention of some intronic sequences on the 5' side of the first
transduced exon. Base substitutions and deletions within the onc domain could
be explained by events that occur before, during, or after transduction and are
hence less problematic.
The prevailing model for conversion of proto-oncogenes to viral transfonn-
ing genes (16,18,226,244,249) recapitulates several of the mutational themes
of this essay.

The initial event is held to be an insertion mutation. During retroviral
infection, the transducing provirus is integrated on the 5' side of the c-onc to
be captured in the same transcriptional orientation. Although direct evi-
dence is lacking, it is attractive to presume that the insertion mutation either
initiates tumorigenesis as proposed above for several retrovirus-induced
tumors or at least stimulates growth; clonal expansion of the cell containing
an insertion mutation appropriate to instigate transduction would markedly
improve the probability that the subsequent rare events also necessary for
transduction will occur.
The second step is proposed to be a deletion mutation that extends from a
site within the provirus to a site within the oncogene. The latter site can be
within either an intron or an exon, accounting for the variety of 5' end points
for host-derived sequences in retroviral genomes. (Smaller deletions or
none are also compatible with the model provided that the polyadenylation
signal in the 3' LTR is occasionally overridden; aberrant splicing or later
deletions during reverse transcription would then have to be invoked to
explain the observed viral genomes.) The proposed deletion creates a hybrid
transcriptional unit, one that should generate a primary transcript with
sequences normally found at the 5' end of viral RNA joined to c-onc
sequences that extend to the 3' end of the gene.

3. The hybrid RNA will be recognized as a substrate both for splicing (to
remove the introns that lie between c-onc exons) and for packaging into
virus particles.
4. To join viral sequences necessary for viral replication to the 3 ' side of onc
sequences, the mechanism that facilitates high frequency recombination
between retroviral genomes is invoked. Although the details of that

CELLULAR ONCOGENES 59 1

mechanism are still in dispute, the central events are agreed to be the
formation of heterodimers during virus assembly and recombination during
reverse transcription, the latter step favorably influenced by homology
between the genomic subunits (33, 100, 103, 244a). Heterodimers com-
posed of a viral-onc hybrid transcript and a wild-type viral RNA from
another provirus in the same cell can be expected to recombine at relatively
low frequency, since any homology at the 3' ends of the RNAs will be
fortuitous. Occasional and irregular recombinational products have been
obtained in cell culture using model substrates (82,84), however, providing
one of the few pieces of experimental support for the transduction mecha-
nism. When partial deletion mutants of v-onc-containing viruses are used to
induce tumors or infect cells in cultures, the missing domain is recovered
from the c-onc at a modest frequency, implying that homology accelerates
the process of transduction (177,204,249,250). Such results are consistent
with the model discussed here and with others as well. It might be possible
to challenge aspects of the model more forcefully by using the few situations
in which the transduction of oncogenes appears to occur in cell culture
without obvious homology (178-1 SO), by attempting to capture metaboli-
cally selectable genes, or by exploiting the recent discovery that myc-
containing FeLV proviruses are produced in a substantial number of FeLV-
associated lymphomas arising in domestic cats (124, 141, 147).

The mechanism proposed for transduction of oncogenes requires further
embroidery to account for a number of retroviral genomes.

1. There are now five instances in which sequences from two apparently
unlinked cellular genes have been found in single viral genomes (Table 2).
This situation could arise if transduction occurred at a site of chromosomal
translocation that had previously fused the two-cellular domains to be
transduced; alternatively, the retrovirus that arises from the capture of one
host sequence might then direct transduction of a second.
2. Two peculiarities at the 3' end of v-src in the genomes of RSV (123, 202,
226, 228) also require more elaborate explanation. First, the sequence that
terminates the v-src coding region originates outside the c-src coding
sequence, about 900 bp downstream from the stop codon in c-src. This
situation probably results from deletion formation in the host chromosome
or during reverse transcription of hybrid RNA; either deletion would be
facilitated by an eight bp direct repeat shown to exist at the joining sites
(228). Alternatively, an aberrant splicing event could have joined the
downstream sequence to the penultimate coding exon. Second, v-src alleles
are flanked by direct repeats of viral sequences (approximately 100 nu-
cleotides in length). These are proposed to result from recombinational
events during reverse transcription: the 3' end of the onc region appears to

592 VARMUS

have been joined to a viral sequence in wild-type RNA that was also present
upstream of the oncogene in the viral-host hybrid RNA.

3. The several nucleotide substitutions and deletions within the transduced
domains of virtually all oncogenes (18,239) can be most simply explained
as the products of the error-prone process of reverse transcription (34), with
selection for any alterations that create a more effective transforming gene.
However, it is conceivable that some of the observed differences in nu-
cleotide sequence arose in the host chromosome in the early stages of
transduction, augmenting the proposed neoplastic effects of the primary
insertion mutations.

The Functional Significance of Differences
between c-onc's and v-onc's

Despite considerable experimental effort, it remains difficult to assign func-
tional attributes to the differences that distinguish v-om's from c-onc's. This is
so in part because it is often difficult to control the intracellular concentration of
gene products and hence to distinguish qualitative from quantitative effects,
and in part because the cell types most readily employed in the laboratory are
not necessarily the target cells for oncogenesis in the animal.
Several c-om's [including c-Ha-rusl (31), c-mos (20, 159), c-fos (137), and
c-myc (114; W. Lee et al, unpublished data)] have oncogenic effects on
cultured fibroblasts when expressed more efficiently than usual under the
influence of viral promoters, even though their protein coding sequence is
unaltered. Furthermore, some of these genes (and others) have been implicated
in rearrangements (Tables 4-6) that appear primarily to affect gene expression,
implying an oncogenic effect based on dosage. But all of the v-om derivatives
of these genes have multiple alteration in the coding sequence that may also be
significant. Most obviously, the differences between c-Ha-rusl and v-Ha-rus
include amino acid changes sufficient to render c-Ha-rasl transforming with-
out the replacement of its native promoter (Table 7). Evidence for the trans-
forming activity of normal c-myc under the control of an LTR has been obtained
in primary rodent embryo cells in the company of a mutant c-Ha-rasl allele; the
various v-myc alleles have yet to be compared to c-myc in a systematic fashion
in the several target cells for oncogenesis by myc genes. There are gross
differences between v-fos and c-fos alleles [the v-fos sequences are either fused
with sequences from two other genes, gag andfox, or altered by a frameshifting
deletion of 104 bp (240,241)], and these could enhance the neoplastic potential
of the genes in ways too subtle to appreciate in cultured fibroblasts. As an
illustration of this possibility, the v-ab1 sequence, expressed as a gag-ab1 fusion
gene in the genome of Ab-MLV, appears to require at least a portion of the

CELLULAR ONCOGENES 593

preceding gag domain for the transformation of lymphocytes but not for the
transformation of fibroblasts (173).
The transforming activity of the v-src gene seems to depend primarily upon
qualitative changesaither the scattered base substitutions throughout the gene
or the concerted change at its 3' terminus. Expression of c-src at a level above
that required for transformation of the same cells by v-src does not induce
transformation (166; H. Hanafusa, personal communication), although it is
possible that some high threshold for transformation by c-src exists. In con-
trast, an amount of v-src protein only slightly higher than that of c-src protein is
transforming, although again the dose is important: the cellular phenotype can
be radically altered with a four-fold change in v-src protein achieved by
manipulating expression with a hormone-responsive promoter (E. Jakobovits,
unpublished data).
In the aggregate, these findings suggest that full activation of proto-
oncogenes through retroviral transduction may depend upon multiple changes
that affect both the structure and expression of the oncogr ne product. Further-
more, these changes mimic many of the individual alterations described in
cellular oncogenes during tumorigenesis. It is likely that retroviral oncogenes,
particularly those with a long passage history, are repositories of a number of
mutations that enhance their neoplastic potential and thus favor their selection
under laboratory conditions. A completely satisfying analysis of the phenotypic
consequences of these iesions has yet to be performed.

INACTIVATION OF INTEGRATED V-OY~C GENES

A

SAMPLER OF MUTATIONAL MECHANISMS

Most of this essay has been devoted to those mutations that activate proto-
oncogenes; what can be said of mutations that cripple active oncogenes?
Numerous mutations of the latter sort have been isolated during the study of
viral oncogenes (l25,234a), but the vast majority of these have been generated
during virus life cycles, with or without the aid of mutagens, or by manipula-
tion of viral DNA in vitro. Hence, although crucial to an understandii g of the
functions of oncogenes, they are not directly informative about the mutations
that occur in eukaryotic chromosomes and fall beyond our purview here.
In a few instances, however, mutant viral oncogenes have been isolated by
seeking phenotypic revertants of virally transformed cell lines that carry only
integrated viral genomes (58, 134, 219, 246). In one particularly large collec-
tion of mutants, assembled from non-transformed derivatives of a rat cell
(called B31) carrying a single integrated RSV provirus, several kinds of lesions
have been shown to.inactivate the v-src gene (132, 158, 245, 246). Among

594 VARMUS

over 100 morphological revertants of B3 1 cells, approximately one-third have
lost the entire RSV provirus, presumably by either chromosomal non-
disjunction or a large deletion; about 5% have suffered deletions (1.5 kb to over
20 kb in length) that encompass the 5' LTR but spare the 3' portion of the
provir,is; two incuried insertion mutations following superinfection with
another retrovirus lacking an oncogene (MLV); at least two mutations involved
the loss or gain of a single base pair in the v-src coding sequence; and the rest,
over half, appear to be due to nucleotide substitutions in v-src.
The insertion and frameshift mutations in the B31 cell line have been
examined in greatest detail, in part because some of them undergo secondary
mutations that restore aspects of the transformed phenotype. Both of the
insertional mutagens are apparently intact MLV proviruses located between the
splice donor and acceptor sites for production of src mRNA in the same
transcriptional orientation as the RSV provirus (245). Mature transcripts from
the mutant loci appear to begin in the RSV 5' LTR and end in the MLV 3' LTR;
thus, v-src is not expressed in mRNA. One of the two insertion mutants reverts
to a wild-type phenotype by homologous recombination between the MLV
LTRs at a frequency of 10-6-10-7 per cell per generation, leaving behind a
single MLV LTR that has little apparent effect on the production of src mRNA.
The residual MLV LTR does not appear to serve as a promoter for v-src;
however, when retrieved from the cell and dissociated from the RSV LTRs by
molecular cloning, it is competent to direct the expression of a heterologous
gene lacking its native promoter (S. Ortiz, unpublished data).

Two frameshift mutations of v-src have been defined by nucleotide sequenc-
ing, and both conform to the dictum that loss or gain of a nucleotide pair is most
likely to occur by slippage of DNA polymerase in regions containing multiple
residues of a single base (221). In one case, the sequence GAZG was converted
to GA3G (132); in the other, the sequence CGSA was converted to CG4A (G.
Mardon, unpublished data). Both frameshift mutants revert to phenotypes
resembling wild type at moderate frequency. One revertant of the +1
frameshift mutant was found to make a v-src protein approximately 8 kd larger
than normal as a result of a spontaneous 242 bp duplication: since the dupli-
cated region included the primary mutation and created an additional + 1 shift
of frame at the boundary between the two copies, three successive i-1
frameshifts ultimately restored the correct reading frame (132). Two revertants
of the - 1 frameshift mutant result from insertion of one bp or the deletion of
two bp downstream from the primary mutation, with a portion of v-src trans-
lated from a fortuitously open reading frame between the primary and secon-
dary mutations (G. Mardon, unpublished data). Neither of these secondary
mutations involves expansion or contraction of pre-existing multiples of a
single nucleotide; in one case, the sequence ACTGAA was converted to
ACAA, and in the other the sequence GGCTG was converted to GGCTTG.

I

CELLULAR ONCOGENES 595

NEW DIRECTIONS TOWARD A MOLECULAR
DEFINITION OF CANCER

Our main business is not to see what lies dimly at a distance, but to do what lies clearly at
hand (Carlyle)

Attending to the main business at hand, the biochemical characterization of
mutations affecting cellular oncogenes in turnor cells, has provided the fuel for
this essay. However, the described oncogenic mutations are no doubt limited in
scope by the procedures available for discovering them; further technical and
theoretical innovation may now be required to provide a satisfactory picture of
oncogenesis, of the normal functions of proto-oncogenes, and of the nature of
somatic mutations in vertebrate oncogenes. This penultimate section considers
some prospects for the more complete view that may lie dimly at a distance.

Recessive Oncogenic Mutations

All of the lesions thought to influence neoplasia and summarized to this point
are presumed or documented to act in a dominant manner. However, there is
abundant rationale for believing that recessive mutations contribute directly to
oncogenesis. On purely speculative grounds, it is possible that cells could
progress to neoplasia because they are defective in functions required for entry
into an irreversible differentiation pathway or required for responsiveness to
external signals that normally retard cell cycling.
Such vague possibilities take on weight in view of genetic evidence for
inherited heterozygous states that strongly predispose to neoplasia in defined
organs, appear to depend on a second mutation for expression (172), and have
been dissociated from some of the known proto-oncogenes by linkage studies
(loa). Direct support for the idea that the second mutation might involve the
normal allele at the same locus on the companion chromosome has recently
emerged from studies of retinoblastomas (13, 30, 142). Individuals with
karyological or enzymatic signs of a small deletion mutation involving 13q14
in germinal cells develop retinoblastomas in which enzymatic assays for
esterase D (a marker closely linked to 13q14) and tests for restriction site
polymorphisms suggest conversion of a heterozygous to a homozygous or
hemizygous state. The studies to date indicate that a number of mechanisms-
including mitotic recombination, chromosomal nondisjunction with or without
duplication, gene conversion, and point mutation-could account for one or
another of the observed cases, although mitotic recombination seems particu-
larly prevalent (30). These findings should stimulate efforts to isolate the
posited retinoblastoma gene (Rb), to define the lesions that produce Rb-/Rb+,
Rb-/Rb-, and Rb-/O genotypes, and to seek better definition of other loci at
which recessive mutations are believed to contribute directly to cancer [e.g. the
Wilms's tumor-aniridia locus on llp13 (69)l.

596 VARMUS

Mutations of Proto-Oncogenes in Genetically
Malleable flosts

The only mutations of proto-oncogenes in vertebrate cells that seem presently
accessible are dominant and apparently oncogenic. Such mutations seem in-
herently unlikely to provide much insight into the normal functions of proto-
oncogenes; the phenotypic requirements may in addition impose a bias toward
certain types of molecular changes (e.g. genetic rearrangements). The discov-
cry of homologues of vertebrate proto-oncogenes, mainly c-onc's, in the
genomes of Saccharomyces cerevisiae and Drosophila melanogajter, tradi-
tionally favored for the induction and isolation of mutants, has enormously
increa: ed the opportunity for obtaining natural, induced, and engineered
mutants in such genes.
Thus far, most of the reported studies of c-onc homologues in yeast and flies
have been confined to structural analysis of such genes [e.g. ras-like genes in
yeast (76, 153, 172a) and src- and ubl-like genes in Drosophila (97,212)], but
genetic approaches are near or at fruition as well.

1. Homologous recombination during DNA transformation of yeast has been
used to inactivate the two genes (RAS1 and RAS2) homologous to Ha- and
Ki-uas genes; disruptive lesions in either locus do not affect viability of
haploids or diploids, but lesions in both loci prevent resumption of vegeta-
tive growth by haploid spores (104a, 230a). Similar mutation of another
yeast gene less closely related to vertebrate ras genes (76) is also a recessive
lethal (N. Segev, D. Botstein, personal communication).
2. Introduction into normal cells of a mutant yeast ras gene with a gly + Val
change in the position that corresponds to amino acid 12 in human c-Ha-
rusl interferes with efficient sporulation (1 04a).
3. Existing mutants that affect progression through the yeast cell cycle seem
likely in some cases to be assigned to the homologues of vertebrate proto-
oncogenes. The product of the cdc28 locus, for example, shows about
25-30% amino acid homology to several c-onc proteins and to cyclic
AMP-dependent protein kinase (128), and the cdc4 and cdc36 loci show
modest similarity to the host-derived sequence, ets, in an avian retrovirus
(170b).
4. By using known deficiencies that encompass the chromosomal positions of
oncogene homologues in Drosophila, it is possible to screen for new
recessive lethal mutants likely to map within loci of interest (M. Simon, M.
Hoffman, personal communication).
5. Striking variations in the mode and level of expression of the src and ab1
homologues occur during development of Drosophila (123a, M. Simon, B.
Drees , personal communication), offering promise of insight into multiple
effects of proto-oncogenes upon ontogeny.

CELLULAR ONCOGENES 597

Identijying Genes Whose Products Might Interact with
Oncogene Proteins

In other experimental systems it has proven instructive to harness genetic as
well as biochemical methods to identify protei is that interact in functionally
important ways (21). This is generally done by seeking extragenic suppressors
of primary mutations. At least two sorts of approaches seem plausible exten-
sions of this general strategy to oncogenes. First, viewing active oncogenes as
mutant proto-oncogenes, it should be possible t9 isolate cells with suppressor
mutations in other genes, e.g. host mutants that prevent transformation by
certain retroviral oncogenes, perhaps by altering targets for critical enzymatic
activities such as protein phosphorylation on tyrosine residues (40). There are
accounts of mutants that might meet these criteria for papovaviral or retroviral
oncogenes (e.g. 86, 101, 152, 187, 220), but the most thoroughly studied are
rat lines resistant to transformation by several, but not all, retroviral oncogenes
(151). Products of the restricted oncogenes-v-Ki-rus, v-Hams, v-fes, and
v-src-are not similar on biochemical grounds, suggesting that the mutation
might affect some relatively late function in neoplastic transformation. Further-
more, analysis of somatic cell hybrids suggests, surprisingly, that resistance is
dominar,t. These features may diminish the chances of using the mutants to
identify a protein that normally comes in direct contact with a product of a
restricted v-onc gene.
A second strategy is to isolate mutant cellular genes that suppress conditional
or non-conditional mutations in active oncogenes. To date, no cellular mutants
of this type have been described, although there exist mutant viral oncogenes
whose behavior is host dependent. One of the mutants of the B31 v-src gene,
for example, produces no apparent phenotypic effect in rat cells (in which the
v-src protein displays no tyrosine kinase activity), but the same gene trans-
forms chicken cells where its product is active as a protein kinase (158, 246).
DNA transformation and molecular cloning procedures theoretically could
permit the isolation of cellular genes that restore the activity of such mutant
proteins.

Assessing Epigenetic Changes
The findings summarized in this review further the argument that cancer has its
origins in genetic rather than epigenetic change. Still, important non-
mutational events seem likely to be intimately involved in oncogenic mecha-
nisms and may be unfairly neglected (191a). Measurement of polyadenylated
c-onc RNA is perhaps the most obvious way to begin a search for relevant
epigenetic change, but even when relatively dramatic, specific, and consistent
increases in c-onc RNA are found in the absence of overt mutations (66,213),
interpretation is uncertain. In particular, it is difficult to distinguish between
effects that directly influence the oncogenic phenotype and those that simply

598 VARMUS

reflect either the transformed state or the position of the tumor cell within a
cellular lineage. As experience broadens with cultured cells in many differenti-
ated states and as the potential increases for transforming such cells with mutant
oncogenes, it may be possible to distinguish between events that are critical for
achieving the transformed state and those that are only consequences of it.

A CONCLUDING PERSPECTIVE

Prior to the intensive pursuit of oncogenes, information about the structural
forms of mutations in eukzryotic chromosomes was derived largely from germ
line mutations of globin genes (130) and from the genetic alterations that
accompany the specialized process of immunoglobulin gene development
(234). Attempts to use bacterial genes introduced into cultured animal cells as
easily recovered targets for somatic mutations have been compromised by the
extremely high frequency of mutational events (26, 183). With molecular
cloning of several cellular genes that confer metabolically selectable pheno-
types in culture, somatic mutations affecting a number of genes in their normal
chromosomal context have been described in molecular terms, if not usually at
the level of nucleotide sequence. Among these are nucleotide substitutions and
deletions affecting the uprt gene (2 1 1); deletions, possible translocations,
amplification, and presumptive nucleotide substitutions affecting the hprt locus
(75); and amplification and deletions affecting the dkfr locus (197, 238).
However, the sheer number of scrutinized somatic mutations that affect proto-
oncogenes cannot be rivalled in other quarters of the eukaryotic genome, and
the analysis to date is certain to affect predictions about forms of mutational
change throughout the genome. Although the types of mutations observed
cannot be considered novel, the drama for geneticist and oncologist alike is to
be found in the startling patterns: the recurrence of certain types of mutant ras
alleles in a wide variety of tumors or the strong association of certain rearrange-
ments with certain tumors (e.g. N-myc amplifications in human neuroblasto-
mas, int-1 or int-2 insertion mutations in mouse mammary tumors, or c-myc
translocations in Burkitt lymphomas). What do such observations imply about
mutational mechanisms? Do some lesions occur with higher frequency than
expected from random mutation? Or can all be explained by selective pressures
during the growth of tumor cells?
What can be said about oncogenic mutations at present is largely descriptive;
with the possible exception of proviral insertion mutations, little is known
about the mechanisms by which such somatic mutations arise. It has been
argued that cells en route to malignancy may be mutators and more liable to
incur either nucleotide substitutions or genetic rearrangements than are normal
cells or cells less advanced on a neoplastic pathway (32). Whether the observa-
tions that sustain this argument are dependent on some specialized mutational

CELLULAR ONCOGENES 599

apparatus or on more frequent action of mechanisms shdred with normal cells is
beyond our power to decide at present.
More importantly for an understanding of cancer, we lack clear insight into
phenotypic consequences of mutations affecting proto-oncogenes. This de-
ficiency reflects in part our imprecise definition of neoplastic change and
present constraints on the ways in which we assay for it. But, as must be evident
from this survey, the difficulty of assigning biological significance to mutations
in tumor cells is implicit in the complexity of the neoplastic genotype, a? well as
the phenotype. Wc have seen several examples of single alleles affected by
multiple mutations and of cells affected by mutations at multiple loci, although
there is little reason to believe that the search for genetic change in neoplastic
cells has been exhaustive. To learn which lesions initiate tumorigenic growth,
which maintain it, and which lead to higher states of malignancy are tasks likely
to occupy students of oncogenic mutations for many years to come.

ACKNOWLEDGEMENTS
I thank many colleagues here and elsewhere for helpful discussions, J. Marinos
for artful preparation of the manuscript, and the NIH and ACS for support.

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Cellular myc oncogene is altered by
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plasmacytomas and is rearranged similar-
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2. Albino, A. P., LeStrange, R., Oliff, A.
I., Furth, M. E., Old, L. J. 1984. Trans-
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3. Alitalo, K., Schwab, M., Lin, C. C.,
Varmus, H. E., Bishop, J. M.  1983.
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