119

Harold Varmus

J.M. Bishop, The Molecular iology of NA Tumor Viruses: A Physician's Guide,

New Eh land Journal of Medic  e 303:67 -682, 1980, "Oncogenes", Xientific
-+ American 2 6r80,rch 19 2; Varmus, 6 Form and Function of Retroviral
Proviruses, Science 21 1982, Weinberg, A Molecular Basis of Cancer,
Sci. Amer., Nov. 1 3;          e Eherging Genetics of Human Cancer, NEE
309:454309, 1983.pT. Hunter, The Proteins of Oncogenes, -=. Amm. 251:70,
Aug. 1984.

Marx, J.C. The Case of the Misplaced Gene. Science 218:983-985, 1982.
The Molecular Biology of Tumor Viruses, Part 11, DNA Tumor Viruses, 1980; Part

IITRNA Tumor Viruses , 1982, Cold Spring Harbor Laboratory.

Review of Medical Microbiology, chapter 40.
Davis, et ale, Microbiology, Chapter 63.

- Cold Spring Harbor Symposium on Quantitative Biology, Vol. 42,  1979.
Bunn et al., Clinical course of retrovirus-associated adult T-cell lymphoma in
%6'~eh'd#&rus Seminar and/or instructor for further readings.

the U.S. New Engl. J. Med. 309:257-264, 1983.

%Ad-. % "F-4 'CS- -___c.------

-. -

/Wd

- 11. Introduction

role

for viruses in the etiology of most human tumors

and biochemical studies suggest that conventional

agents are not important in the majority of

                             to these statements -- evidence that
hepatitis B vir& is a causative factor in hepatoma, that a human retrovirus may
cause a form of T cell leukemia and lymphoma, and that herpes viruses may play
some role in a few human cancers -- will be considered in the herpes irus and
hepatitis virus lectures and in the seminar on tumor viruses. /k A, -

    This lecture is intended primarily to acquaint you with the rapid advances
now being made in the understanding of how viruses cause cancers in animals
other than man and how tumor virology has introduced us to a class of cellular
genes, called proto-oncogenes, that appear to be activated In many human tumors
of unknown etiology. Whether or not human cancer is virus-related,  animal tumor
viruses offer an important opportunity to learn how cells work,  how their
behavior can be altered by a very small number of viral genes, and how cellular
genes related to viral genes might be implicated in human cancer.

-1tclPvf

Y

- 111. General Considerations

A. Transformation

Tumor viruses, by definition, have the capacity to produce tumors upon

infection of appropriate animals.  In practice, most work with tumor viruses is
now conducted in cultured animal cells; in the appropriate cells,  tumor viruses
generally have the capacity to cause an in vitro analogue of tumor production,
referred to as "neoplastic transformation" of cells.
tumor viruses involves a stably inherited change in one or more properties of

Transformation of cells by

-2-

cell behavior ; most commonly, such change includes altered morphology
(transformed cells look more like tumor cells than like normal cells) and
freedom from normal Rrowth restraints (transformed cells,  in contrast to normal

L ells, will grow in random array to a very high density, without attachment to a
  solid surface, and at low serum concentration; they will form a nfocusn of
  piled-up cells, permitting an assay for viral transformation; and they will
  usually produce tumors when injected into animals). In addition, various
  biochemical changes (alteration in properties of cell membranes, production of
  new cellular and viral proteins, reduction of cyclic AMP levels, etc.) may also
  accompany transformation. As discussed below, a major focus of interest in
  tumor virology is an understanding of how a single viral gene can produce such a
  profound effect upon cell behavior.

B. DNA and RNA tumor viruses

Tumor viruses, like other viruses, are classified according to the nature

of their genomes.
exhibit a variety of biological effects, but (fortunately for students) the
biochemical and structural properties of these viruses all appear very similar.
The DNA tumor viruses are more complex, since the major classes of DNA viruses
(papova viruses, hepatitis B viruses, adenoviruses, herpesviruses, and pox
viruses) all have members which are tumorigenic (see appendix A and Table 1).

There are many specific examples of RNA tumor viruses which

f

DNA TUMOR VIRUSES: GENERAI, PROPERTIES

Three size classes:

small (DNAsize 3-5 kb )---papovaviruses (SV40, polyana, papillm

  viruses: other hm and simian isolates) ;
  hepatitis B viruses (man, modchuck)
)--adenoviruses (fm several animal species,
  e.g., chickens, mnkeys, man)

e.g., frogs, chickens, mDnkeys, man);
pox viruses (generally pmduce benign tmrs;
found in several species)

medium (DNA size 40 kb

large (DNA Size 100-15Okb) -l+erpesviruses (frcm several animal species,

C. lysogeny model

Since tumor viruses are able to produce stable alterations in host cells

and since those changes appear to be maintained by the activity of viral genes,
it is not surprising to learn that tumor virus DNA has the capacity to integrate
covalently into the genome of the host cell; thus tumor viruses appear to be at
least partially related to lysogenic bacteriophage (Table 2).  As in the case of
most bacteriophage, the genomes of many tumor viruses appear to pass through a
stage in which they are in the form of double stranded DNA circles; this is even
true of RNA tumor viruses (see below). There is no evidence that tumor viruses
produce repressors of the sort responsible for the maintenance of lysogeny.
However, in many cases, there is limited expression of viral genes in cells
transformed by tumor viruses.

'1 2'1

-2a-

TAELE 2. REL~ANCE OF LYSOGENY MODEL TO TUMOR VIROLOGY

CIRCULAR LlMITED

&DNA VIRAL EXPRE=SSION OF

CLASS ExANpI;E GENaME PHASE INTEGRATION REPRESSOR VIWGENES

Temperate Larnbaaphase LineardSDNA + +

   twr      ,9740      Circular &DNA     + +

Mtumor   Rmssaraxm   LinearssM      + +

Phase

virus

VirUS Virus

FIGURE 1.

ALTERNATIVE CONSEQ~NCES OF SV4O ~~ECTIO~ IN D~FF~~T CEU TYPES

MONKEY CELL

(African green monkey)

sv40

Viral DNA replicated
Early and late genes expressed
Virus produced No virus produced
Cell lysed Cell Transformed

Viral DNA integrated
"Early" genefsf expressed (T antigen]

-3-

D. Permissive nonpermissive cells

-- DNA tumor viruses, with the exception of some herpes viruses and perhaps

hepatitis B viruses, do not normally produce tumors in their natural hosts, and
cells from the natural host are generally permissive for virus replication.
Ordinarily, DNA tumor viruses only transform cells when some component of the
virus or the host cell is defective for replication. Most commonly,
transformation by DNA tumor viruses is studied in foreign (or heterologous) host
cells which, for generally unknown reasons, are defective in their ability to
support viral replication (i.e., are nonpermissivr for that virus).  For example
(Fig. 1) the DNA tumor virus SV40 (simian virus 40) replicates in monkey cells,
produces cytopathic effects (e.g., vacuolization in rhesus monkey cells or lysis
in African green monkey cells) but it transforms rodent cells (e.g., mouse, rat,
hamster) in which it is unable to replicate.

&,..hwoJ

-I__ RNA tumor viruses, by contrast, are frequently implicated in cancers in

their natural hosts, and they are capable of transforming cells while
replicating within them. However, RNA tumor viruses can also transform
heterologous host cells in which they are Unable to replicate. In other worcts,
-- RNA tumor viruses can transform permissive_ or non-permissive host cells.

IV. Properties of RNA Tumor Viruses

R us have several unusual characteristics which are not

")

considered elsewhere in this course; these characteristics have profound'
implications for molecular biology and they have strongly influenced the search
for human tumor viruses.

Structural and genetic features

A* --- --

#I ,e

-s have been identified in a wide variety of animals (e.g.,

viper, fish, birds, rodents, ungulates , cats , and several primates including
man) but their biochemical and structural features are highly similar:

   1. The viruses are enveloped, leaving infected cells by budding through

-/

cytoplasmic membranes and entering cells by interacting with host receptors, as
described for several virus classes in lectures on replication.

2. The genome is in the form of two identical subunits of 5-10 thousand

bases of single stranded RNA.

3. The virus core contains a virus-coded RNA-directed DNA polymerase

("reverse transcriptase") that converts the RNA genome to double stranded DNA
during the virus life cycle.

4.

     Newly synthesized viral DNA forms a closed circle and is then
integrated into host chromosomes by a precise mechanism tht generates a provirus
structurally similar to many transposable elements (see previous lecture). The
genes of the provi
permissive cells a
retroviruses.

xpressed by host cell machinery. Note th t -

ysedfi by

9

stent ly infected -often transformed,

5. Retroviru ion, but many viruses are

defective for replication ore of these three genes)

-4-

123

and must be complemented by a helper virus that supplies the missing functions
in co-infected cells (much as described for defective transducing phages in
previous lectures).  Many of these replication defective viruses carry a gene
that mediates the oncogenic effects of the virus (a viral oncogene, derived from
a normal cellular gene; see below). But retroviruses without oncogenes  can also
produce a variety of tumors and other kinds of pathology (e.g., anemia,
osteopetrosis, etc.).

6. The most commonly studied RNA tumor viruses are:

avian sarcoma viruses (e.g., Rous sarcoma virus)
avian leukosis virus
mouse leukemia virus
mouse sarcoma virus
mouse mammary tumor virus
feline leukemia and sarcoma virus s
simian sarcoma virus -d-
human T cell

dj-

   7. and retroviral
proviruses are thus endogenous to the chromosomes of most if not all species,
including man.

Some properties of endogenous retroviruses are listed below:

e

-- - Genetically transmitted in the form of proviruses
-- May be induced chemically (see Section V1.C below)
-- Often xenotropic (grow well in foreign hosts, poorly

-- Occasionally oncogenic (e.g., murine leukemia virus

-- Probably present in all vertebrates, including man

in species from which they are isolated)

and murine mammary tumor virus) but more often not pathogenic

V. How do Viruses Transform Cells?

   The major attraction of tumor viruses as laboratory tools is their
capacity to alter cell behavior with very few genes.
transformation upon the continued synthesis of the products of these  "viral
oncogenes" has been shown by the use of temperature sensitive mutants with
lesions in these genes (Table 3).

The dependence of

The genome of the most intensely studied DNA virus, SV-40,  is divided into

an "early" region (expressed prior to replication of viral DNA)  and a "late"
region.
proteins, and it is not expressed in transformed cells (which do not produce
virus, see above 1. The "early" region also contaii&overlapping
genes, encoding at least two proteins; genetic studies with viral mutants
indicate that one or more of these "early" proteins are necessary for
transformation of non-permissive cells and for replication of viral DNA in
permissive cells.

The "late" region consists of three overlapping genes for coat

The best studied RNA tumor virus, Rous 8arcoma virus of chickens, has four

genes, only one of which (the viral one gene, called E in Rous sarcoma virus)
mediates transformation.
DNA, is not required for virus replication; in addition, the gene product
transforms permissive as well as non-permissive cells. The product, a

This gene, in contrast to the early region of SV40

124

-5-

phosphoprotein of 60,000 daltons, has the intriguing capacity to phosphorylate
tyrosine residues in certain proteins and it is found mainly in plasma
membranes. Furthermore, this gene has been derived from a normal cellular gene,
called c-E (see below Section VII).

TABLE 3

ExAE.IpLES OF VIW ONCOGENES

Simian Virus 40

Rous Sarccana Virus

Property

src gene

~arly region -

(A gene)

Size of protein ( s ) ca. 85,000 and ca. 60,000

17,000

-red for viral Yes

replication

Required for trans- Yes

formation

No

Yes

Functions of product DNA binding (stk Protein kinase (@os-

lates DNA synthesis) phorylates tyrosine
ATPase

residues)


(mainly)

(Others)

mation of product Primarily nuclear Plasma mn-brane

lar homlogue No Yes (see section

<addition to E, there are over 15 distinguishable retroviral oncogenes,

each of which has been derived from normal cellular genes, called C-E'S or
proto-oncogenes. Several of these other oncogenes also encode protein kinases
specific for tyrosine and found at the plasma membrane, but most produce
proteins that clearly have other kinds of biochemical properties (Table 4).  In
several cases, determination of the nucleotide sequences of the genes show that
they are closely related, suggesting that retroviral oncogenes are derived from
a few families of related cellular genes.
under normal circumstances as regulators of growth and development.
(e) the gene has been shown to be derived from a host gene for a peptide
hormone called platelet-derived growth factor (PDCF); in another case (erb B),
the gene encodes the receptor for the epidermal growth factor (EGF).
importance of these proto-oncogenes in human cancer is discussed further below

Such genes are thought to function
                  In one case

The

(Section VII).

-Sa-

TABLE 4.

A SAMPLING OF RETROVI RAL ONCOGENES :

ANCESTRIES AND PROTEIN PRODUCTS

.I

AMINO ACID
HOMOLOGY

TYR KINASE

J

EGF-R- ERB-B

            MOS
PDGF- SIS

DNA BINDING-MYC I'

FOS

I

SKI

PI OR

CYTOPLASM

~CLEUS

'1 26

VI. Mechanisms of virus rescue

   It is of inherent interest to know whether viral genes can be recovered
from transformed cells which fail to produce virus (e.g., permfssive cells
transformed by defective viruses, or nonpermissive cells transformed by non-
defective viruses). In addition, development of such techniques may be impor-
tant in searches for human tumor viruses.
erally used.

Three types of approaches are gen-

A. Cell fusion(Fig. 3).

A _transformed =-permissive cell is fused with

an uninfected ~ermissive cell to form a heterokaryon (cell with two or more
different nuclei) which provides the factors necessary for virus replication.
This method can be used with both RNA and DNA viruses.

RESCUE OF IU.f<tClZ \iiRUS f:AOt,\

I~~S'T

N 0 I\! - 1' E Rh\ I SS Iv E F IF3 P 0 B 1 A ST

TRANSFOR!AED CELL \%`!Ti+

INFECTED BY Ti'MCR VIRUS INTEGRATED VIRAL GENOMF,

NO VIRUS PRODUCTION

Figure 3

I!

*?

?. 8

-$Lc-,;m=m

-.

H E TE R 0 K A P YO N P 2 0 D !! C t t4 f;

TUMOR VIRUS

FUSION OF TRANSFORMED
M 0 N - P E R M 1 S S I V E C E 11 W 1Ti-i
M OR&' A 1, P E R M I S S I V F C E L L

fparornyxo virus used
as fuzinq agent)

B.

Helper virus.

A permissive cell transformed by a virus defective in

127

-7-

replication function(s) is superinfected by a whelpern virus with normal
replicative powers. The helper virus provides the factors required for
replication of the defective transforming virus, and both are produced by the
cell.  This phenomenon has been observed with both RNA and DNA viruses.

C. Chemical induction.

1)

      Treatment of certain =-permissive cells transformed b~ DNA tumor
viruses with a variety of chemical agents (some of which induce lysogenic phage
in bacteria) results in production of virus, presumably by facilitating excision
of the integrated DNA genome.

2)

Treatment of normal cells with similar inducing agent8 most

commonly halogenated pyrimidines, such as iododeoxyuridine (IUdR) or
bromodeoxyuridine (BUdR) often results in the production of endogenous RNA tumor
viruses (see above Section XV). The mechanism of the induction is unknown.
Only endogenous RNA viruses have been observed; their properties are considerdd
more completely below.
cells may also be augmented by such inducing agents (see Tumor Virus Seminar).

Production of retroviruses from exogenously-infected

VII. Cellular proto-oncogenes

   "Proto-oncogenes" in the genomes of normal cells were first discovered by
molecular hybridization experiments performed with the viral gene (v-~) ahown
to be responsible for the oncogenic properties of Rous sarcoma virus (see part
V, above).
extraordinary result has been obtained: a very similar gene is present in the
normal genome of the animal species from which the virus was isolated.
Moreover, these genes are not part of some endogenous retroviral provirus, but
instead constitute genes recognized as cellular in origin by their architecture
(introns and exons), their mode of expression, and their conservation throughout
evolution.  (All are present throughout vertebrates, including man, and some are
even found in insects, worms, and yeast).
oncogenes are not known, they generally produce proteins very similar to the
protein products of viral oncogenes in small amounts, and it is presumed that
their high degree of conservation bespeaks some fundamental role in growth or
development (see Table 4).
oncogenic RNA viruses implies that the genes were "transduced" by retroviruses
that lacked oncogenes during their passage through host animals.  The fact that
each atember of this group of genes can mediate oncogenic events (transformation
of cultured cells and tumor induction in animals) when it is part of a viral
genome suggests that each might have oncogenic potential in its natural setting,
within the genomes of normal cells,  e.g., if altered by mutations that affect
the level of expression or the nature of the protein product (Fig. 4).

With each of over 15 different retroviral oncogenes the same

Though the functions of most proto-

The appearance of these genes in the genomes of

Several lines of experimentation support this view:

(1 ) In vitro manipulation.

                 It has been shown that a few (though not all)
cellular proto-oncogenes, e .go, c-E and c-F, directly isolated from normal
cell genomes, can transform cells and make t=rs, if' .programed to be expressed
efficiently when reintroduced into normal cells.

(2)

Retroviral insertion mutations activate proto-oncogenes. Some of the

-8-

retroviruses that lac.. their own oncogenes (e.g., Le chicken or mouse leukosis
viruses or mouse mammary tumor virus)  can nevertheless induce tumors of various
types. Tumor production appears to be dependent upon the stimulation of
efficient expression of a "cellular oncogene" by proviral DNA inserted nearby
during infection. This mechanism has been clearly implicated in the case of B
cell lymphomas in chickens, since proviral DNA is almost always found adjacent
to a known proto-oncogene (first identified by its virtual identity to the viral
oncogene in an acute leukemia virus of birds); in these tumors, the proto-
oncogene, c-mx, is expressed about 50 times more efficiently than in normal B .

(3)

Point mutations of cellular ras genes make them oncogenic.

DNA

isolated from some tumors of animals and man, regardless of whether a causative
agent (8.g.) chemical or virus) is known, can transform the behavior of cultured
mouse fibroblasts from a normal to malignant phenotype. The most likely
interpretation of these results is that a mutation has occurred during the
development of the original tumor, rendering a normal cellular proto-oncogene
into a "transforming gene".  Remarkably, several of these "transforming genes",
including some isolated from human tumors of the lung, colon, and bladder, have
proven to be members of the group of proto-oncogenes identified by their
homology with viral oncogenes (Table 6). In.severa1 cases, the mutant
"transformingn genes, all members of the E gene family thus far (Table 41,
have been cloned in bacteria and subjected to nucleotide sequencing.
resul
to M?posistions, codonsmd 61 in the
altered properties of the cehlular genes.

The

show that sinule base substitutions, different in each case but confined

genes, are responsible for the

TABLE 6. Cellular Genes Implicated Tumorigenesis

By the 3T3 Cell Transfordlation Assay ,'

- Gene - Tumor A1 t era t ion

c-Ua-E Bladder CA Single base substitition

Lung CA
Mammary CA

.

C-Ki-E Lung CA, Colon CA, Single. base substitution

N-E Neuroblastoma Single base substitution .
           Myeloid leukemia ..

others

0

Rhabdomyosarcoma

(4)

Rearrangements of proto-oncogenes affects their expression and may
        In a number of human and murine tumors, the cellular

-- render them oncogenic.
homologues of retroviral oncogenes have been found to be grossly altered, either
by (a) chromosomal translocations or (b) gene amplification (some examples are
provided in Table 7.)

(a) The translocations frequently involve the same chromosomal sites

in many tumors of the same type; most attention has been given to translocations
in (human) Burkitt's lymphoma that join c-E on chromosome 8, band q24, to the
immunoglobulin heavy chain locus on chromosome 14, band q32, and translocations

'

129

that join the same loci ( n chromosomes 15 and 12) in mouse plasmacytomas. The
translocations appear to alter the level of expression of c-m> in at least some
cases, but the effect   the translocations upon the neoplastic process has yet

to be defined. J

(b) Amplification has been shown to occur in several types of tumors,

affecting several oncogenes (Table 7).
generally be seen during karyotyping as a homogeneously staining region within a
chromosome or as multiple, small chromosomes lacking a centromere (double minute
chromosomes).
in oncogene expression.
human cancers (e.g., N-E in neuroblastomas) and may be useful in diagnosis.

The amplified unit is large and can

The increase in gene dosage is generally matched by an increment
      Amplification sometimes correlates with the staging of

-- TABLE 7.

CELLULAR GENES IMPLICATED IN NON-VIRAL

HUMAN AND MURINE TUMORS BY REARRANGEMENTS

-----

Gene

-

Tumor

--

Alteration

C-!w Burkitt 's lymphoma    Translocations

C-fi Myeloid leukemia      Translocation

Plasmacytoma

(Ph,iladelphia chromosome)

C-m Myeloid leukemia Amp 1 i f i cation

Apudoma
Small cell lung

carcinoma

c-Ki-~ Adrenocortical Ca Ampli f i ca t ion

N4lU Neuroblastomas Amplification

i2-3

Retinoblasto s

More than one of these var
sometimes found within the same tumor cell. This is consistent with the
generally accepted notion that full fledged cancer cells arise as a result of

A major goal of contemporary cancer research is the explicit
se several steps in the genesis of a  cancer.

P-uAUe4c

mu ations that affect cellular oncogenes are

The general implication of these several findings is that tumor viruses (in

  ticular, retroviruses) may have led investigators to recognize a class of
normal cellular genes (%ellular oncogenes") that may be involved in cancers
initiated by various infectious, chemical, physical, or genetic mechanisms.
Therefore, the normal functions of these genes, the kinds of changes they suffer
during tumorigenesis, and the properties that make them oncogenic under certain
circumstances are all matters of extraordinary interest to oncologists. As
viewed in Table 8, these genes can be thought to have undergone a variety of
"activating" events: transduction to become a viral oncogene, point mutation to
produce a gene that transforms cultured cells; or insertion mutation,
amplification, or translocation that affect gene expression. Lastly, in vitro,

130 - 10 -

r

NORPlAL
CELLULAR
ONCOGENES

some of these genes can be made oncogenic by addition of strong regulatory

           TRANSDUCTION --e RETROVI RAL ONCOGENE

Signals o

+ AMPLIFIED GENE


.) TRANSLOCATED GENE

REARRANGEMENTS

I

L

TABLE 8.

\ IN VITRO

b OVEREXPRESSED GENE

ElAN I PULAT I ON

Appendix A.

Additional Historical Information About DNA Tumor Viruses

Pa ova viruses ---viruses with small, circular (double-stranded) DNA genomes
&.T-mase pairs); "papova" derived from common members of this group:
Papilloma viruses (from several natural hosts); Polyoma virus of mice; and
Zcuolating viruses (causes vacuole formation ups infection of natural hosts 1

which simian virus 40 (SV 40) is most famous example.

therefore not well studied.

Polyoma virus ---common in wild mice in which the virus occasionally produces a
variety of types of tumor (hence *cpolyn "oma," or many tumors); in cell culture,
undergoes lytic, replicative cycle in its natural host (mouse) and transforms
certain heterologous, nonpermissive hosts (e.g., hamster).

SV 40 ---discovered in rhesus monkey cells during development of polio vaccine;
causes tumors in and transforms cells from certain heterologous, nonpermissive


cells.

/-- hosts (e.g., mouse and other rodents); undergoes replicative cycle in monkey

Hepatitis Etype viruses ---discovered first in man (see hepatitis lectures),
later in woodchucks, ground squirrels, and ducks; associated with hepatocellular
carcinomas in man and woodchucks; inability to grow virus in tissue culture has
slowed study of its replication and oncogenicity.

- 11 -

Adenoviruses ---viruses with medium-sized , linear (double-stranded
(ca. 35-40 kilobase pairs); found in many species, including man; the several
human types show low, medium, or high oncogenlcity when used to infect newborn
rodents (especially hamsters) or rodent cells in culture (these are
nonpermissive, heterologous hosts) ; replicate in cells from natural host (man)
and cause mild, acute GI and respiratory illness (see lectures on respiratory
infections) o

DNA genomes

Herpes viruses ---enveloped viruses with large, linear (double-stranded) DNA
genome (ca. 150 kilobase pairs); associated with a varlety of diseases,
including tumors, in the several hosts in which these viruses have been found
(e.g., chicken, frog, subhuman primates, and man); evidence for oncogenicity in
man is not conclusive; virus replicates in cells from natural host, and, after
irradiation to damage replication genes, it can transform certain heterologous
host cells in culture (see herpes virus lecture).

Epstein-Barr virus (EBV) ---a herpes-like virus originally found in cells from
African patient with Burkitt's lymphoma; now commonly seen in human lymphocytes
from normal as well as diseased persons; it is the causative agent of infectious
mononucleosis and has been indirectly implicated in causation of African
Burkitt's lymphoma and nasopharyngeal carcinoma (see herpes virus lecture).

131

FIGURE 4.

THE DOMINANT P,

R

DIGM

NORMAL CELLS --==-> CANCER CELLS

ONCOGENE (SI

MUTATION (S I
BASE CHANGES
REARRANGEMENTS

PROTO-ONCOGENES

1

INAPPROPRIATE AMOUNTS
OR ALTERED KINDS OF
REGULATORY PROTEINS

J,

NORMAL REGULATORS

OF GROWTH