VIRUSMYTH HOMEPAGE
Progress in Nucleic Acid Research and Molecular Biology
43:135-204, 1992
Latent Viruses and Mutated
Oncogenes: No Evidence
for Pathogenicity
PETER
H. DUESBERG AND
JODY
R. SCHWARTZ
Department of Molecular and Cell Biology
University of California at Berkeley
Berkeley, California 94720
IV. Mutated Oncogenes, Anti-oncogenes, and Cancer
A. Mutated Proto-myc Genes and Burkitt's Lymphoma
The transforming gene of the directly oncogenic avian
carcinoma virus MC29 contains a specific coding region, now termed myc
(217), derived from a cellular gene termed proto-myc (218). Thus,
the viral myc gene is a genetic hybrid that consists of a strong
retroviral promoter linked to a coding region that is a hybrid of virus-
and proto-myc derived sequences (219). This viral myc gene,
like synthetic hybrids in which the native proto-myc promoter is
replaced with that of a retrovirus (40, 42), is expressed to about 100-fold
higher levels in all virus-transformed cells in vitro and in viral
tumors than the cellular proto-myc genes (220-222).
The cellular proto-myc gene, located on chromosome
8, is rearranged with immunoglobulin genes from chromosomes 2, 14 and 22
in all (29) or most (30) cell lines derived from Burkitt's lymphomas. However,
direct cytogenetic studies show that chromosome 8 is rearranged in only
about 50% of primary Burkitt's lymphomas (223-226). Analogous rearrangements
have also been observed in the proto-myc genes of mouse plasmacytoma
cell lines (1, 8, 36). The rearrangements do not alter the coding
region of proto-myc genes. Most rearrangements link the proto-myc
coding regions to genetic elements from cellular immunoglobulin genes
in the opposite transcriptional orientation (1, 8, 36). Other rearrangements
in Burkitt's lymphomas do not affect the location and structure of proto-myc
on chromosome 8, but instead rearrange regions 3' from proto-myc (36,
227-232). Because both retroviral myc genes and the rearranged
proto-myc genes of most, but not all, Burkitt's lymphomas differ
from normal proto-myc genes in truncations 5' from the coding region,
and because both were found in cancers, the viral and rearranged cellular
myc genes were proposed to be equivalent oncogenes (6, 8, 29, 30).
The transcriptional activity of the rearranged proto-myc
genes in lymphomas is moderately enhanced, not altered, or even suppressed
in Burkitt's lymphoma cells compared to normal proliferating cells (5,
30, 36, 216, 227). It is thus nearly 100-fold lower than that of viral
myc genes or proto-myc genes artificially linked to retroviral
promoters (40, 42, 220-222, 233).
Moreover, rearranged proto-rnyc genes from Burkitt's
lymphomas do not transform any human or rodent cells upon transfection
(5, 36, 38)-even if they are artificially linked to retroviral promoters
(234, 236). In efforts to develop a system that is more efficient
than transfection for introducing mutated proto-myc genes into cells
or animals, synthetic avian retroviruses with the coding region of the
human proto-myc gene were constructed (233, 237). Since these
viruses transform avian cells, it was concluded that "ungoverned expression
of the gene can contribute to the genesis of human tumors" (237).
However, transformation of human cells was not demonstrated. Moreover,
three independent studies report that murine cells cannot be transformed
by authentic avian (238) and synthetic murine retroviruses with myc
genes (239, 240), signaling a restricted transforming host range of
myc genes.
Several arguments cast doubt on the hypothesis that rearranged
proto-myc genes of Burkitt's lymphomas are functionally equivalent
to retroviral myc genes and thus oncogenic:
1. Rearranged proto-myc genes from Burkitt's lymphomas
or mouse plasmacytomas lack transforming function in transfection assays,
while retroviral myc genes and proto-myc genes driven by
retroviral promoters are sufficient to transform at least avian primary
embryo cells (40, 42, 237). This indicates that the proto-myc genes
from lymphomas and viral myc genes are functionally not equivalent.
2. Since expression of rearranged proto-myc genes
from lymphomas is either the same as, or similar to, that of normal proto-myc
genes, and their coding regions are identical, rearranged proto-myc
cannot be sufficient for lymphomagenesis. By contrast, viral myc
genes are oncogenic, owing to a 100-fold higher level of myc expression.
3. Primary Burkitt's lymphomas with normal chromosome
8, and with rearrangements of chromosome 8 that occur 3' from proto-myc
and thus do not affect the structure and regulation of the proto-myc
gene, indicate that proto-myc translocation is not necessary for
Burkitt's lymphomas.
It follows that rearranged proto-myc genes of human
and animal tumors are transcriptionally and functionally not equivalent
to viral myc genes, and that they are not necessary for lymphomagenesis.
In view of this, the demonstration (237) that human proto-myc
transforms avian cells after it had been converted artificially to a retroviral
myc gene is not relevant to its hypothetical role in human tumors.
This claim is all the more questionable because even retrovirus-promoted
myc genes appear unable to transform non-avian cells. Instead, such
experiments model the genesis of a viral myc gene from a retrovirus
and a cellular proto-myc gene by rare illegitimate recombination
(37). The critical step in this process is the substitution of the weak
cellular promoter by the strong retroviral counterpart (40, 42).
Thus, there is currently only circumstantial evidence
for the hypothesis that rearranged proto-myc genes play a role in
Burkitt's lymphomas. This evidence includes the structural, but not functional,
similarity to viral myc genes, and the approximately 50% incidence
of chromosome-8 rearrangements with breakpoints near proto-myc in
primary lymphomas. In view of this, rearranged proto-myc genes either
may be involved in a mechanism of leukemogenesis that is not analogous
to the viral model, or they may not be involved at all. Since the incidence
of chromosome-8 rearrangements is higher in lymphoma cell lines than in
primary lymphomas, it has been pointed out that the rearrangement may favor
lymphoma cell growth in culture (225).
In efforts to link the proto-myc rearrangements
with a role in tumorigenesis, despite these discrepancies with the one-gene
model, it was postulated that rearranged proto-myc genes may cooperate
with other genes for carcinogenesis (236, 238, 241 ). To test these ad
hoc hypotheses, transgenic mice were constructed that carry rearranged
proto-myc genes linked to artificial promoters and hypothetical
helper genes in every cell of their bodies. However, only some of these
mice developed clonal tumors late in their lives (236). This indicates
that even these combinations are not sufficient for carcinogenesis. Consequently,
further helper genes were postulated (236, 241).
An alternative hypothesis suggests that the appearance
of certain chromosome abnormalities is sufficient for lymphomagenesis.
It is consistent with this proposal that cytogenic studies have identified
chromosome abnormalities in all Burkitt's lymphomas, even in those that
lack rearranged proto-myc genes (224-226). The reason that a high
percentage of these rearrangements include proto-myc and immunoglobulin
genes may be a consequence of the natural functions of these genes in B
cells, namely generating antibody diversity in which proto-myc genes
may play an active or passive role.
B. Rearranged Proto-abl Genes
and Myelogenous Leukemia
Human myelogenous or granulocytic leukemia develops in
two stages. The first is a chronic phase that may last, on average, 3-4
years. During this phase, immature myeloblasts are overproduced in the
bone marrow and appear in the blood, but may differentiate into functional
cells. This hyperplastic stage is followed by a terminal blast crisis of
several months, during which non-functional leukemic cells emerge (242,
243). The leukemic cells of both the chronic and terminal stages in 85-90%
of patients are marked by a reciprocal translocation between chromosomes
9 and 22. The rearranged chromosome 22 is termed the Philadelphia chromosome
(193). In the remaining 10-15% of cases, chromosome 22 is rearranged with
other chromosomes (193, 242-245). The reciprocal translocation between
chromosomes 9 and 22 substitutes the 5' end of the coding sequence of the
proto-abl gene on chromosome 9 with a 5' regulatory and coding element
of a gene of unknown function, termed bcr (for breakpoint cluster
region), from chromosome 22 (33, 246-248). The breakpoints with regard
to the proto-abl gene vary over 200 kb (249), but those within bcr
fall in a range of 5.8 kb (8, 247, 248). The transcriptional activity of
the proto-abl gene is virtually unaffected by the translocation
(8, 246).
The proto-abl gene is the cellular precursor of
the transforming gene of the murine Abelson leukemia virus (6). This virus
is sufficient to cause terminal myelogenous leukemia in susceptible mice
within 3-5 weeks after infection (250, 251). In this virus, the promoter
and 5' coding sequence of proto-abl are replaced by retroviral counterparts.
Since the 5' proto-abl coding regions are substituted by heterologous
genetic elements in both the virus and the leukemias, it has been postulated
that the structurally altered proto-abl gene of the leukemia is
a cellular oncogene that is functionally equivalent to the transforming
gene of Abelson virus (7, 8, 246, 252). However, the Abelson virus or provirus
(253), but not the DNA of human leukemic cells, is capable of transforming
the mouse NIH 3T3 cell line in vitro (8).
The failure of the bcr-proto-abl hybrid
genes to function like the virus could be a technical problem, because
the hybrid genes may not be transfectable due to their large size of over
200 kb (8, 249). Therefore, the transforming function of a cDNA transcribed
from the 8.5-kb mRNA of the bcr-proto-abl was tested in murine
retrovirus vectors. In such vectors, as in wild-type Abelson virus (251),
the transcriptional activity of the abl gene is about 100 times
that of normal or rearranged cellular proto-abl genes (252, 254,
255). One such recombinant virus induced proliferation of lymphoid mouse
cells in vitro (254). Another induced clonal lymphomas when introduced
into the germline of transgenic mice (255). Finally, a myelogenous leukemia
was obtained by infecting bone marrow in vitro with the synthetic
virus and transplanting this marrow into irradiated syngeneic mice (252).
The leukemias appeared after relatively short latent periods of 9 weeks
(252), almost as fast as those caused by the wild-type Abelson virus (250).
The karyotype of this leukemia was not described (252).
Yet several observations cast doubt on the hypothesis
that the rearranged proto-abl gene from human chronic myelogenous
leukemias is functionally equivalent to the transforming gene of Abelson
virus and that it is leukemogenic:
1. The transcriptional activity of the rearranged proto-abl
gene in the leukemias is about 1% of that of wild-type Abelson virus and
those of the synthetic recombinant viruses. Thus, mutated cellular proto-abl
genes and viral abl genes are functionally not equivalent.
2. Given estimates that chromosome translocations occur
spontaneously in human cells in 1 out of 102 to 104 mitoses (37, 256, 257),
it can be calculated that a brc-proto-abl rearrangement would
be much more probable than chronic myelogenous leukemia. The probablity
that a random reciprocal rearrangement falls within the 200-kb 5' region
of proto-abl and the 5.8-kb 5' region of bcr of the 106-kb
human genome is (200 : 106) x (5.8 : 106) or 10-9.
Thus, 1 in 109 translocations would generate a Philadelphia chromosome.
Considering that humans carry about 1010 to 1011 lymphocytes (186), which
are replaced at least six times per year (53), or 420 times in an average
lifetime of 70 years, a human life represents at least 1013 mitoses of
lymphocytes. Making the conservative assumption that a translocation occurs
in 1 out of 104 human mitoses (256, 257), about 109 (1013 : 104)
lymphocytes with rearranged chromosomes are generated in a lifetime. Accordingly,
every human should, by the age of 70, develop 1, and possibly 100, lymphocytes
with a Philadelphia chromosome (109 : 109) and thus leukemia.
However, chronic myelogenous leukemia is observed in only 1 (242) to 2.4
(197, 258) out of 100,000 per year or about 0.1% of people in a 70-year
lifetime. Therefore, a rearranged proto-abl gene appears not to
be sufficient for leukemogenesis.
3. Since in 10-15% of the chronic myelogenous leukemia
cases proto-abl is not rearranged (193, 244), proto-abl mutation
is not necessary for leukemogenesis. According to Nowell, "These variants
appear to have no significance with respect to the clinical characteristics
of the disease, and so it appears that it is the displacement of the sequence
of chromosome 22 that is of major importance, rather than the site to which
it goes" (193).
Thus, a rearranged proto-abl is functionally not
equivalent to the transforming gene of Abelson virus. The rearrangement
appears to be more probable than a leukemia, and is not even necessary
for the leukemia. It is consistent with the first point that the proto-abl
translocation is observed in the rather benign, early stage of chronic
myelogenous leukemia, in which cells can differentiate into functional
myeloblasts (242, 243), whereas the Abelson virus causes a terminal leukemia
within several weeks.
Since the transcriptional activity of retroviral abl
genes is about 100 times that of normal and rearranged proto-abl
genes, and since it is not known whether even a viral abl gene can
transform a human cell, the claims that "retrovirus-mediated expression
of the bcr-proto-abl protein provides a murine model system
for further analysis of the disease" (252) are not realistic. These
claims fail to take into consideration the 100-fold transcriptional discrepancy
between the retroviral and cellular abl genes and the question of
whether the transforming host range of abl genes includes human
cells. Therefore, synthetic proto-abl viruses are just experimental
reproductions of the rare spontaneous genesis of retroviral transforming
genes from normal cellular genes and retroviruses. The critical step in
this process is the recombination of the coding region of a proto-onc
gene with a retroviral promoter (37).
It follows that the 85-90% incidence of proto-abl
rearrangements in chronic myelogenous leukemia and the structural similarity
of the gene to that of Abelson virus are the only evidence to suggest that
proto-abl plays a role in human leukemogenesis. In view of this,
proto-abl either must be involved in human leukemogenesis by a mechanism
that is not analogous to that of the viral counterpart, or it may not be
involved at all.
An alternative hypothesis suggests that alterations of
the normal balance of chromosomes cause the leukemia. According to this
hypothesis, the Philadelphia translocation would only affect the growth
control of the cell. This is consistent with the rather normal function
of cells with the translocation during the 3-4 years prior to the blast
crisis. In one case, a person with a Philadelphia chromosome did not develop
a leukemia for at least 7 years (P. H. Fitzgerald, personal communication,
1985) (245). Indeed, the blast crisis of myelogenous leukemia is accompanied
by further chromosomal abnormalities, which are observed in leukemia with
and without rearranged proto-abl genes (193, 244).
C. Point-mutated Proto-ras Genes
and Cancer
Two laboratories have reported that transfection of the
DNA of a human bladder carcinoma cell line transforms morphologically the
mouse NIH 3T3 cell line (259, 260). Subsequent cloning proved the transforming
DNA to be the coding region of the proto-ras gene, the same gene
from which the coding region of the ras gene of the murine Harvey
sarcoma virus is derived. Sequencing indicated that the 3T3 cell-transforming
proto-ras from the bladder carcinoma cells differs from normal proto-ras
in a point-mutation in codon 12 that changes the native Gly to Val (23,
26, 261).
Further transfection analyses with the 3T3-cell-transformation
assay detected point-mutated proto-ras genes in less than 1% to
about 20% of most common human tumors (1, 6, 36, 262) and in up to 40%
of colon cancers (28, 263, 264). The proto-ras genes from these
tumors were each from a closely related group that includes the Harvey,
Kirsten, and N-ras genes. Like the Harvey gene, the Kirsten proto-ras
gene is named after a sarcomagenic murine retrovirus with a coding region
of that gene (6). Regardless of point-mutation, proto-ras expression
is enhanced 2- to 10-fold in about 50% of tumors compared to normal control
tissues (44, 262, 265, 266). Transcription of normal proto-ras is
also enhanced in normal proliferating cells (36), as, for example, 8-fold
in regenerating rat liver cells (267).
1. The Original ras-Cancer Hypothesis
Postulates a First-Order Mechanism of Transformation
The observations that point-mutated proto-ras genes
from human and some animal tumors transform mouse 3T3 cells became the
basis for the hypothesis that point-mutations of proto-ras genes
cause cancer (23, 26, 27). The hypothesis derived additional support from
the observation that the ras genes of Harvey and Kirsten sarcoma
viruses also differ from normal proto-ras in point-mutations in
codon 12 (5, 39, 268). The hypothesis assumes that point-mutations confer
to proto-ras genes dominant transforming function that is equivalent
to that of sarcomagenic retroviral ras genes (268). Further, it
assumes that the 3T3-cell transformation assay measures a preexisting function
of mutated cellular proto-ras genes. Consequently, point-mutated
proto-ras genes were termed "dominantly acting oncogens"
(4, 5, 9, 46, 259, 260, 269). Subsequently, other proto-onc genes,
such as proto-myc (270, 271) and proto-src and the src
genes of Rous sarcoma virus (275), and even genes that are not structurally
related to retroviral oncogenes, such as certain anti-oncogenes (see Section
IV, E), were also proposed to derive transforming function from point-mutations
(1, 5, 6, 9, 46, 272-274).
Numerous observations designed to test the ras
point-mutation-cancer hypothesis indicate that point-mutation is not sufficient
for carcinogenesis:
1. Point-mutated proto-ras genes from tumors do
not transform diploid embryo cells from rodents or humans, as retroviral
ras genes do (238, 276). However, upon simultaneous transfection
with other viral oncogenes or cellular genes linked to viral promoters,
proto-ras genes transform embyro cells (234, 235, 238). This indicates
that point-mutation is not sufficient to convert proto-ras to a
gene that can transform normal cells.
2. Numerical arguments based on relative probabilities
of point-mutations versus cancer also indicate that point-mutated proto-ras
genes are not sufficient for carcinogenesis. The probability of point-mutations
is 10-9 per nucleotide and per mitosis in eukaryotic cells (37, 38, 47,
277). Since eukaryotes carry about 109 nucleotides per cell (278) and consist
of 1011 (mice) to over 1014 (humans) cells, mice carry 102 (1011 : 109)
and humans carry 105 (1014 : 109) cells with the specific point-mutation
that changes Gly to Val in codon 12 of proto-ras at any time (37,
38). Since the average cell is replaced about 100 times during a human
lifetime of 70 years (37, 277), this number must be multiplied by 100.
Moreover, since at least 50 different point-mutations in at least five
different codons confer transforming function to proto-ras in the
3T3 assay (39, 279), mice would contain 5 x 103 and humans have 5 x 106
such cells.
3. Further, the existence of point-mutated proto-ras
genes in nontumorigenic, hyperplastic tissues (see Section VI) (280-284)
and in transgenic mice (236, 241; R. Finney and J.M. Bishop, 7th Annual
Meeting on Oncogenes, Frederick, Maryland, 1991, personal communication)
indicates that these mutations are not sufficient for carcinogenesis.
4. Point-mutation is not necessary for the transforming
function of Harvey and other murine sarcoma viruses, as mutants without
point mutations in ras and synthetic retroviruses with normal proto-ras
coding regions are almost as oncogenic as those with point-mutations (41,
44, 285). This indicates that viral ras genes derive transforming
function from other virus-specific elements (39, 41, 44) and suggests that
point-mutation may not be sufficient for proto-ras genes to transform.
5. In primary tumors, point-mutated proto-ras genes
are expressed at nearly the same level as normal proto-ras genes
(36, 44, 262, 264, 280, 286). By contrast, point-mutated proto-ras
genes in cells transformed by transfection are expressed like viral ras
genes, which is at a level at least 100-fold higher than native proto-ras
genes (44, 234, 235, 262, 280, 286, 287). Thus, the 3T3-cell-transfection
assay creates proto-ras expression artifacts that are transcriptionally
about 100 times more active than native proto-ras genes from tumors.
Their activity is similar to that of retroviral ras genes.
It appears then that a point-mutated but intact cellular
proto-ras gene is not sufficient for carcinogenesis. Further, it
follows that the transfection assay does not measure a genuine function
of pointmutated proto-ras genes as they exist in tumors, but measures
that of an expression artifact created during the transfection assay. An
analogous functional artifact has been observed upon transfection of an
antioncogene (see Section IV, E) (287a).
Such artifacts could be generated during transfection
by substituting by illegitimate recombination the native proto-ras
regulatory elements by artificial promoters derived from carrier and helper
gene DNA (44). Indeed, transformation of primary cells by cellular proto-ras
genes depends on the presence of added viral helper genes or on other cellular
genes linked to viral promoters (234, 235, 288, 289), or on the presence
of retroviral promoters alone (44). This recombination process is entirely
analogous to the generation of retroviral ras genes, in which coding
regions of normal proto-ras genes are recombined by transduction
with heterologous retroviral promoters that enhance the transcription over
100-fold compared to proto-ras (37, 38, 43, 44). In addition, transfection
generates concatenated DNA multimers, an artificial gene amplification
that would also enhance the dosage of ras transcripts (290-293).
The probable reason that proto-ras genes from tumors
transform 3T3 cells, but not primary cells, is that mouse NIH 3T3 cells
are much more readily transformed by exogenous genes, as well as spontaneously
(294), than are embryo cells (238). Thus, the weak promoters acquired from
random sources during transfection are sufficient to convert proto-ras
genes with point-mutations to 3T3-cell transforming genes, but not to genes
capable of transforming primary cells.
The reason that point-mutated, but rarely normal, proto-ras
genes (261) are detected by transfection assays is that point-mutations
enhance about 10- to 50-fold the transforming function imparted by heterologous
promoters on proto-ras genes (39, 44, 285, 295). Thus, proto-ras
genes derive their transforming function from heterologous promoters, and
certain point-mutations merely enhance this transforming function.
2. Ad Hoc ras-Cancer Hypotheses Postulating
Second- and Higher-Order Mechanisms of Transformation
In view of the evidence that native, point-mutated proto-ras
genes detected in some tumors are not equivalent to viral ras genes
and not sufficient for carcinogenesis, ad hoc ras-cancer
hypotheses have been advanced proposing that cellular ras genes
with point-mutations depend on helper genes for carcinogenesis (6, 28,
46, 236, 238). However, the hypothetical helper genes have not been identified
in most tumors, except for colon cancers.
In the case of colon cancer, it has been postulated that
point-mutated Kirsten and N-ras genes depend on the mutation of
at least three tumor suppressor genes for transforming function (28, 46,
272). Yet the incidence of these mutations in colon cancers is not convincing
proof for their postulated function for the following reasons.
Among primary colon cancers, about 40% carry point-mutated
Kirsten ras genes (28, 263, 264) and some others contain point-mutated
N-ras genes (28). In addition 70% of all carcinomas carry deletions
or mutations in the presumed tumor suppressor gene DCC (deleted in colon
cancer) located on chromosome 18, 75% in the presumed suppressor gene p53
located on chromosome 17 and 30% in the presumed suppressor gene APC (adenomatous
polyposis coli) on chromosome 5 (28). Thus, only about 6% (0.4 x 0.7 x
0.75 x 0.3) of the colon cancers studied carry the genetic constellation
postulated for colon cancer. About 87% carry various combinations of these
mutations, and 7% carry none of the mutations (28). In addition, recent
evidence indicates that mutations on chromosome 5 are scattered over several
hypothetical suppressors or anti-oncogenes (296). Despite these radical
mutational differences among colon carcinomas, the carcinomas do not differ
from each other in any known histological or biological properties. In
addition, all of these mutations alone, and even together, are also observed
in benign colon adenomas (see Section VI) (28). Other tumors with point-mutated
proto-ras genes are also histologically and morphologically indistinguishable
from counterparts without these mutations (262, 297).
In view of such poor correlations and the absence of ras-specific
tumor markers, a functional test is the only method to prove the hypothesis
that point-mutated proto-ras genes have transforming function in
conjunction with helper genes. However, the only functional test currently
available is the 3T3-cell-transfection assay, which generates helper-independent
proto-ras expression artifacts. Thus, the hypothesis that point-mutated
proto-ras genes play a role in carcinogenesis is based only on circumstantial
evidence, namely, structural, but not functional, similarity to viral ras
genes. In addition, it is based on epidemiological evidence that mutated
genes are more common, or are observed more commonly, in tumors than in
normal cells (see Section VI). Moreover, the assumption that mutation of
p53 is obligatory for carcinogenesis has not been confirmed in a recent
study that generated developmentally normal mice without p53 genes (319a)
(see Section IV, E).
It follows either that unrearranged, point-mutated proto-ras
genes are oncogenic by a second- or higher-order mechanism of carcinogenesis
that is not analogous to the first-order mechanism of viral ras
genes and of the transfection artifacts of proto-ras genes, or that
they are not relevant to carcinogenesis. Since constellations of mutated
proto-ras and helper genes that are tumor-specific have not been
found, there is currently no evidence for a role in carcinogenesis.
Therefore, we propose that other events, such as chromosome
abnormalities, which are consistently found in colon carcinomas with and
without mutated oncogenes or anti-oncogenes (28, 47, 192), may cause colon
cancers. The clonal mutations in proto-ras and hypothetical helper
genes could reflect the origin of tumor cells from non-tumorigenic somatic
cells with the same mutations (see Section VI).
D. int Genes with Integrated Mouse
Retroviruses and Mouse Mammary Carcinomas
Mouse mammary tumor virus (MMTV) is one of the many endogenous
retroviruses that are genetically and perinatally transmitted but hardly
ever expressed by most strains of mice (5, 6, 298). However, inbred female
mice of the C3H and GR strains express high titers of mammary tumor virus
in their milk. Approximately 90% of the female offspring of C3H mice develop
mammary tumors between the ages of 7-10 months (299, 300). Foster-nursing
of C3H offspring by virus-free mothers of other strains reduces the risk
of tumors to 20-40% and delays their appearance to 18 to 24 months (299,
301). However, wild mice foster-nursed by a C3H mother fail to develop
mammary tumors (over a spontaneous background of 3% at 2 years of age),
although they are infected by the virus (302, 303).
Virus replication at high titers enhances reversible,
hormone-dependent mammary hyperplasias that are poly- or oligoclonal (304).
Out of these hyperplasias, clonal tumors emerge that are hormone-independent
(304, 305). Thus, infection by milk-borne virus initiates virus replication,
hyperplasias, and frequently tumorigenesis at an earlier age compared to
spontaneous virus activation and tumorigenesis-but only in certain inbred
strains of mice. The virus is replicating in both early and late tumors
(305). The tumors are clonal, defined by specific virus integration sites
and chromosome abnormalities (2). Since only one out of millions of virus-producing
mammary cells becomes tumorigenic, tumorigenesis may be virus-independent,
or may be due to virus-mediated activation, or inactivation of a cellular
gene, in which case, site-specific provirus integration must be postulated.
Site-specific integrations in mammary tumors were originally
observed in three different mouse strain-specific loci, termed int-1
(34), int-2 (306), and int-3 (307). In C3H mice, the provirus
is primarily observed in int-1, in BR6 mice in int-2, and
in some feral mice in int-3. Subsequently, "numerous"
(305) int loci were observed in mouse mammary carcinomas (308).
In light of the observation that proto-myc genes in certain chicken
leukemias are transcriptionally acitvated by retrovirus insertion (31)
these int loci have been postulated to be cellular proto-onc
genes that are activated to cancer genes by the promoter of integrated
MMT provirus (1, 6, 8, 34, 306, 308). This hypothesis is compatible with
the clonality of the mammary tumors.
Integration sites within a given int locus are
spread over 20 kb and occur in both transcriptional orientations (1, 2,
8). Viral integrations into int loci are also observed prior to
tumorigenesis in hormone-dependent hyperplasias (304, 309). Only 1-10 copies
of int RNAs are found in tumor cells that express int genes
(310). By comparison, synthetic (39, 40, 42, 44) and natural (31) retrovirus-promoted
proto-onc genes make about 103 to 104 copies of RNA per transformed
cell. In many viral mammary tumors, the int loci are not expressed,
and in some tumors the int loci are expressed but the MMT provirus
is not integrated at or near int. For example, int loci are
expressed in only 2 out of 9 clonal tumors of GR mice (304), and int
loci are expressed in only 19 out of 46 clonal tumors of C3H mice (305).
It has also been reported that int loci are expressed in tumors
in which MMTV is integrated at non-int sites. Accordingly, there
is no report of int-specific tumor markers.
In view of this, several arguments cast doubt on the int-activation
hypothesis:
1. Since "numerous" int loci are observed
in mammary carcinomas, and since integrations are scattered over 20 kb
within a given locus and occur in both orientations, MMTV integration into
int loci cannot be sufficient for carcinogenesis based on the following
numerical arguments. Given random retrovirus integration (6) and 1 x 106-kb
DNA per mouse genome (278), and assuming only five 20-kb int loci,
about 1 out of every 104 (5 x 20 out of 106) infected mammary cells should
become tumorigenic. Thus, tumors should appear very soon after infection.
Since this is not the case, MMTV integration cannot be sufficient for carcinogenesis.
2. Since MMT proviruses integrate into int genes
prior to tumorigenesis, provirus-mutated int genes cannot be sufficient
for tumorigenesis.
3. Since wild mice are susceptible to the virus and produce
the same hormones as the inbred mice that develop mammary carcinomas, even
the virus-hormone package is not sufficient for tumorigenesis.
4. Provirus integration into different int loci
in different strains of mice indicates that integration is host-directed.
Therefore, the virus is not sufficient for site-specific integration and
thus for tumorigenesis, if site-specific integration proves to be relevant
for tumorigenesis.
5. Since int loci are not expressed in many viral
mammary tumors, transcriptional activation of int genes by any mechanism
is not necessary for carcinogenesis. It is consistent with this view that
proviruses are integrated into int genes in both directions and
integration sites are spread over 20 kb, but retroviral promoters activate
transcription in only one direction and only over limited distances (42).
6. Since the same tumors are observed with and without
integration into int genes, site-specific integration is not necessary
for carcinogenesis, because "clonal, hormone-independent tumors .
. . seem to be the result of mutations that are unrelated to int
activation" (304).
7. The retroviral int-activation hypothesis fails
to account for the clonal chromosome abnormalities of all virus-positive
tumors that have been characterized (2)-except if one makes the additional
odd assumption that MMTV only transforms cells with preexisting chromosome
abnormalities.
It thus appears that the MMTV plays only an indirect role
in tumorigenesis as one of several factors that enhance mammary hyperplasias,
a known risk factor for carcinogenesis (1, 203). This role is similar to
that of other highly expressed animal retroviruses in leukemogenesis. For
example, inbred viremic mice and chickens have been described that develop
virus-induced hyperplasias from which clonal lymphomas or leukemias emerge
(2). Alternatively, high levels of retrovirus expression may just signal
a heritable loss of intracellular suppressors which could themselves predispose
to overgrowth and thus favor carcinogenesis (2). The incidence of 20-40%
carcinomas in foster-nursed C3H mice compared to a background of 3% in
other laboratory mice (303) supports this view. This would be analogous
to the activation of other retroviruses in cells induced to proliferate
by genetic damage from chemicals or radiation (see Section III).
In view of this, we propose that mammary carcinogenesis
is a rare, spontaneous event initiated by chromosome abnormalities that
occur in one out of millions of virus-infected cells. This hypothesis would
explain clonal viral integration sites as accidental consequences of the
clonal chromosome abnormality that created a tumor cell from a normal virus-infected
cell. It would also explain why the carcinomas are not distinguished by
the type of int gene that is mutated. The int-loci would
be strain-preferred provirus integration regions that are not relevant
to tumorigenesis.
E. Constitutive Oncogenes, Mutated Anti-oncogenes,
and Cancer
There are heritable and spontaneous retinoblastomas (45).
Cytogenetic analyses of both have observed that chromosome 13 is either
missing or deleted in 20 to 25% (311,312). In addition, other chromosome
abnormalities have been observed in all retinoblastomas (311, 312). On
this basis, it was proposed that retinoblastoma arises from the loss of
a tumor suppressor or an anti-oncogene, now termed rb, that is part
of chromosome 13 (45). In the familial cases, the loss of one rb
allele would be inherited and the second one would be lost due to spontaneous
mutation. In the spontaneous cases, somatic mutations would have inactivated
both loci. In the retinoblastomas with microscopically intact chromosomes
13, submicroscopic mutations were postulated.
This anti-oncogene hypothesis predicts that normal cells
would constitutively express oncogenes that render the cell tumorigenic
if both alleles of the corresponding suppressor are inactivated. The hypothesis
further predicts that the suppressor genes must be active at all times
in normal cells. In 1986, Weinberg et al. (313) cloned a human DNA
sequence that was missing or altered in about a third of 40 retinoblastomas
and in 8 osteosarcomas. Therefore, the gene encoded in this sequence was
termed the rb gene. Reportedly, the rb gene was unexpressed
in all retinoblastomas and osteosarcomas, even in those without rb
deletions (313). The rb gene measures almost 200 kb, includes 27
exons and encodes, from an mRNA of 4.7 kb, a 110,000-dalton protein (8,
278).
An analysis of 34 primary retinoblastomas undertaken to
test the hypothesis found deletions of the rb gene in only 4 of
34 tumors analyzed and transcripts of the rb gene were found in
12 out of 17 retinoblastomas and in 2 out of 2 osteosarcomas, casting doubt
on the deletion hypothesis (314). The remaining tumors had apparently normal
rb genes. However, subsequent studies of retinoblastomas have observed
point-mutations and small submicroscopic deletions in rb genes that
did not have macrolesions (273, 274, 315, 316). For example, both Weinberg
et al. (273) and Lee et al. (274) reported a point-mutation
in a splice sequence of the rb gene. In view of this, it is now
believed that point-mutations or other minor mutations of the rb
genes are sufficient for tumorigenesis (273, 315). However, Gallie et
al. reported point-mutations and deletions of rb genes in only
13 out of 21 tumors (315). In an effort to develop a functional assay,
a DNA copy of the mRNA of the rb gene was cloned into a retrovirus;
infection by this virus inhibited the growth of a retinoblastoma cell line
in vitro (274, 317). However, two recent studies show that an intact,
synthetic rb gene fails to inhibit tumorigenicity of human retinoblastoma
and breast cancer cells in nude mice (318, 318a).
Clearly, the point-mutation hypothesis of the rb
gene would never have emerged if the original chromosome deletion hypothesis
had been confirmed. It advanced the anti-oncogene hypothesis into a virtually
inexhaustible reservoir of hypothetical cancer genes: Any gene with any
mutation in each of both alleles in a cancer cell could be a tumor suppressor
or anti-oncogene. According to Weinberg, "... one can cast a broad
net for tumor suppressor loci by using a large repertoire of polymorphic
DNA markers to survey ... for repeated instances of LOH (loss of heterozygosity).
Indeed, this genetic strategy has revolutionized the research field"
(287a). Over a dozen deleted or point-mutated anti-oncogenes are now considered
to cause osteosarcomas, breast cancer, bladder cancer, lung cancer, colon
cancer, Wilms' tumor, and neuroblastoma, in addition to retinoblastoma
(8, 9, 46, 287a, 317). For example, a point-mutation in one of three genes
of a colon cancer cell would signal an inactivated hypothetical colon cancer
suppressor gene (272, 296). Further, the range of the rb suppressor
gene has since been extended to other cancers, including small cell lung,
bladder, prostate, and breast carcinomas, and osteosarcoma (8, 317).
The anti-oncogene hypothesis has been difficult to prove
because (a) the oncogenes that are said to be suppressed have not been
named or identified (269) and will be difficult to assay because all normal
cells or animals should suppress them with the corresponding antioncogenes,
and because (b) transfection of an intact rb gene (274, 318) has
failed to revert transformed cells to normal and to suppress their tumorigenicity
(274, 318, 318a). Likewise the hypothetical colon cancer suppressor gene
p53 has failed to revert transformed cells to normal (319) and its complete
absence has not affected the normal development of mice (319a). Nevertheless,
74% of these p53-free mice developed lymphomas and sarcomas at six months
that probably derived from single cells, rather than through a systemic
transformation as the anti-oncogene hypothesis would have predicted (319a).
At this time, the hypothesis suffers from the following
short-comings:
1. The probability of point-mutations and minor mutations
in both alleles of the rb gene appears much higher than the cancers
they are said to cause. Since the rb gene has 27 exons and each
exon is flanked by at least four essential splice nucleotides, at least
108 (4 x 27) point-mutations could inactivate the rb gene. In addition,
one can assume that point-mutations of at least 10% of the 928 amino acids
of the 110,000-dalton rb protein would inactivate the gene (8).
Thus, at least 200 point-mutations should be able to inactivate rb.
Since 1 in 109 human cells contain any possible point-mutation of the human
genome (see Section IV,C), about 1 in 5 x 106 would contain an inactive
rb gene, and 1 in (5 x 106)2 or 2.5 x 1013 would contain two inactive
rb genes in the same cell. This number would be even higher if other
mutations such as minor deletions and chromosome nondysjunctions were included.
Chromosome nondysjunctions are estimated to occur in 1
out of 104 human cells (320, 321). The probability of generating a retinoblastoma
cell from a point-mutation in one rb gene and a missing chromosome
13 would be 1 in 5 x 106 x 104 or 1 in 5 x 1010. Thus, every adult human
consisting of about 1014 cells would contain at least 1 and possibly 5
x 103 cells in which both rb genes are inactivated, and would develop
over 100 to 100,000 such cells in a lifetime of 70 years, which represents
about 1016 cells (37, 277). Since inactive rb genes are now said
to cause retinoblastomas, osteosarcomas, small cell lung, breast, and bladder
carcinomas, etc., and the corresponding tissues represent over 20% of the
human body, one would expect at least 20% of humans to develop such a tumor
per year.
Moreover, 1 in 5 x 106 cells of every person with one
inherited rb mutation should have defects in both rb alleles
due to secondary mutation and 1 in 104 cells due to chromosome nondysjunction.
A recent review on tumor suppressor genes reports exactly the same probabilities
for rb mutations as we do (287a). Thus, all persons with an inherited
rb deletion should develop retinoblastomas and other cancers. Since
this is not the case (45), point-mutation or deletion of both rb
alleles cannot be sufficient for carcinogenesis.
2. Since neither deletion nor minor mutation of rb
genes is observed in all retinoblastomas or other specific tumors, rb
deletion or mutation is not necessary for tumorigenesis.
3. The relevance of the growth inhibitory function of
the artificial retrovirus with an rb coding region to the putative
tumor suppressor function of rb is unclear for several reasons:
(a) Expression from a retroviral promoter enhances the rb protein
concentration at least 100-fold above physiological levels (274) and thus
may not be relevant to its normal function. Similar reservations are expressed
by Weinberg: "... many genes ... will antagonize growth when they
are forced on a cell by ... gene transfer, but this provides no testimony
as to whether these genes are normally used by the cell to down-regulate
its own proliferation...." (287a). (b) Recently, elevated rather than
reduced rb expression was observed in tumor cells (322). (c) Human
retinoblastoma cell lines and breast cancer lines transfected with intact
and artificially overexpressed rb genes are tumorigenic in nude
mice, indicating that the rb gene does not suppress tumorigenesis
by retinoblastoma and mammary carcinoma cells (318, 318a).
It follows that deletion or mutation affecting both alleles
of the rb and p53 genes is not sufficient and probably not necessary
for carcinogenesis since the same retinoblastomas and colon cancers occur
in the presence and absence of these genes. An alternative hypothesis suggests
that the many chromosome abnormalities associated with retinoblastomas
(311,312), other tumors with rb mutations (194) and colon cancers
are to blame for carcinogenesis (see Section VI).
Continu
VIRUSMYTH HOMEPAGE