Progress in Nucleic Acid Research and Molecular Biology
43:135-204, 1992

Latent Viruses and Mutated
Oncogenes: No Evidence
for Pathogenicity


Department of Molecular and Cell Biology
University of California at Berkeley
Berkeley, California 94720

VI. Alternative Hypotheses

A. Latent Viruses as Harmless Passengers

The inactive viruses associated with fatal diseases such as AIDS, hepatitis C, cervical cancer, T-cell leukemia, hepatoma, Burkitt's lymphoma, and encephalitis are all not disease-specific. They are common, like HSV, HPV, EBV, and HBV (3, 12), or rare, like HIV and HTLV-I (54), in healthy persons. The long "latent periods" and the low incidence of "viral" disease among virus carriers indicate that such infections are typically not pathogenic. Although the term "latent period" implies that the virus becomes active thereafter, even this is almost never true (see Section II and III). During the presumably virus-caused diseases, including AIDS, cervical cancer, T-cell leukemia, hepatoma, or panencephalitis, the virus remains typically inactive, leaving pathogenic functions to unnamed cofactors. And there is no cofactor that has been found only during the disease but not prior to it. It is hardly surprising that latent viruses or fragments of their DNAs are still there if their host develops a nonviral disease. Thus, the latent viruses are innocent bystanders or "passengers," rather than drivers, in nonviral disease processes (159).

B. Drugs as Alternatives to Hypothetical Viral Pathogens

The great triumphs in the pursuit of microbial and viral pathogens in the last 100 years have eclipsed, and even led to the ridicule of, alternative, less spectacular, explanations of disease, such as pathogenic drugs and toxins (15, 324-326). Although we are in the middle of a drug-use epidemic in America, the pathology and epidemiology of recreational drugs, and even of some medical drugs such as AZT, are virtually unstudied by the scientific community (155).

The drug-AIDS hypothesis, described in Section II, A, 2, is one example of how drug use could cause AIDS diseases (54, 60, 103). Psychoactive drugs and medical drugs could explain diseases caused by the depletion of many cells, such as the depletion of T-cells in AIDS or of hepatocytes in hepatitis C, much better than can dormant viruses. Indeed, both of these diseases are observed primarily in drug addicts (54, 103, 160). Drug toxicity is also much more compatible with the restriction of these diseases to risk groups, as, for example, AIDS, which is almost exclusively restricted to users of recreational drugs and anti-HIV drugs such as AZT (like lung cancer is to smokers).

Exogenous toxins could also explain the actions of putative viral tumors, such as nitrite inhalants causing Kaposi sarcomas and AZT causing lymphomas (69, 103), smoking possibly causing cervical cancer (198, 204), nutritional toxins causing hepatomas, and radiation possibly causing T-cell leukemia (190) (see Section III). Toxins would also provide a plausible explanation for the lack of contagiousness of these "viral" diseases. The cumulative effects of drug or nutrient toxicity over time are compatible with the appearance of these diseases relatively late in life and at unpredictable intervals after infection by presumed viral causes. By contrast, viruses as self-replicating toxins all cause diseases soon after infection. In light of this theory, hypothetical linkages between infection by a virus and a subsequent onset of disease via long and unpredictable latent periods of up to 55 years would dissipate, because infection and pathogenesis are independent events.

C. Mutated Genes and Latent Viruses as Trivial Genetic Scars of Cancer Cells

The spontaneous or virus-induced mutations in tumor cells are also not disease-specific. For example, point-mutated proto-ras genes have been observed in chemically induced skin hyperplasias of laboratory mice (280) and in spontaneous liver hyperplasias of B6C3FI mice (281) that all spontaneously revert to normal. Further, they have been observed in reversible skin hyperplasias of humans (282, 327) and in human hemopoietic hyperplasias (238, 284). Moreover, a recent study of transgenic mice concluded that "... expression of the mutant [proto-Ha-ras] gene via its own promoter at the normal chromosomal locus is nontransforming" (R. Finney and J.M. Bishop, 7th Annual Oncogene Meeting, Frederick, Maryland, 1991, personal communication). In addition, point-mutations and all other mutations affecting hypothetical tumor suppressor genes are not tumor-specific. They are detected singly and in all combinations, including mutated proto-ras, in benign colon adenomas at about the same rates as in malignant carcinomas (28).

Proto-abl translocations are seen in functional granulocytes that are overproduced during the chronic, hyperplastic phase of myelogenous leukemia (242, 243) (see Section IV). Hormone-dependent mammary hyperplasias with int genes mutated by integrated MMT proviruses have been described (see Section IV). Also, the DNA of hepatitis B virus has been detected and is expressed in non-tumorigenic liver cells more consistently than in hepatomas (196, 211). Inactive and defective HPV DNA is routinely detected in non-tumorigenic tissues with the commercial Vira/Pap test or with the PCR (199). And HTLV-I is almost only detected in normal rather than leukemic carriers (see Section III). Further, viable transgenic mice with mutated proto-abl, proto-myc, and proto-ras, and even with hypothetically cooperating combinations of proto-myc and proto-ras, have been constructed, and some are commercially bred ("OncoMouse-TM shortens the path to knowledge ...," Dupont Co., Wilmington, DE, 1990) (236). This argues either for even more cofactors or for other mechanisms altogether.

Thus, spontaneous and viral mutations of tumor cells are not disease-specific. These findings confirm the above calculations that the probability of these mutations is much higher than the incidence of cancer and that carcinogenesis even among hyperplasias is still a very rare event. In view of this, we agree with Bishop that "the nomenclature for the affected genes [oncogenes] is unfortunate, since it is based largely on occasional [presumed] pathogenic aberrations...." (9).

Nevertheless, since clonal tumors have been observed to emerge from hyperplasias and transgenic animals at a higher-than-normal rate, their mutated genes and latent viruses could play roles in carcinogenesis that are not analogous to those played by the biochemically active models that led to their discovery. For example, they could alter growth control genes and thus generate hyperplasias. However, even this is speculation because the mutations and latent viruses are not consistently found in hyperplastic cells, with the exception of HPV in papillomas (13) (see Section III). Therefore, they must be presumed innocent until proven guilty (326).

In view of this, we propose that the mutations and latent viruses that are found in tumor cells are trivial genetic scars that were picked up by non-tumorigenic somatic cells during many generations of growth in the presence of mutagens or viruses. Because of detection and reporting biases in favor of disease, the mutations and latent viruses would be reported more often in diseased than in healthy carriers. Further, the mutations and viruses would be more readily and more often observed in cancer cells than in non-tumorigenic somatic tissues, because cancers are clonal populations of cells (192, 193, 328) that provide multiple copies of identical mutations, biological equivalents of the PCR. In contrast, such mutations, including latent and fragmented viral DNAs, would not be detectable in mutationally "heterogenous" populations of normal cells, unless individual cells were cloned or their nucleic acids were amplified.

Since many of these somatic mutations could be incompatible with normal fetal development, they would not be seen in the germline (329) and thus not in an average normal cell. The many congenitally and genetically transmitted animal (6) and human retroviruses, including HTLV-I and HIV (54), would be notable exceptions. Apparently, retroviruses are so harmless that they can be accepted as parasites even during development (2).

The hypothesis correctly predicts the same mutations and latent viruses in non-tumorigenic somatic cells and in tumors that emerged from these cells, as, for example, the proto-ras and other mutations or the many "tumor" viruses that are shared by tumorigenic and nontumorigenic cells. Further, the hypothesis correctly accounts for the "too many mutations in human tumors" observed by Loeb (47), perhaps those that were considered irrelevant for carcinogenesis by Heidelberger ("I don't care if cells are 90% transformed, I am only interested in the last 10%.") (330). In view of this hypothesis, the latent viruses and nonactivating mutations of cellular genes in cancer cells would be genetic trivia.

D. Cancer by Somatic Gene Mutation Unconfirmed

The clonality, irreversibility, and predictable course of most cancers all indicate that cancer has a genetic basis. Yet an autonomous cellular cancer gene, or a complement of interdependent ones, that can be activated by the statistically cheap mutations observed in hypothetical oncogenes and anti-oncogenes is improbable on the following grounds.

(i) Nothing could be more terminal for a multicellular organism than a battery of latent cancer genes that are as easy to "activate" as the over 50 putative cellular oncogenes that have been named or the unnamed oncogenes that are said to be activated by inactivation of suppressor genes (6, 8, 9, 331). The activation of just one dominant oncogene would be sufficient to initiate a clonal cancer and thus to kill the organism. By comparison, activation of a hypothetical death gene would kill only a single cell.

Indeed, since each of these oncogenes is thought to be activated via point-mutations, truncations, and virus insertions and since the probability of such mutational events is as high as 1 in 106 per mitosis and gene, and is as high as 1 in 109 per mitosis and nucleotide (see above estimates for proto-ras, proto-abl, and rb) (37, 38, 47, 277), multicellular organisms such as humans, with about 1016 cells per average 70-year lifespan, would generate at least 50 x 1016 : 109 = 5 x 108 cancer cells per lifetime. This number would be even higher if multiple mutational sites for the activation of specific oncogenes and for the inactivation of specific anti-oncogenes were considered (6, 8). It would be further enhanced by the multiplicity of certain oncogenes that exist as large families, including proto-myc and proto-ras (6, 8).

Nevertheless, the numerology of mutations could be reconciled with the real incidence of cancer by postulating adequate numbers of cooperating mutations, as has been attempted in the case of colon cancer (see Section IV). However, this would be analogous to the invention of more and more Ptolomaic epicycles by geocentrists, in the face of Galileo's challenge that the earth was not the center of the solar system. Naturally, the relevance of these efforts to carcinogenesis would depend on functional proof.

(ii) Based on the only proven examples of "mutated cellular" oncogenes, the retroviral oncogenes, a cellular gene would have to become about 100-fold more active than normal to become a cancer gene. However, the odds of truly activating a gene about 100-fold over the level for which it has been optimized during 3 billion years of evolution by spontaneous mutation, must be much lower than the odds of the presumably "activating" point-mutations or truncations or virus insertions that are observed in the hypothetical proto- and anti-oncogenes of tumors. The rare, accidental recombinants with imported retroviral promoters, which in turn have been optimized during virus evolution to override cellular controls, are as yet the only known examples of oncogenic mutations (37).

The odds for activating a cellular gene 100-fold by spontaneous mutations would be particularly low for the many interdependent genes that must determine "how cells govern their replication...." (7), the presumed natural function of proto- and anti-oncogenes (7, 287a). According to Bishop, mutational "damage" to the "relays in regulatory circuitry" (proto-onc genes) and "governors of proliferation" (anti-oncogenes) is considered a "gain of function" sufficient to produce cancer (9). These oncogenic functions are postulated to be "dominant because ... evil overrides good" (9). However, "damage" of the kind observed in putative oncogenes naturally inactivates genes causing diseases such as sickle cell anemia and hemophilia (320, 332). Such damage is a loss of function and thus recessive, because the remaining "good" gene overrides "evil." Ironically, the same kind of somatic mutations or damages to genes thought to "activate" oncogenes are said to perform conventional gene inactivations when they affect anti-oncogenes.

Indeed, it is one of the most common misconceptions that cancer is a consequence of unrestricted growth, because unrestricted growth produces benign hyperplasias, not cancer. According to Cairns, "It is a common mistake to assume that cancer cells multiply faster than the normal cells from which they were derived .... The fact is that the cells of most cancers divide at about the normal rate, and some even less frequently than their normal counterparts, but they are able to increase in number because a greater proportion of the cells' progeny remain in the dividing pool than is normally allowed" (277).

(iii) There is no functional proof for cellular oncogenes, because according to Stanbridge "... despite intensive efforts to transform normal human fibroblasts or epithelial cells with varying combinations of activated cellular oncogenes, the results have been uniformly negative" (269). Moreover, their presence, unlike that of related viral oncogenes, does not determine the character of a given type of tumor. Likewise, unmutated anti-oncogenes fail to revert tumor cells to normal, and mutated anti-oncogenes fail to distinguish tumors from those in which they are normal (see Section IV).

The somatic mutation hypothesis owes much of its popularity to the fact that, in the 1960s and 1970s, many carcinogens were found to be mutagens (335, 338), although substantial non-correlations between carcinogens and mutagens were also noted (335, 337). In the 1980s, the hypothesis derived further notoriety from the consensus that proto-onc genes and anti-oncogenes are the critical targets among the anonymous genes that are mutated by carcinogens (9, 287a). Says Weinberg: "Mutations that potentiate the activities of proto-oncogenes create the oncogenes that force the growth of tumor cells. Conversely, genetic lesions that inactivate suppressor genes liberate the cell ... yielding the unconstrained growth of the cancer cell" (287a). However, not even one of the many somatic mutations observed to date in cancer cells has been shown to function as a cancer gene. According to Pitot: "... that carcinogens are mutagenic or may be converted to mutagens is important but not direct evidence for the genetic origin of neoplasia" (16).

In sum, the gene mutation hypothesis of cancer is numerically and evolutionarily implausible and is functionally unconfirmed. Similar conclusions were reached by Rous (203, 333) and Rubin (334) after studying oncogenic viruses and cancer for over 50 and 30 years, respectively. Rous concluded: "A favorite explanation has been that oncogens (Rous' term for carcinogens) cause alterations in the genes of the ordinary cells of the body ... somatic mutations as these are termed. But numerous facts, when taken together, decisively exclude this supposition" (203). "A hypothesis is best known by its fruits. What have been those of the somatic mutation hypothesis? ... It acts as a tranquilizer on those who believe in it, and this at a time when every worker should feel goaded now and again by his ignorance of what cancer is" (333). Likewise, Cairns "... suggests that most human cancers are not caused by conventional mutagens ..." (335).

E. Chromosome Abnormalities as Causes of Cancer

But if there are no cellular genes that are converted to cancer genes by somatic mutations, cancer would have to be caused by normal cellular genes. Perhaps a cell could become transformed by gross numerical imbalances of normal genes, e.g., via chromosome abnormalities, just as a computer could be rendered uncontrollable by deleting, duplicating, and misplacing intact chips, or by altering the operating software. To test this hypothesis, it would be necessary to determine how probable such abnormalities are compared to cancer and whether abnormalities exist that are cancer-specific.

Indeed, chromosome abnormalities are the oldest, and, as yet, the only consistent observation made on cancer cells. It was postulated by Boveri in 1914, prior to the discovery of DNA and point-mutations, that cancer would be caused by abnormal chromosomes (194, 336). The clonal origin of tumors, the stemline concept predicted by Boveri and defined by Winge in 1930 (336), is the strongest support for the view that clonal chromosome abnormalities are the causes, rather than consequences, of carcinogenesis.

This abnormal chromosome-cancer hypothesis would explain why chromosome abnormalities are consistently found in tumors with or without mutated cellular oncogenes and with or without latent viruses.

The hypothesis predicts that diploid cancers that differ from normal cells only in mutated oncogenes or anti-oncogenes are not observed, because certain chromosome abnormalities instead of somatic mutations of specific genes are carcinogenic. Tumor progression would be a consequence of further discontinuous chromosome abnormalities. The hypothesis would readily resolve the paradox that all "viral" tumors presumably caused by HTLV-I, HBV, HPV, HSV, and MMTV have clonal chromosome abnormalities. By contrast, all virus-cancer hypotheses would have to make the odd assumption that only cells with preexisting chromosome abnormalities are transformed by these "tumor" viruses.

Our hypothesis also explains why "... despite intensive efforts to transform normal human fibroblasts or epithelial cells with varying combinations of activated cellular oncogenes, the results have been uniformly negative" (269). In addition, the hypothesis explains why mutated proto-onc genes and anti-oncogenes do not distinguish tumors by their presence. According to our hypothesis, accidental somatic mutations generated by chromosome translocations, such as rearranged proto-myc or proto-abl genes, would be as irrelevant to carcinogenesis as other mutations of specific genes, such as point-mutated ras genes. Further, the hypothesis would explain why transgenic mice with activated oncogenes are breedable and why retinoblastoma cells remain carcinogenic for mice, even if they are infected by a retrovirus that overexpresses its presumed suppressor, rb anti-oncogene (see Section IV). Our hypothesis would also resolve the discrepancy between the rather high probability and incidence of mutation or "activation" of proto-onc genes compared to the much lower probability and incidence of cancer (see Section IV) (37, 337).

We have previously proposed another alternative to the oncogene hypothesis. It holds that cancer genes are generated by substituting the normal promoters of proto-onc genes via rare illegitimate recombinations by strong heterologous promoters from viruses or from cellular genes (37). As yet, the retroviral oncogenes are the only proven examples of this hypothesis (40, 43, 44). The relevance of this hypothesis to virus-free tumors depends on whether the cell contains promoters that are as strong as those of viruses. *