Viruses and Mutated Oncogenes: No Evidence for Pathogenicity
Peter H. Duesberg and Jody R. Schwartz
in Nucleic Acid Research and Molecular Biology 43:135-204, 1992
I. New Technology
and Old Theories in the Search for the Causes of Disease
A New Generation of Virologists Presents Latent Viruses as Pathogens
From Retroviral to Cellular Oncogenes-The Oncogene Hypothesis
From Autonomous Pathogens to Multifactorial Causes of Disease
The Search for Alternative Hypotheses
Viruses and Diseases Resulting from the Loss of Cells
Human Immunodeficiency Virus (HIV) and AIDS
The Virus-AIDS Hypothesis
The Drug-AIDS Hypothesis
The Drug- versus the Virus-AIDS Hypothesis
Hepatitis C Virus and Non-A Non-B Hepatitis
Measles Virus, HIV, and Subacute Scleroting Panencephalitis
Phantom Viruses and Neurological Disease
as Causes of Clonal Cancer
Human T-cell Leukemia Virus and Adult T-cell Leukemia
Herpes Virus, Papilloma Viruses, and Cervical Cancer
Hepatitis B Virus and Liver Carcinoma
Epstein-Barr Virus and Burkitt's Lymphoma
Oncogenes, Anti-oncogenes, and Cancer
Mutated Proto-myc Genes and Burkitt's Lymphoma
Rearranged Proto-abl Genes and Myelogenous Leukemia
Point-mutated Proto-ras Genes and Cancer
The Original ras-Cancer Hypothesis Postulates a First order
Ad hoc ras-Cancer Hypotheses Postulating Second- and Higher-order
int Genes with Integrated Mouse Retroviruses and Mouse Mammary
Constitutive Oncogenes, Mutated Anti-oncogenes, and Cancer
Evidence That Latent Viruses and Mutated Cellular Genes Are Pathogenic
Helper Genes and Cofactors to Close the Activity, Infectivity, and
Specificity Gaps of
Latent Viruses as Harmless Passengers
Drugs as Alternatives to Hypothetical Viral Pathogens
Mutated Genes and Latent Viruses as Trivial Genetic Scars of Cancer
Cancer by Somatic Gene Mutations Unconfirmed
Chromosome Abnormalities as Causes of Cancer
evidence is a very tricky thing," answered Holmes, thoughtfully.
"It may seem to point very straight to one thing, but if you
shift your point of view a little, you may find it pointing in an
equally uncompromising manner to something entirely different....
There is nothing more deceptive than an obvious fact...."
Conan Doyle, in The Boscombe Valley Mystery, 1928
community has been virtually unanimous in admiring its recent triumphs
in biotechnology-above all, the detection and amplification of minute
amounts of materials into workable and marketable products. However,
in clinical diagnostic applications, the new detection methods have
become a mixed blessing, which benefits medical scientists but not
necessarily their clients. Since rare signals have become just as
detectable as abundant ones, many latent viruses have been detected
and have been assumed to be just as pathogenic as active prototypes
(1-3). Likewise, cellular mutations have become detectable that
do not, or just barely, affect the function and activity of genes.
Yet when the affected genes are structurally related to retroviral
oncogenes, they are assumed to be just as oncogenic as highly active
retroviral oncogenes (1, 4-8). However, the evidence for these hypotheses
is only circumstantial-based on structural similarities to classical
pathogenic viruses and viral oncogenes. Thus, without direct proof,
these hypotheses may open the doors to psychologically harmful prognoses
and clinically harmful prevention programs, termed "molecular
genetics at the bedside" by Bishop (9).
I. New Technology
and Old Theories
in the Search for the Causes of Disease
A. A New Generation
Presents Latent Viruses as Pathogens
viral epidemics have all but disappeared in the Western world since
polio was eliminated with vaccines in the 1950s, the number of viruses
currently discovered and studied by virologists has reached epidemic
proportions. For example, zealous virus hunters have been able to
detect by ultrasensitive biological and biotechnical methods latent
viruses that are neutralized by antiviral immunity in diseases such
as AIDS, leukemias, lymphomas, hepatomas, hepatitis, cervical cancers,
encephalitis, and many others (1,3). Their proposals that latent
viruses cause these diseases are widely accepted, because from the
days when only the most pathogenic and abundant viruses were detectable,
all viruses still have the reputation of being pathogens.
diseases with which these newly discovered latent viruses are associated
are not contagious-unless one makes bizarre assumptions. One assumption
postulates that these viruses are "slow viruses" or "lentiviruses"
causing diseases only up to 55 years after infection and only after
they are neutralized by antibodies (see Sections II and III). Yet
all of these viruses replicate and are immunogenic within weeks,
not years, after infection just like conventional viruses. Another
assumption is that these viruses can shift from a nonpathogenic
dormant state to a pathogenic state without increasing their biochemical
activity or abundance.
A case in
point is the assumption that AIDS is caused by a virus. There were
over 160,000 AIDS patients in the U.S. in the last 10 years, and
there is no antiviral vaccine or drug. Yet at the time of this writing
there is not even one confirmed case of a health care worker who
contracted AIDS from a patient, nor of a scientist who contracted
AIDS from the "AIDS virus" that is propagated in hundreds
of research laboratories! The AIDS virus is just as inactive in
patients as it is in asymptomatic virus carriers (see Section II).
are not compatible with classical criteria of viral pathogenicity.
Conventional viruses are very active, abundant and replicating in
many cells that are killed or transformed when they cause diseases
such as polio, flu, measles, mumps, hepatitis, herpes, Rous sarcoma,
and many others (3, 10-12). Likewise, SV40 and adenoviruses inundate
many cells with viral T-antigens when they cause tumors, even though
the respective host animals are not permissive for viral replication
(13). Pathogenicity by these classical viruses results from high
biochemical activity in large numbers of cells. These viruses are
not pathogenic when they are latent or infect only small numbers
of cells. Indeed, even the most pathogenic viruses depend for their
survival on asymptomatic infections in which they are highly active
in small numbers of cells before they are stopped by antiviral immunity,
the reason that such infections are asymptomatic (3).
all conventional viruses are maximally pathogenic within weeks or
months after infection before they are neutralized by antiviral
immunity, causing disease as soon as they reach pathogenic thresholds
in the host (10-12). In rare cases, they may be reactivated to resume
replication, and hence pathogenicity, long after they are neutralized
by antiviral immunity (e.g., the herpes simplex virus). Reactivation
typically follows a transient immunodeficiency acquired by another
primary disease or other immunosuppressive conditions (12). Except
for these instances of viral reactivation, there are no known examples
of viruses that cause diseases only after a long latent period and
only after they have been neutralized by antibodies.
evidence that latent viruses can be pathogenic is only circumstantial,
based on structural similarities between latent viruses and active,
pathogenic viral prototypes. Further, these hypotheses are based
on the epidemiological evidence that latent viruses occur, or appear
to occur, in diseases at a higher rate than would be expected from
random infection (3, 14, 15) (see Section V).
B. From Retroviral
to Cellular Oncogenes-
The Oncogene Hypothesis
detecting point-mutations, deletions, and truncations of cellular
genes and latent or defective viruses put new life in the somatic
mutation hypothesis of cancer (16). It was postulated in 1969 by
Huebner and Todaro that latent viruses and covert cancer genes preexist
in normal cells and are "activated" to cancer genes and
cancer viruses by mutation (17). The proposal became known as the
oncogene hypothesis. The discoveries in 1970 of retroviral oncogenes
(18, 19) and in 1973 of cellular genes from which the coding regions
of retroviral oncogenes are derived (20-22) put the oncogene hypothesis
to its first test. It was proposed that mutation turns those genes
from which the coding regions of retroviral oncogenes are derived
into equivalents of viral oncogenes (6). These genes are now called
either proto-onc genes or cellular oncogenes (1, 5-8, 23,
24) or even "enemies within" the cell (25). And mutated
cellular oncogenes are euphemistically termed "activated"
cellular oncogenes (1, 5-8).
"activated" oncogenes are point-mutated proto-ras
genes that are thought to be bladder or colon cancer genes (23,
26-28), truncated proto-myc genes that are thought to be
Burkitt's lymphoma genes (29, 30), proto-myc genes with retroviruses
integrated upstream (31) and downstream (32) that are thought to
be avian lymphoma genes, and rearranged proto-abl genes that
are thought to be myelogenous leukemia genes (7, 8, 33). By analogy
to proto-onc genes, even genes that are not related to retroviral
onc genes are now thought to be "activated" oncogenes
if mutated by provirus integration, like the int genes of
mouse mammary tumors with retroviruses integrated within or nearby
(5, 8, 34).
proto-onc genes and int genes with integrated retroviruses
are either just as active or only slightly more active than their
normal counterparts (see Section IV). Moreover, the mutant genes
from tumors do not transform cells upon transfection. By contrast,
proviral DNA copies of retroviral oncogenes transform susceptible
cells and are about 100 times more active than normal proto-onc
genes (24, 35-38). During the last 5 years, the transforming function
of retroviral oncogenes, including those of Rous sarcoma, Harvey
sarcoma, and MC29 and MH2 carcinoma viruses, has been shown to depend
absolutely on transcriptional activity, rather than on mutations
in the coding region (39-44). This high transcriptional activity
of retroviral oncogenes results from retroviral promoters.
modification of the oncogene hypothesis, the antioncogene hypothesis,
proposes that constitutively active, but as yet unnamed, oncogenes
are "activated" by mutational inactivation of tumor suppressors
or anti-oncogenes (8, 9, 45). Examples are the retinoblastoma and
p53 anti-oncogenes that are thought to cause retinoblastoma (45)
and colon cancer (46) if they are inactivated by point-mutation,
truncation, or deletion. However, unmutated antioncogenes do not
revert tumor cells to normal (see Section IV).
evidence for these hypotheses is only circumstantial, based on structural
similarities between mutated "cellular oncogenes" displaying
a normal level of activity and about 100 times more active viral
oncogenes. Further, these hypotheses are based on the epidemiological
evidence that mutated genes occur, or appear to occur, in diseases
at a much higher rate than would be expected from spontaneous mutation
(4, 5, 7, 28, 47) (see Section V).
C. From Autonomous
to Multifactorial Causes of Disease
In view of
the apparent non-equivalence between the postulated pathogens and
their prototypes, the original hypotheses have been supplemented
by ad hoc hypotheses. Typically, these ad hoc hypotheses
postulate second- or even higher-order mechanisms of pathogenesis
that include cofactors and helper genes, in contrast to the classical
prototypes, which all follow first-order mechanisms of pathogenesis.
Moreover, the putative helper genes, like the putative primary pathogens,
are not disease-specific, because they are also found in asymptomatic
subjects. Indeed, "cofactors" are euphemisms for new hypotheses,
which grant face-saving roles to failing incumbents with large constituencies.
D. The Search
for Alternative Hypotheses
In the following,
we have reinvestigated the evidence for the claims that latent viruses
and mutated genes are pathogenic. Since the available evidence for
pathogenicity is insufficient, we conclude that the latent viruses
and mutated genes must be considered innocent until proven guilty.
creates a vacuum, we have attempted to present brief alternatives,
drawing in most cases from published work. However, in the case
of AIDS, we have documented an alternative to the virus-AIDS hypothesis
more extensively, because there is hardly any mention of alternatives
in the over 60,000 papers published on the AIDS virus and AIDS since
1983 (48). By challenging currently unproductive hypotheses and
by providing falsifiable alternatives, we hope to contribute to
the search for what really causes these diseases.
Viruses and Diseases
Resulting from the Loss of Cells
A. Human Immunodeficiency
Virus (HIV) and AIDS
AIDS is a
new syndrome of 25 previously known diseases (49-52). In America,
61% are microbial diseases such as pneumonia, candidiasis, tuberculosis,
cytomegalovirus, and herpes virus disease (50, 52) that result from
immunodeficiency due to a severe depletion of T-cells (49, 51).
The remaining 39% of AIDS diseases are dementia, wasting disease,
Kaposi sarcoma, and lymphoma, which are not consistently associated
with immunodeficiency and microbes (52-54). In the U.S., 32% of
AIDS patients are intravenous drug users (52, 55), about 60% are
male homosexuals (52) who frequently used drugs as aphrodisiacs
(54, 56-64, 103), and most of the remainder have severe clinical
or congenital deficiencies, including hemophilia (52, 54, 61). Over
80% of the American AIDS patients are 20- to 44-year olds, of which
about 90% are males (52). Different AIDS-risk groups have different
AIDS diseases. For example, homosexuals have 20 times more Kaposi
sarcoma than other AIDS patients (65), intravenous drug users have
a proclivity for tuberculosis (66, 67), "crack" (cocaine)
smokers for pneumonia (68), and users of the cytotoxic DNA-chain-terminator
AZT, prescribed to inhibit HIV, for anemia, nausea, and lymphoma
of all American AIDS patients are currently confirmed to have antibodies
to a retrovirus, termed human immunodefieieney virus (HIV) (51,
54, 72). However, all AIDS diseases occur in all risk groups in
the absence of HIV (see Section II,A,3) (54). In the U.S., HIV is
fixed to an extremely constant reservoir of about 1 million carriers,
ever since 1985, when it became possible to detect antibody against
HIV (the "AIDS test") (54, 73). HIV is naturally transmitted
from mother to child, like other retroviruses, at an efficiency
of about 50% (54). This efficiency might be higher than serological
tests indicate, because some proviruses of other perinatally transmitted
human retroviruses only become immunogenic with advanced age (54)
(see Section III). Sex is another natural mode of transmission.
However, it is highly inefficient, depending on an average of about
1000 sexual contacts (54, 74), because there is no HIV provirus
detectable, even with the polymerase chain reaction (PCR), in semen
in 24 out of 25 HIVpositive men (75). Since 1987, when AIDS was
given its current definition (50), about 30,000, or 3% of the 1
million Americans infected by HIV (53, 54, 73), develop AIDS annually
most medical scientists believe that AIDS is caused by HIV (51).
The hypothesis assumes: (i) that AIDS is new because HIV is thought
to be new in all countries with AIDS (14, 51); (ii) that AIDS is
acquired by sexual and parenteral transmission of HIV; (iii) that
HIV causes immunodeficiency by killing infected T-cells; (iv) that
50-100% of HIV infections lead to fatal AIDS diseases; (v) that
AIDS occurs on average only 10 years after antibodies to HIV appear
(a positive "AIDS test"), to reconcile the low (3%) morbidity
with the large number of asymptomatic HIV carriers; (vi) that antibodies
to HIV do not neutralize the virus (53, 76, 77), to reconcile AIDS
with antibodies to HIV; and (vii) that all unrelated AIDS diseases
are caused by the same HIV (49, 51, 54, 78).
In view of
this hypothesis, AIDS has been defined exclusively by the association
of the 25 indicator diseases with antibody to HIV (50, 51, 54).
Further, "safe sex" (49, 51) and "clean injection
equipment" for recreational drugs (55) are recommended as AIDS
prophylaxis for uninfected persons, and the cytotoxic DNA-chain-terminator,
3'azidothymidine (AZT) is prescribed to infected healthy, as well
as sick, persons to inhibit HIV (51, 71, 80a, 79, 80). The presence
of antibody to HIV in a healthy person is interpreted as a prognosis
for AIDS. Testing and counseling are provided routinely to applicants
of the U.S. Job Corps (81). Several countries, including the U.S.
and China, bar entry to HIV-positive persons. And a negative "AIDS
test" for antibodies to HIV has become mandatory in the U.S.
since 1985 for the approximately 12 million blood donations that
are collected annually (82) by the American blood banks and the
Red Cross (Irwin Memorial Blood Bank, San Francisco, personal communication,
1990) and for admission to the U.S. Army (73, 83).
Each of the
seven assumptions of the virus-AIDS hypothesis can be challenged
on epidemiological and virological grounds:
all new microbes spread exponentially in a population (11), the
complete failure of HIV to spread from its 1985 level, when it became
first detectable, indicates that the American "HIV epidemic"
is old. This is particularly compelling if one considers that there
is no antiviral vaccine and no antiviral drug. Thus, HIV is not
new in the U.S.
that procreative sex is about 10% efficient (3 days per month) and
sexual transmission of HIV only 0.1%, it follows that HIV depends
on perinatal transmission for its survival (54). If HIV survives
naturally via perinatal transmission, it cannot be pathogenic by
itself, just like all other perinatally transmitted parasites (12)-except
if one assumes latent periods that exceed the normal generation
time of humans. Indeed, chimpanzees experimentally inoculated and
health care workers accidentally inoculated with HIV do not develop
AIDS (51, 54). Thus, sexual transmission of HIV cannot be a sufficient
cause for AIDS.
no more than 1 in 500 T-cells of AIDS patients ever contains a DNA
provirus of HIV and over 99% of infected T-cells survive infection
(84), and since about 1 in 25 T-cells is regenerated during the
2 days it takes a retrovirus to infect a cell, HIV infection cannot
be responsible for the loss of T-cells in AIDS (53). Thus, HIV,
like all other retroviruses, does not kill cells (53, 85, 86). Indeed,
HIV is propagated commercially for the "AIDS test" in
cultured lines of the same human T-cells that it is said to kill
in vivo (87).
4. The assumption
that HIV is 50-100% fatal within 10 years cannot be correct, because
about 1 million Americans carry HIV since 1985 but only about 30,000
develop AIDS annually since 1987, when AIDS received its current
definition (50). Instead, it would take 33 years for all U.S. HIV
carriers to develop AIDS diseases based on the current data (3%
per year). An average latent period of 10 years would predict that
100,000 Americans would develop AIDS in 1 year.
viruses, as self-replicating toxins, are all fast immunogens and
thus potentially fast pathogens, but AIDS diseases are estimated
to occur on average only 10 years after HIV is neutralized by antiviral
antibodies, the assumption that HIV needs 10 years to cause AIDS
is arbitrary. The long intervals between infection and AIDS probably
indicate that HIV is not even necessary for AIDS, because there
is no "late" HIV activity, and because antibodies continue
to neutralize the virus during AIDS (53, 54).
6. The complete
absence of free HIV in nearly all AIDS patients (53, 54, 88)-the
reason that the isolation of HIV had escalated into an international
scandal (89, 90)-invalidates the assumption that antibodies to HIV
do not neutralize HIV. Indeed, antiviral immunity effectively restricts
HIV in AIDS patients (91, 92) to 1 provirus in about 500 T-cells,
and viral activity to less than 1 in 10,000 T-cells (53, 54, 84).
7. Since all
AIDS diseases occur in the absence of HIV in intravenous drug users,
homosexuals, and hemophiliacs, HIV is not even necessary for AIDS
diseases-except for their classification as AIDS (53, 54).
the many virological and epidemiological inconsistencies of the
virus-AIDS hypothesis, some, notably Montagnier (93) and recently
Maddox (94-96), have proposed that HIV is not sufficient for AIDS.
Accordingly, a number of "cofactors" such as mycoplasmas
(85, 93) and other viruses (15, 76) have been postulated as helping
HIV to cause AIDS. However, there is no consensus at this time about
a specific cofactor that would be sufficient to cause AIDS in combination
with HIV (76, 93). Moreover, there is not even one plausible hypothesis
as to how a latent retrovirus such as HIV, which is present in no
more than 1 in 500 T-cells, could possibly help another microbe
to cause AIDS that, by itself, is not able to do so.
are at least six inconsistencies between AIDS and infectious disease:
there is not even one case reported in the scientific literature
of a health care worker who contracted AIDS from a patient, although
there were over 200,000 AIDS patients in the U.S. in the last 10
years (52). Likewise, not even one scientist contracted AIDS from
the "AIDS virus" or from other microbes from AIDS patients,
which are propagated in hundreds of research laboratories and companies
(53, 54, 87).
2. All new
infectious diseases spread exponentially in susceptible populations
(11). However, despite widespread alarm, AIDS claims since 1987
only about 30,000 or 0.03% per year from a reservoir of over 100
million susceptible, sexually active Americans. This is particularly
paradoxical for a presumably infectious syndrome, because conventional
venereal diseases are increasing in the U.S. (97) and because there
is no anti-HIV vaccine and no anti-HIV drug.
3. The distribution
of all infectious venereal diseases is almost even between the sexes
(98). By contrast, 90% of American AIDS is restricted to males since
1981 (52). This is incompatible with infectious venereal disease.
all (94%) of the Americans who develop AIDS have been subject to
abnormal health risks (52). These risks include either long-term
consumption of recreational, psychoactive, and aphrodisiac drugs
and anti-HIV drugs such as the cytocidal DNA chain-terminator AZT
(see below) or congenital or acquired deficiencies such as hemophilia
(52, 54). This indicates that specific health risks are necessary
5. The observations
that distinct AIDS-risk groups have distinct AIDS diseases-e.g.,
homosexuals having 20 times more Kaposi sarcoma than HIV carriers
from other risk groups (65), intravenous drug users having a proclivity
for tuberculosis (66, 67), "crack" (cocaine) smokers for
pneumonia (68), and AZT users for anemia, nausea, and lymphoma (69-71)-are
also difficult to reconcile with a single infectious cause.
6. All AIDS
diseases occur in all AIDS-risk groups in the absence of HIV (54).
In view of
these inconsistencies between AIDS and infectious disease and the
total lack of a common active microbe in AIDS, several investigators,
including us, have concluded that AIDS may not be infectious (54,
hypothesis proposes that American AIDS diseases, above their normal
background, are the result of the long-term consumption of (a)
intravenous and (b) oral recreational drugs, and (c)
anti-HIV drugs (54, 60, 103). The following epidemiological and
drug-toxicity data support this hypothesis.
Recreational Drugs. Currently, 32% of the American AIDS
patients come from groups that use intravenous drugs such as heroin,
cocaine, and others (52, 55). This group includes about 75% of the
heterosexual AIDS cases, 71% of the females with AIDS, and over
10% of the male homosexuals and hemophiliacs with AIDS (52, 55).
In addition, about 50% of American children with AIDS were born
to mothers who are confirmed intravenous drug users and another
20% to mothers who had "sex with intravenous drug users"
and are thus likely users themselves (52, 55). Likewise, 33% of
European AIDS patients are intravenous drug users (104).
Recreational Drugs. Approximately 60% of the American AIDS patients
are 20- to 44-year-old male homosexuals (52). The following evidence
indicates that they come from groups who use oral psychoactive and
aphrodisiac drugs. A survey of 3916 self-identified American homosexual
men, the largest of its kind, reported in 1990 that 83% had used
one, and about 60% two or more, drugs with sex during the previous
6 months (105). These drugs include nitrite and ethylchloride inhalants,
cocaine, amphetamines, methaqualone, lysergic acid, phenylcyclidine,
and more (59, 61-63, 101,105-112). A study of 359 homosexual men
from San Francisco reported in 1987 that 84% had used cocaine, 82%
alkylnitrites, 64% amphetamines, 51% quaaludes, 41% barbiturates,
and 20% injected drugs, and 13% shared needles (107). This group
had been randomly selected from a list of homosexuals who had volunteered
to be investigated for hepatitis B virus infection and to donate
antisera to hepatitis B virus between 1978 and 1980.
and possibly other drugs are preferred by male homosexuals as aphrodisiacs
because they facilitate anal intercourse (105, 111, 113, 114). For
example, an early CDC study that included 420 homosexual men found
nitrite use far more frequent among homosexuals than among heterosexuals
and correlating directly with the number of different homosexual
partners (57). Surveys studying the use of nitrite inhalants in
San Francisco found that among homosexual men 58% were users in
1984 and 27% in 1991 compared to less than 1% among heterosexuals
and lesbians of the same age group (115).
are directly toxic as oxidants of biological molecules such as hemoglobin,
and are effective mutagens (101, 103). The National Institute on
Drug Abuse reports correlations from 69% (116) to virtually 100%
(101, 113) between nitrite inhalants and Kaposi sarcoma and pneumonia,
which are diagnosed as AIDS in the presence of antibody to HIV (50,
51, 54). In view of this, a causal link between nitrite inhalants
and Kaposi sarcoma and pneumocystis pneumonia in homosexuals was
first suggested in 1982 by the CDC (57) and other investigators
(56, 58). As a consequence, the sale of nitrite inhalants was banned
by the U.S. Congress in 1988 (Public Law 100-690) (117, 118). The
direct and indirect toxicity associated with the long-term use of
other recreational drugs has been described elsewhere (103).
Drugs. About 80,000 Americans and 120,000 persons worldwide
with and without AIDS currently take the cytocidal DNA chain-terminator
AZT (54) and an unknown number take other DNA chain-terminators
such as ddI and ddC (71). AZT has been prescribed since 1987 to
symptomatic (51, 70, 79, 119), and since 1990 to asymptomatic, carriers
of HIV, including babies and hemophiliacs (80, 120), in an effort
to inhibit HIV DNA synthesis (121). Thus, an unknown, but possibly
high, percentage of the 30,000 Americans that currently develop
AIDS per year (52) have used AZT prior to or after the onset of
AIDS. For instance, 249 out of 462 HIV-positive, AIDS-free homosexual
men from Los Angeles, included in the above survey (105), are on
AZT or ddI (122).
is an inhibitor of HIV DNA synthesis, it is not a rational medication
for persons with antibodies to HIV for the following reasons: (i)
There is no proof that HIV causes AIDS. (ii) Since no detectable
RNA-dependent viral-DNA synthesis occurs, and since the number of
infected cells remains stable once the virus is neutralized by antibodies
(53, 54) only cell DNA with and without proviruses of HIV is terminated
by AZT treatment. Further, since AZT cannot distinguish infected
from uninfected cells, and only 1 in 500 T-cells is infected in
AIDS patients and asymptomatic carriers (54, 84), it kills 500 uninfected
cells for every infected cell. Thus, AZT is inevitably toxic, killing
500 times more uninfected than infected cells. (iii) In view of
the hypothesis that HIV causes AIDS by killing T-cells (49, 51),
it is irrational to overkill infected cells with AZT.
from an inhibitor of DNA synthesis, many studies report AZT-mediated
toxicity. Anemia, neutropenia, and leukopenia occur in 20-50%, with
about 30-50% requiring transfusions within several weeks (70, 71,
123-125). Severe nausea from intestinal intoxication is observed
in up to 45% (70, 71, 80) and severe muscle atrophy in 6-8% (70,
126-128). Acute hepatitis, insomnia, headaches, dementia, seizures,
and vomiting are also reported effects of AZT (71). Lymphomas appear
in about 9% within 1 year on AZT (69). AZT is also mutagenic and
carcinogenic in mice (129, 130) and transforms cells in vitro
as effectively as methylcholanthrene (131). AZT toxicity varies
a great deal with the patient treated, due to differences in kinases
involved in its uptake and in AZT metabolism (71, 121, 131, 132).
All of these results explain Temin's profound observation that "...
the drug generally becomes less effective after six months to a
AZT is thought to have serendipitous therapeutic benefits based
on the only placebo-controlled study of its effects on AIDS patients
(70, 119). The study was sponsored by Burroughs Wellcome, the manufacturer
of AZT (70, 119). In this study, T-cell counts were observed to
increase from 4 to 8 weeks and then to decline to pretreatment levels.
Above all, AZT was claimed to "decrease mortality" because
only 1 out of 143 in the AZT-treated group died compared to 19 out
of 135 in the placebo group.
out of the 143 in the AZT group depended on multiple transfusions
for survival from anemia, compared to only 5 out of the 135 in the
placebo group. Since the number of subjects in the AZT group who
would have died from anemia if untreated (30) was larger than the
AIDS deaths and anemias of the control group combined (19 + 5),
the claim of decreased mortality is not realistic (70, 119). Moreover,
66 in the AZT group suffered from severe nausea and 11 from muscle
atrophy, compared to only 25 and 3 in the control group. The lymphocyte
count decreased over 50% in 34% of the AZT group and in only 6%
of the control. The study is further compromised by "concomitant
medication" (70), the failure to consider the effects of recreational
drug use and of patient-initiated randomizations of blinded AZT
and placebo controls (135). The brief AZT-induced gain of T-cells
may reflect compensatory hemopoiesis and random killing of pathogenic
parasites (132) and the influence of concomitant medication (70).
In view of
the inevitable toxicity of AZT, its popularity as an anti-HIV drug
can only be explained by the widespread acceptance of the virus-AIDS
hypothesis and the failure to consider the enormous difference between
the viral and cellular DNA targets. This may also be the reason
that long-term studies of AZT in animals compatible with human applications
have not been published (71).
Drug- Versus the Virus-AIDS Hypothesis
between HIV and drugs as causes of AIDS, it is necessary to determine
whether HIV carriers develop AIDS only when they use drugs, and
whether HIV-free drug users develop AIDS indicator diseases.
Use Necessary for AIDS in Presumed or Confirmed Carriers of HIV.
(i) Epidemiological correlations suggest that nitrites are necessary
for Kaposi sarcoma. (a) A 27- to 58-fold higher consumption of nitrites
(111, 115) correlates with a 20-fold higher incidence of Kaposi
sarcoma in male homosexuals compared to all other AIDS patients
of the same age group (65). (b) Among male homosexuals, those with
Kaposi sarcoma have used nitrite inhalants twice as often as those
with other AIDS diseases (101). (c) During the last 6-8 years, the
use of nitrite inhalants among male homosexuals decreased (e.g.,
from 58% in 1984 to 27% in 1991 in San Francisco) (115). In parallel,
the incidence of Kaposi sarcoma among American AIDS patients decreased
from a high of 37% in 1983 (136) to a low of 10% in 1990 (52). In
fact, nitrites may be sufficient causes for these diseases, because
there is no evidence that HIV was even present in any of these studies.
correlations indicate that nitrites are necessary for AIDS. The
first five cases diagnosed as AIDS in 1981, before HIV was known,
were male homosexuals who had all consumed nitrite inhalants and
presented with pneumocystis pneumonia and cytomegalovirus infection
(137). Early CDC data indicate that, in 1981 and 1982, 86% of male
homosexuals with AIDS had used oral drugs at least once a week and
97% occasionally (57, 138), and that every one of 20 Kaposi sarcoma
patients had used nitrites (56). The National Institute on Drug
Abuse reports correlations from 69% (116) to virtually 100% (101,113)
between nitrite inhalants and Kaposi sarcoma and pneumonia. Again,
drugs may have sufficed to cause these diseases, because HIV was
not diagnosed (50, 51, 54).
incidence of AIDS diseases among 297 HIV-positive, asymptomatic
intravenous drug users over 16 months was three times higher in
those who persisted than in those who stopped injecting drugs (139).
(iv) The T-cell
count of 65 HIV-infected drug users from New York dropped over 9
months in proportion with drug injection-on average, 35%-compared
to controls who had stopped (140).
(v) A placebo-controlled
study, investigating AZT as AIDS prophylaxis in HIV-positive, AIDS-free
25- to 45-year-old male homosexuals and intravenous drug users,
indicates that AZT induces diseases from within and without the
AIDS definition (80). During 1 year of taking 500 mg of AZT per
day, a group of 453 developed 11 AIDS cases, and a group of 457,
taking 1500 mg of AZT per day, developed 14 cases. The placebo group
of 428 developed 33 AIDS cases.
price for the presumed savings of 22 and 19 AIDS cases with AZT
was high, because 19 more cases of anemia, neutropenia, and severe
nausea appeared in the 500-mg AZT group, and 72 more such cases
appeared in the 1500-mg AZT group, than in the placebo group. This
indicates cytocidal effects of AZT on hemopoiesis and on the intestines.
Although these AZT-specific diseases were not diagnosed as AIDS,
neutropenia generates immunodeficiency. Surprisingly, in view of
its toxicity on leukocytes and red cells, a consistent loss of T-cells
was not observed in this study. A recent study investigating AZT
as AIDS prophylaxis observed leukopenia, e.g., T-cell depletion,
in 82% within 1 to 1.5 years of AZT treatment (140a). The study
is further compromised by the failure to report and to consider
the recreational drug-use histories and the many AZT-treatment adjustments
of the subjects analyzed.
48 weeks on AZT, 172 (56%) out of 308 AIDS patients developed additional
AIDS diseases, including pneumonia and candidiasis (125). This indicates
that AZT induces AIDS diseases within less than 1 year, and thus
much faster than the 10 years HIV is said to need to cause AIDS
(54). Likewise, no therapeutic benefits were observed for 365 French
(123) and 4 Norwegian AIDS (133) patients after 6 months on AZT.
annual lymphoma incidence of AZT-treated AIDS patients was reported
to be 9% by the National Cancer Institute and was calculated to
be 50% over 3 years (69). The lymphoma incidence of untreated HIV-positive
AIDS-risk groups is 0.3% per year and 0.9% per 3 years, derived
from the putative average progression rate of 10 years from HIV
to AIDS (54, 141,142) and the 3% incidence of lymphoma in AIDS patients
(52). Thus, the lymphoma incidence is 30-50 times higher in AZT-treated
than in untreated HIV-positive counterparts. In addition, "during
the past three years [of AZT therapy] a progressive increase in
the number of [AIDS] patients dying from lymphoma, ..." to
a current level of 16%, was noted in 1991 in a group of 346 AIDS
patients in London, most of whom were on AZT (143).
It is likely
that the chronic levels of the mutagenic AZT, at 10-30 µM (500-1500
mg/person/day), were responsible for the lymphomas. The alternative
proposal that HIV-induced immunodeficiency was responsible for the
lymphomas (69) is unlikely, since cancers do not reflect a defective
immune system (53, 144).
out of 11 HIV-positive, AZT-treated AIDS patients recovered cellular
immunity after discontinuing AZT in favor of an experimental HIV
vaccine (145), indicating that AZT sufficed for immunodeficiency.
out of five AZT-treated patients recovered from myopathy 2 weeks
after discontinuing AZT; two redeveloped myopathy on renewed AZT
(x) Four patients
with pneumonia developed severe pancytopenia and bone marrow aplasia
12 weeks after the initiation of AZT therapy. Three out of four
recovered within 4-5 weeks after AZT was discontinued (124), indicating
that AZT was sufficient for pancytopenia.
Use Sufficient for AIDS Indicator Diseases in the Absence of HIV.
(i) Among intravenous drug users in New York, representing a
"spectrum of HIV-related diseases," HIV was observed in
only 22 out of 50 pneumonia deaths, 7 out of 22 endocarditis deaths,
and 11 out of 16 tuberculosis deaths (66).
was diagnosed in 6 out of 289 HIV-free and 14 out of 144 HIV-positive
intravenous drug users from New York (146).
54 prisoners with tuberculosis in New York State, 47 were street-drug
users, but only 24 were infected with HIV (67).
(iv) In a
group of 21 heroin addicts, the ratio of helper to suppressor T-cells
declined within 13 years from a normal of 2 to less than 1, which
is typical of AIDS (50, 51), but only 2 were infected by HIV (147).
and immunodeficiency were diagnosed in 15 intravenous drug users
on average 10 years after they became addicted, but 2 were not infected
with HIV (148).
reactivity and abundance was depressed by long-term injection of
drugs not only in 111 HIV-positive but also in 210 HIV-free intravenous
drug users from Holland (149).
same lymphadenopathy, weight loss, fever, night sweats, diarrhea,
and mouth infections were observed in 49 out of 82 HIV-free and
89 out of 136 HIV-positive, long-term intravenous drug users from
New York (150), and in about 40% of 113 intravenous drug users from
France, of which 69 were HIV-positive and 44 were negative (151).
The French group had used drugs for an average of 5 years.
six HIV-free male homosexuals with Kaposi sarcoma, five reported
the use of nitrite inhalants (152).
neurological deficiencies were observed among 12 HIV-infected and
16 uninfected infants from drug-addicted mothers (153).
long-term use of recreational and anti-HIV drugs appears necessary
in HIV-positives and sufficient in HIV-negatives to induce AIDS
indicator and other diseases.
that the drug-AIDS hypothesis is epidemiologically and pathologically
better grounded than the virus-AIDS hypothesis. About 32% of American
AIDS patients are confirmed intravenous drug users, probably 60%
use aphrodisiac drugs orally, and an unknown but large percentage
of both behavioral and clinical AIDS-risk groups use AZT. Moreover,
the consumption of recreational drugs by AIDS patients is probably
underreported, because the drugs are illicit, and because medical
scientists and support for research are currently heavily biased
in favor of viral AIDS (68, 154, 155). The pathogenicity of these
drugs is empirically known for all, and mechanistically for some,
drugs, notably for AZT and nitrites (103).
evidence for the role of drugs in AIDS is rejected by proponents
of the virus-AIDS hypothesis (15, 77, 105). This is certainly one
reason why despite the current drug-use epidemic, there are no studies
that investigate the long-term effects of psychoactive drugs and
AZT in animals, compatible with the time periods and dosages used
by AIDS patients (155).
to the near complete correlation between drugs and AIDS, antibodies
to HIV are confirmed in only about 50% of AIDS patients (51, 72),
and it is a complete mystery how HIV acts as a pathogen, despite
enormous research efforts (14, 15, 54, 156).
hypothesis resolves all scientific paradoxes posed by the prevailing
1. In America,
HIV is a long-established, endemic virus, but AIDS is new-because
the drug epidemic is new.
2. AIDS is
restricted for over 10 years to 10,000 (52) or 0.01% of the over
100 million sexually active heterosexual Americans per year, and
to 20,000 (52) or 0.25% of the 8 million homosexuals, estimated
at 10% of the adult male population (109, 111). But conventional
venereal diseases are on the rise in the U.S. (97), and there is
no vaccine or drug against HIV. This is because AIDS is due to drug
consumption rather than sexual activity.
3. Over 72%
of American AIDS cases are 20- to 44-year-old males (52)-although
no AIDS disease is male-specific (50, 51)-because males of this
age group consume over 80% of all "hard" psychoactive
and aphrodisiac drugs (101, 103, 111, 115, 157, 158).
AIDS diseases occur in distinct risk group-because they use distinct
drugs (e.g., users of nitrites get Kaposi sarcoma, users of intravenous
drugs get tuberculosis, and users of AZT get leukopenia and anemia).
AIDS occurs, on average, 10 years after HIV infection (51, 53, 54),
although infectious agents, being self-replicating toxins, typically
strike within weeks or months after infection (11, 12). Indeed,
HIV is immunogenic, and may be mildly pathogenic in humans within
weeks after infection and is then "effectively and rapidly
limited" by antiviral immunity (91, 92). This is because HIV
infection and AIDS are unrelated events. The duration and toxicity
of drug consumption and individual thresholds for disease determine
when AIDS occurs, irrespective of when and whether HIV infects.
6. HIV, as
well as many other parenterally and venereally transmitted microbes
and viruses, are mere markers for AIDS and AIDS risks (54, 107,
159)-because the higher the consumption of unsterile, injected drugs
(140, 151) and sexual contacts mediated by aphrodisiac drugs, the
more microbes are accumulated.
7. Some old
diseases of hemophiliacs, other recipients of transfusions, and
the general American population are called AIDS-if they coincide
with perinatal or parenteral HIV infection (54).
8. Old African
diseases such as slim disease, fever, diarrhea, and tuberculosis
are called AIDS now, although they are clinically and epidemiologically
very different from American AIDS. They occur in adolescents and
adults of both sexes that are subject to protein malnutrition, parasitic
infections, and poor sanitary conditions (53). Only because HIV
is endemic in over 10% of Central Africans are over 10% of old African
diseases now called AIDS (51, 53, 54).
hypothesis predicts that the AIDS diseases of the behavioral AIDS-risk
groups in the U.S. and Europe can be prevented by controlling the
consumption of recreational and anti-HIV drugs, but not by "safe
sex" (51) and "clean injection equipment" (55) for
unsterile (!) street drugs. According to the drug-AIDS hypothesis,
AZT is AIDS by prescription. Screening of blood for antibodies to
HIV is superfluous, if not harmful, in view of the anxiety that
a positive test generates among the many believers in the virus-AIDS
hypothesis and the toxic AZT prophylaxis, prescribed to many who
test "positive." Eliminating the test would also reduce
the cost of the approximately 12 million annual blood donations
in the U.S. (82) by $11 each (Irwin Memorial Blood Bank, personal
communication, 1990) and would lift travel restrictions for antibody-positives
to many countries, including the U.S. and China. The drug-AIDS hypothesis
is testable epidemiologically and experimentally by studying AIDS
drugs in animals.
C Virus and Non-A Non-B Hepatitis
hepatitis is observed primarily in recipients of transfusions and
in intravenous drug users (3, 12, 160). It has been postulated to
be a viral disease because inoculation of plasma or serum (3-75
ml) from hepatitis patients into chimpanzees induced some biochemical
markers of hepatitis, such as alanine aminotransferase, in half
of the animals (160). However, none of the animals developed hepatitis
(161, 162). Trace amounts of presumably viral RNA have recently
been detected in the liver of hepatitis patients. In addition, "nonneutralizing"
antibodies to "nonstructural epitopes," from an apparently
latent RNA virus, have been identified mostly in asymptomatic carriers
(160). Cloning and sequencing indicated that the RNA is directly
coding and measures about 10 kb. Therefore, the suspected virus
has been tentatively classified as a togavirus (160). Viral RNA
was only detectable after amplification with the PCR in 9 out of
15 non-A non-B hepatitis patients, and non-neutralizing antibodies
were found in only 7 of the 9 RNA-positive and in 3 of the 6 RNA-negative
patients (163). Likewise, liver tissues from chimpanzees inoculated
with sera from hepatitis patients contain only one viral RNA molecule
per ten cells (160).
In view of
this evidence, the putative virus has been termed hepatitis C virus
(HCV) to indicate that it is the cause of the hepatitis. Subsequently,
the Food and Drug Administration has recommended, and the American
Association of Blood Banks has mandated, as of 1990, the testing
of the approximately 12 million annual blood donations in the U.S.
(82) for antibodies to HCV at an approximate cost of $5 per test.
The test was developed by Chiron Co., Emeryville, California (Irwin
Memorial Blood Bank, personal communication, August 15, 1991).
arguments cast doubt on the hypothesis that HCV causes hepatitis:
sera or plasma from hepatitis patients does not cause hepatitis
if inoculated into chimpanzees, indicating that HCV is not sufficient
to cause the disease. Moreover, since the virus has not been propagated
in culture and isolated in a pure form, the possibility exists that
the biochemical markers of hepatitis that are observed in chimpanzees
inoculated with plasma were induced by another agent. Thus, HCV
is not likely to be a sufficient cause of hepatitis in humans.
2. The presence
of HCV in asymptomatic subjects at the same concentration and activity
as in hepatitis patients also indicates that the virus is not sufficient
to cause hepatitis.
3. The absence
of viral RNA in 6 out of 15 hepatitis C patients indicates that
the virus is not necessary for the disease.
that HCV either causes disease by unprecedented mechanisms with
as little as one RNA molecule per 10 liver cells in some and even
less in other carriers, or that the virus is not the cause of non-A
non-B hepatitis. By contrast, the concentration of viral RNAs made
by conventional pathogenic viruses, including togaviruses, ranges
from 103 to over 104 per cell (10). Therefore, it seems plausible
that a latent passenger virus was identified that survives by establishing
chronic asymptomatic infections at very low, nonpathogenic titers
Virus, HIV, and Subacute Scleroting Panencephalitis
In 1967, a
cytocidal measles virus was proposed to be the cause of a very rare,
subacute scleroting panencephalitis of children (165), based on
correlations with antibodies to the virus or trace amounts of virus
(3, 10, 12). The encephalitis is observed only 1-10 years after
an acute primary infection, in the face of antiviral immunity, and
in only about 1 out of 1 million children infected by the virus
(3, 10, 12). The virus can only be isolated from the brains of 2
out of 8 encephalitis patients after cocultivation of brain cells
with susceptible human cells (166). Thus, only a few intact virus
particles are present in the brains of some, but apparently not
in all, children with encephalitis. Viral gene expression in brain
autopsies is 10- to 200-fold lower than in virus-replicating control
cells, amounting to as few as 10 mRNAs per cell (167). Moreover,
mutations and deletions were observed in these viral RNAs compared
to wild-type measles virus (168). Accordingly, some viral RNAs are
not even translated (3). By contrast, the wild-type virus causes
measles within weeks after infection, at very high virus titers,
and prior to antiviral immunity (10, 12).
virus-encephalitis hypothesis has a number of epidemiological and
the disease does not occur concurrently with, or instead of, the
conventional measles disease during a primary infection, and since
antiviral immunity does not protect against the disease, measles
virus cannot be sufficient to cause the subacute panencephalitis.
2. The virus
cannot be a sufficient cause of the disease because only 1 in 106
infected persons develops panencephalitis, compared to one in a
few if not all who develop measles disease before antiviral immunity
(3, 10, 12).
viruses are self-replicating toxins, all are potentially "fast"
pathogens, but encephalitis is observed only 1-10 years after infection,
measles virus cannot be sufficient for panencephalitis.
4. The absence
of infectious virus in some panencephalitis cases, and the very
low concentration of viral RNA in all cases, suggest that measles
virus is either not causative, or is causative by a mechanism that
is totally different from that causing measles disease. During conventional
measles disease, the virus is abundant, making over 1000 RNA molecules
per cell in large numbers of cells (3, 10, 12, 167, 168).
In view of
these paradoxes, it was suggested that selection of viral mutants
would account for the encephalitis-pathogenicity of the virus (3,
167, 168). However, this seems unlikely, because the virus does
not replicate sufficiently in encephalitis patients to generate
new pathogenic variants, and because natural variants with a neurotropic
specificity would then be expected.
About 15 years
after the measles virus-encephalitis hypothesis was advanced, others
proposed that the encephalitis was caused by a latent retrovirus
closely related to HIV (169). This hypothesis also suffers from
the problem that the presumed viral pathogen is latent (169). In
addition, an encephalopathy is hard to reconcile with the fact that
retroviruses depend on mitosis for infection (170) and the fact
that neurons stop dividing soon after birth (1).
Viruses and Neurological Disease
belief in viruses as causes of diseases has in some instances even
exceeded their very definition. For example, the Nobel Prize in
1976 was given for hypothetical, slow, and unconventional viruses
that would cause neurological diseases such as kuru, Creutzfeld-Jacob's,
and Alzheimer's diseases, after long latent periods of up to 30
years (171). Kuru is a now-extinct neurological disease of a small
tribe of 35,000 in New Guinea that reportedly was transmitted by
ritual cannibalism (3, 12, 171). "Slow and unconventional"
viruses have been postulated because 4 out of 7 chimpanzees had
developed neurological diseases about 1-2 years after they had been
inoculated intracerebrally with brain suspensions from kuru patients
(172). The presumed Creutzfeld-Jacob virus failed to induce neurological
disease if presumably infected materials were inoculated into the
brains of chimpanzees (3). A slow, unconventional virus has also
been claimed as the cause of scrapie, a neurological disease of
sheep (3, 12).
incubation periods from inoculation of brain suspensions from kuru
patients to neurological disease in the animals (1-2 years) and
from presumed infection of humans to kuru (up to 30 years) differ
significantly, it is not clear whether the diseases were caused
by the same agent. Considering the claim that the viruses are naturally
transmitted by cannibalism, it seems inappropriate that the traumatic
intracerebral inoculation was chosen to test the oral transmission
hypothesis. Nevertheless, Gajdusek et al. pointed out, "To
anyone who had the opportunity of observing the unique syndrome
of kuru ... the similarity of its clinical picture and course to
the experimentally induced syndrome ... is dramatically evident"
The slow virus-neurological
disease hypothesis suffers from several shortcomings:
1. None of
these hypothetical viruses has ever been isolated and chemically
analyzed. Their presumed properties all far exceed the known ranges
of conventional viruses and even of known proteins and nucleic acids.
For example, the kuru and Creutzfeld-Jacob viruses are said to resist
boiling water, ionizing gamma radiation, ultraviolet radiation,
and inactivation with formaldehyde (3, 171 ). Moreover, the viruses
are not antigenic, and not visible under the electron microscope,
although available preparations are reported to have titers of 107
lethal doses per milliliter (3). Paradoxically, the slow, unconventional
viruses have since evolved into an infectious protein, termed prion,
"derived from a normal cellular protein ... through an unknown
posttranslational process" (173).
2. The virus-kuru
hypothesis fails to account for the long latent periods between
presumed infection and disease and for the restriction of the disease
to a very specific risk group.
3. A recent
analysis of the original data on kuru transmission casts doubt on
the virus-kuru hypothesis, because the evidence for cannibalism
was fabricated (174).
In view of
this, we agree with a review by Gibbs, a collaborator of Gajdusek,
that "many paradoxes [were] thrust on us by the discovery of
these unconventional viruses as the etiological agents of chronic,
progressive, degenerative diseases of the central nervous system
. . . " and that "toxic or genetic determinants and even
trauma lead to the same pathogenesis ..." (3). Indeed, it seems
plausible that the toxicity and trauma of intracerebral inoculations
of human brain suspensions from kuru patients could cause neurological
diseases without phantom viruses said to be the etiological agents.
The restriction of the slow neurological diseases to specific ethnic
groups or to sporadic cases could reflect genetic and acquired deficiencies
rather than selective and slow viruses.
as Causes of Clonal Cancer
A. Human T-cell
Leukemia Virus and Adult T-cell Leukemia
leukemia virus-I (HTLV-I) was originally discovered in a T-cell
line from a leukemic patient (175). This line, termed HUT 102, only
produced virus after it had been propagated in vitro, in
the absence of the virus-suppressing immune system of the host,
and after it had been treated with mitogens and mutagens such as
iododeoxyuridine, an agent known to activate dormant retroviruses
(6). Since the virus was isolated from a cell line that came from
an adult patient with T-cell leukemia, the virus was proposed to
be the cause of adult T-cell leukemia (ATL), and hence named human
T-cell leukemia virus (175, 176). However, a parallel T-cell line,
termed HUT 78, derived from another patient with T-cell leukemia,
failed to yield a retrovirus (87).
for the hypothesis was derived from epidemiological correlations
between antibodies to HTLV-I and ATL in Japan and the U.S. (3, 37,
176). Based on 30,000 blood donations, the American Red Cross has
reported that in 1986-1987 about 0.025% or 65,000 Americans were
infected with HTLV-I (3, 82), but the American T-cell Leukemia/Lymphoma
Registry had recorded in 1990 in the U.S. no more than 90 ATLs.
Among these, 75 were non-Caucasians (177), a group in which HTLV-I
is often endemic (178). However, the same Registry also reports,
"although most cases of ATL are HTLV-I-associated ... many
are not" (177). As in the U.S., HTLV-I-free ATLs have been
observed in Japan (1 79). A controlled study comparing the incidence
of the leukemia in HTLV-I-positive and -negative control groups
has never been published.
"The diagnosis of ATL is made from the characteristic clinical
findings, the detection of serum antibodies to HTLV-I and, when
necessary, the confirmation of monoclonal integration of HTLV-I
proviral DNA in cellular DNA of ATL cells" (180). According
to this tautology, ATL is defined and distinguished from virus-free
T-cell leukemias solely by the presence of antibody to HTLV-I or
HTLV-I is also postulated to cause an HTLV-I-associated myelopathy
(HAM), which is a neurological disease also defined only by the
presence of HTLV-I (3, 181).
ATL is clonal,
originating from a single cell, like virus-free T-cell leukemias.
The clonality of the leukemia is defined by chromosome abnormalities,
as well as by clonal proviral integration sites (2, 176). However,
there are no specific integration sites of HTLV-I in different leukemias
(2). In leukemic cells, the virus is always latent, suppressed by
antiviral immunity, and sometimes even defective (2). It is for
this reason that the virus was originally discovered only in
vitro, after reactivation from latently infected leukemic cells
grown in culture.
other non-oncogenic retroviruses (6, 54), is naturally transmitted
from mother to child with an efficiency of 22% based on testing
for antiviral antibodies (176, 182, 183). Indeed, latent proviruses
appear to be transmitted perinatally at a higher efficiency than
antibody tests indicate, because the antibody titers increase with
age (176) at a much faster rate than could be accounted for even
by thousands of sexual contacts (183). Thus, this virus, like all
other retroviruses without oncogenes (54), survives from perinatal
transmission. Sex is another, although highly inefficient, mode
of transmission, depending on an average of over 1000 sexual contacts
Based on epidemiological
studies from Japan, HTLV-I is said to cause leukemia in only 1-5%
of all virus carriers in a lifetime (182). The annual incidence
of the leukemia per HTLV-I carrier in Japan is estimated to be only
1 in 1000 (182, 184). Since HTLV-I is a perinatally transmitted
retrovirus, but leukemia typically appears, if at all, only in 50-
to 60-year-olds, the latent period from infection to disease is
estimated at 55 years (176, 185).
epidemiological and virological arguments cast doubt on the HTLV-I-leukemia
to the American Red Cross, "ATL ... as of September 1989, has
not been reported in association with transfusion transmitted HTLV-I
infection," although about 65,000 Americans were infected with
HTLV-I and about 12 million blood donations are annually transfused
to millions of recipients in the U.S. (82). Thus, HTLV-I cannot
be sufficient to cause leukemia.
viruses, as self-replicating toxins, are all potentially fast pathogens,
but leukemia is only observed about 55 years after infection, HTLV-I
cannot be sufficient for leukemia.
that 1% of HTLV-I carriers develop ATL per lifetime in Japan and
about 0.1% (90 : 65,000) in the U.S., that the leukemias are clonal
deriving from single cells, and that each carrier must contain at
least 107 latently infected T-cells (because the limit of provirus
detection by hybridization is 1 in 1000 cells) and that humans contain
1010 to 1011 T-cells that go through at least 420 generations in
a 70-year lifetime (see Section IV) (37, 186), then only 1 out of
102 (Japan) to 103 (U .S.) x 107 x 420 = 1012 infected T-cells become
leukemic. Thus, HTLV-I cannot be sufficient for leukemogenesis.
antibodies that completely neutralize HTLV-I to virtually undetectable
levels (2) do not protect against the leukemia. This also indicates
that HTLV-I is not sufficient for leukemogenesis.
cause either polyclonal tumors via dominant, biochemically active
oncogenes (6, 37), or possibly clonal tumors via site-specific integration
that generates active virus-cell hybrid oncogenes (31, 40, 42).
Yet HTLV-I neither expresses a leukemia-specific gene product that
could function as an active oncogene, nor does it integrate at a
specific site in different "viral leukemias" (2, 187).
Thus, HTLV-I cannot be sufficient for leukemogenesis.
6. The statement
of the American T-cell Leukemia/Lymphoma Registry that "although
most cases of ATL are HTLV-I associated ... many are not" (177)
and the reports of virus-negative leukemias from Japan (179) and
other countries (2) indicate that HTLV-I is not even necessary for
7. The HTLV-I-leukemia
hypothesis fails to explain the clonal chromosome abnormalities
that are consistently found in all ATLs (2, 188)-except if one makes
the additional odd assumption that HTLV-I only transforms cells
with a preexisting chromosome abnormality.
are no virus-determined diagnostic criteria, besides the presence
of antiviral antibodies, nor are there any controlled epidemiological
and virological criteria to support the hypothesis that HTLV-I is
the cause of ATL. Therefore, ad hoc hypotheses have been
advanced proposing "a second oncogenic event, such as a chance
translocation or a second oncogenic virus ..." for viral leukemogenesis
(187). Others estimate five steps in leukemogenesis, of which HTLV-I
is postulated to be an "initiator" (185).
even one transfusion-transmitted leukemia case has been recorded
in the U.S., it seems surprising that a blood test for antibodies
against HTLV-I has become mandatory for members of the American
Association of Blood Banks since February 1989. It raises the cost
of each of the approximately 12 million annual blood donations in
the U.S. (82) by $5-11 (189; Irwin Memorial Blood Bank, personal
communication, 1990). Indeed, an HTLV-I epidemiologist pointed out,
"Ironically, this route of [HTLV-I] transmission is numerically
the least important," considering the 55-year average latent
period from infection to leukemia, "the advanced age of most
U.S. blood recipients, and the observation that as many as 60% of
transfusion recipients may die within approximately 3 years of transfusion
because of their underlying disease" (183). Nevertheless, in
terms of blood testing expenses, HTLV-I has reached cost-parity
with HIV, which adds another $11 test fee to each blood donation
(Irwin Memorial Blood Bank, personal communication, 1990).
hypothesis suggests that spontaneous or perhaps radiation-induced
chromosome abnormalities induce the clonal leukemias (see Section
VI). Nuclear radiation from the Hiroshima and Nagasaki bombs is
blamed for 147 leukemias (190). By proposing that one out of billions
of normal HTLV-I-infected cells is transformed by a spontaneous
chromosome abnormality, our hypothesis readily resolves the paradox
of the clonal chromosome abnormalities in all "viral"
Virus, Papilloma Viruses, and Cervical Cancer
the SV40/adenovirus-cancer models, infection by herpes simplex virus
(HSV) was postulated in the 1970s to be the cause of cervical cancer
based on epidemiological correlations with HSV DNA (3). The virus
is sexually transmitted and is latent in about 85% of the adult
population of the U.S. (3). Infection by intact HSV typically kills
the cell. However, defective and intact viruses that become latent
do not kill cells (3).
DNAs in cervical cancers are defective and integrated with cell
DNA. Cervical cancers with defective HSV DNA are clonal, just like
virus-free cancers (191-194). In agreement with the SV40/ adenovirus
models, HSV does not replicate in the tumors. But, unlike the SV40/adenovirus
models, no set of viral genes is consistently present or expressed
in human cervical cancers. Therefore, the "hit-and-run"
mechanism of viral carcinogenesis was proposed (195). It holds that
neither the complete HSV, nor even a part of it, needs to be present
in the tumor. Obviously, this is an unfalsifiable, but also an unprovable,
by the SV40/adenovirus models, and based on epidemiological correlations,
infection by human papilloma virus (HPV) was postulated in the 1980s
by zur Hausen to be a causative factor in cervical and anogenital
cancers (3, 191, 196).
viruses are transmitted by sexual and other contacts, like the herpes
viruses, and are widespread or "ubiquitous" in at least
50% of the adult population of the U.S. and Europe (3, 191). For
example, using the PCR to amplify sequences of one particular strain
of papilloma virus, 46% of 467 women in Berkeley, California, with
a median age of 22 were found to carry HPV, but none of them had
cervical cancer (199). Many other strains of HPV exist (3, 191)
that could not be detected with the assay used in this study (199).
Like the SV40/ adenovirus models, HPV does not replicate in the
tumors. But, unlike these models, HPV naturally replicates nonlytically
(13), forming polyclonal warts with unintegrated viral DNA plasmids
reports that cervical cancers occur in less than 3% of infected
women in their lifetime, but the incidence in HPV-free controls
was not reported (191). In the U.S., the incidence of cervical cancer
in all women, with and without HPV, per 70-year lifetime is about
1% (197). In a controlled study of age-matched women, 67% of those
with cervical cancer and 43% of those without were found to be HPV-positive
(198). These cancers are observed on average only 20-50 years after
sets and amounts of viral DNA are integrated into cell DNA of different
carcinomas (191), and viral DNA is poorly expressed in some cancers
and not expressed at all in others (3, 191, 201). Moreover, different
HPV strains are found in different cancers (3, 191, 196). Viral
antigens are found in only 1-5% of carcinomas (3). Accordingly,
HPV does not replicate in the cancer cells and there are no reports
of HPV-specific histological or physiological markers that set HPV
DNA-positive apart from negative carcinomas (191). There is also
no virus-specific integration site in HPV DNA-positive cancers (191),
indicating that no specific cellular gene is activated, or that
a tumor suppressor gene is inactivated by integration of viral DNA.
HPV DNA-positive tumors are clonal and carry clonal chromosome abnormalities,
just like virus-negative tumors (191-194).
hypothesis of zur Hausen proposes that HPV encodes a "transforming
factor" that is suppressed in normal cells by a cellular interference
factor (CIF). Inactivation of both CIF alleles by mutation is postulated
to result in viral carcinogenesis (191). The low probability of
developing mutations in both suppressor alleles is said to explain
the long intervals between infection and cancer. This hypothesis
correctly predicts that only a small fraction of infections lead
to cancer. It further predicts clonal tumors with active HPV DNA
and mutations in both alleles of the suppressor genes, and it predicts
no effects on the karyotypes of cells.
al. proposed that a viral protein neutralizes the proteins of
the retinoblastoma and p53 tumor suppressor genes, and that neutralization
of these suppressor proteins causes cancer (202). The proposal is
modeled after the hypothesis that retinoblastoma is caused by a
cellular cancer gene, provided that a complementary suppressor gene,
termed the retinoblastoma gene, is inactivated (see Section IV).
This hypothesis predicts polyclonal tumors.
epidemiological and biochemical arguments cast doubt on these HPV-cancer
allelic mutation of suppressor genes, as postulated by zur Hausen,
predicts a few cancers soon, and more long after infection. Since
cancers only appear 20-50 years after infection, cooperation between
HPV and mutations cannot be sufficient for carcinogenesis.
the proposal of zur Hausen that inactivation of host suppressor
genes is necessary for viral transformation is not compatible with
HPV survival. Since HPV, like all small DNA viruses, needs all of
its 8-kb DNA for virus replication (13), suppression of one or more
HPV proteins by normal cellular genes would effectively inhibit
virus replication in all normal cells. Conversely, if viral transforming
proteins were not suppressed by normal cells, virus-replicating
wart cells should be tumorigenic because all viral genes are highly
expressed in virus replication (1, 13, 191).
3. The clonality
of cervical cancers rules out the Howley hypothesis.
4. The lack
of a consistent HPV DNA sequence and of consistent HPV gene expression
in HPV DNA-positive tumors is inconsistent with the zur Hausen and
Howley hypotheses and indicates that HPV is not necessary to maintain
5. The presence
of HPV in no more than 67% of age-matched women with cervical cancer
(198) also indicates that HPV is not necessary for cervical cancer.
6. The hypothesis
also fails to explain the presence of clonal chromosome abnormalities
consistently seen in cervical cancer (16, 192-194)-except if one
makes the additional odd assumption that only cells with preexisting
chromosome abnormalities are transformed by HPV.
that neither HPV nor HSV plays a direct role in cervical carcinomagenesis.
Moreover, the HPV-cancer hypothesis offers no explanation for the
absence of a reciprocal venereal male carcinoma.
inactive and defective viral DNA from past infections in non-tumorigenic
cells with a commercial hybridization test (Vira/Pap, Digene Diagnostics,
Silver Spring, Maryland) or with the PCR (199) seems worthless as
a predictor of rare carcinomas appearing decades later, in view
of the "ubiquity" (191) of these viruses in women and
the total lack of evidence that cervical cancer occurs in women
with HPV more often than in those without. This test, at $30-150,
is currently recommended for the 7 million Pap smears that appear
"atypical" in the U.S. per year (Digene Diagnostics, personal
communication, 1991). By contrast only 13,000 cervical cancers are
observed annually in both HPV-positive and -negative women in the
U.S. (197). Indeed, the test may be harmful, considering the anxiety
a positive result induces in believers of the virus-cancer hypothesis.
cervical carcinoma hypothesis suggests that rare spontaneous or
chemically induced chromosome abnormalities, which are consistently
observed in both HPV and HSV DNA-negative and -positive cervical
cancers (192-194), induce cervical cancer. For example, smoking
has been identified as a cervical cancer risk (204). The controlled
study of age-matched women described above suggests that 52% of
the women with cervical cancer were smokers compared to only 27%
of those without (198). Indeed, carcinogens may be primary inducers
of abnormal cell proliferation rather than HPV or HSV. Since proliferating
cells would be more susceptible to infection than resting cells,
the viruses would be just indicators, rather than causes of abnormal
proliferation. Activation of latent retroviruses like HTLV-I (Section
III,A) (2), herpes viruses (12), and lambda phages (205) by chemical
or radiation-induced cell damage and subsequent proliferation are
classical examples of such indicators. Indeed, Rous first demonstrated
that the virus indicates hydrocarbon-induced papillomas; it "...
localized in these and urged them on ..." and suggested that
enhanced proliferation is a risk factor for carcinogenesis (203).
to this hypothesis, HPV or HSV DNAs in tumor cells reflect defective
and latent viral genomes accidentally integrated into normal or
hyperplastic cells, from which the tumor is derived. This hypothesis
readily reconciles the clonal chromosome abnormalities with the
clonal viral DNA insertions of the "viral" carcinomas.
The inactive and defective viral DNA in the carcinomas would be
a fossil record of a prior infection that was irrelevant to carcinogenesis.
B Virus and Liver Carcinoma
evidence indicates that chronic hepatitis B virus (HBV) carriers
in Asia have a 250-fold higher risk of developing hepatomas than
do non-carriers (3, 12, 206-208). The virus is typically transmitted
perinatally in Asia and Africa (3, 207). In over 95% of infections
in Asia and 99.9% in the U.S. and Europe the virus is completely
neutralized by antiviral immunity. In people with drug- or disease-induced
immunodeficiencies the virus remains chronically active (12). Approximately
1 out of 70 chronic HBV carriers in Asia develop clonal hepatomas
and 1 out of 300 develop liver cirrhosis in their lifetime (3, 207).
However, the liver tumors appear only in 30- to 60-year-olds. Moreover,
chronic HBV carriers in Asia are "more likely" to develop
hepatomas than those in Europe and the U.S. (12). Inoculation of
HBV into chimpanzees has failed to cause hepatomas (3).
is thought not to kill infected cells and viral DNA is replicated
as a plasmid and thus not typically integrated into the host DNA
(3, 12). However, molecular studies have detected clonal inserts
of HBV DNA randomly integrated into the cellular DNA of liver carcinoma
tissues (196, 209). Viral DNA is defective and not replicated in
HBV DNA-positive hepatomas (209), like SV40 and adenovirus DNAs
in the corresponding viral tumors. By contrast to the SV40/adenovirus
models, no subset of viral DNA is consistently found or expressed
in HBV-positive tumors (209, 210). Only 11-19% of tumors in HBVpositive
patients express some viral antigens, compared to 26-61% expressing
them in surrounding non-tumorous tissues (211). In addition to clonal
inserts of HBV DNA, the hepatomas carry clonal chromosome abnormalities
(16, 193, 196).
On the basis
of these data, it has been proposed that HBV causes liver carcinoma
in a step-wise process that begins with antigenemia, followed by
chronic hepatitis, cirrhosis, and cancer (3, 207, 209). However,
cirrhosis is not a necessary precursor of a hepatoma (3).
epidemiological and biochemical arguments cast doubt on the HBV-hepatoma
1. The long
intervals of 30-60 years between infection and hepatomas indicate
that HBV is not sufficient to initiate carcinogenesis.
2. The evidence
that HBV is naturally transmitted perinatally also indicates that
the virus is not sufficient to cause fatal diseases such as cirrhosis
and hepatomas, because the viruses that depend on perinatal transmission
for survival are not inherently pathogenic.
3. The evidence
that the hepatoma risk among chronic HBV carriers in Asia is higher
than in the U.S. and Europe also indicates that HBV is not sufficient
4. The clonality
of the HBV-positive hepatomas further indicates that HBV is not
sufficient for carcinogenesis, because only one out of billions
of chronically infected liver cells becomes tumorigenic.
5. The absence
of an HBV-specific tumor marker, and of a specific HBV DNA sequence
or integration site in viral hepatomas, both indicate that HBV is
not necessary to maintain hepatomas.
6. The HBV-hepatoma
hypothesis fails to explain the clonal chromosome abnormalities
of hepatomas-except if one makes the additional odd assumption that
HBV only transforms cells with preexisting chromosome abnormalities.
is no convincing evidence that HBV DNA is functionally relevant
for the initiation and maintenance of hepatomas. Its presence in
the tumor could merely reflect that the tumor had originated from
one of probably many liver cells of HBV carriers that contain defective,
biochemically inactive viral DNA integrated randomly into their
chromosomes (196). Therefore, molecular analysis of HBV DNA and
of HBV DNA integration sites (210) is not likely to illuminate carcinogenesis.
replicating HBV may function as an indirect carcinogen in the form
of a long-term source of intoxication, inducing necrosis and tissue
regeneration, a known risk factor for carcinogenesis (1, 196, 203).
This view is consistent with the higher-than-normal incidence of
hepatomas in persons with chronic HBV infection.
hypothesis suggests that chronic HBV infection may only be an indicator
of a chronic nonviral intoxication and immunodeficiency. Indeed,
nonviral factors are involved in hepatomagenesis because the incidence
of the hepatomas per HBV carrier varies with different countries
(12). Intoxication could induce tissue regeneration and immune suppression,
a classical precondition for opportunistic virus infections (see
HPV in Section III, B). According to this view, the hepatoma would
be caused by a rare virus-independent mechanism that generates chromosome
abnormalities in one of many normal cells with HBV DNA inserts.
This hypothesis would readily resolve the presence of the clonal
chromosome abnormalities in all "viral" hepatomas. The
defective and inactive viral DNAs in the hepatomas would be a fossil
record of a prior infection that was irrelevant to carcinogenesis.
Virus and Burkitt's Lymphoma
In the early
1960s, Burkitt suggested that a B-cell tumor, now called Burkitt's
lymphoma, which occurs in 1 out of 10,000 Central African children
per year between 4 and 14 years of age, was caused by a virus (3,
12). Although not detectable in biopsies of lymphoma patients, a
virus was found with the electron microscope in lymphoma cells grown
in culture away from the suppressive immune system of the host (212).
The Epstein-Barr virus (EBV) has since been postulated to be the
cause of Burkitt's lymphoma (3, 8, 12).
Africa, infection with the virus occurs perinatally in the first
months of life in almost 100% of the population (3, 207). In the
U.S. and Europe, infection occurs typically during or after puberty
in about 50% of healthy adults (3, 213). However, the incidence
of lymphomas with EBV in these countries is only less than 1 in
106 per year (3). Moreover, only 30% of otherwise indistinguishable
lymphomas express EBV antigens (3). In America, Burkitt's lymphomas
free of EBV DNA were described in 1973 (214). In China, EBV is also
said to cause nasopharyngeal carcinoma in adults (1, 3).
During a primary
infection, the virus may induce transient, polyclonal lymphoproliferative
diseases, such as mononucleosis, if a large percentage of lymphocytes
are infected prior to immunity. After antiviral immunity is established,
the virus remains chronically associated with the host in a latent
form (3, 12). During the chronic state of infection, viral DNA is
detectable with the PCR in about 1 out of 105 lymphocytes (213)
and viral antigens in only about 1 out of 107 lymphocytes (12).
the virus is also suppressed, producing but a few viral antigens
(3), as the history of its discovery had first indicated. Burkitt's
lymphomas are clonal, deriving from single cells that carry characteristic
chromosome translocations that often rearrange the proto-myc
gene (see Section IV). Since EBV, like other herpes viruses, generally
does not integrate into the host chromosome (1, 3), the time of
infection of tumor cells (e.g., whether infection occured before,
during, or after tumorigenesis) cannot be determined.
hypothesis suffers from numerous epidemiological and biochemical
1. The clonality
of the lymphomas that emerge from a single tumorigenic cell among
billions of non-tumorigenic EBV-infected cells indicates that EBV
is not sufficient for tumorigenesis.
2. The long
intervals between infection and carcinogenesis, averaging 10 years
in Africa, and the incidence of only 1 lymphoma per 10,000 infected
persons also indicate that EBV is not sufficient to initiate tumorigenesis.
3. The lymphoma
incidence varies over 100-fold between African and European or American
EBV carriers, also indicating that EBV cannot be sufficient to cause
4. The lack
of a lymphoma-specific EBV function in symptomatic carriers indicates
that EBV is not necessary to maintain lymphomas.
5. The existence
of EBV-free Burkitt's lymphomas in American and European patients
indicates most directly that EBV is not even necessary for Burkitt's
appears neither necessary nor sufficient for lymphomagenesis. Nevertheless,
it has been argued that EBV plays at least an indirect role in lymphomagenesis,
because only a minority of susceptible cells from EBV-positive patients
are infected in vivo, but virtually all lymphoma cell lines
in culture are infected by the virus (215, 216). However, this could
be an artifact of studying cells in culture, because the virus would
spread unimpaired by immunity from a few infected, normal, or lymphoma
cells to all lymphoma cells that survive in culture.
100% of the Central African and 30-50% of the American population
carries latent EBV, and since EBV-negative Burkitt's lymphomas exist,
it is likely that the correlations between EBV and tumors are accidental
rather than causal. In view of this, an alternative hypothesis has
been advanced, which holds that altered cellular proto-myc
genes are the cause of Burkitt's lymphoma (see Section IV).
Oncogenes, Anti-oncogenes, and Cancer
Proto-myc Genes and Burkitt's Lymphoma
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).
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).
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).
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.
cast doubt on the hypothesis that rearranged proto-myc genes
of Burkitt's lymphomas are functionally equivalent to retroviral
myc genes and thus oncogenic:
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.
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
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.
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
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
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).
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.
Proto-abl Genes and Myelogenous Leukemia
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).
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).
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).
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.
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.
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.
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).
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.
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,
Proto-ras Genes and Cancer
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).
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
Original ras-Cancer Hypothesis Postulates a First-Order Mechanism
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).
designed to test the ras point-mutation-cancer hypothesis
indicate that point-mutation is not sufficient for carcinogenesis:
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.
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.
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.
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
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.
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).
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).
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.
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.
Hoc ras-Cancer Hypotheses Postulating Second- and Higher-Order Mechanisms
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.
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).
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
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).
Genes with Integrated Mouse Retroviruses and Mouse Mammary Carcinomas
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).
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.
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
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
In view of
this, several arguments cast doubt on the int-activation
"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.
MMT proviruses integrate into int genes prior to tumorigenesis,
provirus-mutated int genes cannot be sufficient for tumorigenesis.
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.
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
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).
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"
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.
Oncogenes, Mutated Anti-oncogenes, and Cancer
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.
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).
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,
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).
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
At this time,
the hypothesis suffers from the following short-
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.
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.
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.
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).
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).
That Latent Viruses and Mutated Cellular Genes Are Pathogenic Is
classical prototypes, the presumably latent viruses and mutated
cellular genes all suffer from activity, infectivity, and specificity
gaps: (1) The viruses and genes that are postulated to be pathogenic
or oncogenic are each orders of magnitude less active, and their
products less abundant, than the transcriptionally active, pathogenic
prototypes that have inspired these hypotheses; (2) infections with
the viruses do not cause the postulated diseases, and transfections
of appropriate cells with mutated cellular genes do not transform
normal cells; (3) the hypothetical pathogens are not disease-specific,
because (a) the same latent viruses and mutated cellular genes occur
in symptomatic and asymptomatic subjects (see Section VI) and because
(b) histologically and clinically indistinguishable diseases occur
without disease involving limited numbers of cells also occurs with
classical viral pathogens and is essential for their survival (3,
12). However, it becomes an important deficiency for hypotheses
claiming pathogenicity for viruses that are equally inactive and
restricted in diseased and healthy carriers.
there is only circumstantial or epidemiological evidence for the
role of mutated genes in cancer and latent viruses in disease. Indeed,
Bishop writes that "... on the basis of circumstantial evidence
of considerable variety, damage to diverse proto-onc genes
has been implicated in the genesis of human tumors" (7). Varmus
pointed out: "Although the dividends of conferring the status
of protooncogenes upon these cellular genes have been considerable,
it must be acknowledged that the basis for doing so, the genetic
definition of v-onc's [retroviral oncogenes], has not been uniformly
rigorous" (5). And on the basis of epidemiological correlations,
point-mutations in cancer cells are said to be "smoking guns"
(272). Yet the same tumors occur without this circumstantial evidence
(see Section VI).
Based on just
two as yet unconfirmed studies from 1989, Baltimore and Feinberg
pointed out that "HIV viremia cannot be said to be 'necessary'
for AIDS on the basis of any available data, but the new results
are a consistent feature of AIDS" (77). Further, Blattner,
Gallo, and Temin (14) argued that "... the strongest evidence
that HIV causes AIDS comes from prospective epidemiological studies
..." and Weiss and Jaffe (15) concurred ("the evidence
that HIV causes AIDS is epidemiological...."), although Gallo
(76) conceded that epidemiology is just "one hell of a good
beginning" for proving the virus-AIDS hypothesis. But even
this beginning is flawed by the tautological definition of AIDS,
which only diagnoses AIDS when antibodies to HIV are found (54)
and ignores all AIDS indicator diseases that occur in the absence
of HIV, even in AIDS-risk groups (54). For example, half of all
American intravenous drug users (55) and 25% of all hemophiliacs
(54) are HIV-free, so that their AIDS indicator diseases will not
be reportable as AIDS.
The same is
true for the epidemiological evidence that HTLV-I causes T-cell
leukemia. The leukemia is solely defined by the presence of HTLV-I,
although it has been observed in its absence (Section III). In an
effort to link human myelopathy with latent HTLV-I, it is proposed
that "similarities between HAM (HTLV-I associated myelopathy)
and visna [are] the result of still deeper identities" (323).
Visna is a neurological disease that occurred in the 1930s in a
now extinct strain of sheep. Its cause is believed to have been
a latent retrovirus, termed visna virus, that is present in over
50% of healthy sheep in Europe and the U.S. (53). Likewise, there
is no controlled study to show that the incidence of cervical cancer
is higher in HPV-positive than in matched negative controls (see
viruses and mutated cellular genes are postulated to be pathogenic
only because (i) they structurally resemble active pathogenic viruses
or active viral oncogenes, and because (ii) they occur, or are assumed
to occur, in the respective diseases more often than in normal tissues
(6, 49, 51, 296).
Genes and Cofactors to Close the Activity, Infectivity, and Specificity
Gaps of Hypothetical Pathogens
latent viruses and mutated cellular genes do not behave like the
autonomous pathogens they were originally postulated to be, the
original theories have been supplemented by ad hoc hypotheses.
it has been postulated that the long latent periods, ranging up
to 55 years, of the hypothetical viral pathogens are necessary for
various, unproven cofactors to help the latent viruses to cause
disease. Accordingly, the viruses have been termed "slow viruses"
or "lentiviruses" (3, 12), although the viruses replicate
within a few days and are immunogenic within a few weeks after infection
(13, 37, 91, 92). Further, it has been postulated that antiviral
antibodies consistently fail to neutralize viruses such as HIV (76,
77) or hepatitis C virus (160), although the respective viruses
are almost undetectable for the duration of the diseases they are
said to cause. Moreover, helper genes or cofactors are postulated
for carcinogenesis by mutated genes that are not transcriptionally
activated, including the point-mutated proto-ras genes, the
int genes mutated by provirus insertion, or the rearranged
the cofactors are modern euphemisms for new hypotheses. The ad
hoc hypotheses all assume second- or third-order mechanisms
of pathogenesis relying on unproven cofactors for both latent viruses
and mutated cellular genes to cause disease. Moreover, these ad
hoc hypotheses all lack appropriate precedents because all available
pathogenic viruses and viral oncogenes are helper-independent first-order
pathogens. Yet the ad hoc hypotheses are popular because
they leave arbitrary but face-saving roles for failing incumbents,
which were all originally proposed to cause the diseases by themselves.
In the absence
of functional proof, circumstantial and epidemiological evidence
is only relevant for causation if the respective viruses and cellular
mutations and their hypothetical cofactors are at least disease-specific.
This appears not to be the case.
Viruses as Harmless Passengers
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
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).
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).
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.
Genes and Latent Viruses as Trivial Genetic Scars of Cancer Cells
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
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.
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...."
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.
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
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.
by Somatic Gene Mutation Unconfirmed
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.
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.
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).
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
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.
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).
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).
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
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.
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,
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.
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"
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.
We thank J.
Michael Bishop (San Francisco), Michael Botchan, Eli Canaani (Philadelphia),
Asit Chakraborty, Bryan J. Ellison, David Goodrich (San Antonio),
Robert Hoffman (San Diego), Ruhong Li, Ranu Nandi, Philip Rosen
(Boston), Harry Rubin, Janie Stone, and Charles Thomas, Jr. (San
Diego) for critical reviews, critical information, or both; Jane
A. Byrd for the final draft of this manuscript and its many precursors;
and Cell for providing a free copy of Human Retrovirology: HTLV
(176) for a book review that was not published. P.H.D. is supported
by Outstanding Investigator Grant 5-R35-CA39915-07 from the National
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