The hypothesis according to which some human cancers might be caused
by viruses is placed in an historical perspective. The contrast between
the period 1945-1970, dominated by electron microscopy, and the post-1970
era, dominated by molecular biology is emphasised. Demonstrating association
with viruses is far from enough to establish an etiological relationship.
The overall impression is that possible etiological relationships belong
more to DNA than to RNA viruses. Pitfalls in the molecular approach to
the study of RNA viruses, and in particular of retroviruses, are underlined
in reference to to-day's HIV research.
The hypothesis according to which some human cancers might be caused by
filterable micro-organisms such as viruses is almost one hundred years
old. It was indeed in 1903 that Borrel, in France, suggested such a possible
relationship. To put this hypothesis in an historical perspective one
should refer to the book "The Riddle of Cancer" (1)
which Charles Oberling published in 1952 and in which the possible role
of viruses in human cancer was presented with extensive references to
contributions of initial pioneers such as Rous, Shope, and Bittner. Since
our purpose is, to some extent, focussed on the evolution of methodologies
which cancer researchers utilised in attempts to verify the hypothesis,
one should emphasise that the approach of Rous and his followers was essentially
based on establishing the difference between transmission of tumours and
leukaemias by cell transplants, i.e. grafts, or by cell-free filtrates.
Transmission of tumours in experimental animals by cell-free filtrates
was always interpreted as demonstration of viral etiology.
During the past fifty years, viral oncology has been studied in almost
all cancer research centers, world wide. Practical results in terms of
effective therapy of human malignancies have been nil. But still, recent
issues of all the main oncology publications contain numbers of studies
related to viral oncology, clearly indicating that the hypothesis still
has considerable momentum!
The past fifty years can be analysed in two distinct periods. The first,
between 1945-1970 was dominated by electron microscopy; the second, from
1970 until now, being dominated by molecular biology.
Electron microscopy (EM) contributed a considerable amount of data, which
can briefly be summarised as follows.
- EM can readily demonstrate associations between viruses and cancers
of several laboratory animal species, such as chickens and mice (2,
- However, EM data by themselves do not prove any role of these viruses
in the etiology of the tumours (4). The EM data did, however,
trigger microbiological experiments, based on ultrafiltration, which
frequently brought scientific evidence for etiological relationships.
- As pointed out by André Lwoff et al. (5) in 1962,
electron microscopy is probably the most efficient approach to viral
- Viruses shown to be associated with several cancers of laboratory
animals belong to various families of viruses (herpes, vaccinia, papova,
retroviruses, DNA, RNA,...) and are not restricted to any one family.
- Viruses associated with some cancers and those responsible for infectious
diseases look identical. There is no such thing as a family of oncogenic
viruses, a terminology, which never appears in general classification
of viruses and should actually be regarded as a misnomer.
- Practically, EM is essential to monitor the level of success in the
sequential steps leading to virus isolation and purification. Therefore,
the success of biochemical characterisation of viral markers depends
on electron microscopy to ascertain the purity of viral isolates and
the absence (or minimal amounts) of non-viral contaminants.
- Finding particles with typical viral morphology does not mean that
these viruses are pathogenic. Actually, there are probably many more
non-pathogenic than pathogenic viruses. This point was well illustrated
in a special conference sponsored around 1960 by the New York Academy
of Sciences under the title "Viruses in Search of Disease".
- Viruses, infectious or cancer-associated, rarely satisfy all the Koch
postulates which, incidentally, were presented before viruses were discovered.
- While the association between viruses and numerous malignancies of
laboratory animals has been readily demonstrated by electron microscopy,
and in spite of considerable efforts, similar associations have never
been observed in human cancers (4) (with very rare exceptions
such as common wart and molluscum contagiosum...)
"Of Mice and Men"...
Research in viral oncology changed drastically around 1970, when methods
of molecular biology took the lead, while electron microscopy was relegated
to a distant background.
Dedicated "virus hunters", as Peter Duesberg (6)
would call them, were obviously not discouraged by the negative results
of twenty years of active search for viruses by electron microscopy in
many types of human cancers. To the contrary, large research programs
were initiated, based primarily on the identification of viral, molecular
"markers" such as enzymes, nucleic acids or proteins identified
most frequently in cell cultures derived from human malignancies, rarely
directly from the tumour tissues or blood plasma. The fact that viruses
had never been directly observed in human tumours by electron microscopy
was conveniently explained in terms of virus latency, and/or by integration
of a provirus in the genome of tumour cells.
The most significant example illustrating this drastic change in the approach
is given by the reverse transcriptase enzyme, discovered in 1970 by Temin
in purified Rous sarcoma virus (7) and by Baltimore in Rauscher
mouse leukaemia virus (8). This discovery was regarded as
historical. It resulted in two Nobel prizes and in the renaming of all
RNA tumour viruses as "retroviruses". DNA synthesis from an
RNA template was indeed a very surprising observation in 1970. The enzyme
was initially thought to represent a unique feature of RNA tumour viruses
and was, therefore, regarded as a reliable "marker" for the
presence of "retroviruses", even when retroviruses particles
were never convincingly observed with the EM. We learned, later on, that
reverse transcription is a common phenomenon, that the enzyme (RT) is
present in many different cells (9), and that demonstration
of RT activity is far from enough to substantiate any claim for the isolation
of any "retrovirus".
GENERAL CONSIDERATIONS ON STUDIES RELATED TO THE HYPOTHETICAL VIRAL ETIOLOGY
OF SOME HUMAN CANCERS.
Contemporary viral oncology research is primarily based on the identification
of viral markers such as proteins or nucleic acids.
However, the specificity of viral markers depends on the success of virus
isolation and purification. Without fully demonstrated success in virus
isolation and purification, identification of "viral markers"
is extremely hazardous and can lead to severe misinterpretation of clinical
data. A dramatic illustration of this is to be found in current HIV research.
In this case, the virus (HIV) has never been properly isolated, since
sedimentation in sucrose gradient at the density of 1.16 gm/ml was erroneously
considered to yield "pure virus", systematically ignoring that
material sedimenting at that density contains large amounts of cell debris
and microvesicles (10, 11). Therefore, proteins and nucleic
acids found in such "1.16 bands" are very likely to be of cellular
origin and cannot be used as viral markers. Such a faulty methodology
has had extremely serious consequence, i.e. the world-wide use of HIV-antibody
tests, Elisa and Western Blot, which dangerously lack specificity, as
demonstrated in 1993 by Papadopulos et al. (12), in Australia.
Admitting, however, that some viral markers can be specific, their presence
within tumour cells will probably never show more than an association.
Etiological relationships are unlikely to be demonstrated by the presence
of markers, even if these markers are related to the viral genome. One
has difficulties in following Levin and Levine (13) when
they state that the identification of the viral genome in the tumour cells
is "the strongest evidence for its activity as an oncogenic agent".
This is reminiscent of an old problem when electron microscopy was only
showing association with viruses, but never their etiological significance.
In microbiology, most viral diseases are highly contagious. If some forms
of cancer had viral etiology, how is it that we don't see more cancer
"clusters"? Clusters have been occasionally observed, but their
number is very small and is certainly not compatible with the concept
of primary infections. We know that EBV is a ubiquitous virus. And, as
T. Osato (14) points out, "ubiquity and oncogenicity
are seemingly incompatible". But we are not aware of the ubiquity
of HHV-8, and we don't see any evidence for clusters of HHV-8 associated
An area in which progresses have been highly significant is unquestionably
that of apoptosis. Thirty years ago, viruses were regarded as either cytolytic
or non-cytolytic. This property was considered as an intrinsic characteristic
of the virus itself. Today, factors controlling cell cycle are much better
understood, and the cell cycle appears as a fragile balance between apoptotic
cell death and cell immortalization. Suppression of apoptosis may contribute
to cancer. As studied at the Ludwig Institute in London (15),
it appears, for example, that over-expression of the anti-apoptotic BCL-2
protein is a key event in follicular lymphoma. Factors interfering with
the progression through the cell cycle are many. Some are endogenous,
some are exogenous. Some are chemical in nature, others are physical.
Some could probably be added by the activation of latent viruses, like
EBV. However, all experiments supporting this view are in vitro experiments.
And it will take considerable clinical skill to demonstrate that these
in vitro observations are of any significance in the sudden development
of tumours in latently EBV infected individuals.
If viral markers show only "association", without implying etiology,
this does not mean that the presence of such markers within cancer cells
is not of possible therapeutic usefulness. "Targeting" is an
interesting approach to chemotherapy, or to CTLs lymphocytes. A significant
example for this can be found in the paper by Roskrow et al. (16)
on EBV-specific cytotoxic T lymphocytes for the possible treatment of
patients with EBV-positive relapsed Hodgkin's disease.
But what about antiviral therapy? Could it possibly be that its eventual
success would produce the evidence for oncogenicity of some viruses, which
we are so eagerly trying to establish?
For DNA viruses associated malignancies, we have at hand effective antiviral
agents of manageable toxicity. This is not the case for RNA virus associated
diseases, and in particular for syndromes such as AIDS, hypothetically
associated (6, 12) with infection with the HI-retrovirus.
In these cases, the currently used combined antivirals are unacceptably
toxic, making the so-called "therapy" worse than the disease
itself! Moreover, the effects of anti-retroviral therapy are currently
measured by "quantitative" PCR technology. Unfortunately, Karry
Mullis PCR technology is not reliable to measure what has been erroneously
labelled "viral load" in AIDS patients (17, 18).
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of contaminating cellular proteins found in purified HIV-1 preparations.
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Western Blot proof of HIV infection ? Bio/Technology 1993; 11:696-707.
13) Levin LI, Levine PH. The epidemiology of Epstein-Barr virus-associated
human cancers. In: Osato T, ed. Epstein-Barr virus and Human Cancer, Gann
Monograph on Cancer Research No 45. Basel: S. Karger AG, 1998.
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Research No 45. Basel: S. Karger AG, 1998.
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Advances in Cancer Research 1998; 72:109-40.
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17) Busch MP, Henrad DR, Hewlett IK, et al. Poor sensitivity, specificity,
and reproducibility of detection of HIV-1 DNA in serum by polymerase chain
reaction. J AIDS 1992; 5:872.
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AIDS in hemophilacs: An analysis of their relationship. Genetica 1995;
95:25-50.The full version of this article has originally been published
in the Journal of Hematology Vol. 84/No 5, May 1999. Reprint with kind
permission of the editor.
The author can be contacted at <firstname.lastname@example.org>