lnstitut für Klinische Virologie 852 Erlangen, Loschgestrasse 7
Federal Republic of Germany It is certainly a hard task to summarize
discussions dealing with virological aspects of human leukemia.
The diverging views, the different approaches rendey it almost impossible
to review comprehensively the data presented. It is unavoidable,
in addition, that the biased views of the author are applied to
the issues raised at this meeting. Is there any progress visible
as compared to the last meeting 2 years ago at this very place?
Progress in the elucidation of the role of viruses in human leukemia?
Progress in our understanding of the mechanisms leading to virus-induced
leukemia in general? It appears to be easier to start with the second
question first: many elegant studies were reported dealing with
virus-induced leukemogenesis in avian as well as in mammalian systems.
BALUDA pointed out the importance of the target cell for a specific
response in terms of cell transformation and stressed the acquisition
of new DNA sequences in transformed cells (1). Nontransformed cells
contain DNA which reveals 60 % of homology with avian myeloblastosis
virus (AMV) sequences only. His data were somewhat contrasted by
GRAF's studies who presented evidence for the cell specificity of
various AMY strains, each of them transforming different target
cells (2). Based on his experiments GRAF claims that the different
types of tumors observed in BALUDA's studies are due to a mixture
of different AMV strains present within thc original inoculum. In
this respect it was interesting to learn that the helper RA Y -virus,
present in preparations of defective erythroblastosis virus (2),
induces by itself severe anemia in chicken, but no erythroblastosis.
This may have some relevance for human leukemias, where (as stressed
by MOLONEY) refractory anemia or even pancytopenia frequently is
a preceding disorder . The avian systems was also investigated in
DUESBERG's studies in order to identify the localization of transforming
sequences within the avian sarcoma virus genome (3). Sarcomavirus-specific
sequences were identified in 3 different strains of sarcoma viruses
by selecting specific fragments of partially degraded viral genomes
and subjecting them to fingerprinting after further partial digestion.
These studies come close to experiments reported by BISHOP's group
in isolating sarcoma-specific sequences by hybridization techniques
( 4 ). I hesitate to agree to call such sequences "oncogene" since
transformation of lymphatic cells occurs naturally by leukosis viruses
which appear to lack the respective sequence. BAUER and HOFSCHNEIDER
reported the isolation of a new particle from the allantoic fluid
of embryonic eggs (5). It seems to differ from known avian leukosis
viruses in that it does not share antigens with AMV. It also reveals
distinct properties of its RNA-dependent DNA polymerase. Turning
now to the mammalian systems, the situation becomes increasingly
complex: many of the newly isolated mammalian oncomaviruses offer
the fascinating possibility to study their evolution across the
species-barrier. As explained by TODARO, endogenous viruses of baboons
are also found in a number of cat species (6) and permit a rough
calculation when an infectious process took place from the baboon
to the cat or vice versa. This as well as similar systems may provide
us with an entirely new approach to study the evolution of certain
species. It should not be overlooked, at the same time, that most
of these studies are performed with material derived from laboratory
animals. It is abvious, therefore, that the possibility of inadvertent
contaminants has to be excluded. ]AENISCH presented extremely interesting
data on genetic control of oncomavirus information in the mouse
system (7). He studied the infection of embryos at the 4-8 cell
stage and looked into the presence of virus-specific DNA within
the germ line as well as within somatic cells at later stages of
development. This seems to offer a new approach in the regulation
of virus-specific information in mammals. It was interesting to
learn at this occasion that cells at very early stages of embryonic
development are non-permissive for those viruses he tested (murine
leukemia viruses and SV 40). One wonders whether there exists a
specific mechanism which protects such cells and possibly also germ
line cells against early genetic damage. Studies on the role of
FRIEND leukemia virus in the differentation of mouse pluripotential
stem cells intO erythroblasts were reported by OSTERTAG. HARDESTY
also alluded to this question (8). The ingenious cell separator
used by OSTER TAG, based on laser-beam scanning and computer-directed
deflection of drops, appears to represent an elegant and important
tool in the elucidation of cell differentation. This was also convincingly
demonstrated by GREA VES (9) experiments. OSTERTAG's statements
on the possible role of DMSO in the induction of viral and globin
messengers RNA-synthesis by affecting repressor binding within the
cell may deserve further studies. Transfection experiments revealing
the existence of DNA proviruses were rather briefly discussed at
this meeting. BENTVELZEN made the interesting observation that DNA
from spleens of Rauscher virus leukemic mice transfects and transforms
efficiently when applied under appropriate conditions. In this respect
it seems interesting to note that similar studies have not yet been
reported with human leukemic cell DNA. One could imagine that similar
events may take place in tissue culture or by transfecting cells
of primates in vivo with DNA originating from human leukemia cells.
Interesting new aspects were contributed by BURNY in his studies
on the viral etiology of bovine lymphosarcoma (11). The epidemiologyof
this disease resembles the spread of feline leukemias which were,
unfortunately, not discussed at this meeting. It is of interest
to note that lOO % of animals developing disease revealed antibodies
to viral antigens. This in part to such an extent that they can
be measured by relatively insensitive immunoprecipitation methods.
This appears to contrast markedly the situation in human leukemia,
where the demonstration of even leukemia-specific antigens, as pointed
out by GREAVES (9), is presently either impossible or requires difficult
manipulations. The presence of bovine oncornaviruses in commercially
available batches of calf serum, as observed by BURNY (11 ). should
be another word of caution in claims of new oncornaviruses from
tissue culture cells maintained with such reagents. Turning now
to human leukemia and lymphosarcoma, isolates from human disease
naturally require special attention. Two claims of successful oncornavirus
isolations were reported at this meeting (12) and others are found
in the literature (13, 14, 15). GALLO described extensively the
successful isolation of such viruses from a patient with acute myelogenous
leukemia (12). According to his studies the agent appears not to
be an endogenous virus of man or certain primates. It shares many
characteristics with the simian sarcoma virus and it is not yet
entirely clear whether it can be differentiated at all from this
agent. Although repeatedly isolated from the same patient, there
are some disturbing observations which are difficult to reconcile
with a role of this virus in human myelogenous leukemia :
(i) recent studies reveal the presence of two different oncornaviruses
in these isolates. One of them appears to be idential with simian
sarcoma virus, the other shares features with baboon endogenous
(ii) no convincing levels of antibodies directed against these isolates
can be demonstrated in the patient, nor in other individuals suffering
from the same disease, or in healthy control persons (17).
(iii) DNA-sequences related to these agents have not been demonstrated
in the DNA derived from spleen cells of the patient from whom the
viruses were recovered. Thus, there remains the possibility, as
remote as it may be, of a laboratory contamination. Further studies
appear to be essential to clarify the origin of the isolated agents.
The second isolate was reported by NOOTER. It has been obtained
from a child with lymphosarcoma. This virus has not yet been further
characterized. Although the data seem to be intriguing, the use
of rat XC-cells for plaquing this agent raises some questions. Endogenous
rat oncornaviruses have recently been found in XC-cells. The third
group isolating putative human oncornaviruses was not represented
at this meeting. KIRSTEN and P ANEM were able to recover a simian
sarcoma virus-like agent from human embryonic lung fibroblasts (13).
It is obvious that each of these isolations requires great interest.
It appears to be a long way to clarify whether they indeed represent
human viruses. If so, it will be an even longer way before they
can be implicated in human leukemic disease. SPIEGELMAN reported
the presence of specific DNA sequences, as determined in his endogenous
reaction, in almost every kind of human tumors (18). The significance
of these findings should be further elucidated, since they are also
found in two human malignancies most probably induced by a DNA containing
virus (19). The various isolations of oncornaviruses from primates
should support attempts to recover similar agents from human leukemias
and lymphomas. It is of particular interest that oncornaviruses
have been isolated from acute myelogenous leukemias in gibbons.
There are, however, certain features of most human leukemias which
are presently difficult to reconcile with an oncornavirus-induction.
Although anologies to animal leukemias sponsor intensively the current
interest in oncornaviruses, it may be worthwhile to consider some
of the diverging aspect:
(i) In contrast to most animal oncornavirus-induced tumors it appears
to be extremely difficult to demonstrate any kind of oncornavirus-specific
molecules in human leukemic cells. This is also shown in GREA VE's
study on antigens specific for acute lymphatic leukemia (9).
(ii) Sera derived from leukemic patients appear to lack antibody-activities
against known oncornaviruses. This certainly includes the woolly
monkey isolate. In regard to all known naturally occurring oncornavirus-induced
leukemias and lymphomas it would be exceptional if man would respond
without antibody production.
(iii) Human leukemias and lymphomas represent, at least in their
vast majority, monoclonal diseases. Thus, the continuous production
of transforming particles appears to be somewhat unlikely.
(iv) The failure to demonstrate viral particles in human leukemic
cells certainly contrasts the situation in animal systems. In this
respect it was somewhat surprising that the only virus known to
be oncogenic in man and consistently associated with specific lymphatic
diseases, the Epstein-Barr virus (EBV), played a minor role at this
meeting. This DNAcontaining herpes group virus was briefly discussed
by DIEHL showing that NULL cells apparently lack receptors for EBV-infection
(20). It has to be remembered that EBV is found in virtually 100%
of African Burkitt's lymphoma cells, as well as in very few cases
of similar histology outside of the African tumor belt; that it
infects and transforms specifically B-lymphocytes, but is also found
in every epithelial tumor cell of human nasopharyngeal carcinoma
(19). This virus induces lymphoproliferative disease in marmosets
and transforms and "immortalizes" human lymphocytes efficiently
(19). The most potent and effective leukemogenic agent in primates,
herpesvirus saimiri, was also discussed in one presentation only
(21). LAUFS reported on prevention of saimiri-induced oncogenesis
by prior inoculation of heat- and formaline-inactivated vaccines.
It should be noted that herpesvirus saimiri induces lymphomas or
acute lymphatic leukemias after short incubation periods in 100%
of inoculated marmosets (22). Returning to human leukemias, there
is presently no good reason to speculate that these diseases are
herpesvirus-induced. In such case it would be, most probably, not
too difficult to detect virus-specific antigens within the transformed
cell or on their surface. The entire lack of these "footprints"
in human leukemic cells remains a puzzle in regard to their suspected
viral etiology. It could be relevant in this respect that there
exists a group of transforming viruses which are most difficult
to trace within their transformed host cells, the human papilloma
or wart viruses (23). In spite of numerous attempts it has not yet
been possible to detect papilloma virus-specific T-or surface antigens
within their transformed host cells. Recent results reveal that
there exist several types of human papilloma viruses which can be
differentiated by biochemical methods (zur Hausen and Gissmann,
unpublished results). There may be other candidate viruses along
these lines and it appears tO me to be a good bet that at least
some forms of human leukemias (if they do have a viral etiology
at all) should be due to non-enveloped viruses. I am stating this
because it is my feeling that our intensive search for human analogies
to well established laboratory system in animals may misguide us.
Most probably it will be worthwhile to persue also different avenues
in our search for a viral etiology of human leukemia. If the intensive
search for human oncornaviruses fails to provide conclusive evidence
we should be prepared to look as well into the role of other agents
in the induction of this malignant disease.
1. Baluda, M. A., et al. this volume.
2. Graf, T., et al. this volume.
3. Duesberg, P ., et al. this volume.
4. Stehelin, D., Varmus, H. E., and Bishop, J. M., Detection of
nucleotide sequences associated with transformation by avian sarcoma
viruses. VIIth Int. Symp. Comp. Leukemia Res., Oct. 13-17, 1975,
Copenhagen, in print.
5. Bauer, G., et al. this volume.
6. Todaro, G., this volume.
7. Jaenisch, R., et al. this volume.
8. Hardesty, B., et al. this volume.
9. Greaves, M. F., et al. this volume.
10. Nooter et al. this volume.
11. Kettmann et al. this volume.
12. Gallo, R. C., this volume.
13. Kirsten, W., and Panem, S., Kinetics of type-C virus induction
from normal diploid human fibroblast cell strains. VIIth Int. Symp.
Comp. Leukemia Res. Oct. 13-17,1975, Copenhagen, in print.
14. Gabelman, N., Waxman, S., Smith, W., and Douglas, S. D., Appearance
of C-type virus-like particles after co-cultivation of a human tumor-cell
line rat (XC) cells. Int. J. Cancer 16, 355-369, 1975.
15. Vosika, G. J., Krivit, W., Gerrard, J. M., Coccia, P. F., Nesbit,
M. N., Coalson, J. J., and Kennedy, B. J., Oncornavirus-like particles
from cultured bone marrow cells preceding leukemia and malignant
histiocytosis. Proc. Nat. Acad. Sci. 72, 2804-2808, 1975.
16. Gillespie, D. H., and Gallo, R. C., Concepts concerning the
origin of RNA tumor virus markers in human leukemic cells. VIIth
Int. Symp. Comp. Leukemia Res., Oct. 13-17,1975, Copenhagen, in
17. Kurth, R., Discussion at VIIth Int. Symp. Comp. Leukemia Res.,
Oct. 13-17, 1975, Copenhagen, in print.
18. Spiegelman, S., this volume.
19. zur Hausen, H., Oncogenic herpesviruses, Biophys. Biochem. Acta
417; 25 53, 1975.
20. Diehl, V., this volume.
21. Laufs, R., and Steinke, this volume.
22. Melendez, L. V. (ed.), Symposium on viruses of South American
monkeys: importance of these viruses in oncogenic studies. J. Nat.
Cancer Inst. 49, 209-294, 1972.
23. zur Hausen, H., Gissmann, L., Steiner, H., Dippold, W., and
Dreger, I., Papilloma viruses and human cancer. VIIlh Int. Symp.
Comp. Leukemia Res., act. 13-17,1975, in print.