Tumor Viruses and the Cancer Problem: A Summation
of the Conference
ANDRÉ
LWOFF
(Institut Pasteur, Paris, France]
"The \iruses are actual workmen in the cellular world."
Peyton Rous, 1943
I. INTRODUCTION
In 1943 a group of scientists of the Rockefeller
Institute published a book entitled Virus Dis
eases. Peyton Rous was responsible for the chap
ter dealing with viruses and tumors. This paper,
certainly one of the classics in virology and can
cer, is perhaps too often ignored. Given here to
day, it would appear almost entirely up to date.
I would like to quote one of Peyton Rous's per
tinent statements, namely, that the host of the
oncogenic virus is not the organism but the in
dividual cell. The problem of the tumor virus has
to be considered at the cellular level, and the
study of the interrelations between virus and cell
is one of the essential tasks of virologists. Yet, if
the host of the tumor virus is the cell, the host of
the cell-virus system—the malignant cell—isthe
organism.
An organism may be considered as an inde
pendent unit of integrated and interdependent
structures and functions. The cells, the constitu
tive parts of the organism, are the ultimate units
of integration and reproduction. The functioning
of each cell, its activity, its movements, its
growth and reproduction are controlled by the
organism as a whole. Each cell knows what task
it has to perform and when it has to perform its
task. In a normal organism, each cell receives or
ders and obeys.
It happens that a cell may escape the regula
tion of control mechanism. It multiplies when it
should not multiply. It goes where it should not
go. It invades and destroys the normal tissues,
and, as a result of such a pathological activity,
the organism is finally killed. This is cancer. And
we are assembled here because cancer may arise
as the consequence of a viral infection. Why and
how?
II. REGULATORY MECHANISMS
In order to understand what is going on in a
cell, the best procedure is certainly to study a
microorganism, i.e., a structure in which the
problem of the cell and the problem of the or
ganism are concentrated in one and the same
unit.
For the sake of simplicity, two main types of
molecules will be considered: the enzymes which
catalyze simple reactions and the nucleic acid
which perpetuates the genetic information for
the patternization of amino acids into specific
proteins. Nucleic acids are endowed with a dual
function: (a) they reproduce their own struc
ture, that is they replicate; (b) they are respon
sible for enzyme synthesis.
The studies performed in recent years have
disclosed the essential laws of molecular balance
and control. Schematically, it may be said that
the end-product of the activity of a chain of en
zymes is built into a repressor as a result of the
activity of a "regulator" gene. The repressor it
self acts on the "structural" genes, those genes
which carry the information for the synthesis of
enzymes. The repressors command the expres
sion of the structural genes—i.e., they decide
whether and when the synthesis of the specific
enzymes should start or stop. The repressor is
group-specific: it controls the synthesis of each
member of a chain of enzymes, whether or not
one enzyme is missing as a result of a mutation.
Moreover, it seems as though the repressor syn
thesized by the regulator gene would not act di
rectly on the structural gene. Its action is prob
ably mediated by a third category of gene, the
operator, one operator being common for a group
of structural genes corresponding to a chain of
reactions. The operator might or might not be
sensitive to the repressor. At any rate, it is the
operator which seems to send the order "synthe
size" or "stop synthesizing" to the group of struc
tural genes.
820
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LAYOFF—Tumor
Viruses and Cancer Problem
Another category of particles and mechanisms
certainly plays an important role in cell biology,
namely, the episomes. The episomes are en
dowed with genetic continuity. They can be ab
sent or present, i.e., they are dispensable parti
cles. When present, they are either attached to
the chromosome or free in the cytoplasm. They
can be suppressed by certain chemicals. In bac
teria, they may be responsible for differentiation.
For example, a male bacterium possesses the fer
tility factor F+ which is missing in the female.
When the female bacterium receives the F+ fac
tor as a consequence of copulation, it is turned
into a male. This is a sexual differentiation. The
problem of differentiation in animal cells has
been often discussed. Some biologists consider
that differentiation has a chromosomal basis,
whereas others visualize it as a cytoplasmic
event. Episomes establish the link between nu
clear and cytoplasmic particles and reconcile two
apparently incompatible theories. Their role in
differentiation in the animal cell is for the time
being hypothetical. If one considers, however,
that the activity of an episome is different when
attached to the chromosome or when free in the
cytoplasm, the episome should be seriously con
sidered as a new candidate for a theory of dif
ferentiation.
Episomes are not only important for the prob
lem of cellular balance and cellular differentia
tion, but also as links or intermediaries between
normal cellular constituents and viruses. The ge
netic material of a temperate phage exhibits the
properties of an episome. It is endowed with ge
netic continuity. It may be present or absent. It
may be either attached to the bacterial chromo
some or free in the cytoplasm. When attached to
the chromosome, the genetic material of a bacteriophage behaves as if it were a bacterial gene.
It replicates in harmony with the bacterial chro
mosome. However, the viral functions of the
prophage are not expressed—i.e., bacteriophage
proteins are not synthesized, and no bacterio
phage particles are produced. Sometimes, as a
result of an induction, the prophage ceases to be
a prophage, the vegetative phase is started, bac
teriophage proteins are produced, and infectious
particles are finally organized.
As a result of mutation, one or many of the
viral functions of the genetic material of the bac
teriophage can be lost. The prophage then be
comes defective. Lethal syntheses can be started
as a result of an induction, but the abortive vege
tative phase does not culminate in infectious par
ticles. For all practical purposes, the prophage
does not behave any more like the genetic mate
821
rial of a virus, but like a pathological cellular organelle. A defective prophage is a pathological
episome. Owing to the fact that a number of
viral functions can be lost, a number of interme
diary phases or degrees exist between a virus and
an episome.
Thus, the molecular balance of a microorgan
ism is mainly controlled by a complex system of
repressers involving three main types of genes:
regulator, operator, and structural, which con
trol the synthesis of enzymes by a feed-back
mechanism. The final state of the cell can be
modified by episomal elements which are either
normal cellular organelles, viruses, or mixed, hy
brid, intermediary structures. When one consid
ers bacteria, and especially lysogenic bacteria, it
becomes clear that viruses can play an impor
tant role in the physiological balance of the or
ganism, and this is also true for oncogenic vi
ruses.
III. TUMOR VIRUSES
A. Viruses.—Inthe past the viral nature of tu
mor viruses has sometimes been disputed. How
ever, since several years, our ideas concerning vi
ruses have been clarified, and before discussing
tumor viruses it seems useful to say a few words
about "true" classical viruses.
Viruses represent a specific category of para
sitic entities. In order that these entities can be
classified as viruses, they have to possess a few
characteristic features which are absent in bac
teria, protozoa, and fungi (i.e., in microorgan
isms). These features are the following:
1. A virus has to possess an infectious phase in
its life cycle: the viral particle. If not, it cannot
be recognized as a virus;
2. a virus is devoid of the enzymes necessary
for the synthesis of its building block and of the
enzymes necessary to manufacture the high-en
ergy bonds necessary for biological synthesis;
the corresponding information is absent in the
viral genetic material;
3. viruses possess only one type of nucleic
acid, either deoxyribonucleic acid or ribonucleic
acid;
4. viruses carry a specific viral type of infor
mation for the synthesis of the subunits which
are constitutive parts of the viral coat;
5. the infectious particle of a vims is built of
subunits, mainly proteinic, enclosing and protect
ing the genetic material. The viral infectious par
ticle is metabolically inert. Its structure has no
equivalent among normal cellular organelles;
6. viruses are reproduced from their sole ge
netic material.
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Cancer Research
All the features which have been considered
seem to be correlated, and any one of these prop
erties will probably be enough to identify an in
fectious parasitic entity as a virus. Viruses can
accordingly be defined either as organized infec
tive particles reproduced from their sole genetic
material or as pieces of genetic material carrying
the information for the production of organized
infectious particles.
A viral infection may be defined as the intro
duction into a cell of the genetic material of a
virus. As a consequence of this infection, either
the cell dies or it survives. Those viruses which
always kill the cell they infect are called cellulicidal, and the infection is designated as non
integrative. Those viruses producing an infection
compatible with cell survival are said to be mod
erate (temperate in the case of bacteriophage).
However, the fate of a cell infected by a mod
erate virus is variable. The cell may survive, but
it may also die. When the infected cell survives,
the infection is said to be integrative. Two
"parts," the cell and the virus, have been united
into a whole: this is an integration. A new system
has been formed. The new system, the cell/virus
system which behaves and reproduces as a
whole, is different from the original cell, for the
"infected" cell carries and perpetuates the ge
netic material of the infecting virus. This genetic
material may interfere with normal cellular func
tions.
When, as a consequence of a viral infection, a
cell becomes malignant, the virus is said to be
oncogenic. Tumor viruses may exhibit consider
able differences in size, morphology, morpho
genesis, site of multiplication. Transcending
these nonessential differences, tumor viruses ex
hibit all the common features recognized as
characteristic of the viral category. It is known,
for example, that tumor viruses contain only one
type of nucleic acid and are reproduced from
their sole genetic material. It is also known that
their coat is built of subunits which are organ
ized just like the subunits of classical viruses.
B. The integrative infection.—li the oncogenic
virus would kill all the cells it infects, there
would probably be no virus-induced malignancy.
Viruses as causative agents or rumors may be
conceived only if they can produce integrative
infections, that is, if they are moderate. This does
not mean that the infection has to be always in
tegrative. As in the case of the temperate phage/
bacterium system, the infection of a ceU by a
moderate tumor virus has only a certain proba
bility of being integrative.
The outcome of the infection of a cell by a tu
Vol. 20, June, 1960
mor virus depends on a variety of factors: spe
cies and genetic constitution of the animal, its
age, the nature of the cell and its physiological
state as controlled by its environment.
Thus, a cell infected by a tumor virus may die
or survive. Nevertheless, in those cells which are
going to survive, the vegetative phase is some
times initiated, and infectious particles are pro
duced. An individual fibroblast infected by the
Rous virus and isolated in a micro-drop has been
seen dividing after having liberated virus parti
cles. Why is the viral vegetative phase sometimes
lethal, sometimes not? Cellular death might be
caused by a specific type of protein synthesized
as a product of viral development. The rate of
viral development must also be considered. The
Rous fibroma virus and the Stewart Eddy polyoma virus have a long eclipse phase of 40-24
hours, and the release period lasts many days. If,
however, some cellulocidal viruses, such as, for
example, the poliovirus, exhibit much shorter
eclipse phase, 2 hr. 30 min. to 3 hr., other cellulo
cidal viruses such as adeno-viruses and salivary
gland viruses exhibit a very long eclipse phase.
A long latent phase is, therefore, not a unique
characteristic of tumor viruses. What is perhaps
more important is that the viral production per
cell per unit time is generally small in tumor vi
ruses as compared with cellulocidal viruses. One
of the differences between an integrative and
nonintegrative vegetative phase could be a dif
ference of rate of viral production. In any event,
the outcome of an infection is controlled not only
by the genetic constitution of the cell and the
virus, but also by the environment. The influence
of the environment is particularly striking in the
case of the fibroblast/Rous virus system. More
over, one has to take into account not only the
influence of the culture medium as such, but also
the influence of the culture fluid as modified by
the metabolism of the cellular population which
has developed in this medium.
It is of interest to remember that, under cer
tain conditions, infected cells produce a sub
stance, interferon, which inhibits or blocks nonspecifically the multiplication of many viruses.
Whether the genetic information for the synthe
sis of interferon, which is a protein, comes from
the cell or from the virus is not yet known. How
ever, some indications have been gained recently
concerning the mode of action of this substance.
Cells treated with interferon show a threefold
increase of CO2 and lactic acid production. In
view of the sensitivity of the development of
many viruses to a low pH, the action of inter
feron on viral reproduction could be due to a
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LwoFF—Tumor
Viruses and Cancer Problem
decrease of the cellular pH. Whatever the case
may be, it is important to know that a cell may
react to a viral infection by the production of a
substance which depresses viral multiplication.
C. Loss of infectivity.—The original ability of
a malignant cell to produce infectious particles is
sometimes lost. This is the case in some of the
carcinoma developed from Shope's papilloma.
The original malignant cell produces infectious
particles and viral antigens. After a few transfers,
the malignant cell produces viral antigens but no
infectious particles. Finally, neither infectious
particles nor antigens are formed. A similar situ
ation has been described in hamster tumors in
duced by the polyoma virus: the virus is lost
after a few transfers from hamster to hamster.
It should be remembered that the viral vege
tative phase of ordinary viruses does not neces
sarily culminate in the production of infective
particles. Sometimes, the development is abor
tive. This is the case, for example, when myxoviruses (influenza group of viruses) infect the
chorionic cells of the chick embryo. This also
happens when the fowl plague virus infects the
L strains of mouse fibroblast. The infection is
here abortive, apparently because one of the
proteins produced in the nucleus fails to be re
leased in the cytoplasm where the constituents
of the infectious particles are "normally" assem
bled at the end of a complete vegetative cycle.
The loss of the capacity to produce viral anti
gens or viral particles could also be due to the
selection of a defective viral genetic material.
Lysogenic bacteria provide a model for this type
of event. Defective prophages are known which
perpetuate one or a few mutated genes; as a con
sequence, one or more viral functions are miss
ing, and the vegetative phases do not end with
the production of infectious particles.
Finally, it is theoretically conceivable that, as
a result of the presence of a tumor virus, the cell
has undergone an irreversible hereditary altera
tion responsible for malignancy. In this case, the
viral genetic material could disappear without
the virus-induced malignancy's being lost.
IV. CARCINOGENS AS INDUCING
AGENTS
It has been seen that the response of a cell to
an infection by a tumor virus is controlled by the
environment. Once the integrative response has
taken place, the balance of the cell virus system
can also be modified by extrinsic factors.
If the skin of the domestic rabbit infected with
Shope's papilloma virus is painted with methylcholanthrene,
the cellular proliferation
is in
823
creased. A hyperkeratinization takes place, and
the production of infectious particles is multi
plied by a factor of 10,000. The same type of
action has been observed after x-ray irradiation.
If the papillomatous skin of a domestic rabbit
is rubbed with tar, carcinomas are produced.
When newborn mice of adequate genetic con
stitution are given injections of the Gross virus,
malignant cells appear only after a few months.
In x-radiated animals, malignant cells may ap
pear within a few weeks. Here, x-rays have ac
celerated the transition of the normal cells to
ward malignancy or, let us say, have increased
the probability of the malignant change in the
virus-infected cell.
As a result of the concerted action of tar and
of the fibroma virus, malignant tumors may ap
pear in the domestic rabbit.
What does all this mean? How do the carcino
gens act on the cell virus system?
In order to answer this question, lysogenic
bacteria have to be used once more as a model.
Two phases of the life cycle can be influenced
by carcinogenic agents: the response of the in
fected bacterium and the transition from prophage to vegetative phage.
The fate of a bacterium infected by a bacteriophage is decided within a few minutes. The
response depends on whether a "vegetative" pro
tein or a represser is synthesized first. If it is a
protein, the vegetative phase is started and the
bacterium is lysed. If it is a repressor, the viral
functions are repressed, the infecting genetic ma
terial cannot express its potentialities, and bacteriophage proteins are not synthesized. When
the repressed genetic material of the bacteriophage reaches the specific receptor site of the
bacterial chromosome, the bacterium becomes
lysogenic. From then on the genetic material of
the bacteriophage multiplies in harmony with
the bacterial chromosome and behaves as if it
were a bacterial gene. Agents which block pro
tein synthesis considerably increase the proba
bility of the lysogenic response, i.e., the chance
of an integrative infection. Inducing agents, such
as, for example, ultraviolet light, shift the bac
terial response toward the vegetative phase and
bacterial death, i.e., increase the probability of a
nonintegrative infection.
In order to understand how inducing agents
modify the response toward infection, it is best
to consider the factors which, in a lysogenic bac
terium, control the balance of the bacterium/
prophage system. In a lysogenic bacterium, bac
teriophage proteins are not synthesized. Homol
ogous superinfecting bacteriophages are unable
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Cancer Research
to enter the vegetative phase. Elegant experi
ments have shown that the absence of the syn
thesis of bacteriophage proteins is due to the
presence of a specific represser produced by the
prophage. This repressor is absent in nonlysogenic
bacteria.
Some strains of lysogenic bacteria are inducible: if irradiated with a sufficient dose of U.V.
light, the vegetative phase is started after 20
minutes and infectious particles are formed. How
does the U.V. light exert its inducing activity?
The most probable hypothesis is that inducing
agents block the synthesis of the bacteriophage
repressor.
To account for the controlling action of repressors on the synthesis of enzymes, one has to
assume that repressers have a short half-life, of
the order of a few minutes for a bacterium. If
repressers were stable, it would take a very long
time—many generations—for a bacterium to con
trol its enzymatic balance. This being admitted,
the mechanism of the inducing effect of U.V.
light appears to be simple: as a result of the
inhibition of the synthesis of the new phage re
pressers and of the decay of the preexisting ones,
the repressor level decreases. When the level has
reached a certain critical threshold value, repres
sion ceases and the prophage can express its po
tentialities: proteins are synthesized, and the
vegetative phase is initiated.
Some bacteriophages are known to produce a
large amount of repressor, others to be complete
ly insensitive to repressors, the two functions be
ing controlled by specific bacteriophage genes.
This explains why some lysogenic systems are
inducible whereas others are not, and why inducibility, like any other bacteriophage function,
can be modified by mutation. What is for us here
of utmost importance is that most of the inducing
agents tested so far have proved to be carcino
gens.
U.V. light and x-rays, nitrogen mustard, or
ganic peroxides, epoxides, and ethyleneimines
have been found to act as inducers. All are carcin
ogenic agents. A few water-insoluble carcino
gens have failed to show an inducing action, but
this could of course be due to an absence of
penetration into the bacterium. Finally, ethylurethan is devoid of inducing activity; this sub
stance is, however, not a "complete carcinogen"
but only an "initiating" agent. It should be added
that the inducing agents, when acting on
genic bacteria, induce the vegetative phase
if the bacteria are in a given physiological
called "aptitude." This recalls the fact that
"promoting" agents can induce malignancy
lyso
only
state
some
only
Vol. 20, June, 1960
if an "initiating" agent has been allowed to act
first. Whatever the part of speculation might here
be, it should be remembered that the expression
of viral functions, like the expression of the syn
thesis of enzymes, is controlled by specific re
pressors.1
It is now interesting to speculate about the
possible mode of action of carcinogens in ani
mals. All carcinogens can induce malignancy in
the absence of detectable tumor viruses. The role
of repressors in the molecular balance of the cell
has already been discussed. It is clear that a
block in the synthesis of one or a few repressors
could unleash the expression of a gene, i.e., re
lease the synthesis of a normally blocked enzy
matic system and thus be responsible for a new
type of steady state.
When tumor viruses are involved and when
malignancy is due to the concerted action of a
virus and of a carcinogen, the role of carcinogens
could be the same as when inducers act on lyso
genic bacteria: they would upset the balance of
the cell/virus system by interfering with the
synthesis of repressors.
The part played by hypotheses in the logical
picture of induction and of carcinogenesis which
has been presented is obvious. An attempt at a
unifying concept of two phenomena having so
much in common was, however, felt necessary.
Moreover, the hypotheses might turn out to be
useful, since, it seems, they can be submitted to
experimental tests.
Another hypothesis has to be discussed. Episomal elements, episomes, may play an impor
tant role in cellular physiology and in differentia
tion. I would like to remind you that episomes
are either attached to the chromosome or free
in the cytoplasm and that their activity in one or
the other position is different. The action of car
cinogens could be to shift the position of an
episome.
Finally, it has been shown that the maintenance
of a normal chromosomal outfit in the mammalian
cell is controlled by environmental factors. A
phenotypic change of the cellular balance,
whether caused by a virus or by a physical or
chemical agent, might be indirectly responsible
for aneuploidy, that is, for a permanent genetic
1 Among the inducers are also azaserine and mitomycin
C. Both are known to inhibit the synthesis of DNA, just
as do other carcinogenic agents. And it would be strange
if both azaserine and mitomycin C did not possess carcino
genic activity, just as the other inducers. Nevertheless, the
yet unsolved problem of the nature (nucleic or nucleic
acid-containing ) of the repressor is posed here once more.
The solution is the key to a rational approach of antiviral
and antitumoral therapy.
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LwoFF—Tumor
Viruses and Cancer Problem
825
us today is that the viral expression is repressed.
In a lysogenic bacterium, induction necessarily
leads to bacterial death: the repression has to be
V. VIROGENY
absolute, since the vegetative phase is always a
lethal process. The vegetative phase of a mod
The question has often been raised concerning
erate virus is compatible with the survival and
the existence in animal cells of a situation homol
ogous to lysogeny. All efforts to disclose "virog
multiplication of the cells. Therefore, a highly
eny" with nontumor viruses have so far failed— repressed cell/virus system would be, from a
this despite the fact that, in some cases, such as, practical point of view, analogous to lysogeny.
At any rate, in the two cases considered here:
for example, in the virus sensitizing Drosophila
to CO2, the infection is of the integrative type. (a) the genetic material of the tumor virus is
Nevertheless, many scientists have expressed the present in the cell which continues to divide;
(fo) viral antigens are, as far as we know, not
view that virogeny should exist, but it was ob
vious that its existence could be conclusively produced, and, as a consequence, infectious par
ticles cannot be built; the viral functions of the
proved only by the study of individual cells.
If one considers the data discussed during the viral genetic material are repressed; (c) as a
conference, it seems quite possible that a virog- consequence of a change in the physiology of
eny-like situation does exist in cell/tumor virus the host cell, the vegetative phase of the life
systems. Let us consider Shope's papilloma. In cycle is started, i.e., viral antigens are synthe
sized; the viral functions are now "derepressed";
the skin of the domestic rabbit, viral antigens
cannot be detected in the cells of the basal, mal- ( d ) whether infectious particles are produced or
not is irrelevant—their absence might be due to
pighian layer. Electron microscopy does not re
veal any abnormal particle. The earliest signs of some genetic defects of the viral genetic material
the presence of a virus are to be seen in the or to an unsuitable physiological state of the host
keratohyalin layer, close to the germinal layer. cell; (e) it is of the utmost importance to re
Pathological granules first appear in the nucle- member that carcinogenic agents do modify the
olus, and viral antigens are synthesized. Here a cell/tumor virus system.
We are obviously dealing here with repression
change in the physiology of the cell is responsible
and "derepression" of viral functions, and we
for the onset of the viral vegetative phase. When
the cell grows, multiplies, and does not form have to conclude that, from a practical point of
keratin, viral antigens are not produced. When view, a virogeny-like situation does exist.
From a theoretical point of view, virogeny, the
cellular growth ceases and when keratin is syn
situation
homologous to lysogeny, would imply
thesized, then viral proteins are synthesized.
A similar type of situation has been disclosed in a complete repression and also the attachment of
the genetic material of the tumor virus to a specific
the case of the virus of myeloblastosis. Electron
microscopy does not reveal the presence of viral receptor of the cell, whether chromosomal or not.
It will, of course, be essential to know whether
particles in the myeloblasts present in the blood;
or not the expression of viral functions is essential
but, when grown in vitro, the malignant myelo
for the onset of virus-induced malignancy.
blasts produce virus, around 70 particles/cell/
hour (normal myeloblasts do not multiply in
VI. TUMOR VIRUSES AS CARCINOGENIC
vitro ). Things happen as if a physiological altera
AGENTS
tion resulting from the transfer of myeloblasts
from the blood into a culture medium had "in
As a result of a viral infection, cancer may
duced" the production of viral particles. One develop. Are tumor viruses able to produce ma
can of course speculate about the "absence" of lignant cells by themselves, or does their pres
virus in the blood myeloblasts. It is possible that ence merely increase the probability of the ma
lignant change? In order to answer this question,
the inability to detect infectious particles is a
it is necessary to learn first how a cell becomes
reflection of their rarity due, for example, to a malignant in the absence of a virus, under the
very slow vegetative phase. It is also possible influence of physical or chemical carcinogens.
that the vegetative phase is, in the blood, gen
The change from normality to malignancy is a
erally abortive.
long and complex process involving many steps.
Whether, in a malgnant myeloblast, the virus In this long "progression" two phases have been
is present in a proviral or in a "slow" undetectable
recognized, initiation and promotion. Initiation is
vegetative phase is, from a theoretical point of the process whereby normal cells are changed
view, an essential problem. What is important for into neoplastic cells, either benign or malignant.
alteration which could play a role in the malig
nant alteration.
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Cancer Research
Promotion is a subsidiary process whereby neoplastic cells are stimulated to multiply.
Urethan, for example, is an initiating agent,
devoid of promoting action. Croton oil is essen
tially a promoting agent, although it might show
in some cases a slow initiating activity. Some
substances, such as the oncogenic hydrocarbons,
are "complete oncogens," i.e., have both an ini
tiating and a promoting activity. What is the
position of tumor viruses among carcinogens?
Shope's fibroma virus can be considered as an
initiating agent, the promotion being produced
by tar (see Table 1). Shope's papilloma virus
may behave in some cases like an initiating
agent, sometimes like a promoting agent. Bittner's
virus, the milk agent, can be considered as an
initiating agent, the promoting factor being the
TABLE1
VIRUSES
ASINITIATING
ANDPROMOTING
AGENTS
Vol. 20, June, 1960
the cells—whether they are fibroblasts, epithelial
cells of the iris, or epithelial cells of the kidney.
This change is hereditary.
Thus, a cell has been infected by a tumor virus.
The genetic material of the virus has penetrated
into the cell. The infection has been integrative:
the cell has survived, and it now perpetuates the
genetic material of the tumor virus. Sometimes
the viral genetic material expresses its potentiali
ties, and viral antigens are produced. A yet hypo
thetical provirus could perhaps also be respon
sible for new cellular functions by a mechanism
analogous to the "conversion" of bacteria by a
bacteriophage. As a result of the infection, a new
system has been produced, the cell/virus system :
it perpetuates the viral information which is now
a part of its genetic make-up. As a consequence
of the new functions introduced by the virus,
moreover, the cell is now malignant.
VII. THE MALIGNANT CELL
AND THE PROBLEM OF
thevirus of
Rous'sterminology
P.
CONTACT INHIBITION
by:ShopeInitiation
by:TarEstrogensSpontaneous
1943ProvocativeDeterminingActuating
A. The malignant cell.—Malignancy can prob
virusBittner
fibroma
ably not be ascribed to the alteration of one
(milk"agent")Shope
virus
specific reaction or property. Moreover, the fac
tors are still largely unknown which, in an or
(un
virusTarPeyton
papilloma
known)Shope
ganism, stop or start the multiplication and be
papillomavirusrus;
havior of the specific cellular species. Hormones
are certainly operative. However, it is completely
Rous's sarcoma viPromotion
perma-nent
direct
unknown why a malignant cell does not respond
transformation. The virus is a com
to the unknown factors of cellular coordination.
plete oncogenAction
Let us, however, discuss some data concerning
the difference between a normal and a malignant
estrogen. In all these cases, the concerted action cell. A phenomenon analogous to progression has
of a tumor virus and of a hormone, a carcinogen, been described in plants. Phytomonas tumefaciens
or an unknown factor are necessary in order that can induce different degrees of malignancy, the
a given cell becomes malignant. According to degree depending on the time during which the
Rous's terminology, their action is provocative in
bacterium has exerted its action.
one case, determining in the other. The virus and
A normal plant cell needs to be provided with
the oncogenic agent both increase the probability
a few essential metabolites in order to grow. The
of the change from normal to malignant which malignant cell can grow in the absence of these
might, in some cases, be considered as a somatic substances in the medium, and the number of
essential metabolites needed for rapid multipli
mutation.
Some viruses, however, act as actuating agents, cation decreases with the degree of malignancy.
i.e., as a complete carcinogen. This is the case of The order in which the needs disappear is always
Rous virus and of the polyoma virus. When a the same in all plant species investigated so far.
normal fibroblast is infected with the Rous sar
Whether the cancer has been induced by a bac
coma virus, its morphology is altered within 2 terium, by a virus such as the wound tumor virus,
days. The altered fibroblast, seeded on a layer of by a spontaneous mutation, or by radiation, the
normal cells, behaves like a malignant cell: it result is the same.
exhibits no more contact inhibition.
Let us now consider two cases in which animal
is present in
The type of morphological change induced by viruses are involved. No argüÃ-ase
the virus may be altered by a viral mutation. The the epidermal cells of the rabbit. Arginase is
found when Shope papilloma virus is present. In
infection with a certain mutant virus produces
highly elongated cells regardless of the nature of the liver of the normal chick embryo, no cartilage
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LwoFF—-Tumor Viruses and Cancer Problem
has ever been found. When the chick is infected
with De Ruyck's virus, cartilage appears: the
same phenomenon has been observed in liver
tissue grown in vitro. Whether or not De Ruyck's
virus is the etiologic agent of the human hydatiform mole is, for our purpose, irrelevant.
Finally, I would like to call attention to a veiy
important observation which remains alive thanks
to Peyton Rous. Cells which have absorbed fatsoluble dyes, Sudan 3, or scarlet red behave as
if they were malignant. However, when as a
consequence of growth and division, the dye has
been diluted out, the cells return to their normal
state. The fat-soluble dyes have produced a
phenocopy of a malignant cell.
It seems clear that an extensive search for the
biochemical-physiological modifications induced
in a cell by a tumor virus is of great importance
for our understanding of the malignant change.
B. Contact inhibition.—One of the main charac
teristics of malignant cells is their invasive power.
A normal cell stays where it should stay. A malig
nant cell migrates into forbidden areas. When a
normal fibroblast traveling in one direction makes
contact with another normal fibroblast, it cannot
continue to move in that direction. This "contact
inhibition" takes place neither between two ma
lignant fibroblasts nor between a malignant fibro
blast and a normal one. After infection with the
Rous virus, the infected fibroblasts no longer
respond to contact inhibition.
This contact inhibition probably plays an im
portant role in morphogenesis. Let me recall that
the morphology of the fibroblast is rapidly mod
ified by the Rous virus. Let me recall also that
in some cases a compatibility has been disclosed
between viral protein and some cell proteins: the
proteins of the viral coat can become part of the
cell surface and, for example, confer hemagglutinating power to the infected cell.
A mere change in the surface of the cell as the
direct effect of the virus could perhaps, in some
cases, be responsible for malignancy. The dem
onstration of a difference between the membrane
of a normal and of a malignant cell would, there
fore, be of utmost importance.
Chimeras of different species can be produced.
The testis of duck and mouse can be associated,
and mixed tubuli are produced in which Sertoli
cells of both animals alternate. Mixed bronchiolar
tubules of chick and mouse can also be obtained.
Thus normal cells of widely different animals
recognize themselves. It is clear that any morphogenetic process involves some sort of recognition
and inhibition.
When malignant cells are inoculated on an
827
embryonic tissue, such as, for example, the mesonephros of a chick embryo, they invade the nor
mal organ and destroy it. This type of behavior,
which is, up to now, specific for malignant cells,
might be a useful tool especially to ascertain the
nature of the change produced in various cells
with material originating from human tumors.
VIII. THE ANIMAL HOST
The influence of environmental factors on the
balance and evolution of the cell virus system
has been stressed again and again. We have to
remember that the host of the malignant cell is
the organism. It has been known for a long time
that the physiological state of the organism can
modify the outcome of an infection by a tumor
virus. The role of estrogens in the mammary
carcinoma initiated by Bittner's agent has already
been mentioned. In many cases, the age of the
animal controls the outcome of a viral infection.
Shope fibroma virus produces malignant tumors
in young rabbits; in the adult, only benign tumors
appear which regress spontaneously. Rous sar
coma virus produces a hemorrhagic disease in
young chickens, whereas a sarcoma is initiated
in adult animals.
In some strains of mice, with a low incidence
of leukemia, x-radiation considerably increases
the incidence of the disease. In the nonirradiated
mice, virus cannot be detected, whereas virus is
present in those animals which develop leukemia
after irradiation.
If thymus is removed before or shortly after
x-radiation, leukemia does not develop. Finally,
if newborn mice are given injections of leukemia
virus and thymectomized 1 month later no leukemias develop. Thymus is probably necessary
for the production of a lymphocytosis-stimulating
factor (L.S.F.). The virus is not in the thymus.
How the virus acts is a mystery. It could act by
modifying either the synthesis of the L.S.F. or
the sensitivity of the cells to the L.S.F., or some
thing produced by the thymus could modify the
sensitivity of the cell to the virus.
It is known also that the direct injection of
carcinogens into the thymus increases the inci
dence of leukemia. This is obviously a highly
complex situation which is not yet understood.
IX. CONCLUSIONS
In a microorganism, in a bacterium, the molec
ular balance is controlled by a system of repressors produced by regulator genes. In the absence
of repressors, the synthesis of enzymes can only
be anarchic: one molecular species would out
grow the others. This is what happens in effect
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828
Cancer Research
when the represser system is disturbed by a mu
tation of the regulator gene. One type of mole
cule is synthesized in excess, and the result is a
pathological condition: the diseased bacterium
is outgrown by the normal ones.
The same type of regulatory mechanism is
known to exist in the cells of multicellular organ
isms. In the organism, the cell is, in addition,
submitted to the action of extrinsic substances
produced by other cells. And the problem of the
regulating mechanisms and of developmental
functions in differentiation and morphogenesis
has to be reconsidered today in the light of the
new ideas, brought about by the discovery of the
represser system and of episomal elements. It is
essential to remember that each molecule is a
dependent part of a cell, each cell a dependent
part of the organism, and that the tumor virus is
an anarchic infectious entity which disorganizes
an integrated system of structures and of func
tions. We have to study the parts without ever
forgetting that they belong to a whole. Whatever
the carcinogenic factor might be, malignancy can
only be the consequence of the disturbance of
the regulating system.
An infection by a tumor virus is the introduc
tion into a balanced dynamic system of a new
type of genetic information. The expression of
viral functions, as controlled by "repression" and
"derepression," interacts with the normal cellular
functions. This is exemplified by the altered me
tabolism, the increased rate of multiplication,
the suppression of contact inhibition, and, finally,
invasiveness and invasion. The hereditary cellular
alterations caused by a tumor virus can only be
a disturbance of the normal molecular order. The
most important problems are: (a) to identify
those alterations which are the direct or indirect
consequence of the viral infection of the cell,
(&) to determine the sequence of these altera
tions, (c) to find out which of these alterations
is or are responsible for malignancy.
Every cell/virus system amenable to a bio
chemical/physiological
analysis is suitable for
the purpose. However, the data thus obtained
will reveal their full significance only when the
malignant cell can be compared with the normal
one.
One of the great scientific achievements of our
time is the merging of a number of heretofore
separated disciplines. Cytology, biochemistry,
physiology, genetics are now integrated into a
new discipline, molecular biology, which tran
scends the various individual approaches and
confers a remarkable unity to modern biology.
Developmental functions as well as viral func
Vol. 20, June, 1960
tions are parts of molecular biology, as is also
the pathological malignant function.
No single scientist can dominate the various
disciplines involved. That is why meetings such
as this, in which various viewpoints can be pre
sented, are of utmost utility. Nobody, I hope,
expected that the summing up would provide
the solution of this highly complex problem.
X. APPENDIX
This summing up is, naturally, based on the
reports, papers, and discussions given at the con
ference. The field covered has been wide, and
the main speakers have presented valuable syn
theses of a complex subject: this is exemplified
by a certain degree of overlapping, certainly a
healthy sign of a tendency toward unification
and integration.
I arrived in Rye not completely unprepared,
but, as far as I can judge, without preconceived
views, except perhaps on one specific point. Can
cer may develop either as a result of a viral
infection or as the consequence of the action of
physical or chemical agents. It seemed to me
that the problem of the mode of action of carcin
ogens had perhaps been left somewhat too much
in the background and that a constructive dis
cussion on the subject could be useful. I have
accordingly tried to do something in this direc
tion. For having introduced this personal view
point in the summing up, I have to apologize.
I also have to apologize for something much
more serious. So many scientists have been re
sponsible for important contributions that it was
felt impossible to give credit to anyone for his
achievements and his theoretical views. A selec
tion would have been most arbitrary. Therefore,
no name has been cited in the summing up. Only
one exception has been made—Dr. Peyton Rous
—andfor this no explanation is needed.
While reading the preprints and hearing the
discussions, I was struck by the fact that a certain
number of papers or reviews were not quoted
and that a certain number of data had not been
taken into account. Of course, no one was ex
pected to give an exhaustive bibliography or to
discuss everything. And perhaps some of the pa
pers I have in mind have not been cited because
they were considered as too classical. Yet, some
of them dealing with relatively new data could
have been overlooked. Because I found them
useful for myself, i.e., for an outsider in the field,
and because the proceedings of this conference
might be read by nonspecialists, these "missing"
references are provided herewith. The others are
to be found in the reviews of my colleagues.
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LwoFF—Tumor Viruses and Cancer Problem
XI. LITERATURE
I. Introduction:
II.
III.
B.
C.
829
bactérieslysogènes defectives. I. Déterminisme
génétique
de la morphogenèse chez un bactériophage tempéré.
Ann. Inst. Pasteur, 90:282-302,
1956.
IV. Carcinogens as Inducing Agents:
FOULDS, L. Neoplastic Development. In: W. D.
MCELROY& B. GLASS( eds. ), The Chemical Basis
of Development, pp. 680-700. Baltimore: The
Johns Hopkins Press, 1958.
JACOB,F., and WOLLMAN,E. L. Induction of Phage
Development in Lysogenic Bacteria. Cold Spring
Harb. Symp. Quant. Biol., 18:101-21, 1953.
LWOFF, A. L'induction. Ann. Inst. Pasteur, 84:225-
Rous, P. Viruses and Tumors. In: Virus Diseases, pp.
147-70. New York: Cornell University Press,
1943.
. Concerning the Cancer Problem. Am. Scien
tist, 34:329-58, 1946.
Regulatory Mechanisms and Episomes:
JACOB, F. Genetic Control of Viral Functions, The
Harvey Lectures, Series 54, pp. 1-39. New York:
Academic Press, 1958-1959.
JACOB, F., and MONOD, J. Gènes de structure et
gènesde régulationdans la biosynthèse des pro
41, 1953.
téines.Compt. Rend. Acad. Se., 249:1282-84,
LWOFF, A., and JACOB,F. Induction de la produc
tion de bactériophages et d'une colicine par les
1949.
JACOB, F.; SCHAEFFEB,P.; and WOLLMAN, E. L.
peroxydes, les éthylèneimineset les halogénoalEpisomic Elements in Bacteria. Xth Symp. Soc.
coylamines. Compt. Rend. Acad. Se., 234:2308Gen. Microbiol., London, 1960.
10, 1952.
JACOB,F., and WOLLMAN,E. L. Les épisomes,élé
OTSUJI, N.; SEKIGUCHI,M.; IIJIMA, T.; and TAKAGI,
ments génétiques
ajoutés.Compt. Rend. Acad.
Y. Induction of Phage Formation in thé
Lysogenic
Se., 247:154-56, 1958.
Escherichia coli K12 by Mitomycin C. Nature,
MONOD,J. Biosynthese eines Enzyms. Information,
184:1079-80, 1959.
Induktion, Repression. Angew. Chemie, 71:685VII. The Malignant Cell and the Problem of Contact Inhi
91, 1959.
bition
Tumor Viruses:
ABERCROMBIE,M. Exchanges between Cells. In:
The integrative infection.
W. D. MCELROY& B. GLASS(eds.), The Chemi
ISAACS,A. Metabolic Effects of Interferon on Chick
cal Basis of Development, pp. 318-28. Baltimore:
Fibroblasts. Virology, 10:144-46, 1960.
Johns Hopkins Press, 1958.
Loss of infectivity.
SCHNEIDER,N. Sur les possibilitésde propagation
d'un
sarcome de souris sur des organes embryon
JACOB, F., and FUEHST, C. R. The Mechanism of
Lysis by Phage Studied with Defective Lysogenic
naires de poulet à différentsstades du développe
Bacteria. J. Gen. Microbiol., 18:518-26, 1958.
ment. Arch. Anat. Microsc. et morphol. expér.,
JACOB, F.; FUERST, C. R.; and WOLLMAN, E. L.
47:573-604, 1958.
WOLFF, E., and WOLFF, E. Les résultatsd'une
Recherches sur les bactérieslysogènesdefectives.
II. Les types physiologiques liés
aux mutations du
nouvelle méthodede culture de cellules cancé
reuses "in vitro." Rev. franc, étudesclin, et biol.,
prophage. Ann. Inst. Pasteur, 93:724-53, 1957.
JACOB,F., and WOLLMAN,E. L. Recherches sur les
111:945-51, 1958.
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Tumor Viruses and the Cancer Problem: A Summation of the
Conference
André Lwoff
Cancer Res 1960;20:820-829.
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