CANCER
VOLUME
24
RESEACH
AUGUST
1964
NUMBER
7
Biochemical Perspectives in Cancer Research*
VAN
RENSSELAER
POTTER
(McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin)
INTRODUCTION
It is a great privilege to be asked to present the Fourth
Annual G. H. A. Clowes Memorial Lecture.
I speak as a
biochemist and, rather diffidently, as a molecular biologist,
a cell biologist, and an oncologist. I feel that all or any of
these appellations would have pleased the man who is
being honored this evening.
Dr. Clowes obtained his Ph.D. in chemistry at Gottingen
University at the age of 22 and came at once to the New
York State Institute for the Study of Malignant Diseases
in 1900. He was a charter member of the American Asso
ciation for Cancer Research, which was organized in 1907,
and he recalled those early days in a historically impor
tant essay entitled “CancerResearch Fifty Years Ago
and Now― which was published at the invitation of Dr.
Harold Rusch in 1956 (13). He noted that the main
topics of discussion in 1907 were (a) whether the patholo
gists were willing to designate as malignant each of the
new transplantable
animal tumors that were being in
troduced at that time, (b) the nature of the immune forces
brought into play by such tumors, and (c) the possible
role played by parasites, viruses, or other extraneous agents
in the development of cancer.
It is a sobering thought to
realize that none of these problems have become settled
by 50 years of research.
World War I took Dr. Clowes to the Chemical Warfare
Service in 1918 and thence to Eli Lilly and Company,
where he was Director of Research from 1920 to 1945.
As Emeritus Director at age 68 he continued to publish
up to the year of his death at age 81 in 1958.
Such a life
has many facets, but after a thorough study of his list of
publications, his chief contribution to the field of cancer
research in my opinion remains that series of studies on
Arbacia eggs, in which he provided model experiments on
the attempted correlation between a biochemical measure
ment and a biological observation over a wide range of
drug concentrations.
In 1934, when Dr. Clowes was 57, the first of a long
series of papers by Krahl and Clowes (30) reported the
stimulation of oxygen uptake and concomitant inhibition
of cell division by substituted phenols. Curiously enough,
the origin of Dr. Clowes' interest in these compounds is
*
Presented
as
the
Fourth
Annual
G.
H.
A.
Clowes
Memorial
Lecture, on April 20, 1964, at the 55th Annual Meeting of the
American Association for Cancer Research, in Chicago, Ill.
Received for publication April 23, 1964.
@
not revealed in his publications, which were among the
first to be concerned with the dinitrophenols,
and I sus
pect that he must have encountered them during World
War I, when the biological interest in them began (52).
In 1934 ATP had been known for only 5 years,
the Embden-Meyerhof
scheme of glycolysis had just been
published, the Krebs citric acid cycle was undiscovered,
and oxidative phosphorylation
wasn't even a theoretical
possibility.
It was not until about 17 years later that
the relation between oxygen uptake and oxidative phos
phorylation was well established and the dinitrophenols
were categorized as “uncouplers―
of oxidation from phos
phorylation,
as may be noted in the excellent symposia
on Phosphorus Metabolism edited by McElroy and Glass
in 1951 and 1952 (33). And it was in 1951 that Dr.
Clowes, at age 74, at long last was able to publish a key
report that pulled together his considerable experience
with the nitro- and halophenols for the New York Academy
of Science.
The typical experiment was graphed as shown
in Chart 1 (12). It was found that the compounds that
stimulated
oxygen
cell division.
uptake
caused
a decrease in the rate of
This result remained anomalous until the
“uncoupling―
action of the dinitrophenols was understood.
When it was realized that oxygen uptake is regulated by
functional demand expressed in terms of ATP breakdown,
and that the dinitrophenols short-circuit this regulatory
process and make oxidative energy unavailable for useful
work, the experiments of Dr. Clowes began to have a
rational explanation. The excitement of this period is
suggested by the Addendum in which Dr. Clowes reported
that cell-free particulate
systems that were capable of
oxidative phosphorylation had been obtained from Arbacia
eggs and that
the compounds
which stimulated
oxygen
uptake and repressed cell division were effective in un
coupling
phosphorylation,
whereas
the compounds
that
were ineffective in the intact cells exerted no effect on
oxidative phosphorylation.
In his final paragraph the
hope was expressed that “A
correlation of data obtainable
with these cell-free phosphorylating systems with those
available regarding inhibition of cell division may ulti
mately serve to throw light on the mechanism of cell
division, both normal and pathological.―
Experiments
in my laboratory
have in a sense justified
some of Dr. Clowes' hopes for the dinitrophenols and along
with many others have provided at least a partial explana
tion for his results. What was perhaps the most satisfy
1085
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Cancer Research.
1086
@
Cancer Research
Vol. 24, August
ing experiment ever done in my laboratory was in almost
the identical pattern seen in Dr. Clowes' experiment.
This experiment in 1953 by Siekevitz and Potter (50) is
shown in Chart 2 and includes measurements of oxygen
uptake of rat liver mitochondria as a function of dinitro
phenol (DNP) concentration, just as in Chart 1. But in
addition, the balance between ATP, ADP, AMP, and
inorganic phosphate for the total system (flask plus mito
chondria) was measured.
The results show that, as the
oxygen uptake was increased due to uncoupling,
the
amount of ATP declined.
A similar experiment by Aisen
berg and Potter (2) showed another example of increased
oxygen uptake associated with a decreased synthetic
a:
U
..1
z 150(
Ui
0
I-
‘000
25
4
I-
zUi
C,
>.
0
I-
013
0
20
DNP
CHART 3.—Stimulation
30
Mx105
of oxygen
uptake
and inhibition
of ace
tate activation in rat liver mitochondria as a function of the con
function, in this case the formation of acetyl-CoA which
is a necessary prelude to the conversion of acetate to CO2
or other products (Chart 3). More recently Aisenberg
centration of dinitrophenol.
Data from Aisenberg and Potter (2).
TABLE 1
EFFECT
OF
DINITROPIIENOL
LABELED
THYMIDINE
(DNP)
ON
INTO DNA
Results in counts/mm/mg DNA.
QNOa
1964
INCORPORATION
OF TUMOR
OF
SLICES
Data from Aisenberg (1).
III
256
CARCINOSARCOMAAerobic
LYMPROSARCOIIAWALKER
CoxmTloNsMuxpzy-S@vit
3.@
counts!
counts!
counts!
counts/
mmAnaerobicnunAerobic mmAnaerobic
mm0.015
M glucose
(control)
0.015 Mglucose + 2 X 10@M
“In
I,
0
10851620 10102210
9121120
924
DNP2240
2.'
(1) has come closer to the Clowes experiment
by adding
dinitrophenol to tumor tissue slices that could incorporate
labeled thymidine into DNA. The experiments showed
0
1.4
0
0
.
I
1
2
CHART 1.—Stimulation
I
3
0
1
of oxygen
2
uptake
30
and
1
inhibition
2
3
of cell
division in fertilized Arbacia eggs as a function of the molar con
centration of various nitrophenols (X 10'). Data from Clowes
that in the presence of glucose the glycolyzing tumors were
able to make labeled DNA in the presence of dinitrophenol
at about the same rate aerobically or anaerobically (Table
1). These data are in harmony with Dr. Clowes' finding
with tumor homogenates in which it was found that the
glycolytic phosphorylating
mechanism (14) was resistant
to uncoupling by the dinitrophenols
that were effective
in blocking cell division in Arbacia eggs. Since the
Arbacia eggs showed a complete block in cell division in
the presence of dinitrophenol we must conclude that they
are unable to obtain their division energy by anaerobic
means.
This conclusion is supported by Clowes' experi
(12).
ments with cyanide inhibition or reduced oxygen tension
(12).
In his final paper, published posthumously in 1958 (15)
in his 81st year, Dr. Clowes was still concerned with the
effect of the dinitrophenols on the energy-yielding processes
in glycolyzing tumors. It is my opinion that much is
still to be learned by the use of the dinitrophenols in the
study of energy-utilization
for cell division.
New experi
ments with cells in tissue culture, and measuring DNA
synthesis by chemical means as well as by autoradiographic
technics, may help us to decide whether the ATP generated
in different cell compartments—nuclear,
mitochondrial,
and soluble fraction—is equally available to the processes
of DNA synthesis and mitosis. The fact that the Arbacia
CHART
2.—Stimulation
of oxygen
uptake
and
uncoupling
ATP
synthesis in rat liver mitochondria as a function of the concentra
tion of dinitrophenol. Data from Siekevitz and Potter (50).
eggs tend to pile up at the prophase stage (12) under the
influence of dinitrocresol suggests a preferential action on
mitosis.
It is clear that the work begun by Dr. Clowes
will not be ignored but will be carried on in new environ
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1087
PoTTER—Biochemical Perspectives in Cancer Research
ments and with new technics,
enlarging theoretical framework.
BIOCHEMICAL
Perhaps
research
supported
TABLE
by an ever
BACKGROUND
that
I recently
1913-1963
(45)
Biochemistry1913—1923Enzyme
BiochemistryGeneral
PeriodCancer
in the context of Dr. Clowes' 50 years of cancer
we may refer to a table
2
FIFTY YEARS OF CANCER BIOCHEMISTRY
differences1918,
prepared
to cover almost the same span of time on the occasion of
the 50th Anniversary of the Netherlands Cancer Institute
(45). In Table 2 I have indicated the developments
in
the biochemistry of cancer in parallel with developments in
general biochemistry.
Following Bergel (4) the three
main theories of the biochemistry of cancer are listed as
Aerobic Glycolysis,
which was Warburg's
idea; the
Hypothesis
of Convergence, which has been associated
with the name of Dr. Jesse Greenstein; and the Deletion
Hypothesis, which has been advanced by James and Eliza
beth Miller (34) and by me, as has been chronicled else
where (4, 43, 45). This table is reproduced here for several
reasons.
First, it is emphasized
that advances
in cancer
biochemistry are closely related to the parallel and ante
cedent
developments
in general biochemistry.
How
could Warburg or Greenstein be expected to devise a
hypothesis of cancer that could have more than descriptive
qualities when we consider the state of knowledge in their
time?
How can we do more than indicate our faith in
the general trend of biochemistry in 1964 when we attempt
to develop guiding hypotheses for the immediate future?
Second, it is important
to recognize “howeach of the
older hypotheses has proved inadequate
and how each
retains elements that still command our respect― (45).
It is here emphasized
that neither the Warburg
Hypothesis nor the Greenstein Hypothesis was concerned
with the mechanism of how normal cells are converted to
cancer cells. They were purely descriptive, and in the
case of the Warburg Hypothesis it was assumed that the
correlation
between aerobic glycolysis and malignancy
was universal.
Since there are now available a class of malignant tumors
that do not exhibit the property of aerobic glycolysis the
Warburg
generalization
does not hold (45), and the
property cannot be said to be essential for malignancy,
although it is still relevant to the problem of chemo
therapy.
The new class of malignant tumors was dis
covered in 1959 (46) when we obtained a variety of trans
plantable
hepatomas
from Dr. Harold Morris of the
National Cancer Institute, and an attempt has been made
to encourage
the use of these new tumors
in as
many laboratories as possible.
I believe it is safe to say
that the widespread study of these tumors has ushered
in a new era in cancer biochemistry—an era in which we
will never again be content with hypotheses that give
no hint as to possible mechanisms of conversion of normal
cells to cancer cells. Both Warburg
and Greenstein
appear to have assumed that any enzyme difference that
could be demonstrated between a cancer tissue and normal
tissues was ipso facto relevant to the carcinogenic change.
In the new era, any change is suspected as possibly not
essential to the carcinogenic change, and each emerging
generalization is in danger of being modified by data from
a new strain of minimal-deviation
tumor.
As we look back upon the Warburg and Greenstein
6-P1923—1933Aerobic
3' RNA-tides
1918, Fructose 6-P
1922, Glucose
glycolysis
(respiratory defect)1924,
1929,
1930,
scheme1933—1943Aerobic
1933,
Slice technic
ATP
Deoxyribose
Glycolytic
TPN, DPN
Homogenate technic
1937, Citric cycle
phosphorylation1943—1953Convergence1950,
1939, Oxid.
glycolysis
1936,
defect)1934—35,
(respiratory
Alter. pathways
1951, 5' RNA-tides
1952, Pentose
cycle1953-1963Catabolic
deletion
(alternative path
1956,
1957,
1961,
1962,
ways)1953,
sites1963—Feedback
DNA structure
Feedback
Repression
Messenger RNA
Regulatory
deletion
eras, we can advocate
we can suggest
that
several new points of view.
certain
enzymes
that
First,
are peculiar
to a
given normal cell, which we can call “marker―
enzymes,
should be looked for in tumors that are supposed to be
derived from the normal cell, and we should recognize
that, unless one or more “markerenzymes― are present,
we cannot claim to know the normal cell of origin. Thus
we may hope to devise better controls for studies on cancer
tissue (45).
Second, we now propose to recognize that the conversion
of a normal cell to a cancer cell can proceed in stages, and
that the biochemistry
of carcinogenesis can conceivably
be deduced by comparing early stages with the cells of
origin, but not by comparing late stages with the cells of
origin, especially if the late stages have undergone so many
changes that the cell of origin can no longer be specified.
We recognize that the biochemistry of the late stages is
relevant to the problem of chemotherapy
but question
the relevance to the problem of carcinogenesis.
We be
lieve that the minimal-deviation
hepatomas
represent
examples
of the earliest
stages
of cancer
cells available
in
transplantable
form up to this time and also represent
cancer cells for which the cell of origin is identified and
readily available (45).
Third, owing to the complexity of the cancer problem,
the diversity of cells of origin, the diversity of enzyme
patterns compatible with cancer, and the multiplicity of
properties that make up the totality of properties that we
call cancer, we should recognize an obligation to relate any
biochemical differences that we can observe to mechanisms
affecting specific cancer properties such as growth rate,
invasiveness,
metastatic
tendency, or uncontrolled
cell
division, since there may be many biochemical differences
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1088
Cancer Research
that are not critical for any of the properties mentioned
(45).
Referring again to Table 2, I must say that The Cata
bolic Deletion Hypothesis, which was my special contribu
tion to the general Deletion Hypothesis of the Millers
(34), may be just as outdated for the early steps in car
cinogenesis as the Warburg or the Greenstein hypotheses,
although it did have the merit of having a mechanistic
basis, and many investigators contributed data in support
of it (42, 45). So what is the meaning of all the biochemi
cal differences between normal and cancer cells that have
been recorded up to this time?
Is there any thread of
continuity that will enable us to give meaning to the differ
ences that have been observed?
I believe that the War
burg generalization, the Greenstein Hypothesis of Conver
gence, and the Potter hypothesis of Catabolic Deletion all
take on meaning in terms of the concept of Tumor Pro
gression, which has been most prominently
associated
with the name of Foulds (20), but which has received
powerful support from the work of Furth, Rous, Beren
blum, Hauschka, Rusch, and Boutwell, as detailed else
where (44). In terms of tumor progression, the develop
ment of aerobic glycolysis, the convergence to a more or
less uniform pattern, and the deletion of catabolic enzymes
are not initial steps in the conversion of normal cells to
cancer cells but instead are secondary steps that are coin
patible with survival and which may lead to competitive
advantage in the struggle with normal host cells, as has
been emphasized also by Weber (59) and by Quastel (49).
Thus we must begin anew to develop a biochemical
hypothesis for the initial steps in carcinogenesis, and I
have chosen to call your attention in Table 2 to the hypoth
esis of Feedback Deletion, which is the best term we can
devise in the present historical
framework
including
columns 2 and 3 in Table 2. More will be said about
Feedback Deletion later.
Numerous investigators
have
expressed this concept of cancer since the basis for it has
become elegantly demonstrated
in bacterial and animal
systems.
This is a time for steady nerves and abiding faith in the
ethics and methods of our scientific community.
As
oncologists we are on a collision course with an army of
molecular biologists, biochemists,
embryologists,
micro
biologists, immunologists,
cytologists, and many others.
I believe that the possibility of a general understanding
of the nature of the cancer problem may be very near at
hand or even already published
(32). Although new
developments
will no doubt produce some startling
insights, we cannot afford not to make every possible
effort to understand
where we stand today.
We wifi
proceed from an earlier attempt in 1958 (42). What new
developments have occurred in 6 years?
Messenger RNA
and allosteric regulatory sites on enzymes are certainly
two of the most important
new developments
in bio
chemistry in relation to the cancer problem (37). In
the narrower sense, the discovery and exploitation
of
minimal-deviation
hepatomas
got under way in 1960,
and the experimental
approach to the separation of es
sential from nonessential
biochemical
changes in rein
tion to carcinogenesis really dates from about 1961 (43).
Before discussing the “meaning of biochemical differ
Vol. 24, August 1964
ences―between normal and cancer cells it seems desirable
to assess the present status of the mechanism of carcino
genesis, since I intend to emphasize the need for looking
for biochemical differences in cancer cells that differ as
little as possible from their cell of origin. This need is
in fact the whole point in developing the concept and
experimental
approach of the minimal-deviation
hepa
toma.
I intend to discuss carcinogenesis as a process that
consists of a number of irreversible steps, some of which
can be facilitated by certain biochemical changes that
are reversible (9). In the case of viral carcinogenesis it
will be conceded that in certain cases, of which the Rous
Sarcoma Virus may be a unique example, the virus appears
to be able to carry in sufficient genetic information
to
encompass all the necessary steps to produce a malignant
tumor (57). Similarly, it appears that, when overwhelm
ing doses of carcinogen are given, all the necessary ir
reversible changes can occur in a few single cells in a very
short time as shown by Huggins (26).
BIOLOGICAL
BACKGROUND:
IN GENOME
CHANGES
The concept that the initial step in carcinogenesis was
irreversible was never seriously doubted until recently,
when Monod and Jacob (35) and Pitot and Heidelberger
(37) published papers that I will discuss a little later.
Since cancer cells have always been understood
to be
able to continue to grow in the absence of the inciting
agent, it has been assumed that an irreversible change
had been produced, and many had accepted the idea that
the change was of the nature of a somatic mutation.
This concept of somatic mutation was clearly stated
by a committee consisting of S. Bayne-Jones,
Ross G.
Harrison, C. C. Little, John Northrop, and J. B. Murphy
as long ago as 1938 (quoted in ref. 40) and the idea goes
back at least as far as the proposal by Boveri in 1912
([10], cited in [28]). The con@tmittee produced a series
of five statements on the nature of cancer, of which the
fifth was “Thenew property of the cell appears to de
velop suddenly, becomes a fixed character, and is trans
mitted to its descendants.
It gives evidence of being a
somatic mutation.― In many studies cited in support of
the theory of somatic mutation much weight has been
given to abnormal chromosome numbers or dimensions
(28). As newer evidence revealed tumors without gross
chromosome abnormalities
(23) it has been necessary to
assume that mutations in individual codons (nucleotide
triplets) had occurred and that gross aberrations were not
required for carcinogenesis but were merely the result of
secondary changes in the genetic apparatus.
Virus theory.—The general acceptance of the mutation
theory of carcinogenesis was probably slowed by the emer
gence of the virus theory of carcinogenesis and the con
tinuing belief that the virus theory and the somatic
mutation theory were mutually exclusive.
In a paper of
utmost historic significance Peyton Rous, stimulated by a
summary of events at the 1958 International
Cancer
Congress in London (47), wrote an impassioned rebuttal
of the somatic mutation theory (48). Rous noted that
“WhenBoveri first outlined his idea that tumors are
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1089
POTTER—Biochemical Perspectives in Cancer Research
consequent
on somatic
mutations
he pointed
out that
traneous.
Thus,
in 1963 Hilleman
(25) listed
TABLE 3
it
was not incompatible
with the assumption
that their
primary cause is parasitic in nature,― but he questioned
this view. He noted the phenomenon of transduction
in
bacteria and then rejected it as a possible analogy to the
production
of cancer cells. He raised the question of
“whether this reconciling (sic) assumption
(of transmis
sible mutagens)
may not apply to viruses producing
growths which fall somewhat short of the standard for
typical neoplasms― and went on to say “Thesejustified
questions carry no implication that viruses (in general)
are harmless to the genetic apparatus of cells. They may
well play havoc with it. But to assume this in the case of
neoplastic cells is less than necessary (sic).― With regard
to the somatic mutation hypothesis he concluded, “It
has resulted in no good thing as concerns the cancer prob
lem, but in much that is bad.―
However, since these words were written the “reconcil
ing assumption― has moved closer and closer to experi
mental verification,
although the workers in the virus
field still seem to regard other theories as somehow ex
the follow
lug theories of carcinogenesis: The irritation hypothesis,
the embryonic rest theory, somatic mutation of spon
taneous origin or mediated by irradiation
or chemical
mutagens,
extrachromosomal
mutation,
immunologic
loss of identity antigens, hormonal alteration, ribonucleic
acid template deletion hypothesis, transformation of car
bohydrate
metabolism, and the virus theory.
He con
cluded “Ofall the theories, the best supported and, to
some workers, the most intellectually
satisfying, is the
virus theory.― Although seeming to brush aside the
somatic mutation theory, Hileman was not unaware of
the “reconcilingassumption,― since he regarded the virus
as “an
extrinsic source of genetic material which may alter
the genetic constitution
of the cell―and also suggested
that “Thevirus genetic material might be lost completely
after causing significant alteration in the genetic appara
tus of the host― (cf. Table 3). Indeed, his final conclusion
is that “Contemporary data support the concept that
malignant growth is a single cell phenomenon and that
cancer cells possess heritable abnormal genetic qualities
which allow them to escape the homeostatic mechanisms of
the host and permit the establishment of a varying degree
of autonomy.― Despite his conclusion, Hilleman did not
consider the possibility that there may be cancers that
are not caused by any virus, or that somatic mutations
represent the general case, with particular cases “of
spon
taneous origin or mediated by irradiation
or chemical
mutagens― or caused by viruses.
The reconciling assumption.—With the development
of
the modern concepts of molecular biology the question
of whether the “reconcilingassumption― that viral nucleic
acid could cause a somatic mutation must be re-examined
in the light of new information
and new experimental
methods. It is clear that virology is no longer a purely
biological science; it has become a biochemical science as
well.
As a matter of fact, the possibility of a “reconciling
assumption― seemed at first to recede as more and more
knowledge of nucleic acid function came to light. Reasons
THE
RECONCILING ASSUMPTION: HERITABLE, IRREVERSIBLE
CHANGE
IN GENOME
WITH OR WITHOUT
VIRUS
RECOVERY
1. Virus reproduces
or 2. Virus incorporated into genome
or 3. Virus fragments incorporated into genome
or 4. Virus causes change in genome
for this included the fact that at least one tumor virus,
the Rous sarcoma virus (RSV), had been shown to be of
the RNA type (16), plus the fact that the Central Dogma
of Crick (17) had been widely interpreted to mean that
genetic information passed from DNA to RNA to protein
and never in the reverse direction.
All the available data
from cell-free systems appear to substantiate
this re
stricted view of the original postulate,
although what
Crick actually said was that information should be able
to pass from nucleic acid to nucleic acid and from nucleic
acid to protein but not from protein to nucleic acid.
Thus, although Crick left the possibility of an RNA
directed DNA synthesis is open, the experimental progress
during the past 5 years has demonstrated
only that mam
malian cells contain a DNA-directed
DNA polymerase
(6) and a DNA-directed
RNA polymerase
(27, 61).
With these two enzymes on a firm basis, no additional
nucleic acid-synthesizing
mechanisms
in normal mam
malian cells seemed necessary, although they were not
excluded.
The problem of how RNA viruses could be
synthesized in mammalian cells could be adequately ex
plained by the synthesis of RNA-directed
RNA polymer
ase, which in the case of L cells infected by Mengo virus is
apparently programed by the virus, which is of the RNA
type (21) (Chart 4).
Another obstacle to the acceptance of the reconciling
assumption was the continued production of virus par
tides in tumor cells, which was apparently not doubted
by Rous in 1958 (48, p. 1357). If the presence of a virus
in a cell could produce a metabolic imbalance that re
sembled neoplasia, there would be no need to assume a
somatic mutation.
More recently,
there have been
cases of tumor viruses that disappeared from tumor cells.
According to Vogt and Dulbecco (58), in the “late―
trans
formed cells the polyoma (DNA) virus is integrated in a
“non-extractablenon-inducible form or is altogether ab
sent.― The absence of a DNA virus could be explained
in terms of integration into the host genome, but it was
somewhat surprising when the Rous Sarcoma (RNA)
Virus was found to be capable of being carried in the host
cells in such a way that no virus or other infectious mate
rial could be found (54). In these cases, the information
needed to produce virus was carried in a form called a
provirus, which was considered to be the information
necessary for virus production (55). Since the expression
of this information was blocked by actinomycin
D—in
contrast to the synthesis of Mengo virus RNA by an
RNA-directed
RNA polymerase (21)—Temin suggested
that the provirus either is DNA or is located on DNA
(55). When DNA synthesis was blocked with amethop
tern or FUdR, the provirus was not formed, but cells
containing provirus could form virus, again suggesting
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Cancer Research
1090
DNA
@
Actinomycin D
blocks —@
L
DNA-.drected
DNA-polymerase
—@:_.@___3.
Vol. 24, August 1964
flEWDNA
DNA-directed
RNA polymerase
new RNA
I
I
@
(RNA.-cHrected
DNA-polymerase)
@\s%%%%S\\
RNA
CHART 4.—Categories
of nucleic
acid
synthesis
according
to what
template
is
used (DNA or RNA) and what building blocks are used (ribotides or deoxy
ribotides).
The
RNA-directed
DNA
polymerase
is
still
hypothetical;
the
others are recognized. See text.
that the provirus contains DNA (57), in which case it
presumably
would have to be synthesized by a special
RNA-directed
DNA polymerase
so far undiscovered.
There is thus pending the distinct possibility that both
RNA and DNA tumor-producing
viruses may become in
tegrated into the genome of normal cells in order to convert
them to tumor cells, and the production of virus particles
would not be required for the continuing cell division seen
in malignant tumor cells. One may ask whether the ge
nome for specific virus production is a requirement, and
at this point it seems likely that it is not. The question
is whether the virus genome is incorporated
in tote,
whether it is incorporated in pieces, or whether it merely
produces changes (mutations)
in the host genome.
If
the latter can occur in some cases, then the “reconciling
assumption― would be realized, and the conversion of
normal cells to cancer cells could be seen as an alteration
of the cell genome either by alteration of existing genetic
material (somatic mutation) or by addition of new genetic
material which would not be mutation in the usual mean
lag of the term but would be transformation,
transduction,
or conversion (Table 3).
In contrast to this view, Zilber (62) finds it “hardto
believe that processes resulting in essentially
similar
changes of the cell might be induced once by a change in
the genetic information of the cell proper, while in other
instances by additional genetic information received by
the cell from outside.― He is inclined to look for latent
viruses even in the case of carcinogenesis by chemical or
physical factors.
Zilber regards the additional
genetic
information
as responsible
for hereditary
metabolic
changes resulting in proteins of another antigenic struc
ture being synthesized.
“These,as well as some other
changes, such as enzymatic ones, release the cell from
subordination
to systems which regulate cellular growth,
and inadequate cell reproduction results in the formation
of a tumor.―
On the other hand, Temin (56) suggests that perhaps
any virus under circumstances
appropriate
to it may be
capable of causing a tumor by “beingone of a chain of
events leading to the production of the new genes re
quired for carcinogenesis.― He suggests that we can only
hope to find the remains of virus as pieces of foreign in
formation in the cell and that the classical biological pro
cedures of virology (i.e., Koch's Postulates) may be inade
quate or need to be supplemented
in the cases in which
the information
for whole virus reproduction
has been
lost. It is clear that both Zilber and Temin and many
others now regard the alteration of the host genome as
the primary event, whether it be by addition of all or a
part of the virus genome, or by an alteration of the host
genome by virus or other means.
In either case it would
appear that we are moving closer to the “reconciling
assumption― and that the somatic mutation theory and
the virus theory are not mutually exclusive.
In both
cases an irreversible change in the hereditary apparatus
of the cell is postulated
(Table 4). There remains the
very important question of just what alteration in genetic
material or what new genetic material needs to be brought
in to effect the conversion of a normal cell to a tumor cell,
and it is here that the immunologist and the biochemist
re-enter the picture.
FEEDBACK
DELETION:
WITH
OR
CHANGES IN GENOME
WITHOUT
Just as the somatic mutation
theory and the virus
theory seemed to be agreeing on the genetic basis of the
malignant change (Table 4) biochemistry and molecular
biology became aware of many new ways in which genetic
expression may be controlled.
After the basic essentials
of the Central Dogma (17) involving DNA:RNA:Protein
were learned the great new advance was in the field of
feedback controls (Chart 5).
Perhaps the simplest expression of the application of
the new possibilities was my own statement in 1958 (42),
as quoted by Luria (31), who said, “Themost productive
hypothesis is that the basic controls . . . are ‘systems re
sponsible for negative feedback on specific enzyme-form
lag systems required for cell-division'
(42). . . .“Luria
continued,
“Thefull-fledged cancer cell has lost these
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
POTTER—Biochemical Perspectives in Cancer Research
regulatory systems, so that its ability to divide has be
come unrestricted.
In addition it has acquired a variety
of new properties that render it destructive to the organ
ism as a whole.― . . . “Severalchanges may be needed
before the full neoplastic powers of an altered cell are
expressed.― Luria (31) was clearly in favor of the “recon
ciling assumption― referred to above, and felt that the
somatic mutation theory and the virus theory are not
“alternative and mutually exclusive― but are likely to
come together in terms of “thestructural
biochemistry
of genetic materials― and “thebiochemistry of gene action
and cellular regulation.― Luria regarded the permanent
cellular change as either genetic or epigenetic, the latter
being changes in the expression of genetic potentialities,
which could include “alterationsin self-maintaining sk@ady
skite mechanisms regulated by metabolic feedback― (31)
(italics mine).
The latter possibility was expanded by Monod and
Jacob (35), who demonstrated
the ease with which one
could “classifyand define a priori the main types of cellu
lar regulatory mechanisms, including any likely or plaus
ible (s@) mechanism which may or may not have been
actually observed. . . .“
After describing a variety of “modelscapable, in pm
ciple, of accounting for virtually any type of differentia
tion― Monod and Jacob commented as follows:
“Theseobservations
may have some bearings on the
problem of the initial event leading to malignancy. Malig
nant cells have lost sensitivity to the conditions which
control multiplication
in normal tissues.
That the dis
order is genetic cannot be doubted.
That, following an
TABLE 4
FEEDBACK
DELETION
(ALTERATION)
WITH OR WITHOUT CFIANGES
IN THE GENOME
II. No Change in Genome
PROTEIN DELETION
I. Change in Genome
SOMATIC
MUTATION
of Feedback
in Feedback
Target
or Feedback
Message
Message
VIRUS THEORY
trend toward I.
initial event, mutations within the cellular population are
progressively
selected, leading towards
greater
hide
pendence, i.e., heightened malignancy, is now quite clear,
due in particular to the work of Klein and Klein (1958)
(29). But while the initial event, responsible for setting
up the new selective relationships,
may of course be a
genetic mutation, it might also be brought by the transient
action of an agent capable of complexing or inactivating
temporarity a genetic locus, or a repressor, involved in the
control of multiplication.
It is clear that a wide variety
of agents, from viruses to carcinogens, might be responsible
for such an initial event.― This statement will be dis
cussed further in connection with experimental data pro
vided by Boutwell (below).
These stimulating ideas were further expanded and dis
cussed by Pitot and Heidelberger (37), who pointed out
that, “bya suitable application of [the Monod and Jacob]
theories, it is possible to explain how a cytoplasmic interac
tion of a carcinogen and a target protein [italics mine]
could lead to a permanently altered and stable metabolic
situation without the necessity of any direct interaction
of the carcinogen and genetic material.
It is furthermore
a consequence of this theory that, under the proper cir
cumstances and before chromosomal alterations occurred,
the process might be reversed and lead to the production
of a normal from a tumor cell―(cf. Chart 6). It appears
that this paper was an attempt to explore the connotations
of the Jacob and Monod feedback possibilities with the
reservation that there was no attempt “torule out or
deny the possibility that chemical carcinogenesis
is a
consequence of the direct interaction
of the compound
with genetic material.― However, in my opinion the
reservations are likely to be overshadowed by the numer
ous statements suggesting that “earlycancer could be con
sidered as a phenotypic rather than a genotypic disease,―
that “a
reversion from the malignant to the nonmalignant
state is well within reason,― or that “Sucha picture fits
the known concepts of chemical carcinogenesis and shows
in a theoretical manner how a malignant cell may be
produced in the absence of a genetic (DNA) change.―
Finally, it should be noted that Pitot and Heidelberger
stated that, in discussing the effect of “metabolicchanges
CH3
D_T,DEsII,@!:!_R-@r1DE5
@
DNA:DNA>
SRNA
r@oaJ@
DNA@—LDNA:RNA
1 \@Si.._CORTISONE?
r RNA
,,,j@s@—-—.-RIBOTCES
EFS'@―Pc@Y5OME)
t@idO
_____________
[@NVIRON@@j
______
PROTEIN
(PROTEIN)n
SUBSTRATE@t
_1
CHART 5.—Present
concepts
1091
of gene expression
CIRCU@RY
and modulation
I
with
the chemical
environment indicated to show possible interaction between cells (45).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
Cancer Research
1092
Vol. 24, August 1964
refinements
of the experiments
on the concept that
chemical carcinogenesis involves stages of initiation and
promotion as originally proposed by Rous, Rusch, Beren
GeneI
Gene II
blum, and others (cf. 9). Boutwell clearly confirmed the
DNA for Synthesis
DNA for control
operational basis for stating that the initiating action of
carcinogens is irreversible.
This was done by applying a
single dose of a carcinogen at a sub-threshold
level for
I
4
carcinogenesis in the absence of further treatment.
The
I
RNA
c0)(@@,
RNA
promoter (croton oil) was then applied for a period of 16
I
C—,,
weeks either immediately
following the carcinogen or
after a delay of 16 weeks. The incidence of skin papillo
mas was almost identical (near 100 per cent of animals)
I
UGENE
PROTEIN@——@
in both groups, but the group with the delayed croton
PRODUCT―
I
I
oil developed papillomas almost exactly 16 weeks later
I (RNA,
proteIn,
metabollte)
I
than the group that was treated immediately
following
I
I@,
ii,
the carcinogen application as seen in Chart 7 ([9], Fig. 10).
I
@ocess
of DNAReplication,
UnspecIfiedmet-.
From this type of experiment Berenblum, Boutwell, and
I
CellDIvIsion
abolicFunctIons
others have concluded that the initial step in the conver
I
I
sion of a normal cell to a cancer cell is an irreversible
L@..___
@_ ——
@@ncti@n_
__
_ J
process, and in view of the small numbers of papillomas
(about seven per mouse in Chart 7) formed from some mil
P@•ot.In
DeleHon by Comphxtng wIth
I Carcinogens,
Pvomotsrs,
Hormones,
orNatural lions of mouse skin cells subjected to treatment with the
carcinogen it seems likely that the initial event is a single
cell phenomenon, that it is irreversible for as long as the
Inactive Repressor
cell may live, and that the initiated cell is not yet in a
CHART 6.—Release
of cell division
by feedback
deletion
with no
state of autonomous growth.
change in genome (cf. 35, 37). If a “Gene
Product―from Gene I
These experiments show that chemical carcinogenesis
can block the production of the protein corresponding
to Gene II,
can
be experimentally
divided into at least two stages,
a temporary deletion of this protein would be self-perpetuating.
which may be referred to as Initiation and Promotion.
Although both repressor functions are 8hown as blocking at the
level of “Transcription―
from DNA to RNA, the inhibition could 1\Iany years ago, in 1945 to be exact, I discussed such
also be at the level of enzyme synthesis or enzyme activity as in
experiments in terms of an Induction Period, a Critical
dicated in Chart 5. A rather clear-cut example of the inhibition
Period, and a Period of Progression (41). The Critical
of the synthesis of an enzyme on the DNA synthetic pathway
Period was considered to be the time during which the
(Tdr
Kinase)
by a protein
formed
by a second
gene has
been demonstrated by McAuslan (32), but the data did not include potential cancer cells “aresusceptible to the influence of
the repression of Gene II by a “Gene
Product―from Gene I. This the host and are restrained by the normal cells.― It
chart can also be used to discuss release of cell division by might be suggested that Initiation
is a single-cell phe
RELEASEOF CELL DIVISION BY FEEDBACK DELETION WITH
NO CHANGE IN GENOME IN A RECIPROCATING SYSTEM (33,35)
r@-@-@i
I
@
@L-@'
I@
I PI'oducts
from
other
Genes
Feedback Deletion With changes in the Genome, since mutations
either
Gene I or Gene II could
lead to alterations
in either
in
the
feedback target or the feedback message (see Table 4), and other
nomenon
and that
chemistry
of the entire cell population
Promotion
is an alteration
in the bio
in the environment.
mutations could modify the communication between target and
message.
INITIATION: IRREVERSIBLE
100
@
@
brought about by the temporary interaction of the car
cinogen and a cytoplasmic protein,― “we
are here dealing
only with the earliest changes in carcinogenesis.― “Once
the altered regulation is established (possibly within miii
utes or hours) other effects appear . . . which are probably
secondary to the primary change.―
It may be noted that Monod and Jacob also referred to
the initial event which they regarded as setting the stage
for the selection of further mutations, and they regarded
this “stage-setting―as possibly due either to a genetic
mutation or to the temporary inactivation of a gene locus
by the mechanisms which they and Pitot and Heidelberger
were emphasizing.
Stages of carcinogenesis: Initiation and Promotion.—I
feel that it is important to ask whether these theoretical
considerations are being applied to single-cell phenomena
or to populations of cells (22) and, moreover, to relate
them to the experimental
facts which have been estab
lished by Boutwell in the case of skin cancer (9).
Boutwell's studies have been concerned with certain
73
PA
75
50
25
‘-0WEEKS S
16
24
32
01111
1
I
I
Cf
CHART
7.—Irreversibility
of
“Initiation―
following
a single
treatment of mouse skin with 25 @g.of dimethylbenzanthracene
(at arrows) followed by repetitive applications of croton oil (short
vertical
Numbers
marks).
Croton
oil
alone
produced
no
on curves show final number of papillomas
papillomas.
per mouse.
Data from Boutwell (9).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
PoTTER—Biochemical Perspectives in Cancer Research
However, it is only in the initiated cells that these changes
result in autonomous growth.
We must now ask whether the changes in the Initiated
Cells can be defined at the molecular level. Are the
changes genetic or epigenetic in the sense of the Luria
discussion (31)—i.e., are they somatic mutations or “al
terations
in self-maintaining
steady-state
mechanisms
regulated by metabolic feedback?― (31). Similarly, are
the changes during the critical period genetic or epigene
tic? Could promotion
be the result of alterations
in
feedback relations between initiated cells and host cells
which permit further genetic changes in the initiated cells?
In short, do the theories of Monod and Jacob apply to
both the stages of initiation and the stages of promotion,
or do they apply to feedback relations between initiated
cells and host cells? Is feedback more important at the
level of the single cell, or should we consider feedback in
terms of the interactions between a cell and its neighbors,
as Jacob Furth has emphasized (22)? One suspects that
we are still lacking the key information that determines
the “expression of genetic potentialities― (Luria [31])
and that until this information is available a clear decision
between genetic and nongenetic changes in initiated cells
will not be possible.
However, we must try to dissect
the processes of carcinogenesis into as many experimental
stages as possible, and it appears that a good beginning
has been made in the case of initiated cells that have had a
single exposure to a carcinogenic hydrocarbon
at the
proper dose level.
It is clear that, whatever the carcinogen is capable of
doing under continuous application, in the single applica
tion it does convert a few normal cells to potential cancer
cells, and it does not give them autonomous grmj,th. Yet
it seems clear that Pitot and Heidelberger were thinking
in terms
of a mechanism
that
would
result
in autonomous
growth when they said (37, p. 1698) “.
. . Thus, according
to this model, the protein to which the carcinogen is
bound is the repressor of this growth process; . . .“(italics
mine) (cf. Chart 6).
It is difficult to reconcile the proposal that the change
caused by initiating doses of the carcinogen in the initial
minutes or hours of contact with normal cells releases
PROMOTION:
Average
Frequency
them from a growth repressor with the operational fact
that these cells do not appear to assume immediate auton
omy. At least they do not multiply at a rate that is
sufficient to produce either papillomas or carcinomas in the
type of experiment that has been done with mouse skin.
To me it would seem more reasonable to try to relate the
Monod and Jacob feedback mechanisms to the process of
promotion, which has long been considered to involve
release from a growth repressor in skin (9). Indeed, the
appearance of tumors in areas of wound-healing following
appropriate
pretreatments
(9) is a strong argument in
favor of looking for an explanation of certain aspects of
promotion along the lines used by Pitot and Heidelberger
to explain
the initial
molecular
and to seek other explanations
effects
of the carcinogens
for the process of initia
tion.
This discussion is not a matter of semantics, and it is
basic to the next phase of the biochemical attack on the
nature of cancer.
The contributions
of Monod and
Jacob and of Pitot and Heidelberger
are not idle
speculations,
but they should be related if possible to
experimental
systems that are already operational
in
terms of Tumor Progression.
At this point I wish to return to the work of Boutwell,
and in particular to his work on the reversible nature of
promoter action.
Earlier workers had mentioned
the
need for continuous application of the promoter and the
noncumulative
nature of the lower doses, but it remained
for Boutwell to actually emphasize the word reversible
and to carry out a variety of experiments to demonstrate
reversibility.
These experimental
results are shown in
Charts 8, 9, and 10 ([9], Figs. 12, 13, 14). They clearly
show that the effects of the promoter croton oil are re
versible and nonadditive and that an optimum frequency
of repetitive
applications
can be demonstrated.
In sharp contrast to these results, experiments with the
carcinogen dimethylbenzanthracene
(DMBA) in divided
doses with a constant co-carcinogenic dose of croton oil
showed that the action of the carcinogen was irreversible
and additive : the same incidence of tumors was obtained
with 1 @g.of DMBA whether it was applied as a single
dose, or in four parts given over a period of 2 weeks or 8
REVERSIBLE
number PA. / mouse
Per cent mice with PA.
Amount / applic.
once / week
125
@g.
once / 2 wks.
125
@g.
once!
125
@g
4 wks.
Promotor:
CHART
1093
1.5
8.—The
mg.
reversible
none
croton
nature
none
Initiator:
oil
of the
process
of promotion.
75 gtg. DMBA once
Data
from
Boutwell
(9).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
1094
Vol. 24, August 1964
Cancer Research
PROMOTION:
NON-ADDITIVE
Average number PA. / mouse
Frequency
Per cent mice with PA
Amount / applic.
Mg.
once/week
125
twice/week
31.25
0.05
6.25
10 times /wk
P romotor:
1•
1.5
mg.
croton
CHART 9.—The nonadditive
from Boutwell
(9).
oil
nature
Initiator:
of the process
75
@g. DMBA
of promotion
by croton
once
oil.
INITIATION:
Tumor
lOx/wk.
lOx/week
ix/week
lx/4weeks
CHART
10.—Optimal
dosage
lxlwk.
for
% at
32
weeks
in (
(1.8)
@//@
lxl4wks.
y
ri,@
i@(
r%
°/0Croton Oil
A
BC
.06
.01 02
.2
.6
.1
.8 24
.4
required
in
of PA. / mouse
(1.4)
.@
ADDITIVE
incidence
Av. No.
Data
promotion
by
croton
oil. Data from Boutwell (9).
weeks, as shown in Chart 11 ([9], Fig. 11). The demon
stration of irreversibility in the case of the carcinogen and
reversibility in the case of the promoter constitutes strong
evidence in favor of the idea that the two processes are
intrinsically different.
Since it has been shown that cer
tain carcinogens can produce cancer at higher dosages
without any auxiliary treatments,
it is clear that if both
initiation and promotion are essential the classic car
cinogens must have both reversible and irreversible effects
on cells. It seems possible that the observed combina
tions of carcinogens with proteins are evidence of their
reversible effects in populations of cells, some of which
have been irreversibly affected at the DNA level.
It is suggested that the difference between an autono
mous, rapidly growing cancer cell and its normal cell of
origin involves a number of gene mutations.
If such a
cell arises by progression, then each new mutation occurs
in a previously mutated cell.
According to this view the operationally
defined initi
ated cell may be one in which two or more gene mutations
have occurred but in which the number of gene mutations
is not great enough to achieve autonomy.
Promotion is
regarded as a kind of titration of a renewable substance
(“PROTEIN― in Chart 6?) that when complete favors
an accumulation
of further mutations until greater and
greater autonomy is achieved.
none
1Mg.0.25
@g.once4
times
twice/wk.4
Initiator:
once/2
wks.once
DMBA
Promotor:
CHART
@g.0.25
@g.0.25
times
1%
11.—Additive
nature
croton
of
oil
small
in benzene
doses
of
dimethyl
benzanthracene because of irreversible changes produced, with in
dication of a threshold at very low dosage. Data from Boutwell
(9).
(If there are some stable RNA templates
that can
function through many cell generations
without DNA
information,
the
mutation
of the
gene
would
not
be
felt
unless the stability of the RNA template were decreased
or unless cell division had diluted it to less than one unit
per cell (cf. Beale [31).)
The proposed interpretation
differs from the epigenetic
mechanisms of Monod and Jacob and Pitot and Heidel
berger mainly by emphasizing the difference between the
initiating stage and the stage or stages of promotion.
I
would lean toward changes in the genome in the case of
initiation and toward protein deletion in the case of pro
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
1095
PoTTER—Biochemical Perspectives in Cancer Research
motion.
It is clear that the purpose of the above authors
was to show how epigenetic
mechanisms
could
operate
in
molecular terms at the level of initiation, recognizing the
paucity of data on DNA changes and the availability of
considerable data on protein binding (37). The unresolved
question
at the
present
is whether
protein
binding
can
produce cancer cells in the absence of genetic changes
(Chart 6 and Table 4).
Everyone seems to agree that in general terms some
kind of a feedback deletion or modulation must occur in
any case, and it may be emphasized that the assumption
of a break in a feedback loop does not have to be ipso facto
genetic or epigenetic.
Perhaps by next year at this time
experiments of the type carried out by Berwald and Sachs
(5) will have provided an experimental system in which
this 50-year-old question can be resolved.
These authors
have “transformed―as many as 18 per cent of clonable
cells in 9 days in tissue culture exposed to benzpyrene and
5 per cent in 2 days (in a separate experiment).
Trans
formed cells were identified by morphology and are being
assayed in vivo. It is to be hoped that these technics could
also be used to resolve the distinction between “Initiation―
and “Promotion.―
intact but many kinds of control imbalances were present
in the hepatomas.
In a continued search for a generalization
that would
apply to all minimal-deviation
hepatomas,
Cho, Pitot,
and Morris (11) have suggested that the enzyme-forming
systems
that
are substrate-stimulated
appear
to have
a
shorter half-life in the hepatomas
in adrenalectomized
animals in comparison with normal liver under the same
conditions, and Pitot and Peraino have suggested a defect
in the endoplasmic reticulum as a common denominator
(39). A defect in the endoplasmic
reticulum and Dr.
Pitot's suggestion that it might be brought about by an
epigenetic alteration
is an exciting new development
worthy
of much
further
study,
and
Webb and Dr. Blobel have found
reticulum may be altered (59).
my
colleagues
indications
Dr.
that
the
3000
_J
A
@2OO0-@
FEEDBACK
DELETION
IN MINIMAL
DEVIATION
HEPATOMAS
@
@,
The concept of Tumor Progression raises the question
whether all differences between cancer cells and normal
cells are essential to the fact of malignancy.
We have
approached this problem by means of the minimal-devia
tion hepatomas.
It was proposed that, if a series of
strains of hepatomas were found to lack a given enzyme
and one strain of hepatoma was found to contain the
enzyme, it would have to be concluded that the loss of
the enzyme was not essential to the conversion of a normal
cell to a cancer cell, though the loss might influence growth
rate.
In
synthesis
tion
all
these
appear
in feedback
hepatomas,
to be partially
is not yet identified
The minimal-deviation
tain
@
many
the
of the enzymes
enzymes
released,
hepatomas
peculiar
for
DNA
but the altera
chemically.
were found to con
to normal
liver
cells
(43, 46), and a major breakthrough
was achieved by
Pitot (38), who demonstrated
that the hepatomas did not
respond to the procedures that would induce tryptophan
pyrrolase in normal liver. With further research it was
found that a few hepatomas
were able to respond to
tryptophan
pyrrolase induction to varying degrees (11,
19), although all seemed dependent on intact adrenals
(11). One could then ask whether the hepatomas that
failed to respond to tryptophan
pyrrolase induction also
failed to respond to other induction procedures, such as
those that lead to great increases in threonine dehydrase.
Again a generalization of parallel responses could not be
made (7, 45).
In a study of two enzymes that could be varied over
wide ranges in a reciprocal fashion, Bottomley,
Pitot,
Potter, and Morris (8) showed that a spectrum of minimal
deviation hepatomas under standard dietary conditions
showed all of the variations that liver could be made to
assume under special conditions (Charts 12 and 13). It
was as if the liver structural genes for these enzymes were
1000 -
@_M
ala
1000
MM
2000
3000
C-KETOBJTYRATE
/HR/G. LIVER
CHART 12.—Threonine dehydrase
activity
4000
(as a-ketobutyrate)
and glucose 6-phosphate dehydrogenase activity (as TPN reduc
tion) in normal rat liver under maximal conditions of induction.
Each point represents both enzymes in a given sample and shows
that where one is high in activity the other is low in activity. Data
from Bottomley et at. (8).
rF000
tI000
z
x
LA
.@A
14
I
1000 2000 3000 4000
@M@-KETOBUTYRATE
/HR/G.
CHART 13.—Threonine
phate dehydrogenase
tomas on a standard
dehydrase
TUMOR
activity
and
glucose
6-phos
activity in various minimal-deviation
hepa
diet plotted as in Chart 12. In some hepa
tomas one enzyme is high in activity and the other is low in as
tivity, whereas in others the converse is true, indicating
that the
control mechanism is similar to that in normal liver (Chart 12) but
that the internal milieu of the various hepatomas is not the same.
Data from Bottomley ci at. (8).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
1096
@
Cancer Research
In contrast to the idea of a common denominator, the
remarkable diversity of the minimal-deviation
hepatomas
can be illustrated in the case of threonune dehydrase in
Chart 14, in which not only the initial values are shown to
deviate from normal but also the responses show marked
deviation (Chart from [45], data from [7]). A similar
chart for tryptophan
pyrrolase would show that the re
spouses of individual hepatoma strains to the separate
inducers are not proportional.
In the case of threonine
dehydrase, we do not believe that the level of this enzyme
or the rate of its synthesis is essential to the malignant
property.
My present interpretation
of the data from
ndniinal-deviation
hepatomas
is that their diversity is
explained by assuming that they have two or three es
sential mutations
accompanied
by one or more nones
sential changes, as in Chart 15. Another way of looking
at it is to assume that certain mutations or epigenetic
changes affect not one but many enzyme-forming
sys
tems.
Thus, the proposal by Pitot (39) that the endo
plasmic reticulum may be altered, thereby affecting the
stability and function of all enzyme-forming systems that
function on its surface is an attractive
suggestion, al
though I believe the data suggest that changes in specific
enzyme systems would have to be superimposed on such
a general change. Thus, a control gene may act specific
ally on an individual enzyme-forming
system or it may
act on a number of enzyme-forming systems.
Regardless
of whether one accepts the concept of so-called regulator
or operator genes in mammalian tissues, it is clear that
there must be heritable control mechanisms
that de
terinine how or when an enzyme-forming
system will
function (cf. McAuslan [32]).
The question has frequently been asked whether the
minimal-deviation
hepatomas are the least altered of any
available cancer cell. It is clear that there are stages
Vol. 24, August 1964
HYPOTHETICAL COMBINATIONSOF DEFECTh'E REGULATOR
OR OPERATOR GENES IN MINIPML. DEVIATIONHEP@ITOMAS
A
B
C
D
E
F
OPERATORGENES
MINMA.L 0EV. â€ẫ€”
r@@oi
E@H@1
r@@i
Li@1(@i@ift1
IGGN
I
r@-iL@1L@J
L@i
MNIVtALXV. + I -
-DEA, -DEB,-DEC,-DEF
MINI@P4AL.
DEV.+2=
-OEB,-DE@-DEAF-DEBC,-DEBF-DECF
REGULATOR
OR
D@CONTROL OF ENZYMES FOR ThYMIDINE
TRIPHOSPHATE SYNTHESIS AND USE
E=CONTI@L OF ENZYMES FOR MITOSIS
A=CONT@L CF TRYPTOPHAN PYRROLASE
B@CONTROL OF ThREONINE DEHYDRASE
C—
CONTROL OF TYROSINE TRANSAMINASE
F—
CONTROL OF ENZYMES FORGbCOGEN SYNTHESIS AND USE
CHART 15.—Hypothetical
indirect
regulating
combinations
of defective
genes in minimal-deviation
direct
hepatomas
or
(45).
Changes in Genes A, B, C, or F are considered to be fortuitous and
nonessential
for the production
of minimal-deviation
but they are considered relevant to properties
hepatomas,
shown in Charts 13
and 14.
that are closer to normal cells than the minimal-deviation
hepatomas
studied thus far. The present tumors are
malignant
and autonomous.
It should be possible to
find hepatomas that are benign and some that are de
pendent in the sense that they will not grow unless certain
hormones are in the chemical environment
in proper
concentration,
as shown by Furth (22). Recently, an
example of a hormone-dependent
and possibly minimal
deviation hepatoma has been found by Heston (24). The
spontaneous hepatomas in (C3H X YBR) F1 male mice
were found to occur in 100 per cent of intact mice but in
0 per cent of mice hypophysectomized
before 6 weeks of
age. They are therefore presumably dependent on growth
hormone or some other hormone controlled by the pi
tuitary gland.
Although it is only an assumption that in the minimal
deviation hepatomas there has been a break in the feed
back control of cell division—forexample, in the control
or synthesis of an enzyme needed for DNA synthesis—a
rather clear-cut example of faulty feedback has been
actually demonstrated
in the case of cholesterol synthesis
by a minimal-deviation
hepatoma.
Siperstein and Fagan
(51) have found that cholesterol biosynthesis from labeled
acetate was completely unaffected by exogenous cholesterol
at levels that suppressed synthesis in normal liver to near
zero.
anism
Aside from evidence of a break in a control mech
the significance
of this finding is as yet unknown.
The continued study of minimal-deviation
tumors in
liver and other organs and the further dissection of the
stages
of carcinogenesis
in terms
of Initiation,
Promotion,
and Progression should make it possible to find tumors
with fewer and fewer differences from their cells of origin
and finally to determine which changes are necessary for
autonomous growth, for invasion, and for metastasis, and
to determine what role is played by hormones, wound
TIME ON 91% PROTEIN DIET
healing, irritation, and caloric restriction in cancer de
CHART 14.—Threonine dehydrase activity in various minimal
velopment.
The question of whether the biochemistry of
deviation hepatomas after shifting the host animal to a high pro
cancer
can
be
studied more advantageously
in the mini
tamdiet. Noneofthehepatomasshowsa normalliverresponse,
mal-deviation
hepatomas provided by Morris (36), the
and each strain of hepatoma
shows a different
response.
Data
hypophysis-dependent
hepatomas
demonstrated
by
replotted from Bottomley, Pitot, and Morris (7).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
PoTTER—Biochemical Perspectives in Cancer Research
Heston
(24), the epidermal
cancers studied
(9), the hormone-dependent
tations in Rat Hepatomas. VI. Substrate-Hormone
Relation
ships in Tryptophan
Pyrrolase Induction. Cancer Res., 24:
by Boutwell
systems of Furth (22), the
Rous sarcoma system as studied by Temin (57), the tissue
cultures developed by Berwald and Sacks (5), or in other
systems remains to be seen. However, I must say that
I do not believe that useful information on carcinogenesis
will be obtained by biochemical studies on the classical
transplantable
solid or ascites tumors, and I hope that the
relatively small amount of biochemistry now being done
on the favorable systems can be tremendously
expanded
in many laboratories throughout the world. As I asked
myself which was the better system the thought
to me that the enzyme pattern
of a chemically
cancer cell has not been arrived
occurred
induced
at by eons of evolution,
1097
52—58,1964.
12. CLOWES, G. H. A. The Inhibition
tuted
Phenols
with
Special
of Cell Division by Substi
Reference
to
the
Metabolism
of
Dividing Cells. Ann. N. Y. Acad. Sci., 51:1409—31,1951.
13.
. Editorial: Cancer Research Fifty Years Ago and Now.
Cancer Res. 16:2—4,
1956.
14. CLOWES,G. H. A., ANDKELTCH, A. K. A Non-particulate,
Di
nitrocresol-resistant,
Glycolytic, Phosphorylating
Mechanism
Present in Malignant and Certain Normal Tissues. Proc. Soc.
Exp. Biol. Med., 77:369—77,
1951.
15. CLOWES, G. H. A. ; WALTERS,C. P. ; ANDK.ELTCH,A. K. Tern
perature Dependence of Dinitrocresol Stimulation of Aerobic
and Anaerobic Lactate Production in Ascites Tumor Cells.
Proc. Soc. Exp. Biol. Med., 99:415—18,1958.
16. CRAWFORD,
L. V., ANDCRAWFORD,
E. M. The Properties of
Rous Sarcoma Virus Purified by Density Gradient Centrifuga
tion. Virology, 13:227—32,1961.
17. CRICK, F. H. C. On Protein Synthesis. Symp. Soc. Exp. Biol.
XII., pp. 138—63.
New York: Academic Press, 1958.
18. DEMEREC, M. Selfer Mutants of Salmonella typhinzurium.
Genetics, 48:1519—31,1963.
19. DYER, H.M.; GTJLLINO,P.M.; ANDMORRIS,H. P. Tryptophan
Pyrrolase Activity in Transplanted
“Minimal Deviation―
and therefore it cannot be assumed to have a “rational―
reason for every enzyme it contains, as we have come to
expect in cells that have survived through ages of natural
selection.
Since tumor cells do not have surviving
progeny after the death of the host, their evolution is not
progressive.
However, it is possible that in the case of
the Rous virus, the extraneous
information
has been
eliminated by selection, and the changes may be all highly
revelant
to the malignant
changes.
In this discussion I have grossly neglected chemo
therapy, but I have indicated that the biochemistry
of
carcinogenesis and the biochemistry of chemotherapy
in
advanced cancers are separate subjects, since the latter
must
contain
so many
and such diverse
As we look at the biochemistry
today
we must
be optimistic
enzyme
patterns.
of cancer as it stands
about
the
chances
for the
early resolution of many of the problems raised in Tables
3 and 4 and Charts 4, 5, and 6. But it must give us a
real feeling of humility to realize how much has changed
since
Dr.
Clowes
passed
from
the
scene.
How
little
did
he know what was to come ! How little do we know of
what the future holds I
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Biochemical Perspectives in Cancer Research
Van Rensselaer Potter
Cancer Res 1964;24:1085-1098.
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