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[CANCER RESEARCH 33, 2537-2550,
November 1973]
Carcinogenesis—Cellular Evolution as a Unifying Thread:
Presidential Address1
Emmanuel Farber2
Fels Research Institute and Departments of Pathology and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Let me illustrate with an example. We all applaud
enthusiastically
the recent advances made in cancer
chemotherapy, as reviewed by our president of last year, Dr.
Frei (32). Yet lurking in the background is the ever present
fear that some of these patients, especially the children, may
develop new cancers or other diseases in the years to come as a
consequence of the treatment. Naturally, this risk is entirely
justified. However, would it not be far preferable to treat such
patients in ways that might be less hazardous? For example,
we know that occasionally some cancers can "cure
themselves" by undergoing differentiation to mature cells. It is
Dr. Creech, fellow members, and guests, I would like to take
this opportunity to thank you for the honor of being your
president for the past year. It has been a year of mixed
blessings for cancer research, a year that has brought elements
of uncertainty
and even foreboding to science despite
increased commitment to cancer. We here all realize full well,
probably more than any other single group, the extent of our
ignorance when we attempt to explain or discuss the essence
of how a cancer differs from a collection of normal cells and
how it came about. Although great practical advances have on
occasion been made in medicine without much understanding
of the problem at hand and the same has occurred and will no
doubt continue to occur in cancer, nevertheless, it is also
apparent that knowledge relating to the problem offers
opportunities to modify, innovate, or even radically change in
a manner that would be almost impossible on a random hit or
miss basis. As a physician, I am naturally interested in the
most rapid advance toward the prevention and cure of cancer.
As a scientist, I also realize that the only hope for the best
way to prevent and cure is more knowledge. This can be
obtained only through scientific analysis of this biological
process.
1Supported in part by Grant BC-7N from the American Cancer
Society and Grants CA-10439, CA-11218, and AM-14882 from the
NIH.
2American Cancer Society Research Professor.
Received August 10, 1973;accepted August 10, 1973.
NOVEMBER
well within the realm of thinking in 1973, without indulging in
too extreme a form of dreaming, that in the future we shall be
able to force or at least encourage cancer cells to differentiate
and thereby cure the disease. Such a more subtle approach is
theoretically feasible but will require a much greater depth of
understanding of the control of differentiation of normal cells.
Will the research funding by our Federal Government
encourage this or discourage it? We are repeatedly told that
the policy is the former, yet the practice so far as we all know
is the latter.
The area of cancer research I would like to discuss with you
this evening lies in my view at the heart of neoplasia. How do
the many hazards being identified in our environment, both
biological and nonbiological, induce cancer? My approach to
this fundamental challenge in cancer research is predicated
upon the belief that it may be possible to outline in essential
detail one or more series of molecular events that are
intimately related to and responsible for the conversion of a
normal population
of cells to one showing malignant
neoplastic behavior. The ultimate focal point of such
sequences is the cell, as the smallest integrating unit in
biology: a pseudo-intelligent computer that receives, screens,
changes, reacts to, and adapts to a host of environmental
signals, all of this activity apparently designed, through
evolution, for cell survival and host survival. It is my
conviction that such knowledge is basic (a) to the development
of the safest means of preventing cancer in the face of the
continual presence of carcinogens in our environment, a
situation that may well last for a long time, and (b) to the
development of nonhazardous means of treating those forms
of cancer not amenable to the current approaches.
Before coming to grips with this major problem, it might be
well to look briefly at what we know concerning the two most
critical phases of cancer, the beginning and the end, the
"causes" of cancer and the essential nature of cancer.
Parenthetically, I do not belong to that group who advocate
that we abandon the word "cancer" for something more
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Emmanuel Farber
approaches are of fundamental importance in the development
of a rational scientific view of carcinogenesis.
Studies of many different types, too numerous to name, all
suggest that any single neoplasm is not an end stage but only a
step in a long evolutionary history which is part of what
Foulds (31), in his astute analysis of neoplasia, calls
descriptive term to encompass all the many conditions in progression. In the life history of any single neoplasm,
which cells proliferate for whatever reason in a more or less progression toward more aggressive growth is commonly seen
uncontrolled manner, invade tissues, and set up satellite in animals and in man. Presumably, the different cell
growths in other organs. The overall result of such a process, if populations in the patient with melanoma, showing different
host-response patterns and different behavior, are the result of
left undisturbed, is almost always the death of the host.
such an evolutionary selection process.
However, as is evident from the previous illustrations, we
Properties of Neoplastia Cell Populations
also see another feature of neoplasms, namely, varying degrees
of differentiation. Neoplastic cells as a population not only
What do we know about the essence of how a malignant proliferate but differentiate. In this respect, a fascinating
neoplastic cell differs from its normal homolog? I must stress phenomenon is what might be called "self-cure of cancer by
Pierce (77-80)
has some outstanding
at this point that cancer is not a unity but rather a whole differentiation."
biological spectrum which varies quite widely with the nature examples: epidermal cells and primitive embryonal cells, each
of the tissue and cell involved as well as with the physiological of which becomes transformed into nonproliferating cells by
state of the host. Yet despite this spread, most examples of virtue of their differentiation.
A few examples of such
malignant neoplasia do fall fairly easily into a category that behavior are well documented in man (26). Also, Braun (10)
allows one to classify them as cancer.
has an impressive study of the same phenomenon in some
As stated clearly by Prehn (87), neoplasia "comprises two plant neoplasms.
separate alterations in cell physiology: (1) a form of abnormal
Thus, neoplastic cells often retain the ability to be
somatic cell variation that represents somatic mutation and/or transformed into functionally normal or reasonably normal
aberrant and defective differentiation, and (2) a pathologic differentiated cells with loss of the prime property of a
form of hyperplasia." Although some examples of cancer seem neoplasm, cell proliferation.
However, neoplastic cells may show a less meaningful form
to have an unusually uniform cell population suggestive of a
monoclonal origin, this is often not the case. In fact, one is of differentiation as well, such as the production of hormones
frequently impressed by the multiple nature of the cell (9, 65, 66), of isozymes (108), of fetal antigens (1,3, 24,38),
populations that comprise many malignant neoplasms. Let me and even of antigens that are characteristic of other types of
illustrate this by a few slides borrowed from Dr. Clark, who cells in the same organism (76). This behavior is difficult to
has been studying human melanoma in its several forms for consider as normal differentiation and must be viewed as
many years (14, 15, 70). Grossly, patients with melanoma may abnormal or aberrant. Included among this group of changes is
show both highly pigmented and nonpigmented lesions. the acquisition of neoantigenicity (56) unrelated so far to any
Microscopic examination of the lesions in one such patient known antigen in the history of the cells. These so-called
disclosed
two
quite
different
cell populations,
a "tumor antigens" are unfortunately variable with respect to
melanin-synthesizing
population and a population with no presence or degree in many neoplasms. When present, they
apparent pigment formation (Figs. 1 to 5). Metastasis hopefully will allow the selective inhibition of tumor growth
apparently occurred only from the latter. Of course, this by immunotherapy. Their nature and significance and the true
approach cannot prove that one is dealing with more than one biological meaning of unique versus common antigens offers
type of cell that is breeding true. However, studies by Dr. one of the urgent challenges in the study of the neoplastic cell
Clark of a variety of such tumors by electron microscopy has today.
shown that some non-pigment-producing
neoplastic cell
Malignant neoplasms often but not invariably are associated
populations have the organelle for pigment production, the with karyotype abnormalities in both number and structure.
melanosome, while others do not have the organelle at all Also, studies by Harris et al. (44) with cell fusion and of Sachs
(Figs. 6 to 9). These seem to breed true. I present these to et al. (46, 47) with revertants indicate that the behavior
illustrate in graphic form the varying cell populations that one pattern of neoplastic cells is very much a function of the
often sees in malignant neoplasms. How did these two balance between different numbers or sets of chromosomes,
populations in a single neoplasm arise?
some patterns converting neoplastic to nonneoplastic and
Studies during the past several years with the use of glucose others the reverse. Consistent with this is the evidence for
6-phosphate dehydrogenase isoenzymes as genetic markers chromosomal instability in those human diseases associated
have suggested that several types of benign and malignant with a strong predisposition for the development of various
neoplasms may be monoclonal in origin (for recent reviews, types of cancer (36).
see Refs. 29 and 35). Conceivably, the indication for more
Another property of malignant neoplasms relates to their
than one cell type in an advanced state of a neoplasm, such as longevity. Virtually all normal cells have a more or less built-in
in the patient with melanoma, suggests as a minimum either a life-span characteristic of the cell type and species. Neoplastic
multiclonal origin or more probably a progressive but cells seem to be immortal. However, this is not unique to
restricted evolution from a monoclonal origin. Clearly, such frankly malignant cells, since aneuploid cell lines that show no
precise. Precision is essential for scientific analysis, yet the
essence of the cancer problem is just that. We do not know
what the components are that allow the cellular behavior we
see. Until we know that, we had best use an imprecise term;
otherwise, we are just as likely to pick a wrong precise one. In
the context of this presentation, "cancer" is an imprecise
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Carcinogenesis and Cellular Evolution
overt malignant behavior may show the same property. Unlike
the latter, however, the malignant cells show the ability to pile
up and to continue to grow under conditions of serum
concentration, etc., which discourage the nonmalignant cells.
The essential basis for this so-called contact inhibition or
topoinhibition (23) remains to be clarified.
Another equally interesting series of properties are the
surface changes in cancer cells as measured by their response
to lectins such as wheat germ agglutinins and concanavalin A
(97). Although normal cells show similar properties at one
phase of their cell cycle, the malignant cells seem to retain
them throughout. Again, the underlying mechanism and the
relation of these changes to the properties of growth, invasion,
and metastasis remain exciting areas for further study.
Although metastasis is often considered to reflect, at least in
part, the host response to a neoplasm, recent studies by Fidler
(30) suggest that this key property may be, at least in part, an
inherent one of the malignant cell. He has shown that mouse
melanoma cells can be selected on the basis of different
abilities to induce pulmonary metastasis, even though the cells
appear to be very similar by several other criteria such as cell
size, viability, etc. This system could prove to be very useful in
the ultimate identification of those properties of neoplastic
cells that favor metastasis. Another system using herpesvirus
type 1-transformed cells, in which metastasis is reproducibly
enhanced by prior immunization (22), may offer a different
perspective into the interplay between innate modulations of
the neoplastic cell and the response of the host in the distant
spread of cancer.
Before leaving this topic, I would like to emphasize that we
are apparently not dealing with a fixed immutable program of
behavior in a given collection of malignant cells but rather a
modifiable
program, one that can be modulated
by
environment. For example, the addition of 2,3-cyclic adenylic
acid (51, 54, 86), of testosterone (51), of 5-bromodeoxyuridine (95, 98), or of dimethyl sulfoxide (Ref. 95; A.
Bendich, personal communication)
may cause a radical
change in appearance and growth characteristics of certain
malignant cells toward a more normal pattern. This
modulation is striking and, although reversible, nevertheless
emphasizes in a different way one of the key points I would
like to leave with you, that the behavior of cancer cells is very
much a property not only of the inherent information content
of the cell but equally of their environment. This brings us
right back to the question of differentiation of cancer cells as a
way to turn off malignant behavior. Hopefully, these new
leads will be pursued vigorously and may give us further
insight into the relationship between the overt expression of
cancer cells and the balance between that part of the basic
programming of the information
of the cell directing
malignant behavior and that part counterbalancing such an
orientation.
Conceivably, the observations of Sachs and
coworkers and of Harris, KJein, and their coworkers already
referred to are an expression of these counterbalancing forces
at the chromosome level.
Thus, overall, in many neoplasms, we seem to be dealing
with two groups of properties that appear antagonistic, at least
superficially: some properties that indicate the ability to
differentiate more or less normally under some circumstances
and abnormally under others and other properties that point
NOVEMBER
to some basic genetic defects. Somehow, somatic mutation
and altered differentiation are both characteristics of many
neoplasms of man and of animals and it is my feeling that only
through the reconciliation of these two points of view can a
rational basis for malignant neoplastic behavior be developed.
Another facet that I think is important to emphasize in this
context is that the biological properties that seem to be
characteristic of cancer are not necessarily a single unitary
package of biological aberration but rather seem to be a
number of discrete units that can be acquired separately
during its life history. This concept of progressive acquisition
of the alterations that go to make up a neoplasm at any stage
in its evolution is, in my view, a fundamental one that 1 shall
return to later in greater detail.
Causes of Cancer
Let us now return to the second of the initial phases of
cancer, the "causes" of cancer. It is evident that living things
including man live on a planet full of environmental health
hazards, some man-made and some natural, and that many of
these can induce cancer in one or more experimental animals
and some also in man. They are readily grouped into two
categories: those with nucleic acid that can be translated by
target cells, given the universality of the genetic code (viruses,
etc.); and those that can only alter or destroy the preexisting
information content of the host cell (chemicals, irradiation).
Interestingly, the former, both RNA and DNA viruses, persist
throughout the whole carcinogenic process and may even be
required for the continual neoplastic behavior of the affected
cells. The latter are for the most part "hit and run" agents.
Although an occasional chemical carcinogen may persist
through the period of cancer development and may even be
found in the cancer (25), I believe this will prove to be the
exception, rather than the rule. Certainly with some chemicals,
their biological half-life can be measured in hours or days, not
months or years. The time-span with irradiation is even
shorter. Do these differences between viruses and other agents
imply that the pathogenesis of cancer is fundamentally
different with these two groups of carcinogenic stimuli or that
the "hit and run" agents play a facilitating role as a prelude to
takeover by a virus?
The Carcinogenic Process
Given a plethora of potential carcinogenic stimuli and a
susceptible host, how does cancer develop? There is almost
universal agreement that more than one step is essential before
we can observe with any method now available an identifiable
cancer. This stepwise process is best observed in the skin with
chemical carcinogens (6—8, 104) but is now amply docu
mented in many different systems in man and in animals.
Although instances of a direct conversion of a normal target
cell to growing cancer may be found, the majority of examples
thus far studied indicate a more complex process.
The initial interaction between a chemical carcinogen or one
of several forms of irradiation and the target tissue may be of
very short duration and irreversible. We are now beginning to
obtain some insight into this phase, initiation, and I shall
return to this shortly. The subsequent steps are still very much
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2539
Emmanuel Farber
unclear. There are at least two major possibilities. Hypothesis
1 states that the process of initiation induces a cancer cell and
that all the subsequent steps, however many they may be, are
concerned with encouraging the growth of this cellular species.
In other words, according to this hypothesis, the problem is
essentially the development or evolution of a cancer cell
(Chart 1). Alternatively, Hypothesis 2 states that the process
of initiation induces an altered but nonneoplastic cell that now
can evolve into cancer, acquiring seriatim as it does the
properties that endow the cancer with its characteristic
behavior pattern. In other words, according to this second
hypothesis, the problem is the development or evolution to a
cancer cell (Chart 2). This whole process leading to the first
appearance of a malignant neoplastic cell population, together
with the subsequent evolution of an already formed cancer,
would all be included in what Foulds (31) calls progression.
Although these distinctions between the two hypotheses may
seem trivial, they are far from it, both theoretically and
especially practically. How one would attempt to analyze the
process in order to prevent the appearance of a cancer could
differ greatly depending upon which hypothesis one subscribes
to.
Let us look at these concepts a little more closely.
Hypothesis
1. It has often been suggested that the
neoplastic cell induced by the initiation event(s) may remain
dormant for short or long periods of time (6-8). Although the
exact properties of such a cell have not been described, since it
EVOLUTION
OF A CANCER CELL
CARCINOGENIC
TARGET
STIMULUS
CELL
NEOPLASTIC
CELL
CANCER
»
Chart 1. Postulated Evolution of a cancer cell induced by a carcinogen.
EVOLUTION
TO A CANCER CELL
CARCINOGENIC
TARGET
NEW
STIMULUS
CELL
CELL
POPULATIONS
GROWTH
I
ETC
BENIGN NEOPLASIA
CANCER
-•
OR
CONDITIONAL NEOPLASIA
Chart 2. Postulated evolution of a nonneoplastic precursor cell to
cancer.
2540
has never been seen, it is implied that the cell already has
many or all of the properties needed for it to become a cancer.
The process of emergence is considered to be greatly hastened
by noncarcinogenic stimuli that are grouped together under
the heading of promoting agents. These are often considered as
mainly exerting a proliferative response that now encourages
the growth of the transformed but dormant cell or cells. If this
were valid, then one should observe the appearance, at least
microscopically, of growing collections of neoplastic cells
whenever cell proliferation appropriate to that cell type is
induced. So far this has not been described (88, 106).
Pertinent to this discussion is an interesting idea, first
suggested by Thomas in 1959 and subsequently championed
by Burnet (12), suggesting that neoplastic transformation is a
common phenomenon perhaps occurring all the time in all of
us. According to this view, it is our immunological compe
tence, especially that part of it related to homograft rejection,
which protects us from this continual hazard. In fact, Thomas
and Burnet suggested it is the need for defense against
neoplasia that has encouraged the evolution of a homograft
rejection mechanism in vertebrates. The neoplastic cell
induced in the experimental animal by an initiating agent
would theoretically also be subject to the same type of
protective mechanism.
Although this idea has attractive features, there is no hard
evidence in its favor, yet this hypothesis remains conceptually
so important as to demand a vigorous scrutiny of its validity
by critical tests.
Hypothesis 2. The bulk of the studies on carcinogenesis,
both experimental and human, favor an alternate view of
neoplastic development. Repeated studies with a variety of
impure or pure chemical agents, many on skin (33,37,39,40,
75, 90, 91) but an increasing number on many other tissues or
organs such as liver, breast, bladder, kidney, etc. (cf. ReÃ-s.27,
28, 31, 50, and 92), and studies with various forms of
irradiation, especially ionizing (see Refs. 2 and 16), all point to
the presence of many tissue and cellular changes that precede
the overt appearance of a malignant neoplasia. The same has
been found to be true of many studies of human cancer where
cellular and tissue lesions have been described preceding the
appearance of cancer in various organs (e.g. Refs. 11,31, 52,
and 89).
By and large, these changes consist of cell proliferation and
often cytological alterations, especially nuclear and even
chromosomal (36, 89). Although not always seen, they occur
sufficiently often to warrant the tentative hypothesis that
some at least are not merely phenomena parallel to the
pathway for development of malignant neoplasia but are
integral participants in the process. This, I stress, remains a
hypothesis, albeit an attractive one.
If a multistep process is the usual, what is its meaning in
relation to cancer? What is the essential need for a series of
steps, each probably occurring in a new cell population, in the
development of a malignant neoplastic process? The simplest
hypothesis states that a cell or group of cells that display all
the major properties we associate with cancer have several
"biological units" of aberration or deviation from the original
target cell population and that each of these units is usually
acquired separately in a new cell population in a stepwise
fashion (Chart 3). Although, conceivably, some circumstances
CANCER RESEARCH VOL. 33
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Carcinogenesis and Cellular Evolution
CARCINOGENIC
STIMULUS
INITIAL CELL
REVERSIBLE
CELL
-^GROWTH
—¿
IRREVERSIBLECELL
—¿Â»-GROWTH
(NODULAR,FOCAL;
|REGENERATIVE?i
INVASIVENESS,
CYÃŽOLOGIC ABERRATION,
METASTASIS
(NUCLEAR)
PROGRESSION
THE .VAEBURG
"CANCER CELL
Chart 3. Some probable steps in the carcinogenic process.
may exist whereby all the units can be induced simultane
ously, this appears to be the exception rather than the rule, at
least with chemical carcinogens, with irradiation, and with
inert plastic sheets. If this concept has any validity, we would
anticipate as a minimum three or four major steps, since
growth, invasion, metastasis, and cytological alteration appear
to be easily recognizable characteristics of many forms of
cancer (see Ref. 57).
Obviously, these are only gross behavioral properties of a
malignant cell population.
There must be many more
alterations when one views the population at metabolic and
molecular levels. In addition, each property has a quantitative
range, often very wide, so that growth may be slow or fast,
invasion may be minimal or extensive, etc. This is seen clearly
not only in cancer of different tissues but also in cancer of the
same type at different phases of its development.
This view of carcinogenesis implies that the transformation
of an original target cell population to an overt cancer consists
of a number of discrete cell populations, each of which in their
makeup and properties is one step closer to the final goal. In
other words, carcinogenesis may be nothing more or less than
equivalent to a process of progressive cellular evolution, which
stops only with the demise of the host.
This overall view of carcinogenesis in essence is a very old
one but one that has been formulated most recently in an
unusually lucid manner by Foulds (31). Regrettably, although
his term progression is frequently used, the essence of this
concept of carcinogenesis is still not to a sufficient degree an
integral part of our thinking in cancer. As stated by one of our
former presidents, Jacob Furth (34), in his quotation from
Seneca, "what is never sufficiently learned is never too often
repeated." Apparently, this concept falls easily into this
category.
It is no doubt evident to many of you that what I have been
discussing has also been formulated in essence by Potter (82,
83) as the concept of "minimal deviation." The fruits of this
idea have been many, although regrettably they do not include
a clear experimental demonstration of the essential biochem
istry of the cancer cells, as was hoped. Nevertheless, the
increasingly availability of a whole spectrum of discrete
cancers of the liver and more recently of kidney, as developed
by Morris and Wagner (74), has offered the cancer research
worker a group of models that may continue to be extremely
valuable in the study of cellular evolution in neoplasia.
NOVEMBER
An Alternate View
I must hasten to add that the viewpoint just presented,
although championed by some, is by no means the only
reasonable one. It has focused exclusively upon the neoplastic
cell and its precursors without regard to the host in which
these changes are presumably occurring. An equally extreme
alternate way to view the whole process is to assume that the
malignant neoplastic transformation is a more or less tightly
coupled package which is acquired late in time after exposure
to a carcinogenic stimulus and that it is the differentiation of
the host protective mechanisms that determines which
properties will or will not be expressed at any time period. To
name but one such possible system, the immunological system,
it is becoming increasingly evident that the immunological
response to a neoplastic cell population is no more static than
is the neoplasm. This system shows varying properties, both
cell mediated and humoral, that are now beginning to be
dissected and partially understood. Conceivably, quantitative
or qualitative changes in this system could be the major
determinants of biological behavior of a neoplastic cell
population. Whether a cancer invades, metastasizes (e.g., Ref.
22), grows, regresses, etc., may be just as much a resultant of
the activities of the host tissues as of the neoplasm. As with so
much in cancer, immunoselection as a major force in the
carcinogenic process remains attractive but unproven.
Very probably, as in so many biological systems, what we
actually observe at any one moment in time is the resultant of
the interplay of these antagonistic forces.
Steps in Carcinogenesis
Having briefly presented some overall views concerning cells
and their evolution in carcinogenesis, what can now be said
about the detailed steps in the process?
Initiation. Assuming an evolutionary progression from
initial target cell to cancer, what is the essential nature of the
initiating events and what are their roles in subsequent steps?
As was briefly alluded to in the discussion of the neoplastic
cell, two apparently alternate views have been entertained,
somatic mutation and altered or aberrant differentiation.
According to the first, the evolution to neoplasia is initiated
by one or more irreversible changes in the genome of the
target cell. Since DNA represents the dynamic repository of
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Emmanuel Farber
the information in the cell, a somatic mutation is often viewed
as a change in DNA. According to the second, the initiating
event or events need not be a fixed change in the genome but
something else, perhaps even epigenetic, that is analogous to
the periodic events occurring during fetal and postfetal
development that are responsible for the progressive differenti
ation of cells. Since these are often dramatic and largely
irreversible, e.g., the differentiation to liver that subsequently
makes liver that makes liver, etc., they could also participate in
the "differentiation" of a normal target cell to a neoplastic cell
and in the aberrant differentiation that seems to be associated
with many neoplasms.
In my view, as of 1973, these two viewpoints are not
antagonistic but could have much in common. Firstly, we do
not know the nature of the irreversible steps in differentiation.
They could very well be associated with subtle alterations in
DNA, such as methylation or deamination (94), perhaps not at
all unlike those associated with some somatic mutations.
Secondly, whether an initiated cell evolves into a neoplasm is
largely determined by the environment of the cell. Viewed in
this way, initiation creates the potential for cancer develop
ment but does not induce cancer. The cell or cells so altered
are now subject to many of the same controlling and
modulating forces that control the behavior of normal cells
but that in this context favor the development toward
neoplasia. This could be true whether the process of initiation
is genetic or epigenetic.
A striking example of this comes to mind in regard to an
antischistosomal drug, hycanthone. This drug is quite effective
in a single dose in treating schistosomiasis, a very widespread
disease. Hycanthone is a mutagen in several systems and breaks
liver DNA in vivo. A single injection, as used in treating the
human disease, is without evident effect in the normal mouse.
However, when given in the same manner to a mouse with
schistosomiasis, it induces liver cancer (41).
An attractive and provocative resolution of these different
approaches in which the concepts of altered differentiation
and somatic mutation are discussed in relation to cancer has
been presented by Kauffman (55). The limitation of time does
not allow me to detail in any way Kauffman's views. However,
he presented a compelling series of arguments that allow a
rational reconciliation of somatic mutation and aberrant
differentiation as both playing an important role in carcinogenesis. I commend those of you who are interested to read his
interesting and challenging views.
Regardless of the theoretical considerations relating to
initiation, it is now clear that many forms of irradiation and
chemical carcinogens produce alterations of all the major
macromolecules of the target cells, including protein, RNA,
and DNA (Chart 4). Although some chemicals are active per
se, the majority require prior metabolic activation to highly
reactive products, ultimate carcinogens. This phase of chemical
carcinogenesis has been comprehensively reviewed by Miller
(72) in his Clowes Memorial Lecture in 1970 and requires no
elaboration at this time. All the work of the Millers and others
since then (e.g., Refs. 53 and 110) with several carcinogens
have added increasing evidence for the essential validity of the
hypothesis presented by Miller.
For the past year, our laboratory has been studying DNA
damage and repair in the liver of the intact animal under
2542
Carcinogen
Ptecutsor
" Proximate
Carcinogen
. electrophilic
reactant
(active
carcinojen)
Carcinogen
Chart 4. Interaction of chemical carcinogens
derivatives with various cellular molecules.
or their active
conditions in which cancer can be induced. The purposes of
the study have been manifold but two of the most important
have been (a) the attempt to understand more about the
possible role, if any, of altered DNA and of its repair in
carcinogenesis and (¿>)the development of a rapid in situ
bioassay of potential carcinogens for different organs or
tissues.
In arriving at a decision to concentrate upon DNA as an
attractive and probably relevant target in carcinogenesis, we
have been influenced by the following considerations: (a)
where studied, carcinogenic stimuli interact with all the major
cellular macromolecules including DNA, RNA, and protein;
(b) carcinogenic stimuli are almost all mutagenic (4, 71) in
bacteriophage, in microorganisms, and probably also in many
eukaryotic cells and the only macromolecule that can be
related today to such an action is DNA; (c) altered DNA as a
basis for initiation is the simplest hypothesis that does not
require a complex network of indirect interlocking effects as
do other hypotheses that focus on protein or RNA (81); (d)
carcinogenic processes, especially those initiated by a brief
exposure to a carcinogen, show an eclipse period which may
last for weeks or even months. During this period, no overt
manifestations of altered cell behavior may be evident at a
level now visible to the observer. It is difficult to consider
molecules, such as most proteins or RNA's that are apparently
not self-duplicating and that show continual or intermittent
turnover, as being capable of storing information for such
relatively long periods of time; and (<?)in one human disease,
xeroderma pigmentosum, which is characterized by a very high
incidence of skin cancers, some alteration in DNA metabolism
may well be involved in the clinical manifestations of the
disease.
I will not bore you with all the details. However, it now
appears that every liver carcinogen so far studied, with the
possible exception of ethionine, induces within a matter of
hours measurable single-strand damage to liver DNA in vivo as
monitored by centrifugador! in an alkaline sucrose gradient
(17, 18, 20, 100). This applies to chemical carcinogens
whether they are known to induce liver cancer specifically or
not. However, the ones active for the liver show a slow
prolonged repair, the DNA strands being still completely
broken or incompletely rejoined (18,20, 100) at 14 days after
a single administration. In contrast, those not known to be
carcinogenic for the liver but only for other organs show much
more rapid repair, usually within 48 to 72 hr (18, 20). These
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Carcinogenesis and Cellular Evolution
tentative conclusions had to be modified considerably when it
was found by Dr. Stewart in our laboratory, in collaborative
work with Dr. Mirvish of the Eppley Institute in Omaha, that
nitrosodihydrouracil,
one of the few nitrosamides carcinogenic
for liver, induced not only rapid damage to liver DNA but
equally rapid apparent repair (101). However, there was one
striking difference; this compound, unlike most of the others
studied, induced both single- and double-strand breaks, as
measured on a neutral sucrose gradient. Meanwhile, Dr. Sarma,
at our Institute, had been studying the effects of liver
regeneration on carcinogen-induced damage of DNA and from
these studies formulated the interesting hypothesis that
initiation of carcinogenesis, with at least some compounds,
might consist of the induction of double-strand breaks in DNA
with the resultant high probability of errors during repair (93).
Two activated forms of 2-acetylaminofluorene, the TV-hydroxy
and jV-acetoxy derivatives, as well as nitrosomorpholine and
nitrosodihydrouracil,
all potent liver carcinogens, induced
double-strand breaks in liver DNA (Table 1).
Also, with some chemicals, a single injection can initiate
carcinogenesis in the liver if given during liver regeneration
(13, 19, 48, 69, 84). In our view, the regeneration may well be
converting single-strand damage of DNA to double-strand
damage, thereby preventing complete restitution of the
original DNA base sequence.
These observations now suggest an attractive working
hypothesis. Initiation with chemicals may consist of perma
nent damage to DNA by virtue of the induction of
double-strand DNA damage followed by faulty repair as well
as by the induction of nonreparable alterations. This would
suggest that some chemicals may be similar to some forms of
irradiation in their functional effect on DNA. If this
hypothesis has any validity, one should be able to induce liver
cancer in an intact animal with a reasonable incidence by a
single injection of those chemicals that induce double-strand
DNA damage.
Recent work by Dr. Stewart in our laboratory also indicates
that the interaction with DNA by a carcinogen like dimethylnitrosamine is not random but seems to select some portions
of the DNA over others. The work in vitro of Weinstein étal.
(109) with 2-acetylaminofluorene
and of Powers and Holley
(85) with dimethyl sulfate and tRNA indicates that chemical
interactions need not be random but can be highly specific for
even single bases in complex macromolecules like tRNA. The
recent observations that fibrosarcomas induced in Chinese
hamsters and rats by a virus and by a polycyclic aromatic
hydrocarbon
are associated with reproducible nonrandom
chromosome variation (73) is potentially exciting evidence
consistent with this idea. Also, this concept is not at all in
conflict with the interesting data and ideas of Knudson et al.
(57—59) concerning mutations and some forms of human
cancer.
Thus, it may now become possible to relate, at least with
some reasonable working hypotheses, molecular effects with
corresponding biological consequences. In this regard, it is
conceivable that inert plastic sheets, by virtue of the disturbed
local environment (e.g., possible anoxia, etc.), may also induce
alterations in DNA that are analogous to those induced by
chemically reactive carcinogens or irradiation without direct
chemical alteration of the DNA.
NOVEMBER
Before leaving this aspect of carcinogenesis, I would like to
mention in passing that the measurement of DNA damage and
repair by physical means may well allow a new approach to
the bioassay of potential carcinogens for specific organs or
tissues. It now becomes possible to measure effects of such
hazards on any one of many tissues and at quite low levels and
to begin to explore whether one can specify possible target
sites with this approach. It also appears feasible to study
possible additive effects of very low doses of more than one
carcinogen on an important cell macromolecule, DNA.
Further Steps in Carcinogenesis. Although some reasonable
postulates can be made concerning initiation, as we have
already seen, this becomes more difficult to do for the
subsequent history of the carcinogenic process. However, one
cellular activity stands out prominently in the development
phase of most carcinogenic processes, proliferation of nonneoplastic cells. The vast majority of studies on the tissue changes
seen during carcinogenesis show such hyperplasia to be a
common phenomenon. These include skin, liver, bladder,
mammary gland, kidney, etc. (2, 6, 8, 11, 27, 31, 33,37,39,
43,45, 50, 52, 75,88-92,
104, 106). An impressive aspect of
this hyperplasia is its focal nature. In the liver there is now
experimental evidence to support the clonal nature of this
proliferation (27, 96). Thus, the earlier initiation events have
presumably altered scattered cells throughout the tissue or
organ so as to encourage their proliferation without significant
hyperplasia of the remaining cells.
We do not yet have any clear notion as to the number of
different cell populations involved during any carcinogenic
process. In both liver (28) and skin and probably some other
tissues as well (31, 45), there appear to be at least two focal
proliferative lesions that precede the appearance of cancer, one
a reversible hyperplasia and another a more irreversible
population. At least in the liver, the latter appears con
siderably later than the reversible populations (27, 28) (see
Chart 5).
However, it would appear that the exact number is a
function of the experimental circumstances. With a single
exposure to at least some liver carcinogens, the first nodule
population to appear seems to be an irreversible one, although
Table 1
Carcinogens thai induce strand breaks in liver DNA in vivo
Double strand
Single strand only
Dime thy Initrosamine
Methylazoxymethanol
A'-Methyl-jV-nitrosourea
/V-MethyWV-nitroso-iV'-nitroguanidine
Methyl nitrosourethan
Methyl methanesulfonate
Diethylnitrosamine
yV-Ethyl-yV-nitrosourea
Ethyl methanesulfonate
2-Acetylaminotluorene
yV-Nitrosopiperidine
/V.jV-Dinitrosopiperazine
4-Nitroquinoline 1-oxide
4-Hydroxyaminoquinoline
oxide
/V-Hydroxy-2-acetylaminofluorene
Af-Aeetoxy-2-acetylamino!1uorene
/V-Nitrosomorpholine
/V-Nitrosodihydrouracil
3-Hydroxyxanthine
1-
1973
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2543
Emmanuel Farber
ORIGINAL
CELL
CELL
CELL
POPULATION POPULATION POPULATION
i
n
m
INITIATION
SELECTION
ACTIVE
CARCINOGEN
(ELECTROPHILIC
REACTANT)
PROGRESSION
SELECTION FOR
INCREASING
MALIGNANCY
INCLUDING
METASTASIS
CYTOTOXIC ,
"OR
IMALIGNANCY)
ATIN
RESISTANCE
EXPOSURE
TOCARCINOGEN
DMADAMAGE
_
DNA"RÈPAÌR
"
Chart 5. Evolution of liver cancer through different cell precursor
populations.
a transitory reversible phase has not yet been ruled out. The
process may somehow by-pass a possible step.
What might the mechanisms be for the encouragement of
the growth of these selected populations without involving the
whole organ or tissue population? We know very little about
this as yet. However, we are just beginning to obtain some
insight into one such population, the hyperplastic nodule
population in the liver of animals fed carcinogenic diets (27,
28). There are many old observations, going back over 30 or
more years, that indicate that carcinogens, among other
properties, are not infrequently cytotoxic and are often
inhibitory to some cell functions (40, 105). In the liver, this
can readily be seen by inducing cell proliferation by partial
hepatectomy. When exposed to hepatic carcinogens, the liver
cells fail to respond to such a stimulus, measured either by
mitosis or by DNA synthesis (64, 67, 68). However, scattered
focal islands of liver cells have escaped somehow this
inhibitory influence and do show cell proliferation (27, 28, 64,
67, 68). Thus, even at an early stage in carcinogenesis, some
new cells show resistance to the cytotoxic effects of
carcinogens, a property often shown by cancer cells under
similar circumstances (40, 105).
These observations suggested that we might begin to
identify early during carcinogenesis the existence of carcino
gen-resistant populations by the use of acute toxic agents. For
example, when animals fed the carcinogen 2-acetyl-aminofluorene for 3 weeks are given an acute cytotoxic dose of
dimethylnitrosamine,
they show extensive necrosis with no
generalized cell proliferation. However, scattered throughout
the liver are isolated islands of proliferation, even in the
centers of areas of necrosis. Are these islands of proliferating
cells original resistant cells or are they mutant-like populations
now selectively encouraged to proliferate by virtue of the
selection pressure imposed?
I present this to you, not because of the details but because
I believe they may illustrate a principle we must begin to
apply. // we are to understand the essential nature of
neoplastia development, we must begin to identify, isolate,
and study the different cell types that seem to be the key
precursors from which the malignant neoplasm is ultimately
derived. The techniques for cell culture and cell study are
reaching the point where such studies are now feasible. In my
2544
view, this approach, in combination with studies on in vitro
carcinogenesis, offer new and exciting possibilities to analyze
the carcinogenic process in a new dimension.
Our experience with liver, as well as the experience of
others with other systems, such as the skin (88, 106), raise
serious doubt as to whether one can explain promotion simply
on the basis of cell proliferation. Under some circumstances, a
general cytotoxic effect as well as a hyperplastic effect might
well be the key components. I cannot help thinking that the
requirements for promotion are going to vary much with the
nature of the target tissue and that selective rather than
general hyperplasia may be important.
In this respect, it is important to recall that many types of
differentiation or cellular modulation require an episode of
DNA synthesis for the new property to be initiated. Since
differentiation seems to be related somehow to neoplasia, I am
becoming increasingly impressed with this aspect as perhaps
being as important as cell proliferation per se at certain steps
in the evolution of a neoplastic process.
Time of Expression of Properties Seen in a Cancer
It is now clear that many malignant neoplasms express
phenotypic properties not seen in the corresponding normal
adult cell or tissue. These include "new" antigens (possibly
fetal in origin) (5, 56), isozymes (108), hormones (9, 65,66),
and other fetal antigens (1, 3, 24, 38). When in the cellular
evolution of a neoplastic process do these begin to be
expressed? This subject has not yet received the attention it
deserves. However, there are a few early studies that are highly
significant (Chart 6).
As to new antigens, this has been explored to my knowledge
in the skin and mammary gland only in Prehn's laboratory (62,
63, 99). In both instances, with chemical carcinogens,
hyperplastic premalignant mammary gland lesions (99) and
skin papillomas (62, 63) showed neoantigenicity. This neoantigenicity persisted essentially unchanged through years of
transplantation
and was still present in the malignant
neoplasms that subsequently developed. Thus, this property
CARCINOGENIC
STIMULUS
INCREASING
PROBABILITY
OF TRANSFORMATION
TO MALIGNANCY
Chart 6. Appearance of different populations of neoplastic cells at
different stages of the cellular evolution of cancer.
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Carcinogenesis and Cellular Evolution
appears to be acquired relatively early and well before the
progression to cancer.
Isozyme patterns often show quite marked differences
between normal and neoplastic tissues (108). In the liver,
many hepatomas have characteristic patterns indicating by and
large a tendency to lose those seen in well-differentiated liver
as the growth rates increase. Studies with Dr. Weinhouse and
Mrs. Shatton of isozyme patterns in preneoplastic liver lesions
showed no consistent indication that they are acquired early
during carcinogenesis.
In contrast, studies in several laboratories (21,60,61,
107)
indicate that the rise in serum a-fetoprotein in animals on
various carcinogens coincides with the early appearance of
nodular hyperplasia. Dr. Okita in our laboratory has found
that the later reappearance of this fetal protein often occurs
before any recognizable malignant neoplasia appears. Some
hyperplastic nodules in such circumstances show the protein
by fluorescence microscopy. Thus, again, a property of a
malignant neoplasm appears well before cancer can be
detected. To my knowledge, no studies have appeared on the
possible inappropriate synthesis or secretion of hormones by
precursor cell populations.
A cellular property that seems to be very important in
malignant neoplasms is surface membrane changes. These are
often considered to be closely related to the invasive
properties of neoplastic cells. A critical consideration becomes
the time at which these are acquired. If they are late
phenomena, their relation to invasion is more likely. However,
if they appear early, it might suggest that they are related to
other characteristic properties of the cancer cell. Dr. Shinozuka at our Institute has been looking at the plasma membrane
of hyperplastic nodule cells early in carcinogenesis. Although
this study is only preliminary, he has found clear-cut
membrane changes very early in the process. One cannot help
but wonder whether the suggestion of Holley (49) concerning
cell membrane changes as the fundamental alteration in the
development of malignant neoplasia might not have some
support from these observations.
Carcinogenesis
Viruses and Carcinogenesis
Some of the most challenging yet obscure facets of
carcinogenesis are those relating to viruses and virus vis-Ã -vis
other carcinogens.
As to viral carcinogenesis, it is not at all clear whether there
is a similar need for a series of progressively altered cell
populations prior to the appearance of a cancer. This may very
well depend upon the nature of the virus. Temin (102, 103)
and others have suggested that a few of the RNA tumor
viruses, such as Rous sarcoma virus, have all the information to
convert a susceptible target cell to a malignant neoplastic cell
by essentially a one-step process. This system could then be
placed at one end of a spectrum. One could then visualize a
whole series of viruses each progressively less able to transform
rapidly in a single step until the small DNA viruses, such as
polyoma virus and SV40, which seem to require more than
one step, are reached.
This concept fits in very well with that of many
investigators and the one I have discussed in this presentation
of a neoplasm with n number of essential aberrations from the
normal that must be acquired from the target cell population.
The nature of the input by the carcinogen would then
predetermine the minimum number of steps necessary.
The most difficult question is the most important for all of
cancer research today. Is there an obligatory virus or viral-like
component in all carcinogenic processes and, if so, how does
one relate chemicals, irradiation, and plastic films to this virus
(Chart 7)? I pose this as a major challenge to cancer research
today. If, as already mentioned, the virus or viral genome
persists as an essential component of the neoplastic cell in
contrast to a chemical or exposure to irradiation, the question
posed is Number 1 in priority if we are to understand the
development of cancer as a basis for both prevention and cure.
An intriguing new development in this regard is the recent
report that, whereas a virus or irradiation induced in mice
in Vitro
As I have already mentioned, the study of carcinogenesis in
vitro with many agents offers unusual opportunities to dissect
the process as to both cell populations and evolution and
molecular events. In attempting to relate the in vitro
observations to in vivo ones, it becomes difficult so far to fit
the processes together. Since the in vitro studies begin with a
cell population that has already been selected for growth, to
what cell population does this correspond in vivo? Also, in
vivo, since one can often remove the exposure to the
carcinogen before a highly proliferating cell population has
been induced, it is tempting to think that perhaps the order in
which certain events occur prior to the appearance of overt
neoplasia is not critical. A highly proliferating cell population
seems to be generally as susceptible to a carcinogen as is a
nonproliferating or slowly proliferating population as occurs in
vivo. Conceivably, the hormone-induced dependent or condi
tional neoplasia is analogous to the in vitro system in having
stimulated cell proliferation
precede rather than follow
NOVEMBER
exposure to some carcinogenic stimulus. These are very
intriguing considerations that deserve closer scrutiny.
CHEMICAL •¿
RADIATION
CELL
POPULATION
INITIAL
TARGET
IRREVERSIBLE
MALIGNANT
NEOPLASIA
Chart 7. Hypothetical sequence of cellular evolutionary events in
which both chemical or irradiation and viral components participate in
carcinogenesis.
1973
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2545
Emmanuel Farber
leukemia of T-lymphocyte origin, a chemical, 7,12-dimethylbenz(a)anthracene, induced leukemia of B-lymphocyte origin
(42). If this proves to be of general validity, the problem of
virus versus other carcinogens becomes further complicated.
Obviously, this emphasizes in a different but dramatic way the
essential need to identify "clean cells" just as urgently as
"clean chemicals," to paraphrase an aphorism of Dr. Ephraim
Racker.
Concluding Remarks
In this presentation, I have perforce touched briefly on a
wide spectrum of facets of the puzzle that remains as the
object of cancer research. Although each of us must
concentrate on very small aspects of the problem if we are to
build a base on a solid foundation, we must also periodically
attempt to crawl out of our cozy nest to obtain a more
panoramic view of our problem. I have been fortunate in that I
have had a captive audience to share with me or perhaps to
tolerate my sight seeing. However, all of us must do this
periodically if we ever hope to obtain the overall view that I
think is necessary for our mental health. I hope all of you will
have the opportunity and privilege I have had this evening.
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Fig. 1. A transection across a portion of a malignant melanoma of the superficially invasive type. M, an area of pigment-synthesizing cells. AM,
an area of amelanotic melanoma. The electron micrographs in Figs. 6 and 8 were taken from the areas labeled M and the electron micrograph in
Figs. 7 and 9 is taken from the area labeled AM.
Fig. 2. The one area of the base of the tumor illustrated in Fig. 1. M, melanotic cells or pigment-synthesizing cells; AM, amelanotic cells.
Fig. 3. Still higher magnification of an interface between melanotic (M) and amelanotic (AM) cells. At this magnification you can note that the
host lymphocytic response is entirely confined to the amelanotic cells.
Fig. 4. Higher magnification of a melanotic area (M) showing no host cells admixed with the tumor cells.
Fig. 5. Higher magnification of the amelanotic cells (AM) showing the admixture of host lymphocytes.
Fig. 6. Electron micrograph from the cells labeled M in Figs. 1 through 5. The cells show large numbers of melanosomes of the granular type
which show pigment synthesis.
Fig. 7. Electron micrograph from the amelanotic (AM) area illustrated in Figs. 1 through 5. There are no melanosomes. This is a type 4
melanoma cell and is one of the variants of amelanotic cells characterized by the complete absence of melanosomes.
Fig. 8. Higher magnification of the melanotic cells (M) showing the granular melanosomes.
Fig. 9. Higher magnification of the amelanotic cells (AM) showing the absence of melanosomes.
2548
CANCER RESEARCH
VOL. 33
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Carcinogenesis and Cellular Evolution
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2549
Emmanuel Farber
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CANCER RESEARCH
VOL.33
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Carcinogenesis−−Cellular Evolution as a Unifying Thread:
Presidential Address
Emmanuel Farber
Cancer Res 1973;33:2537-2550.
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