Cellular Adaptation in the Origin and Development of Cancer
Emmanuel Farber and Harry Rubin
Cancer Res 1991;51:2751-2761.
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Copyright © 1991 American Association for Cancer Research
(CANCER RESEARCH 51, 2751-2761. June 1. I991|
Perspectives in Cancer Research
Cellular Adaptation in the Origin and Development of Cancer1
Emmanuel Farber and Harry Rubin
Departments of Pathology and of Biochemistry, Medical Sciences Building, University of Toronto, Toronto. Ontario, Canada M5S IA8 [E. F./, and Department of
Molecular and Cell Biology and I'irus Laboratory, Stanley Hall, University of California, Berkeley, California 94720 [H. R.]
Introduction
A dominant view today in cancer research assumes that the
major need for our understanding of cancer resides in the
thorough documentation of the many alterations in gene struc
ture, organization, and arrangement that we know are present
in some malignant cell populations and in their immediate
precursors. While attractive from some points of view, espe
cially in the hope of delineating a relatively simple cause-effect
relationship between the genome and cancer, this simple for
mulation largely ignores a fundamental property of all living
forms, the ability to respond and adapt to many environmental
perturbations. Given the widespread occurrence of cancer in
many species and given the unusually prolonged nature of the
process of cancer development in most instances in humans
and in animals, often lasting from one-third to two-thirds of
the life span of the organism, it would indeed be unlikely and
unbiological for cancer development to proceed without the
participation of many active responses, including adaptive ones,
to the various new cellular phemomena that are part of the
carcinogenic process. Immunological surveillance has been a
popular speculation proposed to account for this prolonged
process. However, immunological responses are only a small
part of this spectrum of reactions, with considerable doubt (1)
that they participate actively prior to the relatively late appear
ance of unequivocal cancer with its many new biological com
ponents such as tissue destruction with cell death, inflamma
tion, ulcération,etc.
All living forms have evolved in a largely unfriendly or even
hostile environment in which protective or adaptive responses
to many different forms of potential injury or harm have been
essential for survival and reproduction. In fact, it appears that
the acquisition of mechanisms for many different adaptive
responses could be considered to be just as important for
survival and reproduction as the development of the essential
pathways for the basic physiological needs of living organisms
such as for energy and for the syntheses of the small and large
molecules that are the component parts of the pathways. It is
widely recognized that a variety of physiological adaptive re
sponses to varying altitudes, other environmental influences,
and hormonal modulations as well as in aging are fundamental
properties of living organisms. In addition, the vast array of
different xenobiotic chemicals and organisms as well as radia
tions to which all living forms have been exposed since the early
forms evolved some 2 to 3 billion years ago would require
highly versatile protective systems to be developed. Such pro
tective or adaptive mechanisms are known to be present
throughout the whole spectrum of living forms from single cell
microorganisms to highly differentiated eukaryotes.
Received 9/18/90; accepted 3/25/91.
' The authors' research included in this article was supported by grants from
the National Cancer Institute of Canada and the Medical Research Council of
Canada (MT-5994) (E. F.): and from National Institutes of Health. CA 15744.
and grant 1948. Council for Tobacco Research. USA (H. R.).
Despite the obvious importance of such systems, their study
in disease has received little current emphasis, especially with
a mechanistic orientation. Since disease processes are largely
expressions of how living organisms react and respond to
perturbations in the external and internal environments, adap
tive or protective responses and their modulations and mecha
nisms would seem to be of the greatest concern in fundamental
studies of disease pathogenesis. Although such considerations
have been the subject of occasional deliberations over the years
(e.g., Refs. 2-8), they have received little attention in the
detailed studies of chronic disease in general and cancer in
particular, especially from the viewpoint of mechanism.
In the face of the many hazards, including some carcinogenic
ones, to which biological systems have been exposed for mil
lions of years, it seems appropriate to ask the question,"What
mechanisms might have evolved to preserve biological conti
nuity?" Probably many earlier species have been eliminated by
exposure to one or more of such hazards. In those that have
survived and endured, what is the nature of the adaptations
that have evolved and could a knowledge of these be useful in
designing new approaches to the prevention of cancer or even
its treatment (9)? What essential roles, if any, do these adaptive
processes play in the development of different cancers?
Adaptive Responses during Cancer Development
Cancer development with the different known etiological
agents, chemicals, radiations, DNA viruses, and some RNA
viruses without oncogenes is a very prolonged process as already
mentioned. The only apparent exceptions to this are the special
carcinogenic processes induced with oncogene-containing retroviruses (10). The long period of development is characteristi
cally associated with the presence of various types of focal
proliferations of an entirely benign nature (some designated as
"benign neoplasms") that may show a slow cellular evolution
to cancer (9).
During this prolonged process, there appear to be a minimum
of three general types of cellular adaptive responses: type A,
early acute transitory responses, usually of a diffuse, zonal, or
regional nature, largely reversible; type B, clonal constitutive
adaptation with the development of a new cell population that
is associated with a resistance of the organ or tissue and of the
organism to environmentally induced cell damage; and type C,
patterns of adaptation of cells to a variety of environmental
modulations during neoplastic transformation. The main sub
stance of this communication is to discuss briefly each of these
three types of adaptations and to suggest how they might be
important as one set of guidelines for cancer prevention and
cancer therapy. This overview is limited to cellular responses
that seem to participate actively in the step-by-step sequence of
events leading to malignant neoplasia. Adaptive responses of
the whole organism, especially the late ones in response to fullyformed cancers, are omitted.
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
Acute Transitory Adaptive Responses
The known responses in this category relate mainly to carcinogenesis with radiations and with mutagenic chemicals. Al
though it is our conviction that similar adaptive responses might
be found in cancer development with cancer-related DNA vi
ruses and some RNA viruses (e.g., Refs. 11 and 12), data in
this area are scanty.
Reversible Inductions of Metabolizing Enzymes. One of the
most general and basic forms of adaptation to xenobiotic chem
icals, including, of course, the majority of drugs, is induction
of enzyme patterns that relate to the metabolism and possible
detoxification of the chemical (13-17). This type of response is
seen throughout the living world including the simplest bacte
ria. In mammals, this often includes two major aspects of
detoxification: metabolic activation to more polar derivatives,
such as via the cytochrome P-450 system and other oxidative
as well as reductive pathways (phase I); and conjugation of
metabolic products for excretion or other pathways of removal
(phase II). The enzymes involved often show transitory in
creases in both transcription and translation and usually return
to their baseline levels after the exposure to the xenobiotic is
over.
It is well documented that these acute responses are fre
quently associated with the more rapid excretion of the poten
tially toxic xenobiotic with an obvious protective result (1320). Apparently, the multiple nature of the responses to so
many xenobiotics facilitates both metabolism and excretion.
Included in the xenobiotic chemicals are a smaller group of
actual or potential mutagenic ones, genotoxic carcinogens, that
form a significant percentage of chemical carcinogens. These
agents also induce a spectrum of enzymes in liver and other
organs that encompass both phase I and phase II components
(21, 22). The obvious roles of those components in the genesis
of more toxic mutagenic derivatives, most often electrophilic
reactants, are well established. However, any role of these
reactive derivatives in the survival of the organism is not evident
at this time.
Acute Reactive Proteins. Another reaction that is often seen
in the higher organisms is the synthesis and secretion of acute
reactive proteins, especially by the liver (23). These proteins are
transitorily elevated in the blood during an episode of injury
and return to their baseline values when their injury is over.
They are considered to play a protective role in the acute injury.
Heat Shock Proteins. A third general adaptive response to
heat, toxic chemicals and a wide spectrum of other environ
mental perturbations is the synthesis of "heat shock proteins"
(24-26). This response, like the xenobiotic-induced enzyme
induction, is present in Escherichia coli, in yeast, and in a wide
spectrum of organisms including insects and mammals. There
is an overall agreement that these responses are often protective
and should be considered as adaptive (26).
Thus, at least three different adaptive response patterns can
be distinguished on exposure to many different xenobiotics and
at least two, Responses 1 and 3, are present in a wide range of
organisms, from single cell to highly complex multicellular.
Responses that span such a broad spectrum of living organisms
probably play an important role in maintaining the integrity
and function of the different living forms, especially as to how
they react to and survive environmental disturbances (27-30).
DNA Repair Enzymes. There is no doubt that the develop
ment of a variety of mechanisms for the repair of DNA, altered
by mutagenic chemicals and radiations and perhaps viruses, is
a major adaptive set of responses of many different organisms
from single cell to the human (31-34). The mechanisms include
photoreversal of pyrimidine dimers, mismatch repair acting on
incorrectly paired bases from unfaithful DNA replication, en
zymatic alterations with recombinant events and excision re
pair. Interesting enzymes are the alkyltransferases which re
move methyl or other alkyl groups from the O6 position of
guanine, the O* position of thymine, or the phosphate groups
of DNA and transfer the alkyl groups to a cysteine S in the
transferase itself. In E. coli, the transfer is part of a very
interesting adaptive response to alkylating agents associated
with the ada gene.
The DNA repair systems are very widespread in nature and
their adaptive protective role is clearly seen in many systems
including, of course, humans. The susceptibility to skin cancer
of humans with xeroderma pigmentosum, with their several
types of deficiencies in the repair enzyme systems, is a striking
example of the importance of the DNA repair systems for
survival.
Cianai Constitutive Adaptation
During a systematic study of the sequence of steps in the
carcinogenic process in the liver (35), it became evident that
another form of adaptation exists, one that originates in rare
single hepatocytes scattered throughout the liver and that is
constitutive, rather than diffuse and readily reversible (9, 36).
This response pattern has been seen with genotoxic chemical
carcinogens. It involves the induction of a new resistance phenotype in a rare hepatocyte and the expansion of these rare
hepatocytes to form focal proliferations (hepatocyte nod
ules).The recognition of this new adaptive phenomenon has
important potential implications not only for a new conceptual
framework for the development of some cancers but also in
tumor promotion, in cancer prevention, in the analysis of
resistance of many primary cancers to chemotherapy, and in
the analysis of how some living forms have adapted, presumably
through evolution, for survival in a hostile environment.
Background. During the first few years of our study of the
mechanisms of chemical carcinogenesis in the liver, we gradu
ally became impressed by five considerations, each of which
suggested that some new approach was needed.
(a) Cancer development in humans and animals with chem
icals, DNA viruses and some RNA viruses, radiations, and diet
is an inordinately long process, requiring from one-third to
two-thirds of the life span of the organism (9, 37). This is
interesting and puzzling, since it can be clearly shown in exper
imental animals and in some humans that exposure to a carcin
ogen began or occurred many years prior to the first appearance
of cancer and was brief and transitory. Although a dominant
role for immunological surveillance was first suggested by
Thomas in 1959 (38, 39), the evidence for this during chemical
carcinogenesis is unimpressive (1). Alternative hypotheses for
the very prolonged nature of the carcinogenic process are nec
essary if we are to develop rational approaches to cancer
prevention.
(b) Autonomous or semiautonomous growth of initiated cells
is a property acquired late in the carcinogenic process. Initiation
with one of many different carcinogens is at no time followed
by spontaneous or autonomous proliferation of any cells in the
liver or in other organs. Only with large doses of carcinogens
and periods of exposure much longer than are required for
initiation does one see focal lesions with autonomous cell
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
proliferation. If the initiated altered cells induced by exposure
to a carcinogen do not grow spontaneously, how might a
differential stimulation of their growth be created? The growth
of the rare altered cell leading to focal proliferations ("benign
neoplasms") is a key phenomenon in promotion in virtually all
experimental carcinogenesis and in many human systems of
cancer development.
(c) Virtually every chemical carcinogen is an inhibitor of cell
proliferation (mitoinhibition)
(see Ref. 40 for references
therein). Haddow (41) suggested in 1938 that inhibition of cell
proliferation could be an early effect of carcinogens and that,
in such an environment, resistant cells might arise and be
encouraged to proliferate. Recently, it has been reported that
two non-carcinogenic promoting agents for hepatocarcinogenesis, phénobarbital(42-45) and orotic acid (46-48), also show
considerable inhibition of hepatocyte proliferation, not unlike
that seen with the carcinogens.
(</) The response of tissues or organs during carcinogenesis,
most relevant as potential precursors for cancer, is focal, not
general. In the majority of instances of cancer development in
humans or in animals in which a precursor cell population or
lesion has been identified or proposed, the "preneoplastic" and
"precancerous" changes are always focal, often clonal, involving
only a very small number of altered cells (9. 36, 37). A selection
pressure must be established if the altered cells are to proliferate
selectively, relative to the surrounding cells (49).
(e) Carcinogens (chemicals, viruses, non-ionizing and ioniz
ing radiations) are widespread in nature. Humans and animals
have been exposed for millions of years to carcinogens, both
present in the external environment, including the food, and
generated endogenously. How has nature handled carcinogens
and carcinogenesis? What systems have evolved that could
allow for the relatively long life span of so many species? Could
any insight into these questions generate clues to mechanisms
and interpretation of the carcinogenic processes?
The Resistant Hepatocyte as an Early Response to Carcino
gens. With well over 75 different chemical carcinogens of quite
different chemical structure and properties as well as in hepatocarcinogenesis seen in rats fed a choline-devoid diet, a resist
ant hepatocyte is a common accompaniment of initiation (9,
35). A seemingly similar phenotype appears in isolated hepatocytes on infection with v-H-ras and v-ra/(12) and in cultured
rat liver epithelial cells during transformation induced by ex
posures to /V-methyl-yV'-nitro-A'-nitrosoguanidine (50, 51).
The resistance phenotype (52) is manifested at three levels of
organization: (a) physiological (behavior), resistance to inhibi
tion of cell proliferation (mitoinhibition); (b) cellular, resistance
to visible cytological responses to some cytotoxic environments;
and (c) biochemical-molecular, the presence of a biochemical
pattern that diminishes or counteracts possible toxic effects of
xenobiotics.
(a) At the physiological level, the key manifestation of the
resistance phenotype in the rare hepatocyte is the ability to
proliferate vigorously in response to a proliferative stimulus in
an environment, such as that created by the presence of one of
many carcinogens and some promoters, in which the vast
majority of hepatocytes, the nonresistant ones, are inhibited.
This is a common basis for the promoting ability of many
hepatocarcinogens and may also be important with two noncarcinogenic promoting agents, the drug, phénobarbitaland the
normal metabolite and obligatory precursor of pyrimidine nucleotides, orotic acid (42-48).
(b) The hepatocyte nodules show resistance to cytotoxic
effects of the agents used in their generation and cross-resist
ance to other xenobiotics and to a dietary deficiency to which
that rat was not exposed (35, 36).
(c) The resistant hepatocytes induced with quite different
regimens show an unusually common biochemical pattern (35,
36). This pattern shows consistent decreases in several phase I
components including total cytochromes P-450 and several
mixed function oxygenases, consistent increases in many phase
II conjugating systems, characteristic alterations in glucose
metabolism, and a reproducible altered pattern of iron and
heme metabolism (36).
It is important to emphasize that many different enzymes
and components contribute to this phenotype. For example,
the decrease in total cytochromes P-450 is so large (60-80%)
that several different ones must be involved (53, 54). This
multiplicity of active components in the resistant hepatocytes
could readily allow for considerable variation in the levels of
one or more components in different clones derived from these
hepatocytes (55, 56) without compromising their overall resist
ant behavior in an appropriate selecting environment.
It is interesting and noteworthy that the resistance phenotype
in the hepatocyte nodules has several resemblances to the
phenotype of some human cancer cells that are resistant to
chemotherapeutic agents (57-59).
Nodules of Resistant Hepatocytes as a Type of Adaptation
with Survival Value. What is the evidence that the nodules of
resistant hepatocytes which we designate as "hepatocyte nod
ules" are better considered as a form of physiological adaptive
response (9) rather than as collections of abnormal cells, namely
benign neoplasms, that are part of a pathological sequence
specifically related to the development of cancer? According to
the latter view, the nodules of hepatocytes derived by clonal
expansion of potential initiated hepatocytes are abnormal from
the outset and represent foreign cell populations derived on the
basis of abnormal genes and gene products.
The most telling evidence that points overwhelmingly to the
physiological adaptive nature of the hepatocyte nodules exists
at three levels of organization: (a) the cell and tissue architec
ture and organization; (b) the whole organism; and (c) the
biochemical-molecular.
(a) The hepatocytes in the nodules are organized differently
than are the hepatocytes in the mature normal and surrounding
liver. In contrast to the hepatocytes in the latter which are
arranged predominantly as single cell plates, those in the nod
ules are in the form of double cell plates and acini (60).
However, this difference in organization and architecture is not
an expression of abnormality, since the hepatocytes in the
nodules undergo spontaneously a radical differentiation with
restructuring and remodelling (61, 62). This restructuring,
clearly genetically programmed, involves the cell to cell orga
nization and the blood supply as well as the biochemical pattern.
This phenomenon is seen in several models of hepatocarcinogenesis. In every respect, this is truly a process of differentia
tion, analogous to other examples of differentiation in normal
development.
(b) Rats with hepatocyte nodules are unusually resistant to a
lethal dose of a potent hepatotoxic agent, carbon tetrachloride
(63). Rats with nodules show complete resistance to a dose of
CC14 that is lethal for 100% of normal rats.
This demonstration of survival value indicates that the ge
netic information necessary to develop hepatocyte nodules and
to have them remodel by differentiation could have been favored
during the long period of evolution.
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
Another aspect, possibly related to survival, is the resistance
of the hepatocytes in nodules to the cytotoxic effects of some
hepatotoxic xenobiotics in vitro and in vivo (see Refs. 35 and
64).
(c) At the biochemical-molecular level, the biochemical pat
tern can readily account for the resistance of the nodules to
cytotoxic effects of xenobiotics and for the more efficient ex
cretion of at least one carcinogen, 2-acetylaminofluorene (65).
A marked decrease in activation associated with the large
decrement in phase I components, coupled with the more
efficient conjugation and excretion of active moieties generated,
associated with the several conjugating systems in phase II,
match quite well (65).
The virtual certainty that this pattern is physiologically pro
grammed is indicated by its manifold nature with at least 25 to
30 different enzymes and other components and by the obser
vations that some agents such as lead nitrate, an interferon,
butylated hydroxyanisole, and butylated hydroxytoluene induce
a biochemical pattern in normal livers very similar to that seen
in the hepatocyte nodules but in a reversible or transient manner
(36). This special biochemical pattern is not seen in fetal,
neonatal, or regenerating liver but seems to be uniquely asso
ciated with the hepatocyte nodule induced by one of many
different carcinogens.
These results suggest that hepatocyte nodules represent a new
state of differentiation, better adapted to survive in a hostile
environment. The resistance, coupled with the capacity of the
majority of nodules to undergo spontaneous differentiation to
normal-appearing mature liver, indicate that the clonal expan
sion seen in the early phase of carcinogenesis is genetically
programmed and is part of a normal physiological pattern of
adaptation to some types of xenobiotics (9, 35, 36).
Is Clonal Adaptation Also Probable in Other Carcinogenic
Systems? Superficially, there are several similarities in the
overall patterns among many experimental and human systems
for cancer development (9, 37). These include: (a) the very long
so-called "incubation period" during which the cellular evolu
tion of malignancy from nonmalignant precursor lesions is
occurring; (b) the apparent reversibility of many putative preneoplastic or precancerous lesions; (c) the common biological
behavior and appearance of early focal proliferations such as
polyps, papillomas, and nodules with many different carcino
gens and promoting environments. Interestingly, the colon
system shows a very large number of changes in the mRNA
patterns between normal mucosa, polyps, and multiple polyposis (66). This is quite consistent with the suggestion that
polyps may represent a different state of differentiation. It
would seem prudent, therefore, to explore some of these systems
from the point of view of clonal adaptation. In our opinion, it
is quite likely that this type of adaptation, as well as perhaps
others, might well be important in cancer development in
several systems.
The most telling is in the case of malignant melanoma in
humans. Clark et al. (67) have shown that the genesis and
behavior of nevi and their role in the development of melanoma
have many similarities to the hepatocyte nodules in rat liver
carcinogenesis. The nevus shows at least two options, differ
entiation to nerve endings as a major pathway and persistence
with further cellular evolution to cancer as a minor one.
Whether the resemblances between rat hepatocyte nodules and
human nevi extend to biochemical-molecular mechanisms re
mains a fascinating problem for study.
Relevance of Clonal Adaptation to Carcinogenesis and Cancer.
Tumor promotion, despite its frequent association with an
element of mystique, is basically clonal expansion of initiated
cells. In the liver, this leads to the genesis of hepatocyte nodules.
The concept of clonal adaptation places tumor promotion in
the liver in quite a different perspective. This part of the
carcinogenic process is only in part and perhaps only periph
erally related to the ultimate development of cancer but becomes
an important way in which the living organism responds in an
adaptive fashion to commonly occurring xenobiotics in the
environment. On this basis, scientifically, the neoplastic deriv
ative, the cancer, is perhaps only incidental while the adaptive
component is the most important from the perspective of
biology. In this regard, the "turning on" of a new phenotype in
a rare cell during initiation becomes the key to what is seen in
promotion.
How a mutagenic carcinogen induces a new common con
stellation of biochemical components that go to make up the
resistance phenotype is not understood. Although the induction
of mutations in a rare cell by mutagenic carcinogens or metab
olites is well known, it remains to be established that this is the
molecular mechanism for the induction of the resistant hepa
tocyte. Altered regulatory genes, rare gene rearrangements,
gene translocations, or gene amplifications are some possibili
ties. Hypomethylation of some genes, the protein products of
which show considerable elevation in the hepatocyte nodules,
has been found (68-72). Altered metabolic patterns [see Fox
and Radicic (73) and Dean and Hinshelwood (5)] coupled with
"progressive state selection" (see below) offer possible epigenetic alternatives to the mutation hypothesis.
It would appear that the study of the control of transcription
of several of the discrete components of the resistance phenotypes, such as glutathione 5-transferase 7-7 (P), 7-glutamyltranspeptidase, D-T diaphorase (quinone reducÃ-ase), and dis
crete relevant cytochromes P-450, to name but a few, could in
turn suggest possible underlying mechanisms for clonal adap
tation including altered methylation.
An interesting aspect of clonal adaptation of great practical
importance relates to the resistance of many cancers to chemo
therapy (74). It is often implied that resistance to chemotherapy
might be a property acquired during the schedule of treatment
as a mutation or some other genomic alteration including gene
amplification. The experience in the liver and the existence of
clonal adaptation suggest the possibility that resistance may be
a primary property of the cancer in some instances. Given the
derivation of cancer by cellular evolution from a nodule with a
resistance phenotype that was used mainly for clonal expansion,
it is certainly conceivable that many cancers in some tissues
may begin with a resistance phenotype. The presence in cancer
precursor cell populations of P-glycoprotein, as the product of
mdr gene and other components of resistance to xenobiotics
including chemotherapeutic agents (52, 57-59), would suggest
that some cancers may begin with a resistance phenotype and
may have to lose this phenotype as a prerequisite for a positive
cytotoxic response to some chemotherapeutic agents.
In view of the widespread occurrence of many potential
carcinogens in the environment, it remains important to under
stand how evolution has enabled organisms to adjust or to
adapt to this ever-present hazard. The long life span of many
species in the face of this hazard attests to the efficiency with
which evolution has succeeded. The long precancerous period,
lasting as long as one-third to one-half or more of the life span,
has largely relegated neoplasia to the postreproductive period.
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
The acquisition of a mechanism to adapt, such as by clonal
adaptation, would seem to be an important way in which this
has been accomplished. If this viewpoint is valid, one would
have to consider neoplasia as mainly a manifestation of the
imperfection of the adaptive process and cancer as a deviant of
adaptation (75). Such a viewpoint might open several new
avenues for the development of novel ways to interrupt the
carcinogenesis process and thereby prevent cancer.
Finally, the presence of a major adaptive component during
carcinogenesis would offer an interesting new perspective to the
dominant current paradigm of carcinogenesis, the progressive
acquisition of abnormal genes and gene products without any
regard to the adaptive ability and talent of biological systems.
Our current view would envision the appearance of new phys
iological phenotypes during initiation either by mutation or by
other mechanisms including epigenetic, and this phenotype
would play a central role in the long adaptive process during
carcinogenesis. Once a malignant change supervenes as a rare
derivative relatively late in the development of cancer, a se
quence of altered genes could then begin which might be a
reasonable basis for some of the late changes seen in the further
evolution of cancers. "Progressive state selection" (see below)
could play an important role in this progression as well as in
its modulation. A formulation more consistent with modern
dynamic biology, such as here proposed, not only seems attrac
tive theoretically but also offers many possible practical ap
proaches to the ultimate prevention and treatment of cancer.
Neoplastic Transformation of Cells in Culture
Initiation or the First Stage of Carcinogenesis in Culture. The
concepts of initiation and promotion of neoplastic transfor
mation and the associated two stage model of carcinogenesis
were developed in the early 1940s from studies of chemically
induced tumors in the skin of rabbits and mice (76, 77). In the
mouse, the first, or initiating, stage was induced by a single
application of carcinogens such as coal tars or purified polycyclic hydrocarbons and persisted indefinitely thereafter in the
treated area, which generally retained its normal appearance.
Repeated application to that area of a promoting agent such as
croton oil or TPA,2 which by themselves are not carcinogenic,
resulted in the second stage defined by the appearance of
tumors. Largely because of the speed of induction of the initi
ated stage, its persistence after removal of the carcinogen, and
the focal origin of the ensuing tumor, the first stage was
generally considered to be mutational in origin (78, 79). The
requirement for repeated application of promoting agents and
the slowness of onset of the second stage led to the belief that
it was nonmutational, or epigenetic, in origin. In fact, however,
whole animal systems were too complex and cumbersome to
provide definitive evidence of these widely accepted explana
tions for the two stages. To add to the complications, it has
been pointed out that the two-stage model refers only to the
number of manipulations used and that many more than two
steps occur in the two stage system in animals (49).
The availability of cell culture systems that could be induced
to undergo neoplastic transformation by exposure to carcino
gens offered a much simpler, easily manipulated, and readily
quantified means for settling the question of the nature of twostage carcinogenesis. Primary cultures of Syrian hamster em2The abbreviations used are: TPA, IJ-O-tetradecanoylphorbol-IJ-acelate:
DMBA. 7.12-dimethylbenz[a)anthracene.
bryos at cloning densities were the first to be used successfully
in studies of in vitro carcinogenesis by chemicals (80). Up to
25% of the clones of these cells underwent morphological
transformation during a 9-day treatment with carcinogenic
polycyclic hydrocarbons, and some of the transformed clones
went on to produce sarcomas when transplanted back into
Syrian hamsters. The high percentage of clones which became
morphologically transformed indicated to the authors that the
transformation was not the result of "the usual type of randomly
occurring mutation" (80). However, since the scoring of trans
formation was subjective, and only a single stage could be
discerned, the mechanism(s) of the two stage models could not
be resolved in this system.
In 1970, Mondai and Heidelberger (81) carried out a classic
series of experiments on chemical carcinogenesis in a line of
mouse prostate cells. They found that every cell exposed to
methylcholanthrene gave rise upon cloning to a population in
which a small minority of the cells initiated transformed foci.
By contrast, only 6% of control cells gave rise to clonal popu
lations which had some transformed foci. The fact that 100%
of carcinogen-treated cells produced progeny with an increased
probability for transformation argued strongly against a muta
tional origin for the transformation.
The work in culture was continued with the C3H10T'/2 line
of mouse embryo cells, hereafter referred to as 10T'/2 cells. This
line was widely used by many investigators to study carcinogen
esis induced by a variety of chemical carcinogens (82) and by
X-irradiation (83). A finding reported by all who used the
system was that the frequency of transformation (the number
of transformed foci divided by the number of cells initially
seeded) was inversely proportional to the number of cells seeded
(82-88). One particular version of this type of experiment was
to administer 400 to 600 rads of X-irradiation to a low density
of 10T'/2 cells, allow them to grow to confluency, transfer them
on many dishes over a wide range of concentrations, and score
foci 4 weeks after each cell concentration reached confluency
(83). A consistent finding was that the number of foci per dish
(usually less than one per culture) was independent of the
number of cells seeded in the transfer. It was concluded that
most, if not all of the cells originally exposed to the X-rays had
been altered by the treatment in such a manner as to transmit
to their progeny an increased probability for transformation. A
similar conclusion was reached for chemically induced trans
formation of the 10T'/2 cells (87) and of retrovirus-infected rat
cells (89).
The production of heritable change in an entire population
is a radical departure from the quantitative expectation of
specific genetic mutation, which usually occurs at a rate of 10~6/
cell division or less. Furthermore, this finding of high frequency,
heritable increase in focus-forming capacity initiated by X-rays
is consistent with other reports that most or all of the cells
which survive X-irradiation but receive sublethal hits have
heritable changes in growth rate (90, 91) and sensitivity to
mutagenic treatment (92). A correlated finding is that the total
size of the transformation targets of ionizing radiation at high
linear energy transfer is as much as 1000 times higher than that
of the targets for specific gene mutations, which are themselves
larger than that for a single gene (93). Large as it is, the target
size based on transformation may be an underestimate for the
initiation process, since only a small minority of initiated cells
undergo transformation (94).
Several types of experiments with tumor initiators in animals
showed that enduring changes occur in a high proportion of
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
carcinogen-treated normal cells just as they do in cell lines. In
one case, the skin of mice was treated with the carcinogen
DMBA and 5 weeks later almost all the basal cells in the
DMBA-treated area responded to croton oil by a faster onset
of DNA synthesis than did the basal cells of mice which had
not been treated with the carcinogen (95). Since epidermis is a
renewing tissue with continuous turnover of cells, the persistent
DMBA-induced responsiveness indicated a heritable increase
in the reactivity to promoting agents of all the basal cells in the
area. In another case, DMBA was repeatedly applied to hamster
cheek pouches, resulting in the appearance of localized carci
nomas (96). There was a large increase in proteolytic activity
in the carcinomas and in the surrounding, normal-appearing
tissues, indicating that a high frequency change had occurred
over the entire exposed area. Several studies on tumor induction
in normal diploid cells show that heritable change occurs in a
high proportion of them after a single dose of X-rays. Xirradiation of thyroid cells freshly removed from the rat con
verts 1 of 32 of the clonogenic cells into tumorigenic cells (97).
A similar result was obtained with mammary epithelium re
moved from rats that had been treated 24 h earlier with a
chemical carcinogen (98). At least 1 in 250 of the cells initiated
a mammary carcinoma when dispersed and grafted into the fat
pads of isologous recipients. Taken together, these findings
clearly show that carcinogenic agents produce heritable changes
in a much higher proportion of treated cells than would be
expected of conventional mutations. The implication is either
that damage to any one of a very large number of genes can
produce the neoplastic transformation or that the primary
targets are not genetic. An important finding favoring the latter
interpretation is that X-ray-initiated 10T'/2 cells are irreversibly
reverted to their original uninitiated condition by treatment
with protease inhibitors begun as late as 10 days, or 13 cell
divisions after irradiation (99). There is certainly no reason to
expect that protease inhibitors would revert genetic mutations
in a whole population of cells, particularly at so long a time
and so many divisions after X-irradiation.
Transformation or the Second Stage of Carcinogenesis in Cul
ture. The promotion stage is defined as that which leads to
tumor formation. Transformation is the cell culture analogue
of tumor formation and can be considered the second stage of
carcinogenesis in vitro (94). Although all the X-irradiated 1OT'/2
cells appear to be initiated, only a small fraction of them (about
10~6) actually produce transformed foci (94). It was concluded
that the second step in transformation is a very rare event.
Fluctuation analysis of clone size distribution of transformed
cells agreed with the hypothesis that the second event is a
spontaneous mutation, with a constant small probability of
occurring every time a cell divides (94). This conclusion was at
odds with an earlier finding that no cells capable of producing
foci on transfer appeared in carcinogen-treated lOT'/z cultures
until about 3 weeks after they had become confluent and net
growth had long since stopped (84). It also seems inconsistent
with the transforming effect of the tumor promoter, TPA, on
10T'/2 cells exposed to 100 rads of X-rays, which is insufficient
radiation to significantly increase the frequency of the rare
focus found among control cultures (100). The addition of TPA
to the lightly X-rayed cells increased focus production to the
extent that almost every culture arising from a single cell had
1 or more foci. The effect of TPA was therefore similar to
increasing the dose of X-rays to 400-600 rads, in that it
increased the focus-forming capacity of almost every cell. On
this basis, the promoting or second stage in these cells, like the
initiating or first stage, could be considered a high frequency
process, even though its visible manifestation occurs with low
probability.
The question of the nature of the second or transforming
step has since been taken up in the NIH 3T3 cell system, which
can be considered an initiated line, since foci of transformed
cells develop if the cultures are left in the confluent state for 10
days or longer in a given range of serum concentrations (101103). This capacity for focus formation is highly dependent on
the passage history of the cells. If they are passaged frequently
at low density in high serum concentration, their capacity for
transformation steadily diminishes to the point that no trans
formed cells arise, even after several successive postconfluent
incubations.' Passage of the original cells at low density but in
low serum concentration gives rise to focus-forming cells, just
as prolonged incubation at confluency does. Both conditions
which evoke the transformation constrain the growth and over
all metabolism of cells, and it was surmised that the transfor
mation represents an adaptive response to that constraint.
However, growth constraint imposed by the very different
method of limiting the availability of glutamine actually inhibits
transformation instead of evoking it (104). The observation that
transformation occurs only under certain specified physiologi
cal conditions shows that it is not a spontaneous event in the
sense that genetic mutations are. The fact that it was favored
by physiological conditions which constrain rather than en
courage multiplication was further evidence against a mutational origin of the transformation.
Although only a small fraction (usually less than 1%) of the
NIH 3T3 cells became focus formers when exposed to growthconstraining conditions, the entire population actually under
went a heritable increase in growth capacity. This was expressed
in a substantial increase in saturation density of the entire cell
population after 3 short passages in low serum concentration
and even greater increases in further passages (102). Clonal
analysis of a similarly treated population revealed that all the
cells had acquired an increased capacity for focus formation,
although less than 1% of the cells seeded from the uncloned
parental population initiated foci in a direct assay (102, 105).
There was an extremely wide range of capacities to produce
foci among the clones, and some of them drifted up or down in
that capacity as they were repeatedly passaged and assayed for
focus formation (102). It is apparent that the population does
not fall into two simple classes of transformed and nontransformed cells; rather it consists of a mosaic of cells with a very
large range of capacities for transformation in confluent cul
tures. There may be an almost continuous spectrum of poten
tialities or probabilities for transformation among cells when
the appropriate conditions are applied. In addition, the expres
sion of the transformed state in the development of foci depends
on the initial state of the cells, a variety of factors in the assay,
and the differential in growth rates among the cells at
confluency.
These considerations indicate that there is an optimal degree
of constraint for eliciting the adaptive growth response that
characterizes transformation. If the serum concentration in
confluent cultures is too low or too high or if cells are seeded
at such high concentrations that they become crowded as soon
as they attach and spread on the dish, the number of foci per
seeded cell is reduced. It is important to take these factors into
account when comparing the results obtained in cell culture
with those obtained in the animal. In cell culture, sparse cell
3 H. Rubin, unpublished observations.
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
populations in high serum concentrations are growing and
metabolizing at a maximal rate and the adaptive response
associated with transformation is brought on when constraints
associated with confluency or low serum concentrations are
introduced. In the animal, most cell types are normally under
the constraint introduced by the 3 dimensional contiguity of
the organized tissue. For example, very few cells of the intact
liver are synthesizing DNA at any moment, and less than 0.01 %
are in mitosis (106). If the liver epithelial cells are dispersed
and seeded at low density in culture, 80% of them enter DNA
synthesis and mitosis within 48 h (107). Therefore, in the
animal, the optimal level of growth and metabolism for neoplastic transformation of initiated cells may come only when
they are stimulated to multiply. This appears to be the case in
liver carcinogenesis, where nodule formation by carcinogentreated cells is brought on by compensatory growth (49). By
contrast, transformation of rat liver cells in culture is enhanced
by keeping the liver cells in the confluent, relatively inhibited
state for 3 weeks at each month passage, instead of serially
passaging them once a week just as they reach confluence (50).
Thus, growth stimulation in vivo and growth inhibition in vitro
may converge on the optimal condition for transformation.
Just as reversal was demonstrated for the initiated state, so
has it been shown for the transformed state. It was first reported
that cultivation of transformed Syrian hamster cells at low
density reversed several of their in vitro growth properties and
reduced their capacity for tumor formation (108). Then it was
found that the same type of low density growth fully reversed
the morphological and tumor-producing properties of a clone
of 10T'/2 cells that had been transformed by X-irradiation (109).
Similar treatment of spontaneously transformed NIH 3T3 cells
resulted in a loss of their focus-forming capacity and of their
ability to form tumors in nude mice (102, 110). In both the
10T'/2 and NIH 3T3 cells, the transformed phenotype was
perpetuated by maintaining the cells at high density. The re
versibility of the transformed state of a whole culture considered
alongside its induction under conditions of constraint, and the
graded response of the entire population leave little room for
doubt that the transforming step, like that of initiation, is in its
inception a nongenetic process.
Adaptive Origin of Spontaneous Transformation. The diverse
evidence for the adaptive origin of "spontaneous" transforma
tion of NIH 3T3 cells can be summarized as follows:
1. No cells are capable of producing dense foci under standard
conditions if the cells had been repeatedly transferred at low
density in high serum concentrations. Focus-forming cells can
be detected, however, by reassaying cells from the standard
assay within a few days after the culture becomes confluent and
net increase in cell number has ceased. Cells capable of forming
foci in the standard assay also arise even if cultures are passaged
at low density, if the serum concentration in the passages is
maintained at a low level. Unlike mutations, therefore, "spon
taneous" transformation is not really spontaneous but occurs
only when the appropriate inducing conditions obtain.
2. Although the capacity to form foci arises in only a fraction
(less than 1%) of the population repeatedly passaged at low
density in low serum, the bulk of the population gains the
capacity to multiply to higher saturation densities. Each cell
also gives rise to a clone with an increased probability to form
foci in the standard assay. The involvement of the bulk of the
population in these changes in growth capacity is inconsistent
with a mutational origin.
3. There are many degrees of adaptation to multiply in low
serum concentrations or at high cell densities. We cannot, in
fact, exclude the possibility that the variation in these cellular
capacities is continuous and therefore distinctive from the dis
crete nature of mutations.
4. The capacities for focus formation and tumor development
are reversible for the entire population of transformed cells.
Although adaptation criteria 1 and 4 have been demonstrated
in cells other than the NIH 3T3 line, it is only in this cell that
all 4 criteria have been demonstrated.
Speculative Mechanism of Carcinogenesis. Given the evidence
that the growth properties of a high proportion of cells treated
with carcinogens are heritably altered, the question arises of
how such pervasive change in whole populations might be
elicited. First, however, it must be noted that heritability among
somatic cells of the capacity for tumor formation differs in
several respects from that associated with transmission of spe
cific gene mutations in organisms. Not only does transforma
tion in culture occur with high frequency in response to carcin
ogens but the phenotype is heterogeneous, involves the gradual
loss of many of the differentiated properties of the treated cells,
and can be prevented in the 10T'/2 line by protease inhibitors,
which have no effect on mutation frequency in mutagen-treated
cells (111). It appears, therefore, that proteases play an essential
role in the initiation of transformation in lOT'/z cells.
Protease activity in yeast cells responds to a change in sub
strates. Yeast cells multiply exponentially until they exhaust
their main substrate, glucose, and remain at a constant number
until they start utilizing another substrate (112). The second
phase of growth is much slower than the first, and it is accom
panied by a 300-fold increase in protease activity. An inactivat
ing mutation in a key protease results in a failure of other
proteolytic and hydrolytic enzymes to be processed to an active
form. If a mutant cell is exposed to wild type cytoplasm through
mating or heterokaryon formation, the mutant segregants can
maintain indefinitely the wild type content of active proteases
and hydrolases normally dependent on the now inactive pro
tease (112). The yeast system demonstrates clearly that an
altered phenotype can be transmitted to progeny by epigenetic
means without the involvement of cytoplasmic genes (113). The
demonstration of the epigenetic persistence of wild type pro
tease content may have particular significance for neoplastic
transformation since: (a) the initiated state in lOT'/z cells can
be reversed by protease inhibitors (99, 111); (b) there is a large
increase in protease content in carcinomas and their surround
ing normal appearing tissue of the carcinogen-treated hamster
cheek pouch (96); (c) the addition of proteases to densityinhibited chick embryo cells stimulates them to multiply (114);
(d) a serine protease that is assayed as an activator of plasminogen is elevated in primary tumor cells, in cells transformed by
viruses, and in a number of tumorigenic cell lines (115); (e) the
induction by TPA of secretion of the protease which activates
plasminogen has been used to order histologically distinct
classes of human colonie adenomas from the most benign to
the most advanced premalignant state (116). It seems plausible,
therefore, that persistent perturbation of the concentration or
localization in cells of proteases and other hydrolytic enzymes
might result in a transmissible defect of growth regulation. It
is not unlikely that such perturbation would increase the genetic
instability of the cells, introducing genetic changes which might
contribute to tumor progression.
It remains a question what the target for carcinogens might
be that could initiate and propagate such epigenetic changes in
cells. One possibility is carcinogen-induced damage to the mem-
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
branes of lysosomes which contain most of the cellular hydrolases. It is noteworthy that UV light, which is a common skin
carcinogen, can in very small doses cause hemolysis of human
erythrocytes which are virtually without DNA or RNA (117).
The hemolysis, which represents the leakage of hemoglobin
from the erythrocyte, is apparently the result of a direct effect
of U V light on its membrane, and it has been conjectured that
the mechanism underlying the changes in skin during carcinogenesis by UV light is closely comparable to that which in
creases the permeability of the erythrocyte (117). Mitochondria
of yeast cells are also disrupted by UV light (118). This effect
is of special interest in cancer where the most commonly
observed biochemical change is increased glycolysis, which was
considered a regulatory response to defective respiration (119).
A relation between damaged mitochondrial membranes and
defective respiration became quite plausible with the advent of
the chemiosmotic model of oxidative phosphorylation and its
dependence on transmembrane gradients of electrons and pro
tons (120). Nongenetic changes in surface structures can be
perpetuated for hundreds of cell generations, as has been dem
onstrated by direct visualization in Paramecium (121). Other
methods must be developed to demonstrate long term mem
brane effects in metazoan cells; thus the proposal is as yet
hypothetical. It is an interesting fact, however, that the carcin
ogenic polycyclic aromatic hydrocarbons are highly lipophilic
and probably localize to a large extent in membranes (122).
Although speculative, the suggestion of carcinogenesis through
membrane damage would appear to be testable by studies of
the effects of carcinogens on, for example, membrane traffick
ing, cell permeability, erythrocyte hemolysis, protease distri
bution, and mitochondrial function. It is well to keep in mind,
however, that the definitive evidence in cell culture against a
mutational origin of both the initiation and promotion stages
came from quantitative experiments on the growth behavior of
living cells. Much remains to be done at that level to obtain a
fuller understanding of neoplastic transformation.
General Considerations. As noted earlier, the two stage model
of cancer refers to the manipulations used in experimental
carcinogenesis, but many more than two steps are involved (49).
Foulds (123) believed that "initiation establishes an expanse or
region of incipient neoplasia that is coextensive with the area
of exposure to carcinogenic action and has a permanent, replicable new capacity for neoplastic development which can be
augmented by repetition of the carcinogenic stimulus." In this
view, the initiated cells, as observed in the classical skin carci
nogenesis model (17, 78), are not dormant tumor cells but are
cells with an increased reactivity to further applications of
carcinogens or of promoters. The changes effected by initiation
are not restricted to the places where tumors emerge at a later
time. Foulds' view coincides with the evidence presented here
that the carcinogenic stimulus affects the entire population of
exposed cells, although only a small fraction progress to the
overtly transformed state. If the initiation step is seen as en
dowing cells with an increased reactivity, the promotional treat
ment may differ only in degree rather than in kind; i.e., pro
moting agents may be effective only on cells previously sensi
tized by a more potent, initiating agent. That this is the case is
suggested by the previously mentioned observation that mouse
epidermis initiated by treatment with DMBA enters DNA
synthesis more rapidly when treated with croton oil than does
noninitiated epidermis (95). Similarly, TPA fails to induce the
secretion of plasminogen activator in primary cultures of nor
mal human colon but does so in cultures of adenoma cells with
increasing efficiency depending on their progression to malig
nancy (116). It has also been reported that tumor promoters
enhance the proliferation of preneoplastic rat liver cells to a
significantly greater extent than normal unaltered liver cells
(124). Finally, there is the finding discussed earlier that TPA
has the same transforming effect on 10T'/2 cells that have
received 100 rads of X-rays as does raising the X-ray dose to
400-600 rads (100). Thus, promoting and initiating agents may
ultimately produce the same result in cells, although the path
way through which they act may differ. That only a rare cell in
an initiated field is the actual antecedent of the tumor would
follow from the intrinsic heterogeneity of growth capacities in
the cell population (102, 103, 125, 126).
An interpretation similar to that developed here was pro
posed by Blum to account for epidermal cancer induced in mice
by UV light (117, 127). Results from a large series of carefully
quantitated experiments indicated that successive doses of UV
light progressively accelerate the proliferation of the epidermal
cells. Blum concluded that the results could not be explained
by separable induction and growth phases or by somatic muta
tion. Rather, the tumor cells could be considered as differing
from normal cells only in the quantitative sense of proliferating
more rapidly in the regulated context of the epidermis with
each successive exposure to UV light. Although UV light un
doubtedly damages cells, the major response of the epidermis
is an increase in the rate of mitosis and a consequent increase
in thickness of the skin (117). If the radiation is not repeated,
the epidermis returns to its normal thickness. The increased
mitosis can be viewed as an overshoot of the adaptive response
to cell damage. If the radiation is repeated frequently, the skin
remains thickened and carcinomas and sarcomas appear within
a few months. Blum designated the cellular response to the
repeated UV irradiation "progressive acceleration of growth."
He indicated that all the cells exposed to the treatment were
incrementally altered but noted that very slight differences in
their accelerated growth rates would, over a period of months,
result in the progeny of one cell outgrowing the others to form
a tumor. In fact, however, there were signs of the beginning of
several tumors on as small an irradiated expanse as the mouse
ear, and sometimes carcinomas and sarcoma tissue were found
in the same tumor mass (117).
Progressive acceleration of growth can account for the pro
gressive increase in number and thickness of transformed foci
of NIH 3T3 cells repeatedly passaged at high density (110) or
in stepwise decreases in serum concentration (105). To this
should be added the selective effect of maintaining cells under
conditions of moderate growth constraint. It has been proposed
that such constraint selects not only for individual cells with
increased growth capacity but also for metabolic states within
cells that are subject to continuous phenotypic fluctuation in
growth capacity (102, 105, 110, 126). Successive repetitions of
this process would lead to relatively stable increases in growth
capacity under conditions of moderate constraint, and the proc
ess was called "progressive state selection" (102). Those con
straints, which are known to lower metabolic rates as well as
growth, could lead to an increase in protease, as occurs in yeast
with the transition from exponential growth to the stationary
phase, and with the switch from rich to minimal medium (128).
Such changes in previously sensitized, i.e., initiated, cells would
lead to increased growth capacity and heterogeneity, just as
exposure to a carcinogen or promoter would, thereby resulting
in repeated selection of cells with the greatest growth potential
and ultimately in neoplasia. The combined effects could be
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CELLULAR ADAPTATION AND THE ORIGIN OF CANCER
characterized as: destabilization + state selection —»
progressive
growth acceleration.
The adaptive nature of the response is illustrated in the
protective value of the nodules from a liver initiation-promotion
regimen against the lethal effect of xenobiotics in the rat (63)
and in the protection against sunlight provided by the thicken
ing of epidermis after UV irradiation (117). A generalized
model for adaptation can be inferred in the continued multipli
cation and function of focus-producing cells in confluent cul
tures where the rest of the cells become strongly inhibited, and
many die. Unfortunately, the adaptive response reveals its less
beneficent aspect in neoplasia when the stress becomes too
severe or prolonged. As one prophetic investigator put it some
15 years ago, "oncogenesis may be adaptive ontogenesis" (129).
Acknowledgments
We greatly appreciate the manuscript preparation by Lori Cutler and
Dawn Davidson.
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