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Clearly, if one assumes that promotion involves mitogenesis, then there are multiple ways to cause cells to divide.  One can kill cells with both genotoxic and non-genotoxic agents. Stem cells remaining in tissues which have been injured via surgery, burns, viral lysis will be stimulated to go into regenerative hyperplasia.  In addition, normal growth factors and hormones needed for growth and development can trigger mitogenesis in the targeted tissues.  Lastly, there are many chemicals, which are not mutagenic, that act as mitogens (at least for initiated cells) at not cytotoxic levels (Trosko and Chang, 1988a).

In solid tissues, cells are normally contact-inhibited; therefore there is very little cell division.  Promotion must involve a mechanism which overcomes contact inhibition.  It has been hypothesized that the inhibition of gap junctional intercellular communication might be the unifying cellular mechanism for any and all kinds of tumor promotion.  The function of tumor promotion would appear to increase the target size of the cells containing the first genetic hit, assuming that a cell must accrue at least two, possibly more hits (Potter, 1981a; Trosko and Chang, 1980; Trosko and Chang, 1985).  If we assume that the probability of activating an oncogene and losing a tumor suppressor gene in a single cell is the product of two rare independent events, then the probability would be the product of those rare probabilities.  If a cell with one hit is clonally amplified, then the longer the promotion period, the greater the number of such cells.  Now the probability of a second hit would involve the multiplication of the product of the independent probabilities and the number of cells.  This has led to the prediction of the “I-P-I”model of carcinogenesis (Potter, 1981a; Potter, 1984).  Experimental evidence seems to be consistent with the hypothesis (Hennings et al., 1983; Reddy and Fialkow, 1987; Scherer et al., 1984). Continue Reading »

If all the preceding concepts have some relevance to the understanding of the carcinogenic process, they must somehow be integrated since not one of them can completely explain this complex biological process.  In addition, if one ever hopes to develop a biologically-based risk assessment model to predict or explain cancer risks to individuals or populations exposed to various endogenous and exogenous factors at different times in their lives, at different concentrations and combinations throughout their lives, then the mechanisms by which these genetic and environmental factors affect cell proliferation and differentiation must be understood.   The concept of intercellular communication might be speculated to provide the integrating link between the multi-stage nature of cancer, the stem cell theory of cancer, the “nature and nurture” theory of cancer, and the oncogene and tumor suppressor gene theory (Trosko et al., 1984; Trosko et al., 1987; Trosko and Chang, 1988b; Trosko et al., 1989; Trosko et al., 1990b; Trosko et al., in press).

The ultimate understanding of how a multicellular organism, such as the buman being, maintains its ability to regulate cell growth, proper differentiation, adaptive responses and wound-healing will necessitate adhering to the principle that an organism is “greater than the sum of its parts” (Eagle, 1965).  This holistic concept, together with the hierarchical (Brody, 1973) and cybernetic (Potter, 1974) concepts, has been utilized to explain how the regulation of ions and molecules is achieved at the molecular/biochemical levels, at the cellular level and at the organ and system levels.  The idea that there exists a cybernetic-like control system in multicellular organisms to regulate cell functions within and between tissues was created by Claude Bernard (1878). W.B. Cannon coined the term, homeostasis, to describe this regulatory process (1929).  In addition, Weiss and Kavanan (1957) postulated the existence of growth regulators of cell proliferation, while Mazia (1961) suggested that “removal of a block” was necessary for cell division, Later, Potter (1983) integrated both the positive and negative factors into a scheme whereby, locally and systematically, they could regulate growth.

The explanation of how a constant “milieu interieur” is maintained was provided by the conceptualization of positive and negative factors, produced by cells of one type/tissue which affects distal cells of another type/tissue.  It predated the discovery of most of the actual molecular entities involved in homeostasis.  Eventually, this concept of homeostasis by a cybernetic process was fleshed out with the discovery of various hormones, biologically active peptides, lymphokines and neurotransmitters.  Note, that oncogenes code for these kinds of molecules (and their receptors).  The bilolgical process by which cells influence control over each other in the multicellular organism is referred to as intercellular communication.  In general there are three forms of intercellular communication; extracellular communication would refer to how cells communicate with distal cells via the secretion of a molecular signals (e.g., hormone, peptide, growth regulator, neurotransmitter); intracellular communication would refer to those transmembrane-signalling elements triggered by the extracellular signals; and Intercellular communication would refer to the transfer of ions and small regulatory molecules from contiguous cells through a protein channel on the cell membrane, called the gap junction (Loewenstein, 1990).

This interconnected communication network allows a multicellular organism to control its fundamental cellular, tissue, organ and system functions.  For example, a hormone secreted by the hypothalamus (extracellular) could trigger intracellular biochemical changes in distal cells of another organ, which in turn, could either increase or decrease intercellular communication in that tissue which has a receptor for that hormone.  By increasing intercellular communication, these hormone triggered cells might now differentiate or if already differentiated, produce a substance unique to that hormone signal.  Alternatively, if the biochemical changes occurring in the targeted cells cause a decrease in gap junction-mediated intercellular communication, then the cells have an opportunity to divide. Continue Reading »

By phrasing the question in this fashion, one can see that the paradigm to understand the cancer problem is fundamentally wrong.  Our current understanding of genetics shapes the concept that DNA, the genetic information, must interact with environmental signals in order to express that information.  In other words, the phenotype of an organism is the result of the genotype interacting with the environment, e.g. nature and nurture, rather than nature versus nurture.  There is no question that genes are involved in carcinogenesis, since it has long been known that there were many genetic syndromes associated with predispositions to cancer, i.e., Down syndrome, xeroderma pigmentosum, Fanconi’s anemia, etc.  On the other hand, physical and chemical agents, such as ultraviolet light and cigarette tar condensates, were known to induce cancers in animals.  Xeroderma pigmentosum, a skin cancer predisposed syndrome, lacks, genetically, enzymes in the cells which are needed to repair damage done in the DNA of ultraviolet light-exposed skin cells.  Cells from normal individuals can repair ultraviolet induced DNA damage under normal conditions, preventing is damage from causing alterations, i.e., mutations, in the newly replicated DNA.  Without these repair enzymes, these ultraviolet induced DNA lesions can cause mutations (Glover et al., 1979; Maher and McCormick, 1976).

In addition, new evidence has identified specific genes, which if mutated, over expressed, abnormally expressed or lost, can influence the carcinogenic process.  These genes, the proto-oncogenes and tumor suppressor genes play critical roles in normal cells by regulating cell proliferation or differentiation (Sager, 1986; Weinberg, 1985).  When altered, these two fundamental processes, which are needed for the survival of all multicellular organisms, contribute to the general characteristics of all cancer cells, namely, these cannot control their growth, nor can they terminally differentiate under normal conditions.  Returning to the xeroderma example, by inheriting mutated genes at conception, the xeroderma individual cannot repair its DNA if exposed to sunlight.  If the cell are not exposed to sunlight, the DNA will not be damaged (ignore for the moment, damage done to these cells by UV-mimicking chemicals).  If the DNA is damaged, mutations will occur in many genes, including the proto-oncogenes [Nature and Nurture].  By inheriting a mutation in this case, more mutations can occur in the body after exposure to the environmental agent, sunlight.  Some of these mutations then can start the cancer process (Glover et al., 1979; Maher and McCormick, 1976).  That is, these mutations, if they occur in the proper oncogene in a stem cell, could prevent the terminal differentiation of the stem cell when it is stimulated to divide and differentiate.  If the amount of UV-induced DNA damage is severe in the terminally-differentiated cells, these cells would die since the XP cells do not repair their DNA very efficiently.  This cell death of the differentiated cell population would force the surviving initiated stem cell to proliferate, but not to terminally differentiate (Trosko, 1981).  This clonal expansion of the initiated cell would be the result of a cell death-type of promotion process.  As these initiated cells accumulate on the skin surface, additional exposure could induce another mutation in one of the initiated cells, thereby providing a second “hit” which might be needed to complete the carcinogenic process in this case.  In other words, xeroderma pigmentosum might represent an “initiation-promotion-progression” or “complete carcinogen” syndrome.

Skin cancer can, of course, be induced in non-xeroderma pigmentosum individuals.  When normal individuals get exposed to massive amounts of ultraviolet light, such that their DNA repair capacities are unable to repair all the damage, some of the damage would then act as substrates for mutations as in the case of xeroderma pigmentosum.  Either repeated exposures to large amounts of sunlight to induce cell killing to act as promoting and progression agents or exposure to non-cytotoxic mitogenic agents would ultimately bring about the skin cancer.

This xeroderma pigmentosum syndrome can further illustrate the “nature and nurture” model when one examines the internal tumors of these individuals.  While all the cells of these XP individuals are deficient in DNA repair, and while there have been reports of internal tumors found in these individuals (Kraemer, 1980), the numbers are small in comparison to the skin tumors.  Some would argue that is because they do not live long enough to develop internal tumors.  While that is probably true, it points out that the environmental exposure to chemical mutagens is far lower than exposure to the physical mutagen, sunlight.  Chemical mutagen exposure would probably never exceed levels which would induce massive cell killing as exposure to sunlight would do.  Therefore, internal initiated cells in the XP individual would have to be exposure to a high enough level, in a sustained, chronic manner, of noncytotoxic mitogenic stimuli to act as a promoter.

Recent molecular findings on the retinoblastoma, Wilms’ and Li-Frameni syndromes might suggest that these could represent “initiator-prone” syndromes (Malkin et al., 1990), if one assumes that the inheritance of one mutated gene through the germ line as the “initiating” event.  On the other hand, Downs and neurofibromatosis syndromes, which have not been sh;own to have defects in DNA repair (Trosko et al., 1985; Yotti et al., 1980), might be conceptualized as “promoter-prone” syndromes (Trosko et al., 1985).  In general, there are genes which could enhance or prevent environmental mutagen-induced DNA damage (e.g.,albinism or dark pigmented individuals, respectively) and genes which stimulate or reduce cell proliferation in certain tissues (e.g. genetic imbalanced growth regulators and hormones).

With the foregoing as a brief background, an examination of the carcinogenic process in both experimental animals and human beings must be made.  The evolution of the neoplastic cell appears to start from a single normal cell, one that obviously has division potential, such as a stem or progenitor cell, which, during the carcinogenic process, involves the acquisition or loss of important phenotypes (Abercrombie, 1979; Nicolson, 1984).  Cancer cells all seem to be characterized by the inability to terminally differentiate, loss of growth control or contact inhibition, the inability to perform gap junctional intercellular communication and to invade distal tissues (Chang et al., 1987).  Although the ultimate tumor tissue appears to be the result of the clonal expansion of a single transformed cell, great heterogeneity exists because of genetic and epigenetic changes occurring during that evolution (Nicolson, 1984).

The concepts of initiation, promotion and progression were derived from animal experiments to help explain phenomenologically distinct processes occurring during carcinogenesis (Bout well, 1974).  Operationally, on the whole animal level, initiation refers to the stable or apparent irreversible conversion of a normal stem or progenitor cell (see later discussion of the de-differentiation hypothesis) to a “premalignant” cell (Trosko et al., 1990b).  This premalignant or initiated cell has, by definition, several features; namely, it has the inability to terminally differentiate (Trosko et al., 1988); it has the potential to proliferate and maintain its inability to terminally differentiate; and lastly, it has the potential to acquire all the other genetic/epigenetic changes needed to become a neoplastic, invasive and meta-static cell.  Promotion, on the other hand, is that phase of the multi-stage process which enhances the frequency and earlier appearance of tumors in the initiated animal (Bout well, 1974).  Progression refers to the conversion of one of the promoted initiated cells to the neoplastic cell.

Although the mechanisms underlying each of these three phases are not vet known, some hypotheses to explain the apparent irreversible, initiation phase, the potentially interruptible or reversible promotion phase and the irreversible progression phase have been proposed.  Since mutagenesis is an irreversible event, and since known mutagens, such as ionizing and ultraviolet radiations, have been shown to be initiators in several initiation-promotion animal model systems (Trosko and Chang, 1985), it would be consistent to hypothesize that initiation is based on a mutagenic mechanism.  Many chemicals, considered to be mutagens based on interpretations of a wide range of cell-free and in vitro/in vivo data, are also speculated to act as initiators because of their presumed mutagenic action.  However, as will be discussed later, just because an animal can have some of its cells promoted after it has been exposed to a known or suspected mutagen, and just because either DNA lesions or DNA damage are found in tissues of the exposed animal, it does not prove that the mutation ultimately found in the tumor cell was induced by the agent that was used to induce the irreversible or initiation event.  In addition, conceivably, an agent, which could induce a stable epigenetic change, could be thought to be an initiating agent.  In short, at present we can only speculate that initiation, induced either by a mutation or stable epigenetic event, is that process which prevents a stem or progenitor cell from terminally differentiating but not from dividing.  This must be the case, for if an affected cell has the ability to terminally differentiate under normal conditions; it could not give raise to a cancer (see later discussion).

Promotion has been postulated to be that process by which a single initiated cell is selectively amplified by one of several mitogenic stimuli.

Here again, the underlying mechanisms are not known.  However, several lines of evidence have been used to hypothesize that promoting agents and conditions are mitogens, not mutagens (Ames and Gold, 1990b; Cohen and Ellwein, 1990; Trosko and Chang, 1988a).  Many natural chemicals, such as phorbol esters and palyiotoxins, endogenous factor, such as hormones and growth factors, nutrients, such as unsaturated fatty acids, pollutants, such as polybrominate biphenyls, pesticides, such as DDT, dieldrin and aldrin, cigarette condensates, and drugs, such as Phenobarbital, have been demonstrated to act as promoters in in vivo model systems (Trosko and Chang, 1988a).  The weight of the other experimental results with these chemicals is that they are not initiators in in vivo animal systems, and do not induce DNA damage or mutations in systems which simulate the in vivo conditions.  Other promoting conditions include normal cell growth (as might be evidenced by initiating young animals prior to their normal growth), cell removal (surgery or wounding), cell killing, and solid state object-induced hyperplasia (Trosko et al., 1990d).  In the animal systems, the mitogenic stimulation must be sustained (Sisskin et al., 1982).  In other words, even if an animal is initiated and exposed to mitogenic stimuli for a short time or for irregular times or at concentrations not sufficient to cause proliferation of the initiated cell (Bout well, 1974), then tumors will not appear at earlier times or higher frequencies.

While the implied cellular basis of promotion is mitogenesis, it does not necessarily mean that all promoting agents will stimulate cell division in all cells exposed to the agent.  By definition, if a tumor is clonally derived from a single cell during the carcinogenic process, then a promoter needs only to stimulate the initiated cell.  This has some important implications to those who wish to identify promoters from non-promoters.  If an agent can be shown to stimulate cell division in an animal tissue, it might have the potential of being a promoter in humans if it can be shown that it also can stimulate human cells.  Also, while a chemical could stimulate normal cells to proliferate, there is no guarantee that an initiated cell would also be stimulated to divide.  Conversely, if a chemical does not stimulate normal tissues to divide, it does not necessarily means it would not cause an initiated cell to proliferate.  In the case where a promoting agent causes both the normal and initiated cell to divide or to stimulate only the initiated cell to divide, the net effect is that the initiated cells will selectively accumulate in the tissue, since the normal progenitor cells will terminally differentiate, whereas the initiated cell will not.  These terminally differentiated cells would eventually be replaced, whereas the initiated cell will have the potential to further proliferate, thereby increasing their numbers.  At this point, a single initiated cell has increased its numbers by sustained mitogenic stimuli, such that a large mass of these undifferentiated cells occupy a tissue.

During that clonal expansion period whereby cells containing a stable alteration in a gene which affects terminal differentiation (This could be a number of genes; possibly a different gene for each tissue of different embryonic origin.), additional mutations or stable epigenetic changes could occur, allowing the initiated cell to acquire all the phenotypic alterations needed to become promoter independent (Trosko et al., 1983a).  Clearly, it at least two or more rare events are needed for complete neoplastic conversion, the probability that these two or more independent events occurring in one initiated cell of the body would be very small.  On the other hand, if one increases that number by promoting the single initiated cell, the larger that mass of initiated cells become, the chances for one of those cells containing one genetic “hit” to acquire the second or additional “hit” increase (Potter, 1981a; Trosko and Chang, 1980; Trosko and Chang, 1985).  Agents, which are thought to be effective at this stage, have been speculated to be able to induce stable genetic or epigenetic events (Hennings et al., 1983; jaffe et al., 1987; Reddy and Fialkow, 1987; Taguchi et al., 1984).  Once this has occurred the cell no longer would need an exogenous stimulator of cell division or of a blocker of cell suppression.

As a summary of the multi-stage multi-mechanism concept of carcinogenesis, three distinct steps have been identified in experimental animal systems.  In reality, for the human being, we are never exposed in unique sequence to those physical and chemical agents and conditions which are known to be initiators, promoters and Progressors.  Humans are constantly exposed to a wide and changing mixture of initiators, promoters and Progressors.  Rarely are we exposed to physical or chemical agents at cytotoxic levels, except possibly the ultraviolet rays of sunlight, alcohol and therapeutic radiation or chemical treatments.  We are all exposed to our growth factors at discreet periods of our lives, to chemicals which could interact additively, synergistically or antagonistically with each other (Aylsworth et al., 1989; Hirose et al., 1991; Warngard et al., 1987), as well as to the different and changing physiological endogenous chemicals of our bodies, due to genetic, sex developmental, life style and dietary factors.

How wonderful it would be for both the scientist and policy regulator if, in fact, cancers, in their full-blown form, were “caused” by a single exposure to an agent, which is currently called a “carcinogen”.  However, this is naïve, since current experimental cancer studies in rodents (Siaga et al., 1982), in vitro studies with human cells (McCormick and Maher, 1989), cancer-prone human syndromes (i.e., retinoblastoma)(Levine et al., 1991; Moolgavkar and Knudson, 1981), and epidemiological studies (Armitage and Doll, 1954), all point to multiple and discreetly unique steps involved in carcinogenesis.  Indeed, it is fairly well accepted by most that a variety of genetic factors can influence the ultimate appearance of a cancer, that the developmental stage and sex of the individual will influence the effect of the agent contributing to the process, that dietary habits, as well as exposure to a wide variety and class of chemicals and drugs, could modify the effluence of the agent thought to be the primary “carcinogen” (Trosko and Chang, 1978a).  This, then leads to our first conceptual problem; namely, “What is a ‘carcinogen’?”

To begin the process of trying to understand the carcinogenic process, introduction to several current hypotheses will be necessary; namely, the ‘initiation promotion-proression’ hypothesis (Pitot et al., 1981); the stem cell theory or the ‘disease of differentiation’ theory[“ontogeny as partially blocked ontogeny”] (Markert, 1968; Pierce, 1974; Potter,1988; Trosko and Chang, 1989a); the nature and nurture theory (Trosko and Chang,1978b); oncogenes and tumor suppressor gene theory (Bishop,1983; Sager, 1986); and the blocked intercellular communication hypothesis (Trosko et al., 1983a).  To some, to muse on the theoretical nature of carcinogenesis will get nowhere.  One only needs either to test the suspected cancer-causing agent in an animal system, short term assay designed presumably to test for a suspected mechanism in carcinogenesis or do epidemiological analyses on humans exposed to the agent.  There are all kinds of inherent problems with all of these approaches, as has been demonstrated with recent challenges to the current procedures to test for “cancer-causing” chemicals (Trosko, 1988; Trosko, 1989).  The basic nature of the problem seen by some is that the animal bioassay procedure, by not being designed with the multi-stage nature of carcinogenesis in mind, cannot provide information as to how the chemical could contribute to the multi-stage nature of carcinogenesis (i.e., Is the chemical a mutagen/initiator or is it a nitrogen/promoter?) (Ames and Gold, 1990b; Cohen and Ellwein, 1990).  On the other hand, because of so many fundamental artifacts and limitations of short-term tests (most assumed only to measure mutations (Trosko, 1988; Trosko,1989)), extrapolation of these results (positive or negative) from an artificial in vitro single cell system to an interacting multicellular rodent, let alone a human being, is truly stretching our current scientific knowledge.

To defend the necessity of trying to understand the mechanistic/theoretical bases of carcinogenesis, it seems that those who claim one needs only empirical animal and short-term tests fail to mention that these tests themselves are based on many implicit assumptions and hypotheses concerning the nature of carcinogenesis and “carcinogens”.  Therefore, in order for an epidemiologist to design a study to check if chemical x or radiation y, shown to induce a positive result in either a short term assay for mutagenesis or animal bioassay for tumor genesis, an understanding of how that particular agent contributes to the multi-stage, multimechanism carcinogenic process will be necessary for the reasons to be shown later.  In the same sense, any agent found by an epidemiologist to be associated with human cancers cannot automatically be assumed to be a mutagen/initiator of carcinogenesis as is implied with the concept, “carcinogen as mutagen” (Ames et al., 1973).

At this point, we have introduced several terms which need to be defined; namely, carcinogen; mutagen; initiator; and promoter.  In the process of defining these terms, the terms of mitogen, cytotoxicant, epigenetic and progressor will b=have to be defined.  Based on the fact that carcinogenesis ins a multi-stage, multi-mechanism process, the implied definition of a “carcinogen” is that it is an agent which “causes” cancer by interacting with a normal cell, transforming it through all the stages, via all of the different mechanisms, and altering all the required genetic factors needed to escape biological defense mechanisms against cancer.  The word, “carcinogen”, itself, creates the wrong impression in the minds of lay persons, risk assessors and scientists alike.  In other words, “carcinogen” does not reflect our current understanding of the multi-stage nature of carcinogenesis.  If, in fact, an agent is truly associated with the ultimate appearance of a cancer, the real question is “Which step in the multi-stage process of cancer did it influence and by what mechanism did it act?”  Possibly, we would do well if the word was banished from the language (Trosko et al., 1983b).

Now when an agent influences the carcinogenic process, it interacts with the organism/human ultimately at the cell level (after various routes fo entry and survival/chemical transformation via pharmokinetic action).  At this point, the biological consequence of the agent’s interaction at the molecular/biochemical level could be: mutagenesis; cell death; or epigenetic alteration (altered gene expression).