Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[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 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 2537 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 2538 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 2541 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 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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"RÈPAÃŒR " 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. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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-à -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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 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. REFERENCES 1. Abelev, G. I. Alpha-Fetoprotein in Ontogenesis and Its Associa tion with Malignant Tumors. Advan. Cancer Res., 14: 295-358, 1971. 2. Albert, R. E., Phillips, M. E., Bennett, P., Burns, F., and Heimbach, R. The Morphology and Growth Characteristics of Radiation-Induced Epithelial Skin Tumors in the Rat. Cancer Res., 29: 658-668, 1969. 3. Alexander, P. Foetal "Antigens" in Cancer. Nature, 235: 137-140,1972. 4. Ames, B. N., Gurney, E. G., Miller, J. A., and Bartsch, H. Carcinogens as Frameshift Mutagens: Metabolites and Derivatives of 2-Acetylaminofluorene and Other Aromatic Amine Carcino gens. Proc. Nati. Acad. Sei. U. S., 69: 3128-3132, 1972. 5. Baldwin, R. W. Immunological Aspects of Chemical Carcinogenesis. Advan. Cancer Res., in press. 6. Berenblum, I. Carcinogenesis and Tumor Pathogenesis. Advan. Cancer Res., 2: 129-175, 1954. 7. Berenblum, I. The Two-Stage Mechanism of Carcinogenesis asan Analytical Tool. In: O. Mühlbockand P. Emmelot (eds.), Cellular Control Mechanisms and Cancer, pp. 259-267. Amsterdam: Elsevier Publishing Co., 1964. 8. Boutwell, R. K. Some Biological Aspects of Skin Carcinogenesis. Progr. Exptl. Tumor Res., 4: 207-250, 1964. 9. Bower, F. F., and Gordan, G. S. Hormonal Effects of Non-Endocrine Tumors. Ann. Rev. Med., 16: 83-118, 1965. 10. Braun, A. C. The Cancer Problem. New York: Columbia University Press, 1969. 11. Burdette, W. J. Carcinoma of the Colon and Antecedent Epithelium. Springfield, 1111.:Charles C Thomas, Publisher, 1970. 12. Burnet, M. Somatic Mutation and Chronic Disease. Brit. Med. J., 1: 338-342, 1965. 13. Chernozemski, I. N., and Warwick, G. P. Liver Regeneration and Induction of Hepatomas in B6AF, Mice by Urethan. Cancer Res., 30: 2685-2690,1970. 14. Clark, W. H., Jr., From, L., Bernardino, E. A., and Mihm, M. C. The Histogenesis and Biologic Behavior of Primary Human 2546 Malignant Melanomas of the Skin. Cancer Res., 29: 705-726, 1969. 15. Clark, W. H., Jr., Ten Heggeler, B., and Bretton, R. Electron Microscope Observations of Human Cutaneous Melanomas Corre lated with Their Biologic Behavior. In: W. H. McCarthy (ed.), Melanoma and Skin Cancer, pp. 121-141. New South Wales, Australia: V. C. N. Blight Gov. Printer, 1972. 16. Cole, L. J., and Nowell, P. C. Radiation Carcinogenesis: The Sequence of Events. Science, 150: 1782-1786, 1'965. 17. Cox, R., Damjanov, I., Abanobi, S., and Sà rma, D. S. R. A Method for Measuring DNA Damage and Repair in the Liver in Vivo. Cancer Res.,.?.?.- 2114-2121, 1973. 18. Cox, R., Damjanov, Ivan and Irving, Charles C. Damage and Repair of Hepatic DNA by Ethylating Carcinogens. Proc. Am. Assoc. Cancer Res., 14: 28,1973. 19. Craddock, V. M. Liver Carcinoma Induced in Rats by Single Administration of Dimethylnitrosamine after Partial Hepatectomy. J. Nati. Cancer Inst.,47: 899-905, 1971. 20. Damjanov, I., Cox, R., Sarma, D. S. R., and Farber, E. Patterns of Damage and Repair of Liver DNA Induced by Carcinogenic Methylating Agents in Vivo. Cancer Res., 33: 2122-2128, 1973. 21. de Néchaud,B., and Uriel, J. AntigènesCellulaires Transitoires du Foie de Rat. III. Mode de Réapparitionde 1' a-Foetoproteine au 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Cours de PHépatocarcinogénèse Chimique. Intern. J. Cancer, 11: 104-115,1973. Duff, R., Doller, E., and Rapp, F. Immunologie Manipulation of Métastases due to Herpes Virus Transformed Cells. Science, 180: 79-81,1973. Dulbecco, R. Topoinhibition and Serum Requirement of Trans formed and Untransformed Cells. Nature, 227: 802-806, 1970. Embryonic and Fetal Antigens in Cancer, Vol. 2, Proceedings of the Second Conference, Oak Ridge, Tenn., 1972. Epstein, E. M., McNary, J., Bartus, B., and Farber, E. Chemical Carcinogenesis: Persistence of Bound Forms of 2-FluorenyIacetamide. Science, 762. 907-908, 1968. Everson, T. C., and Cole, W. H. Spontaneous Regression of Cancer. Philadelphia: W. B. Saunders Co., 1966. Faiber, E. Hyperplastic Liver Nodules. In: H. Busch (ed.), Methods in Cancer Research, Vol. 7, pp. 345-375. New York: Academic Press, Inc., 1973. Farber, E., Sarma, D. S. R., Rajalakshmi, S., and Shinozuka. Liver Carcinogenesis: A Unifying Hypothesis. In: F. F. Becker (ed.), Principles of Liver Disease. New York: Marcel Dekker, in press. Fialkow, P. J. Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females. Advan. Cancer Res., 15: 191-223, 1972. Fidler, I. J. Selection of Successive Tumor Lines for Mestastasis. Nature New Biol.,242: 148-149, 1973. Foulds, L. Neoplastic Development, Vol. 1. London: Academic Press, Inc., 1969. Frei, E., III. Combination Cancer Therapy: Presidential Address. Cancer Res., 32: 2593-2607, 1972. Friedewald, W. F., and Rous, P. The Initiating and Promoting Elements in Tumor Production. An Analysis of the Effects of Tar, Benzpyrene, and Methylcholanthrene on Rabbit Skin. J. Exptl. Med., SO: 101-126, 1944. Furth, J. A Meeting of Ways in Cancer Research: Thoughts on the Evolution and Nature of Neoplasms. Cancer Res., 19: 241-258, 1959. Gartier, S. M. Utilization of Genetic Markers as Tumor Tracers. In: J. German (ed.), Chromosomes and Cancer, in press. German, J. Genes Which Increase Chromosomal Instability in Somatic Cells and Predispose to Cancer. Progr. Med. Genet., 8: 61-101, 1972. Glücksmann,A. The Histogenesis of Benzpyrene-Induced Epider mal Tumors in the Mouse. Cancer Res., 5: 385-400, 1945. CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Carcinogenesis and Cellular Evolution 38. Gold, P. Embryonic Origin of Human Tumor-Specific Antigens. Progr. Exptl. Tumor Res., 14: 43-58, 1971. 39. Guldberg, G. Experimental Researches on Precancerous Changes in the Skin and Skin Cancer. Acta Pathol. Microbio!. Scand. Suppl.,S: 1-223,1931-1932. 40. Haddow, A. Cellular Inhibition and the Origin of Cancer. Acta UnióIntern. Contra Cancrum, 3: 342-352, 1938. 41. Haese, W. H., Smith, D. L., and Bueding, E. Hycanthone-Induced Hepatic Changes in Mice Infected with Schistosoma monsoni, i. Pharmacol. Exptl. Therap., ¡86:430-440, 1973. 42. Haran-Ghera, N., and Peled, A. Thymus and Bone Marrow Derived Lymphatic Leukemia in Mice. Nature, 241: 396-398, 1973. 43. Hard, G. C., and Butler, W. H. Cellular Analysis of Renal Neoplasia: Light Microscope Study of the Development of Interstitial Lesions Induced in the Rat Kidney by a Single Carcinogenic Dose of Dimethylnitrosamine. Cancer Res., 30: 2806-2815,1970. 44. Harris, H., Miller, O. J., Klein, G., Worst, P., and Tachibana, T. Suppression of Malignancy by Cell Fusion. Nature, 223: 363-368,1969. 45. Hicks, R. M., and Wakefield, J. St. J. Rapid Induction of Bladder Cancer in Rats with /V-Methyl-Af-Nitrosourea. I. Histology. Chem.-Biol. Interactions, 5: 139-152, 1972. 46. Hitotsumachi, S., Rabinowitz, Z., and Sachs, L. Chromosomal Control of Reversion in Transformed Cells. Nature, 231: 511-514,1971. 47. Hitotsumachi, S., Schaki, R., Padeh, B., Rabinowitz, Z., and Sachs, L. The Formation of Variants with a Reversion of Properties of Transformed Cells. VII. Chromosomal Numbers and Re-Reversion in Subtetraploid Variants. Intern. J. Cancer, 10: 9-13, 1972. 48. Hollander, C. F., and Bentvelzen, P. Enhancement of Urethan Induction of Hepatomas in Mice by Prior Partial Hepatectomy. J. Nati. Cancer Inst., 41: 1303-1306, 1968. 49. Holley, R. W. A Unifying Hypothesis Concerning the Nature of Malignant Growth. Proc. Nati. Acad. Sei. U. S., 69: 2840-2841, 1972. 50. Horava, A., and Skoryna, S. C. Observations on the Pathogenesis of Neoplasia. Can. Med. Assoc. J., 73: 630-638, 1955. 51. Hsie, A. W., and Puck, T. T. Morphologic Transformation of Chinese Hamster Cells by Dibutyryl Adenosine Cyclic 3':5'-Monophosphate and Testosterone. Proc. Nati. Acad. Sei. U. S., 68: 358-361,1971. 52. Mutter, R. V. P., Foote, F. W., Jr., and Farrow, J. H. In Situ Lobular Carcinoma of the Female Breast, 1939-1968. In: Anderson Hospital (eds.), Breast Cancer Early and Late, pp. 201-226. Chicago: Year Book Publishers, 1970. 53. Irving, C. C. Interaction of Chemical Carcinogens with DNA. Methods Cancer Res., 7. 189-244, 1973. 54. Johnson, G. S., Friedman, R. M., and Pastan, I. Restoration of Several Morphological Characteristics of Normal Fibroblasts in Sarcoma Cells Treated with Adenosine-3':5'-Cyclic Monophos- 55. 56. 57. 58. 59. phate and Its Derivatives. Proc. Nati. Acad. Sei. U. S., 68: 425-429, 1971. Kauffman, S. Differentiation of Malignant to Benign Cells. J. Theoret. Biol., 31: 429-451, 1971. Klein, G. Tumor-Specific Transplantation Antigens: G. H. A. Clowes Memorial Lecture. Cancer Res., 28: 625-635, 1968. Knudson, A. G., Jr. Mutation and Cancer: Statistical Study of Retinoblastoma. Proc. Nati. Acad. Sei. U. S., 68: 820-823, 1971. Knudson, A. G., Jr. Mutation and Human Cancer. Advan. Cancer Res.,/7. 317-352,1973. Knudson, A. G., Jr., and Strong, L. C. Mutation and Cancer: A Model for Wilms' Tumor of the Kidney. J. Nati. Cancer Inst., 48: 313-324, 1972. NOVEMBER 60. Kroes, R., Williams, G. M., and Weisburger, J. H. Early Appearance of Serum a-Fetoprotein during Hepatocarcinogenesis, Age of Rats and Extent of Treatment with 3'-Methyl-4-dimethylaminoazobenzene. Cancer Res.,32: 1526-1532, 1972. 61. Kroes, R., Williams, G. M., and Weisburger, J. H. Early Appearance of Serum a-Fetoprotein as a Function of Dosage of Various Hepatocarcinogens. Cancer Res., 33: 613-617, 1973. 62. Lappé,M. A. Evidence for the Antigenicity of Papillomas Induced by 3-Methylcholanthiene. J. Nati. Cancer Inst., 40: 823-846, 1968. 63. Lappé,M. A. Tumor Specific Transplantation Antigens: Possible Origin in Premalignant Lesions. Nature, 223: 82-84, 1969. 64. Laws, J. O. Tissue Regeneration and Tumour Development. Brit. J. Cancer, ¡3:669-674, 1959. 65. Lebovitz, H. E. Endocrine-Metabolic Syndromes Associated with Neoplasms, In: Lord Brain and F. H. Norris (eds.), The Remote Effects of Cancer on the Nervous System, pp. 104-111. New York: Gruñe& Stratton, 1965. 66. Lipsett, M. B. Hormonal Syndromes Associated with Neoplasia. Advan. Metab. Disorders, 3: 111-152, 1968. 67. Maini, M. M., and Stich, H. F. Chromosomes of Tumor Cells. II. Effects of Various Liver Carcinogens on Mitosis of Hepatic Cells. J. Nati. Cancer Inst., 26: 1413-1427, 1961. 68. Maini, M. M., and Stich, H. F. Chromosomes of Tumor Cells. III. Unresponsiveness of Precancerous Hepatic Tissues and Hepatomas to a Mitotic Stimulus. J. Nati. Cancer Inst., 28: 753-762, 1962. 69. Marquardt, H., Sternberg, S. S., and Philips, F. S. Dimethylbenz(a)anthracene and Hepatic Neoplasia in Regenerating Rat Liver. Chem.-Biol. Interactions, 2: 401-403, 1970. 70. Mihm, M. C., Clark, W. H., Jr., and From, L. The Clinical Diagnosis, Classification and Histogenetic Concepts of the Early Stage of Cutaneous Malignant Melanomas. New Engl. J. Med., 284: 1078-1082,1971. 71. Miller, E. C., and Miller, J. A. The Mutagenicity of Chemical Carcinogens: Correlations, Problems and Interpretations. In: A. Hollander (ed.), Chemical Mutagens, Principles and Methods for Detection, Vol. 1, pp. 83-119. New York: Plenum Publishing Corp., 1971. 72. Miller, J. A. Carcinogenesis by Chemicals: An Overview-G.H.A. Clowes Memorial Lecture. Cancer Res., 30: 559-576, 1970. 73. Mitelman, F., Marks, J., Levan, G., and Levan, A. Tumor Etiology and Chromosome Pattern. Science, 776: 1340-1341, 1972. 74. Morris, H. P., and Wagner, B. P. Induction and Transplantation of Rat Hepatomas with Different Growth Rate (Including Minimal Deviation Hepatomas). In: H. Busch (ed.), Methods in Cancer Research, Vol. 4, pp. 125-152. New York: Academic Press, Inc., 1968. 75. Mottram, J. C. On the Correlation between Malignancy and the Rate of Growth of Tar Warts in Mice. Am. J. Cancer, 22: 801-830,1934. 76. Olenov, J. M., and Fei, V. J. The Antigenic Structure of Tumor Cells as Material for a Study of Tissue Differentiation. J. Embryol. Exptl. Morphol., 19: 299-309, 1968. 77. Pierce, G. B. Teratocarcinoma: Model for a Developmental Concept of Cancer. Current Topics Develop. Biol., 2: 223-246, 1967. 78. Pierce, G. B. Differentiation of Normal and Malignant Cells. Federation Proc., 29: 1248-1254, 1970. 79. Pierce, G. B., and Johnson, L. D. Differentiation and Cancer. In Vitro, 7: 140-145, 1971. 80. Pierce, G. B., and Wallace, C. Differentiation of Malignant to Benign Cells. Cancer Res., 31: 127-134,1971. 81. Pilot, H. C., and Heidelberger, C. Metabolic Regulatory Circuits and Carcinogenesis. Cancer Res., 23: 1694-1700, 1963. 82. Potter, V. R. Biochemical Perspectives in Cancer .Research. Cancer Res., 24: 1085-1098, 1964. 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 2547 Emmanuel Farber 83. Potter, V. R. Biochemical Studies on Minimal Deviation Hepatomas. In: O. Mühlbockand P. Emmelot (eds.), Cellular Control M^'hanisms and Cancer, pp. 190—210. Amsterdam: Elsevier Publishing Co., 1964. 84. Pound, A. W. Carcinogenesis and Cell Proliferation. New Zealand Med. J., 67. 88-99, 1968. 85. Powers, D. M., and Holley, R. W. Selective Chemical Methylation of Yeast Alanine Transfer RNA. Biochim. Biophys. Acta, 287: 456-46*, 1972. 86. Prasad, K. N. Cyclic AMP Induced Differentiated Mouse Neuroblastoma Cells Lose Tumourigenic Characteristics. Cytobios,6: 163-166, 1972. 87. Prehn, R. T. Neoplasia. In: M. F. Lavia and R. B. Hill, Jr. (eds.), Principles of Pathobiology, pp. 191-241. New York: Oxford University Press, 1971. 88. Raick, A. N. Ultrastructural, Histological, and Biochemical Alterations Produced by 12-O-Tetradecanoyl-phorbol-13-acetate on Mouse Epidermis and Their Relevance to Skin Tumor Promotion. Cancer Res., 33: 269-286, 1973. 89. Richart, R. M. Natural History of Cervical Intraepithelial Neoplasia. Clin. Obstet. Gynecol., 10: 748-784, 1967. 90. Rous, P., and Beard, J. W. The Progression to Carcinoma of Virus-Induced Rabbit Papillomas (Shope). J. Exptl. Med., 62. 523-548,1935. 91. Rous, P., and Kidd, J. G. Conditional Neoplasms and Subthreshold Neoplastic States. A Study of the Tar Tumors of Rabbits. J. Exptl. Med., 73: 365-390, 1941. 92. Rusch, H. P. Stages in Cancer Research. Texas Rept. Biol. Med., «.-207-214,1950. 93. Sarma, D. S. R., Rajalakshmi, S., and Farber, E. Interactions of Carcinogens with Nucleic Acids. In: F. F. Becker (ed.), Cancer: A Comprehensive Treatise. New York: Plenum Publishing Corp., in press. 94. Scarano, E. The Control of Gene Function in Cell Differentiation and in Embryogenesis. Advan. Cytopharmacol., 1: 13-24, 1971. 95. Scher, W., Preisler, H. D., and Friend, C. Hemoglobin Synthesis in Murine Virus-Induced Leukemia Cells in Vitro. III. Effects of 5-Bromo-2'-Deoxyuridine, Dimethylformamide and Dimethylsulfoxide. J. Gen. Physiol.,81: 63-70, 1973. 96. Scherer, E., and Hoffmann, M. Probable Clonal Genesis of Cellular Islands Induced in Rat Liver by Diethylnitrosamine. European J. Cancer, 7: 369-371, 1971. 97. Sharon, N., and Lis, H. Lectins: Cell Agglutinating and Sugar-Specific Proteins. Science, 777: 949-959, 1972. 98. Silagi, S., and Bruce, S. A. Suppression of Malignancy and Differentiation in Melanatic Melanoma Cells. Proc. Nati. Acad. Sci. U.S. ,66: 72-78, 1970. 99. Slemmer, G. Host Response to Premalignant Mammary Tissues. Nati. Cancer Inst. Monograph, .7.5: 57-71, 1972. 100. Stewart, B. W., and Farber, E. Strand Breakage in Rat Liver DNA and Its Repair following Administration of Cyclic Nitrosamines. Cancer Res., in press. 101. Stewart, B. W., Farber, E., and Mirvish, S. S. Induction by an Hepatic Carcinogen, l-Nitroso-5,6-Dihydrouracil, of Single and Double Strand Breaks of Liver DNA with Rapid Repair. Biochem. Biophys. Res. Commun., 53: 773-779, 1973. 102. Temin, H. M. Malignant Transformation of Cells by Viruses. Perspect. Biol. Med., 14: 11-26, 1970. 103. Temin, H. M. Mechanism of Cell Transformation by RNA Tumor Viruses. Ann. Rev. Microbiol.,25. 609-648, 1971. 104. Van Duuren, B. L. V. Tumor Promoting Agents in Two-Stage Carcinogenesis. Progr. Exptl. Tumor Res., II: 31-68, 1969. 105. Vasiliev, J. M., and Guelstein, V. I. Sensitivity of Normal and Neoplastic Cells to the Damaging Action of Carcinogenict Substances: A Review. J. Nati. Cancer Inst., 31: 1123-1141, 1963. 106. Vasiliev, J. M., and Guelstein, V. I. Local Cell Interactions in Neoplasms and in Foci of Carcinogenesis. Progr. Exptl. Tumor Res.,«. 26-65, 1966. 107. Watabe, H. Early Appearance of Embryonic a-Globulin in Rat Scrum during Carcinogenesis with 4-Dimethylaminoazobenzene. Cancer Res., 31: 1192-1194, 1971. 108. Weinhouse, S. Glycolysis, Respiration and Anomalous Gene Expression in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture. Cancer Res., 32: 2007-2016, 1972. 109. Weinstein, I. B. Modifications in Transfer RNA during Chemical Carcinogenesis, In: Genetic Concepts and Neoplasia, The Univer sity of Texas M. D. Anderson Hospital and Tumor Institute at Houston, pp. 380-408. Baltimore: The Williams & Wilkins Co., 1970. 110. Weisburger, J. H., and Weisburger, E. K. Biochemical Formation and Pharmacological Properties of Hydroxylamines and Hydroxamic Acids. Pharmacol. Rev., 25: 1-66, 1973. 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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Carcinogenesis and Cellular Evolution AM M - . mffî%P1$ î: '$$$ fvU,\r'' ^V^V*f*; ^; 'V''" / •¿ NOVEMBER 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 2549 Emmanuel Farber l 2550 CANCER RESEARCH VOL.33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Carcinogenesis−−Cellular Evolution as a Unifying Thread: Presidential Address Emmanuel Farber Cancer Res 1973;33:2537-2550. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/11/2537.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.