Mechanisms for the Initiation and Promotion of Carcinogenesis: A Review and a New Concept
1984; Elsevier BV; Volume: 59; Issue: 2 Linguagem: Inglês
10.1016/s0025-6196(12)60244-4
ISSN1942-5546
AutoresRobert E. Scott, John J. Wille, Marjorie L. Wier,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoCarcinogenesis in humans is a multistage process, and the two major stages have been designated initiation and promotion. Although the biochemical basis for initiation and promotion remains to be established, recent research has provided important insights into potentially significant biologic mechanisms. These data are reviewed, and a new concept of carcinogenesis is presented. This concept suggests that the initiation of carcinogenesis may result from cellular immortalization and the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated stem cells develop aberrant autoregulatory growth-control properties. Carcinogenesis in humans is a multistage process, and the two major stages have been designated initiation and promotion. Although the biochemical basis for initiation and promotion remains to be established, recent research has provided important insights into potentially significant biologic mechanisms. These data are reviewed, and a new concept of carcinogenesis is presented. This concept suggests that the initiation of carcinogenesis may result from cellular immortalization and the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated stem cells develop aberrant autoregulatory growth-control properties. Carcinogenesis in humans and animals results from the development of a sequence of defects in stem cells which are the targets of transforming agents.1Pierce GB Shikes R Fink LM Cancer: A Problem of Developmental Biology. Prentice-Hall, Englewood Cliffs, New Jersey1978: 68-87Google Scholar In model systems, two general steps in the process of carcinogenesis have been established: initiation and promotion.2Berenblum I Sequential aspects of chemical carcinogenesis: skin.in: Becker FF Cancer: A Comprehensive Treatise. Vol 1. Plenum Press, New York1975: 323-344Google Scholar, 3Berenblum I Sequential aspects of chemical carcinogenesis: skin.in: Becker FF Second edition. Cancer: A Comprehensive Treatise. Vol 1. Plenum Press, New York1982: 451-484Google Scholar Figure 1 illustrates this concept. For carcinogenesis to develop, stem cells must be initiated by chemical or physical carcinogens or perhaps by certain viruses. Thereafter, such cells must be exposed to a promoting agent to induce complete neoplastic transformation, and the sequence of these events is of critical importance if malignant transformation is to develop. This simple two-stage model of carcinogenesis has endured more than 35 years of scrutiny;4Berenblum I Shubik P A new, quantitative, approach to the study of the stages of chemical carcinogenesis in the mouse's skin.Br J Cancer. 1947; 1: 383-391Crossref PubMed Scopus (386) Google Scholar only recently, however, has it been possible to begin further dissection of the steps that are necessary for initiation and promotion.5Slaga TJ Fischer SM Nelson K Gleason GL Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion.Proc Natl Acad Sci USA. 1980; 77: 3659-3663Crossref PubMed Scopus (401) Google Scholar, 6Cooper GM Cellular transforming genes.Science. 1982; 217: 801-806Crossref PubMed Scopus (400) Google Scholar The following example describes the results of a typical experiment that supports the two-stage model of carcinogenesis.7Hennings H Shores R Wenk ML Spangler EF Tarone R Yuspa SH Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters.Nature. 1983; 304: 67-69Crossref PubMed Scopus (283) Google Scholar, 8Hennings H Devor D Wenk ML Slaga TJ Former B Colburn NH Bowden GT Elgjo K Yuspa SH Comparison of two-stage epidermal carcinogenesis initiated by 7, 12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine in newborn and adult SENCAR and BALB/c mice.Cancer Res. 1981; 41: 773-779PubMed Google Scholar If a low dosage of 7,12-dimethylbenz[a]anthracene (DMBA) is applied to mouse skin in vivo, no malignant tumors develop. If, however, 12-O-tetradecanoylphorbol-13-acetate (TPA) is thereafter applied to the same area, a substantial incidence of malignant tumors becomes evident, as does the formation of some benign tumors. In contrast, if TPA is applied before DMBA or if TPA is applied alone, no malignant tumors develop. Therefore, a low concentration of DMBA initiates carcinogenesis, and TPA promotes carcinogenesis. In in vitro studies, investigators have also shown that low doses of ultraviolet irradiation can act as an initiation agent and that TPA can act as a promoting agent for murine embryo cells.9Mondal S Heidelberger C Transformation of C3H/10T1/2 CLB mouse embryo fibroblasts by ultraviolet irradiation and a phorbol ester.Nature. 1976; 260: 710-711Crossref PubMed Scopus (157) Google Scholar The application of very high dosages of carcinogens can, however, also act as a complete carcinogen to both initiate and promote tumorigenesis. This fact does not detract from the conclusion that cancer results from specific cellular defects that can be separated into initiation and promotion phases. Initiation is generally accepted as the process whereby an irreversible lesion is induced in stem cell genomic DNA which makes it susceptible to a unique effect of a promoting agent and thus the expression of the transformed phenotype.10Becker FF Presidential address: recent concepts of initiation and promotion in carcinogenesis.Am J Pathol. 1981; 105: 3-9PubMed Google Scholar, 11Boutwell RK Verma AK Ashendel CL Astrup E Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.Carcinog Compr Surv. 1982; 7: 1-12PubMed Google Scholar In general, the initiated state is stable and can persist for many months or years, but the precise mechanism by which initiators act has not been established. Although disagreement has arisen about whether the initiated state is phenotypically expressed, in vivo clinical studies in humans and studies in animals provide strong evidence that the initiated state can be detected as preneoplastic conditions. For example, lesions in the class designated as "severe dysplasia/carcinoma in situ" are undoubtedly preneoplastic, noninvasive lesions that can persist for many years before converting into a malignant tumor,12Johnson LD Nickerson RJ Easterday CL Stuart RS Hertig AT Epidemiologic evidence for the spectrum of change from dysplasia through carcinoma in situ to invasive cancer.Cancer. 1968; 22: 901-914Crossref PubMed Scopus (55) Google Scholar and cells of such lesions definitely express a distinct phenotypic character. For example, Figure 2 illustrates the comparative morphologic features of normal squamous epithelium and severe dysplasia/squamous cell carcinoma in situ. It shows that, in normal epithelium, stem cells are localized to the basal regions of the tissue near the basement membrane and that cell division occurs in this region. Once basal cells divide, a certain proportion differentiate and eventually keratinize, whereas the others retain their proliferative capacity. In normal epithelium, therefore, a precise equilibrium seems to regulate the processes of cell proliferation and cell differentiation. In contrast, in severe dysplasia/carcinoma in situ, dividing and proliferating cells can be observed throughout the tissue, and differentiation seems to be abrogated because it occurs to a notably decreased extent and it does not seem to be integrated with the control of stem cell proliferation.13Ackerman LV Rosai J Surgical Pathology. Fifth edition. CV Mosby Company, St. Louis1974: 773-780Google Scholar These observations suggest that the initiated state may indeed be phenotypically expressed as a defect in the integrated control of cell proliferation and differentiation. Other preneoplastic lesions in humans are also typified by apparently similar defects in the integrated control of cell differentiation and proliferation. Preleukemia is one such excellent example. In this disease state, pronounced changes in hematopoietic stem cell differentiation occur long before the development of evidence of aberrant stem cell proliferation.14Kass L Leukemia: Cytology and Cytochemistry. JB Lippincott Company, Philadelphia1982: 1-117Google Scholar In vitro studies performed in our laboratory, which will be described in detail in the subsequent material, also provide experimental support for the concept that the initiation of carcinogenesis may be related to the development of specific defects in the integrated control of cell proliferation and differentiation. Once a stem cell is initiated, it is then susceptible to the effects of promoting agents that cause the fully malignant phenotype to be expressed. Promoting agents are, in general, not mutagenic, and they do not induce malignant tumors by themselves; however, they can cause hyperplasia and, in some cases, the development of benign tumors.7Hennings H Shores R Wenk ML Spangler EF Tarone R Yuspa SH Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters.Nature. 1983; 304: 67-69Crossref PubMed Scopus (283) Google Scholar, 10Becker FF Presidential address: recent concepts of initiation and promotion in carcinogenesis.Am J Pathol. 1981; 105: 3-9PubMed Google Scholar, 11Boutwell RK Verma AK Ashendel CL Astrup E Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.Carcinog Compr Surv. 1982; 7: 1-12PubMed Google Scholar Some studies have suggested that promoters may act through epigenetic mechanisms and that most of their biologic and biochemical effects are reversible even though their tumor-promoting effects are not.11Boutwell RK Verma AK Ashendel CL Astrup E Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.Carcinog Compr Surv. 1982; 7: 1-12PubMed Google Scholar, 15Yun K Scott RE Biological mechanisms of phorbol myristate acetate-induced inhibition of proadipocyte differentiation.Cancer Res. 1983; 43: 88-96PubMed Google Scholar, 16Farber E Chemical carcinogenesis: a biologic perspective.Am J Pathol. 1982; 106: 269-296Google Scholar, 17Farber E Carcinogenesis—cellular evolution as a unifying thread: presidential address.Cancer Res. 1973; 33: 2537-2550PubMed Google Scholar Indeed, investigators have shown that initiated cells must typically be exposed to promoting agents for long intervals before complete transformation results and that specific threshold dosages of promoters must be used.11Boutwell RK Verma AK Ashendel CL Astrup E Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.Carcinog Compr Surv. 1982; 7: 1-12PubMed Google Scholar Promoters include a diverse array of compounds, such as phorbol myristate acetate,4Berenblum I Shubik P A new, quantitative, approach to the study of the stages of chemical carcinogenesis in the mouse's skin.Br J Cancer. 1947; 1: 383-391Crossref PubMed Scopus (386) Google Scholar, 7Hennings H Shores R Wenk ML Spangler EF Tarone R Yuspa SH Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters.Nature. 1983; 304: 67-69Crossref PubMed Scopus (283) Google Scholar, 11Boutwell RK Verma AK Ashendel CL Astrup E Mouse skin: a useful model system for studying the mechanism of chemical carcinogenesis.Carcinog Compr Surv. 1982; 7: 1-12PubMed Google Scholar saccharin,18Trosko JE Dawson B Yotti LP Chang CC Saccharin may act as a tumour promoter by inhibiting metabolic cooperation between cells.Nature. 1980; 285: 109-110Crossref PubMed Scopus (87) Google Scholar and phenobarbital,10Becker FF Presidential address: recent concepts of initiation and promotion in carcinogenesis.Am J Pathol. 1981; 105: 3-9PubMed Google Scholar, 19Peraino C Fry RJM Staffeldt E Effects of varying the onset and duration of exposure to phenobarbital on its enhancement of 2-acetylaminofluorene-induced hepatic tumorigenesis.Cancer Res. 1977; 37: 3623-3627PubMed Google Scholar and specific classes of these promoters seem to affect different stem cell types. As with initiating agents, the critical mechanism of action of tumor promoters in carcinogenesis has not been established. Many promoting agents have, however, been shown to have a mitogenic effect on specific target cells and to induce a pleiotropic response.20Colburn NH Tumor promotion and preneoplastic progression.Carcinog Compr Surv. 1980; 5: 33-56PubMed Google Scholar In one case, it was suggested that a purified growth factor can act as a promoting agent.21Harrison J Auersperg N Epidermal growth factor enhances viral transformation of granulosa cells.Science. 1981; 213: 218-219Crossref PubMed Scopus (41) Google Scholar Indeed, the promotion phase of carcinogenesis is the interval during which increased cellular proliferation occurs and initiated cells clonally expand. In this regard, increased proliferation per se could possibly cause malignant transformation of stem cells; however, such a conclusion is incompatible with clinical and experimental observations. Clinically, disease states have been identified which are characterized by excessive cell proliferation (for example, psoriasis), but such states are benign and show no predilection for the development of malignant tumors. Experimentally, stimuli that induce rapid cell proliferation have also been shown not to cause malignant transformation independently. This finding is true for a variety of growth-promoting factors, some of which are similar to the products of specific oncogenes such as the "sis" oncogene product.22Doolittle RF Hunkapiller MW Hood LE Devare SG Robbins KC Aaronson SA Antoniades HN Simian sarcoma virus one gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.Science. 1983; 221: 275-277Crossref PubMed Scopus (983) Google Scholar In this regard, oncogenes are regions of DNA which are involved in cellular transformation and whose expression commonly results in a proliferogenic cellular response.6Cooper GM Cellular transforming genes.Science. 1982; 217: 801-806Crossref PubMed Scopus (400) Google Scholar Although originally the expression of such gene products was thought to be the sole requirement for transformation of cells, recently this theory has been refuted because, for example, the "ras" oncogene has been shown to be expressed in hyperplastic conditions and related oncogenes have been shown to be reversibly expressed during embryogenesis23Müller R Slamon DJ Tremblay JM Cline MJ Verma IM Differential expression of cellular oncogenes during pre- and postnatal development of the mouse.Nature. 1982; 299: 640-644Crossref PubMed Scopus (380) Google Scholar and during tissue regeneration.24Goyette M Petropoulos CJ Shank PR Fausto N Expression of a cellular oncogene during liver regeneration.Science. 1983; 219: 510-512Crossref PubMed Scopus (214) Google Scholar Thus, the proliferative response, which can be induced by a variety of promoting agents, probably is important in carcinogenesis only if it is irreversibly activated in initiated stem cells. On the basis of these and related data, we developed a hypothesis that suggests that the initiation of carcinogenesis may result from the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated stem cells are irreversibly induced to express autoregulatory and aberrant mechanisms for the control of cell growth. Although this hypothesis is obviously oversimplified, it can explain many aspects of carcinogenesis and it can be experimentally tested in a straightforward manner, as will be discussed. To perform studies to test the aforementioned concept of carcinogenesis, we had to use a noninitiated, nontransformed, cloned stem cell line that could be grown in vitro, that could be transformed, and whose characteristics of proliferation and differentiation could be physiologically regulated. At the outset, we also had to establish how these stem cells normally regulate their characteristics of proliferation and differentiation. We chose to use the murine 3T3 T cell line because it is an established and well-characterized mesenchymal stem cell that is not fully initiated or transformed.25Scott RE Boone CW Promotion of smooth surface tumorigenicity by phorbol myristate acetate in Balb/3T3 cells and Balb/3T3 T proadipocytes.J Natl Cancer Inst. 1981; 66: 733-736PubMed Google Scholar, 26Wille Jr, JJ Maercklein PB Scott RE Neoplastic transformation and defective control of cell proliferation and differentiation.Cancer Res. 1982; 42: 5139-5146PubMed Google Scholar In this regard, 3T3 T cells may, however, be considered to be partially initiated because they are immortalized (see subsequent discussion). Nonetheless, this stem cell line can be induced to differentiate into striated muscle, cartilage, vascular pericytes, and macrophages under physiologic conditions.27Boone CW Scott RE Plate-induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes, chondrocytes, and fibroblasts.J Supramol Struct. 1980; 14: 233-240Crossref PubMed Scopus (11) Google Scholar, 28Krawisz BR Florine DL Scott RE Differentiation of fibroblast-like cells into macrophages.Cancer Res. 1981; 41: 2891-2899PubMed Google Scholar In vitro, however, these stem cells typically show a predilection to differentiate into adipocytes; therefore, most investigators have described them as proadipocyte stem cells. The differentiation of low-density 3T3 T proadipocyte stem cells into adipocytes can be induced by culturing them in heparinized medium containing human plasma;29Krawisz BR Scott RE Coupling of proadipocyte growth arrest and differentiation. I. Induction by heparinized medium containing human plasma.J Cell Biol. 1982; 94: 394-399Crossref PubMed Scopus (45) Google Scholar Figure 3 illustrates the appearance of undifferentiated and differentiated 3T3 T cells. The process is rapid and occurs in a relatively synchronous manner. For example, if rapidly growing low-density 3T3 T proadipocytes are placed in heparinized medium containing human plasma, more than 90% of the cells show growth arrest in the G1 phase of the cell cycle within 24 to 36 hours, and thereafter they differentiate into adipocytes within as few as 48 hours under optimal experimental conditions.30Yun K Hoerl BJ Scott RE Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: evidence for differentiation without DNA synthesis.J Cell Physiol. 1983; 117: 249-259Crossref PubMed Scopus (8) Google Scholar These observations show that, in proadipocyte stem cells, growth arrest in G1 precedes differentiation. G1 arrest has also been shown to mediate control of cellular proliferation, which occurs when cells are placed in a crowded environment, in growth factor-deficient medium, or in nutrient-deficient culture conditions.31Pardee AB Dubrow R Hamlin JL Kletzien RF Animal cell cycle.Annu Rev Biochem. 1978; 47: 715-750Crossref PubMed Scopus (484) Google Scholar We therefore first attempted to determine whether a state exists in G1 in which the control of both cell proliferation and cell differentiation is integrally regulated and whether such a state is distinct from other G1 arrest states. Figure 4 illustrates the results of a complex series of studies on this topic.32Scott RE Florine DL Wille Jr, JJ Yun K Coupling of growth arrest and differentiation at a distinct state in the G1 phase of the cell cycle: GD.Proc Natl Acad Sci USA. 1982; 79: 845-849Crossref PubMed Scopus (138) Google Scholar It shows that there is indeed a distinct state in G1 at which the integrated control of proliferation and differentiation is regulated, and this state is designated GD. As will be discussed subsequently, within the GD state is a complex of substates that control nonterminal differentiation (GD,) and terminal differentiation (TD). More specifically, Figure 4 shows that GD is distinct from two G1 arrest states induced by growth factor deficiency. These states are designated GC and GS and result from so-called contact inhibition and serum deprivation, respectively. Furthermore, Figure 4 shows that GD is also distinct from another G1 growth arrest state, which is induced by nutrient (isoleucine) deprivation and labeled as GN. The relative topography of the GD, GC, GS, and GN arrest states in G1 is only one characteristic that distinguishes GD.33Wille Jr, JJ Scott RE Topography of the predifferentiation GD growth arrest state relative to other growth arrest states in the G1 phase of the cell cycle.J Cell Physiol. 1982; 112: 115-122Crossref PubMed Scopus (34) Google Scholar We have shown, in addition, that only cells at the GD state can differentiate without DNA synthesis,30Yun K Hoerl BJ Scott RE Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: evidence for differentiation without DNA synthesis.J Cell Physiol. 1983; 117: 249-259Crossref PubMed Scopus (8) Google Scholar only cells at the GD state are responsive to the mitogenic effect of methyl isobutyl xanthine,34Scott RE Yun K Florine DL Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state.Exp Cell Res. 1983; 143: 405-414Crossref PubMed Scopus (12) Google Scholar only highdensity cells at the GD state can be lysed by 8-bromocyclic adenosine monophosphate,35Florine DL Hoerl BJ Scott RE Growth arrest of proadipocytes at a distinct predifferentiation G1 state associated with cytotoxic responsiveness to 8-bromo cyclic AMP.Cell Differ. 1982; 11: 195-202Crossref PubMed Scopus (5) Google Scholar and only cells at the GD state have prominent cell-surface microvilli.36Boman BM Maercklein PB Hoerl BJ Scott RE Cell surface characteristics of proadipocytes growth arrested at the predifferentiation GD state: defects associated with neoplastic transformation.Lab Invest. 1983; 48: 199-204PubMed Google Scholar More recent preliminary data further suggest that cells at the GD state possess specific proteins that can be detected by two-dimensional electrophoretic analysis. We next attempted to determine whether multiple steps serve to integrate the control of cell proliferation and differentiation at the GD state. The schematic model in Figure 5 summarizes the results of these studies.37Scott RE Hoerl BJ Wille Jr, JJ Florine DL Krawisz BR Yun K Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation.J Cell Biol. 1982; 94: 400-405Crossref PubMed Scopus (76) Google Scholar It shows that cells first demonstrate growth arrest at the GD state (arrow 1). Cells at GD have the potential to remain quiescent or to reinitiate proliferation (large arrow 5). They can also differentiate; if this process occurs, it consists of at least two steps. First, the cells undergo nonterminal differentiation (arrow 2) and show growth arrest at a nonterminal state of differentiation, designated GD,. GD, cells are fully differentiated and contain large fat droplets and high levels of lipogenic enzymes; they do, however, retain their ability to perform a variety of physiologic functions. For example, cells at the GD, state can undergo terminal differentiation (TD) (arrow 3), they can reinitiate proliferation while remaining differentiated (small arrow 5), or they can be induced to first lose the differentiated phenotype (arrow 4) and then reinitiate proliferation (large arrow 5). The integrated control of cell proliferation and differentiation can therefore be mediated at both GD and GD, because, at both states, cells can remain quiescent, proliferate, or differentiate. In support of this model for the integrated control of proadipocyte stem cell proliferation and differentiation at the GD and GD, states, we have presented evidence that specific physiologic and pharmacologic factors can control each process represented by the designated arrows in Figure 5. Specific fractions of human plasma have been isolated and partially purified which induce GD arrest (arrow 1), nonterminal differentiation (arrow 2), or terminal differentiation (arrow 3).37Scott RE Hoerl BJ Wille Jr, JJ Florine DL Krawisz BR Yun K Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation.J Cell Biol. 1982; 94: 400-405Crossref PubMed Scopus (76) Google Scholar, 38Wier ML Hoerl BJ Scott RE The integrated control of stem cell proliferation and differentiation (abstract).Fed Proc. 1983; 42: 388Google Scholar In addition, growth factors have been identified which can stimulate the proliferation of GD arrested cells (large arrow 5) or of nonterminally differentiated (GD') arrested cells (small arrow 5). Furthermore, certain vitamins and drugs have been identified which can induce GD, cells to lose the differentiated phenotype and revert to the GD state.37Scott RE Hoerl BJ Wille Jr, JJ Florine DL Krawisz BR Yun K Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation.J Cell Biol. 1982; 94: 400-405Crossref PubMed Scopus (76) Google Scholar, 38Wier ML Hoerl BJ Scott RE The integrated control of stem cell proliferation and differentiation (abstract).Fed Proc. 1983; 42: 388Google Scholar, 39Hoerl BJ, Wier ML, Scott RE: Biological mechanisms for the loss of the differentiated phenotype by nonterminally differentiated adipocytes (submitted for publication)Google Scholar Using this highly developed and controllable model system to study the mechanisms for the integrated control of cell proliferation and differentiation, we have performed experiments that have succeeded in answering two additional questions: Do cells have to undergo DNA synthesis before differentiation,40Holtzer H Pacifici M Tapscott S Bennett G Payette R Dlugosz A Lineages in cell differentiation and in cell transformation.in: Revoltella RP Pontieri GM Basilico C Rovera G Gallo RC Subak-Sharpe JH Expression of Differentiated Functions in Cancer Cells. Raven Press, New York1982: 169-180Google Scholar, 41Holtzer H Sanger JW Ishikawa H Strahs K Selected topics in skeletal myogenesis.Cold Spring Harbor Symp Quant Biol. 1972; 37: 549-566Crossref Google Scholar and do cells have to undergo what has been designated a "commitment" process before terminal differentiation?42Ham RG Veomett MJ Mechanisms of Development. CV Mosby Company, St. Louis1980: 317-428Google Scholar In this regard, "commitment" is the process whereby the proliferative capacity of a stem cell is limited, and its differentiation potential is directed along a specific pathway after short-term exposure to a differentiation-inducing agent. Our data clearly showed that neither of these processes is required in proadipocyte stem cells. To prove that DNA synthesis is not necessary for differentiation to occur, we isolated pure populations of metaphase proadipocyte stem cells and then placed them in differentiation-promoting media for appropriate intervals and under conditions that considerably limited the capacity of cells to undergo DNA synthesis and cell cycle traverse. The results clearly showed that 80% or more of these cells can be induced to differentiate without traversing the S phase of the cell cycle.30Yun K Hoerl BJ Scott RE Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: evidence for differentiation without DNA synthesis.J Cell Physiol. 1983; 117: 249-259Crossref PubMed Scopus (8) Google Scholar We also established that there is no detectable state at which the commitment to terminal differentiation occurs in proadipocytes. That is, we have obtained evidence that growth arrest at GD or at GD, does not predispose proadipocyte stem cells to undergo differentiation subsequently or to limit their proliferative capacity.43Wier ML, Seibel-Ross El, Scott RE: Unpublished dataGoogle Scholar The latter observation, although true for proadipocyte stem cells, may not be typical of normal stem cells because, although proadipocyte stem cells are normal in many ways, they do possess karyotypic abnormalities, and they are immortalized and therefore grow as a cell line in vitro. In summary, we suggest that for noninitiated, nontransformed stem cells, the primary physiologic mechanism for growth regulation is mediated by the integrated control of cell proliferation and differentiation at the GD and GD, states. We further suggest that growth control mediated at GC, GS, or GN serves as backup or fail-safe processes that prevent proadipocyte stem cells from growing in adverse environmental conditions, such as when they are too crowded or when the microenvironment is deficient in growth factors or nutrients. We do not suggest that all stem cells will behave in a manner precisely identical to that of proadipocyte stem cells, and we do not suggest that the factors that govern the integrated control of proadipocyte stem cell proliferation and differentiation will necessarily mediate similar processes in other stem cell systems. We do, however, suggest that the system we have developed will serve as an excellent model for study of the processes that integrate the control of stem cell proliferation and differentiation. In support of this conclusion, we have used this stem cell model system to determine the relationship between the development of defects in the integrated control of cell proliferation and differentiation and carcinogenesis. Our first approach attempted to determine whether transformed proadipocyte clones showed selective defects in their abil
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