12-O-tetradecanoylphorbol-13-acetate-induced Apoptosis Is Mediated by Tumor Necrosis Factor α in Human Monocytic U937 Cells

Abstract

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-α (TNFα) in U937 cells, and exogenously added TNFα induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFα antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFα and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFα in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFα is an autocrine-stimulating factor for the induction of apoptosis in these cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-α (TNFα) in U937 cells, and exogenously added TNFα induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFα antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFα and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFα in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFα is an autocrine-stimulating factor for the induction of apoptosis in these cells. Apoptosis, a morphologically distinguished form of programmed cell death, plays important roles not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases including cancer, autoimmune disease, viral infection, and neurodegenerative disorder (1Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar, 2Williams G.T. Smith C.A. Cell. 1993; 74: 777-779Abstract Full Text PDF PubMed Scopus (903) Google Scholar, 3Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar, 4Martin S.J. Green D.R. Crit. Rev. Oncol. Hematol. 1995; 18: 137-153Crossref PubMed Scopus (154) Google Scholar, 5Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6174) Google Scholar, 6White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1322) Google Scholar). Moreover, various chemicals and drugs for treatment of cancers are known to destroy tumor cells through apoptosis (7Eastman A. Curr. Opin. Oncol. 1990; 2: 1109-1114Crossref PubMed Scopus (11) Google Scholar, 8Miyashita T. Reed J.C. Blood. 1993; 81: 151-157Crossref PubMed Google Scholar). The precise mechanisms that control apoptosis have not been elucidated; it appears that this form of cell death is regulated by a genetic program involving both inducers and repressors (6White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1322) Google Scholar). The tumor suppressor p53 is one of the key proteins affecting the fate of cells exposed to external insults; activation of p53 leads to apoptosis or cell cycle arrest. Inactivation of p53 by either a mutation of the gene or interaction with oncogenic viral or cellular proteins is a common event in the development of malignancies (9Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2553) Google Scholar). On the other hand, recent studies have found pathways of apoptosis independent of p53 (10Yonish-Rouach E. Experientia. 1996; 52: 1001-1007Crossref PubMed Scopus (57) Google Scholar).Protein kinase C (PKC) 1The abbreviations used are:PKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetateTNFαtumor necrosis factor-αIL-1βinterleukin-1βTNFR1TNF receptor 1MAPKmitogen-activated protein kinaseERKextracellular signal-regulated protein kinaseMEKmitogen-activated/extracellular-regulated kinasekbkilobase(s)SPPsphingosine-1-phosphateRbretinoblastoma susceptibility gene product 1The abbreviations used are:PKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetateTNFαtumor necrosis factor-αIL-1βinterleukin-1βTNFR1TNF receptor 1MAPKmitogen-activated protein kinaseERKextracellular signal-regulated protein kinaseMEKmitogen-activated/extracellular-regulated kinasekbkilobase(s)SPPsphingosine-1-phosphateRbretinoblastoma susceptibility gene product plays important roles in physiological events such as cell differentiation and proliferation (11Weinstein I.B. Cancer Res. 1988; 48: 4135-4143PubMed Google Scholar). PKC phosphorylates and activates Raf-1, which leads to the activation of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase (ERK) (12Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1153) Google Scholar). By contrast, studies have shown that proliferation of cells is also inhibited by PKC activation; phorbol esters that can activate PKC have been shown to inhibit the phosphorylation of retinoblastoma susceptibility gene product (Rb) (13Sasaguri T. Ishida A. Kosaka C. Nojima H. Ogata J. J. Biol. Chem. 1996; 271: 8345-8351Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 12-O-tetradecanoylphorbol-13-acetate (TPA) is a novel activator of PKC; previous studies (14de Vente J.E. Kukoly C.A. Bryant W.O. Posekany K.J. Chen J. Fletcher D.J. Parker P.J. Pettit G.J. Lozano G. Cook P.P. Ways D.K. J. Clin. Invest. 1995; 96: 1874-1886Crossref PubMed Scopus (66) Google Scholar, 15de Vente J. Kiley S. Garris T. Bryant W. Hooker J. Posekany K. Parker P. Cook P. Fletcher D. Ways D.K. Cell Growth Differ. 1995; 6: 371-382PubMed Google Scholar, 16Chen C.Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar) have reported that TPA induces apoptosis in MCF-7 breast cancer cells, U937 cells, and Jurkat T-lymphoma cells. TPA is also known to modulate apoptosis induced by various agents (17Jarvis W.D. Povirk L.F. Turner A.J. Traylor R.S. Gewirtz D.A. Pettit G.R. Grant S. Biochem. Pharmacol. 1994; 47: 839-852Crossref PubMed Scopus (95) Google Scholar, 18Walker P.R. Kwast-Welfeld J. Gourdeau H. Leblanc J. Neugebauer W. Sikorska M. Exp. Cell Res. 1993; 207: 142-151Crossref PubMed Scopus (117) Google Scholar). However, the effects of TPA on induction of apoptosis seem to depend upon the type of cells; treatment of cells with inhibitors of PKC resulted in apoptosis in lymphocytes and HL60 cells, and TPA blocked apoptosis induced by VP-16 or interleukin withdrawal (18Walker P.R. Kwast-Welfeld J. Gourdeau H. Leblanc J. Neugebauer W. Sikorska M. Exp. Cell Res. 1993; 207: 142-151Crossref PubMed Scopus (117) Google Scholar, 19Lucas M. Sanchez-Margalet V. Sanz A. Solano F. Biochem. Pharmacol. 1994; 47: 667-672Crossref PubMed Scopus (45) Google Scholar, 20Jarvis W.D. Turner A.J. Povirk L.F. Traylor R.S. Grant S. Cancer Res. 1994; 54: 1707-1714PubMed Google Scholar, 21Rusnak J.M. Lazo J.S. Exp. Cell Res. 1996; 224: 189-199Crossref PubMed Scopus (39) Google Scholar). Studies have suggested that this different response to TPA in apoptosis is, in part, due to the existence of multiple PKC isotypes (22Cuvillier O. Rosenthal D.S. Smulson M.E. Spiegel S. J. Biol. Chem. 1998; 273: 2910-2916Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 23Mizuno K. Noda K. Araki T. Imaoka T. Kobayashi Y. Akita Y. Shimonaka M. Kishi S. Ohno S. Eur. J. Biochem. 1997; 250: 7-18Crossref PubMed Scopus (118) Google Scholar).Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine that was originally identified on the basis of its ability to induce hemorrhagic necrosis in murine transplantable tumors and of its direct cytotoxic effects against transformed cells lines in vivo (24Carswell E.A. Old L.J. Kassel R.L. Green S. Fiore N. Williamson B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3666-3670Crossref PubMed Scopus (3723) Google Scholar, 25Helson L. Helson C. Green S. Exp. Cell Biol. 1979; 47: 53-60PubMed Google Scholar, 26Fransen L. Ruysschaert M.R. Van der Heyden J. Fiers W. Cell Immunol. 1986; 100: 260-267Crossref PubMed Scopus (88) Google Scholar). TNFα plays a physiological role in oncogeny and in the control of major inflammatory and immune reactions affecting both myelocytic and nonmyelocytic cells (27Fletcher J.R. Arch. Surg. 1993; 128: 1192-1196Crossref PubMed Scopus (46) Google Scholar). It is primarily produced by activated macrophages and exerts its effect through two distinct cell surface receptors, denoted TNFR1 and TNFR2. These two TNFα receptors are present on all nucleated cell types (28Hohmann H.P. Remy R. Brockhaus M. van Loon A.P. J. Biol. Chem. 1989; 264: 14927-14934Abstract Full Text PDF PubMed Google Scholar, 29Vandenabeele P. Declercq W. Vanhaesebroeck B. Grooten J. Fiers W. J. Immunol. 1995; 154: 2904-2913PubMed Google Scholar). TNFα preferentially kills transformed cells in vivo although it is not cytotoxic to normal diploid cells (26Fransen L. Ruysschaert M.R. Van der Heyden J. Fiers W. Cell Immunol. 1986; 100: 260-267Crossref PubMed Scopus (88) Google Scholar, 28Hohmann H.P. Remy R. 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Science. 1966; 154: 604-612Crossref PubMed Scopus (718) Google Scholar, 52Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12726) Google Scholar). When cells are exposed to external insults, negative regulation of the cell cycle or apoptosis occurs; cells which fail to repair DNA are eliminated through apoptosis (53Lotem J. Cragoe Jr., E.J. Sachs L. Blood. 1991; 78: 953-960Crossref PubMed Google Scholar, 54Imamura J. Miyoshi I. Koeffler H.P. Blood. 1994; 84: 2412-2421Crossref PubMed Google Scholar). In this study, we have investigated the mechanisms of apoptosis by a potent stimulator of PKC, TPA in the human monocytic cell line U937 and the myeloblastic cell line KY821. Our results show that TPA induces apoptosis, at least in part, through a pathway which requires endogenous production of TNFα in these cells.Our study showed that TPA stimulated production of TNFα in these cells, and pretreatment of cells with anti-TNFα neutralizing antibody abrogated apoptosis induced by TPA. Moreover, exogenously added TNFα also induced apoptosis in U937 cells. TPA also increased levels of IL-1β mRNA in U937cells. IL-1β is a cytokine, which is also produced by cells of monocytic lineage, and TNFα and IL-1β share a number of biological properties although they are molecularly distinct cytokines (55Le J. Vilcek J. Lab. Invest. 1987; 56: 234-248PubMed Google Scholar). However, treatment with anti-IL-1β antibody did not affect apoptosis induced by TPA. TNFα induces apoptosis through binding to TNFR1 (56Baker S.J. Reddy E.P. Oncogene. 1996; 12: 1-9PubMed Google Scholar). TNFR1 belongs to the same receptor family as Fas (APO/CD95) which is known to trigger apoptosis in a number of different cell types upon ligand binding (57Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4537) Google Scholar). Studies have shown that both receptors contain an approximately 70-amino acid domain in the cytoplasmic region known as the “death domain,” which is required for the cytotoxic effects (58Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Abstract Full Text PDF PubMed Google Scholar, 59Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1162) Google Scholar). In this study, TPA increased levels of TNFR1 mRNA in U937 and KY821 cells. Moreover, the overexpression of TNFR1 enhanced the apoptosis induced by TPA in U937 cells whereas TPA did not affect levels of Fas. The cytotoxic effect of TNFα on neoplastic cells has been shown also to be synergistically enhanced by TPA in vitro (34Wang A.M. Creasey A.A. Ladner M.B. Lin L.S. Strickler J. Van Arsdell J.N. Yamamoto R. Mark D.F. Science. 1985; 228: 149-154Crossref PubMed Scopus (484) Google Scholar). Taken together, these results suggest that endogenous production of TNFα plays a crucial role in apoptosis of these cells.Death domain receptors including TNFR1 are constitutively expressed in various types of cells; these receptors are maintained in inactive state in the absence of ligands (60Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar, 61Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (999) Google Scholar). Prior studies (60Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar) have suggested that it is difficult to generate stable cell lines overexpressing death domain receptors because the overexpression leads to receptor aggregation and constitutive apoptosis. We stably transfected U937 cells with a human TNFR1 expression vector and obtained several clones with different levels of TNFR1 mRNA. The most highly TNFR1-overexpressing U937 cells showed an approximately 6-fold TNFR1 level, as compared with that of control-transfected cells, with a slightly but significantly elevated basal level of apoptosis. A recent study has found a protein that protects against ligand-independent signaling by TNFR1 and other death domain receptors (60Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar). This protein, silencer of death domain (SODD), inhibits the intrinsic self-aggregation properties of the death domain and maintains TNFR1 in an active monomeric state; TNF reduces levels of silence of death domain bound to TNFR1 in U937 cells (60Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar). The mechanism responsible for the overexpression of TNFR1 in our U937 cells remains to be elucidated but may involve the expression of silence of death domain at a high level in these U937 cells.Recent studies have demonstrated that an activation of PKC-modulated apoptosis though results are contradictory; TPA induces apoptosis in certain cells including U937 cells but inhibits apoptosis induced by various agents including ceramide (18Walker P.R. Kwast-Welfeld J. Gourdeau H. Leblanc J. Neugebauer W. Sikorska M. Exp. Cell Res. 1993; 207: 142-151Crossref PubMed Scopus (117) Google Scholar, 21Rusnak J.M. Lazo J.S. Exp. 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Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 66Mayne G.C. Murray A.W. J. Biol. Chem. 1998; 273: 24115-24121Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These results suggest that whether apoptosis is induced in response to TPA depends on the type of cell. In this study, TPA induced apoptosis in U937 cells; these results were consistent with previous studies (15de Vente J. Kiley S. Garris T. Bryant W. Hooker J. Posekany K. Parker P. Cook P. Fletcher D. Ways D.K. Cell Growth Differ. 1995; 6: 371-382PubMed Google Scholar, 23Mizuno K. Noda K. Araki T. Imaoka T. Kobayashi Y. Akita Y. Shimonaka M. Kishi S. Ohno S. Eur. J. Biochem. 1997; 250: 7-18Crossref PubMed Scopus (118) Google Scholar). We also showed that TPA stimulated MAPK activity and inhibition of PKC or MAPK pathway almost completely prevented the production of TNFα and the induction of apoptosis by TPA. Activation of the MAPK signaling pathway via PKC is an important mechanism for several biological events such as apoptosis, and PKC regulates the MAPK pathway, alone or with other mechanisms (67Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar,68Hall-Jackson C.A. Jones T. Eccles N.G. Dawson T.P. Bond J.A. Gescher A. Wynford-Thomas D. Br. J. Cancer. 1998; 78: 641-651Crossref PubMed Scopus (23) Google Scholar). Our results indicate that activation of the PKC and MAPK-signaling cascade leads to the accumulation of TNFα, which is required for the induction of apoptosis by TPA. However, much evidence accumulated suggests that TPA and TNFα exert opposing effects on the induction of apoptosis in U937 cells (60Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar).TNFα stimulates sphingomyelin degradation with the subsequent formation of ceramide, and ceramide induces apoptosis in leukemia cells (69Dressler K.A. Mathias S. Kolesnick R.N. Science. 1992; 255: 1715-1718Crossref PubMed Scopus (367) Google Scholar, 70Wiegmann K. Schutze S. Kampen E. Himmler A. Machleidt T. Kronke M. J. Biol. Chem. 1992; 267: 17997-18001Abstract Full Text PDF PubMed Google Scholar, 71Yanaga F. Watson S.P. FEBS Lett. 1992; 314: 297-300Crossref PubMed Scopus (57) Google Scholar, 72Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1598) Google Scholar, 73Dbaibo G.S. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar, 74Jarvis W.D. Fornari F.A. Traylor R.S. Martin H.A. Kramer L.B. Erukulla R.K. Bittman R. Grant S. J. Biol. 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Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). stimulates MAPK activity and activation of MAPK cell J. Spiegel S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. M. J. M.E. Science. 1995; 270: PubMed Scopus Google Scholar). Taken together, these studies suggest that activation of MAPK may different pathways responsible for the opposing effects on apoptosis and cell cell lines including U937 and HL60 cells of and of cells that at different of differentiation D.K. Cancer Res. 1987; 47: Google Scholar). A recent has shown that certain PKC isotypes were in cells K. Noda K. Araki T. Imaoka T. Kobayashi Y. Akita Y. Shimonaka M. Kishi S. Ohno S. Eur. J. Biochem. 1997; 250: 7-18Crossref PubMed Scopus (118) Google Scholar). we evidence that TPA induced apoptosis through production of TNFα in U937 cells, the mechanism for the studies by other and remains Studies suggest that the the levels of ceramide and is for the fate of cells (65Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 66Mayne G.C. Murray A.W. J. Biol. Chem. 1998; 273: 24115-24121Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). these the may be of cell type the Furthermore, the levels of of PKC TNFα, and may be in U937 cell line, to opposing effects on programmed cell have shown that U937 cells have type p53 or a mutation that A at the of results in a of the p53 mRNA K. H. R. K. K. F. F. Y. H. Blood. 1992; PubMed Google Scholar, A. M. Br. J. 1990; PubMed Scopus Google Scholar). Our p53 protein in U937 cells, and TPA did not induce p53. apoptosis can in cells for p53 or with p53 M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). TNFα may be of for apoptosis in U937 cells that lack p53. Furthermore, in this study, U937 cells constitutively underwent apoptosis to an Moreover, the constitutive apoptosis was by the overexpression of TNFR1 in these cells. the TNFα protein in the of cells was the level of showed that cell of U937 cells TNFα protein and produced TNFα may induce apoptosis, in part, through in the Apoptosis, a morphologically distinguished form of programmed cell death, plays important roles not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases including cancer, autoimmune disease, viral infection, and neurodegenerative disorder (1Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar, 2Williams G.T. Smith C.A. Cell. 1993; 74: 777-779Abstract Full Text PDF PubMed Scopus (903) Google Scholar, 3Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar, 4Martin S.J. Green D.R. Crit. Rev. Oncol. Hematol. 1995; 18: 137-153Crossref PubMed Scopus (154) Google Scholar, 5Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6174) Google Scholar, 6White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1322) Google Scholar). Moreover, various chemicals and drugs for treatment of cancers are known to destroy tumor cells through apoptosis (7Eastman A. Curr. Opin. Oncol. 1990; 2: 1109-1114Crossref PubMed Scopus (11) Google Scholar, 8Miyashita T. Reed J.C. Blood. 1993; 81: 151-157Crossref PubMed Google Scholar). The precise mechanisms that control apoptosis have not been elucidated; it appears that this form of cell death is regulated by a genetic program involving both inducers and repressors (6White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1322) Google Scholar). The tumor suppressor p53 is one of the key proteins affecting the fate of cells exposed to external insults; activation of p53 leads to apoptosis or cell cycle arrest. Inactivation of p53 by either a mutation of the gene or interaction with oncogenic viral or cellular proteins is a common event in the development of malignancies (9Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2553) Google Scholar). On the other hand, recent studies have found pathways of apoptosis independent of p53 (10Yonish-Rouach E. Experientia. 1996; 52: 1001-1007Crossref PubMed Scopus (57) Google Scholar). kinase C (PKC) 1The abbreviations used are:PKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetateTNFαtumor necrosis factor-αIL-1βinterleukin-1βTNFR1TNF receptor 1MAPKmitogen-activated protein kinaseERKextracellular signal-regulated protein kinaseMEKmitogen-activated/extracellular-regulated kinasekbkilobase(s)SPPsphingosine-1-phosphateRbretinoblastoma susceptibility gene product 1The abbreviations used are:PKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetateTNFαtumor necrosis factor-αIL-1βinterleukin-1βTNFR1TNF receptor 1MAPKmitogen-activated protein kinaseERKextracellular signal-regulated protein kinaseMEKmitogen-activated/extracellular-regulated kinasekbkilobase(s)SPPsphingosine-1-phosphateRbretinoblastoma susceptibility gene product plays important roles in physiological events such as cell differentiation and proliferation (11Weinstein I.B. Cancer Res. 1988; 48: 4135-4143PubMed Google Scholar). PKC phosphorylates and activates Raf-1, which leads to the activation of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase (ERK) (12Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1153) Google Scholar). By contrast, studies have shown that proliferation of cells is also inhibited by PKC activation; phorbol esters that can activate PKC have been shown to inhibit the phosphorylation of retinoblastoma susceptibility gene product (Rb) (13Sasaguri T. Ishida A. Kosaka C. Nojima H. Ogata J. J. Biol. Chem. 1996; 271: 8345-8351Abstract Full Text Full Text PDF PubMed Scopus (41)