A Functional Role for Nicotine in Bcl2 Phosphorylation and Suppression of Apoptosis

Abstract

Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by diverse stimuli including chemotherapeutic drugs. However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is unclear. Bcl2 is a potent antiapoptotic protein and tumor promotor that is expressed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. It is possible that nicotine may regulate Bcl2 to stimulate cell survival. Here we report that nicotine can induce Bcl2 phosphorylation exclusively at the serine 70 site in association with prolonged survival of SCLC H82 cells expressing wild-type but not the phosphorylation-deficient S70A mutant Bcl2 after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16). Nicotine induces activation of PKCα and the MAPKs ERK1 and ERK2, which are physiological Bcl2 kinases. Furthermore, ET-18-OCH3, a specific phospholipase C (PLC) inhibitor, blocks nicotine-stimulated Bcl2 phosphorylation and promotes apoptosis, suggesting that PLC may be involved in nicotine activation of Bcl2 kinases. Using a genetic approach, the gain-of-function S70E mutant, which mimics Ser70 site phosphorylation in the flexible loop domain, potently enhances chemoresistance in SCLC cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. Therefore, novel therapeutic strategies for lung cancer in which Bcl2 is expressed may be used to abrogate the anti-apoptotic activity of Bcl2 by inhibiting multiple upstream nicotine-activated pathways. Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by diverse stimuli including chemotherapeutic drugs. However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is unclear. Bcl2 is a potent antiapoptotic protein and tumor promotor that is expressed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. It is possible that nicotine may regulate Bcl2 to stimulate cell survival. Here we report that nicotine can induce Bcl2 phosphorylation exclusively at the serine 70 site in association with prolonged survival of SCLC H82 cells expressing wild-type but not the phosphorylation-deficient S70A mutant Bcl2 after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16). Nicotine induces activation of PKCα and the MAPKs ERK1 and ERK2, which are physiological Bcl2 kinases. Furthermore, ET-18-OCH3, a specific phospholipase C (PLC) inhibitor, blocks nicotine-stimulated Bcl2 phosphorylation and promotes apoptosis, suggesting that PLC may be involved in nicotine activation of Bcl2 kinases. Using a genetic approach, the gain-of-function S70E mutant, which mimics Ser70 site phosphorylation in the flexible loop domain, potently enhances chemoresistance in SCLC cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. Therefore, novel therapeutic strategies for lung cancer in which Bcl2 is expressed may be used to abrogate the anti-apoptotic activity of Bcl2 by inhibiting multiple upstream nicotine-activated pathways. nicotinic acetylcholine receptor small cell lung cancer non-small cell lung cancer protein kinase C mitogen-activated protein kinase C-Jun N-terminal protein kinase extracellular signal-regulated kinase staurosporine PD98059 phospholipase c wild type mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Lung cancer is currently the leading cause of cancer mortality worldwide and has a strong etiological association with smoking (1Parker S.L. Tong T. Bolden S. Wingo P.A. CA-Cancer J. Clin. 1997; 47: 5-27Crossref PubMed Scopus (2333) Google Scholar). Growing evidence suggests that tobacco use can also affect the responsiveness of cancer cells to treatment, especially for lung, head, and neck cancers (2Rocher C. Dishe S. Saunder M.I. Clin. Oncol. 1992; 4: 214-216Abstract Full Text PDF Scopus (54) Google Scholar, 3Browman G.P. Wong G. Hodson I. Sathya J. Russel R. McAlpine L. Skingley P. Levine M.N. N. Engl. J. Med. 1993; 328: 159-163Crossref PubMed Scopus (459) Google Scholar). Nicotine is a major component in tobacco and exists at high concentrations (90-1000 nm) in the blood of smokers (4Benowitz N. N. Engl. J. Med. 1988; 319: 1318-1330Crossref PubMed Scopus (830) Google Scholar). Chronic exposure to nicotine can lead to sustained activation of growth-promoting pathways and may facilitate the development of lung cancer and potentially reduce the efficacy of anti-cancer agents by activating survival pathways (5Heusch W.L. Maneckjee R. Carcinogenesis. 1998; 19: 551-556Crossref PubMed Scopus (195) Google Scholar). Nicotine signaling can occur as a result of activation of either the classical and/or nonclassical nicotine receptor pathways (6Wright S.C. Zhong J. Zheng H. Larrick J.W. FASEB J. 1993; 7: 1045-1051Crossref PubMed Scopus (171) Google Scholar, 7Garnier M. Lamacz M. Tonon M.C. Vaudry H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11743-11747Crossref PubMed Scopus (29) Google Scholar, 8Giovannelli A. Grassi F. Mattei E. Mileo A.M. Eusebi F. Giovannelli A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10069-10073Crossref PubMed Scopus (31) Google Scholar, 9Zhang S. Day I.N.M. Ye S. Physiol. Genomics. 2001; 5: 187-192Crossref PubMed Scopus (82) Google Scholar). Classical nicotine signaling occurs through the nicotinic acetylcholine receptor (nAChR)1 superfamily, which consists of α, β, γ, ε, and δ subunits (7Garnier M. Lamacz M. Tonon M.C. Vaudry H. Proc. Natl. Acad. Sci. U. S. 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Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (253) Google Scholar). This reaction produces two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which mediate the intracellular Ca2+release and activation of protein kinase C (PKC) that can trigger a downstream kinase cascade leading to activation of the Raf/MEK/ERKs cell growth pathway (16Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar). Bcl2 is a cellular proto-oncogene that functions as a potent antiapoptotic molecule, and its dysregulation can be oncogenic (18Linette G.P. Hess J.L. Sentman C.L. Korsmeyer S.J. Blood. 1995; 86: 1255-1260Crossref PubMed Google Scholar). A correlation between heavy cigarette smoking and increased expression of Bcl2 in patients with lung, head, or neck cancer suggests that Bcl2 may be a target of carcinogens found in tobacco smoke (19Gallo O. Bianchi S. Porfirio B. J. Natl. Cancer Inst. 1995; 87: 1024-1025Crossref PubMed Scopus (29) Google Scholar). We previously discovered that phosphorylation of Bcl2 at Ser70 by growth factor-activated protein kinases including PKC and the MAPKs (ERK1/2) can positively regulate the anti-apoptotic function of Bcl2 (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar,21Ruvolo P.P Deng X. Carr B.K. May W.S. J. Biol. Chem. 1998; 273: 25436-25442Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Recent reports indicate that nicotine can activate both PKC (22Maneckjee R. Minna J.D. Cell Growth Differ. 1994; 5: 1033-1040PubMed Google Scholar) and MAPK (ERK2) in association with prolonged survival of human lung cancer cells in culture (5Heusch W.L. Maneckjee R. Carcinogenesis. 1998; 19: 551-556Crossref PubMed Scopus (195) Google Scholar). However, the downstream survival substrate(s) of nicotine-activated PKC and MAPK/ERK2 has not been identified. Therefore, we tested whether nicotine-mediated enhancement of tumor cell survival may occur, at least in part, through the functional phosphorylation of Bcl2 and how nicotine-induced Bcl2 phosphorylation affects the chemoresistance of lung cancer cells. Anti-Bcl2, Bax, PKCα, ERK1, ERK2, JNK1, p38, and phospho-specific ERK p38 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nicotine, etoposide (VP-16), and cisplatin were obtained from Sigma. ET-18-OCH3 was purchased from Calbiochem. NCI-H69 cell line and human Bcl2 cDNA were obtained from ATCC (Manassas, VA). All reagents used were obtained from commercial sources unless otherwise stated. NCI-H82 and NCI-H69 cells were maintained in RPMI 1640 with 10% fetal bovine serum. Nucleotides corresponding to each serine or threonine residue inBcl2 cDNA were substituted to create a conservative alteration to alanine or glutamic acid with a site-directed mutagenesis kit (Clontech). Each single mutant was confirmed by sequencing of the cDNA and was then cloned into the pCIneo mammalian expression vector (Promega). The pCIneo plasmid containing each Bcl2 mutant cDNA was transfected with NCI-H82 cells by electroporation. Clones stably expressing WT or mutant Bcl2 were selected in a medium containing G418 (0.6 mg/ml). The expression levels of exogenous Bcl2 were compared by Western blot analysis. Three separate clones for each mutant expressing similar amounts of exogenous Bcl2 were selected for further analysis. Cells were washed with phosphate-free RPMI medium and metabolically labeled with [32P]orthophosphoric acid for 60 min. After agonist or inhibitor addition, cells were washed with ice-cold phosphate-buffered saline and lysed in detergent buffer, and Bcl2 was immunoprecipitated as described previously (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). The were to to a and to for the at Bcl2 phosphorylation was by The was then by Western blot with a Bcl2 and by as described previously T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). PKCα was immunoprecipitated from cell with PKCα after nicotine PKCα was in of kinase containing of and The was for at The reaction was by the of and of the for min. The were by The of PKCα were by Bcl2 phosphorylation activated PKCα ERK1, or was as described previously (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar). The and cells were kit to the The of cells of or cells of was the obtained by cell as described X. L. F. Ruvolo P. May W.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Cell was also confirmed the T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Nicotine is a survival agonist that inhibits apoptosis after various (6Wright S.C. Zhong J. Zheng H. Larrick J.W. FASEB J. 1993; 7: 1045-1051Crossref PubMed Scopus (171) Google Scholar). However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is not Bcl2 is the survival involved in the of apoptosis Cell. 87: Full Text Full Text PDF PubMed Scopus Google Scholar). levels of Bcl2 are expressed in human lung cells that SCLC and N. M. Minna J. Cancer 1994; Google Scholar). phosphorylation of Bcl2 can positively regulate the anti-apoptotic function of Bcl2 (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar), is possible that nicotine-induced cell survival may be with Bcl2 phosphorylation. NCI-H69 cells expressing high levels of Bcl2 N. M. Minna J. Cancer 1994; Google Scholar) were metabolically labeled with [32P]orthophosphoric acid and with The result that nicotine can potently stimulate Bcl2 phosphorylation in human SCLC NCI-H69 cells and are currently the for treatment of patients with lung cancer Oncol. 2001; PubMed Scopus Google Scholar). the of nicotine on the regulation of apoptosis, NCI-H69 cells expressing high levels of Bcl2 were with cisplatin or in the or of nicotine for various Cell was as described The that nicotine cell survival after treatment with either cisplatin or These that nicotine-induced increased cell survival is with Bcl2 phosphorylation. the of indicate that the growth can induce Bcl2 phosphorylation exclusively at Ser70 and potently cell survival (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). However, as the exposure of cells to Bcl2 can also be at multiple including and K. H. Korsmeyer S. Cell. Biol. 19: PubMed Scopus Google Scholar). nicotine-induced phosphorylation a of alanine including and was to abrogate each site phosphorylation. WT and alanine were stably transfected in type of SCLC NCI-H82 cells. We cell line NCI-H82 cells levels of Bcl2 N. M. Minna J. Cancer 1994; Google Scholar). Thus, the survival function of Bcl2 be possible from Bcl2 on cell survival be Three clones for each mutant expressing similar levels of exogenous WT and mutant Bcl2 were selected and and for one are nicotine induces the phosphorylation of and but not S70A Bcl2 indicate that nicotine-induced Bcl2 phosphorylation occurs exclusively at the and are not nicotine can and but not S70A cells from apoptosis after treatment with the chemotherapeutic cisplatin These that Bcl2 phosphorylation at Ser70 may be for nicotine-induced lung cancer cell survival. further whether Ser70 site Bcl2 phosphorylation affects chemoresistance in human SCLC a serine mutant at Ser70 was to Ser70 site phosphorylation. It is that at the serine phosphorylation site can the by phosphorylation N. M. S. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. E. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, S70E Bcl2 mutant Ser70 site Bcl2 phosphorylation. cells expressing S70E mutant Bcl2 a gain-of-function anti-apoptotic that mimics nicotine-stimulated survival These genetic evidence that Ser70 site phosphorylation potently enhances the anti-apoptotic function of which may result in chemoresistance in human SCLC cells. indicate that nicotine can induce Bcl2 phosphorylation at Ser70 in human lung cancer cells However, the protein involved is not PKCα and MAPK can Bcl2 in and in 17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar), we tested whether nicotine activate either of Bcl2 kinases to Bcl2 in SCLC cells. indicate that nicotine induces activation of PKCα and with a at A and nicotine has on either or p38, C and These that nicotine-induced Bcl2 phosphorylation may occur through activation of PKCα and/or PKC and but not and p38 protein which can be by staurosporine and NCI-H82 cells expressing WT Bcl2 were with nicotine for various PKCα was immunoprecipitated from cell and with in in kinase as described were by by or Western blot PKCα cells were with nicotine for various as described in the for A. The cells were and lysed in detergent at the Western blot was to or by a phospho-specific ERK or a of ERK1 and The was with phospho-specific JNK1, phospho-specific p38 or JNK1, p38 and cells were with nicotine in the or of various concentrations of PD98059 or for min. and were as described in is a potent PKC inhibitor, PD98059 can ERK1 or activation T. H. I. M. F. Biochem. Biophys. PubMed Scopus Google Scholar, A. P. D.T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). either PD98059 or can nicotine-induced activation and PKC may be upstream of in the nicotine-stimulated kinase These that nicotine-induced phosphorylation may be on PKC further whether PKCα and are involved in nicotine-induced Bcl2 phosphorylation and cell NCI-H82 cells expressing WT Bcl2 were metabolically labeled with [32P]orthophosphoric acid and with nicotine in the or of or PD98059 or The that or PD98059 potently nicotine-induced Bcl2 phosphorylation a inhibitor, has on nicotine-induced Bcl2 phosphorylation we can that PKCα and but not p38 are involved in nicotine-induced Bcl2 phosphorylation. and but not reduce nicotine-induced survival after treatment with the chemotherapeutic cisplatin These that nicotine-activated PKC and may be for Bcl2 and of PKC and/or MAPK kinase in SCLC cancer cells may be for the suppression of nicotine-induced Bcl2 phosphorylation and survival. which type of may be involved in nicotine-induced Bcl2 phosphorylation in human SCLC S.J. N. 2001; PubMed Scopus Google Scholar) and ET-18-OCH3 phospholipase C D. S.H. S.H. C.L. Cancer 2000; Google Scholar) were tested for on Bcl2 phosphorylation. ET-18-OCH3 but not inhibits both nicotine-induced activation of and Bcl2 phosphorylation in a A and These that nicotine-induced Bcl2 phosphorylation may occur through the nonclassical nicotine receptor that phospholipase C to trigger the downstream physiological Bcl2 kinase cascade and in SCLC cells. ET-18-OCH3 but not also nicotine-induced survival after treatment with cisplatin Thus, ET-18-OCH3 may use as a novel anti-cancer in SCLC that Nicotine induces Bcl2 phosphorylation at Ser70 in the flexible loop in association with cell but the mechanism is not Recent reports that phosphorylation not only the of Bcl2 to with the (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar), but also the Bcl2 protein that to X. F. H. May W.S. Blood. 2001; Scholar, S. K. J. A.M. J. Med. PubMed Scopus Google Scholar). whether nicotine-induced Bcl2 phosphorylation affects H82 cells expressing WT Bcl2 and were with nicotine in the or of various concentrations of Bcl2 was by a Bcl2 Bcl2 (i.e. was by After Western blot the of Bcl2 or was by indicate that of Bcl2 with growth and that the Bcl2 is increased to of Bcl2 after nicotine treatment This compared with (i.e. nicotine can induce Bcl2 phosphorylation at Ser70 and increased association may occur, at least in part, through Ser70 phosphorylation. whether of Bcl2 phosphorylation affects ET-18-OCH3 was used to that ET-18-OCH3 can reduce association in a occurs in Bcl2 ET-18-OCH3 can potently nicotine-induced Bcl2 phosphorylation at is possible that phosphorylation at Ser70 may be for association was in H82 cells expressing the gain-of-function mutant S70E or S70A that of S70E Bcl2 with growth only of S70A Bcl2 The Bcl2 levels of S70E and S70A are the These that phosphorylation of Bcl2 at the may be for association with and one mechanism by which Bcl2 phosphorylation its antiapoptotic Bcl2 and are key of cell or apoptosis, a for and E. Korsmeyer S.J. Blood. 88: PubMed Google Scholar, J.M. S. 1998; PubMed Scopus Google Scholar). Recent reports indicate that phosphorylation of Bcl2 in the flexible loop can positively regulate the anti-apoptotic function of Bcl2 and that growth (i.e. or growth phosphorylation is with cell survival T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google W.S. T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, M. T. T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation at Ser70 may be for potent antiapoptotic and nicotine induces Bcl2 phosphorylation exclusively at Ser70 nicotine may growth or survival and activate Bcl2 by phosphorylation. nicotine promotes survival in cells expressing WT but not the phosphorylation-deficient S70A Bcl2 mutant, we that the nicotine-induced cell survival and chemoresistance that occur in human SCLC cells may at least in part, from nicotine-stimulated Ser70 Bcl2 phosphorylation. A report that nicotine can also Bcl2 in but the mechanism (5Heusch W.L. Maneckjee R. Carcinogenesis. 1998; 19: 551-556Crossref PubMed Scopus (195) Google Scholar). It is possible that nicotine-induced of the anti-apoptotic activity of Bcl2 from potently enhances the of Bcl2 protein X. F. H. May W.S. Blood. 2001; S. K. J. A.M. J. Med. PubMed Scopus Google Scholar). Nicotine both PKCα and but has on and p38 and PD98059 not only nicotine-induced Bcl2 phosphorylation but also cell These that nicotine-induced Bcl2 phosphorylation occurs through a pathway activation of PKC and/or the kinases. PLC is and described of cell survival and in both SCLC and cells D. S.H. S.H. C.L. Cancer 2000; Google Scholar). Nicotine has been to induce PLC activation through a nonclassical nicotine receptor pathway (7Garnier M. Lamacz M. Tonon M.C. Vaudry H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11743-11747Crossref PubMed Scopus (29) Google Scholar). PLC phosphatidylinositol to both phosphatidylinositol and diacylglycerol (7Garnier M. Lamacz M. Tonon M.C. Vaudry H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11743-11747Crossref PubMed Scopus (29) Google Scholar, 9Zhang S. Day I.N.M. Ye S. Physiol. Genomics. 2001; 5: 187-192Crossref PubMed Scopus (82) Google Scholar). by from the which may facilitate activation of classical S. Day I.N.M. Ye S. Physiol. Genomics. 2001; 5: 187-192Crossref PubMed Scopus (82) Google Scholar). which can trigger a kinase cascade to induce Bcl2 phosphorylation B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. The American Society for Cell Biology, Garland, NY1994Google Scholar, 17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar). ET-18-OCH3 is a inhibitor that can nicotine-induced MAPKs activation and Bcl2 phosphorylation in association with apoptosis, a inhibitor D. S.H. S.H. C.L. Cancer 2000; Google Scholar), has These indicate that nicotine-induced survival and/or chemoresistance may occur through activation of the nonclassical pathway in SCLC cells. Nicotine not activate or p38 and the inhibitor has on nicotine-induced Bcl2 phosphorylation and indicate that and p38 are not involved in survival Thus, and are events that may be ET-18-OCH3, and PD98059 to nicotine-induced Bcl2 phosphorylation. These may in strategies to in patients with Bcl2 expressing lung with (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 20Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, X. L. F. Ruvolo P. May W.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and M. T. T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (65) Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), phosphorylation of Bcl2 induced by agents (i.e. has been to the anti-apoptotic function of Bcl2 in K. H. Korsmeyer S. Cell. Biol. 19: PubMed Scopus Google Scholar, Korsmeyer S.J. M. Y.S. Cell. Biol. 1998; PubMed Scopus Google Scholar). were the is to how of Bcl2 can cell survival after exposure to S. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (65) Google Scholar). genetic evidence not the that phosphorylation Bcl2 expression of S70E Bcl2 mutant potently inhibits lung cancer cell cells expressing WT or alanine in the of nicotine These that Ser70 site phosphorylation enhances the anti-apoptotic function of which may be with increased chemoresistance in SCLC cells. We that phosphorylation of Bcl2 at its with (17Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar), a for cell M. T. K. G. C. Korsmeyer S. 2001; PubMed Scopus Google Scholar). and nicotine not only Bcl2 phosphorylation but also a mechanism currently to the action of C.L. Korsmeyer S.J. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). ET-18-OCH3 also potently inhibits both nicotine-induced Bcl2 phosphorylation and in association with apoptosis, further suggesting that of Bcl2 phosphorylation may facilitate which may trigger the function of the a novel nicotine-stimulated survival pathway that involves Bcl2 phosphorylation in human SCLC cells. Nicotine may activate PLC through G protein-linked activation (7Garnier M. Lamacz M. Tonon M.C. Vaudry H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11743-11747Crossref PubMed Scopus (29) Google Scholar) that a downstream kinase cascade (i.e. PKC and to Bcl2 and cell survival These may to a novel for treatment of human SCLC by the nicotine-activated Bcl2 kinase We for NCI-H82 human lung cancer cells.