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

Haiqiang MaiDagger, W. Stratford May, Fengqin Gao, Zhaohui Jin, and Xingming Deng§

From the Shands Cancer Center and Department of Medicine, University of Florida, Gainesville, Florida 32610-0232

Received for publication, September 4, 2002, and in revised form, November 5, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PKCalpha 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lung cancer is currently the leading cause of cancer mortality worldwide and has a strong etiological association with smoking (1). Growing evidence suggests that tobacco use can also affect the responsiveness of cancer cells to treatment, especially for lung, head, and neck cancers (2-3). Nicotine is a major component in tobacco and exists at high concentrations (90-1000 nM) in the blood of smokers (4). 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 (5). Nicotine signaling can occur as a result of activation of either the classical and/or nonclassical nicotine receptor pathways (6-9). Classical nicotine signaling occurs through the nicotinic acetylcholine receptor (nAChR)1 superfamily, which consists of alpha , beta , gamma , epsilon , and delta  subunits (7). These nAChRs are transmembrane cationic channels that are activated on cholinergic stimulation (7). The nonclassical nicotine receptor pathway is mediated through a noncholinergic metabotropic receptor, which is not sensitive to acetylcholine (7, 10, 11). The nonclassical receptor type belongs to the G protein-linked receptor superfamily, which contains seven hydrophobic transmembrane domains and is positively coupled to PLC (7). The hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (12), through the direct action of phospholipase C is one of the earliest key events in the regulation of various cell functions by extracellular signaling molecules (12-15). 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 (16, 17).

Bcl2 is a cellular proto-oncogene that functions as a potent antiapoptotic molecule, and its dysregulation can be oncogenic (18). 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 (19). 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 (17, 20, 21). Recent reports indicate that nicotine can activate both PKC (22) and MAPK (ERK2) in association with prolonged survival of human lung cancer cells in culture (5). 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Anti-Bcl2, Bax, PKCalpha , 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.

Cell Lines, Plasmids, and Transfections-- NCI-H82 and NCI-H69 cells were maintained in RPMI 1640 with 10% fetal bovine serum. Nucleotides corresponding to each serine or threonine residue in Bcl2 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.

Metabolic Labeling, Immunoprecipitation, and Western Blot 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 (17, 20). The samples were subjected to 10-20% gradient SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film for the times indicated at -80 °C. Bcl2 phosphorylation was determined by autoradiography. The same filter was then probed by Western blot analysis with a Bcl2 antibody and developed by using an ECL Kit (Amersham Biosciences) as described previously (20).

Assay of PKCalpha Activity and Bcl2 Phosphorylation in Vitro-- PKCalpha was immunoprecipitated from cell lysates with PKCalpha antibody after nicotine treatment. Immunoprecipitated PKCalpha was suspended in 50 µl of kinase assay buffer containing 20 mM Hepes, pH 7.4, 100 µM CaCl2, 10 mM MgCl2, 200 µg/ml histone-1, 100 µM ATP, 100 µg/ml phosphatidylserine, 2 µCi of [gamma -32P]ATP, and 0.03% Triton X-100. The mixture was incubated for 30 min at 30 °C. The reaction was stopped by the addition of 2 × SDS sample buffer and boiling of the sample for 5 min. The samples were separated by SDS-PAGE. The activities of PKCalpha were determined by autoradiography. Bcl2 phosphorylation using purified activated PKCalpha (Panvera, Madison, WI), ERK1, or ERK2 (Calbiochem) was performed as described previously (17).

Cell Viability Assay-- The apoptotic and viable cells were detected using an ApoAlert Annexin-V kit (Clontech) according to the manufacturer's instructions. The percentage of annexin-Vlow cells (percentage of viable cells) or annexin-Vhigh cells (percentage of apoptotic cells) was determined using the data obtained by fluorescence-activated cell sorter analysis as described (23). Cell viability was also confirmed using the trypan blue dye exclusion method (20).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nicotine Induces Bcl2 Phosphorylation in Human SCLC Cells and Is Associated With Suppression of Apoptosis-- Nicotine is a survival agonist that inhibits apoptosis after various stresses (6). However, the intracellular signal transduction mechanism(s) involved in nicotine suppression of apoptosis is not clear. Bcl2 is the first identified survival gene involved in the control of apoptosis (24). High levels of endogenous Bcl2 are expressed in some human lung carcinoma cells that include SCLC and NSCLC (25). Because phosphorylation of Bcl2 can positively regulate the anti-apoptotic function of Bcl2 (17, 20), it is possible that nicotine-induced cell survival may be associated with Bcl2 phosphorylation. To test this possibility, NCI-H69 cells expressing high levels of endogenous Bcl2 (25) were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine. The result indicates that nicotine can potently stimulate endogenous Bcl2 phosphorylation in human SCLC NCI-H69 cells (Fig. 1). Cisplatin and VP-16 are currently the most useful clinical drugs for treatment of patients with lung cancer (26). To test the effect of nicotine on the regulation of apoptosis, NCI-H69 cells expressing high levels of endogenous Bcl2 were treated with cisplatin or VP-16 in the absence or presence of nicotine for various times. Cell death was assessed as described under "Experimental Procedures." The viability curve shows that nicotine significantly prolongs cell survival after treatment with either cisplatin or VP-16 (Fig. 1B). These findings reveal that nicotine-induced increased cell survival is closely associated with Bcl2 phosphorylation. Data represent the mean ± S.D. of three determinations.


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Fig. 1.   Nicotine induces Bcl2 phosphorylation in association with suppression of apoptosis in human SCLC cells. A, NCI-H69 cells expressing high levels of endogenous Bcl2 were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine for 30 min. Bcl2 was immunoprecipitated by using Bcl2 antibody. Phosphorylation of Bcl2 was determined by autoradiography (upper). Western blot analysis was performed to confirm and quantify Bcl2 protein (lower). B, NCI-H69 cells were treated with VP-16 (25 µM) or cisplatin (4 µM) in the absence or presence of nicotine for various times as indicated. At the indicated times, samples were harvested and analyzed for annexin-V and phosphatidylinositol binding by flow cytometry. Cell viability was determined by fluorescence-activated cell sorter as described previously (23). Data represent the mean ± S.D. of three determinations.

Phosphorylation of Bcl2 at Ser70 May Be Required for Nicotine-induced Survival-- Our previous findings indicate that the growth factor interleukin-3 can induce Bcl2 phosphorylation exclusively at Ser70 and potently promote cell survival (17, 20). However, under certain stress situations such as the exposure of cells to paclitaxel, Bcl2 can also be phosphorylated at multiple sites including Thr69, Ser70, and Ser87 (27). To identify nicotine-induced phosphorylation sites, a series of serine/threonine right-arrow alanine mutants, including T69A, S70A, and S87A, was created to abrogate each individual site phosphorylation. WT and alanine mutants were stably transfected in another type of SCLC NCI-H82 cells. We chose this cell line because NCI-H82 cells contain very low levels of endogenous Bcl2 (25). Thus, the survival function of individual Bcl2 mutants could be more accurately evaluated because any possible effect from endogenous Bcl2 on cell survival could be avoided. Three clones for each mutant expressing similar levels of exogenous WT and Ala mutant Bcl2 were selected and tested, and representative results for one clone are shown. Because nicotine induces the phosphorylation of WT, T69A, and S87A, but not S70A Bcl2 (Fig. 2A), these findings indicate that nicotine-induced Bcl2 phosphorylation occurs exclusively at Ser70, whereas the Thr69 and Ser87 phosphorylation sites are not involved. Importantly, nicotine can protect WT, T69A, and S87A but not S70A Bcl2-expressing cells from apoptosis after treatment with the clinically useful chemotherapeutic drug, cisplatin (Fig. 2B). These findings suggest that Bcl2 phosphorylation at Ser70 may be required for nicotine-induced lung cancer cell survival. To further demonstrate genetically whether Ser70 site Bcl2 phosphorylation affects chemoresistance in human SCLC cells, a serine right-arrow glutamate mutant at Ser70 was created to mimic Ser70 site phosphorylation. It is well known that substitution at the serine phosphorylation site using glutamate can mimic the charge conferred by phosphorylation (28, 29). Therefore, S70E Bcl2 mutant could functionally mimic Ser70 site Bcl2 phosphorylation. Interestingly, cells expressing S70E mutant Bcl2 represent a gain-of-function anti-apoptotic phenotype that mimics nicotine-stimulated survival (Fig. 2). These comparative results provide genetic evidence that Ser70 site phosphorylation potently enhances the anti-apoptotic function of Bcl2, which may result in chemoresistance in human SCLC cells.


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Fig. 2.   Phosphorylation of Bcl2 at Ser70 may be required for nicotine-induced survival. A, NCI-H82 cells expressing WT, T69A, S70A, or S87A Bcl2 mutants were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine for 30 min. Phosphorylation of Bcl2 was analyzed as in Fig. 1A. B, NCI-H82 cells expressing WT, T69A, S70A, or S87A Bcl2 were treated with cisplatin in the absence or presence of nicotine at the indicated time. Cell viability was assessed as in Fig. 1B. Similar results were obtained in all these studies using three separate clones for each mutant expressing similar amounts of exogenous Bcl2. Representative results for one clone are presented.

Nicotine Stimulates Activation of PKCalpha and MAPK ERK1/2-- Our findings indicate that nicotine can induce Bcl2 phosphorylation at Ser70 in human lung cancer cells (Fig. 2). However, the protein kinase(s) involved is not clear. Because PKCalpha and MAPK ERK1/2 can directly phosphorylate Bcl2 in vitro and in vivo (Fig. 3; Ref. 17), we tested whether nicotine might activate either of these Bcl2 kinases to phosphorylate Bcl2 in SCLC cells. Time course experiments indicate that nicotine induces activation of PKCalpha and ERK1/2 with a peak at 30 min (Fig. 4, A and B). By contrast, nicotine has no effect on either JNK1 or p38, respectively (Fig. 4, C and D). These findings suggest that nicotine-induced Bcl2 phosphorylation may occur through activation of PKCalpha and/or ERK1/2.


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Fig. 3.   PKCalpha and ERK1/2 directly phosphorylate Bcl2 in vitro. Bcl2 was immunoprecipitated from cell lysates and incubated with purified activated PKCalpha , ERK1, or ERK2 in an in vitro kinase assay as described under "Experimental Procedures." Phosphorylation of Bcl2 was determined by autoradiography.


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Fig. 4.   Nicotine activates PKCalpha and ERK1/2 but not JNK and p38 protein kinases, which can be inhibited by staurosporine and PD98059. A, NCI-H82 cells expressing WT Bcl2 were treated with nicotine (1 µM) for various times. PKCalpha was immunoprecipitated from cell lysates and incubated with purified histone-1 in an in vitro kinase assay as described under "Experimental Procedures." Reaction mixtures were analyzed by SDS-PAGE followed by autoradiography or Western blot using PKCalpha antibody. B-D, cells were treated with nicotine for various times as described in the legend for Fig. 1A. The cells were harvested, washed, and lysed in detergent buffer at the indicated times. Western blot analysis was performed to detect phosphorylated ERK1/2 (p-ERK1/2) or total ERK1/2 by using a phospho-specific ERK antibody or a mixture of ERK1 and ERK2 antibodies. The same filter was reprobed with phospho-specific JNK1, phospho-specific p38 or JNK1, p38 antibodies, respectively. E and F, cells were treated with nicotine (1 µM) in the absence or presence of various concentrations of PD98059 or Stauro for 30 min. Phosphorylated ERK1/2 and total ERK1/2 were analyzed as described in B.

Staurosporine (Stauro) is a potent PKC inhibitor, whereas PD98059 can specifically inhibit MEK-1-induced ERK1 or ERK2 activation (30, 31). Because either PD98059 or Stauro can inhibit nicotine-induced ERK1/2 activation (Fig. 4, E and F), PKC may be indirectly upstream of ERK1/2 in the nicotine-stimulated kinase cascade. These pharmacological data strongly suggest that nicotine-induced ERK1/2 phosphorylation may be dependent on PKC activity.

Staurosporine and PD98059 Inhibit Nicotine-induced Bcl2 Phosphorylation and Survival-- To further test whether PKCalpha and MAPK/ERK1/2 are involved in nicotine-induced Bcl2 phosphorylation and cell survival, NCI-H82 cells expressing WT Bcl2 were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine in the absence or presence of Stauro or PD98059 or their combination. The results show that Stauro or PD98059 potently inhibit nicotine-induced Bcl2 phosphorylation (Fig. 5A). Because SB20219, a p38-specific inhibitor, has no effect on nicotine-induced Bcl2 phosphorylation (Fig. 5A), we can conclude that PKCalpha and ERK1/2 but not p38 are involved in nicotine-induced Bcl2 phosphorylation. More importantly, Stauro and PD98059, but not SB20219, significantly reduce nicotine-induced survival after treatment with the clinically relevant chemotherapeutic drug, cisplatin (Fig. 5B). These findings reveal that nicotine-activated PKC and EPK1/2 may be required for Bcl2 phosphorylation, and inhibition of PKC and/or MAPK ERK1/2 kinase activities in SCLC cancer cells may be critical for the suppression of nicotine-induced Bcl2 phosphorylation and survival.


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Fig. 5.   Staurosporine and PD98059 blocks nicotine-induced Bcl2 phosphorylation and survival. A, NCI-H82 cells expressing WT Bcl2 were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine in the absence or presence of Stauro, PD98059, or SB20219, or all in a combination. Bcl2 phosphorylation was analyzed as in Fig. 1A. B, NCI-H82 cells expressing WT Bcl2 were treated with cisplatin (Cis, 4 µM) in the absence or presence of nicotine, Stauro, PD98059 (PD), or SB20219 (SB) for 6 days as indicated. Cell viability was assessed as described in the legend for Fig. 1B.

Phospholipase C Inhibitor ET-18-OCH3 Inhibits Nicotine-Induced ERK1/2 Activation and Bcl2 Phosphorylation and Promotes Apoptosis-- To identify which type of receptor(s) may be involved in nicotine-induced Bcl2 phosphorylation in human SCLC cells, hexamethionium (a nAchR blocker) (32) and ET-18-OCH3 (a selective phospholipase C inhibitor) (33) were tested for their effects on Bcl2 phosphorylation. Interestingly, ET-18-OCH3 but not hexamethionium inhibits both nicotine-induced activation of ERK1/2 and Bcl2 phosphorylation in a dose-dependent manner (Fig. 6, A and B). These findings suggest that nicotine-induced Bcl2 phosphorylation may occur through the nonclassical nicotine receptor that couples phospholipase C to trigger the downstream physiological Bcl2 kinase cascade (PKC and ERK1/2) in SCLC cells. Importantly, ET-18-OCH3 but not hexamethionium also blocked nicotine-induced survival after treatment with cisplatin (Fig. 6C). Thus, ET-18-OCH3 may have potential clinical use as a novel anti-cancer drug in treating SCLC that expresses Bcl2.


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Fig. 6.   ET-18-OCH3 but not hexamethionium inhibits nicotine-induced ERK1/2 activation as well as Bcl2 phosphorylation and enhances apoptosis. A, NCI-H82 cells expressing WT Bcl2 were treated with nicotine in the absence or presence of various concentrations of ET-18-OCH3 or hexamethionium for 30 min. Phosphorylated ERK1/2 and total ERK1/2 were assessed as described in the legend for Fig. 4B. B, NCI-H82 cells expressing WT Bcl2 were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine in the absence or presence of hexamethionium or ET-18-OCH3 for 30 min. Bcl2 phosphorylation was assessed as described in the legend for Fig. 1A. C, NCI-H82 cells expressing WT Bcl2 were treated with cisplatin in the absence or presence of nicotine, hexamethionium, or ET-18-OCH3 as indicated. Cell viability was analyzed as described in Fig. 1B.

Nicotine Promotes Bcl2/Bax Association, Which Is Inhibited by ET-18-OCH3, and Phosphorylation at Ser70 Site Is Necessary for Maximal Association with Bax-- Nicotine induces Bcl2 phosphorylation at Ser70 in the flexible loop domain in association with cell survival, but the molecular mechanism is not clear. Recent reports suggest that phosphorylation not only facilitates the ability of Bcl2 to associate with the proapoptotic protein, Bax (17), but also stabilizes the Bcl2 protein that serves to maintain mitochondrial integrity (34, 35). To test whether nicotine-induced Bcl2 phosphorylation affects Bcl2/Bax heterodimerization, H82 cells expressing WT Bcl2 and Bax were treated with nicotine in the absence or presence of various concentrations of ET-18-OCH3. Total Bcl2 was determined by quantitative immunoprecipitation using a Bcl2 antibody. Bax-associated Bcl2 (i.e. bound Bcl2) was co-immunoprecipitated by using Bax antibody. After Western blot analysis, the relative amount of Bcl2 or Bax was determined by densitometry. Results indicate that approximately 40% of total Bcl2 associates with Bax under normal growth conditions and that the Bax-associated Bcl2 is increased to 85% of total Bcl2 after nicotine treatment (Fig. 7A). This increase represents an approximately 2-fold increase compared with control (i.e. 40 versus 85%). Because nicotine can induce Bcl2 phosphorylation at Ser70 (Fig. 2), and enhance Bax/Bcl2 association, this increased association may occur, at least in part, through Ser70 phosphorylation. To assess whether inhibition of Bcl2 phosphorylation affects Bax/Bcl2 association, ET-18-OCH3 was used to test this possibility. Results reveal that ET-18-OCH3 can reduce Bax/Bcl2 association in a dose-dependent manner. Importantly, no change occurs in total Bcl2 level (Fig. 7A). Because ET-18-OCH3 can potently inhibit nicotine-induced Bcl2 phosphorylation at Ser70 (Fig. 6B), it is possible that phosphorylation at Ser70 may be necessary for Bax/Bcl2 association. To test this hypothesis genetically, Bax/Bcl2 association was assessed in H82 cells expressing the gain-of-function mutant S70E or nonphosphorylatable S70A Bcl2. Results reveal that approximately 95% of S70E Bcl2 associates with Bax during growth conditions. By contrast, only 25% of S70A Bcl2 binds Bax (Fig. 7B). The total Bcl2 levels of S70E and S70A are the same (Fig. 7B). These findings suggest that phosphorylation of Bcl2 at the Ser70 site may be necessary for maximal association with Bax and represents one potential mechanism by which Bcl2 phosphorylation regulates its antiapoptotic activity.


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Fig. 7.   Nicotine enhances Bcl2/Bax association that is inhibited by ET-18-OCH3, and phosphorylation at Ser70 site is necessary for maximal association with Bax. A, NCI-H82 cells expressing WT Bcl2 and Bax were treated with nicotine in the absence or presence of various concentrations of ET-18-OCH3 for 30 min. The cells were harvested, washed, and lysed in detergent buffer. The lysates were immunoprecipitated using Bcl2 antibody or Bax antibody, respectively. Total Bcl2 was determined by quantitative immunoprecipitation using a Bcl2 antibody. Bax-associated Bcl2 (i.e. bound Bcl2) was co-immunoprecipitated with Bax antibody and analyzed by Western blotting using Bcl2 antibody. The same filter was reprobed to detect total Bax by using Bax antibody. The percentage of bound Bcl2/total Bcl2 after treatments was determined by densitometry. B, NCI-H82 cells expressing S70E or S70A were lysed in detergent buffer. The lysates were immunoprecipitated using Bcl2 or Bax antibody, respectively. Total Bcl2, bound Bcl2, and Bax and the percentage of bound Bcl2/total Bcl2 were determined as described in the legend for A.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bcl2 and related family members are key regulators of programmed cell death or apoptosis, a natural process required for normal development, malignant transformation, and autoimmune disease (36, 37). Recent reports indicate that phosphorylation of Bcl2 in the flexible loop domain can positively regulate the anti-apoptotic function of Bcl2 and that growth factor (i.e. interleukin-3 or nerve growth factor) -induced phosphorylation is associated with cell survival (20, 38, 39). Because phosphorylation at Ser70 may be required for Bcl2's potent antiapoptotic function, and nicotine induces Bcl2 phosphorylation exclusively at Ser70 (Fig. 2), nicotine may mimic growth factor or survival agonist(s) and functionally activate Bcl2 by phosphorylation. Because nicotine promotes survival in cells expressing WT but not the phosphorylation-deficient S70A Bcl2 mutant, we conclude that the nicotine-induced cell survival and chemoresistance that occur in human SCLC cells may result, at least in part, from nicotine-stimulated Ser70 Bcl2 phosphorylation. A recent report indicates that nicotine can also up-regulate Bcl2 in NSCLC H157 cells, but the mechanism remains unclear (5). It is possible that nicotine-induced up-regulation of the anti-apoptotic activity of Bcl2 results from phosphorylation, because this post-translational modification potently enhances the stability of Bcl2 protein (34, 35).

Nicotine activates both PKCalpha and ERK1/2 but has no effect on JNK1 and p38 activities (Fig. 4). Staurosporine and PD98059 not only block nicotine-induced Bcl2 phosphorylation but also promote cell death (Fig. 5). These data suggest that nicotine-induced Bcl2 phosphorylation occurs through a pathway involving activation of PKC and/or the ERK1/2 kinases.

PLC is an important and well described molecular mediator of cell survival and proliferation in both SCLC and NSCLC cells (33). Nicotine has been reported to induce PLC activation through a nonclassical nicotine receptor signal pathway (7). Activated PLC hydrolyzes phosphatidylinositol bisphosphate to generate both phosphatidylinositol 1,4,5-trisphosphate and diacylglycerol second messengers (7, 9). Phosphatidylinositol 1,4,5-trisphosphate increases cytosolic Ca2+ concentration by releasing Ca2+ from the endoplasmic reticulum, which may facilitate activation of classical PKCs (9). Diacylglycerol directly activates PKC, which can trigger a Raf/MEK/ERK1/2 kinase cascade to induce Bcl2 phosphorylation (10, 17). ET-18-OCH3 is a PLC-specific inhibitor that can block nicotine-induced MAPKs ERK1/2 activation and Bcl2 phosphorylation in association with apoptosis, whereas hexamethonium, a nAChR inhibitor (33), has no effect (Fig. 6). These data indicate that nicotine-induced survival and/or chemoresistance may occur through activation of the nonclassical nicotine-receptor signal transduction pathway involving PLC/PKC/ERK/Bcl2 in SCLC cells. Nicotine does not activate JNK1 or p38 and the p38-specific inhibitor SB20219 has no effect on nicotine-induced Bcl2 phosphorylation (Figs. 4 and 5); these findings indicate that JNK and p38 are not involved in nicotine/Bcl2 survival signaling. Thus, PLC, PKC, and ERK1/2 are nicotine-specific events that may be therapeutically targeted using ET-18-OCH3, Stauro, and PD98059 to inhibit nicotine-induced Bcl2 phosphorylation. These findings may have potential clinical relevance in strategies designed to enhance chemosensitivity in patients with Bcl2 expressing lung cancer.

In contrast with our (17, 20, 23) and others' findings (39-41), multisite phosphorylation of Bcl2 induced by microtubule damaging agents (i.e. paclitaxel) has been proposed to inactivate the anti-apoptotic function of Bcl2 in some systems (27, 42). If this were the case, it is difficult to reconcile how forced overexpression of Bcl2 can significantly prolong cell survival after exposure to paclitaxel (40). Our genetic evidence does not support the notion that phosphorylation inactivates Bcl2 because expression of S70E Bcl2 mutant more potently inhibits lung cancer cell death than cells expressing WT or nonphosphorylatable alanine mutants in the absence of nicotine (Fig. 2). These findings directly demonstrate that Ser70 site phosphorylation enhances the anti-apoptotic function of Bcl2, which may be associated with increased chemoresistance in SCLC cells.

We recently reported that phosphorylation of Bcl2 at Ser70 stabilizes its heterodimeric interaction with Bax (17), a proapoptotic molecule required for cell death (43). Our data support and extend these findings, because nicotine stimulates not only Ser70 site Bcl2 phosphorylation but also Bcl2/Bax heterodimerization (Fig. 7), a mechanism currently thought to block the death action of Bax (44). ET-18-OCH3 also potently inhibits both nicotine-induced Bcl2 phosphorylation and Bcl2/Bax heterodimerization in association with apoptosis, further suggesting that inhibition of Bcl2 phosphorylation may facilitate Bcl2/Bax dissociation, which may trigger the proapoptotic function of Bax.

In summary, the findings reported here identify a novel nicotine-stimulated survival signal pathway that involves Bcl2 phosphorylation in human SCLC cells. Nicotine may activate PLC through receptor-coupled G protein-linked activation (7) that triggers a downstream kinase cascade (i.e. PKC and ERK1/2) to functionally phosphorylate Bcl2 and thereby prolong cell survival (Fig. 8). These findings may help to develop a novel strategy for treatment of human SCLC by blocking the nicotine-activated Bcl2 kinase cascade.


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Fig. 8.   Proposed model of nicotine survival signaling. Nicotine induces phospholipase C activation that triggers PKC/ERK1/2 kinase cascade to phosphorylate survival substrate and Bcl2 and promotes cell survival.


    ACKNOWLEDGEMENT

We thank Dr. Lei Xiao for providing NCI-H82 human lung cancer cells.

    FOOTNOTES

* This work was supported by a Flight Attendant Medical Research Institute Clinical Innovator Award (to X. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China.

§ To whom correspondence should be addressed: University of Florida Shands Cancer Center, 1600 S. W. Archer Rd., Academic Research Bldg., R4-216, P. O. Box 100232, Gainesville, FL 32610-0232. Tel.: 352-392-9232; Fax: 352- 392-5802; E-mail: xdeng@ufscc.ufl.edu.

Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209044200

    ABBREVIATIONS

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; JNK, C-Jun N-terminal protein kinase; ERK, extracellular signal-regulated kinase; Stauro, staurosporine; PD, PD98059; PLC, phospholipase c; WT, wild type; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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