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
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ABSTRACT |
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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
PKC 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 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.
Materials--
Anti-Bcl2, Bax, PKC 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 Assay of PKC 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).
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.
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 Nicotine Stimulates Activation of PKC
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 PKC 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.
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.
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 PKC 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.
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
,
,
,
, and
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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 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.
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).
Activity and Bcl2 Phosphorylation in
Vitro--
PKC
was immunoprecipitated from cell lysates with PKC
antibody after nicotine treatment. Immunoprecipitated PKC
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
[
-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 PKC
were determined by autoradiography. Bcl2 phosphorylation using purified
activated PKC
(Panvera, Madison, WI), ERK1, or ERK2 (Calbiochem) was
performed as described previously (17).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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
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.
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
PKC
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 PKC
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
PKC
and/or ERK1/2.
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Fig. 3.
PKC and ERK1/2
directly phosphorylate Bcl2 in vitro. Bcl2 was
immunoprecipitated from cell lysates and incubated with purified
activated PKC
, 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 PKC
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. PKC
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 PKC
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.
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 PKC
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.
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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.
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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
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.
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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.
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ACKNOWLEDGEMENT |
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We thank Dr. Lei Xiao for providing NCI-H82 human lung cancer cells.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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