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`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`
`Vol. 275, No. 50, Issue of December 15, pp. 39435–39443, 2000
`Printed in U.S.A.
`
`Requirement for ERK Activation in Cisplatin-induced Apoptosis*
`
`Received for publication, May 26, 2000, and in revised form, August 15, 2000
`Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M004583200
`
`Xiantao Wang, Jennifer L. Martindale, and Nikki J. Holbrook‡
`From the Cell Stress and Aging Section, Laboratory of Biological Chemistry, National Institute on Aging,
`National Institutes of Health, Baltimore, Maryland 21224-6825
`
`Cisplatin activates multiple signal transduction path-
`ways involved in coordinating cellular responses to
`stress. Here we demonstrate a requirement for extracel-
`lular signal-regulated protein kinase (ERK), a member
`of the mitogen-activated protein kinase family in medi-
`ating cisplatin-induced apoptosis of human cervical car-
`cinoma HeLa cells. Cisplatin treatment resulted in dose-
`and time- dependent activation of ERK. That elevated
`ERK activity contributed to cell death by cisplatin was
`supported by several observations: 1) PD98059 and
`U0126, chemical inhibitors of the MEK/ERK signaling
`pathway, prevented apoptosis; 2) pretreatment of cells
`with TPA, an activator of the ERK pathway, enhanced
`their sensitivity to cisplatin; 3) suramin, a growth factor
`receptor antagonist that greatly suppressed ERK acti-
`vation, likewise inhibited cisplatin-induced apoptosis;
`and, finally, 4) HeLa cell variants selected for cisplatin
`resistance showed reduced activation of ERK following
`cisplatin treatment. Cisplatin-induced apoptosis was as-
`sociated with cytochrome c release and subsequent
`caspase-3 activation, both of which could be prevented
`by treatment with the MEK inhibitors. However, the
`caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-flu-
`oromethylketone protected HeLa cells against apoptosis
`without affecting ERK activation. Taken together, our
`findings suggest that ERK activation plays an active
`role in mediating cisplatin-induced apoptosis of HeLa
`cells and functions upstream of caspase activation to
`initiate the apoptotic signal.
`
`Cisplatin (cis-diamminedichloroplatinum; CDDP)1 is a po-
`tent inducer of growth arrest and/or apoptosis in most cell
`types and is among the most effective and widely used chemo-
`therapeutic agents employed for treatment of human cancers.
`However, a major limitation of CDDP chemotherapy is serious
`drug resistance. Multiple mechanisms have been implicated in
`the development of CDDP resistance including reduced accu-
`
`mulation of the drug, increased levels of glutathione (GSH),
`enhanced expression of metallothionein, enhanced DNA repair,
`increased levels of Bcl-2-related anti-apoptotic genes, and al-
`terations in signal transduction pathways involved in apoptosis
`(1–3). Apoptosis induced by CDDP is generally considered to be
`the result of its ability to damage DNA (4), but the detailed
`mechanisms by which such DNA damage triggers cell death
`remain unclear. Understanding the molecular basis of CDDP-
`mediated apoptosis could lead to strategies resulting in im-
`proved therapeutic benefits.
`Proteins comprising the mitogen-activated protein kinase
`(MAPK) family constitute important mediators of signal trans-
`duction processes that serve to coordinate the cellular response
`to a variety of extracellular stimuli. Three major mammalian
`MAPK subfamilies have been described: the extracellular sig-
`nal-regulated kinases (ERK), the c-Jun N-terminal kinases
`(JNK, also called stress-activated protein kinase), and the p38
`kinases. Each MAPK is activated through a specific phospho-
`rylation cascade. The ERK pathway plays a major role in reg-
`ulating cell growth and differentiation, being highly induced in
`response to growth factors, cytokines, and phorbol esters (5–7).
`It is also activated by some conditions of stress, particularly
`oxidant injury, and in such circumstances is believed to confer
`a survival advantage to cells (8–10). In contrast, JNK and p38
`are generally only weakly activated by growth factors, but are
`highly activated in response to a variety of stress signals in-
`cluding tumor necrosis factor, ionizing and short wave length
`ultraviolet irradiation (UVC), and hyperosmotic stress. Their
`activation is most frequently associated with induction of
`apoptosis (10–14).
`Many studies have demonstrated an activation of JNK in
`response to CDDP treatment, but how it influences cell sur-
`vival is unclear. Although most of the reports published thus
`far have suggested a role for JNK in the induction of apoptosis
`by CDDP, several studies have suggested that JNK signaling
`plays a role in enhancing survival of CDDP-treated cells (15–
`19). There is also mixed evidence for the role of ERK in influ-
`encing cell survival of CDDP-treated cells. For example, two
`recent studies have suggested that ERK activation is associ-
`ated with enhanced survival of CDDP-treated cells (19, 20).
`However, elevated expression of Ras, an upstream component
`of the ERK signaling pathway, has been connected with en-
`hanced sensitivity to CDDP (21, 22).
`The present study sought to examine the roles of the MAPK
`signaling pathways in regulating CDDP-induced apoptosis in
`HeLa cells. Although ERK, JNK, and p38 were all found
`to be activated in response to CDDP treatment, only ERK
`activity appears to be involved in regulating cell survival.
`Using a variety of strategies to manipulate ERK activity, we
`provide evidence that ERK is important in mediating CDDP-
`induced apoptosis through a cytochrome c release-dependent
`mechanism.
`39435
`
`* The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`‡ To whom all correspondence should be addressed: Laboratory of
`Biological Chemistry, Box 12, NIA, National Institutes of Health, 5600
`Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8446; Fax:
`410-558-8386.
`1 The abbreviations used are: CDDP, cis-diamminedichloroplatinum;
`JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated
`kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-acti-
`vated protein kinase/ERK kinase; NAC, N-acetylcysteine; DTT, dithio-
`threitol; PMSF, phenylmethylsulfonyl fluoride; DAPI, 4,6-diamidino-2-
`phenylindole; PARP,
`poly(ADP-ribosyl)
`polymerase;
`zVAD-fmk,
`benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; TPA, 12-O-tetra-
`decanoylphorbol-13-acetate; UVC, short wave length ultraviolet radia-
`tion; VP16, etoposide; DOX, doxorubicin; EGF, epidermal growth factor;
`GFR, growth factor receptor; GST, glutathione S-transferase; MOPS,
`4-morpholinepropanesulfonic acid.
`
`This paper is available on line at http://www.jbc.org
`
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`FIG. 1. CDDP treatment induces apoptosis in HeLa cells. A,
`CDDP induces apoptosis of HeLa cells in a dose- and time-dependent
`manner. HeLa cells were treated either with different doses of CDDP
`for 24 h (left panel), or 30 mM CDDP for the indicated times (right panel).
`Cells were then fixed with 4% paraformaldehyde, stained with DAPI,
`and apoptotic nuclei examined by fluorescence microscopy. B, cleavage
`of the caspase substrate PARP in 30 mM CDDP-treated HeLa cells. Cells
`were harvested at the indicated times, and PARP cleavage was assessed
`by Western blot analysis using an anti-PARP monoclonal antibody.
`
`cultures were treated with various doses of the agent for 24 h,
`after which they were stained with DAPI and examined micro-
`scopically. As shown in Fig. 1A (left panel), CDDP caused
`apoptosis of HeLa cells in a dose-dependent manner, with a
`concentration of 30 mM CDDP resulting in death of greater than
`90% of the cell population by 24 h of treatment. The kinetics of
`CDDP-induced apoptosis were examined using a 30 mM concen-
`tration (Fig. 1A, right panel). Morphological alterations char-
`acteristic of apoptosis were apparent within 14 h of treatment,
`as was cleavage of poly(ADP-ribosyl) polymerase (PARP), a
`biochemical feature of apoptosis that can be detected by West-
`ern blot analysis (Fig. 1B).
`CDDP Activates the MEK/ERK Signaling Pathway—The
`ERK signaling pathway has been shown to be activated in
`response to certain cellular stresses. To investigate whether
`CDDP treatment led to ERK activation, lysates obtained at
`various times from CDDP-treated cells were subjected to West-
`ern blot analysis using an anti-phospho-ERK antibody to detect
`phosphorylated (and therefore activated) ERK (Fig. 2A). The
`same blots were subsequently stripped and reprobed with an
`antibody that recognizes ERK2 to verify equal amounts of the
`protein in the various samples. As shown in the upper panel, 20
`and 30 mM CDDP, both of which resulted in significant apo-
`ptosis, led to strong activation of ERK. Activation became ap-
`parent at about 6 h following treatment with 30 mM CDDP and
`was sustained over the following 14-h period (Fig. 2A, lower
`panel). Importantly, we did not detect any acute activation of
`ERK (within the first hour of treatment), which frequently
`occurs with growth factor stimulation or treatment with oxi-
`dants (8, 10). MEK1/2, the kinases lying directly upstream of
`ERK, and which are responsible for ERK activation, were also
`phosphorylated by CDDP treatment over the same time frame
`as seen for ERK (Fig. 2B).
`MEK1/2 Inhibitors Block CDDP-induced Apoptosis—Two
`specific inhibitors of MEK1/2, PD98059 and U0126, have been
`developed, which are highly selective in their inhibition of the
`ERK pathway (25–27). These were used, therefore, to evaluate
`whether ERK activation is required for CDDP-induced apopto-
`sis. HeLa cells were pretreated with various doses of PD98059
`or U0126 for 30 min prior to addition of 30 mM CDDP. The
`morphology of cells treated with CDDP 6 the MEK inhibitors
`is shown in Fig. 3A. Cells treated with CDDP alone displayed
`
`EXPERIMENTAL PROCEDURES
`Reagents—Cisplatin, 12-O-tetradecanoylphorbol-13-acetate (TPA),
`hydrogen peroxide, etoposide, doxorubicin, and 4,6-diamidino-2-phe-
`nylindole (DAPI) were purchased from Sigma. Anti-JNK1 polyclonal
`antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,
`CA). The MEK1/2 inhibitor, PD98059, the p38 inhibitors (SB202190
`and SB203580), suramin, and N-acetylcysteine were all obtained from
`CalBiochem (San Diego, CA). The monoclonal anti-PARP and anti-
`cytochrome c antibodies were purchased from PharMingen (San Diego,
`CA); U0126 and the anti-phospho-ERK and anti-phospho-JNK rabbit
`polyclonal antibodies were purchased from Promega (Madison, WI).
`Anti-phospho-p38 and anti-phospho-MEK1/2 (Ser217/221) antibodies
`were purchased from New England Biolabs, Inc. (Beverly, MA), and the
`anti-caspase-3 antibody was from Transduction Laboratories (Lexing-
`ton, KY). The caspase inhibitor zVAD-fmk was purchased from Enzyme
`Systems Products (Livermore, CA). The cisplatin-resistant cell lines,
`HeLa-R1 and HeLa-R3, and parental control line HeLa-C were kindly
`provided by Dr. Gilbert Chu (Stanford University, Stanford, CA) (23).
`Cell Culture—HeLa and A549 cells (American Type Culture Collec-
`tion, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s
`medium supplemented with 10% fetal bovine serum (Gemini BioProd-
`ucts Inc., Calabasas, CA), 100 units of penicillin, and 100 mg of strep-
`tomycin/ml. They were cultured at 37 °C in a humidified chamber
`containing 5% CO2. For the induction of apoptosis, cells were plated in
`60-mm dishes 1 day prior to cisplatin treatment.
`DAPI Staining—DAPI staining was performed as described previ-
`ously (24). In brief, prior to staining, the cells were fixed with 4%
`paraformaldehyde for 30 min at room temperature, then washed with
`PBS. DAPI was added to the fixed cells for 30 min, after which they
`were examined by fluorescence microscopy. Apoptotic cells were iden-
`tified by condensation and fragmentation of nuclei. Percentage of
`apoptotic cells was calculated as the ratio of apoptotic cells to total cells
`counted 3 100. A minimum of 400 cells were counted for each
`treatment.
`Western Blot Analysis—For immunoblot analysis, cells were har-
`vested in 300 ml of lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50
`mM b-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM dithio-
`threitol (DTT), 1 mM phenylsulfonyl fluoride (PMSF), 10 mg/ml leupep-
`tin, 10 mg/ml aprotinin, 1 mM Na3VO4, and 5 mM NaF). The resulting
`lysates were resolved on a 4–12% NuPAGE gels (NOVEX, San Diego,
`CA) (30 mg/lane) and transferred onto polyvinylidene difluoride mem-
`brane (Millipore, Bedford, MA). The membranes were blocked with
`Tris-buffered saline with Tween 20 (10 mM Tris-HCl, pH 7.4, 150 mM
`NaCl, 0.1% Tween 20) containing 5% milk and then hybridized with
`different antibodies. Proteins were detected by using enhanced chemi-
`luminescence (ECL) reagents (Amersham Pharmacia Biotech).
`Release of Cytochrome c—Cells were washed twice with phosphate-
`buffered saline, the pellets collected by centrifugation, and resuspended
`in 500 ml of buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2,
`1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 5 mg/ml each
`aprotinin and leupeptin) containing 250 mM sucrose, and homogenized
`25 strokes on ice with a Dounce homogenizer. Nuclei and unbroken cells
`were removed by centrifugation at 1,000 3 g for 10 min at 4 °C, and the
`supernatants centrifuged again at 14,000 3 g for 20 min at 4 °C. The
`resulting supernatant was used as the soluble cytosolic fraction. Equal
`amounts of lysate were separated by NuPAGE gel (4–12%), transferred
`to polyvinylidene difluoride membranes, and subsequently probed with
`anti-cytochrome c.
`Protein Kinase Assays—JNK activity was measured by an immuno-
`complex kinase assay as described previously (10). In brief, cells were
`lysed in 1 ml of lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM
`b-glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM
`PMSF, 1 mM Na3VO4, 5 mM NaF, 10 mg/ml leupeptin, and 10 mg/ml
`aprotinin). Equal amounts of protein samples were immunoprecipitated
`at 4 °C for 4 h with 5 ml of anti-JNK1 antibody with the addition of 35
`ml of 50% slurry protein A-Sepharose. The beads were washed three
`times each in lysis buffer and kinase assay buffer (20 mM MOPS, pH
`7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, and 0.1% Triton
`X-100). JNK kinase assays were performed using GST-c-Jun-(1–135) as
`a substrate. ERK and p38 MAPK activations were determined by West-
`ern blot analysis using anti-phospho-ERK and anti-phospho-p38 MAPK
`antibodies.
`
`RESULTS
`CDDP Treatment Induces Apoptosis in HeLa Cells—CDDP
`treatment results in apoptosis of many different cell types. To
`examine the ability of CDDP to induce apoptosis in HeLa cells,
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`in other cell types. Accordingly, we examined their activities in
`HeLa cells following exposure to CDDP for various lengths of
`time. JNK activation was assessed both by measuring its ki-
`nase activity using an immunocomplex kinase assay with GST-
`c-Jun-(1–135) fusion protein as a substrate, and by examining
`its degree of phosphorylation by Western blot analysis with
`anti-phospho-JNK1/2 polyclonal antibody. The activation state
`of p38 was likewise determined based on its degree of phospho-
`rylation. Total JNK and p38 protein levels were monitored
`using antibodies capable of recognizing both phosphorylated
`and unphosphorylated forms of the proteins. As shown in Fig.
`5A, both JNK and p38 were activated in response to CDDP
`treatment. Although p38 activation occurred over the same
`time frame seen for ERK, activation of JNK was somewhat
`delayed. To investigate the functional consequences of JNK
`activation, we examined the CDDP responsiveness of HeLa
`cells stably expressing a dominant negative mutant form of
`SEK1 (SEK1-DN), a kinase that contributes largely to JNK
`activation during conditions of stress. We have previously
`shown that these SEK1-DN-expressing cells show attenuated
`JNK activation and reduced apoptosis in response to H2O2
`treatment (10). As shown in Fig. 5B, SEK1-DN-expressing
`HeLa cell lines did not differ from vector control HeLa cells in
`their sensitivity to CDDP. To investigate the influence of p38
`activation on survival of CDDP-treated cells, we utilized the
`pharmacologic agents SB202190 and SB203580, which act as
`specific inhibitors of p38 activity. We have demonstrated that
`the concentrations of SB202190 and SB203580 used in the
`present study result in complete inhibition of p38 kinase activ-
`ity in HeLa cells (10). As shown in Fig. 5C, treatment of HeLa
`cells with these agents during exposure to CDDP did not alter
`the outcome. Taken together, these results indicate that nei-
`ther JNK nor p38 plays a role in regulating CDDP-induced
`apoptosis of HeLa cells.
`Phorbol Ester TPA Enhances CDDP-induced Apoptosis—If
`the activation of ERK plays an important role in mediating
`apoptosis of CDDP-treated cells, then agents capable of stim-
`ulating ERK activity, when combined with CDDP treatment,
`should potentiate apoptosis. To address this possibility, cells
`were treated with CDDP in the presence of the phorbol ester
`TPA. Numerous studies have indicated that TPA is a strong
`activator of the ERK signaling pathway (7, 28–30) at concen-
`trations that do not alter JNK activities in HeLa cells (31, 32).2
`Cells were pre-incubated with or without 50 nM TPA for 1 h,
`followed by addition of 20 mM CDDP. The cells were evaluated
`for apoptosis 12 and 24 h later. When administered alone, TPA
`was not toxic for HeLa cells (Fig. 6A). However, TPA-pre-
`treated cells were much more sensitive to CDDP. This effect
`was attenuated by co-treatment with the MEK inhibitor
`U0126. To confirm that the apoptosis-enhancing effect of TPA
`was related to its ability to activate ERK, Western blot analysis
`was used to assess ERK phosphorylation (Fig. 6B). TPA treat-
`ment alone significantly increased the level of phosphorylated
`ERK, with near maximum ERK activation achieved with 6 h of
`TPA treatment. Addition of CDDP did not appreciably alter
`this level. Furthermore, Fig. 6B shows that significant PARP
`cleavage was observed at 12 h of CDDP treatment when cells
`were sensitized with TPA, earlier than observed for CDDP
`alone (Fig. 1B). However, as seen for CDDP-treated cells, TPA-
`induced ERK activation and PARP cleavage was significantly
`inhibited by the presence of U0126. These results suggest that
`the ability of TPA to enhance CDDP-induced apoptosis of HeLa
`cells is mediated through activation of the ERK signaling
`pathway.
`
`2 X. Wang, and N. J. Holbrook, unpublished observations..
`
`FIG. 2. CDDP activates the MEK/ERK signaling pathway. A,
`dose- and time-dependent activation of ERK by CDDP. Upper panel,
`HeLa cells were treated with 5, 10, 20, or 30 mM CDDP for 12 h, after
`which cell lysates were assessed for ERK activation by Western blot-
`ting. Lower panel, HeLa cells were treated with 30 mM CDDP for the
`indicated times, after which cells were lysed and ERK activation deter-
`mined by Western blotting with an anti-phospho-ERK antibody. In both
`panels, total ERK2 protein levels were detected by Western blot anal-
`ysis using an anti-ERK2 antibody. B, CDDP stimulates MEK1/2 activ-
`ity. HeLa cells were treated with 30 mM CDDP for the indicated times,
`after which MEK1/2 activities were examined by Western blotting
`using an anti-phospho-MEK1/2 antibody.
`
`typical features of apoptosis; shrinkage of the cytoplasm, mem-
`brane blebbing, and condensation of nuclei (Fig. 3A, upper
`panel).
`Interestingly, pretreatment of
`cells with either
`PD98059 or U0126 markedly suppressed these morphologic
`changes induced by CDDP (Fig. 3A, upper panel). Staining of
`the cells with DAPI further confirmed these morphological
`findings (Fig. 3A, lower panel). Quantitation of apoptotic cells,
`summarized in Fig. 3B, demonstrated that the protective in-
`fluence of the MEK inhibitors was dose-dependent and oc-
`curred with doses expected to suppress ERK activation. That
`PD98059 and U0126 do indeed prevent activation of ERK in
`response to CDDP treatment is shown in Fig. 3C.
`Specificity of the Cellular Response to CDDP—CDDP induces
`apoptosis in a variety of cell types. To determine if the anti-
`apoptotic effect of MEK1/2 inhibitors against CDDP in HeLa
`cells also occurs in other cell types, A549 lung carcinoma cells
`were examined for their responsiveness to CDDP treatment. As
`seen with HeLa cells, CDDP treatment resulted in apoptosis of
`A549 cells (Fig. 4A). Pretreatment of these cells with U0126
`significantly reduced CDDP-induced apoptosis, although the
`inhibitory effect was not as great as that seen in HeLa cells
`(Fig. 4A).
`To evaluate the role of the ERK pathway in the induction of
`apoptosis following other stresses, HeLa cells were pretreated
`with the 60 mM PD98059 prior to their exposure to several
`stimuli including UVC (30 J/m2), hydrogen peroxide (600 mM),
`etoposide (VP16; 50 mM), and doxorubicin (DOX; 2 mM). We
`found no inhibitory effect of PD98059 on apoptosis occurring in
`response to any of these treatments (Fig. 4B). In fact, PD98059
`pretreatment enhanced both H2O2- and DOX-induced apopto-
`sis. That ERK was indeed activated in response to UVC, VP16,
`and DOX treatment and could be inhibited in the presence of
`the MEK inhibitor is shown in the bottom panel. We have
`previously reported such findings for H2O2 (10). These results
`are consistent with our previous studies supporting a pro-
`survival role for ERK during oxidant injury (8, 10). Thus,
`activation of the ERK pathway participates in the induction of
`apoptosis by CDDP, but not that occurring with other stresses.
`Roles for JNK and p38 in Regulating CDDP-induced Apo-
`ptosis—JNK and p38 activities increase upon CDDP treatment
`
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`ERK Activation Mediates Cisplatin-induced Apoptosis
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`FIG. 3. MEK1/2 inhibitors block
`CDDP-induced apoptosis and ERK
`activation. A, representative phase con-
`trast microscopy (upper panels) and DAPI
`staining (lower panels) of untreated HeLa
`cells, HeLa cells treated with Me2SO
`(drug vehicle control) plus 30 mM CDDP,
`HeLa cells treated with 60 mM PD98059
`plus 30 mM CDDP, and HeLa cells treated
`with 20 mM U0126 plus 30 mM CDDP for
`24 h. Nuclei of apoptotic cells are frag-
`mented and condensed. B, quantitation of
`apoptosis with DAPI staining. C, inhibi-
`tion of ERK activation by MEK1/2 inhib-
`itors as determined by Western blotting
`with an anti-phospho-ERK antibody.
`DMSO, dimethyl sulfoxide.
`
`Suppression of CDDP-induced ERK Activation and Apoptosis
`through Inhibition of Growth Factor Receptor Signaling Path-
`ways and Antioxidant Treatment—Previous findings from our
`laboratory and others have provided evidence that growth fac-
`tor receptors (GFR) are important in initiating the activation of
`the ERK signaling pathway in response to certain stresses
`(33–35). To test the possibility that growth factor receptors are
`involved in mediating CDDP-induced apoptosis in HeLa cells,
`we examined the ability of the broad spectrum growth factor
`receptor inhibitor suramin to prevent ERK activation and in-
`hibit apoptosis of CDDP-treated cells. As shown, suramin
`markedly reduced the level of ERK phosphorylation occurring
`in response to CDDP treatment (Fig. 7A) and significantly
`inhibited CDDP-induced apoptosis as assessed both by DAPI
`staining (Fig. 7B) and PARP cleavage (Fig. 7C).
`Given that a variety of oxidants have been found to activate
`ERK through growth factor signaling pathways, and the find-
`ing above that growth factor receptor signaling pathways ap-
`pear to participate in ERK activation by CDDP, we investi-
`gated whether oxidative stress contributes to the apoptotic
`effects of CDDP. The influence of two antioxidants, N-acetyl-
`cysteine (NAC) and dimethyl sulfoximide (Me2SO), were
`tested. Treatment of cells with 1 mM NAC completely protected
`cells against CDDP-induced apoptosis. Me2SO likewise inhib-
`ited apoptosis in a dose-dependent manner (Fig. 8A). It is
`important to note that Me2SO is a commonly employed solvent
`and indeed was used for preparation of our stock MEK1/2
`inhibitor solutions. However,
`the concentrations used in
`MEK1/2 inhibitor solutions (0.1%) are lower than those shown
`
`here to inhibit ERK activation. In keeping with the ability of
`NAC to prevent apoptosis as assessed by DAPI staining, the
`antioxidant markedly inhibited ERK activation and completely
`prevented PARP cleavage (Fig. 8B).
`Role of ERK in Mediating Cytochrome c Release and Caspase
`Activation in CDDP-treated Cells—Recent studies have indi-
`cated that cytochrome c participates in activating the cell death
`program (36–38). Cytochrome c normally resides in mitochon-
`dria, but is released into the cytoplasm following exposure of
`cells to certain stresses. In the cytoplasm it binds to Apaf-1,
`resulting in the activation of caspase-9 and downstream
`caspases such as caspase-3 (39–41). Therefore, we sought to
`investigate whether cytochrome c release occurred in response
`to CDDP treatment, and if so, to determine whether it was
`dependent on ERK activation. Cells were treated with CDDP
`for different times in the presence or absence of PD98059 (60
`mM) or U0126 (20 mM), after which cytosolic extracts were
`prepared as described under “Experimental Procedures,” and
`cytochrome c protein levels were measured by immunoblotting.
`As shown in Fig. 9, cytochrome c levels in the cytoplasm in-
`creased in response to CDDP treatment, and this was corre-
`lated with cleavage of both caspase-3 and PARP. These proc-
`esses were all markedly inhibited in the presence of the MEK
`inhibitors, particularly U0126, as was ERK activation (Fig.
`3C). These findings indicate that ERK acts upstream of cyto-
`chrome c release to exert its apoptotic influence in CDDP-
`treated cells. Further evidence that ERK activation is an early
`event in the pathway leading to apoptosis following CDDP
`treatment was supported by findings obtained with the broad
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`FIG. 4. Specificity and selectivity of the cellular response to
`CDDP. A, MEK1/2 inhibitor U0126 also blocks CDDP-induced apopto-
`sis in lung carcinoma A549 cells. Representative DAPI staining of A549
`cells treated with Me2SO plus 40 mM CDDP, or 20 mM U0126 plus 40 mM
`CDDP for 24 h. Lower panel, inhibition of CDDP-induced apoptosis in
`A549 cells was quantitated by DAPI staining 24 h after CDDP treat-
`ment. B, PD98059 does not protect against other forms of stress-in-
`duced apoptosis in HeLa cells. HeLa cells were preincubated with 60 mM
`PD98059 MEK1 inhibitor and subsequently treated with 30 mM CDDP,
`30 J/m2 UVC, 600 mM H2O2, 50 mM VP16, or 2 mM DOX. The percentage
`of apoptotic cells was determined 24 h later by nuclear DAPI staining.
`The bottom panel shows the ability of PD98059 to inhibit ERK activa-
`tion in response to the indicated treatments. DMSO, dimethyl
`sulfoxide.
`
`caspase inhibitor zVAD-fmk. Preincubation of cells with zVAD-
`fmk mostly blocked CDDP-induced PARP cleavage and apo-
`ptosis visualized by DAPI staining (data not shown), but had no
`effect on ERK activation (Fig. 10).
`Resistance to CDDP-induced Apoptosis Is Correlated with
`Reduced ERK Activation—We have presented much evidence
`suggesting that ERK activation plays a key role in mediating
`apoptosis in CDDP-treated HeLa cells. A final piece of evidence
`favoring this view was obtained through the investigation of
`HeLa cell variants selected for enhanced resistance to CDDP.
`HeLa-R1 and HeLa-R3 cell
`lines display 4.8- and 14-fold
`greater resistance to CDDP, respectively, relative to the paren-
`tal cells from which they were derived (23). Although the pre-
`cise mechanisms contributing to this resistance have not been
`determined, it is not due simply to altered uptake of the drug
`(23). To investigate whether the relative level of ERK activity
`seen if the individual variants was related to their sensitivity
`to CDDP, lysates prepared from cells treated with two different
`doses of CDDP were investigated by Western blot analysis for
`
`FIG. 5. Roles for JNK and p38 in regulating CDDP-induced
`apoptosis in HeLa cells. A, CDDP activates JNK and p38. HeLa cells
`were treated with 30 mM CDDP for the indicated times, after which cells
`were harvested, and JNK activities evaluated either by an in vitro
`kinase assay using GST-c-Jun as substrate or Western blotting using
`anti-phospho-JNK antibody. p38 activity was assessed by Western blot-
`ting using anti-phospho-p38 antibody. Total JNK1 and p38 protein
`levels were detected by Western blot analysis using anti-JNK1 and
`anti-p38 antibodies, respectively. B, stable expression of dominant neg-
`ative SEK1 (SEK1-DN) does not block CDDP-induced apoptosis as
`measured by DAPI staining 24 h after addition of CDDP. C, p38 inhib-
`itors SB202190 and SB203580 have no effect on CDDP-induced apo-
`ptosis. HeLa cells were preincubated with the p38 inhibitors and sub-
`sequently treated with 30 mM CDDP for 24 h. The percentage of apo-
`ptotic cells was quantitated using DAPI staining. DMSO, dimethyl
`sulfoxide.
`
`ERK activation and PARP cleavage. The parental strain
`(HeLa-C) showed strong activation of ERK with doses of 10 and
`20 mM CDDP, and clear evidence of PARP cleavage (Fig. 11). In
`keeping with their greater resistance to the drug, the HeLa-R1
`and HeLa-R3 lines displayed significantly less ERK activation
`and reduced PARP cleavage compared with the parental line.
`Indeed, in the most resistant line, HeLa-R3, virtually no ERK
`activation or PARP cleavage was seen at the doses used. All
`three cell lines showed similar activation of ERK in response to
`TPA treatment (data not shown).
`
`DISCUSSION
`The importance of MAPK signaling pathways in regulating
`apoptosis during conditions of stress has been widely investi-
`
`
`Page 5 of 10
`
`Fluidigm
`Exhibit 1010
`
`
`
`39440
`
`ERK Activation Mediates Cisplatin-induced Apoptosis
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 30, 2016
`
`FIG. 6. TPA enhances CDDP-induced apoptosis. A, cells were
`pretreated with or without 50 nM TPA for 30 min followed by treatment
`with either 20 mM CDDP alone, or 20 mM CDDP plus 20 mM U0126 for an
`additional 24 h. The percentage of apoptotic cells was determined by
`nuclear DAPI staining. B, Western blot analysis showing enhanced
`ERK activation and PARP cleavage in TPA-treated cells. DMSO, di-
`methyl sulfoxide.
`
`gated. Many such studies have supported the general view that
`activation of the ERK pathway delivers a survival signal that
`counteracts pro-apoptotic effects associated with JNK and p38
`activation (8, 10–14). Consistent with such a prosurvival func-
`tion for ERK, two groups have reported that an inhibition of
`ERK signaling leads to increased sensitivity of ovarian cancer
`cell lines to CDDP (19, 20). In the present study, however, we
`have provided evidence that activation of ERK is important for
`the induction of cisplatin-induced apoptosis in HeLa cells.
`CDDP treatment resulted in high and sustained activation of
`ERK in these cells. Utilizing various strategies to modulate
`ERK activity, we found that down-regulation of ERK led to an
`inhibition of CDDP-induced apoptosis, whereas enhancement
`of ERK activity accentuated cell death. Furthermore, examina-
`tion of two HeLa cell variants exhibiting enhanced CDDP re-
`sistance revealed a strong correlation between the level of ERK
`activation and sensitivity to CDDP toxicity. This influence of
`ERK activity on CDDP-induced apoptosis was not limited to
`HeLa cells, but also occurred in human lung A549 cells. How-
`ever, it is not a universal feature of mammalian cells as inhib-
`iting ERK activation in CDDP-treated PC3 prostate carcinoma
`cells did not alter their sensitivity to the drug.2
`Although a number of studies have shown that JNK is acti-
`vated in response to CDDP treatment, a role for this signaling
`pathway in determining survival is far from clear. Several
`studies have provided evidence indicating that the JNK path-
`way contributes to CDDP-induced apoptosis (15–17). However,
`others have suggested that JNK signaling is important for cell
`survival (18, 19). Indeed, one study has suggested that JNK
`and ERK act collaboratively to enhance survival of CDDP-
`treated cells, as inhibition of either pathway accentuated the
`
`FIG. 7. Suramin inhibits ERK activation and suppresses apo-
`ptosis of CDDP-treated cells. A, Western
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