Requirement for ERK Activation in Cisplatin-induced Apoptosis*

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma 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 activation, 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 associated 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-fluoromethylketone 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 potent inducer of growth arrest and/or apoptosis in most cell types and is among the most effective and widely used chemotherapeutic 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 alterations in signal transduction pathways involved in apoptosis (1)(2)(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 CDDPmediated apoptosis could lead to strategies resulting in improved therapeutic benefits.
Proteins comprising the mitogen-activated protein kinase (MAPK) family constitute important mediators of signal transduction processes that serve to coordinate the cellular response to a variety of extracellular stimuli. Three major mammalian MAPK subfamilies have been described: the extracellular signal-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 phosphorylation cascade. The ERK pathway plays a major role in regulating cell growth and differentiation, being highly induced in response to growth factors, cytokines, and phorbol esters (5)(6)(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 including 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 survival 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)(16)(17)(18)(19). There is also mixed evidence for the role of ERK in influencing cell survival of CDDP-treated cells. For example, two recent studies have suggested that ERK activation is associated 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 enhanced 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 CDDPinduced apoptosis through a cytochrome c release-dependent mechanism.
* 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.
Cell Culture-HeLa and A549 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gemini BioProducts Inc., Calabasas, CA), 100 units of penicillin, and 100 g of streptomycin/ml. They were cultured at 37°C in a humidified chamber containing 5% CO 2 . 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 previously (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 identified by condensation and fragmentation of nuclei. Percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted ϫ 100. A minimum of 400 cells were counted for each treatment.
Release of Cytochrome c-Cells were washed twice with phosphatebuffered saline, the pellets collected by centrifugation, and resuspended in 500 l of buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 5 g/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 ϫ g for 10 min at 4°C, and the supernatants centrifuged again at 14,000 ϫ 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.

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, cultures were treated with various doses of the agent for 24 h, after which they were stained with DAPI and examined microscopically. As shown in Fig. 1A (left panel), CDDP caused apoptosis of HeLa cells in a dose-dependent manner, with a concentration of 30 M 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 M concentration (Fig. 1A, right panel). Morphological alterations characteristic 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 Western 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 Western 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 M CDDP, both of which resulted in significant apoptosis, led to strong activation of ERK. Activation became apparent at about 6 h following treatment with 30 M 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 oxidants (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)(26)(27). These were used, therefore, to evaluate whether ERK activation is required for CDDP-induced apoptosis. HeLa cells were pretreated with various doses of PD98059 or U0126 for 30 min prior to addition of 30 M CDDP. The morphology of cells treated with CDDP Ϯ the MEK inhibitors is shown in Fig. 3A. Cells treated with CDDP alone displayed  Fig. 3B, demonstrated that the protective influence of the MEK inhibitors was dose-dependent and occurred 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 antiapoptotic 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 M PD98059 prior to their exposure to several stimuli including UVC (30 J/m 2 ), hydrogen peroxide (600 M), etoposide (VP16; 50 M), and doxorubicin (DOX; 2 M). 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 H 2 O 2 -and DOX-induced apoptosis. 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 H 2 O 2 (10). These results are consistent with our previous studies supporting a prosurvival 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 Apoptosis-JNK and p38 activities increase upon CDDP treatment 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 kinase activity using an immunocomplex kinase assay with GSTc-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 phosphorylation. 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 H 2 O 2 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 activity 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 neither 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 stimulating 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 concentrations 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 M 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-pretreated 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 treatment 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, TPAinduced 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. .

Suppression of CDDP-induced ERK Activation and Apoptosis through Inhibition of Growth Factor Receptor Signaling Pathways and Antioxidant
Treatment-Previous findings from our laboratory and others have provided evidence that growth factor receptors (GFR) are important in initiating the activation of the ERK signaling pathway in response to certain stresses (33)(34)(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 inhibit 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 finding above that growth factor receptor signaling pathways appear to participate in ERK activation by CDDP, we investigated whether oxidative stress contributes to the apoptotic effects of CDDP. The influence of two antioxidants, N-acetylcysteine (NAC) and dimethyl sulfoximide (Me 2 SO), were tested. Treatment of cells with 1 mM NAC completely protected cells against CDDP-induced apoptosis. Me 2 SO likewise inhibited apoptosis in a dose-dependent manner (Fig. 8A). It is important to note that Me 2 SO 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 mitochondria, 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 M) or U0126 (20 M), 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 increased in response to CDDP treatment, and this was correlated with cleavage of both caspase-3 and PARP. These processes 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 cytochrome c release to exert its apoptotic influence in CDDPtreated 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 caspase inhibitor zVAD-fmk. Preincubation of cells with zVADfmk mostly blocked CDDP-induced PARP cleavage and apoptosis 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 parental cells from which they were derived (23). Although the precise 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 ERK activation and PARP cleavage. The parental strain (HeLa-C) showed strong activation of ERK with doses of 10 and 20 M 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-

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 M 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 blotting 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 negative SEK1 (SEK1-DN) does not block CDDP-induced apoptosis as measured by DAPI staining 24 h after addition of CDDP. C, p38 inhibitors SB202190 and SB203580 have no effect on CDDP-induced apoptosis. HeLa cells were preincubated with the p38 inhibitors and subsequently treated with 30 M CDDP for 24 h. The percentage of apoptotic cells was quantitated using DAPI staining. DMSO, dimethyl 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 function 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, examination of two HeLa cell variants exhibiting enhanced CDDP resistance 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. However, it is not a universal feature of mammalian cells as inhibiting 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 activated 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 pathway contributes to CDDP-induced apoptosis (15)(16)(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 CDDPtreated cells, as inhibition of either pathway accentuated the drug's toxicity (19). Such differential effects observed from one study to another could reflect cell type specificity. In our own studies using HeLa cells, we found that JNK was indeed activated in response to CDDP treatment. However, down-regulation of this pathway through use of dominant negative SEK1 expression mutants did not alter the sensitivity to CDDP. Likewise, although we also showed that p38 was activated in response to CDDP, its inhibition failed to alter the cellular outcome. Thus, of the three MAPKs, only ERK appears to play a major role in influencing the survival of CDDP-treated HeLa cells.
CDDP-induced apoptosis has been closely tied to its ability to cause DNA damage (4). However, our observation that suramin, a general inhibitor of growth factor signaling pathways, can prevent ERK activation and attenuate CDDP-induced apoptosis argues for an important role of growth factor receptors in initiating these responses. Indeed, we and others have shown that oxidants activate ERK largely by usurping growth factor receptor signaling pathways (34,35,42). It has been reported that increased generation of reactive oxygen species is closely associated with CDDP-induced cytotoxicity (43,44). Consistent with these reports, we observed that pretreatment of cells with the antioxidant NAC effectively blocked CDDPinduced ERK activation and apoptosis. Thus, it is possible that reactive oxygen species generated in response to CDDP treat- ment result in activation of growth factor receptors and the downstream signaling pathway involving Ras, Raf, MEK, and ERK. A model for the CDDP-induced ERK activation and apoptosis is presented in Fig. 12. Whether this occurs after, and is dependent on DNA damage or is the result of an independent effect of CDDP is presently unclear. However, it has been shown that the CDDP-resistant HeLa cell clones, HeLa-R1 and HeLa-R3, contain increased levels of a factor, XPE, that recognizes damaged DNA and may participate in its repair (23). It is possible, therefore, that reduced DNA damage in these cells leads to less reactive oxygen species production. Both the in-volvement of growth factor receptor signaling pathways in mediating ERK activation by CDDP, and the importance of ERK in mediating CDDP-induced apoptosis, are consistent with two earlier reports indicating that EGF receptor activity can influence the response to CDDP (45,46). EGF stimulation was  12. Model of CDDP-induced signaling pathways leading to ERK activation and apoptosis. CDDP is known to cause both DNA damage and apoptosis. In the proposed model, CDDP also has effects on the GFR as well as increasing reactive oxygen species (ROS). Through use of numerous biochemical inhibitors, our studies show that CDDP activates the depicted cascade of events, including MEK1/2 and ERK activation, and ultimately results in cell death. DMSO, dimethyl sulfoxide.
found to enhance the sensitivity of a variety of cancer cell types to CDDP (45), while down-regulation of EGF receptor expression by antisense RNA was correlated with enhanced resistance of breast cancer cells to CDDP (46). Overexpression of Ras also enhances sensitivity to CDDP, whereas inhibition of Ras correlates with resistance (21,22). Important questions remaining are how does ERK activation lead to apoptosis of CDDP-treated cells, what are the downstream effectors involved in mediating the apoptotic effects, and what specifies death rather than survival? Clearly, ERK activation, although necessary, is not sufficient to induce apoptosis, as TPA treatment alone did not result in cell death. Rather, an additional signal generated by CDDP treatment is required to stimulate the death pathway. Indeed, we cannot rule out the possibility that ERK activation acts indirectly to cause the apoptosis. ERK activation is part of a proliferative signaling pathway, yet growth arrest is a common response to treatment with genotoxic agents, believed to be important for allowing time for DNA repair. It could be that it is the generation of such conflicting signals by CDDP treatment that leads to apoptosis. However, in this regard it is important to point out that we have shown that growth factor receptor-mediated activation of ERK in response to hydrogen peroxide treatment constitutes a pro-survival signal in these same cells (Refs. 8 and 10; see also Fig. 4). One important difference between the CDDP-and hydrogen peroxide-induced ERK activations is the timing and duration of activity. In the case of hydrogen peroxide, ERK activation is more rapid (occurring within 15 min of treatment, with maximum activation at 60 min) and transient (activity was reduced within 3 h). With CDDP, no significant activation occurs until 6 h, but the activity remains highly elevated through the remaining time period examined (24 h). Although more often associated with survival, a pro-apoptotic function for the ERK pathway has recently also been suggested in several other model systems. For example, one study demonstrated an association between ERK2 activation and B-cell antigen receptor-induced apoptosis in B lymphoma cells (47), and another provided evidence that elevated ERK contributes to brain injury during focal cerebral ischemia (48).
Two major distinct apoptosis pathways have been described for mammalian cells. One involves caspase-8, which is recruited by the adapter molecule Fas/APO-1-associated death domain protein to death receptors upon extracellular ligand binding (49,50). The other involves cytochrome c release-dependent activation of caspase-9 through Apaf-1 (39,41). Although several studies have suggested that CDDP is capable of up-regulating Fas and Fas ligand (FasL) proteins (51, 52), we did not observe any change in either Fas or FasL expression in CDDP-treated HeLa cells (data not shown). We did, however, observe increased levels of cytochrome c in the cytoplasm of CDDP-treated cells relative to untreated cells, suggesting that cytochrome c release plays a role in mediating CDDP-induced apoptosis. The ability of the MEK inhibitors to diminish this effect suggests that the ERK signaling pathway functions upstream of cytochrome c release in the induction of cell death. Further support for this view was obtained using the broad caspase inhibitor zVAD-fmk, which suppressed apoptosis and PARP cleavage without interfering with ERK activation. It will be of great interest to determine how ERK acts to influence cytochrome c release.
Much evidence has accumulated indicating that the ERK pathway is important in cellular transformation (29,53). Recent studies have even suggested that MEK inhibitors can revert tumor cells to a nontransformed phenotype in vitro and arrest tumor growth in vivo, suggesting that MEK inhibitors represent a primary approach for treatment of human malig-nancies (54). Although this may indeed be true, our findings would indicate a need for caution with respect to the generality of this approach. On the other hand, our findings, coupled with previous studies supporting a role for the ERK signaling pathway in enhancing CDDP-induced apoptosis, would suggest that strategies targeting activation of this cascade could be employed to enhance the therapeutic effectiveness of CDDP. In addition, evaluation of ERK activity could be useful in predicting which tumors will respond most favorably to CDDP therapy.