Cisplatin-induced activation of the EGF receptor - Nature

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Oncogene (2002) 21, 8723 – 8731 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Cisplatin-induced activation of the EGF receptor Moran Benhar1, David Engelberg1 and Alexander Levitzki*,1 1

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Cisplatin (CDDP) is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers. CDDP activates nuclear as well as cytoplasmatic signaling pathways involved in regulation of the cell cycle, damage repair and programmed cell death. Here we report that CDDP also activates a membrane-integrated protein, the epidermal growth factor receptor (EGFR). We show that EGFR is activated in response to CDDP in various types of cells that overexpress the receptor, including transformed human glioma cells and human breast tumor cells. CDDP-induced EGFR activation requires its kinase activity, as it can be blocked by an EGFR kinase inhibitor or by expression of a kinase dead receptor. We also show that CDDP-induced EGFR activation is independent of receptor ligand. CDDP induces the activation of c-Src, and EGFR activation is blocked by Src-family inhibitor PP1, suggesting that Src kinases mediate CDDP-induced EGFR activation. We propose that EGFR activation in response to CDDP is a survival response, since inhibition of EGFR activation enhances CDDP-induced death. These findings show that signals generated by DNA damage can modulate EGFR activity, and argue that interfering with CDDP-induced EGFR activation in tumor cells might be a useful approach to sensitize these cells to genotoxic agents. Oncogene (2002) 21, 8723 – 8731. doi:10.1038/sj.onc. 1205980 Keywords: cisplatin; EGF receptor; Src; Glioblastoma; DNA damage Introduction Cis-diamminedichloroplatinum (CDDP) or cisplatin is a DNA-damaging agent and is among the most active and widely used cytotoxic anticancer drugs (Jordan and Carmo-Fonseca, 2000). Cytotoxicity produced by CDDP has been shown to be a consequence of DNA damage caused by the formation of CDDP-DNA adducts. A major limitation in the clinical use of CDDP is the acquisition of resistance of the initially responsive tumors (Kartalou and Essigmann, 2001).

*Correspondence: A Levitzki; E-mail: [email protected] Received 4 February 2002; revised 14 August 2002; accepted 16 August 2002

Acquired resistance may be due to several mechanisms, including altered cellular drug transport, enhanced intracellular detoxification, increased DNA repair, adduct tolerance and modulation of apoptosis (Kartalou and Essigmann, 2001). Understanding the cellular responses to CDDP is critical for determining mechanisms of drug resistance and for the development of therapeutic approaches for increasing the effectiveness of CDDP or other anticancer drugs that act by similar mechanisms. CDDP has been shown to evoke diverse cellular responses. Recent studies have documented that CDDP triggers the activation of the JNK and p38 mitogenactivated protein kinase (MAPK) cascades in tumor cells or transformed cell lines (Benhar et al., 2001; Deschesnes et al., 2001; Gebauer et al., 2000; Pandey et al., 1996). Activation of JNK or p38 by CDDP was shown to promote apoptotic cell death (Benhar et al., 2001; Deschesnes et al., 2001). In addition, several reports have shown that CDDP activates members of the ERK subfamily of MAPKs (Benhar et al., 2001; Persons et al., 1999; Wang et al., 2000a). Activation of the ERK pathway by CDDP was reported to promote (Wang et al., 2000a) or oppose (Benhar et al., 2001; Persons et al., 1999) cell death induction, possibly reflecting cell type specific differences. The molecular mechanisms involved in genotoxic stress-induced ERK activation are poorly understood. It is unclear, for example, whether membrane receptors take part in this process, as in the case of ERK activation induced by growth factors. Recently, we reported that CDDPinduced ERK activation is significantly elevated in transformed cells, such as NIH3T3 cells that overexpress the epidermal growth factor receptor (EGFR) (Benhar et al., 2001). These findings led us to hypothesize that EGFR may mediate some of CDDP effects. In the present study we sought to explore further this hypothesis. Surprisingly, we find that CDDP and other anticancer agents induce the activation of the EGFR in cells that overexpress the receptor. We also addressed the mechanism responsible for CDDPinduced EGFR activation and found that the intrinsic kinase activity of the EGFR is needed and that Src-family kinases are involved in receptor activation. We also show that blocking receptor activation sensitizes human glioma cells to CDDP. These findings uncover novel aspects of stress signaling in transformed cells and maintain that

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preventing CDDP-dependent EGFR activation should reduce drug resistance and improve clinical efficacy.

paclitaxel (taxol) and vinblastine failed to induce receptor phosphorylation (Figure 1F).

Results

Functional EGFR is involved in the response to CDDP

CDDP-induced activation of EGFR We have previously shown that treatment of NIH3T3 cells that overexpress EGFR (DHER14 cells) with CDDP results in pronounced activation of different MAPKs, namely, JNK, p38 and ERK (Benhar et al., 2001). To substantiate the findings regarding ERK activation, we compared the time course of CDDPinduced ERK activation in DHER14 cells and in parental NIH3T3 cells. As shown in Figure 1A, CDDP-induced ERK activation was markedly enhanced in DHER14 cells compared to NIH3T3 cells. The augmented activation of ERK in cells that overexpress EGFR, together with the observation that down-regulation of EGFR expression or activity leads to reduced CDDP-induced ERK activation (Benhar et al., 2001), led us to speculate that EGFR might be involved in the response to CDDP. To examine this hypothesis we exposed DHER14 cells to CDDP and monitored the level of DEGFR tyrosine phosphorylation. CDDP treatment induced time- and dosedependent EGFR tyrosine phosphorylation (Figure 1B). Immunoprecipitation experiments validated the identity of the 170 kDa band as the EGFR (Figure 1D). CDDP did not have effect on EGFR expression. To examine these findings further, we investigated if a similar response occurs in different types of cells. To this end, we studied U87MG human glioma cells, as well as U87MG.wtEGFR and U87MG.DEGFR glioma cells, which overexpress wild-type or truncated EGFR respectively (Nagane et al., 1998). Exposure of glioma cells to CDDP led to an increase in the level tyrosinephosphorylated EGFR in U87MG.wtEGFR and in U87MG.DEGFR, which express high levels of EGFR, but not in parental U87MG cells, which express low levels of EGFR (Figures 1C,D). The time course of EGFR phosphorylation in glioma cells was similar to that observed in DHER14 cells. High increase in the level of EGFR phosphorylation was observed at 8 h or more, following CDDP treatment (Figure 1E, top panel). A similar pattern of receptor phosphorylation was detected also in human breast cancer cells (MDAMB-468 cells), which express high levels of EGFR, upon treatment with CDDP (Figure 1E, bottom panel). Thus, CDDP dependent EGFR activation occurs in different cell types that overexpress the receptor. The hitherto undocumented capacity of CDDP to induce activation of the EGFR prompted us to examine if other chemotherapeutic drugs may induce receptor activation. To this end, we treated glioma cells with a number of anticancer drugs, and measured the level of tyrosine phosphorylation of the EGFR by immunoblotting. EGFR phosphorylation was increased in response to treatment with the DNA damaging agents doxorubicin and camptothecin (Figure 1F). In contrast, treatment with the cytoskeleton disrupting agents Oncogene

The increase in EGFR phosphorylation following CDDP treatment could be a result of autophosphorylation activity or due to phosphorylation by other kinases. To examine if CDDP-induced EGFR phosphorylation requires the kinase activity of the receptor, we employed AG1478, an EGFR kinase specific inhibitor (Osherov and Levitzki, 1994). The tyrosine phosphorylation of the EGFR following CDDP treatment was greatly diminished by AG1478 (Figure 2A). The kinetics of receptor activation as well as its inhibition by AG1478 correlated with the level of activated ERK1/2. The expression levels of EGFR and ERK1/2 were not altered by CDDP or AG1478. These findings suggest that activation of the EGFR by CDDP requires its kinase activity. Furthermore, CDDP-induced EGFR phosphorylation was abolished in glioma cells that express kinase-dead receptor (U87MG.DK cells, Figure 2B), showing that also in glioma cells receptor kinase activity is needed for its activation by CDDP. Recruitment of the adapter proteins SHC and Grb2 to activated receptor tyrosine kinases (RTKs) is a critical step in RTK signaling. In further support of the functionality of CDDP-induced EGFR activation, we observed that EGFR co-precipitated with SHC from CDDP treated cells (Figure 3A). Concomitantly to EGFR-SHC association, Grb2 association with SHC increased upon CDDP treatment. Also, SHC (in particular, p52 and p66 isoforms) and Grb2 levels were elevated in EGFR immunoprecipitates from CDDP-treated cells (Figure 3B). Overall, the data shown in Figures 2 and 3 shows that CDDP stimulates EGFR activation via an autophosphorylation mechanism, which triggers downstream signaling events. CDDP-induced EGFR activation is ligand independent We considered the possibility that CDDP activates EGFR through induction of autocrine ligand production. U87MG.DEGFR cells express a truncated EGFR, lacking 267 amino acids in its extracellular domain. It has been demonstrated that DEGFR is disabled in ligand binding (Nishikawa et al., 1994). Stimulation of glioma cells with EGF triggered EGFR phosphorylation in U87MG.wtEGFR but not in U87MG.d EGFR cells (Figure 4A) in keeping with previous data (Nishikawa et al., 1994). On the other hand, CDDP induced the phosphorylation of both wt EGFR and DEGFR (Figure 4A). These findings support the idea that CDDP-induced EGFR activation is ligand independent. As an independent approach to determine whether EGFR activation is ligand dependent we examined whether an EGFR neutralizing antibody has an effect on CDDP-induced EGFR phosphorylation. The antibody we used binds to the extracellular domain of the

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EGFR Figure 1 CDDP-induced EGFR activation. (A) DHER14 or NIH3T3 cells were treated with 100 mM CDDP for the time indicated and thereafter were lysed as described in Materials and methods. The activation of ERK1 and ERK2 was measured by immunoblotting, using antibodies that recognize the doubly phosphorylated (activated) forms of ERK1 and ERK2. Identical blots run in parallel were reacted with anti-ERK2 antibodies. Due to cross reactivity the anti-ERK2 antibodies recognize both ERK isoforms: ERK2 (p42, lower band) and ERK1 (p44, upper band). (B) Top: DHER14 cells were treated with CDDP for 16 h at the dose indicated. Cells were lysed and subjected to Western blot analysis using anti-phospho-tyrosine (PY) or anti-EGFR specific antibodies. Bottom: DHER14 cells were treated with 100 mM CDDP for the time indicated. Tyrosine phosphorylation and EGFR expression were analysed by immunoblotting. (C) U87MG, U87MG.wtEGFR or U87MG.DEGFR cells were treated with CDDP for 16 h at the dose indicated. Cells were lysed and subjected to Western blot analysis using anti-phospho-tyrosine (PY) or anti-EGFR specific antibodies. (D) DHER14 or U87MG.DEGFR cells were treated with 100 mM for 16 h. Lysates from control and CDDP treated cells were immunoprecipitated with anti-EGFR antibodies. The level of EGFR tyrosine phosphorylation was determined by immunoblotting. (E) Top: U87MG.wtEGFR cells were treated with 50 mM CDDP for the time indicated. EGFR tyrosine phosphorylation was analysed by immunoblotting. Bottom: MDA-MB-468 cells were treated with 100 mM CDDP for the time indicated. EGFR tyrosine phosphorylation was analysed by immunoblotting. (F) U87MG.wtEGFR cells were treated with 50 mM CDDP, 50 mM doxorubicin, 20 mM camptothecin, 2 mM vinblastine or 2 mM taxol for the time indicated. EGFR tyrosine phosphorylation was analysed by immunoblotting. NS, non-stimulated

EGFR EGFR ligands activity results

and competes for binding of ligands to the on cells. It blocks the biological effects of and does not activate the tyrosine kinase of the receptor (Johnson et al., 1993). The in Figure 4B show that this neutralizing

antibody significantly suppressed EGF-induced EGFR phosphorylation but did not affect CDDP-induced EGFR phosphorylation. These results demonstrate that CDDP-induced EGFR activation is unrelated to the binding of EGFR ligands to the receptor. Oncogene

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Because a significant delay in time was observed between the initiation of CDDP treatment and EGFR phosphorylation, we considered the possibility that EGFR activation was secondary to de novo protein synthesis. We therefore examined the effect of cycloheximide on CDDP-induced EGFR phosphorylation. The combination of cycloheximide and CDDP treatment did not significantly affect EGFR phosphorylation compared with CDDP alone (Figure 4C). To confirm the inhibitory effect of cycloheximide in the experiment, we observed that the induction in p53 was completely abolished by this treatment (Figure 4C). Taken together, our findings show that CDDP-induced EGFR activation is independent of ligand or of de novo protein synthesis. A role for Src-family kinases in CDDP-induced EGFR activation To further explore the mechanism that underlies EGFR activation by CDDP we considered the possible involvement of Src-family kinases. Pp60Src cooperates Oncogene

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Figure 3 Coprecipitation of endogenous EGF receptor, SHC and Grb2 following CDDP treatment. (A) U87MG.wtEGFR cells were stimulated with 100 mM CDDP for 16 h or with 10 nM EGF for 10 min. Immunoprecipitates of SHC were resolved by SDS – PAGE and immunoblotted with anti-EGFR (top panel), antiSHC (middle panel) or anti-Grb2 (bottom panel) as described. The position of EGFR, SHC isoforms and Grb2 are as indicated. (B) U87MG.wtEGFR cells were stimulated as described in (A). Immunoprecipitates of EGFR were resolved by SDS – PAGE and immunoblotted with anti-EGFR (top panel), anti-SHC (middle panel) or anti-Grb2 (bottom panel) as described. The position of EGFR, SHC isoforms and Grb2 are as indicated. NS, nonstimulated

with the EGFR in several signaling settings (Parsons and Parsons, 1997) and is involved in the activation of MAP kinases in response to UV radiation and oxidative stress (Aikawa et al., 1997; Kitagawa et al., 2001). To examine if Src-family kinases are involved in CDDP-induced EGFR activation, we employed PP1, an inhibitor of Src-family kinases (Hanke et al., 1996). Exposure of DHER14 cells to PP1 reduced CDDPinduced EGFR phosphorylation and ERK1/2 activation in a dose dependent manner without affecting the expression level of the EGFR and of ERK1/2 (Figure

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EGFR Figure 4 CDDP-induced EGFR activation is ligand independent. (A) U87MG.wtEGFR or U87MG.DEGFR were treated with 100 mM CDDP for 16 h or with 10 nM EGF for 10 min. Tyrosine phosphorylation of EGFR was determined by immunoblotting. NS, non-stimulated (B) U87MG.wtEGFR cells were treated with EGF or CDDP as in (A). Thirty minutes prior to EGF or during the last hour of CDDP treatment, anti-EGFR neutralizing antibody was added (5 mg/ml) as indicated. Tyrosine phosphorylation of EGFR was determined by immunoblotting. (C) DHER14 cells or U87MG.wtEGFR were treated with 100 mM CDDP for 16 h in the absence or presence of cycloheximide (CHX) as indicated. The level of tyrosine phosphorylated EGFR, total EGFR and p53 were determined by immunoblotting

5A). Experiments performed in glioma cells showed that PP1 suppressed CDDP-induced EGFR phosphorylation in these cells as well (Figure 5B). To examine if CDDP can induce c-Src activity, we immunoprecipitated Src from control, CDDP- or EGF-treated cells, and measured its kinase activity, using denatured enolase as a substrate. CDDP treatment led to a *twofold increase in Src activity, which was similar to the activation by EGF (Figure 5C). Phosphorylation of Src on tyrosine 527 (the target residue of Csk) inhibits its kinase activity (Xu et al., 1999; Young et al., 2001). Immunoblot analysis, using antibodies specific to Src phosphorylated on tyrosine 527, showed that CDDP induced the dephosphorylation of Src (Figure 5D) in agreement with the elevation of its kinase activity. Our findings, that CDDP-induced EGFR phosphorylation is abrogated in cells that express kinasedead receptor or in cells treated with AG1478 (Figure 2), suggest that phosphorylation of the receptor occurs on its autophosphorylation sites. EGFR may be also phosphorylated on tyrosine 845 that is positioned outside the autophosphorylation domain. This site has been shown to be a target for Src kinase (Sato et al., 1995; Tice et al., 1999). As shown in Figure 5E, the phosphorylation of tyrosine 845 was elevated following CDDP treatment in DHER14 cells as well as in glioma cells. Collectively, our findings suggest that Src mediates CDDP-induced EGFR activation.

Inhibition of CDDP-induced EGFR activation sensitizes glioma cells to CDDP Expression of the DEGFR in glioma cells was shown to confer resistance to CDDP through elevation of BclXL and suppression of apoptosis (Nagane et al., 1998). The state of EGFR activation was not determined in that study. As we observed that CDDP induces EGFR activation in glioma cells, we reasoned that blocking EGFR activation would negate its protective activity. To test this possibility, we measured cell survival of U87MG.DEGFR cells following CDDP treatment, in the presence of AG1478 or PP1, which block EGFR activity or Src-mediated EGFR activation respectively (Figures 2 and 5). AG1478 treatment enhanced *2 fold CDDP-induced cell death in U87MG.DEGFR (Figure 6), in agreement with previous data (Nagane et al., 1998). PP1 treatment had a comparable effect to AG1478 in enhancing CDDP-induced cell death (Figure 6). AG1478 and PP1 alone did not induce cell death in glioma cells (data not shown). These data suggest that the activation of the EGFR by CDDP plays a role in promoting survival of glioma cells. Discussion Genotoxic stress induces numerous signaling pathways that affect cell growth and death. The response to DNA damage is initiated by a number of nuclear proteins, including ATM, ATR and DNA-PK, which Oncogene

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Figure 5 Involvement of Src-family kinases in CDDP-induced EGFR activation. (a) DHER14 cells were treated with 100 mM CDDP for 16 h. During the last hour of incubation PP1 was added at the indicated concentrations. EGFR phosphorylation and ERK1/2 activation were analysed by immunoblotting. (b) U87MG.wtEGFR or U87MG.DEGFR cells were treated with 50 mM CDDP for 16 h. During the last hour of incubation PP1 was added at the indicated concentrations. EGFR phosphorylation was analysed by immunoblotting. (C) DHER14 cells were treated with CDDP (100 mM for 16 h) or EGF (10 nM for 10 min). c-Src was immunoprecipitated from 300 mg of lysate and subjected to in vitro kinase assay using denatured enolase as a substrate. Src activity (shown in graph) was calculated as the amount of phosphorylated substrate (measured by phosphor imager) normalized to the amount of immunoprecipitated Src protein. Data shown are from a representative experiment, which was repeated three times with comparable results. (D) DHER14 cells were treated with 100 mM CDDP for 16 h. The level of phosphorylated Src (Y527) was analysed by immunoblotting, using phospho-specific antibodies. (E) DHER14 or U87MG.DEGFR cells were treated with 100 mM CDDP for 16 h. The level of phosphorylated EGFR (Y845) was determined by immunoblotting, using phospho-specific antibodies

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Time (h) Figure 6 Treatment of glioma cells with AG1478 or PP1 sensitizes them to CDDP-induced death. Survival of U87MG.DEGFR cells following treatment with 25 mM CDDP in the presence of AG1478 (20 mM), PP1(20 mM) or vehicle (DMSO). The fraction of surviving cells was determined by the automated microculture methylene blue assay Oncogene

in turn stimulate the activation of Chk1, Chk2 and p53 (Shiloh, 2001; Zhou and Elledge, 2000). In addition to nuclear signaling cascades, the DNA damage response involves cytoplasmic pathways, such as those involving MAPKs (Benhar et al., 2002; Pearce and Humphrey, 2001). Together, these nuclear and cytoplasmic elements control the activation of cell cycle checkpoints as well as apoptosis. Activation of membrane associated components, such as receptor tyrosine kinases, by DNA damaging anticancer drugs has not been reported until now. In this study we show that the response to CDDP involves a membrane signaling protein, namely, the EGFR. CDDP induces EGFR activation and as a consequence its downstream effectors in a number of cell lines (Figures 1 – 3). This discovery expands the repertoire of signaling processes, which involve EGFR. Also, these findings provide a mechanistic basis for the augmented CDDP-induced ERK activation in EGFRtransformed cells, notwithstanding the finding that ERK activation occurs also in the absence of EGFR.

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In contrast to the usual rapid and transient EGFR activation in response to stimulation with its physiological ligands, CDDP-mediated EGFR activation occurs several hours after initiation of treatment. This is also distinct from the rapid EGFR activation by UV, H2O2 and alkylating agents (Knebel et al., 1996). In addition, EGFR phosphorylation remains elevated for a prolonged period of time (24 h after exposure, Figure 1). The delayed EGFR activation, approximately 4 h after initiation of CDDP treatment (Figure 1), is consistent with activation occurring after DNA adduct formation. Also, EGFR activation is triggered by doxorubicin and camptothecin, but not by vinblastine and paclitaxel (Figure 1). These data suggest that EGFR activation is not unique to CDDP, and support the notion that a DNA damage signal activates a signaling pathway that culminates in EGFR activation. The mechanisms linking nuclear events evoked by DNA damaging agents to activation of non-nuclear signaling proteins (e.g. MAPKs) are largely unknown. The tumor suppressor p53 is one candidate for transducing DNA damage signals induced by CDDP (Jordan and Carmo-Fonseca, 2000). Recently, it has been reported that p53 can induce heparin-bindingEGF-like growth factor, leading to EGFR-dependent ERK and Akt activation (Fang et al., 2001). However, we show that EGFR activation is independent of de novo protein synthesis and occurs under conditions where p53 induction is blocked (Figure 4). Hence, elucidation of the mechanisms linking DNA damage to activation of EGFR requires further experimentation. Phosphorylation of EGFR depends on the balance between kinase and phosphatase activity. CDDP dependent EGFR phosphorylation is abolished by treatment with AG1478 or in cells that express kinase dead receptor (Figure 2), suggesting that increased EGFR activity rather than decreased phosphatase activity controls the phosphorylation process. However, we cannot rule out that the activities of protein – tyrosine phosphatases are reduced and may play a part in the process of EGFR activation. Our data provide strong evidence that CDDP induces EGFR autophosphorylation in a ligandindependent mechanism (Figure 4), which involves Src-family kinases (Figure 5). Src-family kinases play a role in the cellular response to stress agents, such as UV light, heat shock and oxidative stress (Aikawa et al., 1997; Lin et al., 1997; Yoshizumi et al., 2000). Furthermore, Src is implicated in cross-communication among different signaling systems, such as EGFR activation by integrins (Moro et al., 2002), EGFR cross talk with Na+/K+-ATPase (Haas et al., 2002) or EGFR activation induced by heterotrimeric G proteins (Luttrell et al., 1997; Maudsley et al., 2000), hydrogen peroxide (Chen et al., 2001), UV irradiation (Kitagawa et al., 2001), and Zn2+ ions (Wu et al., 2002). EGFR activation induced by CDDP involves the phosphorylation of tyrosine 845 (Figure 5C). It has been proposed that phosphorylation of tyrosine 845 stabilizes the activation loop of EGFR, maintains the enzyme in the active state and provides a binding

surface for protein substrates (Tice et al., 1999). Furthermore, it was shown that c-Src is responsible for the phosphorylation of the EGFR on tyrosine 845 (Biscardi et al., 1999). Src-dependent EGFR activation may also involve formation of multi-protein complexes (Maudsley et al., 2000; Moro et al., 2002). Regardless of the precise mechanism, it is evident that Src plays a major role in coupling various extracellular and intracellular signals to EGFR activation and to its downstream signaling pathways. Exposure of cells to CDDP or to other anticancer agents can induce the production reactive oxygen species (ROS) (Benhar et al., 2001; Miyajima et al., 1997; Simizu et al., 1998). In our previous study we demonstrated that CDDP-induced activation of JNK and p38 MAPKs was dependent on the production of ROS (Benhar et al., 2001). Treatment of DHER14 or glioma cells with the antioxidant N-acetyl cysteine partially inhibits CDDP dependent EGFR induction (M Benhar, unpublished data). Thus, it is possible that ROS generated in response to CDDP treatment play a role in EGFR activation. Since ROS can both stimulate Src activation (Aikawa et al., 1997) and inhibit RTK associated tyrosine phosphatases (Knebel et al., 1996), this mechanism is feasible. The involvement of a receptor tyrosine kinase in the response to CDDP, as revealed in this study, expands current view of DNA damage-induced signal transduction. What is the biological significance of EGFR activation by CDDP? A number of reports suggest that EGFR promotes cell survival through the activation of the ERK or the PKB/Akt pathways (Gibson et al., 1999; Moro et al., 1998; Wang et al., 2000b). EGFR suppresses cell death also by upregulating antiapoptotic proteins of the Bcl-2 family (Leu et al., 2000; Stoll et al., 1998). Our data, in agreement with these results, suggests that CDDP-dependent EGFR activation is a survival response that reduces the efficacy of CDDP treatment. We showed that inhibition of this response, directly by AG1478 or indirectly by PP1, sensitizes human glioma cells to CDDP and promotes cell killing (Figure 6). The results of this study together with those of Nagane et al. (1998) strongly argue that interception of EGFR activation should be a useful strategy to enhance the therapeutic effect of CDDP, and possibly other drugs, against glioblastoma and other tumors, which overexpress EGFR. Materials and methods Antibodies and other reagents Antibodies were obtained as follows: anti-ERK2, antiphospho-tyrosine (PY20), anti-EGFR and anti-Grb2 from Santa Cruz Biotechnology; anti-phospho-ERK from Sigma; Anti SHC from Transduction Laboratories; Anti-phosphoSrc (Tyr 527) from Biosource and anti-phospho-EGFR (Tyr 845) from Cell Signaling; Anti EGFR (neutralizing, clone LA1) from Upstate Biotechnology. AG1478 and PP1 were synthesized by Dr Aviv Gazit in our laboratory. CDDP was obtained from ABIC Ltd (Netanya, Israel). All other chemicals were purchased from Sigma. Oncogene

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Cell culture and treatment

Src kinase assay

NIH3T3 cells transformed with the EGFR (DHER14 cells) were described previously (Benhar et al., 2001). U87MG, U87MG.wtEGFR, U87MG.DEGFR and U87MG.DK were described (Nagane et al., 1998). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin and streptomycin and incubated at 378C in 5% CO2. Unless otherwise stated, cells were seeded at 16106 in a 100 mm petri dish with 10 ml growth medium and were treated on the third day as indicated. Stock solutions of kinase inhibitors (AG1478 and PP1) in DMSO were diluted 1 : 1000 prior to use in DMEM which contained 10% fetal calf serum. The concentration of DMSO in the controls was equal to the concentration of DMSO in inhibitor-containing media, and did not exceed 0.05%.

For immunoprecipitation, 300 mg of protein were incubated with anti-Src monoclonal antibodies (Mab 327) for at least 3 h at 48C in a rotating wheel. The immune complex formed was then precipitated by adding 40 ml of 50% protein-G sepharose beads in the lysis buffer and incubating for an additional 1 h at 48C. The beads were pelleted by centrifugation, washed once in lysis buffer and three times in assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2). Finally the beads were suspended in 30 ml of assay buffer to which 2 mg of acid denatured enolase were added. The kinase reaction was initiated by the addition of [g-32P]ATP solution (10 mM, 10 mCi per reaction), and carried out at 308C for 10 min. The reaction mixture was separated by SDS – PAGE, and quantification of phosphorylated enolase was performed by phosphor-imager (Fujifilm, FLA 300).

Cell lysis, immunoprecipitation and immunoblotting

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Cells were lysed in 0.3 ml of lysis buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 0.5% NP-40, 3 mM EDTA, 3 mM EGTA, 10% glycerol, 20 mM b-glycerolphosphate, 1 mM of pnitrophenyl phosphate, 0.5 mM Na3VO4, 1 mM DTT, 2 mg/ ml leupeptin, 2 mg/ml aprotonin and 1 mM AEBSF) for 15 min on ice. Cell debris was removed by centrifugation at 20 000 g for 15 min at 48C. Immunoprecipitation of EGFR or SHC was performed as previously described (Daub et al., 1997). Immunoprecipitates or 30 mg of protein lysates were separated by SDS – PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane. After incubation of the membrane with the appropriate antibodies, specific proteins were visualized using an enhanced chemiluminescence (ECL) detection reagent. For quantitation purposes, several exposures were done for each experiment, and only sub-saturation exposures were further analysed. Densitometry of immunoblots was performed with NIH image 1.61.

Cells were seeded at 3000 cells per well in 96 microculture well plates. After 2 days, cells were treated as indicated and the fraction of surviving cells in the post treatment days was measured using the automated microculture methylene blue assay. Briefly, cells were fixed in 0.05% glutaraldehyde for 10 min at room temperature. After washing, the microplates were stained with 0.1% methylene blue in 0.1 M borate buffer (pH 8.5) for 60 min at room temperature. Thereafter, the plates were thoroughly washed to remove excess dye and then dried. The dye absorbed by the cells was eluted in 0.1 M HCl for 60 min at 378C and read at 630 nm.

Acknowledgments This study was supported by the Ministry of Science of the state of Israel and by the Israel Cancer Association through the estate of the late Alexander Smidoda.

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