Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m011164200
ISSN1083-351X
AutoresJun‐ichi Okano, Anil K. Rustgi,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoPaclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1–1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery. Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1–1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery. mitogen-activated protein kinase Asp-Glu-Val-Asp-aldehyde extracellular signal-regulated kinase c-Jun N-terminal kinase MAPK/ERK kinase poly(ADP-ribose) polymerase benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone epidermal growth factor phosphate-buffered saline Paclitaxel (Taxol) is a chemotherapeutic agent utilized for the treatment of breast, ovarian, lung, and esophageal cancers (1Crown J. O'Leary M. Lancet. 2000; 355: 1176-1178Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 2Weiner L.M. Semin. Oncol. 1999; 26: 106-108PubMed Google Scholar). Paclitaxel exerts its cytotoxic effect by binding and stabilizing microtubules (3Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and induces apoptosis in a variety of cell types (4Moos P.J. Fitzpatrick F.A. Cell Growth Differ. 1998; 9: 687-697PubMed Google Scholar, 5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 6Shen S.C. Huang T.S. Jee S.H. Kuo M.L. Cell Growth Differ. 1998; 9: 23-29PubMed Google Scholar) independently of p53 status (7Zhang Z. Liu Q. Lantry L.E. Wang Y. Kelloff G.J. Anderson M.W. Wiseman R.W. Lubet R.A. You M. Cancer Res. 2000; 60: 901-907PubMed Google Scholar). The mechanisms underlying paclitaxel-induced apoptosis are unclear. Paclitaxel was recently demonstrated to release cytochrome c by direct action on the mitochondria (8Andre N. Braguer D. Brasseur G. Goncalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar), activate small GTP-binding proteins (9Lou P.J. Chen W.P. Lin C.T. Chen H.C. Wu J.C. J. Cell Biochem. 2000; 79: 542-556Crossref PubMed Scopus (9) Google Scholar, 10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), and activate the NF-κB/IκB-α signaling pathway (11Huang Y. Johnson K.R. Norris J.S. Fan W. Cancer Res. 2000; 60: 4426-4432PubMed Google Scholar). Although these actions were implicated as the mechanisms through which paclitaxel induces apoptosis, these studies were impaired by the employment of high concentrations of paclitaxel.Akt (protein kinase B), a serine/threonine protein kinase, has a pivotal role in exerting an antiapoptotic effect against various stimuli (12Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar). Overexpression of Akt was reported to confer resistance to paclitaxel (13Page C. Lin H.J. Jin Y. Castle V.P. Nunez G. Huang M. Lin J. Anticancer Res. 2000; 20: 407-416PubMed Google Scholar). However, it was recently reported that paclitaxel induced apoptosis in human ovarian carcinoma cells independently of Akt (14Mitsuuchi Y. Johnson S.W. Selvakumaran M. Williams S.J. Hamilton T.C. Testa J.R. Cancer Res. 2000; 60: 5390-5394PubMed Google Scholar). Thus, it is unclear whether paclitaxel causes apoptosis through modulation of the Akt survival pathway.The mitogen-activated protein kinase (MAPK)1 family comprises c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). The ERK pathway is known to be activated by various stimuli including growth factors (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), lipopolysaccharide (16Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar), and chemotherapeutic agents (5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). ERK activation may exert either an antiapoptotic (17Bueno O.F. De Windt L.J. Tymitz K.M. Witt S.A. Kimball T.R. Klevitsky R. Hewett T.E. Jones S.P. Lefer D.J. Peng C.F. Kitsis R.N. Molkentin J.D. EMBO J. 2000; 19: 6341-6350Crossref PubMed Scopus (638) Google Scholar, 18Kurokawa H. Lenferink A.E. Simpson J.F. Pisacane P.I. Sliwkowski M.X. Forbes J.T. Arteaga C.L. Cancer Res. 2000; 60: 5887-5894PubMed Google Scholar, 19Remacle-Bonnet M.M. Garrouste F.L. Heller S. Andre F. Marvaldi J.L. Pommier G.J. Cancer Res. 2000; 60: 2007-2017PubMed Google Scholar) or a proapoptotic (5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 20Bhat N.R. Zhang P. J. Neurochem. 1999; 72: 112-119Crossref PubMed Scopus (222) Google Scholar) influence depending upon the cellular context and by as yet unclarified regulatory mechanisms. The JNK and p38 MAPK signaling pathways are also activated by various and overlapping stimuli such as heat or osmotic shock, radiation, and growth factors (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 21Paumelle R. Tulasne D. Leroy C. Coll J. Vandenbunder B. Fafeur V. Mol. Biol. Cell. 2000; 11: 3751-3763Crossref PubMed Scopus (62) Google Scholar). Although JNK and p38 MAPK have been reported to be activated by paclitaxel (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), the precise roles of these activated kinases in this context remain obscure.It has been reported that paclitaxel enhances ERK activity in various cell lines (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar). By contrast, paclitaxel attenuated ERK activity in some reports (24Townsend K.J. Trusty J.L. Traupman M.A. Eastman A. Craig R.W. Oncogene. 1998; 17: 1223-1234Crossref PubMed Scopus (97) Google Scholar, 25Wu-Wong J.R. Chiou W.J. Wang J. J. Pharmacol. Exp. Ther. 2000; 293: 514-521PubMed Google Scholar). Since abrogation of the MEK/ERK pathway delayed paclitaxel-induced apoptosis (23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar) or failed to prevent it (26Kalechman Y. Longo D.L. Catane R. Shani A. Albeck M. Sredni B. Int. J. Cancer. 2000; 86: 281-288Crossref PubMed Scopus (44) Google Scholar), ERK activation by paclitaxel and its relationship to apoptosis requires elucidation. Investigation of the relationship of paclitaxel-induced apoptosis and activation of the MAP kinases serves as the basis of our studies and provides a venue to understand the interrelationship or independence of MAPKs and apoptosis.By utilizing human esophageal squamous cancer cells challenged with physiological concentrations of paclitaxel, we demonstrate herein that paclitaxel induces apoptosis, which is accompanied by delayed JNK and p38 MAPK activation and prolonged ERK activation lasting over 72 h. Our studies, employing pharmacological inhibitors for MEK or caspases, revealed that prolonged ERK activity was not mediated via the same pathway as the apoptosis machinery and that MEK/ERK blockade did not modify the cytotoxicity of paclitaxel, indicating that the prolonged ERK activation may be secondary to cell death. The prolonged ERK activation appeared to be mediated through a Ras- and MEK-dependent pathway, in which neither the MEK-1 nor the MEK-2 isoform was involved. This suggests that a different MEK family member might be responsible for the prolonged ERK activity observed with paclitaxel.DISCUSSIONWe observed that physiologically relevant concentrations of paclitaxel induced cell death with features characteristic of apoptosis. Although pretreatment with a caspase inhibitor, Z-VAD-FMK, inhibited PARP cleavage induced by paclitaxel, cell death was rescued partially by Z-VAD-FMK, implying that paclitaxel caused cell death via both caspase-dependent and -independent pathways. The caspase-independent pathway may be analogous with so-called "slow cell death," which is often observed by chemotherapeutic drugs (39Blagosklonny M.V. Leukemia. 2000; 14: 1502-1508Crossref PubMed Scopus (120) Google Scholar).Since caspase-3 is considered to be central to the apoptosis machinery (Ref. 40Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar; reviewed in Ref. 31Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar), it was surprising that we observed caspase-7, not caspase-3, activation upon paclitaxel treatment. Our novel finding is nevertheless consistent with a report demonstrating that upon VP-16 treatment, PARP was cleaved by caspase-7 but not caspase-3 (30Germain M. Affar E.B. D'Amours D. Dixit V.M. Salvesen G.S. Poirier G.G. J. Biol. Chem. 1999; 274: 28379-28384Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Since caspase-3 knockout mice have normal apoptotic response (41Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1700) Google Scholar), and deficiency of a caspase member can be compensated by activation of other caspases (42Zheng T.S. Hunot S. Kuida K. Momoi T. Srinivasan A. Nicholson D.W. Lazebnik Y. Flavell R.A. Nat. Med. 2000; 6: 1241-1247Crossref PubMed Scopus (271) Google Scholar), there may be regulatory mechanisms that preferentially utilize different caspase members in response to apoptotic stimuli, and caspase-3 activation may be dispensable in some cellular contexts. It is tempting to speculate that caspase-7 may be preferentially activated by certain chemotherapeutic agents.Paclitaxel has been reported to activate various signal transduction pathways including JNK (22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), p38 MAPK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), and ERK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar), but the primary target for the paclitaxel action is not defined. Although microtubules are the well documented cellular targets of paclitaxel (3Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), CD18 (43Bhat N. Perera P.Y. Carboni J.M. Blanco J. Golenbock D.T. Mayadas T.N. Vogel S.N. J. Immunol. 1999; 162: 7335-7342PubMed Google Scholar), Ki-Ras (44Thissen J.A. Gross J.M. Subramanian K. Meyer T. Casey P.J. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and Bcl-2 (45Rodi D.J. Janes R.W. Sanganee H.J. Holton R.A. Wallace B.A. Makowski L. J. Mol. Biol. 1999; 285: 197-203Crossref PubMed Scopus (223) Google Scholar), all have the potential to bind to paclitaxel. In addition, Taxol mediates serine phosphorylation of shc (46Yang C.P. Horwitz S.B. Cancer Res. 2000; 60: 5171-5178PubMed Google Scholar). Our data indicate that paclitaxel activated the ERK pathway at the level of Ras or, not inconceivably, upstream of Ras. However, since Ras and ERK can co-localize with microtubules (47Best A. Ahmed S. Kozma R. Lim L. J. Biol. Chem. 1996; 271: 3756-3762Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 48Gundersen G.G. Cook T.A. Curr. Opin. Cell Biol. 1999; 11: 81-94Crossref PubMed Scopus (364) Google Scholar), it is conceivable that paclitaxel, when bound to microtubules, induces ERK activation directly or via a Ras-dependent signaling pathway.It has been reported that PARP cleavage may be suppressed by a MEK inhibitor, demonstrating that the caspase cascade is downstream of the MEK/ERK pathway (49Mohr S. McCormick T.S. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5045-5050Crossref PubMed Scopus (91) Google Scholar). By contrast, in our model systems, when cells were pretreated with MEK inhibitors or SB202190, which inhibited both JNK and p38 MAPK at 50 μm, cell death induced by paclitaxel was unaltered. When these compounds were added simultaneously prior to paclitaxel treatment, cell viability was also unaffected (data not shown). Kinase assays revealed that these compounds efficiently suppressed ERK activity. JNK and p38 MAPK kinase activities were partially suppressed. Although we cannot exclude the possibility that the residual kinase activity contributed to cell death, we conclude that the delayed or prolonged activation of the JNK, p38 MAPK, and MEK/ERK kinases occurred independently of cell death, which is underscored by several independent experiments. In concert with this notion, anisomycin has been demonstrated to induce JNK, p38 MAPK, and ERK activation independently of apoptosis (50Torocsik B. Szeberenyi J. Biochem. Biophys. Res. Commun. 2000; 278: 550-556Crossref PubMed Scopus (32) Google Scholar).Since we observed that MEK phosphorylation upon paclitaxel treatment showed a distinct migration on Western blotting with the phospho-MEK1/2 antibody from that upon EGF stimulation, we sought to identify the MEK isoform(s) involved. Immunoprecipitation/Western blotting and kinase assays using anti-MEK-1 and MEK-2 antibodies revealed that neither MEK-1 nor MEK-2 appeared to be responsible for ERK activation by paclitaxel. Although among the MEK family members identified thus far, MEK-5 is the member most closely structurally related to MEK-1 and MEK-2, it is not known to activate ERK-1 or ERK-2 (51English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Thus, it is possible that another member of the MEK family, perhaps a novel one, modulates ERK activation that is induced by paclitaxel. The identification of a novel MEK member is currently under investigation.Sustained activation of the MEK/ERK pathway has been reported upon stimulation with growth factors such as nerve growth factor (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), insulin (53Zhu X. Price-Schiavi S.A. Carraway K.L. Oncogene. 2000; 19: 4354-4361Crossref PubMed Scopus (22) Google Scholar), EGF (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and hepatocyte growth factor (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) as well as during recovery from cardiac ischemia (54Punn A. Mockridge J.W. Farooqui S. Marber M.S. Heads R.J. Biochem. J. 2000; 350: 891-899Crossref PubMed Scopus (77) Google Scholar). Postulated roles for sustained MEK/ERK pathway activation include mediation of neuronal cell differentiation (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar) and matrix metalloproteinase-9 induction (38Genersch E. Hayes K. Neuenfeld Y. Haller H. J. Cell Sci. 2000; 113: 4319-4330PubMed Google Scholar). However, the duration of ERK activation in their reports is less than 24 h. We observed that ERK was activated in a MEK-dependent fashion, and to our surprise, it was sustained even after 72 h of paclitaxel treatment. To our knowledge, this is the first report demonstrating such a prolonged ERK activation with some form of extracellular stimulation. Although the time course of MEK activation preceded ERK activation, there was a time lag between MEK and ERK activation, and ERK activation was still observed even after MEK activity decayed (Fig. 6, A andB). The significance of the time lag between MEK and ERK activation and sustained versus transient ERK activation, however, are not clear. Ultimately, the effects of delayed or prolonged activation of different MAP kinases in the face of cell death may be a means by which the host cell is attempting to maintain cellular homeostasis (Fig. 8).Impaired CL100/MKP-1 activation upon cisplatin treatment was recently demonstrated to be responsible for sustained JNK activation (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), and negative feedback inhibition of the ERK pathway has been implicated to be the most important factor for sustained ERK activation (56Brightman F.A. Fell D.A FEBS Lett. 2000; 482: 169-174Crossref PubMed Scopus (189) Google Scholar). Therefore, we speculate that regulators of MAPKs, such as JNK/SAPK-associated protein 1 (JSAP1) (57Kuboki Y. Ito M. Takamatsu N. Yamamoto Ki. Shiba T. Yoshioka K. J. Biol. Chem. 2000; 275: 39815-39818Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and CL100/MKP-1 (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), might be deregulated by paclitaxel and thus contribute to the sustained MAPK activations observed in our study. Since a small GTP-binding protein, Rap1, has been shown to mediate sustained ERK activation induced by nerve growth factor (58York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar), the involvement of a specific molecule in sustained MAPK activations needs to be taken into account as well. The clarification of such modulators may open new avenues to enhance the efficacy of chemotherapeutic agents. Paclitaxel (Taxol) is a chemotherapeutic agent utilized for the treatment of breast, ovarian, lung, and esophageal cancers (1Crown J. O'Leary M. Lancet. 2000; 355: 1176-1178Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 2Weiner L.M. Semin. Oncol. 1999; 26: 106-108PubMed Google Scholar). Paclitaxel exerts its cytotoxic effect by binding and stabilizing microtubules (3Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and induces apoptosis in a variety of cell types (4Moos P.J. Fitzpatrick F.A. Cell Growth Differ. 1998; 9: 687-697PubMed Google Scholar, 5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 6Shen S.C. Huang T.S. Jee S.H. Kuo M.L. Cell Growth Differ. 1998; 9: 23-29PubMed Google Scholar) independently of p53 status (7Zhang Z. Liu Q. Lantry L.E. Wang Y. Kelloff G.J. Anderson M.W. Wiseman R.W. Lubet R.A. You M. Cancer Res. 2000; 60: 901-907PubMed Google Scholar). The mechanisms underlying paclitaxel-induced apoptosis are unclear. Paclitaxel was recently demonstrated to release cytochrome c by direct action on the mitochondria (8Andre N. Braguer D. Brasseur G. Goncalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar), activate small GTP-binding proteins (9Lou P.J. Chen W.P. Lin C.T. Chen H.C. Wu J.C. J. Cell Biochem. 2000; 79: 542-556Crossref PubMed Scopus (9) Google Scholar, 10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), and activate the NF-κB/IκB-α signaling pathway (11Huang Y. Johnson K.R. Norris J.S. Fan W. Cancer Res. 2000; 60: 4426-4432PubMed Google Scholar). Although these actions were implicated as the mechanisms through which paclitaxel induces apoptosis, these studies were impaired by the employment of high concentrations of paclitaxel. Akt (protein kinase B), a serine/threonine protein kinase, has a pivotal role in exerting an antiapoptotic effect against various stimuli (12Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar). Overexpression of Akt was reported to confer resistance to paclitaxel (13Page C. Lin H.J. Jin Y. Castle V.P. Nunez G. Huang M. Lin J. Anticancer Res. 2000; 20: 407-416PubMed Google Scholar). However, it was recently reported that paclitaxel induced apoptosis in human ovarian carcinoma cells independently of Akt (14Mitsuuchi Y. Johnson S.W. Selvakumaran M. Williams S.J. Hamilton T.C. Testa J.R. Cancer Res. 2000; 60: 5390-5394PubMed Google Scholar). Thus, it is unclear whether paclitaxel causes apoptosis through modulation of the Akt survival pathway. The mitogen-activated protein kinase (MAPK)1 family comprises c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). The ERK pathway is known to be activated by various stimuli including growth factors (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), lipopolysaccharide (16Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar), and chemotherapeutic agents (5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). ERK activation may exert either an antiapoptotic (17Bueno O.F. De Windt L.J. Tymitz K.M. Witt S.A. Kimball T.R. Klevitsky R. Hewett T.E. Jones S.P. Lefer D.J. Peng C.F. Kitsis R.N. Molkentin J.D. EMBO J. 2000; 19: 6341-6350Crossref PubMed Scopus (638) Google Scholar, 18Kurokawa H. Lenferink A.E. Simpson J.F. Pisacane P.I. Sliwkowski M.X. Forbes J.T. Arteaga C.L. Cancer Res. 2000; 60: 5887-5894PubMed Google Scholar, 19Remacle-Bonnet M.M. Garrouste F.L. Heller S. Andre F. Marvaldi J.L. Pommier G.J. Cancer Res. 2000; 60: 2007-2017PubMed Google Scholar) or a proapoptotic (5Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 20Bhat N.R. Zhang P. J. Neurochem. 1999; 72: 112-119Crossref PubMed Scopus (222) Google Scholar) influence depending upon the cellular context and by as yet unclarified regulatory mechanisms. The JNK and p38 MAPK signaling pathways are also activated by various and overlapping stimuli such as heat or osmotic shock, radiation, and growth factors (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 21Paumelle R. Tulasne D. Leroy C. Coll J. Vandenbunder B. Fafeur V. Mol. Biol. Cell. 2000; 11: 3751-3763Crossref PubMed Scopus (62) Google Scholar). Although JNK and p38 MAPK have been reported to be activated by paclitaxel (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), the precise roles of these activated kinases in this context remain obscure. It has been reported that paclitaxel enhances ERK activity in various cell lines (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar). By contrast, paclitaxel attenuated ERK activity in some reports (24Townsend K.J. Trusty J.L. Traupman M.A. Eastman A. Craig R.W. Oncogene. 1998; 17: 1223-1234Crossref PubMed Scopus (97) Google Scholar, 25Wu-Wong J.R. Chiou W.J. Wang J. J. Pharmacol. Exp. Ther. 2000; 293: 514-521PubMed Google Scholar). Since abrogation of the MEK/ERK pathway delayed paclitaxel-induced apoptosis (23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar) or failed to prevent it (26Kalechman Y. Longo D.L. Catane R. Shani A. Albeck M. Sredni B. Int. J. Cancer. 2000; 86: 281-288Crossref PubMed Scopus (44) Google Scholar), ERK activation by paclitaxel and its relationship to apoptosis requires elucidation. Investigation of the relationship of paclitaxel-induced apoptosis and activation of the MAP kinases serves as the basis of our studies and provides a venue to understand the interrelationship or independence of MAPKs and apoptosis. By utilizing human esophageal squamous cancer cells challenged with physiological concentrations of paclitaxel, we demonstrate herein that paclitaxel induces apoptosis, which is accompanied by delayed JNK and p38 MAPK activation and prolonged ERK activation lasting over 72 h. Our studies, employing pharmacological inhibitors for MEK or caspases, revealed that prolonged ERK activity was not mediated via the same pathway as the apoptosis machinery and that MEK/ERK blockade did not modify the cytotoxicity of paclitaxel, indicating that the prolonged ERK activation may be secondary to cell death. The prolonged ERK activation appeared to be mediated through a Ras- and MEK-dependent pathway, in which neither the MEK-1 nor the MEK-2 isoform was involved. This suggests that a different MEK family member might be responsible for the prolonged ERK activity observed with paclitaxel. DISCUSSIONWe observed that physiologically relevant concentrations of paclitaxel induced cell death with features characteristic of apoptosis. Although pretreatment with a caspase inhibitor, Z-VAD-FMK, inhibited PARP cleavage induced by paclitaxel, cell death was rescued partially by Z-VAD-FMK, implying that paclitaxel caused cell death via both caspase-dependent and -independent pathways. The caspase-independent pathway may be analogous with so-called "slow cell death," which is often observed by chemotherapeutic drugs (39Blagosklonny M.V. Leukemia. 2000; 14: 1502-1508Crossref PubMed Scopus (120) Google Scholar).Since caspase-3 is considered to be central to the apoptosis machinery (Ref. 40Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar; reviewed in Ref. 31Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar), it was surprising that we observed caspase-7, not caspase-3, activation upon paclitaxel treatment. Our novel finding is nevertheless consistent with a report demonstrating that upon VP-16 treatment, PARP was cleaved by caspase-7 but not caspase-3 (30Germain M. Affar E.B. D'Amours D. Dixit V.M. Salvesen G.S. Poirier G.G. J. Biol. Chem. 1999; 274: 28379-28384Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Since caspase-3 knockout mice have normal apoptotic response (41Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1700) Google Scholar), and deficiency of a caspase member can be compensated by activation of other caspases (42Zheng T.S. Hunot S. Kuida K. Momoi T. Srinivasan A. Nicholson D.W. Lazebnik Y. Flavell R.A. Nat. Med. 2000; 6: 1241-1247Crossref PubMed Scopus (271) Google Scholar), there may be regulatory mechanisms that preferentially utilize different caspase members in response to apoptotic stimuli, and caspase-3 activation may be dispensable in some cellular contexts. It is tempting to speculate that caspase-7 may be preferentially activated by certain chemotherapeutic agents.Paclitaxel has been reported to activate various signal transduction pathways including JNK (22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), p38 MAPK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), and ERK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar), but the primary target for the paclitaxel action is not defined. Although microtubules are the well documented cellular targets of paclitaxel (3Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), CD18 (43Bhat N. Perera P.Y. Carboni J.M. Blanco J. Golenbock D.T. Mayadas T.N. Vogel S.N. J. Immunol. 1999; 162: 7335-7342PubMed Google Scholar), Ki-Ras (44Thissen J.A. Gross J.M. Subramanian K. Meyer T. Casey P.J. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and Bcl-2 (45Rodi D.J. Janes R.W. Sanganee H.J. Holton R.A. Wallace B.A. Makowski L. J. Mol. Biol. 1999; 285: 197-203Crossref PubMed Scopus (223) Google Scholar), all have the potential to bind to paclitaxel. In addition, Taxol mediates serine phosphorylation of shc (46Yang C.P. Horwitz S.B. Cancer Res. 2000; 60: 5171-5178PubMed Google Scholar). Our data indicate that paclitaxel activated the ERK pathway at the level of Ras or, not inconceivably, upstream of Ras. However, since Ras and ERK can co-localize with microtubules (47Best A. Ahmed S. Kozma R. Lim L. J. Biol. Chem. 1996; 271: 3756-3762Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 48Gundersen G.G. Cook T.A. Curr. Opin. Cell Biol. 1999; 11: 81-94Crossref PubMed Scopus (364) Google Scholar), it is conceivable that paclitaxel, when bound to microtubules, induces ERK activation directly or via a Ras-dependent signaling pathway.It has been reported that PARP cleavage may be suppressed by a MEK inhibitor, demonstrating that the caspase cascade is downstream of the MEK/ERK pathway (49Mohr S. McCormick T.S. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5045-5050Crossref PubMed Scopus (91) Google Scholar). By contrast, in our model systems, when cells were pretreated with MEK inhibitors or SB202190, which inhibited both JNK and p38 MAPK at 50 μm, cell death induced by paclitaxel was unaltered. When these compounds were added simultaneously prior to paclitaxel treatment, cell viability was also unaffected (data not shown). Kinase assays revealed that these compounds efficiently suppressed ERK activity. JNK and p38 MAPK kinase activities were partially suppressed. Although we cannot exclude the possibility that the residual kinase activity contributed to cell death, we conclude that the delayed or prolonged activation of the JNK, p38 MAPK, and MEK/ERK kinases occurred independently of cell death, which is underscored by several independent experiments. In concert with this notion, anisomycin has been demonstrated to induce JNK, p38 MAPK, and ERK activation independently of apoptosis (50Torocsik B. Szeberenyi J. Biochem. Biophys. Res. Commun. 2000; 278: 550-556Crossref PubMed Scopus (32) Google Scholar).Since we observed that MEK phosphorylation upon paclitaxel treatment showed a distinct migration on Western blotting with the phospho-MEK1/2 antibody from that upon EGF stimulation, we sought to identify the MEK isoform(s) involved. Immunoprecipitation/Western blotting and kinase assays using anti-MEK-1 and MEK-2 antibodies revealed that neither MEK-1 nor MEK-2 appeared to be responsible for ERK activation by paclitaxel. Although among the MEK family members identified thus far, MEK-5 is the member most closely structurally related to MEK-1 and MEK-2, it is not known to activate ERK-1 or ERK-2 (51English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Thus, it is possible that another member of the MEK family, perhaps a novel one, modulates ERK activation that is induced by paclitaxel. The identification of a novel MEK member is currently under investigation.Sustained activation of the MEK/ERK pathway has been reported upon stimulation with growth factors such as nerve growth factor (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), insulin (53Zhu X. Price-Schiavi S.A. Carraway K.L. Oncogene. 2000; 19: 4354-4361Crossref PubMed Scopus (22) Google Scholar), EGF (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and hepatocyte growth factor (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) as well as during recovery from cardiac ischemia (54Punn A. Mockridge J.W. Farooqui S. Marber M.S. Heads R.J. Biochem. J. 2000; 350: 891-899Crossref PubMed Scopus (77) Google Scholar). Postulated roles for sustained MEK/ERK pathway activation include mediation of neuronal cell differentiation (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar) and matrix metalloproteinase-9 induction (38Genersch E. Hayes K. Neuenfeld Y. Haller H. J. Cell Sci. 2000; 113: 4319-4330PubMed Google Scholar). However, the duration of ERK activation in their reports is less than 24 h. We observed that ERK was activated in a MEK-dependent fashion, and to our surprise, it was sustained even after 72 h of paclitaxel treatment. To our knowledge, this is the first report demonstrating such a prolonged ERK activation with some form of extracellular stimulation. Although the time course of MEK activation preceded ERK activation, there was a time lag between MEK and ERK activation, and ERK activation was still observed even after MEK activity decayed (Fig. 6, A andB). The significance of the time lag between MEK and ERK activation and sustained versus transient ERK activation, however, are not clear. Ultimately, the effects of delayed or prolonged activation of different MAP kinases in the face of cell death may be a means by which the host cell is attempting to maintain cellular homeostasis (Fig. 8).Impaired CL100/MKP-1 activation upon cisplatin treatment was recently demonstrated to be responsible for sustained JNK activation (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), and negative feedback inhibition of the ERK pathway has been implicated to be the most important factor for sustained ERK activation (56Brightman F.A. Fell D.A FEBS Lett. 2000; 482: 169-174Crossref PubMed Scopus (189) Google Scholar). Therefore, we speculate that regulators of MAPKs, such as JNK/SAPK-associated protein 1 (JSAP1) (57Kuboki Y. Ito M. Takamatsu N. Yamamoto Ki. Shiba T. Yoshioka K. J. Biol. Chem. 2000; 275: 39815-39818Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and CL100/MKP-1 (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), might be deregulated by paclitaxel and thus contribute to the sustained MAPK activations observed in our study. Since a small GTP-binding protein, Rap1, has been shown to mediate sustained ERK activation induced by nerve growth factor (58York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar), the involvement of a specific molecule in sustained MAPK activations needs to be taken into account as well. The clarification of such modulators may open new avenues to enhance the efficacy of chemotherapeutic agents. We observed that physiologically relevant concentrations of paclitaxel induced cell death with features characteristic of apoptosis. Although pretreatment with a caspase inhibitor, Z-VAD-FMK, inhibited PARP cleavage induced by paclitaxel, cell death was rescued partially by Z-VAD-FMK, implying that paclitaxel caused cell death via both caspase-dependent and -independent pathways. The caspase-independent pathway may be analogous with so-called "slow cell death," which is often observed by chemotherapeutic drugs (39Blagosklonny M.V. Leukemia. 2000; 14: 1502-1508Crossref PubMed Scopus (120) Google Scholar). Since caspase-3 is considered to be central to the apoptosis machinery (Ref. 40Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar; reviewed in Ref. 31Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar), it was surprising that we observed caspase-7, not caspase-3, activation upon paclitaxel treatment. Our novel finding is nevertheless consistent with a report demonstrating that upon VP-16 treatment, PARP was cleaved by caspase-7 but not caspase-3 (30Germain M. Affar E.B. D'Amours D. Dixit V.M. Salvesen G.S. Poirier G.G. J. Biol. Chem. 1999; 274: 28379-28384Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Since caspase-3 knockout mice have normal apoptotic response (41Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1700) Google Scholar), and deficiency of a caspase member can be compensated by activation of other caspases (42Zheng T.S. Hunot S. Kuida K. Momoi T. Srinivasan A. Nicholson D.W. Lazebnik Y. Flavell R.A. Nat. Med. 2000; 6: 1241-1247Crossref PubMed Scopus (271) Google Scholar), there may be regulatory mechanisms that preferentially utilize different caspase members in response to apoptotic stimuli, and caspase-3 activation may be dispensable in some cellular contexts. It is tempting to speculate that caspase-7 may be preferentially activated by certain chemotherapeutic agents. Paclitaxel has been reported to activate various signal transduction pathways including JNK (22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), p38 MAPK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 22Shtil A.A. Mandlekar S., Yu, R. Walter R.J. Hagen K. Tan T.H. Roninson I.B. Kong A.N. Oncogene. 1999; 18: 377-384Crossref PubMed Scopus (150) Google Scholar), and ERK (10Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 23Lieu C.H. Liu C.C., Yu, T.H. Chen K.D. Chang Y.N. Lai Y.K. Cell Growth Differ. 1998; 9: 767-776PubMed Google Scholar), but the primary target for the paclitaxel action is not defined. Although microtubules are the well documented cellular targets of paclitaxel (3Diaz J.F. Strobe R. Engelborghs Y. Souto A.A. Andreu J.M. J. Biol. Chem. 2000; 275: 26265-26276Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), CD18 (43Bhat N. Perera P.Y. Carboni J.M. Blanco J. Golenbock D.T. Mayadas T.N. Vogel S.N. J. Immunol. 1999; 162: 7335-7342PubMed Google Scholar), Ki-Ras (44Thissen J.A. Gross J.M. Subramanian K. Meyer T. Casey P.J. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and Bcl-2 (45Rodi D.J. Janes R.W. Sanganee H.J. Holton R.A. Wallace B.A. Makowski L. J. Mol. Biol. 1999; 285: 197-203Crossref PubMed Scopus (223) Google Scholar), all have the potential to bind to paclitaxel. In addition, Taxol mediates serine phosphorylation of shc (46Yang C.P. Horwitz S.B. Cancer Res. 2000; 60: 5171-5178PubMed Google Scholar). Our data indicate that paclitaxel activated the ERK pathway at the level of Ras or, not inconceivably, upstream of Ras. However, since Ras and ERK can co-localize with microtubules (47Best A. Ahmed S. Kozma R. Lim L. J. Biol. Chem. 1996; 271: 3756-3762Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 48Gundersen G.G. Cook T.A. Curr. Opin. Cell Biol. 1999; 11: 81-94Crossref PubMed Scopus (364) Google Scholar), it is conceivable that paclitaxel, when bound to microtubules, induces ERK activation directly or via a Ras-dependent signaling pathway. It has been reported that PARP cleavage may be suppressed by a MEK inhibitor, demonstrating that the caspase cascade is downstream of the MEK/ERK pathway (49Mohr S. McCormick T.S. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5045-5050Crossref PubMed Scopus (91) Google Scholar). By contrast, in our model systems, when cells were pretreated with MEK inhibitors or SB202190, which inhibited both JNK and p38 MAPK at 50 μm, cell death induced by paclitaxel was unaltered. When these compounds were added simultaneously prior to paclitaxel treatment, cell viability was also unaffected (data not shown). Kinase assays revealed that these compounds efficiently suppressed ERK activity. JNK and p38 MAPK kinase activities were partially suppressed. Although we cannot exclude the possibility that the residual kinase activity contributed to cell death, we conclude that the delayed or prolonged activation of the JNK, p38 MAPK, and MEK/ERK kinases occurred independently of cell death, which is underscored by several independent experiments. In concert with this notion, anisomycin has been demonstrated to induce JNK, p38 MAPK, and ERK activation independently of apoptosis (50Torocsik B. Szeberenyi J. Biochem. Biophys. Res. Commun. 2000; 278: 550-556Crossref PubMed Scopus (32) Google Scholar). Since we observed that MEK phosphorylation upon paclitaxel treatment showed a distinct migration on Western blotting with the phospho-MEK1/2 antibody from that upon EGF stimulation, we sought to identify the MEK isoform(s) involved. Immunoprecipitation/Western blotting and kinase assays using anti-MEK-1 and MEK-2 antibodies revealed that neither MEK-1 nor MEK-2 appeared to be responsible for ERK activation by paclitaxel. Although among the MEK family members identified thus far, MEK-5 is the member most closely structurally related to MEK-1 and MEK-2, it is not known to activate ERK-1 or ERK-2 (51English J.M. Vanderbilt C.A. Xu S. Marcus S. Cobb M.H. J. Biol. Chem. 1995; 270: 28897-28902Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Thus, it is possible that another member of the MEK family, perhaps a novel one, modulates ERK activation that is induced by paclitaxel. The identification of a novel MEK member is currently under investigation. Sustained activation of the MEK/ERK pathway has been reported upon stimulation with growth factors such as nerve growth factor (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), insulin (53Zhu X. Price-Schiavi S.A. Carraway K.L. Oncogene. 2000; 19: 4354-4361Crossref PubMed Scopus (22) Google Scholar), EGF (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and hepatocyte growth factor (15McCawley L.J. Li S. Wattenberg E.V. Hudson L.G. J. Biol. Chem. 1999; 274: 4347-4353Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) as well as during recovery from cardiac ischemia (54Punn A. Mockridge J.W. Farooqui S. Marber M.S. Heads R.J. Biochem. J. 2000; 350: 891-899Crossref PubMed Scopus (77) Google Scholar). Postulated roles for sustained MEK/ERK pathway activation include mediation of neuronal cell differentiation (52Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar) and matrix metalloproteinase-9 induction (38Genersch E. Hayes K. Neuenfeld Y. Haller H. J. Cell Sci. 2000; 113: 4319-4330PubMed Google Scholar). However, the duration of ERK activation in their reports is less than 24 h. We observed that ERK was activated in a MEK-dependent fashion, and to our surprise, it was sustained even after 72 h of paclitaxel treatment. To our knowledge, this is the first report demonstrating such a prolonged ERK activation with some form of extracellular stimulation. Although the time course of MEK activation preceded ERK activation, there was a time lag between MEK and ERK activation, and ERK activation was still observed even after MEK activity decayed (Fig. 6, A andB). The significance of the time lag between MEK and ERK activation and sustained versus transient ERK activation, however, are not clear. Ultimately, the effects of delayed or prolonged activation of different MAP kinases in the face of cell death may be a means by which the host cell is attempting to maintain cellular homeostasis (Fig. 8). Impaired CL100/MKP-1 activation upon cisplatin treatment was recently demonstrated to be responsible for sustained JNK activation (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), and negative feedback inhibition of the ERK pathway has been implicated to be the most important factor for sustained ERK activation (56Brightman F.A. Fell D.A FEBS Lett. 2000; 482: 169-174Crossref PubMed Scopus (189) Google Scholar). Therefore, we speculate that regulators of MAPKs, such as JNK/SAPK-associated protein 1 (JSAP1) (57Kuboki Y. Ito M. Takamatsu N. Yamamoto Ki. Shiba T. Yoshioka K. J. Biol. Chem. 2000; 275: 39815-39818Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and CL100/MKP-1 (55Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (126) Google Scholar), might be deregulated by paclitaxel and thus contribute to the sustained MAPK activations observed in our study. Since a small GTP-binding protein, Rap1, has been shown to mediate sustained ERK activation induced by nerve growth factor (58York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar), the involvement of a specific molecule in sustained MAPK activations needs to be taken into account as well. The clarification of such modulators may open new avenues to enhance the efficacy of chemotherapeutic agents. We are indebted to members of the Rustgi laboratory for discussion and support.
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