JOURNAL OF

MolecularNeuroscienceEditor-in-Chief: ILLANA GOZES, PhD

JOURNAL OF

MolecularNeuroscienceEditor-in-Chief: ILLANA GOZES, PhD

Volume 25 Number 2, 2005 ISSN: 0895–8696

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In This Issue

Receptor Trafficking

Humanin, Erythropoietin

Ras-Erk BACE1

Synucleins

Signaling Pathways

JMN_25_2_cvr 2/22/05, 2:22 PM1

Roles of Ras-Erk in Apoptosis of PC12 Cells Induced by Trophic Factor Withdrawal or Oxidative Stress

Hao Jiang,1,2,4 Lijie Zhang,1,2 David Koubi,1 Jarret Kuo,1,2 Laurent Groc,1Alba I. Rodriguez,1,2 Tangella Jackson Hunter,1,2 Stephen Tang,1

Philip Lazarovici,6 Subhash C. Gautam,3 and Robert A. Levine*,1,2,4,5

1William T. Gossett Neurology Laboratories, 2Complementary and Integrative MedicineResearch Program, and 3Department of Surgery, Henry Ford Health System, Detroit, MI 48202;

4John D. Dingell Veterans Administration Medical Center, Detroit, MI 48201; 5Department of Pharmaceutical Sciences, Wayne State University School of Pharmacy, Detroit, MI 48201,

and 6Department of Pharmacology and Experimental Therapeutics, School of Pharmacy,Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel

Received June 4, 2004; Accepted August 23, 2004

Abstract

To understand the role of Ras-MAPK (mitogen-activated protein kinase) in trophic factor withdrawal- andoxidative stress-induced apoptotic cell death processes, undifferentiated rat pheochromocytoma PC12 cells anda PC12 variant cell line stably expressing the Ras dominant-negative mutant (M-M17-26) were subjected toserum withdrawal in the absence or presence of H2O2 treatment. The extent of cell death was analyzed by lac-tate dehydrogenase release, internucleosomal DNA fragmentation, and caspase-3 assays. Both serum with-drawal and H2O2 treatment induced apoptotic cell death in PC12 cells, and the extent of cell death was greatlyenhanced in M-M17-26 cells. DNA fragmentation induced by serum withdrawal or H2O2 treatment was blockedcompletely by a general caspase inhibitor, Z-VAD-FMK. A selective MAPK kinase inhibitor, U0126, blocked theH2O2-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinase) in PC12 cells and increased thelevels of active caspase-3 in M-M17-26 under serum withdrawal or H2O2 treatment. In addition, the short-termH2O2 treatment (5–30 min) was sufficient to cause DNA fragmentation in M-M17-26 cells even though H2O2 wasremoved and cells were incubated in regular growth medium with complete serum for 24 h. However, similar,short-term H2O2 treatment of PC12 cells did not induce DNA fragmentation 24 h later. These results suggestthat the Ras-Erk pathway is critical in mediating protection against apoptotic cell death induced by either trophicfactor withdrawal or increased oxidative stress.

Index Entries: PC12; Ras, Erk, apoptosis; oxidative stress; trophic factor; hydrogen peroxide.

Introduction

Oxidative stress has been demonstrated as one ofthe major causes in aging, neuronal injury, and neu-rodegeneration (Olanow and Arendash, 1994).Oxidative stress is characterized by impaired energy

metabolism, mitochondrial dysfunction, increasedlipid peroxidation, and increased DNA and proteindamage (Okuda et al., 1996; Bains and Shaw, 1997).Elevated production of reactive oxygen species(ROS), such as superoxide, hydroxyl free radicals,and H2O2, might lead to oxidative stress. Substantial

Journal of Molecular NeuroscienceCopyright © 2005 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN0895-8696/05/25:133–140/$30.00

Journal of Molecular Neuroscience 133 Volume 25, 2005

*Author to whom all correspondence and reprint requests should be addressed. E-mail: bob-levine@earthlink.net.

ORIGINAL ARTICLE

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evidence suggests that ROS can induce apoptotic celldeath, characterized by membrane blebbing, decreaseof mitochondrial membrane potential, nuclear chro-matin condensation, and DNAfragmentation (Jacob-son, 1996). Above a certain level, ROS might alsocause necrotic cell death (Sakamoto et al., 1996). Reac-tive oxygen species (ROS) induces apoptosis in a vari-ety of cell types through the modulation of variousintracellular signaling pathways. Treatment of PC12cells with low concentrations of H2O2 results in apop-totic cell death (Kamata et al., 1996), which is asso-ciated with activation of caspase-3 (Lee et al., 2000),as well as changes of expression in the Bcl-2 familyof proteins (Maroto and Perez-Polo, 1997; Yamakawaet al., 2000). A selective inhibitor for caspase-3, Z-DEVD-FMK, and a general caspase inhibitor, Z-VAD-FMK (Z-Val-Ala-Asp[OMe]-CH2F), inhibit caspase-3activation as well as H2O2-induced apoptotic celldeath (Yamakawa et al., 2000).

Mitogen-activated protein kinases (MAPKs) con-sist of a family of serine/threonine kinases, includ-ing extracellular signal-regulated kinase (Erk),SAPK/JNK (stress-activated protein kinase/c-JunN-terminal kinase), and p38 MAPK (Tabakman etal., 2004). Mitogen-activated protein kinases(MAPKs) control multiple intracellular biologicalfunctions, including cell growth, differentiation, sur-vival, and death and can be activated in response tovarious extracellular signals (Robinson and Cobb,1997). Different stimuli activate MAPKs or SAPKswith distinct cellular responses. It is generally agreedthat Erk is involved in cell survival, whereasSAPK/JNK and p38 MAPK are participants in thecell death process. It has been reported that H2O2 cansimultaneously activate multiple MAPK pathways(Kyriakis, 1999) and increase the activation of non-receptor kinases, including Ras, protein kinase C,Raf-1, MAPK kinase 1(MEK1), and Src kinase familymembers (Abe et al., 1998, 2000).

PC12 cells have been used extensively as a modelfor studies of neuronal differentiation, neuronalsurvival, and neurotransmitter secretion, as well asdefining the underlying molecular mechanisms(Tischler and Greene, 1978;Rukenstein et al.,1991;Vaudry et al., 2002a, 2002b). UndifferentiatedPC12 cells undergo apoptosis after serum depriva-tion and can be rescued by trophic agents, such asnerve growth factor (NGF), fibroblast growth factor,and dibutyryl cAMP or by overexpression of pro-survival Bcl-2 proteins (Greene, 1978; Lindenboimet al., 1995; Anastasiadis et al., 2001). H2O2-inducedapoptosis in PC12 cells also can be prevented by

NGF, forskolin, and Bcl-2 overexpression (Satoh etal., 1996). In the current studies, we examined theextent of cell death after serum withdrawal or H2O2-induced oxidative stress in wild-type PC12 cells anda variant PC12 cell line (M-M17-26) stably expressinga dominant-negative Ha-Ras (Asn-17) (Szeberenyiet al., 1990), as well as the contribution of the Ras-Erksignaling pathway during this process. We demon-strated that the Ras-Erk signaling pathway might beone of the critical prosurvival signaling pathwaysagainst apoptotic cell death induced by either trophicfactor withdrawal or increase of oxidative stress.

Materials and MethodsMaterials

Hydrogen peroxide (37%) was purchased fromSigma (St. Louis, MO). A general caspase inhibitor,Z-VAD-FMK, and a MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenyl-thio]butadiene) (Favata et al., 1998), were obtainedfrom Calbiochem (La Jolla, CA). Antibodies againstphospho-Erk and Erk1 were obtained from SantaCruz Biotechnology (Santa Cruz, CA). Antibodyagainst cleaved caspase-3 (Asp-175) was obtainedfrom Cell Signaling Technology (Beverly, MA).

Cell Culture PC12 cells were cultured in Dulbecco’s modified

Eagle medium (DMEM), supplemented with 7% fetalbovine serum, 7% horse serum, 100 µg strepto-mycin/mL and 100 U penicillin/mL (Invitrogen,Carlsbad, CA) at 37°C with 5% CO2 in a humidifiedincubator (Jiang et al., 1997b). PC12 cell variant M-M17-26, expressing the Ha-Ras Asn-17 gene underthe transcriptional control of the mouse metalloth-ionein-I promoter, was originally obtained from Dr.Geoffrey M. Cooper (Department of Biology, BostonUniversity, MA) (Szeberenyi et al., 1990). PC12 cellvariants were cultured under the same culture condi-tions as wild-type PC12 cells, as described previously(Lazarovici et al., 1998).

Lactate Dehydrogenase Release AssayPC12 cells (5 × 104 cells/well) were plated into 24-

well plates the day before the experiment. Aftervarious treatments, the amount of lactate dehydro-genase (LDH) released to the medium and total cel-lular LDH were determined using a colorimetricCyto Tox 96 Non-Radioactive Cytotoxicity Assayfrom Promega (Madison, WI). The percentage ofreleased LDH vs total LDH was calculated.

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DNA Fragmentation After various treatments, cells were collected and

subjected to DNA fragmentation, as described pre-viously (Anastasiadis et al., 2001). Briefly, cells werelysed with lysis buffer (0.5% Triton X-100, 5 mMTris buffer at pH 7.4, 20 mM EDTA), and RNA wasremoved by incubation with RNase A (0.8 mg/mL)at 37°C for 30 min. DNA was extracted withphenol/chloroform and precipitated with 1/10 vol3 M sodium acetate (pH 5.2) and 2 vol of 100%ethanol. The DNA pellet was obtained by centrifu-gation at 10,000g for 20 min at 4°C, dried, and finallyresuspended in 25 µL of 1× TAE (40 mM Tris-acetateand 1 mM EDTA). The samples were separated ona 1.2% agarose gel, and DNA laddering wasdetected by incubating the gel with ethidium bromide (1 µg/mL) for 20 min.

TUNEL Staining After various treatments, TUNEL staining was

performed using an ApopTag in situ apoptosis detec-tion kit from Intergen (Purchase, NY). Briefly, afterfixation in 4% paraformaldehyde and incubation inpermeabilization solution (0.1% Triton X-100 in 0.1%sodium citrate), fixed cells were incubated withTUNEL reaction mixture at 37°C for 1 h. TUNEL-stained cells were detected under a fluorescentmicroscope and photographed.

Western Blot AnalysisWestern blot analysis was performed as reported

previously (Jiang et al., 1997a). After various exper-imental treatments, cells were collected by centrifu-gation, washed once in phosphate-buffered saline,and lysed in lysis buffer (20 mM HEPES at pH 7.4,100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxy-cholic acid, 10% glycerol, 1 mM EDTA, 1 mM EGTA,1 mM NaVO3, 50 mM NaF, 1 mM PMSF, 10 µg/mLleupeptin, 10 µg/mL aprotonin) for 30 min on ice.The soluble proteins were obtained by centrifuga-tion at 14,000 rpm for 10 min at 4oC, and concentra-tion of each sample was determined by the Bradfordprotein assay kit (Pierce Biotechnology, Rockford, IL).For immunoblotting, equal amounts of cell lysatewere subjected to electrophoresis on 10% SDS–poly-acrylamide gels. Separated proteins were then elec-trotransferred to Immobilon membranes (Millipore,Bedford, MA). After exposure to specific antibodies,proteins were visualized by use of an enhancedchemiluminescence protein detection kit (PierceBiotechnology, Rockford, IL), according to themanufacturer’s instructions.

Statistical Analysis Data were represented as means ± S.D. Differ-

ences between groups were tested for statisticalsignificance using Student’s t-test. A p-value < 0.05was considered as a significant difference.

ResultsDose- and Time-Dependent Cell Death Induced

by Serum Withdrawal and H2O2 in PC12 Cells To examine the dose-dependent effect of H2O2 on

cell death, PC12 cells were treated with H2O2 at con-centrations of 100, 200, or 500 µM for 16 h under serum-free conditions. LDH release assay showed that thepercentage of LDH released was increased from 4%under control conditions to 35%, 75%, and 79%, at100, 200, or 500 µM of H2O2, respectively (Fig. 1A). Toexamine the time-dependent cell death induced byH2O2, PC12 cells were treated with 200 µM H2O2 for4, 8, and 16 h under serum-free conditions. LDHrelease was increased from 4% under control condi-tions to 59%, 55%, and 69%, after 4, 8, and 16 h of H2O2

Fig. 1. Dose- and time-dependent decrease of cell via-bility in PC12 cells treated with H2O2. The extent of celldeath was measured by LDH release assay. (A) PC12 cellswere treated with 0, 100, 200, and 500 µM H2O2 in serum-free medium for 16 h; (B) PC12 cells were treated with200 µM H2O2 in serum-free medium for 2, 4, 8, and 16 h.Error bars represent S.D. (n = 6; ***, p < 0.001).

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treatment, respectively (Fig. 1B). These results sug-gest that H2O2-induced cell death in PC12 cells is bothdose- and time-dependent. Aconcentration of 200 µMof H2O2 was used in all of the experiments that follow.

Serum Withdrawal and H2O2-Induced ApoptoticCell DeathTUNEL staining was performed to examine if

H2O2-induced cell death is apoptotic. PC12 cellswere treated with 200 µM H2O2 for 16 h underserum-free conditions. Under control conditions,only a few TUNEL-positive cells were detected (Fig.2A,a), but an increasing number of TUNEL-posi-tive cells was detected in H2O2-treated conditions(Fig. 2A,b). As a positive control, PC12 cells werealso treated with 200 nM staurosporine for 8 h,which is known to induce apoptosis in PC12 cells.Staurosporine induced a marked increase in thenumber of TUNEL-positive cells (Fig. 2A,c). Themorphology of the cells in each treatment is shownin Fig. 2A,d–f. Treatment of PC12 cells with 200 µMH2O2 in serum-free medium for 16 h induces DNAfragmentation in PC12 cells, but no apparent DNAfragmentation was observed under serum-freeconditions for 16 h (Fig. 2B). However, serum with-drawal for 16 h caused a great increase of DNAfrag-mentation in M-M17-26 cells, and exposure of cellsto 200 µM H2O2 caused further enhancement ofDNA fragmentation (Fig. 2B). Treatment of PC12cells or M-M17-26 cells with 100 µM general cas-pase inhibitor, Z-VAD-FMK, completely blockedboth serum withdrawal– induced and H2O2-induced DNAfragmentation (Fig. 2B). These resultssuggest that M-M17-26 cells, which contain a Rasdominant-negative mutant, are more sensitive toserum withdrawal or H2O2 treatment. Caspase acti-vation might be involved in these cell deathprocesses.

To examine the specific caspase involved in apop-totic cell death, PC12 or M-M17-26 cells were treatedfor either 5 or 16 h under conditions of serum-containing, serum-free, and serum-free plus 200 µMH2O2 media. Western blot analysis using the anti-cleaved caspase-3 antibody showed a significantincrease in levels of active caspase-3 proteins underserum-free and serum-free plus 200 µM H2O2 con-ditions in M-M17-26 cells, after 5 or 16 h of treatment(Fig. 2C). In PC12 cells, a small increase in levels ofactive caspase-3 was also observed under serum-free and serum-free plus 200 µM H2O2 conditions(Fig. 2C). These results suggest that Ras-mediatedsignaling might be critical in the apoptotic cell death

process under conditions of serum withdrawal orH2O2 treatment.

MEK Inhibitor U0126 Blocked H2O2-InducedErk1/2 PhosphorylationTreatment of PC12 with 200 µM H2O2 induced

activation of Erk1/2 (Fig. 3A). However, 200 µMH2O2 did not induce phosphorylation of Erk1/2 inM-M17-26 cells (Fig. 3A), suggesting that H2O2-induced, Ras-mediated phosphorylation of Erk1/2is impaired in this cell line. It has been shown pre-viously that both NGF and epidermal growthfactor were also unable to induce activation ofErk1/2 in M-M17-26 cells, as measured by kinaseassay using myelin basic protein as the substrate(Lazarovici et al., 1997). Treatment of PC12 cellswith 10 µM U0126 not only reduced the basal levels

Fig. 2. H2O2-induced apoptotic cell death in PC12 andM-M17-26 cells. (A) TUNEL staining of PC12 cells leftuntreated, or treated with H2O2 (200 µM) for 16 h or stau-rosporine (200 nM) for 8 h. Panels a–c represent TUNELstaining of PC12 cells untreated, or treated with H2O2and staurosporine, respectively; Panels d–f represent thecorresponding phase-contrast images of a–c, respectively.(B) PC12 and M-M17-26 cells were untreated or treatedwith 200 µM H2O2 for 16 h in the absence or presence of100 µM general caspase inhibitor Z-VAD-FMK. DNA frag-mented assay was performed. (C) Western blot analysis ofactive caspase-3 under conditions of serum-containing,serum-free, or serum-free plus H2O2 treatment (200 µM)for 5 or 16 h.

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of Erk1/2 phosphorylation but also blocked H2O2-induced Erk1/2 phosphorylation (Fig. 3B).

MEK Inhibitor U0126 Enhanced H2O2-InducedApoptosisThe effects of U0126 on H2O2-induced cell death

were determined by LDH release assay (Fig. 4A).Under serum-free conditions, 16 h of H2O2 treatmentinduced LDH release to 16.7% from the control levelsof 4.6%; however, in the presence of U0126, H2O2-induced LDH release was increased further from16.7% to 25%. U0126 treatment by itself showedapprox 5% of LDH release, which was similar to thecontrol levels of 4.8%. These results suggest that thepresence of MEK inhibitor U0126 might enhanceH2O2-induced cell death.

To further examine the role of U0126 in the apop-totic cell death process, Western blot analysis of activecaspase-3 was performed in PC12 cells using celllysates derived from samples treated with serum-free or serum-free plus 200 µM H2O2 for 16 h, in theabsence or presence of 10 µM U0126 (Fig. 4B). In thepresence of U0126, marked increase of active cas-pase-3 proteins was observed under serum with-drawal or H2O2 treatment. These results suggest thatthe MEK inhibitor U0126 can enhance apoptotic cell

death induced by serum withdrawal or H2O2treatment through the increase of active caspase-3.

Short-Term Treatment of H2O2 on the Extent of Cell Death To examine the short-term effects of H2O2 treatment

on DNAfragmentation, PC12 and M-M17-26 cells weretreated with 200 µM H2O2 in serum-free medium for10 min. Medium was removed and cells were incu-bated in regular growth medium with complete serumfor 24 h. No DNAfragmentation was observed in PC12cells, but enhanced fragmentation was observed inM-M17-26 cells (Fig. 5A). In addition, M-M17-26 cellswere treated with 200 µM H2O2 in serum-free mediumfor 5, 15, and 30 min and incubated with regular growthmedium with complete serum for an additional 24 h.Increased DNAfragmentation was observed (Fig. 5B),and DNAfragmentation was maximal with either 15 or30 min of treatment with H2O2. These results suggest

Fig. 3. Effects of MEK inhibitor U0126 on H2O2-inducedErk1/2 phosphorylation in PC12 M-M17-26 cells. (A) West-ern blot analysis of Erk1/2 phosphorylation in PC12 andM-M17-26 cells treated with 200 µM H2O2 for 0, 1, 3, and5 h; (B) Western blot analysis of phospho-Erk1/2 and basalErk1/2 levels in PC12 cells subjected to conditions of serum-free and serum-free plus 200 µM H2O2 media for 1 h, inthe absence or presence of 10 µM U0126. Fig. 4. Effects of MEK inhibitor U0126 on H2O2-induced

apoptosis in PC12 cells. PC12 cells were subjected to serum-containing, serum-free, and serum-free plus 200 µM H2O2treatment for 16 h in the absence or presence of 10 µMU0126. (A) LDH release assay of H2O2-induced cell deathin PC12 cells; *, significant difference between control andH2O2-treated groups; *∆, significant difference betweenH2O2-treated and U0126 plus H2O2-treated groups; p <0.05;n = 6; (B) Western blot analysis of active caspase-3 levels.

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that M-M17-26 cells are more sensitive to short-termoxidative stress.

DiscussionOur studies show that (1) serum withdrawal and

H2O2 treatment result in apoptotic cell death, whichcan be detected within hours under stress condi-tions; (2) PC12 cells stably expressing Ras dominant-negative mutant are more sensitive to either serumwithdrawal or H2O2 treatment; (3) a novel MEKinhibitor, U0126, further enhances apoptotic celldeath induced by serum withdrawal or H2O2 treat-ment. Our results are consistent with other studiesshowing that both serum withdrawal and H2O2 caninduce apoptosis in PC12 cells (Park et al., 1996). Weobserved dose- and time-dependent apoptosisinduced by serum withdrawal and oxidative stressin PC12 cells and a great enhancement of apoptosis

in a variant PC12 cell line stably expressing the Rasdominant-negative mutant. These observations sug-gest that the Ras-mediated signaling pathway playsa critical role in promoting cell survival under trophicfactor withdrawal and oxidative stress conditions.The enhancement of apoptosis after MEK inhibitorU0126 treatment further suggests that Ras-mediatedErk1/2 activation might be involved in the apop-totic cell death process. In addition, the enhance-ment of DNAfragmentation by H2O2 under serum-freeconditions suggests that trophic factor withdrawal-and oxidative stress-induced apoptosis might uti-lize the same signaling pathway to activate the celldeath process. This pathway is likely to be caspase-dependent, as blocking the caspase activities usingthe general caspase inhibitor Z-VAD-FMK results incomplete inhibition of the DNA fragmentation. Theinvolvement of caspases is further confirmed by theobservation of an increase of the cleavage of pro-caspase-3 to active caspase-3 after serum deprivationor H2O2 treatment.

Exposure of PC12 cells with H2O2 has been shownto activate ERK2, and this activation can be blockedby either inducible or constitutive expression of thedominant-negative Ras-N-17 in PC12 cells (Guytonet al., 1996). It shows further that PC12 cells express-ing Ras dominant-negative mutant were more sen-sitive to H2O2 toxicity than wild-type PC12 cells.Constitutively active MEK, when expressed in NIH-3T3 cells, is more resistant to H2O2 toxicity (Guytonet al., 1996). It has been reported that PD98059 canattenuate H2O2-induced cell death by approx 50%,and both PD98059 and U0126 can inhibit H2O2-induced JNK activation in HT29 cells (Salh et al.,2000). Our finding of an enhancement of cell deathin the presence of MEK inhibitor U0126 suggestedthat the Ras-Erk signaling pathway might be criti-cal for cell survival. Similar studies have shown thatblocking Erk1/2 activation with 10 µM U0126enhanced H2O2-induced toxicity when H2O2 wasused at the concentration 100–300 µM in culturedcortical neurons (Crossthwaite et al., 2002). In addi-tion, transient activation of Erk, or basal, constitu-tive Erk activity, has been suggested to participatein the induction of apoptosis or the maintenance ofcell survival, respectively (Ishikawa and Kitamura,1999). Taken together, these results demonstrate theimportance of the Ras-Erk signaling pathway in pre-venting apoptosis under trophic factor withdrawalor oxidative stress conditions.

The Ras-Erk signaling pathway has been reportedto be involved in many cellular biological functions,

Fig. 5. Short-term effects of H2O2 in PC12 and M-M-17-26 cells. (A) PC12 and M-M17-26 cells were left untreatedor treated with 200 µM H2O2 for 10 min, and the mediumwas replaced with regular growth medium with serum for anadditional 24 h; (B)M-M17-26 cells were untreated or treatedwith 200 µM H2O2 for 5, 15, and 30 min, and the mediumwas replaced with regular growth medium with serum for anadditional 24 h. DNA fragmentation assay was performed.

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including cell growth, differentiation, and cell sur-vival (Vaudry et al., 2002b). Erk1/2 activity mightbe closely related to the expression of members ofthe Bcl-2 family of proteins and subsequent modu-lation of the caspase cascade. It has been reportedthat U0126 inhibited activities of ERKs, resulting inthe down-regulation of expression of the Bcl-xL pro-tein and subsequently apoptosis in a human ery-thropoietin (EPO)-dependent leukemia cell lineUT-7/EPO (Mori et al., 2003). Treatment of murineB-cell lymphoma cells with MEK inhibitors PD98059,U0126, and PD184352 led to a loss of ERK1/2 phos-phorylation, a reversal of NF-κB1 nuclear p50homodimer DNA binding, and a decrease in Bcl-2protein expression (Kurland et al., 2003). It has beenreported that U0126 can markedly sensitize TRAIL-induced apoptosis in human melanoma cells andincrease caspase-3 activation after cotreatment(Zhang et al., 2003). These observations suggest thatthe Ras/MEK/Erk pathway acts upstream of andregulates the expression of members of the Bcl-2family of proteins. Inhibition of this pathway mightresult in down-regulation of the expression of pro-survival members of the Bcl-2 family of proteins,such as Bcl-2 and Bcl-xL, which in turn leads to theincrease of activation of the downstream caspasecascade, such as caspase-3.

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