Portal de Periódicos da CAPES

Central Role of Fas-associated Death Domain Protein in Apoptosis Induction by the Mitogen-activated Protein Kinase Kinase Inhibitor CI-1040 (PD184352) in Acute Lymphocytic Leukemia Cells in Vitro

2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês

10.1074/jbc.m304793200

1083-351X

Xue Meng, Joya Chandra, David A. Loegering, Keri Van Becelaere, Timothy Kottke, Steven D. Gore, Judith E. Karp, Judy Sebolt-Leopold, Scott H. Kaufmann,

NF-κB Signaling Pathways

Because the MAPK pathway plays important roles in cell proliferation and inhibition of apoptosis, this pathway has emerged as a potential therapeutic target for solid tumors and leukemia. At the present time there is little information about activation of this pathway and the consequences of its inhibition in acute lymphocytic leukemia cells (ALL). In the present study, constitutive MAPK pathway activation, as evidenced by phosphorylation of ERK1 and ERK2, was observed in 8 of 8 human lymphoid cell lines and 33% (8:24) of pretreatment ALL bone marrows. Inhibition of this pathway by the MEK inhibitors CI-1040 and PD098059 induced apoptosis through a unique pathway involving dephosphorylation and aggregation of Fas-associated death domain protein followed by death receptor-independent caspase-8 activation. Jurkat cell variants lacking Fas-associated death domain protein or procaspase-8 were resistant to CI-1040-induced apoptosis, as were Jurkat or Molt3 cells treated with the O-methyl ester of the caspase-8 inhibitor N-(Nα-benzyloxycarbonylisoleucylglutamyl) aspartate fluoromethyl ketone. In contrast, CI-1040-induced apoptosis was unaffected by blocking anti-Fas antibody, soluble tumor necrosis factor-α-related apoptosis-inducing ligand decoy receptor, or transfection with cDNA encoding the anti-apoptotic Bcl-2 family member Mcl-1 or dominant negative caspase-9. Collectively, these results identify the MAPK pathway as a potential therapeutic target in ALL and delineate a mechanism by which MEK inhibition triggers apoptosis in ALL cells. Because the MAPK pathway plays important roles in cell proliferation and inhibition of apoptosis, this pathway has emerged as a potential therapeutic target for solid tumors and leukemia. At the present time there is little information about activation of this pathway and the consequences of its inhibition in acute lymphocytic leukemia cells (ALL). In the present study, constitutive MAPK pathway activation, as evidenced by phosphorylation of ERK1 and ERK2, was observed in 8 of 8 human lymphoid cell lines and 33% (8:24) of pretreatment ALL bone marrows. Inhibition of this pathway by the MEK inhibitors CI-1040 and PD098059 induced apoptosis through a unique pathway involving dephosphorylation and aggregation of Fas-associated death domain protein followed by death receptor-independent caspase-8 activation. Jurkat cell variants lacking Fas-associated death domain protein or procaspase-8 were resistant to CI-1040-induced apoptosis, as were Jurkat or Molt3 cells treated with the O-methyl ester of the caspase-8 inhibitor N-(Nα-benzyloxycarbonylisoleucylglutamyl) aspartate fluoromethyl ketone. In contrast, CI-1040-induced apoptosis was unaffected by blocking anti-Fas antibody, soluble tumor necrosis factor-α-related apoptosis-inducing ligand decoy receptor, or transfection with cDNA encoding the anti-apoptotic Bcl-2 family member Mcl-1 or dominant negative caspase-9. Collectively, these results identify the MAPK pathway as a potential therapeutic target in ALL and delineate a mechanism by which MEK inhibition triggers apoptosis in ALL cells. ERK1 1The abbreviations used are: ERKextracellular signal-regulated kinaseALLacute lymphocytic leukemiaAMLacute myelogenous leukemiaCRcomplete responseDEVD-AFCNα-acetylaspartylglutamylvalinyl-aspartyl-7-amino-4-trifluoromethylcoumarinDRdeath receptorEGFPenhanced green fluorescent proteinFADDFas-associated death domain proteinFasLFas ligandIETD(OMe)-fmkN-(Nα-benzyloxycarbonylisoleucylglutamylthreonyl)aspartic acid fluoromethyl ketoneMAPKmitogen-activated protein kinaseMEKmitogen-activated kinase/extracellular signal-regulated kinase kinasePh+Philadelphia chromosome-positiveTRAILtumor necrosis factor-α-related apoptosis-inducing ligandZVAD(OMe)-fmkthe O-methyl ester of N-(Nα-benzyloxycarbonylvalinylalanyl) aspartic acid fluoromethyl ketoneRTreverse transcriptasePARPpoly(ADP-ribose) polymeraseHSP90heat shock protein 90.1The abbreviations used are: ERKextracellular signal-regulated kinaseALLacute lymphocytic leukemiaAMLacute myelogenous leukemiaCRcomplete responseDEVD-AFCNα-acetylaspartylglutamylvalinyl-aspartyl-7-amino-4-trifluoromethylcoumarinDRdeath receptorEGFPenhanced green fluorescent proteinFADDFas-associated death domain proteinFasLFas ligandIETD(OMe)-fmkN-(Nα-benzyloxycarbonylisoleucylglutamylthreonyl)aspartic acid fluoromethyl ketoneMAPKmitogen-activated protein kinaseMEKmitogen-activated kinase/extracellular signal-regulated kinase kinasePh+Philadelphia chromosome-positiveTRAILtumor necrosis factor-α-related apoptosis-inducing ligandZVAD(OMe)-fmkthe O-methyl ester of N-(Nα-benzyloxycarbonylvalinylalanyl) aspartic acid fluoromethyl ketoneRTreverse transcriptasePARPpoly(ADP-ribose) polymeraseHSP90heat shock protein 90. and ERK2 are the final kinases in a signal transduction pathway that involves binding of ligands to cell surface receptors, activation of Ras isoforms, recruitment and activation of Raf kinases, and phosphorylation of the MAPK kinases MEK1 and MEK2 (1Sebolt-Leopold J.S. Oncogene. 2000; 19: 6594-6599Crossref PubMed Scopus (298) Google Scholar, 2Dent P. Grant S. Clin. Cancer Res. 2001; 7: 775-783PubMed Google Scholar, 3Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3439) Google Scholar). A number of cytokines, including steel factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5, signal through this pathway in hematopoietic cells (4Okuda K. Sanghera J.S. Pelech S.L. Kanakura Y. Hallek M. Griffin J.D. Druker B.J. Blood. 1992; 79: 2880-2887Crossref PubMed Google Scholar, 5Welham M.J. Duronio V. Sanghera J.S. Pelech S.L. Schrader J.W. J. Immunol. 1992; 149: 1683-1693PubMed Google Scholar). Once activated by dual phosphorylation on a threonine and nearby tyrosine, the ERKs in turn translocate to the nucleus, where they phosphorylate and activate a number of transcription factors, including Elk-1, c-Jun, and c-Myc (6Pulverer B.J. Kyriakis J.M. Avruch J. Nikolakaki E. Woodgett J.R. Nature. 1991; 353: 670-674Crossref PubMed Scopus (1187) Google Scholar, 7Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1104) Google Scholar, 8Seth A. Alvarez E. Gupta S. Davis R.J. J. Biol. Chem. 1991; 266: 23521-23524Abstract Full Text PDF PubMed Google Scholar). In addition, activated ERKs phosphorylate and enhance the activity of the anti-apoptotic Bcl-2 family members Bcl-2 and Mcl-1 (9Deng X. Ruvolo P. Carr B. May Jr., W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar, 10Domina A.M. Smith J.H. Craig R.W. J. Biol. Chem. 2000; 275: 21688-21694Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Collectively, phosphorylation of these substrates enhances proliferation and inhibits apoptosis, providing two alterations that are thought to be critical for neoplastic transformation (11Green D.R. Evan G.I. Cancer Cell. 2002; 1: 19-30Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar). Consistent with these observations, expression of a constitutively active MEK1 allele is sufficient to transform cells (12Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar, 13Mansouer S.J. Mathen W.T. Hermann A.S. Candida J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1254) Google Scholar).In view of the involvement of this pathway in both proliferative and anti-apoptotic signaling, there has been considerable interest in exploring the effects of MEK inhibitors in a variety of cell types (1Sebolt-Leopold J.S. Oncogene. 2000; 19: 6594-6599Crossref PubMed Scopus (298) Google Scholar, 2Dent P. Grant S. Clin. Cancer Res. 2001; 7: 775-783PubMed Google Scholar, 14English J.M. Cobb M.H. Trends Pharmacol. Sci. 2002; 23: 40-45Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The aminoflavone derivative PD098059 has been shown to stabilize an inactive MEK conformation, thereby inhibiting the activity of MEK1 and MEK2 in vitro and in vivo (15Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 7686-7689Crossref Scopus (2584) Google Scholar, 16Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar). As a presumed consequence of this inhibition, PD098059 inhibits proliferation and induces apoptosis in solid tumor cell lines in vitro (1Sebolt-Leopold J.S. Oncogene. 2000; 19: 6594-6599Crossref PubMed Scopus (298) Google Scholar, 17Boucher M.J. Morisset J. Vachon P.H. Reed J.C. Laine J. Rivard N. J. Cell. Biochem. 2000; 79: 355-369Crossref PubMed Scopus (363) Google Scholar, 18Hoshino R. Tanimura S. Watanabe K. Kataoka T. Kohno M. J. Biol. Chem. 2001; 276: 2686-2692Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Additional studies have demonstrated that CI-1040 (previously designated PD184352), an orally bioavailable MEK inhibitor derived from a chemical series structurally distinct from PD098059, inhibits ERK phosphorylation in colon cancer xenografts for up to 12 h after administration to tumor-bearing mice (19Sebolt-Leopold J.S. Dudley D.T. Herrera R. Van Becelaere K. Wiland A. Gowan R.C. Tecle H. Barrett S.D. Bridges A. Przybranowski S. Leopold W.R. Saltiel A.R. Nat. Med. 1999; 5: 810-816Crossref PubMed Scopus (889) Google Scholar). Studies with CI-1040 (19Sebolt-Leopold J.S. Dudley D.T. Herrera R. Van Becelaere K. Wiland A. Gowan R.C. Tecle H. Barrett S.D. Bridges A. Przybranowski S. Leopold W.R. Saltiel A.R. Nat. Med. 1999; 5: 810-816Crossref PubMed Scopus (889) Google Scholar), as well as PD098059 (18Hoshino R. Tanimura S. Watanabe K. Kataoka T. Kohno M. J. Biol. Chem. 2001; 276: 2686-2692Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), have suggested that the antiproliferative effects of MEK inhibitors are greater in cells with higher levels of MAPK pathway activation.Previous studies (20Towatari M. Iida H. Tanimoto M. Iwata H. Hamaguchi M. Saito H. Leukemia (Baltimore). 1997; 11: 479-484Crossref PubMed Scopus (132) Google Scholar, 21Kim S.-C. Hahn J.-S. Min Y.-H. Yoo N.-C. Ko Y.-W. Lee W.-J. Blood. 1999; 93: 3893-3899Crossref PubMed Google Scholar, 22Milella M. Kornblau S.M. Estrov Z. Carter B.Z. Lapillonne H. Harris D. Konopleva M. Zhao S. Estey E. Andreeff M. J. Clin. Investig. 2001; 108: 851-859Crossref PubMed Scopus (285) Google Scholar) have demonstrated constitutive ERK activation in 50–70% of clinical AML specimens. Importantly, ERK phosphorylation correlates with the ability of CI-1040 to induce apoptosis in AML in vitro (22Milella M. Kornblau S.M. Estrov Z. Carter B.Z. Lapillonne H. Harris D. Konopleva M. Zhao S. Estey E. Andreeff M. J. Clin. Investig. 2001; 108: 851-859Crossref PubMed Scopus (285) Google Scholar). In AML cell lines, CI-1040-induced apoptosis is preceded by a p27Kip1-mediated G1 arrest and by down-regulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL (22Milella M. Kornblau S.M. Estrov Z. Carter B.Z. Lapillonne H. Harris D. Konopleva M. Zhao S. Estey E. Andreeff M. J. Clin. Investig. 2001; 108: 851-859Crossref PubMed Scopus (285) Google Scholar). The latter changes suggest that CI-1040 might induce apoptosis through the mitochondrial pathway, a series of reactions involving cytochrome c translocation to the cytoplasm, ATP-dependent assembly of the procaspase-9/Apaf-1 holoenzyme, and subsequent proteolytic activation of caspases-3, -6, and -7 (reviewed in Refs. 23Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (385) Google Scholar and 24Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2249) Google Scholar). Potential involvement of other caspases in MEK inhibitor-induced killing has not been examined previously.Because the number of ALL specimens examined previously was limited, less is known about MAPK pathway activation in this disorder. ALL occurs about 1/3 as often as AML in adults and remains a difficult malignancy to treat. Most adult ALL patients respond to initial therapy but then relapse and die of this disease (25Litzow M.R. Curr. Treat. Options Oncol. 2000; 1: 19-29Crossref PubMed Scopus (7) Google Scholar, 26Velders M.P. ter Horst S.A. Kast W.M. Leukemia (Baltimore). 2001; 15: 701-706Crossref PubMed Scopus (12) Google Scholar, 27Martin T.G. Gajewski J.L. Hematol. Oncol. Clin. N. Am. 2001; 15: 97-120Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Accordingly, there is considerable interest in identifying mechanisms of drug resistance and developing new therapies for this disorder.In the present study, we examined the biochemical consequences of MAPK inhibition in ALL. In particular, in view of data presented below showing constitutive ERK phosphorylation in clinical ALL samples, we examined the effect of the MEK inhibitor CI-1040 on cell cycle progression and survival in lymphoid cell lines. Results of these studies differed substantially from previously reported effects of MEK inhibition in solid tumor and AML cell lines. In particular, CI-1040 induced S phase arrest in 6 of 8 lymphoid cell lines examined. This was followed by FADD dephosphorylation and aggregation, which led to receptor-independent caspase-8 activation. Collectively, these observations indicate that the MAPK pathway is frequently activated in ALL cells and identify a novel mechanism by which MEK inhibitors can kill these cells.EXPERIMENTAL PROCEDURESMaterials—The broad spectrum caspase inhibitor ZVAD(OMe)-fmk (28Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar) and caspase-8-selective inhibitor IETD(OMe)-fmk were from Enzyme Systems Products (Dublin, CA). The caspase substrate DEVDAFC and the MEK1/2 inhibitor PD098059 were purchased from Biomol (Plymouth Meeting, PA) and Alexis (San Diego, CA), respectively. Other reagents were obtained as described (29Kottke T.J. Blajeski A.L. Meng X. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar).Rabbit sera that specifically recognize the phosphorylated threonine and tyrosine residues of ERK as well as separate sera that recognize ERK irrespective of phosphorylation state were from Promega (Madison, WI) or Cell Signaling Technology (Beverly, MA). Additional antibodies were obtained from the following suppliers: murine monoclonal antibodies that recognize XIAP, Mcl-1, Bcl-xL, FADD, and caspases-2, -3, and -8 from BD Biosciences; monoclonal anti-Fas (Apo-1–1) from Alexis (San Diego, CA); monoclonal anti-Bcl-2 from Dako (Carpenteria, CA); rabbit anti-Bax and monoclonal anti-Cdc25a from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti-Myc from Covance (Richmond, CA); rabbit anti-Bak and murine anti-Fas (CH-11 agonistic antibody and ZB4 blocking antibody) from Upstate Biotechnology (Lake Placid, NY); and rabbit anti-cleaved poly(ADP-ribose) polymerase (PARP) from Promega (Madison, WI). Murine monoclonal antibodies that recognize PARP and HSP90 were gifts from Dr. G. Poirier (Laval University, Ste-Foy, Quebec, Canada) and David Toft (Mayo Clinic, Rochester, MN), respectively. Rabbit antisera that recognize neoepitopes at the C termini of the caspase-3, -8, and -9 large subunits were characterized previously (29Kottke T.J. Blajeski A.L. Meng X. Svingen P.A. Ruchaud S. Mesner Jr., P.W. Boerner S.A. Samejima K. Henriquez N.V. Chilcote T.J. Lord J. Salmon M. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 2002; 277: 804-815Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30Mesner Jr., P.W. Bible K.C. Martins L.M. Kottke T.J. Srinivasula S.M. Svingen P.A. Chilcote T.J. Basi G.S. Tung J.S. Krajewski S. Reed J.C. Alnemri E.S. Earnshaw E.C. Kaufmann S.H. J. Biol. Chem. 1999; 274: 22635-22645Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Peroxidase-coupled and phycoerythrin-coupled secondary antibodies were obtained from Kirkegaard & Perry (Gaithersburg, MD) and Southern Biotechnology Associates (Birmingham, AL), respectively.Tissue Culture, Drug Treatment, and Analysis—Cell lines were obtained from the following sources: Jurkat (T cell ALL) and JM14A5 (a Jurkat clone lacking Fas expression (31Eischen C.M. Kottke T.J. Martins L.M. Basi G.S. Tung J.S. Earnshaw W.C. Leibson P.J. Kaufmann S.H. Blood. 1997; 90: 935-943Crossref PubMed Google Scholar)) from Paul Leibson (Mayo Clinic); SKW6.4 (B lineage), CEM (T cell ALL), H9 (Sezary syndrome), Molt3 (T cell ALL), I2.1 (a Jurkat clone lacking FADD), and I9.2 (a Jurkat clone lacking procaspase-8) from American Type Culture Collection (Manassas, VA); BJAB (Burkitt's lymphoma) from Marcus Peter (University of Chicago, Chicago); JB-6 (a Jurkat derivative lacking procaspase-8 (32Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (274) Google Scholar)) from Shigekazu Nagata (Kyoto University, Kyoto, Japan); and Daudi and Raji (both Burkitt's lymphoma) from Adele Fielding (Mayo Clinic). All lines were maintained at ≤1.0 × 106 cells/ml in RPMI 1640 medium containing 100 units/ml penicillin G, 100 μg/ml streptomycin, 2 mm glutamine, and 15 (H9, I2.1, I9.2, JB-6) or 10% heat-inactivated fetal bovine serum.Unless otherwise indicated, log phase cells were treated for the indicated lengths of time with 10 μm CI-1040, a concentration chosen to approximate serum levels of the drug and its active metabolite observed in a phase I clinical trial of CI-1040 in solid tumor patients (33LoRusso P.M. Adjei A.A. Meyer M.B. Wozniak S.M. Gadgeel S.M. Hanson L.J. Reid J.M. Mitchell D.Y. Bruzek L.M. Leopold J.S. Herrera R. Van Becelaere K. Carlson T. Packard C. Gulyas S.W. Erlichman C. Proc. Am. Soc. Clin. Oncol. Annu. Meet. 2002; 21: 81Google Scholar). After treatment, cells were harvested for assessment of cell cycle distribution and apoptosis.To determine cell cycle distribution, cells were sedimented at 200 × g for 10 min, washed in ice-cold calcium- and magnesium-free Dulbecco's phosphate-buffered saline, fixed in 50% ethanol, treated with RNase A, and stained with 50 μg/ml propidium iodide in 0.1% sodium citrate (34Bible K. Kaufmann S.H. Cancer Res. 1997; 57: 3375-3380PubMed Google Scholar). After 20,000 cells were analyzed on a FACScan flow cytometer using an excitation wavelength of 488 nm and an emission wavelength of 617 nm, data were analyzed using ModFit software (BD Biosciences).Apoptosis was assessed by using one of two techniques. For morphological analysis, cells were sedimented at 200 × g for 10 min, fixed in 3:1 (v/v) methanol/acetic acid, stained with 1 μg/ml Hoechst 33258 in 50% (v/v) glycerol, and examined under epi-illumination using a Zeiss Axioplan microscope. At least 300 cells/sample were scored for apoptotic changes (peripheral chromatin condensation or nuclear fragmentation) as illustrated previously (35Martins L.M. Mesner P.W. Kottke T.J. Basi G.S. Sinha S. Tung J.S. Svingen P.A. Madden B.J. Takahashi A. McCormick D.J. Earnshaw W.C. Kaufmann S.H. Blood. 1997; 90: 4283-4296Crossref PubMed Google Scholar). Alternatively, the sedimented cells were subjected to flow microfluorimetry essentially as described above. After 10,000 events were collected, data were analyzed for the percentage of subdiploid cells using CellQuest software (Verity Software House, Topsham, ME). In experiments where both methods of quantitating apoptotic cells were employed, the results agreed within 3–5%.Samples for immunoblotting were treated with CI-1040 for the indicated length of time. For whole cell lysates, cells were sedimented at 200 × g for 10 min, washed in buffer A (RPMI 1640 medium containing 10 mm HEPES (pH 7.4 at 4 °C)), lysed in alkylation buffer (6 m guanidine hydrochloride, 250 mm Tris-HCl (pH 8.5 at 21 °C), and 10 mm EDTA supplemented before use with 1 mm α-phenylmethylsulfonyl fluoride and 150 mm β-mercaptoethanol), and prepared for SDS-PAGE as described previously (36Kaufmann S.H. Svingen P.A. Gore S.D. Armstrong D.K. Cheng Y.-C. Rowinsky E.K. Blood. 1997; 89: 2098-2104Crossref PubMed Google Scholar). To evaluate FADD solubility, washed cells were extracted at 4 °C for 15 min with DISC buffer (1% (w/v) Triton X-100, 30 mm Tris-HCl (pH 7.5 at 4 °C), 150 mm NaCl, 1% (v/v) glycerol, 1 mm α-phenylmethylsulfonyl fluoride, 100 mm NaF, 1 mm Na2VO4, 20 nm microcystin, and 10 μg/ml leupeptin and pepstatin A) and sedimented at 15,000 × g for 15 min. Aliquots containing 50 μg of total cellular protein or the Triton-soluble versus -insoluble fractions were separated on SDS-polyacrylamide gels and probed with antibodies as described above. To assess caspase activation, cytosol (100,000 × g supernatant) was prepared and incubated with DEVD-AFC exactly as described (31Eischen C.M. Kottke T.J. Martins L.M. Basi G.S. Tung J.S. Earnshaw W.C. Leibson P.J. Kaufmann S.H. Blood. 1997; 90: 935-943Crossref PubMed Google Scholar).Analysis of Cell Surface Fas Expression—1 × 106 cells were stained with mouse Apo-1-1 anti-Fas on ice for 45 min. After washing, cells were incubated with phycoerythrin-conjugated anti-mouse IgG for an additional 30 min on ice. Following washing, cells were fixed in 1% paraformaldehyde and stored in the dark at 4 °C until analyzed by flow cytometry.Transient Transfections—Plasmids encoding EGFP alone (pEGFPN1), a dominant negative caspase-9/EGFP fusion protein, a dominant negative FADD/EGFP fusion protein, or Mcl-1 were obtained from Clontech (Palo Alto, CA), Emad Alnemri (Thomas Jefferson University, Philadelphia), Greg Gores (Mayo Clinic), and Ruth Craig (Dartmouth Medical School. Hanover, NH), respectively. cDNAs encoding full-length FADD or procaspase-8 were, respectively, cloned into pEGFP-N1 or pIRES-Stag-Rad9-EGFP (from Larry Karnitz, Mayo Clinic) (37Roos-Mattjus P. Vroman B.T. Burtelow M.A. Rauen M. Eapan A.K. Karnitz L.M. J. Biol. Chem. 2002; 277: 43809-43812Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) after removing Rad9. Plasmids were sequenced to confirm their integrity. Log phase Jurkat, JB-6, or I2.1 cells were transfected in the buffer described by van den Hoff et al. (38van den Hoff M.J.B. Moorman A.F.M. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Crossref PubMed Scopus (380) Google Scholar) using a T820 square wave electroporator (BTX, San Diego, CA) delivering a 240-V pulse for 10 ms. After a 24-h incubation, 30–45% of the cells displayed green fluorescence. The brightest 10–12% of the total cell population was isolated by fluorescence-activated cell sorting, exposed to drug or diluent as indicated in each figure legend, fixed, and examined for apoptotic morphological changes.RT-PCR—After log phase Jurkat cells were treated with 10 μm CI-1040 for the indicated length of time, total RNA was isolated (RNeasy™ mini kit, Qiagen, Valencia, CA). cDNAs were synthesized using a Superscript™ First-strand Synthesis kit (Invitrogen). One-twentieth of the cDNA product was used for each amplification reaction.Using the primers described in Table I, PCRs were performed using Expand™ high fidelity PCR reagents from Roche Applied Science following the supplier's instructions. Following amplification, products were electrophoresed on a 1% agarose gel containing 0.5 μg/ml ethidium bromide in 1× TAE buffer (30.7 mm Tris, 20 mm sodium acetate, and 1 mm EDTA), visualized on a UV transilluminator, and sequenced to confirm their identity.Table IPrimers used for RT-PCRTranscriptForward primerReverse primerβ-Actin5′-TCCTGTGGCATCCACGAAACT-3′5′-ATCGTCCACCGCAAATGCTTC-3′c-Myc5′-CTACTGCGACGAGGAGGAGAAC-3′5′-CGCAGATGAAACTCTGGTTCAC-3′Cdc25a5′-AGCCCCAAAGAGTCAACTAATCCAGA-3′5′-CCGGTAGCTAGGGGGCTCACA-3′p27Kip15′-GAGGACACGCATTTGGTGG-3′5′-GAGGCAGATCATTTAAGAGTG-3′p215′-CCTGCCGAAGTCAGTTCC-3′5′-CTGTGGGCGGATTAGGGC-3′Mcl-15′-CGGTAATCGGACTCAACCTC-3′5′-CCTCCTTCTCCGTAGCCAA-3′Bcl-25′-CTGAAGAGGACCTGGACC-3′5′-ACAATTGCAGAGCCATAGG-3′Bcl-xL5′-TGGCACCTGGCAGACAGC-3′5′-CGGAGGATGTGGTGGAGC-3′TNF-R15′-ACCAAGTGCCACAAAGGAAC-3′5′-TCTGGCGTAGGCACAACTTC-3′TNF-αaTNF-α, tumor necrosis factor-α5′-AGCCCATGTTGTAGCAAACC-3′5′-GGAAGACCCCTCCCAGATAG-3′Fas5′-CAAGGGATTGGAATTGAGGA-3′5′-GACAAAGCCACCCCAAGTTA-3′Fas ligand5′-GGCCTGTGTCTCCTTGTGAT-3′5′-TCATCATCTTCCCCTCCATC-3′DR45′-CTGAGCAACGCAGACTCGCTGTCCAC-3′5′-TCCAAGGACACGGCAGAGCCTGTGCCAT-3′DR55′-GCCTCATGGACAATGAGATAAAGGTGGCT-3′5′-CCAAATCTCAAAGTACGCACAAACGG-3′TRAIL5′-GGAACCCAAGGTGGGTAGAT-3′5′-TCTCACCACACTGCAACCTC-3′GAPDHbGAPDH, glyceraldehyde-3-phosphate dehydrogenase5′-GGCAAATTCCATGGCACCGTCAGG-3′5′-GGAGGCATTGCTGATGATCCTGAGG-3′a TNF-α, tumor necrosis factor-αb GAPDH, glyceraldehyde-3-phosphate dehydrogenase Open table in a new tab Clinical Samples—In conjunction with an institutional review board-approved treatment protocol (39Kaufmann S.H. Karp J.E. Burke P.J. Gore S.D. Leuk. & Lymphoma. 1996; 23: 71-83Crossref PubMed Scopus (12) Google Scholar), heparinized bone marrow aspirates were obtained from the posterior iliac crests of newly diagnosed ALL patients prior to the initiation of induction chemotherapy. Within 2 h of aspiration, samples were sedimented on ficoll-Hypaque step gradients (density = 1.077 and 1.119 g/cm3). Cells collected from the upper interface were diluted with buffer A, sedimented at 200 × g for 10 min, and resuspended in buffer A. After aliquots were removed for counting and cytospins, samples were sedimented at 200 × g for 10 min, solubilized in alkylation buffer, prospectively prepared for electrophoresis as described above, and lyophilized in multiple single-use vials (36Kaufmann S.H. Svingen P.A. Gore S.D. Armstrong D.K. Cheng Y.-C. Rowinsky E.K. Blood. 1997; 89: 2098-2104Crossref PubMed Google Scholar). Aliquots containing 5 × 105 marrow mononuclear cells were subjected to electrophoresis on gels containing a 5–15% acrylamide gradient. To provide a positive control, HL-60 cells, which are known to have constitutively phosphorylated ERK1 and ERK2 (40Meighan-Mantha R.L. Wellstein A. Riegel A.T. Exp. Cell Res. 1997; 234: 321-328Crossref PubMed Scopus (17) Google Scholar), were prepared for electrophoresis in an identical fashion. After SDS-PAGE, samples were transferred to nitrocellulose and probed with primary antibodies followed by horse-radish peroxidase-coupled secondary antibodies as described previously (41Kaufmann S.H. Kellner U. Methods Mol. Biol. 1998; 80: 223-235Crossref PubMed Scopus (11) Google Scholar). Blots were quantified on a FluorS Max Multimaging System (Bio-Rad) and considered positive for ERK phosphorylation if the integrated phospho-ERK signal was ≥20% of the total ERK signal.RESULTSMAPK Pathway Activation in Lymphoid Cell Lines and Clinical ALL—To assess MAPK pathway activation in lymphoid cells, whole cell lysates were subjected to immunoblotting with a rabbit antiserum that specifically recognizes dually phosphorylated ERK1 and ERK2. In contrast to solid tumor cell lines, some of which fail to exhibit constitutive MAPK activation (19Sebolt-Leopold J.S. Dudley D.T. Herrera R. Van Becelaere K. Wiland A. Gowan R.C. Tecle H. Barrett S.D. Bridges A. Przybranowski S. Leopold W.R. Saltiel A.R. Nat. Med. 1999; 5: 810-816Crossref PubMed Scopus (889) Google Scholar), immunoblotting readily demonstrated phosphorylated ERK1 and ERK2 in all lymphoid cell lines examined (Fig. 1A and data not shown). Collectively, these lymphoid lines provide a model system to examine the effect of MEK inhibition in the studies described below.To determine whether the MAPK pathway is also constitutively activated in the clinical setting, 24 pretreatment ALL bone marrow samples that contained >80% blasts + lymphocytes (median 88% blasts) and additional samples from patients with other leukemias were examined using identical techniques. Results of this analysis are summarized in Table II. Constitutive MAPK activation was not detectable in specimens of chronic lymphocytic leukemia (Table II), an indolent lymphoid malignancy with an extremely low proliferative index. In contrast, 12 of 33 ALL marrows displayed constitutive ERK phosphorylation, as illustrated in Fig. 1B. Reprobing of the same blots with antiserum that recognized ERK regardless of phosphorylation state confirmed the presence of ERK1 and ERK2 in all samples at similar levels (Fig. 1B, middle panel). Collectively, these results not only provide evidence for MAPK activation in 1/3 of pretreatment ALL samples but also demonstrate that this activation is independent of ERK overexpression, a factor that has been implicated in MAPK activation in some AML samples (21Kim S.-C. Hahn J.-S. Min Y.-H. Yoo N.-C. Ko Y.-W. Lee W.-J. Blood. 1999; 93: 3893-3899Crossref PubMed Google Scholar).Table IIFrequency of constitutive ERK phosphorylation in pretreatment marrow samplesSamples analyzedNo. (%) positive for ERK phosphorylationCLL23aRepresents three bone marrows and 20 peripheral blood samples from CLL patients. All other samples in this table were obtained from bone marrow0Plasma cell leukemia11 (100%)ALL3312 (36%) Newly diagnosed248 (33%) Ph+40 (0%) B lineage165 (31%) T lineage83 (38%) Relapsed94 (44%)CML90 (0%) Lymphoid blast crisis40 (0%)AML (newly diagnosed)3110 (32%)a Represents three bone marrows and 20 peripheral blood samples from CLL patients. All other samples in this table were obtained from bone marrow Open table in a new tab When possible relationships between constitutive ERK phosphorylation and various clinical parameters were examined, there was no