JNK-dependent Release of Mitochondrial Protein, Smac, during Apoptosis in Multiple Myeloma (MM) Cells (2003)

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH2-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells. Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH2-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells. poly(ADP-ribose) polymerase second mitochondria-derived activator of caspases cytochrome c multiple myeloma 2-methoxyestradiol proteasome inhibitor-341 stress-activated protein kinase c-Jun NH2-terminal kinase antibody monoclonal Ab dominant-negative green fluorescence protein 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide glutathione S-transferase The cellular response to diverse classes of stress inducers includes growth arrest and induction of apoptosis. Apoptosis is triggered through a controlled program that is associated with distinctive morphological changes including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1Willie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6724) Google Scholar). The induction of apoptosis involves a cascade of initiator and effector caspases that are activated sequentially (2Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar, 3Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar). Caspases, a family of cysteine proteases with aspartate substrate specificity, are present in cells as catalytically inactive zymogens (2Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar). Once activated, the effector caspases induce proteolytic cleavage of various cellular targets, including poly(ADP-ribose) polymerase (PARP)1 (4Oberhammer F.A. Hochegger K. Froschl G. Tiefenbacher R. Pavelka M. J. Cell Biol. 1994; 126: 827-837Crossref PubMed Scopus (302) Google Scholar), DNA-dependent protein kinase, protein kinase C-δ (2Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar), and other substrates, ultimately leading to cell death. One of the major caspase cascades is triggered by the release of mitochondrial apoptogenic protein, cytochrome c(cyto-c) (5Liu X. Naekyung Kim C. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4462) Google Scholar, 6Yang J. Liu X. Balla K. Kim C.N. Ibardo N.M. Cai J. Peng T.i. Jones D.P. Wang X. Science. 1997; 275: 1129-1136Crossref PubMed Scopus (4410) Google Scholar). Cytosolic cyto-c binds to the CED-4 homolog Apaf-1 and induces caspase-9-dependent activation of caspase-3 (7Hu Y. Wu D. Inohara N. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4386-4391Crossref PubMed Scopus (497) Google Scholar). Recent studies have identified another important regulator of apoptosis, Smac (second mitochondria-derived activator of caspase) or DIABLO, which is released from mitochondria into the cytosol during apoptosis (8Srinivasula S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2924) Google Scholar) and functions by eliminating inhibitory effects of inhibitor of apoptosis proteins on caspase-9 (9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2924) Google Scholar, 10Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1971) Google Scholar). Our prior studies showed that 2ME2-induced apoptosis in MM cells involves release of both cyto-c and Smac (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar). However, the upstream signal that triggers the 2ME2-induced mitochondrial apoptotic pathways in MM cells is unclear. A recent study has shown that stress-activated protein kinase (SAPK) or c-Jun NH2-terinal Kinase (JNK) mediates the release of cyto-c during apoptosis (12Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1542) Google Scholar). JNK has been linked to apoptosis (13Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 14Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 15Verheij M. Bose R. Lin X.H. Yhao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1715) Google Scholar). Specifically, two serine residues (Ser63 and Ser73) in the amino-terminal transactivation domain of c-Jun are substrates for JNK (13Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 14Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar), and previous studies have shown that stress stimuli (e.g.irradiation, tumor necrosis factor, sphingomyelinase, and UV light) activate JNK (13Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 14Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 15Verheij M. Bose R. Lin X.H. Yhao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1715) Google Scholar). In the present study, we show that 2ME2-induced apoptosis in MM cells is, at least in part, mediated by JNK activation, and JNK-dependent release of Smac from mitochondria to cytosol. Our findings provide the first evidence for the requirement of JNK in triggering Smac-mediated apoptosis. Human MM.1S MM cells (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. Mononuclear cells were isolated from a patient with MM (PCL cells) by Ficoll-Hypaque density gradient centrifugation and incubated with HB-7 (anti-CD38) MoAb-biotin-streptavidin and 2H4 (anti-CD45RA) MoAb-fluorescein isothiocyanate on ice. Tumor cells (96 + 2% CD38 + 45RA-) were isolated using an Epics C cell sorter (Coulter Electronics, Hialeah, FL), washed, and resuspended in regular growth medium. Cells were treated with 3 μm of 2ME2, or 10 nm proteasome inhibitor PS-341, as described previously (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar, 16Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliott P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar). Cells were also treated with 3 μm 2ME2 in the presence or absence of JNK-specific inhibitor SP600125 (17Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2228) Google Scholar). Dual fluorescence staining with DNA-binding fluorochrome Hoechst 33342 and propidium iodide was used to quantitate the percentage of apoptotic (propidium iodide-Hoechst 33342+) cells by flow cytometry (The Vantage, BD Biosciences), as described previously (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar). In vitro immune complex c-Jun or ATF2 kinase assays were performed as described previously (18Chauhan D. Pandey P. Ogata A. Teoh G. Treon S. Urashima M. Kharbanda S. Anderson K.C. Oncogene. 1997; 15: 837-843Crossref PubMed Scopus (160) Google Scholar). MM.1S or patient MM cells were washed twice with phosphate-buffered saline, and the pellet was suspended in 3 volumes of ice-cold buffer A (20 mm HEPES, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin, aprotinin, and pepstatin A) containing 250 nm sucrose. The cells were homogenized using a Dounce homogenizer, and cytosolic or mitochondrial extracts were isolated as described previously (19Chauhan D. Hideshima T. Rosen S. Reed J.C. Kharbanda S. Anderson K.C. J. Biol. Chem. 2001; 276: 24453-24456Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Proteins were separated from cell lysates by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Smac (kindly provided by Dr. Xiaodong Wang, University of Texas Southwestern Medical Center, Dallas), anti-cyto-c(Sigma), anti-Hsp60 (Stressgen), anti-phospho-specific JNK (New England Biolabs), as well as anti-PARP (Pharmingen), or anti-JNK and anti-SHP2 (Santa Cruz Biotechnology, Santa Cruz, CA) Abs. The blots were developed by enhanced chemiluminescence (ECL), using the manufacturer's protocol (Amersham Biosciences). MM.1S cells were transiently transfected using “cell line NucleofectoTM kit V,” according to manufacturer's instructions (Amaxa Biosystems), with vector alone or DN-JNK and cotransfected with vector containing green fluorescence protein (GFP) alone. Following transfections, GFP-positive cells were selected by flow cytometry, treated with 2ME2 (3 μm) and analyzed for cell viability as described above. Additionally, cytosolic extracts from these cells were also analyzed for the accumulation of Smac by Western blotting as described previously. Cell viability was assessed by MTT (Chemicon International Inc., Temecula, CA) assay (Roche Molecular Biochemicals), and trypan blue exclusion, as described previously (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar). MM.1S cells were treated with 2ME2 in the presence or absence of JNK inhibitor (SP600125) for 12 h, harvested, and analyzed for mitochondrial membrane potential (ΔΨm). 2ME2 or dexamethasone-treated MM cells were stained with lipophilic cationic dye CMXRos (Mitotracker Red) (Molecular Probes, Eugene, OR) in phosphate-buffered saline for 20 min at 37 °C and analyzed by flow cytometry to assay for alterations in ΔΨm. To examine whether 2ME2 induces apoptosis in the MM.1S MM cells, we performed flow cytometric analysis using propidium iodide and Hoechst staining. Treatment of cells with 3 μm 2ME2 for 24 h significantly increased the percentage of apoptotic cells (51% ± 3.6%, p < 0.005, n = 3), as in our prior studies (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar). Previous studies have shown that activation of JNK plays an important role in apoptosis (14Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 15Verheij M. Bose R. Lin X.H. Yhao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1715) Google Scholar). To examine whether 2ME2 induces activation of JNK, MM.1S MM cells were exposed to 2ME2 for various time intervals, and JNK activity was analyzed byin vitro immune complex kinase assays using GST-Jun as a substrate. Analysis of anti-JNK immunoprecipitates demonstrated significant (8–10-fold) increase in GST-Jun phosphorylation, indicating the activation of JNK (Fig.1A, upper left panel). Moreover, cotreatment of MM.1S cells with SP600125, a specific inhibitor of JNK (17Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2228) Google Scholar), blocks 2ME2-induced JNK activity (Fig.1A, upper left panel). This activation of JNK in response to 2ME2 does not alter JNK protein levels (Fig. 1A,lower left panel). Increase in JNK activity was detectable as early as 1h after 2ME2 treatment and peaked at 12 h/24 h (Fig. 1A, upper right panel). Our findings are in concert with those of Verheij et al. (15Verheij M. Bose R. Lin X.H. Yhao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1715) Google Scholar), which demonstrate a role of JNK in ceramide- and tumor necrosis factor-α-induced apoptosis. Moreover, coordinate regulation of JNKs, p38, and extracellular signal-regulated kinase (ERK) kinases facilitates apoptosis induced by withdrawal of growth factor, suggesting that activation of JNK may be essential for apoptosis (20Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5034) Google Scholar). We next examined the mechanism whereby JNK mediates its apoptotic effects during 2ME2-induced apoptosis. Translocation of JNK from cytosol to the nucleus has been shown in response to osmotic stress (21Kawasaki H. Moriguchi T. Matsuda S. Li H.Z. Nakamura S. Shimohama S. Kimura J. Gotoh Y. Nishida E. Eur. J. Biochem. 1996; 241: 315-321Crossref PubMed Scopus (39) Google Scholar). Other studies showed that JNK translocates to mitochondria after genotoxic stress and inhibits the of proteins to family S. S. K. Pandey P. M. Wang K. A. T. J. R. C. D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, G. Reed J.C. 2000; 6: PubMed Scopus Google Scholar), the release of mitochondrial apoptogenic proteins to cytosol and activation of caspase We next whether 2ME2 induces translocation of JNK to mitochondria or nucleus in MM cells. and nuclear extracts from and MM.1S cells were for the JNK protein in 2ME2 significantly JNK protein in mitochondrial as assessed by of mitochondria was by the blots with an antibody protein (Fig. panel). in JNK protein levels in the nuclear were in cells not these findings demonstrate that 2ME2-induced apoptosis is associated with both activation of JNK and its translocation to These a potential of mitochondria during 2ME2-induced apoptosis in MM cells. two of apoptosis, Smac (second mitochondria-derived activator of caspase) or DIABLO, which are released from mitochondria into the cytosol during apoptosis (5Liu X. Naekyung Kim C. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4462) Google Scholar, 6Yang J. Liu X. Balla K. Kim C.N. Ibardo N.M. Cai J. Peng T.i. Jones D.P. Wang X. Science. 1997; 275: 1129-1136Crossref PubMed Scopus (4410) Google Scholar, S.M. Datta P. Fan X.J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2924) Google Scholar, J. C. Wu S. Wang X. Y. Nature. 2000; PubMed Scopus Google Scholar). The upstream that triggers the release of these proteins from mitochondria to cytosol is unclear. A recent study showed that activated JNK is for the cyto-c release and associated apoptosis (12Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1542) Google however, the of JNK on the release of Smac is that 2ME2 JNK in MM cells and JNK translocates to mitochondria, we next assessed whether the release of Smac and whether JNK mediates the release of MM.1S cells were treated with 2ME2, in the presence or absence of JNK inhibitor cytosolic extracts were and to with anti-Smac in 2ME2 induces the release of Smac, as in our previous study (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar). cotreatment with SP600125 significantly inhibits the release of Smac (Fig. upper panel). the with anti-SHP2 protein (Fig. panel). These findings demonstrate that 2ME2-induced JNK activation is for the mitochondrial release of of the effects of 2ME2 on the release of as well as requirement of JNK for the release of demonstrated To we transiently transfected MM.1S cells with either dominant-negative mutant of JNK or treatment with 2ME2, cytosolic extracts were analyzed for accumulation of Smac in the cytosol. in of DN-JNK abrogates the 2ME2-induced release of Smac from mitochondria to cytosol. To the of JNK and DN-JNK were with anti-JNK antibody (Fig. second panel). with JNK antibody showed a in 2ME2-induced JNK activity in cells transfected with DN-JNK with vector (Fig. which the of with and anti-SHP2 demonstrated and protein (Fig. and To the of we examined lysates from DN-JNK transfected cells for both and JNK activity using and GST-Jun as substrates, in an in vitro immune complex kinase DN-JNK 2ME2-induced JNK, not activity not these demonstrate that JNK is for the release of Smac during 2ME2-induced apoptosis in MM cells. We next the of 2ME2-induced JNK MM.1S cells were transiently transfected with either DN-JNK or vector and treated with in after treatment with 2ME2, MM.1S DN-JNK significantly cells transfected with vector viability was and after 2ME2 treatment of vector transfected and after 2ME2 treatment of DN-JNK transfected MM.1S cells = as by were in MM.1S cells treated with 2ME2 in the presence or absence of JNK inhibitor (Fig. a for of a JNK inhibitor SP600125, we also treated cells with 2ME2 in the presence or absence of in in to SP600125, inhibitor does not 2ME2-induced Moreover, of JNK also inhibits 2ME2-induced a event during apoptosis Other studies have that a in mitochondrial membrane potential release of Smac and cyto-c (5Liu X. Naekyung Kim C. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4462) Google Scholar, 9Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2924) Google Scholar), and we whether 2ME2-induced apoptosis with changes in and whether of JNK the ΔΨm. in 2ME2 triggers a significant in and cotreatment with JNK inhibitor SP600125 2ME2-induced in these that of JNK 2ME2-induced apoptosis. Our prior study demonstrated that 2ME2-induced apoptosis is associated with activation of and (11Chauhan D. Catley L. Hideshima T. Li G. Leblanc R. Gupta D. Sattler M. Richardson P. Schlossman R.L. Podar K. Weller E. Munshi N. Anderson K.C. Blood. 2002; 100: 2187-2194Crossref PubMed Scopus (109) Google Scholar), and both are to activate caspase-3 and In to apoptotic may of cyto-c or Smac is that the may be in response to 2ME2 the of which may for the of provided by DN-JNK 2ME2-induced apoptosis. Moreover, other upstream JNK may Smac and studies are to these our present findings demonstrate that JNK, at least in part, mediates 2ME2-induced apoptosis in MM cells. To whether other apoptotic inducers in MM cells the we treated MM.1S cells with a proteasome inhibitor in the presence or absence of SP600125, and cell extracts were analyzed for both JNK activity and accumulation of Smac in the cytosol. treatment of MM.1S cells triggers significant JNK as well as release of Smac (Fig. A upper with JNK inhibitor significantly blocks the release of Smac (Fig. and upper the requirement of JNK for release of were patient MM cells were exposed to 2ME2 (Fig. JNK has been shown to mitochondrial proteins during apoptosis G. Reed J.C. 2000; 6: PubMed Scopus Google however, not apoptotic a activation of JNK and mitochondrial pathways E.S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google N. K. H. T. Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Our prior studies showed that apoptosis in MM.1S of JNK activation, is associated with Smac release (18Chauhan D. Pandey P. Ogata A. Teoh G. Treon S. Urashima M. Kharbanda S. Anderson K.C. Oncogene. 1997; 15: 837-843Crossref PubMed Scopus (160) Google Scholar). The that 2ME2 induces JNK activation in MM.1S cells that JNK is not in these cells. This is in concert with other studies that apoptosis is associated with the release of cyto-c in cells, apoptosis in these cells JNK activation for release of cyto-c (12Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1542) Google Scholar). These findings that both activation of JNK and its requirement for the release of mitochondrial proteins are our present study that two apoptotic in MM cells induce JNK, which activation translocates to mitochondria and triggers the release of of JNK activation by either DN-JNK or a specific inhibitor of JNK Smac release and associated apoptosis. our findings have important and that Smac the apoptotic activity of J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), with our present that induce apoptosis via release of mitochondrial Smac, that Smac or Smac may MM cells to 2ME2 or cell death. We Dr. Xiaodong Wang for and as well as J. Kyriakis and L. Zon for