Apaf-1/Cytochrome c-independent and Smac-dependent Induction of Apoptosis in Multiple Myeloma (MM) Cells

Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c(cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced caspase-9 activation and apoptosis in MM cells. Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c(cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced caspase-9 activation and apoptosis in MM cells. inhibitor of apoptosis second mitochondria-derived activator of caspases apoptotic protease-activating factor-1 ionizing radiation cytochrome c multiple myeloma dexamethasone interleukin polyacrylamide gel electrophoresis antibody monoclonal antibody Hoechst 33342 propidium iodide immunoblotting fluoromethylketone p-nitroanilide The cellular response to diverse classes of stress inducers includes growth arrest and activation 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 (6725) 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). Effector caspases, such as caspase-3, are activated by initiator caspases, such as caspase-9. Once activated, the effector caspases induce proteolytic cleavage of various cellular targets, inducing poly(ADP-ribose) polymerase (4Oberhammer F.A. Hochegger K. Froschl G. Tiefenbacher R. Pavelka M. J. Cell Biol. 1994; 126: 827-837Crossref PubMed Scopus (302) Google Scholar, 5Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2347) Google Scholar), DNA-dependent protein kinase, protein kinase C-δ, and other substrates (6Emoto Y. Manome Y. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar), ultimately leading to cell death. Recent studies have shown that the inhibitor of apoptosis (IAP)1 family of proteins suppresses apoptosis by directly binding to and inhibiting caspases (7Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2281) Google Scholar,8Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). For example, XIAP, c-IAP-1, and c-IAP-2 bind to procaspase-9 and prevent its activation (9Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar), thereby blocking the downstream apoptosis-related events such as proteolytic cleavage of caspase-3, -6, and -7 (10Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar). One of the major caspase cascades is triggered by the release of mitochondrial apoptogenic protein, cytochrome c(cyto-c) (11Liu X. Naekyung Kim C. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 12Yang 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, 13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar). Cytosolic cyto-c binds to the CED-4 homolog Apaf-1 and induces caspase-9-dependent activation of caspase-3 (14Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar, 15Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1799) Google Scholar, 16Hu Y. Ding L. Spencer D.M. Nunez G. J. Biol. Chem. 1998; 273: 33489-33494Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17Bossy-Wetzel E. Green D.R. Mutat. Res. 1999; 434: 243-251Crossref PubMed Scopus (129) 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 (18Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (710) Google Scholar, 19Srinivasula 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, 20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar) and functions by eliminating inhibitory effects of IAPs on caspases (20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar,21Verhagen 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 (1972) Google Scholar). Our prior study demonstrated that dexamethasone (Dex)-induced apoptosis is independent of cyto-c release and associated with caspase-3 activation (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar, 23Chauhan 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). In the present study, we examined the upstream signaling leading to caspase-3 activation. The results demonstrate that Dex-induced apoptosis in MM cells is mediated by Smac, which activates caspase-9 by binding to and inhibiting XIAP. Interleukin-6 (IL-6), a growth factor for MM, blocks Dex-induced release of Smac and apoptosis. Taken together, this study provides evidence for an Apaf-1-cyto-c-independent pathway mediating caspase-9 activation via Smac. Moreover, these findings also demonstrate a functional role of Smac in IL-6-mediated block during Dex-induced apoptosis. Human MM.1S (Dex-sensitive) and MM.1R (Dex-resistant) multiple myeloma cells (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar, 24Chauhan D. Pandey P. Hideshima T. Treon S. Raje N. Davies F.E. Shima Y. Tai Y.-T. Rosen S. Avraham S. Kharbanda S. Anderson K.C. J. Biol. Chem. 2000; 275: 27845-27850Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) were grown in RPMI 1640 media 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) mAb-biotin-streptavidin and 2H4 (anti-CD45RA) mAb-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 media. Cells were treated with 10 μm Dex (Sigma) in the presence or absence of 100 ng/ml of IL-6. γ-Radiation (IR) was performed as described previously (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar). Cells were also treated with anti-Fas as described previously (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar). MM.1S or PCL cells were washed twice with PBS, and the pellet was suspended in 3 volumes of ice-cold buffer A (20 mm HEPES, pH 7.5, 1.5 mmMgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and aprotinin and pepstatin A) containing 250 nmsucrose. The cells were homogenized using a Dounce homogenizer, and cytosolic or mitochondrial extracts were isolated as described previously (12Yang 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, 25Pandey P. Saleh A. Nakazawa A. Kumar S. Srinivasula S.M. Kumar V. Weichselbaum R. Nalin C. Alnemri E.S. Kufe D. Kharbanda S. EMBO J. 2000; 19: 4310-4322Crossref PubMed Scopus (488) Google Scholar). Proteins were separated from cell lysates by SDS-PAGE, transferred to nitrocellulose, and probed with anti-cyto-c (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar), anti-Smac (kindly provided by Dr. Xiaodong Wang), anti-tubulin (Sigma), anti-Hsp60 (Stressgen, Victoria, British Columbia, Canada), anti-XIAP (Transduction Laboratories), as well as anti-caspase-9, anti-casapase-8, and anti-caspase-3 (PharMingen) Abs. The blots were developed by enhanced chemiluminescence (ECL) using the manufacturer's protocol (Amersham Pharmacia Biotech). MM.1S cells were transiently cotransfected with FLAG-Apaf-1 and T7-Apaf-1 using SuperfectTM (Qiagen, Santa Clarita, CA) and treated with 10 μm Dex for 24 h. Lysates from transfectants were then incubated with dATP and subjected to immunoprecipitation with anti-FLAG M2 (Eastman Kodak Co.). The immunoprecipitates were then analyzed by immunoblotting with anti-T7 (Novegen, Madison, WI) or anti-FLAG. MM.1S cells were also transiently transfected with pcDNA3-Myc-XIAP vector (26Mesner 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. Krajeewski S. Reed J.C. Alnemri E.S. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 1999; 274: 22635-22645Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Lysates were subjected to immunoprecipitation with anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), and the immunoprecipitates were then analyzed by immunoblotting with anti-caspase-9, anti-Smac, or anti-XAIP antibodies. Caspase-9 activation was performed using LEHD-pNA as a substrate, as per the manufacturer's instructions (colorimetric assay kit, Biovision, Palo Alto, CA). MM.1S MM cells were also treated with Dex (10 μm) in the presence or absence of caspase-9 inhibitor LEHD-FMK (5 μm) for 24 h and then analyzed for apoptosis. Flow cytometric analyses: dual fluorescence staining with DNA-binding fluorochromes Hoechst 33342 (HO) and propidium iodide (PI) was used to quantitate the percentage of apoptotic (HO+PI−) cells using flow cytometry (The Vantage, Becton Dickinson), as described previously (24Chauhan D. Pandey P. Hideshima T. Treon S. Raje N. Davies F.E. Shima Y. Tai Y.-T. Rosen S. Avraham S. Kharbanda S. Anderson K.C. J. Biol. Chem. 2000; 275: 27845-27850Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). DNA fragmentation assays were also performed as described previously (24Chauhan D. Pandey P. Hideshima T. Treon S. Raje N. Davies F.E. Shima Y. Tai Y.-T. Rosen S. Avraham S. Kharbanda S. Anderson K.C. J. Biol. Chem. 2000; 275: 27845-27850Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) To determine whether Dex-induced apoptosis in MM cells is associated with the release of Smac, MM.1S MM cells were treated with Dex for various times, and cytosolic and mitochondrial extracts were analyzed for the levels of Smac. The results demonstrate that Dex treatment is associated with an increase in Smac levels in the cytosol at 24 and 48 h, with a concomitant decrease in mitochondrial Smac levels (Fig. 1 A). Dex-induced increase in cytosolic Smac and a corresponding decrease in mitochondrial Smac levels were specific, because no change was observed in the levels of tubulin protein and mitochondrial matrix protein, Hsp60, respectively (Fig. 1 A). Similar results were obtained when patient MM cells were exposed to Dex (Fig. 1 B). These results suggest that Dex-induced apoptosis is accompanied by accumulation of Smac in the cytosol. Smac is known to promote caspase activation in the cyto-c/Apaf-1/caspase-9 pathway; therefore, we next examined the release of cyto-c triggered by Dex or IR in MM.1S MM cells. The cytosolic extracts from Dex- or IR-treated cells were subjected to immunoblot analyses with anti-cyto-c and anti-Smac. As in our previous findings (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar), treatment of MM.1S MM cells with Dex did not induce release of cyto-c in the cytosol (Fig. 1 C, upper panel); in contrast, γ-radiation (IR) stimulated the release of cyto-c (Fig.1 C, upper panel), demonstrating that the release cyto-c is functional in these cells. To assay for Smac release these immunoblots were then stripped and reprobed with anti-Smac. As seen in Fig. 1 C, both Dex and IR induced release of Smac in the cytosol. Furthermore, low to undetectable cytosolic Smac or cyto-c levels were observed in the untreated cells. Reprobing the immunoblots with anti-tubulin confirms equal protein loading (Fig. 1 C). Since Dex-induced apoptosis is associated with Smac release, but not cyto-c release, we next determined whether Apaf-1 oligomerization is required for Smac-related signaling. For these experiments, we utilized the same MM.1S MM cells model (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar) to determine whether IR or Dex induce Apaf-1 oligomerization. Cells were transiently cotransfected with FLAG-Apaf-1 and T7-Apaf-1 or empty vector and treated with Dex or IR. Cell lysates were incubated with dATP. As shown in Fig. 1 D, IR (lane 3), but not Dex (lane 2), induces Apaf-1 oligomerization in MM.1S MM cells (Fig. 1 D, lane 3). The finding that IR induces Apaf-1 oligomerization in MM.1S MM cells indicates that the Apaf-1 oligomerization system is functionally intact and served as a positive control. Taken together, these findings suggest that Dex-induced apoptosis in MM cells is mediated by Smac and is independent of cyto-c/Apaf-1 mechanism. To examine whether Dex-induced Smac release and apoptosis are associated with processing of caspase-9, the cytosolic extracts from Dex-treated cells were subjected to immunoblot analysis with anti-caspase-9. The results demonstrate that treatment of MM.1S cells with Dex induces proteolytic cleavage of procaspase-9 into 37- and 35-kDa fragments (Fig. 2 A,upper panel). Reprobing the immunoblot with anti-tubulin confirms equal protein loading (Fig. 2 A, lower panel). We next assayed for catalytic activity of caspase-9 using LEHD-pNA conjugated substrate in a colorimetric protease assays (26Mesner 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. Krajeewski S. Reed J.C. Alnemri E.S. Earnshaw W.C. Kaufmann S.H. J. Biol. Chem. 1999; 274: 22635-22645Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Incubation of cytosolic extracts from Dex-treated MM.1S cells with LEHD-pNA was associated with efficient cleavage of LEHD-pNA (Fig.2 B). Although LEHD-pNA may be cleaved by caspases other than caspase-9, our study indicates that activated caspase-8 in MM.1S MM cells does not cleave this substrate (Fig. 2 B), further supporting its specificity for caspase-9. Taken together, these findings demonstrate that treatment of MM.1S cells with Dex is associated with activation of caspase-9. We next asked whether caspase-9 activation is an obligatory event during Dex-induced apoptosis. MM.1S MM cells were cultured with Dex in the presence or absence of caspase-9 tetrapeptide inhibitor LEHD-FMK for 24 h and then assayed for proteolytic cleavage of caspase-9 and caspase-3. LEHD-FMK abrogates Dex-induced cleavage of both caspase-9 and caspase-3 (Fig. 2 C, left andright panel). In contrast, LEHD-FMK did not inhibit anti-Fas-induced caspase-8 or caspase-8-mediated caspase-3 cleavage in MM.1S MM cells (data not shown), further indicating the selectivity of LEHD-FMK for caspase-9. We next determined whether blocking caspase-9 activation affects Dex-induced apoptosis, MM.1S MM cells were cultured with Dex in the presence or absence of caspase-9 inhibitor LEHD-FMK for 24 h and then assayed for apoptosis using flow cytometric analysis with PI and HO dual staining to determine the percentage for PI− and HO+ apoptotic cells. Dex-induced apoptosis (51 + 3% apoptotic cells (n = 3)) was significantly inhibited in cells pretreated with caspase-9 inhibitor (27 + 2% apoptotic cells (n = 3)) (Fig.2 D). Other studies have demonstrated that caspase-9 proteolytically cleaves and activates procaspase-3 (14Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar). In that context, our previous studies have shown that Dex triggers caspase-3 activation in MM.1S MM cells (22Chauhan D. Pandey P. Ogata A. Teoh G. Krett N. Halgren R. Rosen S. Kufe D. Kharbanda S. Anderson K.C. J. Biol. Chem. 1997; 272: 29995-29997Crossref PubMed Scopus (174) Google Scholar, 23Chauhan 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). Taken together, these results suggest that Dex induces sequential activation of Smac → caspase-9 → caspase-3 and is independent of cyto-c·Apaf-1 apoptosome complex formation. We next determined the mechanism of Dex-induced caspase-9 activation. for activation have (18Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (710) Google Scholar). the release of cyto-c to Apaf-1 which then activates Smac with IAPs of apoptosis such as and eliminates the inhibitory effects of IAPs on caspase-9 Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; PubMed Scopus Google Scholar). Since Dex-induced apoptosis is not associated with cyto-c release or Apaf-1 we asked whether XIAP with Smac during Dex-induced apoptosis. MM.1S MM cells were transiently transfected with and treated with Dex for 24 h. Cytosolic extracts were subjected to immunoprecipitation with anti-Myc and immunoblotting with anti-caspase-9, anti-Smac, or As shown in Dex treatment induces an XIAP and Smac. Dex treatment also to of XIAP from caspase-9 (Fig. 2 levels of transfected XIAP protein were by the with anti-XIAP (Fig. 2 These findings are in with other studies demonstrating that Smac promotes caspase activity of initiator caspase-9 by binding to and inhibiting IAPs (7Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2281) Google Scholar, 20Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, 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 (1972) Google Scholar). To the functional of Smac release during Dex-induced apoptosis, we utilized a known inhibitor of apoptosis in MM cells. abrogates Dex-induced apoptosis in MM.1S MM cells A, 23Chauhan 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 and D. Anderson K.C. 6: PubMed Scopus Google Scholar), as by DNA fragmentation To determine whether affects Dex-induced release of Smac, MM.1S MM cells were treated with Dex in the presence or absence of and cytosolic extracts were analyzed for Smac The Dex-induced increase in cytosolic Smac was significantly in the cells pretreated with B). IL-6-mediated block in the Dex-induced Smac levels was specific, because no change was observed in the levels of tubulin (Fig. 3 B). evidence for supporting the role of Smac during Dex-induced signaling was obtained by MM cells. MM.1R cells were treated with Dex or IR for 48 h, and cytosolic extracts were analyzed for Smac As shown in Fig. 3 C, but not induces Smac The finding that IR induces release of Smac from mitochondria to cytosol in MM.1R MM cells indicates that the Smac release system is functionally intact and served as a positive control. The that prevents Dex-induced Smac release and apoptosis, with the of Smac release and apoptosis by Dex in MM.1R (Dex-resistant) a role for Smac in mediating Dex-induced apoptosis and is with the known to Dex treatment in MM with and levels of D. Anderson K.C. 6: PubMed Scopus Google Scholar, R. 1995; PubMed Google Scholar). the present study that Dex-induced apoptosis in MM cells is associated with Smac release and caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. In contrast, treatment of cells with Dex to induce release of Smac and apoptosis. Furthermore, blocks Dex-induced apoptosis in MM cells and Smac release, thereby Taken together, these findings evidence for a activation of caspase-9 and apoptosis, independent of and suggest both Smac and XIAP. We Dr. Xiaodong Wang for Smac-related and We also Dr. S. Alnemri for and T7-Apaf-1 We the of and

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