Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death.

BACKGROUND AND AIMS: Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy. METHODS: Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well. RESULTS: Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis. CONCLUSIONS: The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.     

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity.Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.     

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells

In present studies, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 microg/mL of Apo-2L (95.0% +/- 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53(wt) function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L-induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L-induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-x(L). Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L-induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L-induced apoptosis. (Blood. 2000;96:3900-3906)     

Expression of TNF-related apoptosis-inducing Ligand receptors and antitumor tumor effects of TNF-related apoptosis-inducing Ligand in human hepatocellular carcinoma

AIM: To investigate the expression of TNF-related apoptosis -inducing Ligand (TRAIL) receptors and antitumor effects of TRAIL in hepatocellular carcinoma (HCC). METHODS: Expression of TRAIL receptors was determined in 60 HCC tissues, 20 normal liver samples and two HCC cell lines (HepG2 and SMMC-7721). The effects of TRAIL on promoting apoptosis in HCC cell lines were analyzed after the cells were exposed to the recombinant TRAIL protein, as well as transfected with TRAIL-expression construct. In vivo effects of TRAIL on tumor growth were investigated by using nude mice HCC model of hepG2. RESULTS: Both death receptors were expressed in all HCC tissues and normal hepatic samples. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant TRAIL alone was found to have a slight activity as it killed a maximum of 15 % of HCC cells within 24 h. Transfection of the TRAIL cDNA failed to induce extensive apoptosis in HCC lines. In vivo administration of TRAIL gene could not inhibit tumor growth in nude mice HCC model. However, chemotherapeutic agents or anticancer cytokines dramatically augmented TRAIL-induced apoptosis in HCC cell lines. CONCLUSION: Loss of DcR (especially DcR1) in HCC may contribute to antitumor effects of TRAIL to HCC.HCC is insensitive towards TRAIL-mediated apoptosis, suggesting that the presence of mediators can inhibit the TRAIL cell-death-inducing pathway in HCC. TRAIL and chemotherapeutic agents or anticancer cytokines combination may be a novel strategy for the treatment of HCC.     

Targeting MEK/MAPK signal transduction module potentiates ATO-induced apoptosis in multiple myeloma cells through multiple signaling pathways

We demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in human myeloma cell lines (HMCLs) and in tumor cells from patients with multiple myeloma (MM) through a caspase-dependent mechanism. In HMCLs retaining a functional p53, PD treatment greatly enhances the ATO-induced p53 accumulation and p73, a p53 paralog, cooperates with p53 in caspase activation and apoptosis induction. In HMCLs carrying a nonfunctional p53, cotreatment with PD strikingly elevates the (DR4 + DR5)/(DcR1 + DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated cells. In MM cells, irrespective of p53 status, the combined PD/ATO treatment increases the level of the proapoptotic protein Bim (PD-mediated) and decreases antiapoptotic protein Mcl-1 (ATO-mediated). Moreover, Bim physically interacts with both DR4 and DR5 TRAIL receptors in PD/ATO-treated cells, and loss of Bim interferes with the activation of both extrinsic and intrinsic apoptotic pathways in response to PD/ATO. Finally, PD/ATO treatment induces tumor regression, prolongs survival, and is well tolerated in vivo in a human plasmacytoma xenograft model. These preclinical studies provide the framework for testing PD325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.     

肿瘤坏死因子相关的凋亡诱导配体受体在白血病细胞中的表达及其意义

目的研究肿瘤坏死因子相关的凋亡诱导配体(TRAIL)受体在白血病细胞中的表达及其临床意义。方法应用RT-PCR方法对76例白血病患者的骨髓,包括15例急性淋巴细胞性白血病(急淋)、10例急性淋巴细胞性白血病缓解期(急淋缓解)、16例急性非淋巴细胞性白血病(急非淋)、11例急性非淋巴细胞性白血病缓解期(急非淋缓解)、12例慢性粒细胞性白血病急性变期(慢粒急变期)和12例慢性粒细胞性白血病慢性期(慢粒慢性期)以及25例正常人骨髓或外周血白细胞表面TRAIL受体的表达进行检测。结果急淋、急淋缓解、急非淋、急非淋缓解、慢粒急变期和慢粒慢性期患者骨髓的白细胞表面死亡受体(deadreceptor,DR)DR4和DR5表达高,而诱骗受体(decoyreceptor,DcR)DcR1和DcR2表达低,且DR4的表达高于DR5的表达;缓解期DR4和DR5的表达高于患者组;正常人骨髓或外周血白细胞中DR4和DR5表达低,而DcR1和DcR2表达高。结论TRAIL受体在不同类型的白血病细胞中的表达具有明显的差异性。     

TRAIL及其受体在正常心脏组织的免疫组织化学定位

目的 :观察正常人心脏组织中 TRAIL 及其受体 DR4、DR5、Dc R1和 Dc R2的表达分布情况 ,为进一步明确这些分子在心肌损伤中的作用机制提供形态学依据。方法 :用抗 TRAIL及其受体的单克隆抗体 （m Ab） ,应用免疫组织化学染色技术 ,检测正常人心脏组织中 TRAIL及其受体 DR4、DR5、Dc R1和 Dc R2的表达。结果 :TRAIL及其受体 DR4、DR5、Dc R1和 Dc R2在正常人心脏组织中均有表达 ,表达产物主要分布于胞浆内 ,胞膜上也有表达。结论 :TRAIL及其受体可能参与某些病理状态下心肌细胞的凋亡过程     

Localization of TRAIL／TRAILR in fetal pancreas

AIM: To observe the localization of TRAIL/TRAIR (DR4, DR5,DcR1, DcR2) in the fetal pancreas.METHODS: Fetal pancreas of 32 weeks of pregnancy were obtained from induced abortions, embedded in paraffin, and 4-μm sections were prepared. The localization of TRAIL/TRAILR in fetal pancreas was investigated by fluorescence immunohistochemical method combined with laser scanning confocal microscopy.RESULTS: TRAIL immunoreactive cells were mainly located on the periphery of the pancreas islets. There were a few DcR1 and DcR2 positive cells whereas there were no immunoreactive cells of DR4 and DR5 in the pancreas islets.In the acini and the ducts of the exocrine pancreas there were no TRAIL/TRAILR immunoreactive cells.CONCLUSION: This study not only describes the distribution of TRAIL/TRAILR in the fetal pancreas, but also provides a morphological basis for deducing the function of TRAIL/TRAILR in pancreas, suggesting that in normal pancreatic islets, the pancreatic cells are resistant towards apoptosis too.     

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand

Background/Objectives

Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2.

Methods

Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA.

Results

Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium.

Conclusions

TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.     

Identification of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and its receptors in adult rat ventral prostate

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha (TNF-alpha) family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in adult rat hormonosensitive ventral prostate. TRAIL and its receptors were identified in the rat ventral prostate in terms of protein and mRNA. TRAIL and its receptors were immunolocalized in prostatic epithelial cells.     

Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. The occurrence of DR5-responsive tumor cells indicates that a DR5 receptor-specific TRAIL variant will permit tumor-selective therapies. By using the automatic design algorithm FOLD-X, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even wild-type TRAIL-insensitive ovarian cancer cell lines could be brought into apoptosis. In addition, our results demonstrate that there is no requirement for antibody-mediated cross-linking or membrane-bound TRAIL to induce apoptosis through DR5.     

Human soluble TRAIL/Apo2L induces apoptosis in a subpopulation of chemotherapy refractory nodal diffuse large B-cell lymphomas, determined by a highly sensitive in vitro apoptosis assay

Resistance to chemotherapy in therapy-refractory diffuse large B-cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death-receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy-refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy-refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B-cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl-2 and/or X-linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L-sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy-refractory nodal DLBCL and that disruption of the intrinsic apoptosis-mediated pathway and expression of Bcl-2 and XIAP did not confer resistance to hsTRAIL/Apo2L-induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy-refractory DLBCL should be considered.     

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Burkitt's lymphoma cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of TRAIL receptors on malignant cells. Here we have characterized the TRAIL sensitivity of a panel of Burkitt's lymphoma (BL) cell lines. Overall, 5/12 BL cell lines and 1/2 lymphoblastoid cell lines were sensitive to TRAIL-induced apoptosis, although only one BL cell line approached the sensitivity of Jurkat cells, a widely used model for TRAIL-induced apoptosis. Whereas, 4/5 of the Epstein-Barr virus (EBV)-negative cell lines were TRAIL sensitive, only 1/7 EBV-positive BL cell lines were TRAIL sensitive. However, isogenic BL cell lines with different EBV status were not differently sensitive to TRAIL, indicating that EBV is not a major determinant of TRAIL sensitivity. All cell lines expressed the death receptor (DR)5 TRAIL receptor, whereas expression of DR4 was more variable. Differences in the expression of downstream signalling molecules [Fas-associated death domain protein (FADD), caspase 8] and inhibitors [decoy receptor 1 (DcR1), cellular FLICE-like inhibitory protein (c-FLIP)] did not correlate with TRAIL sensitivity. Therefore, a subset of BL cell lines are sensitive to TRAIL-induced apoptosis, however, the molecular mechanism that determines responsiveness remains to be identified.     

Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor –related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAILinduced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.     

TRAIL induces apoptosis of chondrocytes and influences the pathogenesis of experimentally induced rat osteoarthritis

OBJECTIVE: To investigate whether TRAIL influences the pathogenesis of osteoarthritis (OA). METHODS: A recombinant adenoviral vector system (Ad-TRAIL) was used. Expression of TRAIL in a rat chondrocyte cell line (RCJ3.1C.18) and alterations in the expression of death and decoy receptors after Ad-TRAIL infection were measured by Western blot assay. To explore the underlying mechanism, Western blot assays (to detect caspase 8, poly[ADP-ribose] polymerase [PARP], and caspase 3 activation), mitochondrial membrane potential (DeltaPsim) measurement, Hoechst staining, and DNA electrophoresis were conducted. Next, expression of TRAIL and death and decoy receptors was examined by immunochemistry in primary cultured chondrocytes and on cartilage obtained from rats with experimentally induced OA. RESULTS: Ad-TRAIL infection induced expression of TRAIL in RCJ3.1C.18 cells, increased expression of death receptor 4 (DR4), and decreased expression of DR5 and decoy receptor 1 (DcR1). Ad-TRAIL, at doses of 10 and 100 multiplicities of infection, decreased the viability of chondrocytes 4 days after infection. Reduction of DeltaPsim, cytochrome c release, nuclear condensation, activation of caspase 3 and PARP, and DNA fragmentation proved the induction of apoptosis. Activation of caspase 8 was also observed. Ad-TRAIL also induced apoptosis in primary cultured chondrocytes, in which alterations in expression of TRAIL and death receptors were similar to those observed in RCJ3.1C.18 cells. Cartilage obtained from rats with experimentally induced OA showed increased expression of TRAIL and DR4 and decreased expression of DR5 and DcR1 compared with control cartilage. CONCLUSION: TRAIL induces chondrocyte apoptosis, and TRAIL-induced chondrocyte apoptosis may play a role in the pathogenesis of OA.     

Apoptosis induced by FasL and TRAIL/Apo2L in the pathogenesis of thyroid diseases.

FasL and TRAIL/Apo2L participate in cell-mediated cytotoxicity by inducing apoptosis in susceptible cells via respective cell surface receptors. Normal and neoplastic thyroid tissues are resistant to FasL-induced apoptosis but are sensitized by Th-1-type cytokines. In Hashimoto's thyroiditis, both FasL and its receptor, Fas, are strongly upregulated and their interaction leads to the suicidal/fratricidal death of thyrocytes. In Graves' disease, FasL expression in thyroid follicular cells is induced by thionamides and kills infiltrating lymphocytes. In this condition, Th-2-type cytokines upregulate the anti-apoptotic molecules FLIP and Bcl-x(L) and protect thyrocytes from apoptosis. FasL is expressed by neoplastic thyrocytes and induces apoptosis of infiltrating lymphocytes. TRAIL/Apo2L kills thyroid carcinoma cells but spares normal thyrocytes, thus providing a potential therapy for thyroid cancer.     

Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma

It has been reported that interferons (IFNs) may have antitumor activity in multiple myeloma (MM). The mechanism for their effect on MM, however, remains elusive. This study shows that IFN-alpha and -beta, but not -gamma, induce apoptosis characterized by Annexin V positivity, nuclear fragmentation and condensation, and loss of clonogenicity in 3 MM cell lines (U266, RPMI-8266, and NCI-H929), and in plasma cells from 10 patients with MM. Apo2 ligand (Apo2L, also TRAIL) induction was one of the earliest events following IFN administration in U266 cells. Treatment of these cells with TRAIL, but not with Fas agonistic antibodies, induces apoptosis. Cell death induced by IFNs and Apo2L in U266 cells was partially blocked by a dominant-negative Apo2L receptor, DR5, demonstrating the functional significance of Apo2L induction. This study shows that IFNs activate caspases and the mitochondrial-dependent apoptotic pathway, possibly mediated by Apo2L production. Thus, IFN-alpha and -beta induce cytochrome c release from mitochondria starting at 12 hours, with an amplified release seen at 48 hours. Moreover, Bid cleavage precedes the initial cytochrome c release, whereas the late, amplified cytochrome c release coincides with changes in levels of Bcl-2, Bcl-X(L), and reduction of mitochondrial membrane potential. These results link the Apo2L induction and modulation of Bcl-2 family proteins to mitochondrial dysfunction. Furthermore, IFNs and Apo2L induce cell death of CD38(+)/CD45(-/dim) plasma cells, without significant effect on nonplasma blood cells, in a caspase and Bcl-2 cleavage-dependent manner. These results warrant further clinical studies with IFNs and Apo2L in MM.