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

Introduction Multiple myeloma (MM), currently an incurable disease, is the second most common blood cancer. It is characterized by the presence of malignant plasma cells predominantly located in bone marrow. 1 Interferons (IFNs), a family of pleiotropic cytokines, have been used for the treatment of MM alone or in combination with other chemotherapeutic drugs. Apoptosis is a genetically regulated cell death process. Cells undergo apoptosis by default, and all the critical components for apoptosis are compartmentalized within distinct subcellular organelles. Once committed to death, the cell undergoes the relatively stereotypic execution and degradation phases involving chromatin condensation, phosphatidyl-serine externalization, and selective proteolysis by a family of cysteine proteases, named caspases. The Bcl-2 family of proteins plays a pivotal role in regulating cyt c release and apoptosis. Of the many disparate death stimuli, the signaling pathway initiated by tumor necrosis factor and the related Fas ligand (also Apo-1) is best characterized. 27 These cytokines are expressed as membrane-bound ligands that can be cleaved to a soluble form. Engagement by these ligands of their respective receptors, followed by recruitment of an adapter molecule, such as FADD (Fas-associated death domain protein 28,29 ) leads to direct activation of the caspase cascade. This activation is accomplished by recruitment of caspase 8, followed by its proteolytic activation. Once activated, caspase 8 can proteolytically cleave Bid, with the truncated Bid targeting mitochondria and inducing cyt c release and further amplification of the caspase cascade. Materials and methods Cell culture, bone marrow plasma cell isolation, and treatments Human U266, NCI-H929, and RPMI-8226 MM cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. Clonogenic cell survival assays Cellular clonogenicity was measured as previously described. Apoptosis assays Apoptosis was either measured by nuclear fragmentation and condensation examined by fluorescence microscopy after Hoechst 33258 staining or by examining phosphatidylserine exposure on cell membranes, using Annexin V, as described. The caspase protease assays were performed as described, 13,33 using the acetyl-Ile-Glu-Thr-Asp-pNA (IETD) and acetyl-Asp-Glu-Val-Asp-pNA (DEVD) p-nitroanilide (pNA)-derived chromogenic substrates for caspase 8 and 3 activity, by enzyme-catalyzed release of pNA monitored at 405 nm in a 96-well microtiter plate reader (Spectramax 340; Molecular Devices, Sunnyvale, CA). Caspase peptide inhibitors (z-VAD-fmk, benyloxycarbonylVal-Ala-Asp-fmk), or substrates were from BioMol (Plymouth Meeting, PA); all other chemicals were from Sigma (St Louis, MO), unless otherwise specified. To determine of the role of the Apo2L pathway, a FLAG epitope-tagged dominant-negative Apo2L receptor, DR5⌬ (TRAIL-R2DN) was used as described 30 for stable transfection of U266 cells. DR5⌬ lacks the death domain and has been shown to function as a dominant-negative inactivating the function of the endogenous DR5. Immunoblotting Western immunoblotting was performed as described. Mitochondrial isolation and membrane potential Cell fractionation was done as previously reported. RNA analyses Total RNA was isolated from U266 cells at various intervals after IFN treatment by using the TRIZOL reagent (GIBCO BRL). To determine the steady-state levels of RNA, the RiboQuant system (Pharmingen) for RNase protection was used, as described