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* Blood and Marrow Transplantation Program and
Hematological Malignancies Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612; and
Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088
Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients’ stroma-conditioned medium, we determined that soluble factor(s) produced by stroma–myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.
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1 This work was supported in part by National Cancer Institute Grant CA077859 (to W.D.), Cancer Center Support Grant P30-CA76292-08, Moffitt Aging and Cancer Program Development (5 P20 CA103676-03), and academic development funds from H. Lee Moffitt Cancer Center. L.E.P. is supported by a National Heart, Lung, and Blood Institute K08 Career Development Award (1 K08 HL77662-01A1).
2 Address correspondence and reprint requests to Dr. Lia Elena Perez, 12902 Magnolia Drive, West Clinic Building-BMT 2nd floor Blood and Marrow Transplantation Program, Tampa, FL 33612. E-mail address: lia.perez{at}moffitt.org
3 Abbreviations used in this paper: Apo2L, Apo2 ligand; BM, bone marrow; BMS, bone marrow stroma; CHX, cycloheximide; c-FLIPL, cellular FLIP long form; CM, conditioned medium; EM-DR, environmental mediated-death resistance; EM-IR, environmental mediated-immune resistance; FN, fibronectin; HS5-GFP, HS5 cell line that expresses GFP; PARP, poly(ADP-ribose) polymerase; 7-AAD, 7-amino actinomycin-D; TW, Transwell; TW + HS5, Transwell with HS5 stromal cells; TW – HS5, Transwell without HS5 stromal cells.
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