Elucidation of apoptotic pathway of human B-cell lymphoma and its therapeutic application

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We established a new human Burkitt's lymphoma cell line (HBL-9) which shows a unique feature of spontaneous apoptosis in vitro culture. The spontaneous apoptosis began at 36h after incubation and progressed rapidly. We analyzed bcl-2 family and caspases mainly using Western blot method to elucidate the signaling pathway for execution of spontaneous apoptosis of the HBL-9 cell line. Immunohistochemical stein and Western blot analysis revealed no expression of bcl-2 family (bcl-2, bcl-XィイD2LィエD2, bax and bad protein), Apaf-1, and cytochrome C on each harvested cell sample at the indicated times (0h, 12h, 24h, 36h, 48h). Western blot analysis revealed cleaved proteolytic fragments of caspase-2, caspase-3, caspase-8 at 36h and 48h after incubation but no cleaved proteolytic fragments of caspase-1, caspase-4, caspase-6, caspase-9, caspase-10. As protein substrates by caspase during the execution of spontaneous apoptosis, PARP, DF45/ICAD and lamin B were found but not lamin A. Caspase inhibitors Z-VAD-FMK and Asp-CHィイD22ィエD2-DCB inhibited both apoptosis and the processing caspase-2, caspase-3, caspase-7, caspase8, Caspase-2 inhibitors Ac-VDVAD-CHO and caspase-3/7 inhibitors Ac-DEVD-CHO inhibited the processing of caspase-2 and caspase-3 respectively but not apoptosis.These data indicates the following on the apoptotic pathway of HBL-9 cells;(1) The possibility that a signaling pathway, bcl-2 family→Apaf-1→cytochromeC→caspase-9, does not a company the execution phase of spontaneous apoptosis of HBL-9 cells.(2) The requirements for activations of at least four caspases (caspase-2, caspase-3, caspase-7 and caspase-8) for spontaneous apoptosis.(3) The possibility that a key effector protease upstream of caspase-2, caspase-3, caspase-7 and caspase-8.