田崎 和洋 福島県立医科大学, 医学部, 助手 (70244382)
NAKAMURA Naoya Fukushima Medical University, School of Medicine, Assistant Lecturer, 医学部, 講師 (50227922)
冨永 邦彦 福島県立医科大学, 医学部, 講師 (20145626)
ONO Nobutaka Fukushima Medical University, School of Medicine, Instructor, 医学部, 助手 (80233584)
HOSHI Sayuri Fukushima Medical University, School of Medicine, Instructor, 医学部, 助手 (20285026)
HASHIMOTO Yuko Fukushima Medical University, School of Medicine, Instructor, 医学部, 助手 (60305357)
|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.