|Budget Amount *help
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
We analyzed bcl-2 family molecules, caspase and TRF molecules using Western blot method and Northern blot method (ribonuclease protection assay) to elucidate the signaling pathway for execution of spontaneous apoptosis of HBL-9 cells. In addition, we examined the NF-kB activity in HBL-9 cells using electrophoretic mobility shift assay (EMSA) to investigate the relationship between NF-kB activation and TRAF molecules or bcl-2 molecules. HBL-9 cells showed the expression of caspase-2, caspase-3, caspase-7 and caspase-8 at the protein and gene (mRNA) levels, and the processing of those caspase molecules was demonstrated. HBL-9 cells displayed the gene expression (mRNA) of bcl-XL, bax, bad and bid and expression of Bax, Bad proteins but no expression of Bcl-2 and Bcl-XL proteins. HBL-9 cells expressed the mRNAs ofTRAF 1, TRAF 2, TRAF 3 and TRAF 4, and showed the expression of TRAF 2 and TRAF 3 proteins but no expression of TRAF 1 and TRAF 4 proteins. NF-kB activity was not detected.
Those data indicate the following on the molecular mechanism for the execution of spontaneous apoptosis of HBL-9 cells ;
1) At least four caspases (caspase-2, caspase-3, caspase-7 and caspase-8) are activated for the spontaneous apoptosis of HBL-9 cells, and an unknown upstream caspase of those caspases might play a role as a key caspase in the caspase cascade.
2) A signaling pathway, bcl-2 family - cytochrome C - Apaf-1 - caspase-9, is not associated with the execution of the spontaneous apoptosis of HBL-9 cells.
3) Disturbance of antiapoptotic mechanism involving lack of Bcl-2 and Bcl-XL molecules and inactivation of NF-kB can cause the induction of the spontaneous apoptosis of HBL-9 cells.