Molecular mechanism of the signaling pathway for execution of spontaneous apoptosis of human B-cell lymphoma

我们采用Western blot方法和北方印迹方法（核糖核酸酶保护试验）分析bcl-2家族分子、caspase和TRF分子，以阐明HBL-9细胞自发凋亡的信号通路。此外，我们还用电泳迁移率变动分析（EMSA）检测了HBL-9细胞中NF-κ B的活性，以探讨TRAF分子和bcl-2分子与NF-κ B活性的关系。HBL-9细胞在蛋白和基因水平上表达caspase-2、caspase-3、caspase-7和caspase-8，并显示这些caspase分子的加工过程。HBL-9细胞表达bcl-XL、bax、bad和bid的基因（mRNA），表达Bax、Bad蛋白，而不表达Bcl-2和Bcl-XL蛋白。HBL-9细胞表达TRAF 1、TRAF 2、TRAF 3和TRAF 4的mRNA，TRAF 2和TRAF 3蛋白表达，TRAF 1和TRAF 4蛋白不表达。这些结果提示：HBL-9细胞自发性凋亡的分子机制可能是：1）至少4个半胱天冬酶（半胱天冬酶-2、半胱天冬酶-3、半胱天冬酶-7和半胱天冬酶-8）被激活用于HBL-9细胞的自发凋亡，而这些半胱天冬酶的一个未知的上游半胱天冬酶可能在半胱天冬酶级联反应中起关键的半胱天冬酶的作用。2）一个信号通路，bcl-2家族-细胞色素C-Apaf-1 -半胱天冬酶-9，Bcl-2和Bcl-XL分子缺失及NF-κ B失活等抗凋亡机制的紊乱可诱导HBL-9细胞自发凋亡。

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.