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

采用Western blot法和Northern blot法（核糖核酸酶保护实验）分析bcl-2家族分子、caspase和TRF分子，阐明HBL-9细胞自发凋亡的信号通路。此外，我们利用电泳迁移位移法（EMSA）检测了HBL-9细胞中NF-kB的活性，以研究NF-kB活化与TRAF分子或bcl-2分子之间的关系。HBL-9细胞在蛋白和基因（mRNA）水平上表达caspase-2、caspase-3、caspase-7和caspase-8，并证实了这些caspase分子的加工过程。HBL-9细胞表达bcl-XL、bax、bad和bid基因mRNA，表达bax、bad蛋白，但不表达Bcl-2和bcl-XL蛋白。HBL-9细胞表达TRAF 1、TRAF 2、TRAF 3和TRAF 4 mrna，表达TRAF 2和TRAF 3蛋白，但不表达TRAF 1和TRAF 4蛋白。未检测到NF-kB活性。这些数据提示了HBL-9细胞自发凋亡的分子机制：1)至少有4种caspase （caspase-2、caspase-3、caspase-7和caspase-8）被激活参与HBL-9细胞的自发凋亡，这些caspase的上游未知caspase可能在caspase级联中发挥关键作用。2) bcl-2家族-细胞色素C - Apaf-1 - caspase-9信号通路与HBL-9细胞自发凋亡的发生无关。3)缺乏Bcl-2和Bcl-XL分子以及NF-kB失活等抗凋亡机制的干扰可诱导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.