Molecular mechanism of cell cycle arrest-induced apoptosis

研究表明，细胞周期与细胞凋亡有关。细胞周期阻滞对细胞凋亡的影响在细胞类型或刺激方面存在争议。先前我们发现，在包括小鼠B淋巴瘤系WEHI-23 i在内的多种类型的细胞中，l -氨基糖素或Kip 1的强制表达可诱导细胞周期阻滞于g1期和细胞凋亡。但迄今为止，细胞周期与细胞凋亡之间的分子机制尚不清楚。因此，我们试图分离与细胞周期阻滞诱导的凋亡相关的基因。我们利用逆转录病毒载体从wehi - 231细胞或脾细胞中获得cDNA文库，并将其导入wehi - 23i细胞。用l -氨基葡萄糖处理细胞，用PCR法从活细胞中提取完整的cDNA片段。我们获得了三个独立的部分c-Myc cDNA片段，以反义方向插入到载体中。这些构建体在wehi - 231细胞中的表达抑制了c-Myc的表达并抑制了Kip i诱导的细胞凋亡。此外，过表达c-Myc增强了Kip 1诱导的细胞凋亡，而通过强制表达Mad4抑制c-Myc则逆转了这种凋亡。这些结果表明，c-Myc是细胞周期阻滞时决定细胞命运的关键分子。此外，我们获得了抑制细胞周期阻滞诱导的细胞凋亡的cDNA片段。对这些碎片的分析正在进行中。

It has been shown that cell cycle has relevance to apoptosis. Effect of cell cycle arrest on apoptosis is controversial upon cell types or stimuli. Previously we found that treatment with L-mimosine or enforced expression of Kip 1 induced cell cycle arrest at G 1 phase and apoptosis in various types of cells including mouse B lymphoma line WEHI-23 I. But so far, molecular mechanism of the connection between cell cycle and apoptosis remains unclear. Therefore, we tried to isolate genes related to cell cycle arrest-induced apoptosis. We generated that cDNA library from WEHI-23 1 cells or, spleen cells by using retrovirus vectors and introduced into WEHI-23 I cells. Cells were treated with L-mimosine and integrated cDNA fragments were recovered from the live cells by PCR. We obtained three independent partial c-Myc cDNA fragments were inserted into the vector as antisense direction. Expression of these constructs in WEHI-23 1 cells repressed c-Myc expression and inhibited Kip i-induced apoptosis. Moreover, overexpression of c-Myc augmented Kip 1-induced apoptosis and inhibition of c-Myc by enforced expression of Mad4 reversed it. These results indicated that c-Myc is a crucial molecule which decide cell fate upon cell cycle arrest. Furthermore, we obtained cDNA fragments that inhibit cell cycle arrest-induced apoptosis. Analyses of these fragments are in progress.