N'cho M, Brahmi Z
Department of Microbiology/Immunology, Indianapolis, IN 46202-5200, USA.
Hum Immunol. 2001 Apr;62(4):310-9. doi: 10.1016/s0198-8859(01)00214-2.
Cell death by apoptosis is an efficient mechanism of eliminating unwanted or aberrant cells. Triggering of Fas, a member of the tumor necrosis factor (TNF) receptor superfamily, by anti-Fas antibodies or by the Fas ligand (FasL), has been shown to cause cell death by apoptosis. A recent study from our laboratory has demonstrated that Fas crosslinking leads to the dephosphorylation of the tumor suppressor retinoblastoma protein (Rb) and that this dephosphorylation is inhibited by calyculin A, a serine/threonine phosphatase inhibitor. In this investigation, we compared the effect of Fas crosslinking by CH11, an anti-Fas mAb, with two cyclin-dependent kinase (CDK) inhibitors, a peptide that specifically inhibits CDK2 (cdk2 inh) and roscovitine, which inhibits CDK2, CDC2, and CDK5. We illustrate that roscovitine induced DNA fragmentation, whereas cdk2 inh did not. In contrast to Fas-induced apoptosis, roscovitine-induced apoptosis was resistant to calyculin A. Both cdk2 inh and roscovitine induced cleavage of poly (ADP-ribose) polymerase (PARP) within 2 h. Roscovitine, however, led to the degradation of Rb, whereas cdk2 inh did not. Furthermore, both CH11 and roscovitine caused cell cycle arrest in S phase. In contrast, cdk2 inh did not have any effect on Jurkat cell cycle progression. Taken together, our results strongly suggest that the maintenance of Rb in its hyperphosphorylated form during S phase may be necessary for cell survival and that Rb dephosphorylation during S phase may constitute a crucial step in Fas-induced apoptosis.
细胞凋亡是清除不需要的或异常细胞的有效机制。抗Fas抗体或Fas配体（FasL）触发肿瘤坏死因子（TNF）受体超家族成员Fas，已被证明可导致细胞凋亡。我们实验室最近的一项研究表明，Fas交联导致肿瘤抑制蛋白视网膜母细胞瘤蛋白（Rb）去磷酸化，并且这种去磷酸化被丝氨酸/苏氨酸磷酸酶抑制剂花萼海绵诱素A所抑制。在本研究中，我们比较了抗Fas单克隆抗体CH11交联Fas与两种细胞周期蛋白依赖性激酶（CDK）抑制剂的效果，一种是特异性抑制CDK2的肽（cdk2 inh），另一种是抑制CDK2、CDC2和CDK5的roscovitine。我们发现roscovitine诱导DNA片段化，而cdk2 inh则不会。与Fas诱导的凋亡相反，roscovitine诱导的凋亡对花萼海绵诱素A具有抗性。cdk2 inh和roscovitine在2小时内均诱导聚（ADP - 核糖）聚合酶（PARP）的裂解。然而，roscovitine导致Rb降解，而cdk2 inh则不会。此外，CH11和roscovitine均导致细胞周期停滞在S期。相比之下，cdk2 inh对Jurkat细胞周期进程没有任何影响。综上所述，我们的结果强烈表明，在S期将Rb维持在其高度磷酸化形式可能是细胞存活所必需的，并且在S期Rb去磷酸化可能是Fas诱导凋亡的关键步骤。
