Yeung M C, Lau A S
The Moses Grossman Infectious Diseases Laboratory, Department of Pediatrics, San Francisco General Hospital and University of California, San Francisco, California 94110, USA.
J Biol Chem. 1998 Sep 25;273(39):25198-202. doi: 10.1074/jbc.273.39.25198.
Despite what is known about the early signaling events in tumor necrosis factor (TNF) alpha-induced apoptosis, characterization of the downstream events remains largely undefined. It is now known that a cross-talk exists between the interferon and TNF-alpha pathways. This linkage allows recruitment of the cell proliferation suppressor PKR (dsRNA-dependent protein kinase) from the interferon pathway to play a pivotal role in TNF-alpha-induced apoptosis. In this study, we took advantage of the differential TNF-alpha susceptibilities of human promonocytic U937 subclones, deficient in or overexpressing PKR, to further characterize the role of PKR in apoptosis. By reverse transcription-polymerase chain reaction, we demonstrated that TNF-alpha transiently induces the tumor suppressor p53 in U937 cells. This p53 induction lags behind the TNF-alpha induction of PKR by 1 h. By cell viability determination, ultrastructural studies, apoptotic DNA laddering, and antisense techniques, it was shown that inhibition of p53 expression in PKR-overexpressing U937 cells abrogates the TNF-alpha-induced apoptosis in these cells. Conversely, overexpressing wild type p53 in PKR-deficient U937 cells confers the susceptibility of these cells to TNF-alpha-induced apoptosis. This latter result indicates that p53 induction is an event downstream of TNF-alpha-induced up-regulation of PKR, thereby further establishing the critical role of p53 in TNF-alpha-induced apoptosis in U937 cells. PKR-overexpressing U937 cells were found to possess a constitutively higher level of p53, which partly explains why these cells spontaneously undergo apoptosis even without TNF-alpha treatment. Finally, a model is presented on the interplay between PKR and p53 in effecting TNF-alpha-induced apoptosis in U937 cells.
尽管人们已经了解肿瘤坏死因子（TNF）α诱导细胞凋亡的早期信号事件，但对下游事件的特征描述仍大多不明确。现在已知干扰素和TNF-α信号通路之间存在相互作用。这种联系使得细胞增殖抑制因子PKR（双链RNA依赖性蛋白激酶）从干扰素信号通路被募集过来，在TNF-α诱导的细胞凋亡中发挥关键作用。在本研究中，我们利用人原单核细胞U937亚克隆对TNF-α敏感性的差异（这些亚克隆中PKR表达缺失或过表达），进一步明确PKR在细胞凋亡中的作用。通过逆转录-聚合酶链反应，我们证明TNF-α可在U937细胞中短暂诱导肿瘤抑制因子p53的表达。这种p53的诱导比TNF-α诱导PKR的表达滞后1小时。通过细胞活力测定、超微结构研究、凋亡DNA梯状条带分析和反义技术，结果表明在PKR过表达的U937细胞中抑制p53的表达可消除TNF-α诱导的这些细胞的凋亡。相反，在PKR缺陷的U937细胞中过表达野生型p53会使这些细胞对TNF-α诱导的凋亡敏感。后一个结果表明p53的诱导是TNF-α诱导PKR上调后的一个事件，从而进一步确立了p53在U937细胞TNF-α诱导的细胞凋亡中的关键作用。发现PKR过表达的U937细胞具有组成性较高水平的p53，这部分解释了为什么这些细胞即使未经TNF-α处理也会自发发生凋亡。最后，提出了一个关于PKR和p53在U937细胞中影响TNF-α诱导的细胞凋亡过程中相互作用的模型。
