Molecular mechanism of senescence and immortalization in cultured human diploid fibroblasts.

为了研究细胞衰老和永生化的分子机制，我们建立了sv40t抗原转化的人二倍体成纤维细胞MRC-5的永生化和预永生化细胞克隆。利用该实验系统，我们得到了以下结果：1)在危机中会发生一定的遗传事件。2)在永生化过程中，I型胶原酶基因的表达急剧走向危机，几乎完全丧失。这些发现表明，胶原酶基因的转录调控与细胞衰老和永生的机制是耦合的。3)胶原酶表达的这些变化涉及位于上游约1.7 kb的100bp区域的两个易永生化的顺式作用元件ISE1和ISE2的功能。两种ise2结合因子（假设的激活因子Pluto和推定的抑制因子Orpheus）的相互作用对胶原酶基因在体外衰老和永生过程中的调控至关重要。4)对俄耳甫斯的候选基因，通过西南筛选分离出cDNA克隆，鉴定其编码结构特异性识别蛋白1 （SSRP1）。我们证明了SSRP1在体外衰老和永生相关的方式中介导胶原酶基因的转录抑制。5)冥王星结合到ISE2的正调控元件上，ISE2是假定的Ets家族和ap -1结合位点的串联阵列，并且在ISE2中检测到一个ap -1相关的复合物。冥王星的结合谱与活化T细胞核因子（NFAT）相似。对于冥王星的候选基因，我们分离了编码NFAT基因家族新成员的cDNA克隆。对这种新基因功能的分析正在进行中。这些永生化易感因子的精确表征将为我们了解体外衰老和永生化的内在机制提供更多信息。少

To study the molecular mechanism of cellular senescence and immortalization, we established genetically matched pairs of preimmortalized and immortalized cell clones of SV40 T antigen-transformed human diploid fibroblast MRC-5. By using this experimental system, we obtained the following results : 1)A certain genetic event responsible for immortalization occurs in crisis. 2)Expression of the type I collagenase gene was dramatically induced towards crisis and almost completely lost in the process of immortalization. These findings suggest that the transcriptional regulation of the collagenase gene is coupled with the mechanism of both cellular senescence and immortalization. 3)These changes of the collagenase expression involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The interplay of two ISE2-binding factors, a putative activator Pluto and a putative repressor Orpheus, was suggested to be cru … More cial for regulation of the collagenase gene accompanying in vitro aging and immortalization. 4)For a candidate gene of Orpheus, we isolated a cDNA clone by southwestern screening and identified that it encoded SSRP1 (structure-specific recognition protein 1). We demonstrated that SSRP1 mediated transcriptional repression of the collagenase gene in in vitro aging-and immortalization-associated manner. 5)Pluto bound to the positive regulatory element in ISE2, which was a tandem array of putative Ets family-and AP-1-binding sites, and an AP-1-related component was detected in as a complex with Pluto. This binding profiles of Pluto was similar to those of NFAT (nuclear factor of activated T cell). For a candidate gene of Pluto, we isolated cDNA clones that encoded a novel member of NFAT gene family. The analyzes of the function of this novel gene is now in progress. Precise characterization of these immortalization-susceptible factors will give us much information concerning the intrinsic mechanismof in vitro aging and immortalization. Less