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SV 40 T抗原转化的人二倍体成纤维细胞MRC-5的永生化前和永生化细胞克隆的遗传匹配对。利用这个实验系统，我们得到了以下结果：1）在危机中发生了一个决定永生化的遗传事件。2)I型胶原酶基因的表达被戏剧性地诱导向危机，几乎完全丧失在永生化的过程中。这些发现表明胶原酶基因的转录调控与细胞衰老和永生化的机制相结合。3)这些胶原酶表达的变化涉及两个永生化敏感的顺式作用元件，ISE 1和ISE 2，位于上游约1.7 kb的100 bp区域的功能。两个ISE 2结合因子，一个假定的激活因子Pluto和一个假定的抑制因子Orpheus的相互作用被认为是关键的。 ...更多信息 cial用于调节胶原酶基因伴随体外衰老和永生化。4)针对Orpheus的一个候选基因，我们通过西南筛选法分离到一个cDNA克隆，并鉴定其编码结构特异性识别蛋白1（structure-specific recognition protein 1，SSRP 1）。我们证明，SSRP 1介导的胶原酶基因的转录抑制在体外衰老和永生化相关的方式。5)Pluto与ISE 2中的正调控元件结合，ISE 2是推定的Ets家族和AP-1结合位点的串联阵列，并且在与Pluto的复合物中检测到AP-1相关组分。Pluto的这种结合模式与活化T细胞核因子（NFAT）的结合模式相似。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。目前对该基因的功能分析正在进行中。这些永生化易感因子的精确表征将为我们提供有关体外衰老和永生化内在机制的更多信息。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