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

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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