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

培养的人二倍体成纤维细胞衰老和永生化的分子机制。

• 批准号: 05834014
• 负责人: TAKANO Toshiya
• 金额: $ 1.47万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05834014/
• 关键词: cellular senescence; immortalization; type I collagenase gene; regulation of transcription; transcriptional repressor; transcriptional activator

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

为了研究细胞衰老和永生化的分子机制，我们建立了基因匹配的SV40 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对。通过使用这个实验系统，我们得到了以下结果：1）某种负责永生的基因事件在危机中发生。 2)I型胶原酶基因的表达在永生化过程中被急剧诱导至危机并几乎完全丧失。这些发现表明胶原酶基因的转录调控与细胞衰老和永生化的机制相结合。 3)胶原酶表达的这些变化涉及两个永生化敏感的顺式作用元件ISE1和ISE2的功能，它们位于上游约1.7kb的100bp区域。两个 ISE2 结合因子（推定的激活因子 Pluto 和推定的抑制因子 Orpheus）之间的相互作用被认为对于伴随体外衰老和永生化的胶原酶基因的调节至关重要。 4)对于Orpheus的一个候选基因，我们通过西南筛选分离了一个cDNA克隆，并鉴定其编码SSRP1（结构特异性识别蛋白1）。我们证明 SSRP1 以体外衰老和永生化相关的方式介导胶原酶基因的转录抑制。 5)Pluto 与 ISE2 中的正调控元件结合，ISE2 是假定的 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 相关成分。 Pluto 的这种结合特征与 NFAT（活化 T 细胞核因子）的结合特征相似。对于 Pluto 的候选基因，我们分离了编码 NFAT 基因家族新成员的 cDNA 克隆。目前正在对该新基因的功能进行分析。这些永生化敏感因素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。较少的

项目成果

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Sekawa K、Takano T 等人：“细胞周期蛋白 E 增强 P53 介导的反式激活。”

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Imai S,Takano T,et al.: "Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts," Mol.Cell.Biol.14. 7182-71

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Imai S,Saito F,Takano T,et al.: "Escape from in vitro aging in SV40 large T antigen-transformed human diploid cells : A key event responsible for immortalization occurs during crisis." Mech.Aging Dev.69. 149-158 (1993)

Imai S、Saito F、Takano T 等人：“SV40 大 T 抗原转化的人二倍体细胞逃避体外衰老：危机期间发生了负责永生化的关键事件。”

Segawa K,Takano T,et al.: "Abrogation of p53-mediated transactivation by SV40 large T antigen." Oncogene. 8. 543-548 (1993)

Sekawa K、Takano T 等人：“SV40 大 T 抗原消除 p53 介导的反式激活。”