FIBROBLAST AGING AND PROGRAMMED CELL DEATH

该建议旨在研究决定 对衰老人细胞程序性死亡（凋亡）保护 成纤维细胞 我们的反复尝试表明，体外老化的成纤维细胞， 对血清剥夺诱导的细胞凋亡有抗性; 此外，我们已经表明，在小鼠3 T3成纤维细胞中， 细胞失去其他汀存在并获得c-fos的整体表达， c-jun、c-myc、cdc 2、PCNA和RB磷酸化。 凋亡是 可逆的，直到末端蛋白30 kDa（Tp 30）出现在最大值 水平;文化然后致力于死亡，并进一步拯救， 将血清添加回不再可能。 我们建议， 细胞凋亡可分为两个阶段：早期死亡-启动 阶段，通过阻断上述基因的表达来鉴定， 晚期死亡承诺阶段，通过新的蛋白质产品， TP 30。 我们认为，衰老的人类成纤维细胞不能 经历这两个阶段中的任何一个，由于：i.的失调 关键的早期细胞周期事件，和ii. TP 30的缺失。 这两 独特的特征，加上我们的其他发现， 下调衰老成纤维细胞中的bcl 2蛋白水平， 将这些细胞转化为“三管齐下的抗死亡”表型，并赋予它们 有一种非常有效的分子模式来保护自己不死。 这个项目的重点是调查如何这三个叉 反死亡的模式是相互关联的：这三个都需要建立 保护细胞凋亡？ 如果是的话，有没有一种等级制度 在保护细胞免于自我毁灭的三种方法中， 只有他们足够，离开其他两个只是下游 后果？ 最后，分子操作是否可以去除 通过将Tp 30和/或SV-40 T抗原引入细胞， 解除对c-fos表达和RB磷酸化的抑制等，或 通过去除生存基因表达如bcl 2？ 具体目标 包括：（1.）在人成纤维细胞中， 通过研究早期细胞周期基因表达，Tp 30 bcl 2的存在和转录调控;（2.）表征 影响终末蛋白30 kDa存在的生化特性 凋亡;（3.）纯化用于蛋白质测序的末端蛋白多肽 以及分子克隆和测序;（4.）调查功能 TP 30在程序性细胞死亡中的作用;（5.） 研究在衰老的成纤维细胞中， 通过加入SV-40 T抗原对DNA合成的阻断作用也可以解除， 凋亡;和（6.）功能分析如何调节 生存因子bcl 2与终末蛋白的加工有关 转化为TP 30形式，并致力于程序性细胞死亡。 在这里获得的答案将促进我们对监管的认识， 程序性细胞死亡，一种对健康至关重要的基本机制， 有机体。

This proposal aims to investigate the molecular mechanisms determining the protection from programmed cell death (apoptosis) in senescent human fibroblasts. Our repeated attempts show that in vitro aged fibroblasts are resistant to the induction of apoptosis by serum deprivation; furthermore we have shown that, in mouse 3T3 fibroblasts, apoptotic cells lose their statin presence and gain en bloc expressions of c-fos, c-jun, c-myc, cdc2, PCNA, and RB phosphorylation. Apoptosis is reversible until terminin protein 30 kda (Tp30) appears at maximal levels; cultures ar then committed to death, and further rescue by adding serum back is no longer possible. We suggest that the process of apoptosis may be composed by two stages: the early death-initiation stage, identified by the en block expression of the above genes, and the late death-commitment stage, identified by novel protein products such as Tp30. We suggest that senescent human fibroblasts are incapable of undergoing either of these two stages, due to: i. the dysregulation of key early cell cycle events, and ii. the absence of Tp30. These two unique features, compounded by our other finding of the inability to down-regulate the bcl2 protein level in senescent fibroblasts, may lock these cells into a "three-pronged anti-death" phenotype, and endow them with an exceptionally efficient molecular mode of protection from death. The focus of this project is to investigate how the three prongs of this anti-death mode are interrelated: are all three needed to establish the protection from apoptosis? And if so, is there an order of hierarchy among the three in protecting cells from self-destruction, or is one of them alone sufficient, leaving the other two as merely down-stream consequences? And finally, might molecular manipulations remove the blockade from apoptosis, by introducing Tp30 and/or SV-40 T antigen to lift the repression of c-fos expression and RB phosphorylation, etc., or by removing survival gene expressions such as bcl2? Specific aims include: (1.) characterizing in human fibroblasts the initiation and commitment stages by studying early cell cycle gene expressions, Tp30 presence and the transcriptional regulation of bcl2; (2.) characterizing biochemical properties affecting the presence of terminin 30 kda during apoptosis; (3.) purifying terminin polypeptides for protein sequencing and molecular cloning and sequencing; (4.) investigating the functional role of Tp30 in the commitment to programmed cell death; (5.) investigating whether, in senescent fibroblasts, removing the inhibition to DNA synthesis by adding SV-40 T antigen can also remove the block to apoptosis; and (6.) functional analysis of how regulation of the survival factor, bcl2, is related to the processing of terminin protein into the Tp30 form, and to the commitment to programmed cell death. Answers obtained here will advance our knowledge of the regulation of programmed cell death, a fundamental mechanism pivotal to the well-being of organisms.