FIBROBLAST AGING AND PROGRAMMED CELL DEATH

这项建议旨在研究分子机制决定 对衰老人体细胞程序性死亡(细胞凋亡)的保护作用 成纤维细胞。我们反复尝试表明，在体外老化的成纤维细胞 能抵抗血清剥夺诱导的细胞凋亡； 此外，我们还发现，在小鼠3T3成纤维细胞中，细胞凋亡 细胞失去他汀类存在并获得c-fos的整体表达， C-jun、c-myc、cdc2、增殖细胞核抗原和Rb的磷酸化。细胞凋亡是 可逆，直到终止蛋白30kda(Tp30)出现最大值 水平；然后文化致力于死亡，并通过进一步的拯救 不再可能再添加血清。我们建议，这一过程 细胞凋亡可能由两个阶段组成：早期死亡启动 阶段，通过上述基因的EN阻断表达来识别，并且 晚期死亡-承诺阶段，由新的蛋白质产品识别 作为Tp30。我们认为衰老的人成纤维细胞不能 经历这两个阶段中的任何一个阶段的，原因是：一、 关键的早期细胞周期事件，以及II。Tp30的缺失。这两个 独特的特征，再加上我们的另一个发现，即无法 下调衰老成纤维细胞中bcl2蛋白水平，可能会锁定 将这些细胞转化为“三管齐下的抗死亡”表型，并赋予它们 以一种非常有效的分子模式来保护自己免于死亡。 这个项目的重点是调查这三个方面是如何 反死亡模式是相互关联的：是否需要这三种模式来建立 保护细胞免受细胞凋亡？如果是这样的话，有没有等级顺序 在保护细胞不受自毁方面是三个之一，或者是 光是它们就足够了，剩下的两个仅仅是下游的 后果是什么？最后，分子操作是否会移除 通过引入Tp30和/或SV-40T抗原来阻断细胞凋亡 解除对c-fos表达和Rb磷酸化等的抑制，或 通过去除生存基因的表达，如bcl2？具体目标 包括：(1)人成纤维细胞的起始和分化特征 通过研究早期细胞周期基因表达的承诺阶段，Tp30 Bcl2的存在及其转录调控；刻画 影响终止素30KDA存在的生化性质 细胞凋亡；(3)用于蛋白质测序的末端多肽的纯化 以及分子克隆和测序；调查职能部门 Tp30在细胞程序性死亡中的作用； 研究在衰老的成纤维细胞中，是否消除了这种抑制 加入SV-40T抗原对DNA合成也可解除阻断 细胞凋亡；以及(6)对如何监管的功能分析 生存因子bcl2与终末蛋白的加工有关 转化为Tp30形式，并致力于程序性细胞死亡。 在这里得到的答案将促进我们对监管的了解 程序性细胞死亡，一种对人类福祉至关重要的基本机制 关于有机体的。

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.