GENE EXPRESSION IN SV40-TRANSFORMED HUMAN FIBROBLASTS

人类二倍体成纤维细胞(HF)在培养中的寿命有限， 处于有丝分裂后的衰老状态，这已被记录为 细胞老化模型。理解的一种方法 衰老是研究克服衰老的机制。大蛋白 由DNA病毒SV40编码的T抗原(LT)瞬间克服了 衰老细胞的生长停滞，延长细胞的寿命 衰老，而且，在较少的情况下，永久地克服衰老 产生永生细胞。我们建议对这三个人分别进行调查 SV40-细胞相互作用及其相互关系。我们的工作模式 病毒基因表达(即LT)促进细胞增殖 以及细胞基因的改变，这些基因负责克服 以稳定的方式衰老。我们将特别评估 细胞凋亡对SV40转化细胞生长的抑制作用 在所有三种细胞状态下均为HF。为了促进这一分析，我们有 开发了一系列匹配的先仙和不朽SV40转化 细胞系(SV/HF)，包括依赖温度生长的细胞系 行为(SVtsA/HF和AR 5)。有几个问题将在 这一建议是为了努力识别与 战胜衰老。 我们将确定已报道的LT诱导细胞的能力 衰老细胞的DNA合成(DS)而不是有丝分裂实际上是由于 DS中的缺陷，导致G2中的检查站停止。SV40 TL能够 在转化时诱导有丝分裂，但其作用是暂时的。我们 将在实验结束时评估LT、p53和细胞凋亡的作用 SVtsA/HF-C寿命延长，称为危机。在努力定义 新功能导致危机，我们将分析cDNA文库 由SVtsA/HF-C在危机中过度表达的mRNAs生成 在延长寿命的早期。为了评估促有丝分裂素的作用 在延长寿命和永垂不朽的过程中，我们将尝试取代 LT通过利用依赖于温度的线来发挥作用 单独和与c-myc一起引入的生长(AR5) 其他基因，如ras和p53。AR5/cMYC的稀有克隆 克服温度缺陷还将决定 这种效应的细胞基础。总之，这些研究应该 阐明已知因素的作用并定义未知因素 战胜衰老。

Human diploid fibroblasts (HF) have a limited lifespan in culture, ending in a postmitotic state of senescence, which has been documented as a model for cellular aging. One approach toward an understanding of senescence is to examine mechanisms of overcoming it. The protein large T antigen (LT) encoded by the DNA virus SV40 transiently overcomes the growth arrest in senescent cells, extends the lifespan of cells beyond senescence, and, less frequently, permanently overcomes senescence generating immortal cells. We propose to investigate each of the three SV40-cell interactions and their interrelationships. Our working model is that viral gene expression (i.e. LT) promotes cellular proliferation and alterations in cellular genes which are responsible for overcoming senescence in a stable manner. We will particularly assess the role of cell death through apoptosis in limiting the growth of SV40-transformed HF in all three cell states. To facilitate this analysis, we have developed a series of matched pre-immortal and immortal SV40-transformed cell lines (SV/HF) including ones with a temperature-dependent growth behavior (SVtsA/HF and AR 5). Several questions will be addressed in this proposal in an effort to identify effector genes involved in overcoming senescence. We will determine whether the reported ability of LT to induce cellular DNA synthesis (DS) but not mitosis in senescent cells is actually due to a defect in DS, leading to a checkpoint arrest in G2. SV40 TL is able to induce mitosis upon transformation but its effect is temporary. We will evaluate the role of LT, p53, and apoptosis at the end of the extended lifespan of SVtsA/HF-C, termed crisis. In an effort to define novel functions responsible for crisis, we will analyze cDNA libraries generated from SVtsA/HF-C mRNAs overexpressed in crisis as compared to early in the period of extended lifespan. To assess the mitogen role of LT in extended lifespan and immortalization we will attempt to replace LT function by exploiting lines which are temperature-dependent for growth (AR5) into which c-myc has been introduced alone and together with other genes such as ras, and p53. Rare clones of AR5/cmyc which have overcome the temperature defect will also be characterized to determine the cellular basis for this effect. In conclusion, these studies should clarify the role of known factors and define unknown factors in overcoming senescence.