GENE EXPRESSION IN SV40-TRANSFORMED HUMAN FIBROBLASTS

SV40 转化的人类成纤维细胞中的基因表达

• 批准号: 6233925
• 负责人: HARVEY L OZER
• 金额: $ 12.15万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-10 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233925
• 关键词: DNA damage; Drosophilidae; aging; apoptosis; cell cycle; cell senescence; cell transformation; fibroblasts; gene expression; gene therapy; genetic library; laboratory rabbit; mitogens; oncogenes; simian virus 40; temperature sensitive mutant; tissue /cell culture; virus antigen

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.

人二倍体成纤维细胞（HF）在培养中具有有限的寿命， 在有丝分裂后的衰老状态，这已被记录为一个 细胞衰老的模型。 一种理解 衰老是研究克服它的机制。蛋白质大 由DNA病毒SV40编码的T抗原（LT）瞬时克服了 衰老细胞的生长停滞，延长细胞的寿命， 衰老，并且，不太经常地，永久地克服衰老 产生永生细胞 我们建议调查这三个 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， 以稳定的方式衰老。 我们将特别评估 通过细胞凋亡的细胞死亡限制了SV40转化的 三种细胞状态下的HF。 为了便于分析，我们 开发了一系列匹配的预不朽和不朽的SV40-转化 细胞系（SV/HF），包括具有温度依赖性生长的细胞系 行为（SVtsA/HF和AR 5）。 几个问题将在 这项建议是为了确定参与 克服衰老 我们将确定是否报告的LT诱导细胞增殖的能力， 衰老细胞中的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中的检查站逮捕。 SV40 TL能够 在转化时诱导有丝分裂，但其效果是暂时的。 我们 将评估LT，p53和细胞凋亡在治疗结束时的作用。 SVtsA/HF-C的寿命延长，称为危机。 为了定义 负责危机的新功能，我们将分析cDNA文库 产生自SVtsA/HF-C mRNA在危机中过表达， 在延长寿命的早期。 为了评估促分裂剂的作用， LT在延长寿命和永生化，我们将试图取代 LT功能，通过利用温度依赖的线， 生长（AR 5），其中单独引入c-myc和与 其他基因如ras和p53。 AR5/cmyc的罕见克隆， 克服温度缺陷的特点也将确定 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， 克服衰老