CHARACTERIZATION OF SENESCENCE GENES IN HUMAN CELLS

我们在给定的时间内进行了以下两个项目，并获得了一些新的发现，有助于理解人类细胞的衰老和永胞化。人类7号染色体上衰老/永生基因的鉴定我们从含有pSV2neo标记的人类7号染色体的小鼠A7细胞中制备了一组抗g418辐射杂交细胞。通过对FISH、染色体转移和Alu PCR的分析，我们选择了能够诱导遗传互补D的不朽细胞系衰老的辐射杂种，这些杂种具有非常有限的人类DNA。将人类DNA定位到7号染色体，在YAC载体上克隆衰老基因。细胞衰老信号转导途径的表征研究发现cgmp依赖性蛋白激酶抑制剂可阻断SV40T抗原热失活诱导的SV40T转化的不朽人成纤维细胞衰老。使用其中一种抑制剂作为配体进行亲和层析，然后进行Western blot分析，我们发现可以诱导蛋白质磷酸酶并发生特定蛋白质的去磷酸化。用特异性的磷酸酶抑制剂和抗体在体内和体外研究了诱导磷酸酶和蛋白去磷酸化的机制。

We have undertaken the following two projects in a given time, and obtained several new findings that contribute understanding of cellular senescence and imnrotalization in human cells.1. Identification of a senescence / immortalizing gene on human chromosome 7We made a panel of G418-resistant radiation hybrid cells form mouse A7 cells containing human chromosome 7 tagged with pSV2neo. By analysis of FISH,chromosome transfer, and Alu PCR,we selected radiation hybrids that induce senescence in immortal cell lines assigned to genetic colmnplemenation D and have a very limited amount of human DNA.The human DNA was mapped to chromosome 7 to molecularly clone the senescence gene in a YAC vector.2. Characterization of a signal transduction pathway in cellular senescenceInhibitors of cGMP-dependent protein kinases was found to block senescence induced by heat inactivation of SV40T antigen in SV40T-transformed immortal human fibroblasts. Using one of such inhibitors as a ligand for affinity chromatography followed by Western blot analysis, we have found that a protein phosphatase was induced and dephosphorylation of specific proteins occurred. The mechanism of induction of the phosphatase and the proteins dephosphorylated were examined in vino and in vitro using specific inhibitors of phosphatases and antibodies.