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生化。人7号染色体衰老/永生化基因的鉴定我们用pSV 2neo标记人7号染色体，从小鼠A7细胞中制备了一组抗G418辐射的杂交细胞。通过荧光原位杂交、染色体转移和Alu PCR分析，筛选出了能诱导衰老的永生细胞系，并将其定位于第7号染色体，将衰老基因克隆到YAC载体中.细胞衰老信号转导途径的表征发现cGMP依赖性蛋白激酶抑制剂可阻断SV 40 T转化的永生人成纤维细胞中SV 40 T抗原热灭活诱导的衰老。使用一种这样的抑制剂作为亲和层析的配体，然后进行Western印迹分析，我们发现，蛋白磷酸酶被诱导，并发生特定蛋白的去磷酸化。在vino和体外使用磷酸酶和抗体的特异性抑制剂的磷酸酶和蛋白质去磷酸化的诱导机制进行了检查。

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.