cDNA cloning of cellular aging and cellular senescence

人类正常体细胞的增殖寿命有限。用DNA肿瘤病毒的T抗原转化的细胞可以延长其增殖寿命，但不会无限增殖并最终死亡。以前的研究表明，1)有限增殖寿命的表型占优势，2)增殖寿命结束时的衰老细胞在细胞融合后抑制年轻生长细胞细胞核中DNA的合成，最后3)T抗原失活后，延长寿命的转化细胞立即停止增殖。这些结果和其他结果表明，随着增殖年龄的增加，细胞积累了增殖抑制因子。在这个项目中，我们寻找在衰老、正常和延长寿命的转化细胞中积累的cDNA，而在年轻、正常和永生化的细胞中积累的cDNA。对转化增殖的人成纤维细胞的差异杂交文库进行筛选，发现了几个候选的cDNA。其中一种被命名为N10克隆，与正常年轻和永生化细胞相比，在正常衰老和延长寿命的细胞中积累。N10全长1272bp，可能含有232个氨基酸的开放阅读框。该多肽富含亲水性氨基酸，具有典型转录因子的特征结构，如亮氨酸拉链后面的碱基区。当全长基因在表达启动子作用下连接后，转染成纤维细胞后，部分克隆出现早衰现象。Northern印迹分析表明，N10的表达水平与早衰有关。N10是参与细胞衰老的候选基因之一。

Human normal somatic cells have a limited proliferative lifespan. Cells transformed with T antigen of DNA tumor viruses can extend their proliferative lifespan, but do not proliferate indefinitely and eventually die. Previous studies indicated that 1) phenotype of limited proliferative lifespan is dominant over that of unlimited proliferative lifespan, 2) senescent cells at the end of proliferative lifespan inhibit DNA synthesis in nuclei from young growing cells after cell fusion, and finally 3) life-extended transformed cells cease proliferation immediately after inactivation of T antigen. These and other results suggested that the cells accumulate proliferation-inhibitory factor with increasing proliferative age. In this project we search for cDNAs which accumulate in senescent normal and life-extended transformed cells compared with in young normal and immortalized cells. From differential hybridization screening of cDNA library from human fibroblasts which was transformed and proliferating in life-extended stage revealed several candidate cDNAs. One of these, named N10 clone, accumulated in normal senescent and life-extended cells compared with normal young and immortalized cells. Full length of N10 was consisted of 1272 bp which had a possible open reading frame of 232 amino acid. This peptide was rich in hydrophilic amino acids and had such characteristic structure of a typical transcription factor as basic region follwed by leucine zipper. When full length cDNA was connected under expression promotor and transfected into young fibroblasts, premature senescence occurred in some of transfected clones. Northern blot suggested that premature senescence and expression level of N10 was correlated. N10 is one of candidate cDNA which functions in cellular senescence.