cDNA cloning of cellular aging and cellular senescence

人类正常体细胞具有有限的增殖寿命。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。以往的研究表明：1）有限增殖寿命的表型比无限增殖寿命的表型占优势; 2）增殖寿命结束时的衰老细胞抑制细胞融合后年轻生长细胞核中的DNA合成; 3）寿命延长的转化细胞在T抗原失活后立即停止增殖。这些和其他结果表明，随着增殖年龄的增加，细胞积累增殖抑制因子。在这个项目中，我们寻找的cDNA积累在衰老的正常和延长寿命的转化细胞相比，在年轻的正常和永生化细胞。从人成纤维细胞转化增殖的cDNA文库中进行差异杂交筛选，筛选出几个候选cDNA。其中一个克隆N10在正常衰老和寿命延长的细胞中积累，而在正常年轻和永生化的细胞中积累。N10基因全长1272 bp，可能含有232个氨基酸的开放阅读框架。该肽富含亲水性氨基酸，具有典型转录因子的结构特征，即碱性区后接亮氨酸拉链。当将全长cDNA连接到表达启动子下并转染到年轻的成纤维细胞中时，一些转染的克隆发生了早衰。北方杂交结果表明，N10的表达水平与细胞的早衰有关。N10是参与细胞衰老的候选cDNA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.