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cDNA cloning of cellular aging and cellular senescence

细胞衰老和细胞衰老的 cDNA 克隆

基本信息

批准号：
07672362
负责人：
IDE Toshinori
金额：
$ 1.47万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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.

人类正常体细胞的增殖寿命有限。用DNA肿瘤病毒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。先前的研究表明，1）有限增殖寿命的表型比无限增殖寿命的表型占主导地位，2）增殖寿命末期的衰老细胞抑制细胞融合后年轻生长细胞的细胞核中的DNA合成，最后3）延长寿命的转化细胞在T抗原失活后立即停止增殖。这些和其他结果表明，细胞随着增殖年龄的增加而积累增殖抑制因子。在这个项目中，我们寻找与年轻的正常细胞和永生化细胞相比，在衰老的正常细胞和延长寿命的转化细胞中积累的cDNA。对生命延长阶段转化和增殖的人成纤维细胞的cDNA文库进行差异杂交筛选，发现了几个候选cDNA。其中一种名为 N10 克隆，与正常年轻和永生细胞相比，在正常衰老和寿命延长的细胞中积累。 N10全长1272bp，可能的开放阅读框有232个氨基酸。该肽富含亲水性氨基酸，具有碱性区后亮氨酸拉链等典型转录因子的特征结构。当全长cDNA连接到表达启动子下并转染到年轻的成纤维细胞中时，一些转染的克隆出现过早衰老。 Northern印迹表明早衰与N10的表达水平相关。 N10是在细胞衰老中发挥作用的候选cDNA之一。