O'Hare M J, Bond J, Clarke C, Takeuchi Y, Atherton A J, Berry C, Moody J, Silver A R, Davies D C, Alsop A E, Neville A M, Jat P S
Ludwig Institute for Cancer Research-University College London Breast Cancer Laboratory, Department of Surgery, Royal Free and University College School of Medicine, 67-73 Riding House Street, London W1W 7EJ, United Kingdom.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):646-51. doi: 10.1073/pnas.98.2.646.
Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.
关于仅恢复端粒酶活性是否足以使不同类型的人类体细胞永生化，或者是否还需要其他“永生化”基因编码的额外活性，各报告说法不一。在此我们表明，单独异位表达人类端粒酶催化亚基（hTERT）或猿猴病毒40大肿瘤抗原的温度敏感突变体（U19tsA58）不足以使新鲜分离的正常成人乳腺成纤维细胞和内皮细胞永生化。然而，这两种基因的组合可有效产生永生化细胞系，而与它们导入的顺序无关，也与它们是在培养物正常增殖寿命的早期还是晚期导入无关。然而，转导的顺序和时间确实会影响基因组稳定性。核型分析表明，在传代早期首先导入hTERT，然后导入两种转基因，可产生二倍体细胞系。温度转换实验表明，永生化状态的维持取决于功能性U19tsA58大肿瘤抗原的持续表达，仅hTERT无法在U19tsA58大肿瘤抗原的非允许温度下维持生长。这种条件性二倍体细胞系可能为细胞工程以及永生化和体外转化研究提供有用的资源。
