Retinoblastoma protein and simian virus 40-dependent immortalization of human fibroblasts.
Retinoblastoma protein and simian virus 40-dependent immortalization of human fibroblasts.
复制标题
视网膜母细胞瘤蛋白和猿猴病毒 40 依赖性人成纤维细胞永生化。
DOI：
10.1128/jvi.65.6.2845-2852.1991
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发表时间：
1991
影响因子：
5.4
通讯作者：
Ozer,HL
中科院分区：
文献类型：
--
作者：
Resnick-Silverman,L;Pang,Z;Li,G;Jha,KK;Ozer,HL
猴病毒40（SV 40）对人二倍体成纤维细胞的转化和永生化至少是一个两阶段的过程，因为转化体在培养物中具有有限的寿命。我们已经分离出永生化衍生物（AR 5和HAL）与来源缺陷的SV 40基因组编码的热不稳定的大T蛋白（T抗原）转化体，并报告说，无论是preimmortal和不朽的转化体是连续依赖于T抗原功能的增长，确定由温度变化实验。在这项研究中，我们证明了T抗原和视网膜母细胞瘤易感基因产物（Rb）在35 ℃下形成复合物，并观察到在39 ℃下T抗原功能丧失和生长抑制条件下复合物减少。将已知结合Rb的病毒癌基因（多瘤病毒大T蛋白和腺病毒E1 A 12 S蛋白）引入AR 5和HAL细胞中，两者均通过基因转移稳定地和通过病毒载体瞬时地引入。这样的双转化体在39 ℃下仍然不能增殖，尽管证明了与新引入的癌基因形成复合物。我们认为，T抗原与其他细胞的过程中，除了Rb转化和永生化培养的人类细胞相互作用。我们发现，p53-T抗原复合物在AR 5和HAL细胞中也是温度依赖性的，这可能提供了这样一种额外的机制。
Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in culture. We have isolated immortalized derivatives (AR5 and HAL) from transformants generated with an origin-defective SV40 genome encoding a heat-labile large T protein (T antigen) and reported that both preimmortal and immortal transformants are continuously dependent on T antigen function for growth as determined by temperature shift experiments. In this study, we demonstrate complex formation between T antigen and the retinoblastoma susceptibility gene product (Rb) at 35 degrees C and observed a reduction in complexes under conditions of loss of T antigen function and growth inhibition at 39 degrees C. Viral oncogenes (polyomavirus large T protein and adenovirus E1A 12S protein) known to bind Rb were introduced into AR5 and HAL cells, both stably by gene transfer and transiently by virus vectors. Such double transformants are still unable to proliferate at 39 degrees C, although complex formation with the newly introduced oncogenes was demonstrated. We suggest that T antigen interacts with other cellular processes in addition to Rb to transform and immortalize human cells in culture. Our finding that p53-T antigen complexes are also temperature dependent in AR5 and HAL cells could provide such an additional mechanism.