Cole Sara L, Tevethia M J
Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey 17033, USA.
J Virol. 2002 Aug;76(16):8420-32. doi: 10.1128/jvi.76.16.8420-8432.2002.
The simian virus 40 (SV40) large tumor (T) antigen is sufficient to transform cells in cultures and induce tumors in experimental animals. Transformation of primary cells in cultures requires both overcoming growth arrest by stimulating the cell cycle and blocking cell death activities presumably activated by oncogene-mediated hyperproliferation signals. The study presented here examined the ability of specific regions and activities of T antigen to modulate apoptosis in cells treated with the genotoxic agent 5-fluorouracil (5-FU). The results showed that the expression of full-length T antigen rendered rat embryo fibroblasts (REF) sensitive to 5-FU-induced apoptosis. Thus, neither the p53-binding region nor the Bcl-2 homology region of T antigen was sufficient to prevent cell death induced by the DNA-damaging agent. T-antigen-mediated sensitization occurred independently of retinoblastoma protein or p53 and p300 binding. An N-terminal segment containing the first 127 T-antigen amino acids (T1-127) was sufficient to sensitize cells. A C-terminal segment consisting of T-antigen amino acids 251 to 708 (T251-708) also sensitized cells to 5-FU-induced apoptosis. This sensitization did not occur when T251-708 was targeted to the nucleus by inclusion of the SV40 nuclear localization signal. The introduction of mutations into the T-antigen J domain resulted in mutation-specific and variable inhibition of apoptosis. This result suggested that either the structural or the functional integrity of the J domain is required to sensitize cells to apoptosis. Treatment of REF or REF expressing full-length T antigen, an N-terminal segment, or T251-708 resulted in increased expression of the p53-responsive MDM2 gene; apoptosis occurred through a p53-dependent pathway, as p53-null cells expressing these T antigens were resistant to 5-FU-induced apoptosis. Possible mechanisms involved in sensitizing cells to a p53-dependent apoptosis pathway in spite of the ability of T antigen to bind and inactivate the transcriptional transactivating activity of p53 are discussed.
猴病毒40（SV40）大T抗原足以在培养物中转化细胞并在实验动物中诱导肿瘤。在培养物中转化原代细胞既需要通过刺激细胞周期来克服生长停滞，又需要阻断可能由癌基因介导的过度增殖信号激活的细胞死亡活性。本文所述的研究检测了T抗原的特定区域和活性调节经基因毒性药物5-氟尿嘧啶（5-FU）处理的细胞中凋亡的能力。结果表明，全长T抗原的表达使大鼠胚胎成纤维细胞（REF）对5-FU诱导的凋亡敏感。因此，T抗原的p53结合区域和Bcl-2同源区域均不足以防止DNA损伤剂诱导的细胞死亡。T抗原介导的致敏作用独立于视网膜母细胞瘤蛋白或p53及p300结合而发生。包含T抗原前127个氨基酸的N末端片段（T1-127）足以使细胞致敏。由T抗原氨基酸251至708组成的C末端片段（T251-708）也使细胞对5-FU诱导的凋亡敏感。当通过包含SV40核定位信号将T251-708靶向细胞核时，这种致敏作用不会发生。将突变引入T抗原J结构域导致对凋亡的突变特异性和可变抑制。该结果表明，J结构域的结构或功能完整性是使细胞对凋亡敏感所必需的。用REF或表达全长T抗原、N末端片段或T251-708的REF进行处理导致p53反应性MDM2基因表达增加；凋亡通过p53依赖性途径发生，因为表达这些T抗原的p53缺失细胞对5-FU诱导的凋亡具有抗性。尽管T抗原具有结合并使p53的转录反式激活活性失活的能力，但仍讨论了使细胞对p53依赖性凋亡途径敏感的可能机制。
