Ostad M, Shu W P, Kong L, Liu B C
Department of Urology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Cancer Lett. 1996 Jan 2;98(2):175-82.
To investigate the role of oncogene expression in the resistance to tumor necrosis factor-alpha (TNF), we transfected the mutated T24-Ha-ras oncogene into the murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for TNF mediated cytotoxicity. Cellular cytotoxicity of 45% over 48 h occurred with the NRK cells. However, ras transfectant NRK-Ha cells demonstrated 0% cytotoxicity over the same period. Both C127 cells and the ras transfectant HC127 demonstrated 40% and 25% cytotoxicity, respectively, over 48 h when incubated with TNF. Furthermore, DNA isolated from NRK, C127, HC127, but not NRK-Ha cells revealed the presence of DNA fragmentation 'ladders' indicative of successful apoptosis when the cells were incubated with TNF. To determine the possible mechanism in which the ras oncogene may have protected the NRK-Ha cells from TNF mediated cytotoxicity and apoptosis, total nuclear endonucleases from the NRK cells and the ras transfectant NRK-Ha cells were isolated. We determined that the endonuclease activity in the NRK and the ras transfectant NRK-Ha cells was a pH dependent endonuclease. Significant degradation of the target DNA was observed only in pH 4-6 buffers containing the endonuclease. Furthermore, preliminary intracellular pH analysis suggested that while the NRK cells have an intracellular pH of 6.0, the ras transfectant NRK-Ha cells have an intracellular pH of 7.2 and may have abrogated its pH dependent endonuclease. Both the C127 cells and the ras transfectant HC127 cells did not express a pH dependent endonuclease but rather a Ca2+/Mg2+ dependent endonuclease. Furthermore, preliminary intracellular pH analysis suggested that both the C127 and HC127 cells have the same intracellular pH. Our results indicate that in normal rat kidney cells, ras oncogene transfection may cause a disruption in the endonuclease activation involved in apoptosis.
为了研究癌基因表达在对肿瘤坏死因子-α（TNF）耐药性中的作用，我们将突变的T24-Ha-ras癌基因转染到小鼠肾细胞系NRK和另一种小鼠细胞系C127细胞中。对产生的转染细胞NRK-Ha和HC127进行TNF介导的细胞毒性检测。NRK细胞在48小时内出现了45%的细胞毒性。然而，ras转染细胞NRK-Ha在同一时期显示出0%的细胞毒性。当与TNF一起孵育时，C127细胞和ras转染细胞HC127在48小时内分别显示出40%和25%的细胞毒性。此外，从NRK、C127、HC127细胞（但不是NRK-Ha细胞）中分离的DNA显示，当细胞与TNF一起孵育时，存在指示成功凋亡的DNA片段“梯状条带”。为了确定ras癌基因可能保护NRK-Ha细胞免受TNF介导的细胞毒性和凋亡的可能机制，我们分离了NRK细胞和ras转染细胞NRK-Ha细胞的总核内切酶。我们确定NRK细胞和ras转染细胞NRK-Ha细胞中的内切酶活性是一种pH依赖性内切酶。仅在含有该内切酶的pH 4-6缓冲液中观察到靶DNA的显著降解。此外，初步的细胞内pH分析表明，虽然NRK细胞的细胞内pH为6.0，但ras转染细胞NRK-Ha细胞的细胞内pH为7.2，可能已废除其pH依赖性内切酶。C127细胞和ras转染细胞HC127细胞均不表达pH依赖性内切酶，而是表达Ca2+/Mg2+依赖性内切酶。此外，初步的细胞内pH分析表明，C127和HC127细胞具有相同的细胞内pH。我们的结果表明，在正常大鼠肾细胞中，ras癌基因转染可能会导致凋亡过程中涉及的内切酶激活受到破坏。
