HA-RAS ONCOGENE EXPRESSION ABROGATES A PH DEPENDENT ENDONUCLEASE ACTIVITY OF APOPTOSIS IN NORMAL RAT-KIDNEY CELLS

To investigate the role of oncogene expression in the resistance to tu mor necrosis factor-alpha (TNF), we transfected the mutated T24-Ha-ras oncogene into the murine kidney cell line NRK and an alternative muri ne 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 C 127 cells and the ras transfectant HC127 demonstrated 40% and 25% cyto toxicity, respectively, over 48 h when incubated with TNF. Furthermore , DNA isolated from NRK, C127, HC127, but not NRK-Ha cells revealed th e presence of DNA fragmentation 'ladders' indicative of successful apo ptosis when the cells were incubated with TNF. To determine the possib le mechanism in which the ras oncogene may have protected the NRK-Ha c ells from TNF mediated cytotoxicity and apoptosis, total nuclear endon ucleases 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. Si gnificant degradation of the target DNA was observed only in pH 4-6 bu ffers containing the endonuclease. Furthermore, preliminary intracellu lar pH analysis suggested that while the NRK cells have an intracellul ar 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 t he C127 cells and the ras transfectant HC127 cells did not express a p H dependent endonuclease but rather a Ca2+/Mg2+ dependent endonuclease . Furthermore, preliminary intracellular pH analysis suggested that bo th the C127 and HC127 cells have the same intracellular pH. Our result s indicate that in normal rat kidney cells, ras oncogene transfection may cause a disruption in the endonuclease activation involved in apop tosis.