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Ha-ras oncogene expression abrogates a pH dependent endonuclease activity of apoptosis in normal rat kidney cells
1Department of Urology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Abstract:
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.
Insights
The ras oncogene confers resistance to tumor necrosis factor-alpha (TNF)-induced cell death in kidney cells by altering endonuclease activity. This oncogene disrupts pH-dependent endonucleases, preventing apoptosis and promoting cell survival.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Oncogene expression is implicated in cancer development and progression.
- Tumor necrosis factor-alpha (TNF) is a key mediator of apoptosis and inflammation.
- Understanding mechanisms of resistance to TNF-mediated apoptosis is crucial for cancer therapy.
Purpose of the Study:
- To investigate the role of the mutated T24-Ha-ras oncogene in resistance to TNF-mediated cytotoxicity and apoptosis.
- To elucidate the molecular mechanisms underlying ras-induced resistance to TNF.
Main Methods:
- Transfection of murine kidney cell lines (NRK and C127) with the T24-Ha-ras oncogene.
- Assay of TNF-mediated cytotoxicity and apoptosis (DNA fragmentation) in parental and transfectant cells.
- Isolation and characterization of nuclear endonucleases (pH-dependent vs. Ca2+/Mg2+-dependent).
- Intracellular pH analysis of cell lines.
Main Results:
- Ras transfectant NRK-Ha cells showed 0% cytotoxicity to TNF, unlike parental NRK cells (45% cytotoxicity).
- Ras transfectant NRK-Ha cells did not exhibit DNA fragmentation indicative of apoptosis after TNF treatment.
- NRK cells possess a pH-dependent endonuclease, while NRK-Ha cells exhibit an elevated intracellular pH (7.2 vs. 6.0), potentially abrogating endonuclease activity.
- C127 cells and their ras transfectant HC127 utilized a Ca2+/Mg2+-dependent endonuclease.
Conclusions:
- Ras oncogene transfection can induce resistance to TNF-mediated apoptosis in normal rat kidney cells.
- This resistance is associated with a disruption of pH-dependent endonuclease activation, linked to an increase in intracellular pH.
- The findings suggest a novel mechanism by which oncogenes can promote cell survival in the context of TNF signaling.