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Differential induction of apoptosis in oncogene-transformed NIH 3T3 cells by methylmethanesulfonate
1Institute of Toxicology, College of Medicine, National Taiwan University, Taipei.
Abstract:
Cellular oncogenes have been shown to play crucial roles in the cell death process induced by cytotoxic agents. In this study, we have demonstrated that v-H-ras transformed NIH 3T3 cells but not other transformants (v-raf, v-src, v-erbB-2, v-fes and v-mos) exhibited a survival advantage to treatment by a DNA-damaging agent, methylmethanesulfonate (MMS). Subsequently, the biochemical and morphologic criteria of MMS-treated cells were examined. It was found that MMS induced v-H-ras transformants to go through necrosis, but it induced other transformed cells to undergo apoptosis. The levels of glutathione (GSH) within each transformant as well as in NIH 3T3 cells, were determined. The results showed that GSH levels within ras transformants were 2- to 7-fold higher than the levels in other transformants and normal NIH 3T3 cells. By using the GSH synthesis inhibitor buthionine sulfoximine, GSH levels were artificially reduced. This depletion, however, made ras transformed cells more sensitive to MMS killing, but the mode of cell death was still necrosis. Western blot analysis demonstrated that the anti-apoptotic protein Bcl-2 was constitutively expressed in ras transformed cells but not in NIH 3T3 or other transformed cells. The level of Bcl-2 was correlated with the resistant phenotype of ras transformants during MMS treatment. These observations suggest that GSH and Bcl-2 levels may cooperatively confer the resistant phenotype of ras transformants in response to MMS. In addition, the mode of cell death may possibly be determined at least in part by Bcl-2 protein.
Insights
Ras-transformed cells show resistance to DNA damage via increased glutathione and Bcl-2, leading to necrosis instead of apoptosis. Depleting glutathione increases sensitivity but preserves necrosis as the cell death mode.
Area of Science:
- Cellular and Molecular Biology
- Oncology
- Biochemistry
Background:
- Cellular oncogenes influence cell death pathways.
- Cytotoxic agents induce cell death through various mechanisms.
Purpose of the Study:
- To investigate the role of v-H-ras oncogene in cellular resistance to DNA-damaging agents.
- To determine the mechanism of cell death in response to methyl methanesulfonate (MMS) in transformed cells.
Main Methods:
- Treatment of NIH 3T3 cells and various transformants with methyl methanesulfonate (MMS).
- Biochemical analysis of glutathione (GSH) levels.
- Western blot analysis for Bcl-2 protein expression.
- Inhibition of GSH synthesis using buthionine sulfoximine.
Main Results:
- v-H-ras transformed cells exhibited survival advantage against MMS compared to other transformants.
- MMS induced necrosis in v-H-ras transformants, but apoptosis in other transformed cells.
- Ras transformants displayed significantly higher glutathione (GSH) levels and constitutive Bcl-2 expression.
- GSH depletion sensitized ras transformants to MMS, but cell death remained necrotic.
- Bcl-2 expression correlated with the resistant phenotype of ras transformants.
Conclusions:
- Elevated GSH and Bcl-2 cooperatively confer resistance to MMS in ras-transformed cells.
- Bcl-2 protein may play a role in determining the mode of cell death (necrosis vs. apoptosis).