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Ha-rasVal12 oncogene increases susceptibility of NIH/3T3 cells to lovastatin
1Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Abstract:
This study demonstrates that Ha-rasVal12 oncogene overexpression sensitizes NIH/3T3 fibroblasts to lovastatin (LOV) cytotoxicity. This sensitization is through apoptosis, which was characterized by increasing CPP32 (caspase-3) activity and DNA fragmentation. Bcl-2 overexpression increased the resistance of the Ha-ras transformants to LOV and rescued the cells from apoptosis, further confirming that the LOV-sensitive cells died of apoptosis. Further analysis showed that Ha-ras activity inversely correlated with WAF1 activity. LOV treatment suppressed Ha-ras activity but induced WAF1 activity and disrupted the cell population in G0/G1 and S phases. The Ha-ras transformants expressing either dominant negative RasAsn17 or Raf-1CB4 showed reverted susceptibility to LOV. These data confirm the involvement of Ras and demonstrate that Raf-1 signalling is required for LOV-induced cell death. Taken together, the possible action of LOV-induced apoptosis is through suppressing Ha-ras activity and increasing WAF1 activity, which alters cell cycle progression and finally activates suppressed apoptotic pathway in a Fas/Fas-L- and p53-independent fashion.
Insights
Overexpression of the Ha-rasVal12 oncogene sensitizes NIH/3T3 fibroblasts to lovastatin-induced apoptosis. This effect is mediated by Ras and Raf-1 signaling pathways, independent of Fas/Fas-L and p53.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The Ha-rasVal12 oncogene plays a role in cell proliferation and survival.
- Lovastatin (LOV) is a statin drug with potential anti-cancer properties.
- Understanding the mechanisms of drug-induced apoptosis is crucial for cancer therapy.
Purpose of the Study:
- To investigate the role of Ha-rasVal12 oncogene in lovastatin-induced cytotoxicity.
- To elucidate the signaling pathways involved in lovastatin-induced apoptosis.
Main Methods:
- NIH/3T3 fibroblasts were transfected with Ha-rasVal12 or control vectors.
- Cells were treated with lovastatin, and cytotoxicity was assessed.
- Apoptosis was measured by caspase-3 activity and DNA fragmentation.
- Western blotting was used to analyze protein expression (Bcl-2, WAF1).
- Dominant-negative RasAsn17 and Raf-1CB4 were used to study signaling pathways.
Main Results:
- Ha-rasVal12 overexpression sensitized NIH/3T3 fibroblasts to lovastatin-induced apoptosis.
- Bcl-2 overexpression conferred resistance to lovastatin, indicating apoptosis as the cell death mechanism.
- Lovastatin treatment suppressed Ha-ras activity and induced WAF1 activity, disrupting cell cycle progression.
- Inhibition of Ras or Raf-1 signaling reverted lovastatin sensitivity.
Conclusions:
- Ras and Raf-1 signaling pathways are essential for lovastatin-induced apoptosis.
- Lovastatin induces apoptosis by suppressing Ha-ras activity and increasing WAF1 activity, leading to cell cycle alterations.
- This apoptotic pathway is independent of Fas/Fas-L and p53.