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Oncogenic transformation potentiates apoptosis, S-phase arrest and stress-kinase activation by etoposide
1Department of Molecular Biology, The Cleveland Clinic Foundation, Ohio 44195, USA.
Abstract:
The exact mechanisms for the selective toxicity of chemotherapeutic drugs against tumor cells are not fully understood. We designed a series of experiments to test the possibility that the positive proliferative signal initiated by oncogenes might change the sensitivity for apoptosis induction by the anticancer drug etoposide (VP16), an inhibitor of topoisomerase II (Topo II). Treatment with VP16 induced significantly increased apoptosis in NIH3T3 cells transformed by oncogenic src, ras or raf, compared with the normal 3T3 cells. Apopototic changes involved nuclear DNA fragmentation, morphological alterations and decreased viability. Furthermore it was shown that stress-activated protein kinase (SAPK) was activated much more strongly in all three transformed lines compared to untransformed cells by VP16 treatment, while slight activation of extracellular signal-regulated kinase (ERK1) was observed in all four cell lines. In addition, the transformed cells displayed arrest in mid-S-phase following the treatment, whereas NIH3T3 cells were primarily arrested in late S and G2/M phase. Finally, the cyclin-dependent kinase inhibitor p21 WAF1 was induced in all four cell lines, although induction of p53 was not detected in any of these cell lines. Taken together our results demonstrated that oncogenic transformation can sensitize the cells to apoptosis induction, stress kinase activation and cell cycle arrest in response to VP16 treatment. These results may have important implications for understanding the selective toxicity of anti-cancer drugs in tumor cells.
Insights
Oncogenic transformation sensitizes cancer cells to etoposide (VP16), increasing apoptosis and stress kinase activation. This finding aids understanding of chemotherapy
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Mechanisms of selective chemotherapy toxicity in tumor cells remain unclear.
- Oncogene-induced proliferative signals may influence drug sensitivity.
- Etoposide (VP16) is a topoisomerase II (Topo II) inhibitor used in chemotherapy.
Purpose of the Study:
- To investigate if oncogene-driven proliferation alters sensitivity to etoposide (VP16)-induced apoptosis.
- To explore the role of stress-activated protein kinase (SAPK) and extracellular signal-regulated kinase (ERK1) pathways.
- To examine cell cycle arrest patterns and p21 WAF1 induction.
Main Methods:
- Treatment of normal NIH3T3 cells and NIH3T3 cells transformed with oncogenes (src, ras, raf) with etoposide (VP16).
- Assessment of apoptosis via DNA fragmentation, morphology, and viability assays.
- Analysis of SAPK and ERK1 activation using Western blotting.
- Cell cycle analysis using flow cytometry.
- Measurement of p21 WAF1 and p53 induction via Western blotting.
Main Results:
- VP16 induced significantly higher apoptosis in oncogene-transformed cells compared to normal cells.
- VP16 strongly activated SAPK in transformed cells, with minor ERK1 activation in all cell lines.
- Transformed cells arrested in mid-S-phase, while normal cells arrested in late S and G2/M phases.
- p21 WAF1 was induced in all cell lines; p53 induction was not detected.
Conclusions:
- Oncogenic transformation sensitizes cells to VP16-induced apoptosis and SAPK activation.
- VP16 treatment leads to distinct cell cycle arrest profiles in normal versus transformed cells.
- These findings offer insights into the selective toxicity of anticancer drugs against tumor cells.