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Resistance to apoptosis induced by alkylating agents in v-Ha-ras-transformed cells due to defect in p53 function
1Institute of Toxicology, College of Medicine, National Taiwan University, Taipei.
Abstract:
In this study, we examined the susceptibility of various oncogene-transformed NIH/3T3 cells to apoptosis induced by alkylating agents. Only v-Ha-ras-transformed cells showed marked resistance to apoptotic death induced by these drugs. Upon treatment with methylmethane sulfonate (MMS), NIH/3T3 cells exhibited normal G1 checkpoint function accompanied by the accumulation of p53 and p21CIP1/WAF1 protein. However, no such effects were observed in v-Ha-ras-transformed cells. To further examine the functional status of p53 in ras-transformed cells, we determined the DNA sequence, protein half-life, protein-complexing activity, and specific DNA-binding activity of p53. The results showed that ras transformants and parental NIH/3T3 cells had the same p53 protein half-life of 40 min or less, the same normal wild-type p53 cDNA sequence, and the same coimmunoprecipitable cellular proteins complexed with p53. In electrophoretic mobility gel-shift assays, however, nuclear extracts of cells treated with MMS, ras-transformed cells, and normal cells displayed distinct patterns of binding between p53 and its consensus binding site. Furthermore, western blot analysis showed that the bcl-2 and bax proteins were constitutively elevated in ras-transformed cells but not in parental NIH/ 3T3 cells. Heat-shock protein 70 (hsp70), which has been found to be negatively regulated by wild-type p53, was also dramatically induced in ras-transformed cells but not in NIH/3T3 cells in response to MMS. Thus, our data suggest that an activated ras oncogene can suppress alkylating agent-induced apoptotic cell death by means of a defect in the signal transduction pathway regulating p53 function and alteration in the expression of apoptotic (bax) or anti-apoptotic proteins (bcl-2 and hsp70).
Insights
Activated Ras oncogenes confer resistance to alkylating agents by disrupting p53 signaling and altering apoptotic protein expression. This study reveals how Ras impacts cell death pathways, offering insights into cancer drug resistance.
Area of Science:
- Molecular Biology
- Oncology
- Cell Death Research
Background:
- Oncogene activation, particularly by Ras, plays a critical role in cellular transformation and cancer development.
- Alkylating agents are a class of chemotherapy drugs used to induce cancer cell death via DNA damage.
- The tumor suppressor protein p53 is a key regulator of apoptosis and cell cycle arrest in response to DNA damage.
Purpose of the Study:
- To investigate the differential susceptibility of oncogene-transformed NIH/3T3 cells to apoptosis induced by alkylating agents.
- To elucidate the mechanisms by which the Ras oncogene confers resistance to drug-induced apoptosis, focusing on p53 pathway.
- To analyze the expression of apoptosis-related proteins, including bcl-2, bax, and hsp70, in Ras-transformed cells.
Main Methods:
- Treatment of NIH/3T3 and v-Ha-ras-transformed cells with methylmethane sulfonate (MMS).
- Analysis of G1 checkpoint function, p53 and p21CIP1/WAF1 protein accumulation.
- Assessment of p53 DNA sequence, protein half-life, complexing activity, and DNA-binding activity using electrophoretic mobility gel-shift assays.
- Western blot analysis for bcl-2, bax, and heat-shock protein 70 (hsp70) expression.
Main Results:
- v-Ha-ras-transformed cells exhibited marked resistance to MMS-induced apoptosis compared to parental NIH/3T3 cells.
- Ras-transformed cells showed defective G1 checkpoint function and failed to accumulate p53 and p21CIP1/WAF1 upon MMS treatment.
- While p53 protein itself was functional in terms of half-life and complexing, its DNA-binding activity was altered in Ras-transformed cells.
- Ras-transformed cells displayed constitutive elevation of anti-apoptotic bcl-2 and pro-apoptotic bax, and induced hsp70 upon MMS treatment, unlike parental cells.
Conclusions:
- Activated Ras oncogene suppresses alkylating agent-induced apoptosis through a defect in the p53-regulated signal transduction pathway.
- Alterations in the expression of key apoptotic regulators (bcl-2, bax) and anti-apoptotic proteins (hsp70) contribute to Ras-mediated drug resistance.
- These findings highlight a critical role for Ras in modulating cell death responses and suggest potential therapeutic targets for overcoming chemoresistance.