title: Resistance to apoptosis induced by alkylating agents in v-Ha-ras-transformed cells due to defect in p53 function.
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).
authors.affiliation: Institute of Toxicology, College of Medicine, National Taiwan University, Taipei.
authors.forename: M L
authors.initials: ML
authors.lastname: Kuo
authors.forename: Y W
authors.initials: YW
authors.lastname: Chou
authors.forename: Y P
authors.initials: YP
authors.lastname: Chau
authors.forename: T S
authors.initials: TS
authors.lastname: Huang
mesh_terms: D016475:3T3 Cells; D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002472:Cell Transformation, Viral; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D053938:DNA Fragmentation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D019254:Genes, bcl-2; D016158:Genes, p53; D011905:Genes, ras; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D051028:bcl-2-Associated X Protein
chemical_list: D000477:Alkylating Agents; C494827:Bax protein, mouse; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D015689:Oncogene Protein p21(ras)