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Resistance to apoptosis induced by alkylating agents in v‐Ha‐ras‐transformed cells due to defect in p53 function
Author(s) -
Kuo MinLiang,
Chou YiWein,
Chau YatPang,
Huang TuzShing
Publication year - 1997
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/(sici)1098-2744(199704)18:4<221::aid-mc6>3.0.co;2-g
Subject(s) - biology , apoptosis , 3t3 cells , microbiology and biotechnology , oncogene , cell culture , programmed cell death , heat shock protein , dna damage , cell cycle , dna , transfection , biochemistry , genetics , gene
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 G 1 checkpoint function accompanied by the accumulation of p53 and p21 CIP1/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 co‐immunoprecipitable 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 ad 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). Mol. Carcinog. 18:221–231, 1997. © 1997 Wiley‐Liss, Inc.