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).

A Phylogenetically Conserved DNA Damage Response Resets the Circadian Clock

“…To test this, confluent PER2::LuC MEF cells were treated with either 1 mM MMS to induce DNA damage, or a vehicle control, for 2 h at various times after synchronization. A concentration of 1 mM MMS has been shown to induce DNA damage in the MEF cell line NIH3T3 (Kuo et al, 1997). As expected, DNA damage induced only phase advances that were persistent several days after MMS treatment (Fig.…”

“…A concentration of 1 mM MMS has been shown to induce DNA damage in the MEF cell line NIH3T3 (Kuo et al, 1997). As expected, DNA damage induced only phase advances that were persistent several days after MMS treatment (Fig.…”

Inhibition of p53 by Lentiviral Mediated sh RNA Abrogates G1 Arrest and Apoptosis in Retinal Pigmented Epithelial Cell Line

“…Methane methyl sulfonate (MMS), an alkylating agent, has been reported to induce the p53 pathway which in turn results in the upregulation of p21 transcription. 15,[45][46][47] In this study, following MMS treatment, there was up to approximately an 8-fold induction of the p21 promoter in ARPE-19/p21-luciferase cells (Fig. 5, left).…”

Ras and Rheb Signaling in Survival and Cell Death

“…For instance, an early study by Davis et al . reports constitutively elevated Bcl-2 levels upon Ras-transformation of NIH/3T3 cells, presumably mediated via p53 [64]. In accordance with this, ectopic activation of Raf in breast cancer (MCF-7) cells yields an increase in Bcl-2 protein levels [65].…”