Resistance to apoptosis induced by alkylating agents in v-Ha-ras-transformed cells due to defect in p53 function.

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).

Distinct p53-mediated G1/S checkpoint responses in two NIH3T3 subclone cells following treatment with DNA-damaging agents.

N3T3 and P-3T3 cells, originally isolated from a NIH3T3 cell clone on the basis of their negative and positive transformation by v-Abl, v-Src and Bcr-Abl, were previously found to show distinct cyclin activity changes following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, which is anti-mitogenic for N-3T3 cells and mitogenic for P-3T3 cells. We have found in this study that, while the G1/S arrest and cell death induced by serum starvation and TPA treatment in N-3T3 cells did not involve p53-mediated checkpoint or apoptosis, N-3T3 and P-3T3 cells evidently responded differently in these aspects of cell cycle regulation to DNA-damaging agents, methylmethane sulfonate (MMS) and gamma-radiation. In N-3T3 cells, DNA damages elicit cell growth arrest at G1/S transition with concomitant accumulation of p53 and p53-inducible Waf1/Cip1 proteins and also signs of apoptosis such as DNA ladder patterns and apoptotic (subgenomic) peak in flow cytograph. Conversely, P-3T3 cells treated with the DNA-damaging agents showed no cell cycle interruption nor accumulation of p53 or Waf1/Cip1. However, both P-3T3 and N-3T3 cells showed the same p53 protein half-life of 40 min or less, the same wild-type p53 DNA sequence and the same co-immunoprecipitable cellular proteins in complexes with p53, suggesting that an alteration in a signal transduction pathway upstream of p53 might account for the evasion of p53-mediated G1 checkpoint in P-3T3 cells.

Differential induction of apoptosis in oncogene-transformed NIH 3T3 cells by methylmethanesulfonate.

Cellular oncogenes have been shown to play crucial roles in the cell death process induced by cytotoxic agents. In this study, we have demonstrated that v-H-ras transformed NIH 3T3 cells but not other transformants (v-raf, v-src, v-erbB-2, v-fes and v-mos) exhibited a survival advantage to treatment by a DNA-damaging agent, methylmethanesulfonate (MMS). Subsequently, the biochemical and morphologic criteria of MMS-treated cells were examined. It was found that MMS induced v-H-ras transformants to go through necrosis, but it induced other transformed cells to undergo apoptosis. The levels of glutathione (GSH) within each transformant as well as in NIH 3T3 cells, were determined. The results showed that GSH levels within ras transformants were 2- to 7-fold higher than the levels in other transformants and normal NIH 3T3 cells. By using the GSH synthesis inhibitor buthionine sulfoximine, GSH levels were artificially reduced. This depletion, however, made ras transformed cells more sensitive to MMS killing, but the mode of cell death was still necrosis. Western blot analysis demonstrated that the anti-apoptotic protein Bcl-2 was constitutively expressed in ras transformed cells but not in NIH 3T3 or other transformed cells. The level of Bcl-2 was correlated with the resistant phenotype of ras transformants during MMS treatment. These observations suggest that GSH and Bcl-2 levels may cooperatively confer the resistant phenotype of ras transformants in response to MMS. In addition, the mode of cell death may possibly be determined at least in part by Bcl-2 protein.

Characterization of a cloned pR72H probe for Vibrio parahaemolyticus detection and development of a nonisotopic colony hybridization assay.

Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.