Inhibiting effect of murine double minute-2 oncogene on apoptosis induced by retroviral-mediated wild-type p53

Objective To investigate the effects of wild-type p53 (wtp53) on inducing a poptosis by restoring wtp53 expression in pancreatic adenocarcinoma cell li ne (PC-2) which contains mutant p53, and the interaction between murine dou ble minute-2 (MDM2) and wtp53 in pancreatic adenocarcinoma. Methods A recombinant retroviral vector expressing wild-type p53 was constr ucted and packaged by packaging cell line PA317 cells using calcium phospha te coprecipitation method. The supernatant of the packaged cells PA317 was used to transfect the pancreatic carcinoma cell line (PC-2), then a transfo rmed cell line PC-2/swtp53 was established. The recombinant vector pCMV-MDM 2 was transduced into PC-2/swtp53 cell line by lipofectin-mediated method, a double transfected cell line (PC2/swtp53/pCMV-MDM2) was formed. To determ ine the integration and expression of exogenous wtp53 gene in the transfect ed cells, polymerase chain reaction (PCR), Western blot and immunoprecipita tion analyses were performed. Apoptosis was analyzed by means of flow cytom etry, in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) analysis and DNA agarose gel electrophoresis. Results Transduction with the retroviral vector resulted in integration and expression of wtp53 gene in host cells. The apoptotic cells in PC-2/swtp53 and PC2/swtp53/pCMV-neo cell lines were 12.1% - 12.9%, while the double tr ansfected cell line, PC-2/swtp53/pCMV-MDM2, showed less (3.2%) apoptotic ce lls than its parent cell lines. Conclusions Restoration of expression of wildtype p53 with a retroviral vec tor can increase programmed cell death of pancreatic adenocarcinoma cells ( PC-2) containing mutant p53. The overexpression of MDM2 protein has a negat ive regulating role in wtp53-induced apoptosis in PC-2 cell.