The In Vitro Experimental Study Of The Enhancement Of Radiosensitivity Of Recombinant Adenovirus-mediated Wild Type P53 Gene On Human Lung Adenocarcinoma Cell Lines With Different P53 Status

PURPOSE: To investigate the combined effects of adenoviral-mediated p53(Ad-p53) transgene expression and irradiation response in vitro. To determine whether wtp53 sensitizes cells to radiation independently of their p53 status and to investigate the interaction mechanism.METHODS AND MATERIALS: Two human lung adenocarcinoma cell lines, the A549 and GLC-82 (mutant p53) lines, were examined. A recombinant adenovirus vector containing a CMV promoter , wild-type p53-cDNA (Ad-p53) and green fluorescence protein(GFP) gene was used to facilitate p53 transgene expression. Adenovirus vector contained an expression cassette encoding the E.coli -galactosidase gene(Ad-lacZ) instead of wide type p53 cDNA served as a negative control vector. The whole study involved three parts. In the first part, we focus on conforming the difference between the p53 status of the two selected cell lines. In the second part, immunohistochemistry (IHC) stain was carried out to detect P53 expression before and after transinfection. A multiplicity of infection (MOI) of 100 viral particles per cell was used, based on detection for the GFP reporter gene product. In the third part, cell growth curve and clonogenic assays were performed to evaluate the inhibition effect on cell growth and the degree of sensitization to radiation of viral-transducted cells compared with irradiated nontransducted controls. The relative efficacy of these treatments to induce apoptotic cell death and cell cycle changes was determined using the flow cytometry assay.RESULTS: The two cell lines presented different P53 expression, for A549 positive and for GLC-82 negative. By the method of PCR-SSCP technique, the difference of endogenous p53 status of the two cell lines was identified. A549 has a wide type p53 gene while GLC-82 beard mutant p53 on the exon 7. 100MOI was adapted as an optimal titre for gene transfectionaccordingly to the GFP expression. p53 gene could be efficiently expressed in the two cell lines, which could greatly suppress cell growth. This efficiency didn' t depend on the intrinsic p53 genetic status. After irradiation, its function of inducing G1 arrest and apoptosis and suppressed cell growth on GLC-82 cell line was much stronger than the A549 cell line. Combination of irradiation and p53 transfection was more effective than either treatment alone. In both the A549 and GLC-82 cell lines, the combination of Ad-p53 plus radiation resulted in supra-additive apoptosis, above that seen from the addition of the controls, Ad-p53 alone and RT alone.CONCLUSION: The clonogenic survival and apoptosis data demonstrate that p53 transgene expression sensitizes human lung adenocarcinoma cells in vitro to irradiation. As this effect was observed in both the A549 (p53 wild-type) and GLC-82 (p53 mutant) lines, radiosensitization was independent of endogenous p53 status.