Abstract
The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. Loss of functional p53 leads to impaired responses of cancer cells to apoptosis induction and to poor prognosis in patients with certain types of cancer. This study examined whether X-ray and sodium arsenite (SA) sensitivities depend on various p53 gene status in H1299 cells containing stably transfected with neo-, mp53/neo- or wtp53/neo-containing vector, respectively. The sensitivities of transfected cells to X-ray or SA were determined by sulforhodamine B (SRB) cell viability assay at day 5 after treatment. The IC50 for wtp53-H1299 (S#40) cells after 0-4 Gy X-ray or 0-30 μM SA treatment was 2.4 Gy and 16 μM, respectively. The H1299 cells transfected with wild-type p53 gene (wtp53-H1299 cells) showed a dramatic increase in susceptibility to X- ray or SA (1.5-fold to X-ray and 1.4-fold to SA) compared to neo-transfected H1299 cells. In contrast, mp53-H1299 cells showed X-ray- and SA-resistance due to the dominant negative nature of mutant p53 (R273H), compared with wtp53-H1299 cells. The incidence of sub-G1 apoptosis by X-ray or SA was analyzed with flow cytometry at day 3 after treatment. The results of sub-G1 cells after each same dose of X-ray or SA treatment for 3 different cells were consistent with the cell survival data from SRB observation. The sensitivity order for either treatment was S#40>mp53->neo-1299. Data from DNA fragmentation assay at 24, 48 and 72 h after treatment confirmed the sensitivity order for these three cells, S#40>mp53->neo-H1299. The p53 localization of transfected cells after X-ray or SA treatment was analyzed by immuno-fluorescence stain. After treatment, the localization of p53 apparently accumulated into the nuclei in wtp53-H1299 (S#40) cells as compared with neo- and mp53-H1299 cells. Western analyses results showed that wtp53-H1299 (S#40) cells, but not neo- and mp53-H1299 cells, p53 and pro-apoptotic protein Bak were obviously increased after X-ray or SA treatment. Fragmentation of caspase-3 and Poly (ADP-Ribose) Polymerase (PARP) was observed in wtp53-H1299 (S#40) cells, but almost no such fragmentation was observed in neo- and mp53-H1299 cells after same treatment. These results confirmed that the restoration of p53 function by wild type p53 but not mutant p53 in null-p53 H1299 cells enhanced apparently the sensitivity to X-ray and SA and induced the occurrence of cellular apoptotic response through caspase-3 activation. In addition, our data also supported the possible clinical usage for wild type p53 gene therapy for human non small cell lung cancer (NSCLC) patients.