Abstract
H1299 cells have a homozygous partial deletion of the p53 gene, and thus lack expression of p53 protein. In this study, three kinds of p53-transfected H1299 cells having different p53 gene status have been established from original p53-null H1299 cells. For establishment of wtp53-H1299 cells, normal p53 gene was be constructed from p53-SN into pCDNA3 plasmid, and named it pcDNA3-p53 plasmid. pCDNA3-p53 and EGFP-N1 plasmids were transfected into H1299 cells to establish wtp53/EGFP-H1299 cells by lipofectamine. Two other cell lines, mp53/EGFP-H1299 and pcDNA/EGFP-H1299 cells were established by transfecting pcDNA3-mp53 and pcDNA3, respectively, along with EGFP-N1 plasmids into H1299 cells. Stable transfected cells were selected in G418-containing medium. They were characterized by Western blotting to examine the background expression of p53 and p21. P53 protein was detected in both wtp53/EGFP-H1299, and mp53/EGFP-H1299 cells. However, p21 protein was only markedly observed in wtp53-H1299 cells in comparison with those in mp53/EGFP-H1299 and pCDNA3/EGFP-H1299 cells. These indicated that all transfectants were have expected results. The sensitivity to different concentrations of SA in above transfectants was further examined by SRB assay. The IC50 of SA in wtp53/EGFP-H1299, mtp53/EGFP-H1299, and pCDNA3/EGFP-H1299 cells was 3.9 mM, 6.6 mM, and 15.4 mM, respectively. Therefore, the sensitivity to SA was 4-fold and 2.3-fold in wtp53/EGFP-H1299 and mtp53/EGFP-H1299 cells, respectively, in comparison with that in pCDNA3/EGFP-H1299 cells. In other words, expression of wild type p53 could markedly enhance the sensitivity to SA in p53-transfected H1299 cells. However, expression of mutant type p53 also slightly affected the sensitivity to SA in mp3-transfected H1299 cells. We expected above thee transfected cells are suitable in using to investigate the relationship between p53 status and chemosensitivity to various chemotherapeutic agents.