[Specific point-mutate p53 mini-gene transfectimg effects on biological behaviors of a human cancer cell line PG derived from human pulmonary giant carcinoma].

OBJECTIVES: To explore the suppressive effects of a murine genomic p53 minigene containing an Arg-->Leu substitution at its encoding amino acid 172 on biological behaviors of human carcinoma cell and evaluate its potential application in cancer gene therapy. METHODS: By LipofectaMINE and electraporation methods, this mutant p53 gene which lacked of exon 1 and intron 1 expression vector driven by CMV promoter was co-transfected with PCMVneo into PG cell in which dominant negative p53 pre-exists. A wild-type and another kind of genomic mutate-type p53 gene expression vector were transfected. The latter p53 gene encoding protein contained an Arg-->His substitution at the same position, and pBLuscript plasmid was used as control. All transfectants were screened by 500 micrograms/ml geneticin and identified by mouse specific p53 mRNA RT-PCR and Northern blot analysis. After transfection, the biological behavior changes were studied by colony formation and TUNEL test together with in-situ clone regression for chemosensitivity of anti-cancer drugs. RESULTS: The transfecting effects of this unusual p53 gene were surprisingly strong. They were more significant than those of the wild-type p53 and could suppress the formation of transgenic colonies and passage. The transgenic colonies were sensitive to be treated in adromycin and 5-Fu, and the gene transient expression could result in cell apoptosis. CONCLUSION: Codon 172 mutant (Arg-->Leu) p53 genomic DNA exhibited a strong suppressive transfecting effects on carcinoma cell, so it is a possible candidate to be used in cancer gene therapy.