[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.