[A study on the tumor suppressing effect of a specific point mutant p53 minigene in the expression regulated model with a tetracycline-transactivative response promoter].

OBJECTIVE

To establish a tetracycline-regulated expression model and to determine and verify whether a specific point mutant type p53 minigene, containing an Arg-->Leu substitution at amino acid 172, possesses a suppressing effect on human lung cancer.

METHODS

The tumor suppressing effects of inducing apoptosis and inhibition of the formation of G418 resistant colonies of the specific point mutated p53 minigene in a structural expression vector on a human cancer cell line PG with preexisting dominant negative p53 were preliminarily verified. Then the specific p53 minigene was sub-cloned into a tetracycline-transactivative controlled expression vector pBPSTR1 by gene recombination methods. Through LipofectaMINE, the vector was transfected into PG cells under the presence of tetracycline (1.0 mg/ml), and the transfectants were screened in the selecting medium containing 1.5 micrograms/ml puromycin, the p53 minigene expression and tumor suppressing effects were studied dynamically in presence/absence (1.0/0 mg/ml) of tetracycline.

RESULTS

The specific mutant p53 minigenes had a stronger tumor suppressing effect than wild type p53 minigene on colony formation and transient expression could induce PG cell apoptosis (P < 0.05). The tetracycline transactivative p53 minigene-regulated transgene model was successfully established. When tetracycline was absent, a large amount of apoptosis cells in transgenic passage colonies could be detected. Therefore the tumor suppressing effects were further verified.

CONCLUSION

The specific point mutant p53 minigene may be a good candidate for cancer gene therapy. The tetracycline transactivative response promoter was found to be a good regulator of down stream gene expression, this may be useful in future gene therapy.