Expression of Wild-type and Mutant p16 in H460 Cell Line.

Two p16 mutants, P48L and D74N, were obtained by site-directed mutagenesis using two step PCR method. Mutant p16 cDNA and wild-type p16 cDNA were colned into the mammalian expression vector pcDNA3 to construct p16 expression vector pCMV-p16, pCMV-p16P48L and pCMV-p16D74N, respectively. After the introduction of these expression vectors into human lung cancer cell line H460 in which endogenous p16 gene was homozygously deleted, exogenous p16 expression was detected in G418 resistant cells by Northern blotting and immunocytochemistry staining. The results of immunofluorescence and immunocytochemistry staining showed that the P16 protein was located in the cell cytoplasm. The p16 cDNAs amplificated from the genomic DNA of recombinant H460 cell lines indicated that the plasmid p16 cDNA was integrated into the chromosome of cell lines. That the over expression of wild-type p16 caused G1 arrest suggested the wild-type P16 protein expressed in H460 cell line to be a functional protein.