P48L和D74N突变对p16基因功能的影响

The effect of P48L and D74N substitution on the function of p16 tumor suppressor

应用DNA聚合酶链式反应(PCR)技术,对p16抑癌基因(CDKN2)进行体外定点突变,在p16cDNA中引入第48位密码子CCG(Pro)→CTG(Leu)和第74位密码子GAC(Asp)→AAC(Asn)突变,构建了p16-P48L和p16-D74N突变体。野生型和突变型p16 cDNA克隆于pcDNA3构建pCMV-p16、pCMV-p16P48L和pCMV-p16D74N真核表达载体,导入纯合缺失p16基因的人肺癌细胞株H460,经RNA点杂交、RNA印迹和细胞免疫化学染色,检测到P16表达。通过比较表达野生型和突变型P16的H460细胞在~3H-TdR掺入及细胞所在周期的差异,证实P16表达抑制细胞进入S期,而P48L和D74N突变体对细胞进入S期没有什么影响,提示P48L和D74N突变导致P16蛋白功能丧失。

To evaluate the functional effects of mutations in p16 which have been observed in different primary cancer and tumor cell lines, the p16 cDNA was cloned from HeLa cell by RT-PCR. And two p16 mutants, P48L and D74N, were obtained by site-directed mutagenesis using two step PCR method. Mutant p16 genes were recloned into plasmid M13mpl8 and M13mpl9, and sequenced by the didoxy method to confirm that no any other changes were present in their nucleotide sequence except the designed substitutions. Then wild type p16 and mutant p16 cDNA were cloned into the mammalin expression vector pcDNA3 to construct p16 expression vectors pCMV-p16, pCMV-p16P48L and pCMV-p16D74N, respectively. After introduction of these expression vector into human lung cancer cell lines H460 which p16 gene was homozygous deleted, exogenous P16 expression were detected in G418-resistant cell line by Northern bloting and immunohistochemistry staining. Over expression of wild type p16 inhibited cell proliferation by caused G1 arrest. In contrast, both of two mutants defected in their adbility to arrest cell cycle. This result suggested that these two p16 missense mutants were dificency functionally.