Mutant p27Kip1 and its Potential Effect as Hepatocellular Gene Therapy

Background clin-dependent kinase (cdk) inhibitor p27Kip1 is an important regulator of cell cycle progression as it negatively regulates G0/1 progression and plays a major role in controlling the cell cycle. The screening of the p27Kip1 sequence identified many potential phosphorylation sites. To investigate the effects of the overexpression of exogenous p27Kip1 protein lacking the Thr157 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27Kip1 at Thr157 (T157A p27), and transfected into the SMMC7721 cell line with Lipofectamine. Wild-type and mutant p27 plasmids T157A were transfected separately as control groups. s ected the proliferation of SMMC7721 cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of p27Kip1 by Western blotting analysis and cell fractionation. The cdk2 dependent kinase activity was determined by in vitro kinase assay. s eration of SMMC7721 cells was greatly inhibited and cell cycle was arrested in G0/1 phase after exogenous p27Kip1 mutant expression much more than wild-type p27Kip1. The expressed T157A p27Kip1 proteins were translocated from the cytoplasm into nucleus much more compare with wild-type. Compared with pcDNA3.1-Myc control, transient transfection of T157A p27Kip1 decreased expression of cyclin D1 and the phosphorylated form of retinoblastoma protein. sions findings support the potential effectiveness of a PI3 K/Akt-resistant phosphorylated form of p27 in hepatocellular carcinoma gene therapy.