Mutant p27(Kip1) and its potential effect as hepatocellular gene therapy.
BACKGROUND
The cyclin-dependent kinase (cdk) inhibitor p27(Kip1) is an important regulator of cell cycle progression as it negatively regulates G(0/1) progression and plays a major role in controlling the cell cycle. The screening of the p27(Kip1) sequence identified many potential phosphorylation sites. To investigate the effects of the overexpression of exogenous p27(Kip1) protein lacking the Thr157 sites on subcellular localization, cell cycle, and proliferation, a plasmid was constructed containing mutations of p27(Kip1) 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.


METHODS
We detected the proliferation of SMMC7721 cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of p27(Kip1) by Western blotting analysis and cell fractionation. The cdk2 dependent kinase activity was determined by in vitro kinase assay.


RESULTS
Proliferation of SMMC7721 cells was greatly inhibited and cell cycle was arrested in G(0/1) phase after exogenous p27(Kip1) mutant expression much more than wild-type p27(Kip1). The expressed T157A p27(Kip1) proteins were translocated from the cytoplasm into nucleus much more compare with wild-type. Compared with pcDNA3.1-Myc control, transient transfection of T157A p27(Kip1) decreased expression of cyclin D1 and the phosphorylated form of retinoblastoma protein.


CONCLUSIONS
These findings support the potential effectiveness of a PI3K/Akt-resistant phosphorylated form of p27 in hepatocellular carcinoma gene therapy.
Lu
Mudan
M
Department of Pathology, Affiliated Cancer Hospital of Nantong University, Medical College of Nantong University, Nantong, China.
Fei
Min
M
Cheng
Chun
C
Wang
You
Y
He
Song
S
Zhao
Yueming
Y
Gao
Shangfeng
S
Ke
Qing
Q
Li
Peng
P
Cui
Xiaopeng
X
Shen
Aiguo
A
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D050760:Cyclin-Dependent Kinase Inhibitor p27