Related Experiment Video
Updated: Jul 3, 2026
Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine
Published on: November 4, 2018
Mutant p27(Kip1) and its potential effect as hepatocellular gene therapy
Mudan Lu1, Min Fei, Chun Cheng
1Department of Pathology, Affiliated Cancer Hospital of Nantong University, Medical College of Nantong University, Nantong, China.
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.
Insights
Mutating Thr157 in p27(Kip1) protein significantly inhibits hepatocellular carcinoma cell proliferation and promotes nuclear localization. This modified p27(Kip1) shows promise for gene therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- p27(Kip1) is a key cell cycle regulator, controlling G(0/1) progression.
- Phosphorylation sites on p27(Kip1) influence its function.
- Investigating mutations at Thr157 (T157A p27) can reveal insights into p27(Kip1) regulation.
Purpose of the Study:
- To assess the impact of T157A p27(Kip1) overexpression on SMMC7721 cell proliferation and cell cycle.
- To determine the effect of T157A p27(Kip1) on subcellular localization.
- To evaluate the influence of T157A p27(Kip1) on cell cycle-related proteins.
Main Methods:
- Transfection of SMMC7721 cells with wild-type and T157A p27(Kip1) plasmids.
- Cell proliferation assays (Cell Counting Kit, FACS).
- Western blotting and cell fractionation for protein expression and localization analysis.
- In vitro kinase assays for cdk2 activity.
Main Results:
- T157A p27(Kip1) significantly inhibited cell proliferation and arrested the cell cycle in G(0/1) phase compared to wild-type p27(Kip1).
- T157A p27(Kip1) showed increased nuclear translocation compared to wild-type.
- T157A p27(Kip1) reduced cyclin D1 and phosphorylated retinoblastoma protein expression.
Conclusions:
- The T157A mutation enhances the cell cycle inhibitory function of p27(Kip1).
- Nuclear localization of T157A p27(Kip1) is associated with its enhanced activity.
- A PI3K/Akt-resistant form of p27(Kip1) holds potential for hepatocellular carcinoma gene therapy.
