Transfection of p27Kip1 Threonine Residue 187 Mutant Type Gene, Which Is Not Influenced by Ubiquitin-Mediated Degradation, Induces Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cells

Abstract

Objective: It is well known that reduction of the cyclin-dependent kinase inhibitor p27^Kip1 protein correlates with the malignant behavior of various cancers including oral squamous cell carcinoma (OSCC). The loss of p27^Kip1 protein is suggested to be due to the enhancement of its posttranslational degradation. In the present study, to evaluate the effects of p27^Kip1 transfection on the cell cycle, we transfected OSCC cell lines with a high activity of p27^Kip1 degradation with p27^Kip1 threonine 187-to-alanine (T187A) mutant gene, which is not influenced by ubiquitin-mediated degradation, as well as with wild type gene. Methods: We transfected p27^Kip1 T187A mutant and wild type gene into OSCC cell lines (HSC2 and HSC3) by using an ecdysone-inducible gene expression system. Results: After treatment with ponasterone A, we could find an induction of both p27^Kip1 wild type and T187A mutant protein. Both wild type and T187A mutant protein induced by 5 µM ponasterone A inhibited cell growth and increased cell number at the G1 phase. After treatment with 1 µM ponasterone A, ectopic p27^Kip1 protein was degraded in wild type clones, but not in T187A mutant clones. Moreover, transfection of the T187A mutant gene was more effective in inhibiting cell growth even by induction of a small amount of protein. Conclusion: We suggest that the transfection of the p27^Kip1 T187A mutant gene can be a modality of cancer gene therapy for OSCC.