Transfection of p27(Kip1) threonine residue 187 mutant type gene, which is not influenced by ubiquitin-mediated degradation, induces cell cycle arrest in oral squamous cell carcinoma cells.
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 microM ponasterone A inhibited cell growth and increased cell number at the G1 phase. After treatment with 1 microM 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.


CONCLUSIONS
We suggest that the transfection of the p27(Kip1) T187A mutant gene can be a modality of cancer gene therapy for OSCC.
Kudo
Yasusei
Y
Department of Oral Pathology, Hiroshima University, Faculty of Dentistry, Japan.
Kitajima
Shojiro
S
Sato
Sunao
S
Ogawa
Ikuko
I
Miyauchi
Mutsumi
M
Takata
Takashi
T
D000409:Alanine; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D002453:Cell Cycle / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005434:Flow Cytometry; D015316:Genetic Therapy; D006801:Humans; D009062:Mouth Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D009154:Mutation; D013912:Threonine; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D018797:Cell Cycle Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D018844:Cyclin-Dependent Kinases; D000409:Alanine