Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27(Kip1) inhibits cell growth
| Autor: | Mark G. Goebl, Susan A. Brutkiewicz, Jean A. Hurteau, Maureen A. Harrington, Bernadette M. Allison, Qi Wang |
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| Rok vydání: | 2002 |
| Předmět: |
Mutant
Molecular Sequence Data Mutagenesis (molecular biology technique) Cell Cycle Proteins Biology urologic and male genital diseases Transfection Cell Line chemistry.chemical_compound Tumor Cells Cultured Humans Luciferase Amino Acid Sequence Kinase activity Luciferases neoplasms Reporter gene Cell growth Tumor Suppressor Proteins Obstetrics and Gynecology Molecular biology Growth Inhibitors Oncology chemistry Cell culture Mutagenesis Site-Directed biological phenomena cell phenomena and immunity Growth inhibition Protein Kinases Cyclin-Dependent Kinase Inhibitor p27 |
| Zdroj: | Gynecologic oncology. 86(1) |
| ISSN: | 0090-8258 |
| Popis: | Objective. The purpose of this study was to test the hypothesis that the expression of the mutant p27 Kip1 protein enhances cell growth inhibition and is more stable than that of the wild-type p27 Kip1 . Methods. Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27 Kip1 . To study the effects of the p27 Kip1 mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27 Kip1 , or mutant p27 Kip1 using Fugene 6. The transfected cells were lysed and assayed for luciferase activity 24 h later with a dual-luciferase reporter assay system. To further assess the effects of the p27 Kip1 mutant on cell growth, colony count assays were performed. The experiments were repeated in duplicate and a standard two-tailed Student t test was use to analyze the data. Results. Wild-type p27 Kip1 protein has a half-life of approximately 2 h while the p27 Kip1 mutant has a half-life of greater than 12 h. Furthermore, the p27 Kip1 mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27 Kip1 mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27 Kip1 ( P = 0.001). Colony assays revealed that cells expressing the p27 Kip1 mutant had fewer colonies compared to cells expressing the wild-type p27 Kip1 ( P = 0.04). Conclusions. These data are consistent with the hypothesis that the mutated form of p27 Kip1 is more effective in cell growth inhibition than the wild-type p27 Kip1 protein. |
| Databáze: | OpenAIRE |
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