Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27Kip1 inhibits cell growth

Jean A. Hurteau, Susan A. Brutkiewicz, Qi Wang, Bernadette M. Allison, Mark Goebl, Maureen Harrington

Research output: Contribution to journalArticle

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Abstract

Objective. The purpose of this study was to test the hypothesis that the expression of the mutant p27Kip1 protein enhances cell growth inhibition and is more stable than that of the wild-type p27Kip1. Methods. Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27Kip1. To study the effects of the p27Kip1 mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27Kip1, or mutant p27Kip1 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 p27Kip1 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 p27Kip1 protein has a half-life of approximately 2 h while the p27Kip1 mutant has a half-life of greater than 12 h. Furthermore, the p27Kip1 mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27Kip1 mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27Kip1 (P = 0.001). Colony assays revealed that cells expressing the p27Kip1 mutant had fewer colonies compared to cells expressing the wild-type p27Kip1 (P = 0.04). Conclusions. These data are consistent with the hypothesis that the mutated form of p27Kip1 is more effective in cell growth inhibition than the wild-type p27Kip1 protein.

Original languageEnglish
Pages (from-to)19-23
Number of pages5
JournalGynecologic Oncology
Volume86
Issue number1
DOIs
StatePublished - 2002

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Cyclin-Dependent Kinases
Growth
Cyclin-Dependent Kinase Inhibitor p27
Luciferases
Half-Life
Renilla Luciferases
Mutant Proteins
Threonine
Site-Directed Mutagenesis
Alanine
Phosphotransferases
Students

ASJC Scopus subject areas

  • Obstetrics and Gynecology
  • Oncology

Cite this

Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27Kip1 inhibits cell growth. / Hurteau, Jean A.; Brutkiewicz, Susan A.; Wang, Qi; Allison, Bernadette M.; Goebl, Mark; Harrington, Maureen.

In: Gynecologic Oncology, Vol. 86, No. 1, 2002, p. 19-23.

Research output: Contribution to journalArticle

Hurteau, Jean A. ; Brutkiewicz, Susan A. ; Wang, Qi ; Allison, Bernadette M. ; Goebl, Mark ; Harrington, Maureen. / Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27Kip1 inhibits cell growth. In: Gynecologic Oncology. 2002 ; Vol. 86, No. 1. pp. 19-23.
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AU - Goebl, Mark

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N2 - Objective. The purpose of this study was to test the hypothesis that the expression of the mutant p27Kip1 protein enhances cell growth inhibition and is more stable than that of the wild-type p27Kip1. Methods. Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27Kip1. To study the effects of the p27Kip1 mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27Kip1, or mutant p27Kip1 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 p27Kip1 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 p27Kip1 protein has a half-life of approximately 2 h while the p27Kip1 mutant has a half-life of greater than 12 h. Furthermore, the p27Kip1 mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27Kip1 mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27Kip1 (P = 0.001). Colony assays revealed that cells expressing the p27Kip1 mutant had fewer colonies compared to cells expressing the wild-type p27Kip1 (P = 0.04). Conclusions. These data are consistent with the hypothesis that the mutated form of p27Kip1 is more effective in cell growth inhibition than the wild-type p27Kip1 protein.

AB - Objective. The purpose of this study was to test the hypothesis that the expression of the mutant p27Kip1 protein enhances cell growth inhibition and is more stable than that of the wild-type p27Kip1. Methods. Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27Kip1. To study the effects of the p27Kip1 mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27Kip1, or mutant p27Kip1 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 p27Kip1 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 p27Kip1 protein has a half-life of approximately 2 h while the p27Kip1 mutant has a half-life of greater than 12 h. Furthermore, the p27Kip1 mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27Kip1 mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27Kip1 (P = 0.001). Colony assays revealed that cells expressing the p27Kip1 mutant had fewer colonies compared to cells expressing the wild-type p27Kip1 (P = 0.04). Conclusions. These data are consistent with the hypothesis that the mutated form of p27Kip1 is more effective in cell growth inhibition than the wild-type p27Kip1 protein.

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