Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors
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Purpose: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to Mphase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediatesthese dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequatenonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method withwhich to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. Materials and Methods: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histoneH1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructedusing the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. Results: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore,we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay systemwith which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. Thesemethods were confirmed as useful tools for measuring Cdc25B activity. Conclusion: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be convenientlyused as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.
Abstract
Purpose: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to Mphase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediatesthese dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequatenonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method withwhich to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. Materials and Methods: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histoneH1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructedusing the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. Results: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore,we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay systemwith which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. Thesemethods were confirmed as useful tools for measuring Cdc25B activity. Conclusion: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be convenientlyused as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.
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