Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors
| Autor: | Chung Min Lee, Gyong Sik Ha, Chan Wha Kim |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Biochemistry & Molecular Biology Cdk1 Phosphatase Cell Cycle Proteins nonradioisotopic assay HeLa Histones 03 medical and health sciences 0302 clinical medicine CDC2 Protein Kinase Humans cdc25 Phosphatases Cyclin-dependent kinase 1 biology Chemistry Kinase Cell Cycle Retinoblastoma protein General Medicine Cell cycle biology.organism_classification Cdc25B overexpression cell lines anticancer drugs 030104 developmental biology Biochemistry Cell culture 030220 oncology & carcinogenesis biology.protein Original Article Cdc25B Intracellular HeLa Cells |
| Zdroj: | Yonsei Medical Journal |
| ISSN: | 1976-2437 0513-5796 |
| Popis: | Purpose The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which 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 histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using 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 system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods 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 conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs. |
| Databáze: | OpenAIRE |
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