Development of a novel nonradioisotopic assay and Cdc25B overexpression cell lines for use in screening for Cdc25B inhibitors

Gyong Sik Ha, Chung Min Lee, Chan Wha Kim

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)

    Abstract

    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 his-tone 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.

    Original languageEnglish
    Pages (from-to)995-1003
    Number of pages9
    JournalYonsei medical journal
    Volume59
    Issue number8
    DOIs
    Publication statusPublished - 2018 Oct

    Bibliographical note

    Publisher Copyright:
    © Yonsei University College of Medicine 2018.

    Keywords

    • Anticancer drugs
    • Cdc25B
    • Cdc25B overexpression cell lines
    • Cdk1
    • Nonradioisotopic assay

    ASJC Scopus subject areas

    • General Medicine

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