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 journalArticle

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 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.

Original languageEnglish
Pages (from-to)995-1003
Number of pages9
JournalYonsei Medical Journal
Volume59
Issue number8
DOIs
Publication statusPublished - 2018 Oct 1

Fingerprint

CDC2 Protein Kinase
Cell Line
HeLa Cells
Phosphotransferases
cdc25 Phosphatases
Cyclin B
Retinoblastoma Protein
Tetracycline
Phosphoric Monoester Hydrolases
Isotopes
Cell Division
Histones
Cell Cycle
Western Blotting
Phosphates
Pharmaceutical Preparations

Keywords

  • anticancer drugs
  • Cdc25B
  • Cdc25B overexpression cell lines
  • Cdk1
  • nonradioisotopic assay

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors. / Ha, Gyong Sik; Lee, Chung Min; Kim, Chan Wha.

In: Yonsei Medical Journal, Vol. 59, No. 8, 01.10.2018, p. 995-1003.

Research output: Contribution to journalArticle

@article{abaa2db56557499cbd5d65d2e09ffc85,
title = "Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors",
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 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.",
keywords = "anticancer drugs, Cdc25B, Cdc25B overexpression cell lines, Cdk1, nonradioisotopic assay",
author = "Ha, {Gyong Sik} and Lee, {Chung Min} and Kim, {Chan Wha}",
year = "2018",
month = "10",
day = "1",
doi = "10.3349/ymj.2018.59.8.995",
language = "English",
volume = "59",
pages = "995--1003",
journal = "Yonsei Medical Journal",
issn = "0513-5796",
publisher = "Yonsei University College of Medicine",
number = "8",

}

TY - JOUR

T1 - Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors

AU - Ha, Gyong Sik

AU - Lee, Chung Min

AU - Kim, Chan Wha

PY - 2018/10/1

Y1 - 2018/10/1

N2 - 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.

AB - 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.

KW - anticancer drugs

KW - Cdc25B

KW - Cdc25B overexpression cell lines

KW - Cdk1

KW - nonradioisotopic assay

UR - http://www.scopus.com/inward/record.url?scp=85054049122&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054049122&partnerID=8YFLogxK

U2 - 10.3349/ymj.2018.59.8.995

DO - 10.3349/ymj.2018.59.8.995

M3 - Article

C2 - 30187708

AN - SCOPUS:85054049122

VL - 59

SP - 995

EP - 1003

JO - Yonsei Medical Journal

JF - Yonsei Medical Journal

SN - 0513-5796

IS - 8

ER -