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Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor...

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Autores principales: Hauge, Sissel, Naucke, Christian, Hasvold, Grete, Joel, Mrinal, Rødland, Gro Elise, Juzenas, Petras, Stokke, Trond, Syljuåsen, Randi G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355238/
https://www.ncbi.nlm.nih.gov/pubmed/28030798
http://dx.doi.org/10.18632/oncotarget.14089
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author Hauge, Sissel
Naucke, Christian
Hasvold, Grete
Joel, Mrinal
Rødland, Gro Elise
Juzenas, Petras
Stokke, Trond
Syljuåsen, Randi G.
author_facet Hauge, Sissel
Naucke, Christian
Hasvold, Grete
Joel, Mrinal
Rødland, Gro Elise
Juzenas, Petras
Stokke, Trond
Syljuåsen, Randi G.
author_sort Hauge, Sissel
collection PubMed
description Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.
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spelling pubmed-53552382017-04-26 Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading Hauge, Sissel Naucke, Christian Hasvold, Grete Joel, Mrinal Rødland, Gro Elise Juzenas, Petras Stokke, Trond Syljuåsen, Randi G. Oncotarget Research Paper Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects. Impact Journals LLC 2016-12-22 /pmc/articles/PMC5355238/ /pubmed/28030798 http://dx.doi.org/10.18632/oncotarget.14089 Text en Copyright: © 2017 Hauge et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Paper
Hauge, Sissel
Naucke, Christian
Hasvold, Grete
Joel, Mrinal
Rødland, Gro Elise
Juzenas, Petras
Stokke, Trond
Syljuåsen, Randi G.
Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title_full Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title_fullStr Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title_full_unstemmed Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title_short Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading
title_sort combined inhibition of wee1 and chk1 gives synergistic dna damage in s-phase due to distinct regulation of cdk activity and cdc45 loading
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355238/
https://www.ncbi.nlm.nih.gov/pubmed/28030798
http://dx.doi.org/10.18632/oncotarget.14089
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