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Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells

Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybr...

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Autores principales: Kim, Jung-Hyun, Lee, Hee-Sheung, Lee, Nicholas C. O., Goncharov, Nikolay V., Kumeiko, Vadim, Masumoto, Hiroshi, Earnshaw, William C., Kouprina, Natalay, Larionov, Vladimir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924756/
https://www.ncbi.nlm.nih.gov/pubmed/26943579
http://dx.doi.org/10.18632/oncotarget.7854
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author Kim, Jung-Hyun
Lee, Hee-Sheung
Lee, Nicholas C. O.
Goncharov, Nikolay V.
Kumeiko, Vadim
Masumoto, Hiroshi
Earnshaw, William C.
Kouprina, Natalay
Larionov, Vladimir
author_facet Kim, Jung-Hyun
Lee, Hee-Sheung
Lee, Nicholas C. O.
Goncharov, Nikolay V.
Kumeiko, Vadim
Masumoto, Hiroshi
Earnshaw, William C.
Kouprina, Natalay
Larionov, Vladimir
author_sort Kim, Jung-Hyun
collection PubMed
description Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (“loss of signal” assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this “loss of signal” assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this “gain of signal” assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The “gain of signal” assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level.
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spelling pubmed-49247562016-07-13 Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells Kim, Jung-Hyun Lee, Hee-Sheung Lee, Nicholas C. O. Goncharov, Nikolay V. Kumeiko, Vadim Masumoto, Hiroshi Earnshaw, William C. Kouprina, Natalay Larionov, Vladimir Oncotarget Research Paper Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (“loss of signal” assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this “loss of signal” assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this “gain of signal” assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The “gain of signal” assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level. Impact Journals LLC 2016-03-02 /pmc/articles/PMC4924756/ /pubmed/26943579 http://dx.doi.org/10.18632/oncotarget.7854 Text en Copyright: © 2016 Kim et al. http://creativecommons.org/licenses/by/2.5/ 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
Kim, Jung-Hyun
Lee, Hee-Sheung
Lee, Nicholas C. O.
Goncharov, Nikolay V.
Kumeiko, Vadim
Masumoto, Hiroshi
Earnshaw, William C.
Kouprina, Natalay
Larionov, Vladimir
Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title_full Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title_fullStr Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title_full_unstemmed Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title_short Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells
title_sort development of a novel hac-based “gain of signal” quantitative assay for measuring chromosome instability (cin) in cancer cells
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924756/
https://www.ncbi.nlm.nih.gov/pubmed/26943579
http://dx.doi.org/10.18632/oncotarget.7854
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