A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress

I. Chen Li, Chien Yuan Chiu, Chang Lin Wu, Jhih Ying Chi, Siao Ru Jian, Shainn-Wei Wang, Christina L. Chang

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H 2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2- generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

Original languageEnglish
Pages (from-to)86-95
Number of pages10
JournalFree Radical Biology and Medicine
Volume69
DOIs
Publication statusPublished - 2014 Apr 1

Fingerprint

Microsatellite Instability
Oxidative stress
Sulfhydryl Compounds
Microsatellite Repeats
Oxidative Stress
DNA Mismatch Repair
Frameshift Mutation
Acetylcysteine
Repair
Inflammation
Mutation
Assays
Glutathione
Colorectal Neoplasms
Neoplasms
Flow Cytometry
Cell Line
Polymerase Chain Reaction
Flow cytometry
Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Physiology (medical)

Cite this

Li, I. Chen ; Chiu, Chien Yuan ; Wu, Chang Lin ; Chi, Jhih Ying ; Jian, Siao Ru ; Wang, Shainn-Wei ; Chang, Christina L. / A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress. In: Free Radical Biology and Medicine. 2014 ; Vol. 69. pp. 86-95.
@article{81976b01b0fd46ebb27657d941fb4269,
title = "A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress",
abstract = "The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H 2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2- generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.",
author = "Li, {I. Chen} and Chiu, {Chien Yuan} and Wu, {Chang Lin} and Chi, {Jhih Ying} and Jian, {Siao Ru} and Shainn-Wei Wang and Chang, {Christina L.}",
year = "2014",
month = "4",
day = "1",
doi = "10.1016/j.freeradbiomed.2013.12.019",
language = "English",
volume = "69",
pages = "86--95",
journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
publisher = "Elsevier Inc.",

}

A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress. / Li, I. Chen; Chiu, Chien Yuan; Wu, Chang Lin; Chi, Jhih Ying; Jian, Siao Ru; Wang, Shainn-Wei; Chang, Christina L.

In: Free Radical Biology and Medicine, Vol. 69, 01.04.2014, p. 86-95.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A dual-fluorescent reporter facilitates identification of thiol compounds that suppress microsatellite instability induced by oxidative stress

AU - Li, I. Chen

AU - Chiu, Chien Yuan

AU - Wu, Chang Lin

AU - Chi, Jhih Ying

AU - Jian, Siao Ru

AU - Wang, Shainn-Wei

AU - Chang, Christina L.

PY - 2014/4/1

Y1 - 2014/4/1

N2 - The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H 2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2- generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

AB - The DNA mismatch-repair (MMR) system corrects replicative errors and minimizes mutations that occur at a high rate in microsatellites. Patients with chronic inflammation or inflammation-associated cancer display microsatellite instability (MSI), indicating a possible MMR inactivation. In fact, H 2O2-generated oxidative stress inactivates the MMR function and increases mutation accumulation in a reporter microsatellite. However, it remains unclear whether MSI induced by oxidative stress is preventable because of the lack of a sufficiently sensitive detection assay. Here, we developed and characterized a dual-fluorescent system, utilizing DsRed harboring the (CA)13 microsatellite as a reporter and GFP for normalization, in near-isogenic human colorectal cancer cell lines. Via flow cytometry, this reporter sensitively detected H2O2- generated oxidative microsatellite mutations in a dose-dependent manner. The reporter further revealed that glutathione or N-acetylcysteine was better than aspirin and ascorbic acid for suppressing oxidative microsatellite mutations. These two thiol compounds also partially suppressed oxidative frameshift mutations in the coding microsatellites of the hMSH6 and CHK1 genes based on a fluoresceinated PCR-based assay. MSI suppression by N-acetylcysteine appears to be mediated through reduction of oxidative frameshift mutations in the coding microsatellite of hMSH6 and protection of hMSH6 and other MMR protein levels from being decreased by H2O2. Our findings suggest a linkage between oxidative damage, MMR deficiency, and MSI. The two thiol compounds are potentially valuable for preventing inflammation-associated MSI. The dual-fluorescent reporter with improved features will facilitate identification of additional compounds that modulate MSI, which is relevant to cancer initiation and progression.

UR - http://www.scopus.com/inward/record.url?scp=84893627815&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84893627815&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2013.12.019

DO - 10.1016/j.freeradbiomed.2013.12.019

M3 - Article

VL - 69

SP - 86

EP - 95

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

ER -