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Cellular Consequences of Overproduction of DNA Topoisomerase II in an Ataxia-Telangiectasia Cell Line

Cellular Consequences of Overproduction of DNA Topoisomerase II in an Ataxia-Telangiectasia Cell... Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs e.g. , the DNA intercalator amsacrine (mAMSA). To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5–10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions), Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Research American Association of Cancer Research

Cellular Consequences of Overproduction of DNA Topoisomerase II in an Ataxia-Telangiectasia Cell Line

Cancer Research , Volume 49 (5): 1118 – Mar 1, 1989

Cellular Consequences of Overproduction of DNA Topoisomerase II in an Ataxia-Telangiectasia Cell Line

Cancer Research , Volume 49 (5): 1118 – Mar 1, 1989

Abstract

Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs e.g. , the DNA intercalator amsacrine (mAMSA). To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5–10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions), Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells.

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Publisher
American Association of Cancer Research
Copyright
Copyright © 1989 by the American Association for Cancer Research.
ISSN
0008-5472
Publisher site

Abstract

Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs e.g. , the DNA intercalator amsacrine (mAMSA). To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5–10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions), Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells.

Journal

Cancer ResearchAmerican Association of Cancer Research

Published: Mar 1, 1989

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