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

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 IIinhibitor drugs {e.g., the DNA intercalator amsacrine (inAMS \)|. To study such relationships, various SV40and Epstein-Barr Virus-trans formed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to m \ MS A together with direct and indirect measurements of topoisom erase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisom erase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSAdependent 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 proteindenaturing conditions). Xeroderma pigmentosum (group A) cells demon strated normal responses to in VMSA. The results provide evidence that cellular potential for the generation of topoisomerase H-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.