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Topoisomerase II Inhibition and Altered Kinetics of Formation and Repair of Nitrosourea and Cisplatin-induced DNA Interstrand Cross-Links and Cytotoxicity in Human Glioblastoma Cells¹

Section of Molecular Therapeutics, Department of Experimental Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [F. A-O., S. R.], Departments of Neurological Surgery, [F. A.-O., S. R., M. S. B.] and Neurology [A. S.], and The Cancer Center [R. B. L.], University of Washington, Seattle, Washington 96051

² To whom requests for reprints should be addressed, at The University of Texas, M.D. Anderson Cancer Center, Department of Experimental Pediatrics, 1515 Holcombe Boulevard, Houston, TX 77030.

By altering the accessibility of DNA sequences for alkylation or platination, and/or for subsequent repair, topoisomerase II can potentially affect the level of DNA interstrand cross-links induced in cells by Afunctional agents. In this study, we investigated the extent to which inhibition of topoisomerase II activity in a human glioblastoma multiforme cell line alters the kinetics of both the formation and the repair of total genomic DNA interstrand cross-links, as well as the sensitivity of the tumor cells to cis-diamminedichloroplatinum II (cis-DDP) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Cells were incubated with and without 200 µM novobiocin, a known topoisomerase II inhibitor, for 24 h, followed by exposure to 50 µM BCNU and 25 µM cis-DDP. DNA interstrand cross-linking was determined at various time points over 72 h, using a modified ethidium bromide-DNA binding assay. Sensitivity of the cells to cis-DDP and BCNU was also determined with and without novobiocin pretreatment with 200 µM novobiocin. This concentration of novobiocin showed no significant direct cytoxicity, although it inhibited topoisomerase II activity in tumor cell nuclear extracts by 73%. A significant decrease in the rate of repair of both cis-DDP and BCNU induced DNA interstrand cross-links, with a corresponding decrease in the clonogenic survival of the cells, was observed following novobiocin exposure. Although the peak cross-link indices of novobiocin-treated cells relative to controls were not significantly increased, residual DNA cross-linking in the cells after 72 h was increased by 1.4-fold for BCNU and 3-fold for cells treated with cis-DDP, thus, indicating a greater effect of topoisomerase II on cross-link repair than on cross-link formation. These data suggest that inhibition of topoisomerase II may provide a potentially effective clinical strategy for sensitizing human brain tumors, and possibly other tumors as well, to DNA cross-linking anticancer agents.

¹ This research was supported by Grants CA 46410 and CA 555835 from the National Cancer Institute, NIH, and Grant 100-R104 from the Bristol-Myers Squibb Company.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/26/93. Accepted 9/22/93.

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