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Teniposide-resistant CEM Cells, Which Express Mutant DNA Topoisomerase II, When Treated with Non-Complex-stabilizing Inhibitors of the Enzyme, Display No Cross-Resistance and Reveal Aberrant Functions of the Mutant Enzyme¹

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, and Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26). The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance. These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes. In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors. Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26. Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes. Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G₂ arrest, as determined by flow cytometry. Likewise, at-MDR cells continuously treated with VM-26 also arrested in G₂. By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G₂ but then escaped the G₂ block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide. Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage. Given that both wild-type and mutant topo II alleles are expressed in the at-MDR cells (B. Y. Bugg, M. K. Danks, W. T. Beck, and D. P. Suttle. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA, 88: 7654–7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.

¹ This work was supported in part by Grant CA-40570 and Cancer Center Support (CORE) Grant CA-21765 from the National Cancer Institute, DHHS, Bethesda, MD, and in part by American Lebanese Syrian Associated Charities.

² To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.

Received 8/ 2/93. Accepted 10/ 5/93.

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