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Characterization of Novel Human Leukemic Cell Lines Selected for Resistance to Merbarone, a Catalytic Inhibitor of DNA Topoisomerase II¹

Departments of Molecular Pharmacology [H. K., Q. E. R., W. T. B.] and Pathology and Laboratory Medicine [S. C. R.], St. Jude Children's Research Hospital, Memphis, Tennessee 38105; Departments of Pharmacology [W. T. B.] and Pathology [S. C. R.], College of Medicine, University of Tennessee, Memphis, Tennessee 38168; and Hauptlabor, Medizinische Poliklinik der Universität Würzburg, Würzburg, Germany [F. B.]

Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo II, increased topo IIß, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo II may play role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo II. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistance to topo II and topo I inhibitors.

¹ This work was supported in part by Research Grants CA-40570 (to W. T. B.) and CA-49721 (to S. C. R.), Program Grants CA-23099 (to W. T. B.) and CA-20180 (to S. C. R.), and Cancer Center Support CORE Grant CA-21765, all from the National Cancer Institute, Bethesda, MD; in part by Children's Cancer Research Fund (to S. C. R.); and in part by American Lebanese Syrian Associated Charities.

² Present address: Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan.

³ To whom requests for reprints should be addressed, at present address: Cancer Center (M/C 569), University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL, 60607-7173. Phone: (312) 355-0827; Fax: (312) 355-0194.

Received 2/12/96. Accepted 3/26/96.

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