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Genetic Characterization of the Multidrug-resistant Phenotype of VM-26-resistant Human Leukemic Cells¹

Department of Biochemial and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycolmediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC₅₀ for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10^-8 M, and another was 76 x 10^-8 M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC₅₀ values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10^-8 M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using ³²P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 µM VM-26 maximally stimulated DNA cleavage by 11-fold compared to no-drug controls. By contrast, 100 µM VM-26 stimulated DNA cleavage by only 7-fold in extracts from CEM/VM-1 cells. Drug stimulation of cleavage obtained with extracts from a hybrid of VM-26-sensitive and -resistant cells that expressed the drug-sensitive phenotype were also similar to those of the sensitive cells: VM-26 stimulated activity by 11-fold, indicating that the fusion process reconstituted a topoisomerase II-mediated function. We have previously shown that although topoisomerase II activity was decreased in at-MDR cells compared to controls, immunodetection of topoisomerase II in a Western blot assay revealed no differences in enzyme amount in 1.0 M NaCl nuclear extracts from these lines. Similarly, we found here that the hybrids also had about the same amount of immunoreactive topoisomerase II as in the unfused cells. Our data demonstrate that we have reconstituted topoisomerase II activity by the cell fusion protocol and are the first to show that VM-26 resistance in human tumor cells is expressed recessively.

¹ This work was supported in part by Research Grant CA-30103, Cancer Center Support (CORE) Grant CA-21765, both from the National Cancer Institute, NIH, Bethesda, MD; Biomedical Research Grant RR-05584, from NIH; and American Lebanese Syrian Associated Charities (ALSAC).

² To whom requests for reprints should be addressed, at Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, TN 38101.

Received 12/ 1/88. Accepted 2/ 7/89.

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