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Development and Characterization of a WEHI-3B D⁺ Monomyelocytic Leukemia Cell Line Resistant to Novobiocin and Cross-Resistant to Other Topoisomerase II-targeted Drugs¹

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 [G. R., A. L., A. C. S.], and Experimental Oncology 1, Centro Regionale di Riferimento Oncologico, Aviano, Italy [G. R., A. L.]

A novobiocin-resistant subline of WEHI-3B D⁺ murine monomyelocytic leukemia cells was developed by the continuous exposure of cells to this agent in vitro. Sensitive (WEHI-3B/S) and novobiocin-resistant (WEHI-3B/NOVO) sublines were cloned in vitro. WEHI-3B/NOVO cells were stable in the absence of novobiocin for more than 3 months, and the sensitive and resistant clones displayed the same growth rate, cell cycle distribution, cell size, DNA and protein content, and cloning efficiency. Novobiocin has been shown to compete with ATP for the ATP-binding site of topoisomerase II; therefore, intracellular ATP levels can influence the cellular sensitivity to novobiocin. High-performance liquid chromatographic analysis of total cell extracts demonstrated that no difference exists between WEHI-3B/S and WEHI-3B/NOVO cells in the content of ATP. Furthermore, exposure of both cell lines to novobiocin did not affect intracellular ATP levels. In addition to an approximately 2-fold level of resistance to novobiocin, the WEHI-3B/NOVO subline was also 7- and 11-fold cross-resistant to the topoisomerase II-targeted drugs, teniposide and etoposide (VP-16), respectively. A lower level of cross-resistance, comparable to that of novobiocin, was observed in WEHI-3B/NOVO cells for the intercalating topoisomerase II-reactive drugs, doxorubicin, 4'-(9-acridinylamino)methanesulfon-m-anisidide and aclacinomycin A, while the sensitivity to the cytotoxic action of the non-topoisomerase II-acting agents, camptothecin and vincristine, was not altered. After 3–6 h of exposure to 1 µM VP-16, WEHI-3B/S cells accumulated in the S and G₂ + M phases of the cell cycle. Similar changes were detected in WEHI-3B/NOVO cells only after exposure to a 10-fold higher concentration of VP-16. Exposure to 150 µM novobiocin caused an accumulation of WEHI-3B/S cells in the G₀–G₁ phase of the cell cycle but did not affect the cell cycle distribution of WEHI-3B/NOVO cells, while camptothecin induced the same type and extent of changes in the cell cycle distribution of both cell lines. Although the WEHI-3B/NOVO subline appeared to be less responsive to the differentiation-inducing activity of novobiocin and teniposide, the capacity of WEHI-3B/NOVO cells to respond to the differentiation-inducing agent 13-cis-retinoic acid was not significantly different from that of WEHI-3B/S cells. A slight decrease in the accumulation of VP-16 occurred in the resistant cell line, which did not appear to be of sufficient magnitude to account for the 11-fold increase in the degree of resistance to this agent.

Topoisomerase II activity in 0.35 and 1 M NaCl nuclear extracts of WEHI-3B/NOVO cells was equivalent to that of WEHI-3B/S cells. The capacity of VP-16 to inhibit the P₄-unknotting activity of topoisomerase II and the ATP requirement of the enzyme were essentially equivalent in nuclear extracts prepared from WEHI-3B/S and WEHI-3B/NOVO cells. WEHI-3B/NOVO cells exhibit a pronounced degree of resistance to VP-16-induced DNA-topoisomerase II cross-links, as measured by the potassium-sodium dodecyl sulfate precipitation assay. The reduction in DNA-topoisomerase II cross-links occurred mainly in newly synthesized DNA, as measured by the labeling of nascent DNA with [³H]-thymidine. These findings suggest that a decreased capacity of topoisomerase II to interact with chromatin DNA is responsible for the resistant phenotype of WEHI-3B/NOVO cells.

¹ This research was supported in part by U.S.P.H.S. Grants CA-02817, CA-08341, and CA-16359 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

Received 7/24/91. Accepted 3/10/92.