Increased Rate of Adenosine Triphosphate-dependent Etoposide (VP-16) Efflux in a Murine Leukemia Cell Line Overexpressing the Multidrug Resistance-associated Protein (MRP) Gene1

WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resistance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoisomerase II (topo ID-reactive drugs. We have reported previously (Cancer Res. 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounced de crease in VP-16 induced DNA-topo II cross-link formation compared to the parental WEHI-3B/S cell line in intact cells, in the absence of a significant difference in the IM unknotting activity of topo II assayed in nuclear extracts. Because the pattern of cross-resistance was suggestive of a topo H-mediated mechanism, we have ascertained whether a change in topo II can account for the multidrug-resistant phenotype of WEHI-3B/ NOVO cells. No differences existed between WEHI-3B/S and WEHI-3B/ NOVO cells in topo II inKN A and protein levels, as well as in the amount of topo II associated with the nuclear matrix. Neither sensitive nor resistant cells expressed detectable levels of the MDR1 gene; however, VP-16 accumulation in WEHI-3B/NOVO cells was 3-4-fold less than that present in WEHI-3B/S cells, whereas doxorubicin accumulation was the same in both cell lines. Over the first 60 s, no difference existed in the rate of uptake of VP-16 between parental and resistant cells; however, beyond the first 60 s of incubation, | 'll|\ l'-ld accumulated to a greater extent in parental sensitive cells. Thus, an increased rate of efflux of VP-16 was responsible for the lower steady-state concentration of the drug in resistant cells. The efflux Km for VP-16 in WEHI-3B/NOVO cells was 254.7 MMand the Vmax was 10.4 pmol/s/107 cells. In the presence of the inhibitors of energy metabolism, sodium azide and deoxyglucose, the efflux of VP-16 was markedly inhibited; readdition of glucose restored the original efflux rate. Northern blot analyses using the human 10.1 probe for the 3'-terminal region of the multidrug-resistance protein (MRP) cDNA revealed a mKNA species of approximately 6 kb in WEHI-3B/NOVO cells but not in WEHI-3B/S cells. Overexpression was associated with amplification of the cognate gene. To ascertain whether the overexpressed gene in WEHI-3B/ NOVO cells was the murine MRP or a different member of the same superfamily of ATP-binding ABC cassette transporters, a 341-bp MRP cDNA probe was generated from a murine genomic library. The murine probe spanned a region corresponding to nucleotides 4367-4708 of the human cDNA sequence, with which it shared 86% nucleotide sequence homology. Using this probe, Northern blot analyses demonstrated that WEHI-3B/NOVO cells overexpressed the MRP gene relative to WEHI3B/S cells. It has been shown clearly by others that transfection of the human MRP cDNA is sufficient to induce VP-16 resistance in several tumor cell lines. Thus, although the characteristics of the efflux mecha nism in WEHI-3B/NOVO cells differ from that reported for the MRP, it is conceivable that Overexpression of the murine MRP gene in WEHI-3B/ NOVO cells is responsible for the increased rate of VP-16 efflux that Received 8/16/94; accepted 8/3/95. 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 indicale this fact. 1This research was supported in part by United States Public Health Service Grant CA-44597 (D. J. F.). C. V. C. is the recipient of a Berti Fellowship from Champion Industries, Winston-Salem, NC. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. results in decreased accumulation of drug, decreased formation of poten tially lethal topo II-DNA covalent complexes and, ultimately, reduced cytotoxicity.