INCREASED RATE OF ADENOSINE TRIPHOSPHATE-DEPENDENT ETOPOSIDE (VP-16) EFFLUX IN A MURINE LEUKEMIA-CELL LINE OVEREXPRESSING THE MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) GENE

WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resist ance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoiso merase II (topo II)-reactive drugs. We have reported previously (Cance r Res, 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounc ed decrease 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 o f a significant difference in the P4 unknotting activity of topo II as sayed in nuclear extracts. Because the pattern of cross-resistance was suggestive of a topo II-mediated mechanism, we have ascertained wheth er a change in topo II can account for the multidrug-resistant phenoty pe of WEHI-3B/NOVO cells. No differences existed between WEHI-3B/S and WEHI-3B/NOVO cells in topo II mRNA and protein levels, as well as in the amount of topo II associated with the nuclear matrix. Neither sens itive 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 e xisted in the rate of uptake of VP-16 between parental and resistant c ells; however, beyond the first 60 s of incubation, [H-3]VP-16 accumul ated to a greater extent in parental sensitive cells. Thus, an increas ed rate of efflux of VP-16 was responsible for the lower steady-state concentration of the drug in resistant cells. The efflux K-m for VP-16 in WEHI-3B/NOVO cells was 254.7 mu M and the V-max was 10.4 pmol/s/10 (7) cells. In the presence of the inhibitors of energy metabolism, sod ium azide and deoxyglucose, the efflux of VP-16 was markedly inhibited ; readdition of glucose restored the original efflux rate. Northern bl ot analyses using the human 10.1 probe for the 3'-terminal region of t he multidrug-resistance protein (MRP) cDNA revealed a mRNA species of approximately 6 kb in WEHI-3B/NOVO cells but not in WEHI-3B/S cells. O verexpression was associated with amplification of the cognate gene. T o ascertain whether the overexpressed gene in WEHI-3B/NOVO cells was t he murine MRP or a different member of the same superfamily of ATP-bin ding ABC cassette transporters, a 341-bp MRP cDNA probe was generated from a murine genomic library. The murine probe spanned a region corre sponding to nucleotides 4367-3708 of the human cDNA sequence, with whi ch it shared 86% nucleotide sequence homology, Using this probe, North ern blot analyses demonstrated that WEHI-3B/NOVO cells overexpressed t he MRP gene relative to WEHI-3B/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 char acteristics of the efflux mechanism in WEHI-3B/NOVO cells differ from that reported for the MRP, it is conceivable that overexpression of th e murine MRP gene in WEHI-3B/NOVO cells is responsible for the increas ed rate of VP-16 efflux that results in decreased accumulation of drug , decreased formation of potentially lethal topo II-DNA covalent compl exes and, ultimately, reduced cytotoxicity.