OVEREXPRESSION OF THE MULTIDRUG-RESISTANCE GENES MDR1, MDR3, AND MRP IN L1210 LEUKEMIA-CELLS RESISTANT TO INHIBITORS OF RIBONUCLEOTIDE REDUCTASE

L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazo ne class of inhibitors of ribonucleotide reductase. The line is cross- resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cell s expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thio semicarbazone (3-AP), a relatively newly developed inhibitor of ribonu cleotide reductase. The accumulation of [C-14]3-AP by L1210 MQ-580 cel ls was 5- to 6-fold less than by parental L1210 cells. An increased ra te of efflux of 3-AP was responsible for the lower steady-state concen tration of 3-AP in resistant cells. In reverse transcription-polymeras e chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 g ene, compared with parental L1210 cells. Measurement of the steady-sta te concentration of doxorubicin, a potential substrate for both the md r and mrp gene products, demonstrated that L1210 MQ-580 cells accumula ted 4-fold less anthracycline than parental cells. These findings indi cate that drug efflux is a major determinant of the pattern of cross-r esistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effe cts of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfect ants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [C-14]3-AP than their parental mock-transfected count erparts. Moreover, the cytotoxic activity of 3-AP was significantly gr eater in two double mrp gene knockout cell lines than in parental W 9. 5 embryonic stem cells. Thus, the results suggest that 3-AP is a subst rate for both the P-glycoprotein and MRP and that baseline MRP express ion has the capacity to exert a protective role against the toxicity o f this agent. (C) 1997 Elsevier Science Inc.