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 thiosemicarbazone class of inhibitors of ribonucleotide reductase. The line is cross-resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase. The accumulation of [14C]3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells. An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells. In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells. Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells. These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [14C]3-AP than their parental mock-transfected counterparts. Moreover, the cytotoxic activity of 3-AP was significantly greater 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 substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.