The pH sensitive probe 5‐(and‐6)‐carboxyl seminaphthorhodafluor is a substrate for the multidrug resistance‐related protein MRP1

Cellular function is dependent on tight regulation of intracellular pH and numerous reports show cancer cells have abnormal pH values in the cytosol and organelles, such as lysosomes. 5-(and-6)-carboxyl seminaphthorhodafluor (SNARF-1) is a commonly used pH sensitive probe and was used here to determine cytosolic pH of HL-60 leukemia cells and a drug-resistant variant overexpressing multidrug-resistance related protein 1 (MRP1). Resistant cells accumulated significantly less SNARF-1 compared to parental cells but near control levels of probe accumulation were observed by preincubating cells with the specific MRP1 inhibitor MK571. Two new drug-resistant cell lines were generated following exposure to doxorubicin or daunorubicin and these upregulated MRP1 or P-glycoprotein expression, respectively. Experiments in these cells showed that reduced SNARF-1 accumulation was specific to MRP1 overexpression, as cells upregulating P-glycoprotein accumulated control levels of the probe. Confirmation that SNARF-1 is a MRP1 substrate was obtained using K562 and KG1a cells that have been shown to, respectively, constitutively express MRP1 and P-glycoprotein. Together, the data suggest that SNARF-1 is a substrate for MRP1 but not P-glycoprotein, and could therefore be used as a probe to distinguish between expression and activity of these 2 efflux proteins. Finally, we confirm that doxorubicin but not daunorubicin challenged MRP1 overexpressing HL-60 cells have elevated cytosolic pH.