SHORT REPORT The pH sensitive probe 5-(and-6)-carboxyl seminaphthorhodafluor is a substrate for the multidrug resistance-related protein MRP1 Jing Jin and Arwyn T. Jones * Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, United Kingdom 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 expo- sure 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 accu- mulated 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. ' 2008 Wiley-Liss, Inc. Key words: leukemia; multidrug resistance; cytosolic pH; SNARF-1 Regulation of intracellular pH is fundamental for cellular func- tion, and numerous inherent cellular mechanisms maintain pH ho- meostasis in both the cytosol and organelles, such as the nucleus, Golgi and components on the endolysosomal system. There is evi- dence to suggest that cancer cells have abnormal pH values, most notably in the cytosol (pHi) and acidic endocytic compartments. 1 Abnormalities in pHi together with low pH in the extracellular environment of solid tumors may therefore have significant effects on the ionic status, membrane permeability, intracellular localiza- tion and efficacy of anticancer drugs; particularly, those that are weak bases, such as cisplatin and doxorubicin (Dox). 2,3 Anticancer therapy is also compromised when cells upregulate expression of drug efflux pumps, such as members of the ABC transporter family, P-glycoprotein (P-gp) and MRP1. 4 Cells that have been challenged with, and developed resistance to, 1 class of drug often have resistance to other structurally unrelated drugs and are termed multidrug resistant (MDR). Their occurrence in patients is a major factor in the failure of chemotherapeutic agents to achieve complete remission. MDR cells have been shown to have elevated pHi compared to their parental counterparts and this is accompanied by lower pH in lysosomal compartments. 5–7 Over- all this leads to sequestration of drugs, such as cisplatin and Dox, in acidic vesicles, such as lysosomes, thus reducing the amount that gains access to the DNA target in the nucleus. This process could therefore contribute to the MDR phenotype but the impor- tance of this compared to active efflux remains to be elucidated. Two independent studies showed elevated pHi and accompany- ing low-lysosomal pH in vincristine and Dox-resistant leukemic HL-60 cells. 6,7 Although there was agreement between the 2 stud- ies with respects to the pH effects there were significant differen- ces in the actual pHi values (pH 7.26 vs. pH 6.96) of the parental HL-60 cells. This difference could be attributed to differences between the cultures or their use of different methods for quantifi- cation as numerous methods have now been described for meas- uring pHi in single cells, cell populations and in vivo. Several methods utilize membrane permeable fluorophores such as 2 0 7 0 - bis(2-carboxyethyl)-5(6) carboxyfluorescein-acetoxymethylester (BCECF-AM) and 5-(and-6)-carboxyl seminaphthorhodafluor- acetoxymethylester (SNARF-1 AM). Conjugation of the fluores- cent moiety to the acetomethoxyester group enhances their hydro- phobicity and membrane permeability and once inside the cell, nonspecific esterases release the free acid that is then retained and measured. The use of acetoxymethylester derivatives of BCECF and calcein dyes as pH and calcium indicators, respectively, is however, hampered by the fact that some are substrates for P-gp, MRP1 or breast cancer related protein (BCRP). 8–10 Using an MRP1 overexpressing MDR HL-60 cell line, 11 newly generated HL-60 MDR lines expressing MRP1 or P-gp and com- mercial leukemia cell lines, we show that MRP1 but not P-gp over- expression inhibits cellular accumulation of SNARF-1 and that this process is reversed by the MRP1 specific inhibitor MK571. Material and methods General reagents MK571 was from Cayman Europe (Tallinn, Estonia), SNARF-1 AM (Cat No. C1271) was from Invitrogen (Paisley, UK). Doxoru- bicin (Dox), daunorubicin (Dau), verapamil (VPL) 3-[4,5-dime- thylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and antibodies against P-gp (F4), MRP1 (QCRL1), MRP3 and a-tubu- lin were from Sigma-Aldrich (Gillingham, UK). Complete mini- protease inhibitor cocktail was from Roche (Welwyn Garden City, UK). Horseradish peroxidase (HRP) conjugated anti-mouse and - rabbit antibodies (Pierce, Rockford, IL) were detected on nitro- cellulose papers by enhanced chemiluminescence (ECL). Cell culture Human promyelocytic HL-60 and Dox-selected HL-60/ADR cells were a gift from Dr. Margery Barrand, University of Cam- bridge. 11 In-house Dox and Dau resistant sublines HL-60/Dox200 and HL-60/Dau100 were derived from the parental line by initial exposure to 5 ng/ml Dox or Dau followed by 2-fold stepwise increases to final concentrations of 200 ng/ml Dox or 100 ng/ml Dau over a 6-month period. Human KG1a cells were obtained Abbreviations: BCEFC-AM, 2 0 7 0 -bis(2-carboxyethyl)-5(6) carboxy- fluorescein-acetoxymethylester; Dau, Daunorubicin; Dox, Doxorubicin; MRP1, multidrug-resistance related protein 1; P-gp, P-glycoprotein; SNARF-1 AM, 5-(and-6)-carboxyl seminaphthorhodafluor-acetomethyles- ter; SNARF-1, 5-(and-6)-carboxyl seminaphthorhodafluor; VPL, Verapa- mil. Jing Jin’s current address is: Department of Food Biosciences, University of Reading, Reading, RG6 6AP, United Kingdom. Grant sponsor: Leukemia Research Fund Grant; Grant number: 04075. *Correspondence to: Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff, CF10 3XF, Wales, United Kingdom. Fax: 144-2920874536. E-mail: jonesat@cardiff.ac.uk Received 21 February 2008; Accepted after revision 9 July 2008 DOI 10.1002/ijc.23892 Published online 15 October 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 124, 233–238 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer