[CANCER RESEARCH 64, 1242–1246, February 15, 2004] Advances in Brief Pheophorbide a Is a Specific Probe for ABCG2 Function and Inhibition Robert W. Robey, 1 Kenneth Steadman, 1 Orsolya Polgar, 1 Kuniaki Morisaki, 1 Margaret Blayney, 1 Prakash Mistry, 2 and Susan E. Bates 1 1 Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and 2 Xenova Research, Slough, Berkshire, United Kingdom Abstract Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2 / knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649 –15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumit- remorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 M of the cyclin-dependent kinase inhibitor UCN-01 or 1 M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2- mediated resistance to SN-38 and topotecan was abrogated in ABCG2- transfected HEK-293 cells treated with 1 M tariquidar, and ABCG2-transfected cells were 6 –7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 func- tion and expression in clinical samples. Introduction Overexpression of ATP-binding cassette (ABC) transporters in cancer cells is associated with the multidrug resistance phenotype (1). Among the 48 human ABC transporters, three have been found predominantly in cell lines selected in vitro for drug resistance: P-glycoprotein (Pgp), multidrug resistance-associated protein 1 (MRP1), and ABCG2 (1– 4). One of the most reproducible detection methods for Pgp and MRP1 has been the measurement of fluorescent dye efflux in flow cytometry-based functional assays. These func- tional assays have been useful particularly to quantitate expression and function of Pgp and MRP1 in leukemic samples (5). Functional assays for ABCG2 also have been developed for patient samples using the ABCG2 substrates mitoxantrone, BODIPY-prazosin, or topotecan (6 – 8). However, use of these compounds is limited because they also are substrates of Pgp (6). An ABCG2-specific fluorescent substrate would have direct clinical application in determining the contribution of ABCG2 to clinical drug resistance. In a study of mice lacking Abcg2, Jonker et al. (9) noted that mice fed a diet high in alfalfa and housed close to a light source were prone to phototoxic skin lesions. These lesions were believed to be caused by a chlorophyll catabolite, pheophorbide a (PhA), because plasma levels of PhA were 17-fold higher in Abcg2 -/- mice than in wild-type mice. Because Jonker et al. found PhA to be fluorescent, we wanted to determine whether this compound would prove to be an ABCG2-specific substrate. Addition- ally, we sought to develop a flow cytometric assay to determine expression and function of ABCG2 using PhA. Materials and Methods Chemicals. Cyclosporin A, verapamil, etoposide, and probenecid were purchased from Sigma Chemical (St. Louis, MO). PhA was obtained from Frontier Scientific (Logan, UT). Flavopiridol and UCN-01 were obtained from the National Cancer Institute Anticancer Drug Screen (Bethesda, MD). Fumi- tremorgin C (FTC) was isolated by Thomas McCloud, Developmental Ther- apeutics Program, Natural Products Extraction Laboratory, NIH (Bethesda, MD). Calcein AM was obtained from Molecular Probes (Eugene, OR). Tariq- uidar (XR9576) was a gift of Xenova Research (Slough, Berkshire, United Kingdom). Cell Lines. Selected cell lines overexpressing Pgp, MRP1, or ABCG2 were examined and are listed in Table 1. All of the cells were cultured in RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotic, except for MCF-7 cells and sublines, which were maintained in Improved Minimum Essential Medium (IMEM). We also examined HEK-293 cells that were transfected with empty pcDNA3 vector (Invitrogen, Carlsbad, CA) or pcDNA3 vector contain- ing full-length ABCG2 encoding arginine, threonine, or glycine at amino acid 482. ABCG2 transfectants were grown in Eagle’s Minimum Essential Medium (EMEM; American Type Culture Collection, Manassas, VA) supplemented with 10% FCS, glutamine, and antibiotic along with 2 mg/ml G418 (Invitro- gen). The transfected cells have been characterized previously (10). Immunoblot Analysis. Microsomal membrane protein (30 g) was loaded onto a premade 8% (w/v) SDS-polyacrylamide gel, subjected to electrophore- sis, and electrotransferred onto nitrocellulose membranes. Blots were probed sequentially with the monoclonal anti-MRP1 antibody MRPm2 (Kamiya Bio- medical, Seattle, WA), the anti-Pgp antibody C219 (Signet Laboratories, Dedham, MA), and the anti-ABCG2 antibody BXP-21 (Kamiya Biomedical). Flow Cytometry. The flow cytometry studies presented here are based on those described previously (6). Briefly, trypsinized cells were incubated in complete medium (phenol red-free IMEM with 10% FCS) with 10 M PhA with or without 10 M of the ABCG2 inhibitor FTC for 30 min at 37°C in 5% CO 2 . Cells were washed subsequently with cold complete medium and then incubated for 1 h at 37°C in PhA-free complete medium with 10 M FTC to generate the FTC/Efflux histogram, or without FTC to generate the Efflux histogram. The cells subsequently were washed twice with cold PBS and analyzed by flow cytometry. FTC/Efflux - Efflux values, the difference in mean channel numbers between the FTC/Efflux and Efflux histograms, were calculated for each cell line as a measure of FTC-inhibitable PhA efflux. For comparison, ABCG2-mediated transport also was assessed using 20 M mi- toxantrone with 10 M FTC. When assessing Pgp transport, rhodamine 123 (0.5 g/ml) was used as a substrate, and valspodar (3 g/ml) was used as the inhibitor; for MRP1 transport, calcein AM (0.5 M) was used as the substrate, and probenecid (0.5 mM) was used as the inhibitor. When screening potential ABCG2 inhibitors using ABCG2-transfected cells, PhA was used at a concen- tration of 1 M with or without the desired inhibitor [5 g/ml valspodar (PSC-833), 10 g/ml verapamil, 10 M cyclosporin A, 50 M etoposide, 1 M tariquidar, 100 M UCN-01, and 100 M flavopiridol] and 10 M FTC to generate Inhibitor/Efflux peaks. Results are representative of at least two separate experiments. In studies with the anti-ABCG2 antibody 5D3, cells were incubated in 2% BSA/DPBS with either phycoerythrin-labeled negative control antibody (IgG2b) or phycoerythrin-labeled 5D3 antibody (both from eBioscience, San Diego, CA) according to the manufacturer’s instructions, washed with Dul- becco’s Phosphate Buffered Saline (DPBS), and subsequently analyzed. Sur- face expression of ABCG2 was calculated as the difference in mean channel numbers between the 5D3 histogram and the negative control antibody histo- Received 10/20/03; revised 12/5/03; accepted 12/19/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robert W. Robey, Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 12C103, Bethesda, MD 20892. Phone: 301-496-0795; Fax: 301-402-1608; E-mail: robeyr@mail.nih.gov. 1242 Research. on February 20, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from