A definitive test of the frequency of this variant would go beyond the scope of our work. In conclusion, the results presented here confirm our previous finding that the clinically relevant, frequently occurring Q141K SNP impairs transport of the chemotherapeutic agent mitoxantrone by ABCG2 [13]. most extensively studied of these SNPs with potential clinical relevance is usually 421 C A resulting in a glutamic acid to lysine substitution (Q141K) in the protein. The Q141K SNP was identified with varying frequencies in different ethnic groups and was found to be SP-420 the most prevalent in the Japanese populace (30%). Q141K has been associated with lower levels of protein expression and impaired transport in vitro, though some controversies exist in the publications characterizing this SNP [13C17]. The polymorphism has also been studied in vivo; patients carrying the SNP were found to have elevated plasma levels of gefitinib and diflomotecan, and increased bioavailability of oral topotecan [18C20]. Further, Q141K was associated with a higher incidence of diarrhea in non-small cell lung cancer patients treated with gefitinib [21]. Sequencing the gene in the 60 cancer cell lines maintained and used for screening in the National Malignancy Institute by various groups revealed that ten of the cell lines carried the Q141K polymorphism. Interestingly, A549 and SK-OV-3 cells were reported to have an additional polymorphism, a splicing variant occurring at the mRNA level, resulting in the deletion of amino acids 315 and 316 [16]. This splicing variant was also identified in MCF7 and HT-29 cells, which are wild type at residue 141. In the present study, we expanded our previous functional studies around the Q141K SNP [13] using pheophorbide a, a fluorescent compound that was recently reported by our group as an ABCG2-specific substrate [22]. In addition, we examined the effect of the 315C316 variant with or without the Q141K polymorphism on ABCG2 function. Materials and methods Cell culture Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA) were maintained in Minimal Essential Medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine (BioFluids, Rockville, MD), and 100 models/L penicillin/streptomycin (BioFluids) at 37C in 5% CO2. Stably transfected cell lines were maintained in 2 mg/ml G418 (Invitrogen). Mutagenesis and transfection The 315C316 and the Q141K/D315C316 mutants were generated by site-directed mutagenesis in the pcDNA3.1/Myc-HisA(-) vector (Invitrogen) as previously described [23]. The wild type (R482), Q141K, R482G, and vacant vector-transfected (pcDNA) cells were previously reported [13]. All mutations were confirmed by sequencing the plasmids. The SP-420 full-length insert was also sequenced from genomic DNA from one representative clone of each stable transfectant generated in HEK 293 cells. Transfections were performed using TransFast transfection reagent (Promega, Madison, WI). Colonies were selected in 2 mg/ml G418 with frequent removal of lifeless cells and were expanded prior to study. Membrane preparation and immunoblotting Microsomal membrane preparation and immunoblotting were performed as described previously [23]. Briefly, cells were disrupted by nitrogen cavitation (Parr Instrument, Moline, IL) in a hypotonic lysis buffer, and membranes were obtained by ultracentrifugation at 40,000 rpm. Membrane proteins were loaded onto precast SP-420 7.5% (w/v) SDS-polyacrylamide gels (Bio-Rad, Hercules, CA), subjected to electrophoresis, and electrotransferred onto PVDF membranes (Millipore, Bedford, MA). Blots were probed with a 1:250 dilution of the monoclonal anti-ABCG2 antibody BXP-21 (Kamiya Biomedical, Seattle, WA), or a 1:1000 dilution of the 405 polyclonal antibody, followed by incubation in anti-mouse IRDye800? and anti-rabbit IRDye680? secondary antibodies (LI-COR Biosciences, Lincoln, NE) and visualized with the Odyssey Imaging System (LI-COR). Membranes were stained with 0.1% Ponceau S (Sigma, St. Louis, MO) and checked for comparable loading. Flow cytometry Flow cytometry with the anti-ABCG2 antibody, 5D3 (eBioscience, San Diego, CA), was performed as previously described [23]. Briefly, following trypsinization, cells were incubated with phycoerythrin-conjugated 5D3 and control antibodies for 30 min at room heat, washed twice with DPBS, and kept in the dark until analysis. For transport studies, cells were trypsinized, resuspended in complete media made up of 20 M mitoxantrone (Sigma) or 10 M pheophorbide a (Frontier Scientific, Logan, UT) with or without 10 M of the ABCG2-blocker, Fumitremorgin C (FTC), and incubated for 30 min at 37C LAMNB2 in 5% CO2. (FTC was synthesized by Thomas McCloud, Developmental Therapeutics Program, Natural Products Extraction Laboratory, National Institutes of Health, Bethesda, MD.) Cells were then incubated for 1 h at 37C in substrate-free media, continuing with SP-420 or without 10 M FTC, centrifuged and resuspended in DPBS prior to analysis on a FACSort flow cytometer, equipped with both a 488 nm argon laser and.