Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, S1-M1-80, that exhibited an ATP-dependent reduction in drug accumulation. Comprised of 6 transmembrane domains and a single ATP binding domain, it is thought that dimerization is required for activity, and that the gene encodes a half-transporter molecule. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan). Both substrates and inhibitors of ABCG2 have been discovered at an accelerating pace, and the variation in inhibitors rivals that described for P-glycoprotein. There is increasing evidence supporting a role for ABCG2 in oral absorption of pharmacologic agents. We identified a naturally occurring mutation in ABCG2 (R482T; R482G) that alters substrate and inhibitor specificity; and then carried out a sequence analysis of 90 DNA samples representing a global genetic diversity and identified single nucleotide polymorphisms in ABCG2. We then established transfectants in HEK293 cells. Using the wildtype background (R482), clones expressing V12M, Q141K and D620 N were established. Clones with comparable levels of surface expression were selected, and impaired transport was observed in clones bearing the Q141K. Since gastrointestinal absorption of topotecan has been related to ABCG2 expression in the intestinal epithelium, a clear implication of this work is that the Q141K SNP could be associated with increased oral topotecan uptake. To the extent that ABCG2 is involved in drug excretion, this SNP could increase exposure to substrates such as irinotecan and topotecan. In an attempt to identify the mechanism of dimerization of ABCG2, our laboratory has studied a GXXXG dimerization motif in the transmembrane helix 1, finding that while it is critical for normal transport activity, normal cross-linking is retained in cells expressing glycine to leucine mutations at amino acids 406 and 410. Further mutational analysis is ongoing, generating mutations in residues identified by analysis of homologues or by molecular modeling, based on collaborative studies with Michael Dean and Di Xia, respectively. Our goal is to identify residues involved in the dimerization. In collaboration with Balasz Sarkadi, we have generated Sf9 insect vectors to allow transfection in the high expression insect system. This system is more tolerant of misfolded protein and has already been shown to generate functional ABCG2 molecules. This system will allow co-immunoprecipitation studies that are needed to provide confirmation of the impact of our mutations on dimerization. More recently, additional residues highly conserved or predicted to be important in the model generated by Di Xia are being evaluated. Mutation of the highly conserved amino acid residue 553 results in loss of protein expression on the mammalian cell surface, and expression of a nonfunctional protein on the insect cell surface. Quantitative mitoxantrone efflux assays used in characterizing the mutants and the SNPs were developed in part for use in clinical samples. We were able to convincingly show a linear relationship between mitoxantrone efflux and both mRNA and protein expression. Cells are incubated for 30 min at 37??C in mitoxantrone with or without the ABCG2-specific inhibitor, fumitremorgin C (FTC). The cells are then washed and incubated again at 37??C for 1 hr in mitoxantrone-free medium continuing with or without FTC. The cells are then washed and fluorescence quantitated by flow cytometry. The difference in the post-efflux peaks with or without FTC, the "channel -shift value" is linearly related to both mRNA and surface protein expression in the cells selected for drug resistance.