UCSF

The xenobiotic ABC transporters are membrane bound proteins that efflux substrates coupled with ATP hydrolysis. These transporters have a broad substrate specificity and tissue distribution in excretory and/or barrier sites, supporting their role in the distribution and elimination of xenobiotics. Many factors regulate the expression and function of xenobiotic ABC transporters, and a wealth of data suggests genetic variation may be a factor. The overall hypothesis investigated in this dissertation is that genetic variation affects clinical phenotypes via modulation of xenobiotic ABC transporter expression and function. Xenobiotic ABC polymorphisms were identified in healthy populations of ethnically diverse individuals. Seven amino acid changing variants and two haplotypes of P-glycoprotein (P-gp), encoded by ABCB1, were tested for their in vitro effects on P-gp expression and function. The Asn21Asp, Arg669Cys, Ala893Ser, Ala893Thr, Ser1141Thr and Val1251Ile variants, and the Asp21/1236T/Ser893/3435T haplotype showed altered intracellular accumulation of calcein-AM and/or bodipy-FL-paclitaxel. In a substrate-dependent manner, certain P-gp variants showed less sensitivity to cyclosporin A inhibition. ABCB1 3'-untranslated region (UTR) variants were investigated for their effects on ABCB1 mRNA stability. Computational methods predicted the impact of 3'-UTR variants on ABCB1 mRNA stability and a cell-based assay measured the mRNA half-life of ABCB1 reference and +89A>T, +146G>A and +193G>A 3'-UTR variants. The mRNA half-life for the 3'-UTR variants was similar to the reference half-life of 9.4 h. Colon cancer patients (n = 33) demonstrated variable ABCB1 and ABCC1-ABCC3 mRNA expression in matched normal and tumor colon tissue. A preliminary association analysis identified possible trends between specific ABC genotypes and mRNA expression. ABCB1, ABCC1 and ABCC3 showed altered mRNA expression in tumor tissue compared to adjacent healthy tissue, suggesting differential regulation in normal and tumor tissue. The functional relevance of promoter region polymorphisms in ABCB1, ABCC2-ABCC6 and ABCG2 was predicted using two computational methods. The predictions correctly identified three out of five ABCC2 promoter variants (-1450A>G, -1193A>G and -920A>G) that were previously shown to have decreased transcriptional activity, illustrating the potential utility of these computational predictions in guiding future studies. In summary, the results of this dissertation research suggest xenobiotic ABC polymorphisms can alter gene expression and transport function.