Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.

Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

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The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

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The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane The endothelial cells of the brain's capillaries are tightly joined to form a hydrophobic permeability barrier (32) termed the blood-brain barrier. Pgp expression in these cells limits the movement of hydrophobic cationic drugs from the blood into the brain (36,42,43). However, in vitro, these capillary endothelial cells also transport organic anions unidirectionally toward the capillary lumen in an energy-dependent fashion (5, 25, 41). Therefore, the capillary endothelial cells appear to express an unidentified anionic ABC transporter. Currently, it is unknown whether an anionic ABC transporter is expressed at functional levels in vivo in the endothelium of brain capillaries.The ABC transporter Mrp4, originally described as a nucleotide transporter (37), is known to transport a diverse array of compounds (2,7,34) and is capable of transporting organic anions as well as antiviral and antiretroviral compounds that do not easily penetrate the central nervous system (CNS) (2, 3, 9, 27, 37). Mrp4 expression was previously demonstrated on the basolateral membrane of the prostate gland and the apical membrane of the kidney (21, 44). Studies in cultured epithelial cells have demonstrated basolateral localization of Mrp4 (22). Transporters typically route to one surface in polarized cells. For instance, the Mrp (ABCC) subfamily members localize to either the basolateral or apical membrane, but not to both. MRP1 is restricted to the basolateral membrane of the choroid plexus and intestine, whereas MRP2 is found on the apical membrane in the intestine and liver (26,29). Mrp4 might be unique among the Mrp transporters in having cell-or tissuedependent polarized expression, but the biological importance of this unique ability to localize either apically or basolaterally remains unknown.We have developed Mrp4 knockout mice, and here we report their first use to show that Mrp4 is expressed in the lumen of brain capillaries and in the basolateral membrane in the choroid plexus epithelium. In vivo, Mrp4 restricts topotecan movement from the blood into the CSF and from the capillaries into the brain tissue by virtue of its unique ability to traffic to either the apical or basolateral membrane. We further show that Mrp4 overexpression confers resistance to the camptothecin topotecan. These studies have specific therapeutic implications for targeting the CNS that might harbor tumors but have more general impl...

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.