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Class III multidrug resistance (MDR) Pglycoproteins (P-gp), mdr2 in mice and MDR3 in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte. Mice with a disrupted mdr2 gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologically by portal inf lammation, proliferation of the bile duct epithelium, and fibrosis. This disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum ␥-glutamyltransferase (␥-GT) activity. We report immunohistochemistry for MDR3 P-gp, reverse transcription-coupled PCR sequence analysis, and genomic DNA analysis of MDR3 from two progressive familial intrahepatic cholestasis patients with high serum ␥-GT. Canalicular staining for MDR3 P-gp was negative in liver tissue of both patients. Reverse transcriptioncoupled PCR sequencing of the first patient's sequence demonstrated a homozygous 7-bp deletion, starting at codon 132, which results in a frameshift and introduces a stop codon 29 codons downstream. The second patient is homozygous for a nonsense mutation in codon 957 (C 3 T) that introduces a stop codon (TGA). Our results demonstrate that mutations in the human MDR3 gene lead to progressive familial intrahepatic cholestasis with high serum ␥-GT. The histopathological picture in these patients is very similar to that in the corresponding mdr2(؊͞؊) mouse, in which mdr2 P-gp deficiency induces complete absence of phospholipid in bile.

The human multidrug-resistance protein (MRP) gene family contains at least six members: MRP1, encoding the multidrug-resistance protein; MRP2 or cMOAT, encoding the canalicular multispecific organic anion transporter; and four homologs, called MRP3, MRP4, MRP5, and MRP6. In this report, we characterize MRP3, the closest homolog of MRP1. Cell lines were retrovirally transduced with MRP3 cDNA, and new monoclonal antibodies specific for MRP3 were generated. We show that MRP3 is an organic anion and multidrug transporter, like the GS-X pumps MRP1 and MRP2. In Two members of the large family of ABC transporters are known thus far to confer multidrug resistance in human cancer cells. These are the MDR1 P-glycoprotein (1) and the multidrugresistance protein MRP1 (2). Both membrane proteins transport a wide range of drugs with different cellular targets and confer resistance by decreasing the intracellular concentration of drugs. P-glycoprotein transports these drugs in unmodified form, whereas MRP1 can transport drugs either conjugated to anionic ligands such as glutathione (GSH), glucuronide, or sulfate, or in an unmodified form, possibly together with GSH. Well known substrates for MRP1 are, for example, cysteinyl leukotrienes, glutathione disulfide, S-(2,4-dinitrophenyl-)glutathione, ethacrynic acid S-glutathione, etoposide glucuronide, certain steroid glucuronides, and bile salt derivatives (3-6). Transporters with the characteristics of MRP1 are known as GS-X pumps (7) or multispecific organic anion transporters (8).Another GS-X pump is MRP2, a homolog of MRP1. Unlike MRP1, which is nearly ubiquitously expressed (9), MRP2 is present mainly in the canalicular membrane of hepatocytes (10), but is also present in other apical domains of polarized cells such as the epithelial cells of the proximal tubules of the kidney (11). Studies with mutant rats (TR Ϫ ͞GY or EHBR), which lack the MRP2 protein in the canalicular membrane of hepatocytes, have shown that the substrate specificity of MRP2 is very similar to that of MRP1 (12, 13). MRP2 also contributes to transport of anticancer drugs and some metals. The mutant rats showed a reduced biliary clearance of methotrexate (14), of the topoisomerase I inhibitor CPT-11 and its metabolites (15), and of mercury, cadmium, and arsenite (refs. 16 and 17; R.O.E., unpublished observation). Cells transduced with an MRP2 cDNA construct transport the cytostatic drug vinblastine (18). Moreover, overexpression of the MRP2 gene has been found in several cisplatinresistant cell lines (19,20), and transfection of an MRP2-antisense construct into liver cells was reported to confer an increased sensitivity to cytotoxic drugs (21). All these observations strongly suggest that MRP2 may confer multidrug resistance, but whether it does so in cancer patients remains to be established.Besides MRP1 and MRP2, there are at least four MRP homologs expressed in humans, called MRP3, MRP4, MRP5, and MRP6 (20,22). Not much is known about the substrate specificity of these putative new transporters...

SUMMARY:The multidrug resistance protein (MRP) family consists of several members and, for some of these transporter proteins, distinct roles in multidrug resistance and normal tissue functions have been well established (MRP1 and MRP2) or are still under investigation (MRP3). MRP3 expression studies in human tissues have been largely restricted to the mRNA level. In this report we extended these studies and further explored MRP3 expression at the protein level. Western blot and immunohistochemistry with two MRP3-specific monoclonal antibodies, M 3 II-9 and M 3 II-21, showed MRP3 protein to be present in adrenal gland, and kidney and in tissues of the intestinal tract: colon, pancreas, gallbladder, and liver. In epithelia, MRP3 was found to be located at the basolateral sides of cell membranes. In normal liver, MRP3 was detected at lower levels than anticipated from the mRNA data and was found present mainly in the bile ducts. In livers from patients with various forms of cholestasis, MRP3 levels were frequently increased in the proliferative cholangiocytes, with sometimes additional staining of the basolateral membranes of the hepatocytes. This was especially evident in patients with type 3 progressive familial intrahepatic cholestasis. The present results support the view that MRP3 plays a role in the cholehepatic and enterohepatic circulation of bile and in protection within the biliary tree and tissues along the bile circulation route against toxic bile constituents. The possible functional roles for MRP3 in the adrenal gland and in the kidney remain as yet unknown. In a panel of 34 tumor samples of various histogenetic origins, distinct amounts of MRP3 were detected in a limited number of cases, including lung, ovarian, and pancreatic cancers. These findings may be of potential clinical relevance when considering the drug treatment regimens for these tumor types. (Lab Invest 2002, 82:193-201).

Screening for variants in TPMT did not reduce the proportions of patients with hematologic ADRs during thiopurine treatment for IBD. However, there was a 10-fold reduction in hematologic ADRs among variant carriers who were identified and received a dose reduction, compared with variant carriers who did not, without differences in treatment efficacy. ClinicalTrials.gov number: NCT00521950.

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Mice homozygous for a disruption in the Mdr2 gene (Mdr2 (Ϫ/Ϫ) mice) lack the Mdr2 P-glycoprotein (P-gp) in the canalicular membrane of the hepatocyte and are unable to excrete phosphatidylcholine into the bile. These mice develop a nonsuppurative cholestatic liver disease, presumably caused by the high concentrations of free cytotoxic bile acids in bile. We generated transgenic mice that express the human homolog of Mdr2, MDR3, specifically in the liver by the use of an albumin promoter. In these mice the MDR3 P-gp is exclusively located in the canalicular membrane of hepatocytes and phospholipid excretion into bile is restored. Mice that contain the same amount of MDR3 P-gp as that of Mdr2 P-gp in wild-type mice, also excrete the same amount of phospholipids. No histopathological abnormalities were observed in the livers of these mice. In mice that express MDR3 at a higher or lower level, the phospholipid excretion correlated with the amount of MDR3 P-gp. We conclude that the human MDR3 P-gp is functionally homologous to the murine Mdr2 P-gp and that it can fully replace this P-gp in Mdr2 (Ϫ/Ϫ) mice, restoring the excretion of phospholipids into the bile. The phospholipid excretion is limited by the amount of MDR3 or Mdr2 P-gp. The excretion of cholesterol is not tightly coupled to the excretion of phospholipids in these mice, because a very low phospholipid excretion level is sufficient to give almost wild-type cholesterol excretion into the bile. (HEPATOLOGY 1998;28:530-536.) P-glycoproteins (P-gps) are relatively large, glycosylated transmembrane proteins that can actively transport substrates across membranes. P-gps are members of the family of ABC (for Adenosine triphosphate Binding Cassette) transporter proteins. In humans, two genes encoding P-gps have been identified, MDR1 and MDR3 (also called MDR2); in mice there are three P-gp genes, Mdr1a (also called Mdr3), Mdr1b (Mdr1) and Mdr2. 1 P-gps are best known for their ability to actively extrude a range of hydrophobic cytostatic drugs from the cell. In cancer cells, this can cause multi-drug resistance (MDR). In humans, MDR1 P-gp fulfills this function; in mice both Mdr1a P-gp and Mdr1b P-gp can cause MDR. [2][3][4] Class III P-gps, such as human MDR3 P-gp and murine Mdr2 P-gp (91% identical to each other at the amino acid level 5,6 ) cannot cause MDR and are probably unable to transport drugs at significant rates. These P-gps have only been detected in the canalicular membrane of the hepatocyte, but the MDR3 and Mdr2 genes are also expressed at a low level in the B-cell compartment, skeletal muscle, and heart. 7 The function of Mdr2 P-gp was discovered when Smit et al. generated mice which are homozygous for a disrupted Mdr2 gene resulting in the absence of Mdr2 P-gp from the canalicular membrane. 8 These mice do not excrete phospholipid (PL) into bile; they do develop nonsuppurative inflammatory cholangitis, 8,9 most likely caused by the detergent action of free bile salts, not sequestered in mixed micelles with PL. Heterozygous mice, which contain hal...