L van Deemter

Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs

We have generated mice homozygous for a disruption of the mdr1a (also called mdr3) gene, encoding a drug-transporting P-glycoprotein. The mice were viable and fertile and appeared phenotypically normal, but they displayed an increased sensitivity to the centrally neurotoxic pesticide ivermectin (100-fold) and to the carcinostatic drug vinblastine (3-fold). By comparison of mdr1a (+/+) and (-/-) mice, we found that the mdr1a P-glycoprotein is the major P-glycoprotein in the blood-brain barrier and that its absence results in elevated drug levels in many tissues (especially in brain) and in decreased drug elimination. Our findings explain some of the side effects in patients treated with a combination of carcinostatics and P-glycoprotein inhibitors and indicate that these inhibitors might be useful in selectively enhancing the access of a range of drugs to the brain.

Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease

Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.

P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs.

The mouse mdr1a (also called mdr3) P-GP is abundant in the blood-brain barrier, and its absence in mdr1a (-/-) mice leads to highly increased levels of the drugs ivermectin, vinblastine, digoxin, and cyclosporin A in the brain. We show here that the drugs loperamide, domperidone, and ondansetron are transported substrates for the mouse mdr1a P-GP and its human homologue MDR1. Phenytoin is a relatively weaker substrate for each, and the drugs haloperidol, clozapine, and flunitrazepam are transported hardly or not at all. Tissue distribution studies demonstrated that the relative brain penetration of radiolabeled ondansetron and loperamide (and their metabolites) is increased four- and sevenfold, respectively, in mdr1a (-/-) mice. A pilot toxicity study with oral loperamide showed that this peripherally acting antidiarrheal agent gains potent opiatelike activity in the central nervous system of mdr1a (-/-) mice. mdr1a (-/-) mice also showed increased sensitivity to neurolepticlike side effects of oral domperidone. These results point to the possible role that the drug-transporting P-GP(s) may play in the clinical use of many drugs, especially those with potential targets in the central nervous system.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

Multidrug resistance and the role of P-glycoprotein knockout mice

Drug resistance, be it intrinsic or acquired, is a major problem in cancer chemotherapy. In vitro, one well characterised form of resistance against many different cytotoxic drugs is caused by the MDR1 P-glycoprotein, a large plasma membrane protein that protects the cell by actively pumping substrate drugs out. Available evidence suggests that this protein may cause drug resistance in at least some clinical tumours. Drugs inhibiting the MDR1 P-glycoprotein activity are, therefore, co-administered during chemotherapy of these tumours. To predict the biological and pharmacological effects of the blocking of this protein, we have generated mice with a genetic disruption of the drug-transporting mdr1a P-glycoprotein. These mice are overall healthy, but they accumulate much higher levels of substrate drugs in the brain, and have markedly slower elimination of these drugs from the circulation. For some drugs, this leads to dramatically increased toxicity, indicating that P-glycoprotein inhibitors should be used with caution in patients.

Classical and novel forms of multidrug-resistance and the physiological functions of p-glycoproteins in mammals

In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.

Transfection of the int-1 mammary oncogene in cuboidal RAC mammary cell line results in morphological transformation and tumorigenicity.

The int‐1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int‐1 activation in in vitro cell systems, we have constructed recombinant molecules of genomic int‐1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs into cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.

Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells.

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.