Mdr Gene Family Research Articles

Retinol-induced mdr1 and mdr3 modulation in cultured rat Sertoli cells is attenuated by free radical scavengers

The effects of retinol on modulation of mdr genes in Sertoli cells were investigated. The hypothesis that free radical scavengers may attenuate the effect of retinol was also tested. Sertoli cells isolated from 15-day-old Wistar rats were cultured for 48 h and then treated with retinol for 24 h with or without free radical scavengers (1 mM mannitol, 0.1 mM Trolox or superoxide dismutase [200 U/ml]). Expression of mdr1, mdr2 and mdr3 genes was monitored by RT-PCR. Mitochondrial superoxide production was used as an index of ROS production. Expression of mdr1 and mdr3 was inhibited by retinol treatment (7 μM, 24 h), while mdr2 was not detected in response to any of the treatments. We also observed that retinol supplementation (7 μM, 24 h) increased superoxide production. The observed inhibition of mdr genes was attenuated by all co-treatments, suggesting that retinol-induced ROS are required for inhibition of mdr1 and mdr3 expression. The results suggest that retinol may play an important role in the modulation of the mdr gene family in cultured rat Sertoli cells and that these effects appear to be mediated by ROS.

Regional expression of multidrug resistance genes in genetically epilepsy-prone rat brain after a single audiogenic seizure.

The multidrug resistance (mdr) gene family encodes the drug transport macromolecule P-glycoprotein (P-gp), which contributes to the functionality of the blood-brain barrier. Recent evidence suggests that P-gp-mediated drug extrusion may play a facilitatory role in refractory epilepsy. We investigated the regional expression of mdr genes in genetically epilepsy-prone rat (GEPR) brain after a single audiogenic seizure. Three groups of adult male GEPRs (n = 5/group) were exposed to a seizure-inducing audiogenic stimulus and killed at 4 h, 24 h, and 7 days thereafter. A further group (n = 5) served as a stimulus-naïve control. Expression of mdr1a and mdr1b in distinct anatomic brain regions (cortex, midbrain, pons/medulla, hippocampus) was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the presence of competitive internal standards. When compared with control, mdr1a expression in cortex and midbrain was significantly (p < 0.05) increased at 24 h after a single audiogenic seizure. Cortical mdr1a expression remained elevated at 7 days after stimulus. In contrast, mdr1a expression in pons/medulla and hippocampus was unchanged. The mdr1b isoform was quantifiable in hippocampus alone and not influenced by seizure activity. These findings suggest that acute seizures in the GEPR can induce the expression of mdr genes. The pattern of increased expression appears to follow the anatomic pathway of audiogenic seizures in these animals, with initiation in the midbrain and propagation to the cortex. Further studies are required to investigate the effects of recurrent seizure activity and to characterise mdr expression in other experimental seizure models.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

MDR and MRP gene families as cellular determinant factors for resistance to clinical anticancer agents.

AbstractThe constant threat by a countless array of environmental poisons, natural products and synthetic agents, over evolutionary time has led living organisms to develop many elaborate mechanisms that combat the toxic effects of these insults. Among such mechanisms is one that decreases the intracellular accumulation of a toxic substance by directly pumping toxic molecules out of the cells, and another that modifies the metabolism of the toxic substances and effluxes the metabolized compounds. The former mechanism is typified by the mammalian multidrug resistance system mediated by P-glycoproteins (P-gp) that are encoded by the MDR gene family. The second mechanism is exemplified by the multidrug resistance protein (MRP). Both P-gp and MRP contain ATP-binding cassettes and therefore belong to the ABC superfamily of membrane transportersKeywordsMultiple MyelomaAcute Myeloid LeukemiaMultidrug ResistanceAcute Myeloblastic LeukemiaMulti Drug Resistance ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of Pfmdr 1 gene in mefloquine-resistant Plasmodium falciparum.

Drug resistance in Plasmodium falciparum is a serious problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In our present studies, a molecular analysis of pfmdr 1 in isolate strain of P. falciparum, 523a R, from Japanese mefloquine-resistant patient was done to determine the reported association of pfmdr 1 intragenic alleles and mefloquine resistance, and to examine the antimalarial activities of several antimalarial agents against the P. falciparum strain. The antimalarial activities against the strain was decreased susceptibility to mefloquine, artemisinin and halofantrine, in contrast increased susceptibility to chloroquine. The DNA sequence analysis of pfmdr 1 gene in a strain reveled no association of intragenic alleles with mefloquine resistance. Furthermore, the overexpression of pfmdr1 mRNA have been observed and it is about 7.2 times higher than sensitive strain. Our data shows that overexpression of pfmdr1 gene may be associated in mefloquine-resistance mechanism.

A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome.

A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.

Wild-type p53-mediated Induction of Rat mdr1bExpression by the Anticancer Drug Daunorubicin

The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of the multidrug resistance phenotype in animal cells. mdr expression can be induced by many extracellular stimulants including cytotoxic drugs and chemical carcinogens. However, little is known about the mechanisms involved. Here, we report that the expression of the rat mdr1b can be induced by anticancer drug daunorubicin. Further analysis identified a bona fide p53-binding site spanning from base pairs -199 to -180 (5'-GAACATGTAGAGACATGTCT-3') in the rat mdr1b promoter that is essential for basal and daunorubicin-inducible promoter activities. In addition, our results show that wild-type p53 can up-regulate not only the promoter function but also endogenous expression of the rat mdr1b. To the best of our knowledge, this is the first report showing that a specific p53-binding site is involved in the transcriptional regulation of mdr gene by wild-type p53. Since p53 is a sensor for a wide variety of genotoxic stresses, our finding has broad implications for understanding the mechanisms involved in the inducible expression of mdr gene by anticancer drugs, chemical carcinogens, UV light, and other DNA-damaging agents.

NF-κB-mediated Induction of mdr1b Expression by Insulin in Rat Hepatoma Cells

The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of multidrug resistance phenotype in animal cells. However, the mechanisms controlling the expression of these genes have not been well elucidated. Here, we report that the expression of rat mdr1b gene in cultured H-4-II-E hepatoma cells can be induced by insulin. Transient transfection assays using reporter gene constructs containing various 5' mdr1b sequences showed that the sequence located between base pairs -243 and -163 is important for insulin's induction of mdr1b promoter activity. Further analyses revealed that a NF-kappaB-binding site (located between base pairs -167 and -158) is required for insulin-induced promoter activity. Gel mobility shift assay demonstrated that insulin stimulates the binding of nuclear p50/p65 subunits to the mdr1b NF-kappaB sequence. Cotransfection of plasmids expressing either the p50/p65 NF-kappaB subunits or Raf-1 kinase or both resulted in increased expression of the gene containing wild-type but not NF-kappaB site-mutated mdr1b promoter. Finally, expression of either the antisense p65 subunit of NF-kappaB or dominant negative Raf-1 kinase blocked insulin's induction of the mdr1b promoter activity. Taken together, our results suggest that the insulin-induced mdr1b expression is mediated by transcription factor NF-kappaB via the Raf-1 kinase signaling pathway.

Modulation of P-Glycoprotein and mdr1b mRNA Expression by Growth Factors in Primary Rat Hepatocyte Culture

P-glycoproteins encoded by members of the mdr gene family function as membrane-situated transport proteins, isoforms of which are involved in conferring a form of multidrug resistance by participating in secretion of various xenobiotics. In primary rat hepatocytes maintained in serum-free culture, accumulation of immunodetectable P-glycoprotein and mdr1b mRNA occurred in a time-dependent manner and was accompanied by a substantial decrease in retention of the mdr1 substrate rhodamine 123. However, incubation of cells with epidermal growth factor (EGF) or with insulin-like growth factor I (IGF-I) markedly enhanced time-dependent accumulation of P-glycoprotein and mdr1b mRNA. Furthermore, EGF-treated cells exhibited decreased intracellular rhodamine 123 retention, an effect partially inhibited by the chemosensitizer verapamil. These data suggest that an increase in (a) functional transporter(s) eliciting transport of mdr1 substrates occurs under EGF.

In vivo induction of liver P-glycoprotein expression by xenobiotics in monkeys.

P-glycoprotein (pgp), the protein product of the multidrug resistance (mdr) gene family, can confer a multidrug resistance (mdr) phenotype to cells in which it is expressed. One member of the pgp family, pgp2, is located on the hepatocyte biliary pole where it may have a role in biliary excretion. Using primates we sought to determine if mdr gene expression and pgp levels were affected by xenobiotics excreted via the bile in man. Five drugs were studied in male and female rhesus monkeys: erythromycin, rifampicin, tamoxifen, diethylstilbesterol (DES), and probenecid. For each xenobiotic, with the exception of DES, an increase in mdr2 mRNA was observed. The results suggest that expression of mdr2 is responsive to xenobiotics, or their metabolites, that require biliary excretion. We speculate that the mdr2 gene may be a member of a class of xenobiotic responsive genes coding for proteins that actively excrete xenobiotics and/or their metabolites into the bile.

Expression of multidrug resistance-associated protein (MRP) in thyroid cancers.

It was found that the mechanism of anti-cancer drug resistance in anaplastic carcinoma of the thyroid was not explicable only in terms of expression of mdr1 and its gene product, P-glycoprotein. The multidrug resistance-associated protein (MRP), another member of the mdr gene family, may be involved in anti-cancer drug resistance of this carcinoma. The MRP expression was examined immunohistochemically in 8 cell lines and 73 thyroid cancer tissues; its frequency in anaplastic carcinoma (52%) was significantly higher than that in other thyroid cancer types.

Membrane Topology of P-Glycoprotein As Determined by Epitope Insertion: Transmembrane Organization of the N-Terminal Domain of mdr3

P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR). Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain. To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376). Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells. Intracellular and extracellular localization of the tag in the full-length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope. While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance. The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane. These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions. These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6). Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane.

Multiple drug resistance in the pathogenic protozoa

Evidence for the phenomenon of multiple drug resistance (MDR) in the well studied pathogenic protozoa has been examined. This has been placed in the more familiar context of the MDR efflux transporters and the cloned mdr genes of mammalian cells. Homologues of the mdr gene family in protozoa and their possible role in drug efflux have been compared with their mammalian counterparts. Possible mechanisms and models for drug efflux have been considered. The unusual and extensive range of substrates transported by the ATP-binding cassette (ABC) family of transporters which includes the MDRs has been raised. The impact of kinetics, structure and bioenergetics of the MDR family members on mechanisms of transport has been accentuated to argue that MDR efflux considered in isolation appears bizarre but may be better understood in a broader context.

Two multidrug-resistant Friend leukemic cell lines selected with different drugs exhibit overproduction of different P-glycoproteins.

Two Friend leukemic multidrug-resistance (MDR) cell lines were established by exposure to stepwise increased concentrations of rhodamine-123 (RHO) (cell line RR-30) or Adriamycin (ADR) (cell line ARN-15). RR-30 displays preferential resistance to RHO, whereas ARN-15 is more resistant to ADR. The levels of resistance to other MDR drugs and reversibility by verapamil between these two MDR cell lines were somewhat different. Southern blot, RNase protection, and Western blot analysis using gene-specific probes demonstrated that RR-30 and ARN-15 cells preferentially amplified the mdr1 and mdr3 genes, respectively, leading to overexpression of the corresponding P-glycoproteins (p-gp). Our results suggest that members of the mdr gene family can be amplified independently by using different selecting agents, which could be responsible for the differences in the sensitivities to these selecting agents as well as to these MDR drugs.

Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates

P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant ( mdr) gene family. The physiological function of this protein is unknown. Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis. Both obstructive and α-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver. Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain. Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription. α-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes. To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats. Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil. These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.

Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates

P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant (mdr) gene family. The physiological function of this protein is unknown. Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis. Both obstructive and alpha-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver. Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain. Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription. alpha-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes. To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats. Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil. These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.

Progesterone regulates the murine multidrug resistance mdr1b gene.

P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues. In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor. A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.

Amplification of pfmdr 1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand.

Drug resistance in Plasmodium falciparum is an expanding problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In this study a molecular analysis of pfmdr 1 in recent isolates from Thailand was done (1) to further examine the role of pfmdr 1 in drug-resistant isolates and (2) to examine the reported association of pfmdr 1 intragenic alleles and chloroquine resistance. Most of the isolates (10 of 11) were resistant to all compounds tested. Analysis of pfmdr 1 revealed an apparent association between increased gene copy number and increased level of expression of pfmdr 1 and decreased susceptibility to mefloquine and halofantrine. Sequence analysis of pfmdr 1 in these isolates revealed no association of intragenic alleles with chloroquine resistance.

Cloning and regulation of the rat mdr2 gene.

We have cloned the complete cDNA encoding the rat mdr2 gene by a combination of library screening and the polymerase chain reaction. The sequence of rat mdr2 cDNA is highly similar to other members of the mdr gene family but the initiation of transcription, tissue distribution and regulation of expression of rat mdr2 diverge from the other isoforms. Primer extension analysis showed rat mdr2 mRNA to have a major transcription start point at -277 and a minor one at approximately -518. We constructed gene specific probes for rat mdr2 and mdr1b and compared the expression patterns of these two genes. The highest expression of mdr2 mRNA was in the muscle, heart, liver and spleen. Both mdr2 and 1b mRNA levels were elevated in the livers of rats treated with CCl4 or following partial hepatectomies although the time course of induction of each gene differed. Mdr1b increased by 12 to 24 hours while mdr2 did not increase until 48 hours. Treatment of isolated hepatocytes or RC3 cells with cycloheximide did not effect mdr2 mRNA. In contrast, mdr1b expression was increased. These data suggest that rat mdr2, unlike mdr1b, is not regulated by a negative trans-acting protein factor.

Newly formed chromosome-like structures in independent mouse P388 sublines with developed in vivomdr1 gene amplification

Mouse leukemia P388 sublines that acquired the resistance to multiple drugs as a result of treatment in vivo with anthracyclines (rubomycin, ruboxyl) and/or vincristine were studied. The mdr gene amplification was found in all tested cell lines: in four of five sublines all three members of the mdr gene family showed increased copy numbers, and in one cell line, developed after treatment with ruboxyl, mdr1a and mdr1b genes were amplified to the same degree, whereas the mdr2 gene was not amplified at all. The levels of amplification of mdr genes varied in different cell lines from 30-fold to 50-fold. Unusual cytological manifestations--relatively large newly formed chromosomelike structures, were revealed in four of five long-term independent sublines. Some of these structures did not contained C blocks; the others, in contrast, were enriched by C-heterochromatin. In situ hybridization showed the presence of mdr genes in newly formed bodies. In the majority of cases, the formation of chromosomelike structures was preceded by the appearance of other, smaller size, structures: the so-called "small chromatin bodies" (minichromosomes) and/or homogeneously G-positive small ring chromosomes.