Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.

The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Multidrug resistance protein 1 protects the oropharyngeal mucosal layer and the testicular tubules against drug-induced damage.

The multidrug resistance protein 1 (MRP1) gene encodes a transporter protein that helps to protect cells against xenobiotics. Elevated levels of MRP1 in tumor cells can result in active extrusion of a wide range of (anticancer) drugs with different cellular targets, a phenomenon called multidrug resistance (MDR). To explore the protective function of the mouse mrp1 protein during drug treatment, we investigated the toxicity caused by the anticancer drug etoposide-phosphate (ETOPOPHOS) in mice lacking the mrp1 gene (mrp1(-/-) mice). We show here that the lack of mrp1 protein results in increased etoposide-induced damage to the mucosa of the oropharyngeal cavity and to the seminiferous tubules of the testis. The high concentrations of mrp1 that we find in the basal layers of the oropharyngeal mucosa and in the basal membrane of the Sertoli cells in the testis apparently protect wild-type mice against this tissue damage. We also find drug-induced polyuria in mrp1(-/-) mice, which correlates with the presence of mrp1 protein in the urinary collecting tubules, the major site of kidney water reabsorption. Our results indicate that specific inhibitors of MRP1 used to reverse MDR, in combination with carcinostatic drugs transported by MRP1, might lead to drug-induced mucositis, (temporary) infertility, and diabetes insipidus.

Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier.

Multidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

A family of drug transporters: the multidrug resistance-associated proteins.

The human multidrug resistance-associated protein (MRP) family currently has seven members. The ability of several of these membrane proteins to transport a wide range of anticancer drugs out of cells and their presence in many tumors make them prime suspects in unexplained cases of drug resistance, although proof that they contribute to clinical drug resistance is still lacking. Recent studies have begun to clarify the function of the MRP family members. MRPs are organic anion transporters; i.e., they transport anionic drugs, exemplified by methotrexate, and neutral drugs conjugated to acidic ligands, such as glutathione (GSH), glucuronate, or sulfate. However, MRP1, MRP2, and MRP3 can also cause resistance to neutral organic drugs that are not known to be conjugated to acidic ligands by transporting these drugs together with free GSH. MRP1 can even confer resistance to arsenite and MRP2 to cisplatin, again probably by transporting these compounds in complexes with GSH. MRP4 overexpression is associated with high-level resistance to the nucleoside analogues 9-(2-phosphonylmethoxyethyl) adenine and azidothymidine, both of which are used as anti-human immunodeficiency virus drugs. MRPs may, therefore, also have a role in resistance against nucleoside analogues used in cancer chemotherapy. Mice without Mrp1, a high-affinity leukotriene C(4) transporter, have an altered response to inflammatory stimuli but are otherwise healthy and fertile. MRP2 is the major transporter responsible for the secretion of bilirubin glucuronides into bile, and humans without MRP2 develop a mild liver disease known as the Dubin-Johnson syndrome. The physiologic functions of the other MRPs are not known. Whether long-term inhibition of MRPs in humans can be tolerated (assuming that suitable inhibitors will be found) remains to be determined.

The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin.

Mouse fibroblast cell lines lacking functional Mdr1a, Mdr1b, and Mrp1 genes were selected for resistance to topotecan, mitoxantrone, or doxorubicin. Each of the resulting drug-resistant lines showed marked gene amplification of Bcrp1, the mouse homologue of the human ATP-binding cassette transporter gene BCRP/MXR/ABCP, and greatly elevated expression of Bcrp1 mRNA. All three of the resistant cell lines were highly cross-resistant to topotecan and mitoxantrone and, to a variable extent, doxorubicin. All showed greatly reduced cellular accumulation and greatly increased efflux of mitoxantrone that was dependent on cellular ATP and efficiently reversed by the compound GF120918. The mouse Bcrp1 cDNA encodes a 657-amino-acid protein with 81% identity (86% similarity) to the human breast cancer resistance protein (BCRP) and a virtually superimposable hydrophobicity profile. Our data argue strongly that mouse Bcrp1 is functionally comparable with human BCRP, conferring multidrug resistance to topotecan, mitoxantrone, doxorubicin, and related compounds. Mouse models and cell lines should, therefore, be highly informative in understanding the clinical, pharmacological, and physiological roles of BCRP.

Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance.

Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs. To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b. These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection. For substrate drugs, we found that these contributions can indeed be very substantial. Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold). Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold). Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs. An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.

Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies.

Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1). MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established. The MRP1 protein belongs to a family of at least six members. Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR. The substrate specificity of the other family members remains to be defined. Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR. In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues. The panel consists of P3II-1 and P3II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5II-54 for MRP5. All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of cross-reactivity with any of the other family members. Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted. These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.

Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana.

Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana. Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced+oxidized glutathione (GSH+GSSG) in T. pseudonana were much higher than in D. brightwellii. In T. pseudonana, total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii. Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana. Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii, probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii, a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.

The multidrug resistance protein family.

The human multidrug resistance protein (MRP) family contains at least six members: MRP1, the godfather of the family and well known as the multidrug resistance protein, and five homologs, called MRP2-6. In this review, we summarize what is known about the protein structure, the expression in tissues, the routing in cells, the physiological functions, the substrate specificity, and the role in multidrug resistance of the individual members of the MRP family.

Binding of a bZip protein to the estrogen-inducible apoVLDL II promoter.

Activation of the very low density apolipoprotein II (apoVLDL II) gene in chicken liver by estrogen results in the binding of a variety of nuclear proteins including members of the steroid receptor superfamily and the bZip superfamily to the immediate 5' flanking region. In the present study, we have identified a bZip protein from chicken liver as one of the potential binding activities. Its cognate cDNA was cloned from an expression library using a recognition site DNA probe corresponding to part of the apoVLDL II promoter region. By footprinting and gel shift analysis with the recombinant protein from a prokaryotic expression system we have established that the protein binds to at least three different sites in the apoVLDLII promoter region. One of these sites partially overlaps with the major estrogen response element of the gene. Despite the proximity of their binding sites, the estrogen receptor and the bZip protein can bind simultaneously to the very region. Possible implications of this intimate arrangement of binding sites for the activation of the apoVLDL II promoter are discussed.

Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1.

The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.

The potential impact of drug transporters on nucleoside-analog-based antiviral chemotherapy.

Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported. Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs. This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter. The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance. We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.

Characterization of Pax-6 and Hoxa-1 binding to the promoter region of the neural cell adhesion molecule L1.

The neural cell adhesion molecule L1, a member of the immunoglobulin superfamily, mediates cell interactions in the developing and regenerating nervous system of mammals and is also detectable in the immune system and in the epithelia of intestine, skin, lung, and kidney. This diverse pattern of expression begs the question as to the regulatory mechanisms underlying transcription of the L1 gene. We demonstrate here that the paired domain and homeodomain containing Pax-6 protein binds to three different sites in the promoter region of the L1 gene. The promoter proximal binding site is also recognized by Hoxa-1 and lies approximately 60 bp upstream from the transcription start site only few base pairs upstream of a putative binding site for the TFII-I transcription initiation factor. On the basis of this sequence, we have characterized the binding of Pax-6 and explored two modes of its DNA binding activities.

Oestrogen facilitates the binding of ubiquitous and liver-enriched nuclear proteins to the apoVLDL II promoter in vivo.

Using genomic and in vitro DNasel footprinting, we have analyzed protein-DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apoVLDL II. The footprints coincide with previously detected guanosine-protein contacts in vivo. All footprints identified are present in the apoVLDL II-expressing liver exclusively and absent in hormone-naive liver, spleen and oviduct. They comprise recognition sites for the oestrogen receptor, the ubiquitous COUP-transcription factor, the liver-enriched C/EBP and/or DBP and the liver-specific LF-A1. In vitro, binding of protein to the oestrogen response element (ERE) is excluded by the prior binding of a protein, possibly C/EBP or DBP, to an adjacent element. The recognition sequence of the COUP-TF is also a target for LF-A1. The results suggests that oestrogen-dependent liver specific activation of the apoVLDL II promoter is established by the binding of the oestrogen receptor to EREs and multiple liver-enriched factors (C/EBP, DBP and LF-A1) to their nearby recognition sequences. Apparently, several DNA binding nuclear proteins cooperate to keep the promoter in a state that is accessible for the RNA polymerase complex.

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Tissue-specific and steroid-dependent interaction of transcription factors with the oestrogen-inducible apoVLDL II promoter in vivo.

Using in vivo dimethylsulphate footprinting, we have analysed protein--DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apo very low density lipoprotein II (apoVLDL II). Most of the guanosine--protein contacts found, are located within the 230-bp DNA 5' flanking the gene and can be grouped into separate protein-binding sites. Two of these sites resemble the oestrogen-responsive element (ERE) which is the target site for the oestrogen receptor. A third site has some features in common with the chicken ovalbumin upstream promoter element binding the COUP transcription factor. All protein contacts identified are present in the apoVLDL-II-expressing liver exclusively, and are not found in the hormone-naive liver, in erythrocytes or the oviduct tubular gland. Our results demonstrate the binding in vivo of a protein, presumably the oestrogen receptor, to the ERE and suggest that the hormone activates transcription by establishing a transcription complex comprising several factors at the apoVLDL II promoter.

Segment-specific expression of the neuronatin gene during early hindbrain development.

The developing hindbrain is segmented in a series of repetitive bulges called neuromeres or rhombomeres. In the mouse, first molecular evidence for segmentation of the hindbrain came from rhombomeres 3- and 5-specific expression of the Krox-20 gene. The hindbrain segments are linked with the expression of different Hox genes which have a role in patterning the hindbrain and branchial region of the vertebrate head. Here we identified by subtractive hybridization a gene, mouse neuronatin, that is downregulated in P19 embryo carcinoma cells that have undergone a partial differentiation process. Neuronatin encodes putative transmembrane proteins of 54, 55, and 81 amino acids that might serve as protein ligands, cofactors, or small cell adhesion molecules. The neuronatin gene is transiently expressed in rhombomeres 3 and 5 during early hindbrain development and in the floor of the foregut pocket. In addition, expression is observed in the early Rathke's pouch, in the derived adenohypophysis, and in the developing inner ear. During later embryogenesis the neuronatin gene is strongly expressed in the major part of the central and peripheral nervous system. These results suggest that neuronatin participates in the maintenance of segment identity in the hindbrain and pituitary development and maturation or maintenance of the overall structure of the nervous system.

Crumbs homologue 1 in polarity and blindness.

Several retinal dystrophies, including retinitis pigmentosa type 12 and Leber congenital amaurosis, are caused by a large variety of mutations in the CRB1 (Crumbs homologue 1) gene. This discovery led to an increased focus on the function of CRB1 and the Drosophila homologue Crumbs. In the present study, we review the current knowledge on Crumbs and its vertebrate homologues, their function in cell polarity and their pathogenicity in retinal degeneration.

Pax: gene regulators in the developing nervous system.

In recent years, the discovery of Pax genes in mouse has played an invaluable role in furthering our understanding in mouse developmental processes and disorders. To date, eight murine paired box-containing genes have been cloned. Seven of these exhibit a distinct spatiotemporal expression pattern in the developing nervous system implying a role in the regional specification of the developing spinal cord and brain. The Pax genes encode for sequence-specific DNA binding transcription factors that play a key role in embryonic development. Three of these developmental control genes are altered in mutant mice and two are associated with human diseases. Disruption of these Pax genes leads to abnormalities in neural crest derivatives, neuroectoderm, sclerotome or myotome-derived tissues. Disruption of the Pax-3 gene causes the Splotch phenotype in mice and Waardenburg syndrome in humans. Pax-6 mutations result in Small eye mice and the human genetic disorder aniridia. The Pax-1 gene is mutated in undulated mice. Pax proteins can transform cells in culture which then form tumours following injection in nude mice. Consistent with this activity, PAX3 has been recently implicated in the generation of the tumour alveolar rhabdomyosarcoma.

Regulatory elements and DNA-binding proteins mediating transcription from the chicken very-low-density apolipoprotein II gene.

The chicken Very-Low-Density Apolipoprotein II (apoVLDL II) gene is specifically expressed in liver in response to estrogen. In this study, we performed a functional analysis of the 300-base pair region immediately 5' to the gene by gene transfer of chloramphenicol acetyl transferase (CAT) constructs into chicken embryonic hepatocytes (CEH). Two estrogen response elements (EREs) could be distinguished which together form a potent estrogen response unit. Stimulation of transient expression by co-transfection with a plasmid expressing rat C/EBP confirmed that a similar protein in chicken liver may be involved in apoVLDL II transcription. In vitro DNaseI footprinting and band-shift analysis with liver, oviduct and spleen nuclear extract revealed the tissue distribution of the proteins binding to the promoter region. A liver-specific protein bound to multiple sites of which some resembled the recognition sequence of the CCAAT/Enhancer binding protein, C/EBP. Of the other proteins binding to the apoVLDL II promoter, one was identified as the liver-specific LF-A1 by mobility shift analysis, using purified bovine LF-A1, and another as the general COUP-transcription factor, using an antiserum against the human COUP-TF.