EP0900273A2 - Familie von organischen anionentransportproteine , deren nukleinesäuren, zellen die sie enthalten und verfahren zu deren anwendung - Google Patents

Familie von organischen anionentransportproteine , deren nukleinesäuren, zellen die sie enthalten und verfahren zu deren anwendung

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Publication number
EP0900273A2
EP0900273A2 EP97904656A EP97904656A EP0900273A2 EP 0900273 A2 EP0900273 A2 EP 0900273A2 EP 97904656 A EP97904656 A EP 97904656A EP 97904656 A EP97904656 A EP 97904656A EP 0900273 A2 EP0900273 A2 EP 0900273A2
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EP
European Patent Office
Prior art keywords
protein
cmoat
human
cells
nucleic acid
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EP97904656A
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English (en)
French (fr)
Inventor
Ronald Petrus Johannes Oude Elferink
Coenraad Cornelis Paulusma
Piter Jabik Bosma
Piet Borst
Raymond Evers
Marcel Kool
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Introgene BV
VERENIGING HET NEDERLANDS KANKER INSTITUUT
Academisch Medisch Centrum Bij de Universiteit van Amsterdam
Academisch Medisch Centrum
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Introgene BV
VERENIGING HET NEDERLANDS KANKER INSTITUUT
Academisch Medisch Centrum Bij de Universiteit van Amsterdam
Academisch Medisch Centrum
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Priority to EP97904656A priority Critical patent/EP0900273A2/de
Publication of EP0900273A2 publication Critical patent/EP0900273A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention lies in the field of molecular biology and genetic engineering. It is particularly concerned with mechanisms of transport for substances across cell membranes More in particular it is concerned with transport of cytotoxic substances from the inside to the outside of cells
  • MRPl multidrug resistance-associated protein
  • MRP-1 has a different binding specificity in that it transports anionic organic compounds, possibly complexed or conjuged with other substances
  • a protein has been characterized functionally which is an ATP-dependent non-bile salt organic anion transporter called canalicular Multispecific Organic Anion Transporter This protein has been thought to be identical to MRP-1
  • the liver plays a major role in the detoxification of many endogenous and xenobiotic, lipophilic compounds. Detoxification is accomplished by transferase-mediated conjugation with glutathione- , glucuronide-, or sulphate- moieties, resulting in negatively charged, amphiphilic compounds which are efficiently secreted into bile or urine.
  • cMOAT canalicular Multispeci ic Organic Anion Transporter
  • hMRPl Resistance-associated Protein 1
  • the present invention now provides a nucleic acid comprising a sequence encoding at least a part of a member of a family of organic anion transporters, said nucleic acid comprising at least a gene family specific fragment of one of the sequences of fig.la or fig. lb or figs 17, 18 or 19 or the complement thereof, or a sequence having at least 55%, preferably 70%, in particular 90% homology therewith.
  • a family sofar only one member, mammalian MRP was known. We hypothesized that cMOAT might be a liver-specific homologue of MRPl.
  • PCR polymerase chain reaction
  • rat lung cDNA using nested degenerate oligonucleotide primers which were based on the highly conserved first ATP-binding cassette of the hMRPl sequence (see experimental part) .
  • the 213 base pair product obtained shared 83% amino acid sequence identity with the corresponding region of the hMRPl sequence.
  • RNA Northern
  • this PCR fragment hybridized with a single, 9.5-kb, transcript in all Wistar and TR " rat tissues examined, with high expression in lung and testis, but no detectable expression i ⁇ liver.
  • RNA Northern
  • Fig 2A Northern (RNA) blot analysis of rat tissues with a 1-kb restriction fragment of our isolated cDNA, revealed three different transcripts, ranginq from approximately 6 5 to 9 5-kb, with high expression on ⁇ y in liver, and low expression in kidney, duodenum, and lleum (Fig 2A)
  • Fig 2B n liver
  • the 200-kD protein was completely absent from the canalicular membrane fraction of the TR " rat (fig.3), which correlated with the decreased mRNA level in TR " rat liver (Fig. 2B) . Again, this finding was in good agreement with the defect observed in TR " rats which lack a functional transport system for organic anions in the canalicular membrane
  • MRP homolog encodes the canalicular Multispecific Organic Anion Transporter, and that a 1-bp deletion, resulting in a truncated, non-viable, protein, is responsible for impaired transport of organic compounds from liver to bile in the TR " rat.
  • TR " rats have the same phenotype as patients with the Dubin-Johnson syndrome, characterized by mild chronic conjugated hyperbilirubinemia (15) .
  • Isolation of the human homolog of cmoat is required to elucidate the nature of the defect in humans.
  • Overexpression of hMRPl confers resistance of human tumor cells to a number of cytostatic drugs (16, 17), and this resistance is dependent on intracellular glutathione levels (18) .
  • cMOAT like that of MRPl, might also confer resistance to cancer cells against cytostatic drugs or their metabolites.
  • rat cmoat gene we also found and isolated the cDNA encoding the human cMOAT protein. Now that it is known that these two exist other species of this family of transporters can be found using the present invention. These transport mechanisms occur throughout the living world, so family members can be found in bacteria, bacilli, yeasts and fungi, plants, invertebrae, vertebrae, in particular in mammalians.
  • MRP2 MRP2
  • cMOAT MRP2
  • GS-X pumps MRP2
  • MRP4 MRP5
  • EST Expressed Sequence Tag
  • cMOAT expression is elevated in several cell lines, selected for cisplatin resistance, and also in some sublines of the human non-small lung cancer cell line SW1573/S1, selected for doxorubicm resistance.
  • the expression level of cMOAT correlates with the cisplatin but not the doxorubicin resistance of these cell lines.
  • MRP3 and MRP5 were overexpressed in some resistant cell lines, no clear correlation between drug resistance and the expression levels of MRP3 , MRP4, and MRP5 has emerged from these studies as yet
  • Preferred for the purposes of th s invention are closely related members of the members identified by the sequences of fig. la and fig. lb, most preferred those members which transport similar or the same compounds when expressed in a cell, or the closely related family members identified herein as MRP 3, 4 and/or 5.
  • MRP 3, 4 and/or 5 Most preferred is the human cmoat gene or its human family members and their products for their usefulness in for instance gene therapy and for their use in preparing blocking agents to he transporting product .
  • inventions include but are not limited to a vector comprising a nucleic acid according to the invention and suitable means for replication, transduction and/or expression of said nucleic acid.
  • such a vector further comprises a gene encoding a therapeutically beneficial protein, which may be any protein having a beneficial effect under certain circumstances such as giving glutathion elevating activity, which enhances transport of anionic complexes or conjugates by the invented transporters .
  • a therapeutically beneficial protein which may be any protein having a beneficial effect under certain circumstances such as giving glutathion elevating activity, which enhances transport of anionic complexes or conjugates by the invented transporters .
  • Such vectors include vectors wherein the gene encodes at least a functional part of a gamma glutamyl cysteine synthetase or a UDP-glucuronosyltransferase.
  • Other vectors according to the invention include vectors wherein the therapeutically beneficial protein is another multidrugresistance related protein such as MDR1
  • the invention further provides a cell comprising a nucleic acid or a vector according to the invention Said cells may be any cells, preferred are bone marrow progenitor cells, in particular hematopoietic stem cells
  • said vector thus not encode additional desired functionalities apart from the cMOAT activity as disclosed above, said activity may be present on a separate vector to be introduced into said cell
  • the invention also provides a method for providing cells with Canalicular MultisoecifIC Organic Anion Transport protein activity, comprising transducing said cell with a nucleic acid or a vector according to tne invention, as well as a method for enhancing Canalicular Multispecific Organic Anion Transport protein activity of cells according to the invention, comprising increasing the intracellulax level of glutathion, glucuronide and/or sulphate This may be done by contacting the cell with for instance glutathion esters, but also by providing additional genetic material as disclosed above. This may be done by cotransducmg UDP- glucosedehydrogenase or sulphotransferase or any other means of enhancing such activity.
  • the invention also encompasses methods for enhancing Canalicular Multispecific Organic Anion Transport protein activity of cells according to the invention, comprising enhancing the conjugating capacity and/or the complexing activity of said cell for sulphate, glutathion, glucuronide and the like.
  • the invention provides a method for reducing Canalicular Multispecific Organic Anion Transport protein activity and/or the multidrug resistance of a cell comprising providing said cell with an antisense construct of a nucleic acid or a vector according to the invention, which antisense constructs are thus also part of the present invention .
  • Proteins encoded by a nucleic acid according to the invention or obtainable by expression of a vector according to the invention are of course also part of the present invention, in particular proteins having Canalicular Multispecific Organic Anion Transport protein activity or Canalicular Multispecific Organic Anion Transport protein specific antigenicity comprising at least part of the sequence of fig 4 or being encoded by at least a part of the sequences of MRP 2, MRP 3 or MRP 4 (as given in the accompanying figures) or derivatives thereof having the same or similar function
  • the invention enables the use of a nucleic acid according to the invention or a protein according to the invention in the diagnosis of Dubm-Johnson disease, Rotor disease or another disease involving Canalicular Multispecific Organic Anion Transport protein, as well as the use of a nucleic acid according to the invention or a protein according to the invention in the treatment of Dubin-Johnson disease, Rotor disease or another disease involving Canalicular Multispecific Organic Anion Transport protein.
  • nucleic acids according to the invention can be used as a selectable marker gene.
  • the members of the gene family disclosed herein have several useful applications in the context of gene therapy.
  • the concept of gene therapy has a very broad range of applications with one common denominator and that is the transfer of additional, new or corrected genetic information into cells which have a genetic or acquired defect.
  • genetic disorders eligible for gene therapy are cystic fibrosis, Duchenne's Muscular Dystropy, cancer, Gaucher disease, C ⁇ gler Najjar and Dubin-Johnson syndrome
  • acquired diseases are cancer, viral and parasitic diseases.
  • gene transfer can augment the efficacy of conventional therapies.
  • Vehicles for the transfer of genes into target cells and tissues include vectors of viral and non-viral origin Among the viral vectors munne based retroviruses and human based adenoviruses are the preferred embodiments
  • Retroviruses are RNA viruses which efficiently integrate their genetic information into the genomic DNA of infected cells via a reverse-transcribed DNA intermediate as a proviral copy. Integration into the host's genome and the tact that parts of their genetic material can be replaced by foreign DNA sequences make retroviruses one of the more lucrative vectors for gene delivery n human gene therapy procedures, most notably for gene therapies which rely on gene transfer into dividing tissues. Recombinant urine retroviruses have been the vectors of choice since the start of gene therapy and several clinical trials using recombinant retroviruses are ongoing. In order to generate a recombinant retrovirus which carries the cDNA sequence of a particular gene one needs to introduce the retroviral construct into an appropriate packaging cell line.
  • the retroviral construct carries the cDNA of interest and the cis acting elements for packaging and transcription of the viral RNA genome.
  • the packaging cell line provides the trans acting factors needed for packaging: the gag, pol and env genes. Expression of the retroviral construct into the packaging cell line results in the production of recombinant retroviral particles capable of transducing susceptible target cells and transferring a particular therapeutic gene.
  • the recombinant retrovirus is stably integrated into the target cell genome and conferred to its daughter cells upon cell division.
  • Adenoviruses are non-enveloped DNA viruses.
  • the genome consists of a linear, double stranded DNA molecule of about 36 kb.
  • Recombinant adenovirus vectors have been generated for gene transfer purposes.
  • Recombinant adenoviruses can be generated by co-transfection of two El -deleted recombinant adenoviral DNA constructs, one of which comprising the sequences of interest, into an El-expressing cell line.
  • adenoviruses do not integrate into the host cell genome, are able to infect non-dividing cells and are able to efficiently transfer recombinant genes in vivo.
  • AAV Adeno Associated Virus
  • adenoviruses and retroviruses are preferred embodiments, because of the extensive experience with these viruses in gene therapy concepts .
  • Vectors comprising nucleic acids encoding and expressing functional members of the family of organic anion transporters disclosed in the present invention are of particular importance for the treatment of diseases caused by defects in these transporters .
  • diseases include Dubin-Johnson syndrome, Rotor syndrome and other cholestatic disorders.
  • the human Dubin-Johnson syndrome The earliest evidence for distinct canalicular transport systems for bile acids and non-bile acid organic anions came with the reco g nition of the human Dubin-Johnson syndrome; this is a rare congenital chronic conjugated hyperbilirubinemia.
  • liver histology of this syndrome is characterized by lysosomal pigment accumulation
  • Preferred target tissues for the genetic treatment of these diseases include the liver, gut and kidney
  • Retroviral vectors comprising the nucleic acid sequences disclosed in this invention are constructed as exemplified in EP/95 201211 0 incorporated herein by reference Recombinant retrovirus supernatant stocks are produced by introduction of the retroviral constructs in appropriate retroviral packaging cell lines .
  • Adenoviral vectors comprising the nucleic acid sequences disclosed in this invention are constructed as exemplified in EP/95.202213 5 incorporated herein by reference
  • Adenovirus stocks are produced by transfectmg the adenoviral construct into appropriate El complementing cell lines.
  • HSC Hematopoietic stem cells
  • the gene transfer efficiency into human HSC is insufficient for the treatment of most hematopoietic diseases This forms the bottle-neck for a broader application of bone marrow gene therapy Therefore, it is preferred to provide the recombinant retroviral vector with a marker sequence for positive selection of transduced cells. Selection for the presence of this sequence can be performed in vitro by culturing the transduced cells in the presence of a selective drug. Another approach is to select for transduced cells in vivo, following transplantation of transduced HSC. Both approaches can be taken by inclusion in the recombinant retroviral construct genes encoding transporter proteins conferring resistance to cytostatic drugs.
  • the members of the family of organic anion transporters disclosed in the present invention are important examples of genes that can be used for this purpose.
  • Another important embodiment of the present invention is the use of the disclosed members of the family of organic anion transporters to provide the hematopoietic system of cancer patients with resistance to chemotherapeutic drugs .
  • This makes increased dose-intensity in the chemotherapeutic treatment of cancer possible.
  • increasing the dose-intensity results in increased response rates and a higher proportion of cures.
  • Dose-intensity is the amount of drug administered per unit time, and can be augmented either by increasing the chemotherapy dose or by reducing intervals between cycles.
  • Dose-intensive chemotherapy can produce complete regressions and improve survival in patients with historically refractory solid tumors and non-Hodgkin's lymphomas.Dose-response relationships have been demonstrated for many anticancer drugs.
  • the major dose-limiting toxicity of many anticancer drugs is myelosuppression, which thus prevents optimum dose- intensity administration. Severe myelosuppression makes the patient particularly prone to opportunistic infections and is a frequent reason for curtailing chemotherapy before an adequate therapeutic response has been obtained.
  • DHFR dihydrofolate reductase
  • MDR1 drug pump Members of the family of drugs extruded from the cell by the MDR1 drug pump are e.g. anthracyclines, vinca alkaloids, podophyllotoxins, and colchicine. Etoposide, a commonly used podophyllotoxin of which the dose-limiting toxicity is restricted to the hematopoietic system, is also pumped by the Rl encoded drug pump albeit only poorly.
  • the MDR related drugs have in common that they are lipophilic compounds derived from various natural products. In general, MDR cells are not cross-resistant to alkylating agents (e.g., chlorambucil and cyclophosphamide) , antimetabolites (e.g.
  • cisplatin carboplatin or melphalan.
  • the members of the family of organic anion transporters disclosed in the present invention efficiently extrude organic anion compounds from the cell, including GS-DNP and chemotherapeutic agents such as the conjugated forms of cisplatin, carboplatin, etoposide, chlorambucil, and melphalan. This is shown in a nonlimiting example for cisplatin below.
  • Example 1 Identification and isolation of the rat cmoat (mrp2 ) .
  • a 213-bp PCR product was obtained from rat lung cDNA after first round amplification with degenerate primers corresponding to ammo acid residues 678-648 (forward) and 770-776 (reverse), and subsequent second round amplification v/ith nested primers corresponding to am o acid residues 694-700 (forward) and 760-766 (reveise) of the M4RP1 sequence (5)
  • Partial cDNA clones were isolated from a rat hepatocyte cDNA library (23) which was screened with the 213-bp probe according to standard procedures (24) From a 4 5-kb positive clone a 5 '-located, 0 6-kb HphI restriction fragment was used to screen a gtlO 5 '-stretch rat liver cDNA library (Clontech, Palo Alto) A 0 8-kb overlapping clone was obtained from which a 0 6-kb Avail probe was isolated to rescreen the same library, resulting in the isolation of another overlapping clone.
  • cDNA synthesis was carried out with 5 ⁇ g of total RNA isolated from Wistar rat liver and random hexamer primers using Superscript Reverse Transcriptase II. After purification the cDNAs were tailed with a synthetic oligonucleotide anchor sequence using a 5 ' -RACE kit (Life Technologies, Gaithersburg) .
  • Hybridization of the filters was performed at 65°C in 0.5 M NaP0 4 (pH 7 0), 2 mM EDTA, and 7% SDS (hybridization solution), for 20 hours. Filters were washed four times in 2x SSC, 1% SDS for 30 min at 65°C, and autoradiographed. Nucleotide sequences were determined by the dideoxy-nucleotide chain method [F.Sanger, S. Nicklen, A.R. Coulson, Proc. Natl . Acad. Sci . U.S.A. 74, 463 (1977)] . The cmoat sequence is being submitted to the Genbank database and is available under accession number L49379.
  • a fusion gene consisting of the gene for the Escherichia Coli maltose-binding protein, and the 3' part of the cmoa t cDNA corresponding to amino acid residues 1340-
  • Hybridomas were screened by ELISA with the glutathione-S-transferase-cMOAT fusion protein and subsequently tested for positivity in Western blots .
  • cmoat cDNA was amplified from liver, kidney, ileum and duodenum from both Wistar and TR " rats using primers corresponding to amino acid residues 366-375 (forward) and 451-458 (reverse) of the cmoat sequence.
  • the obtained PCR product was digested with Nialll . In all PCR products from TR " rat digestion produced two bands of 206 and 66 bp whereas in the Wistar three bands of 83, 122 and 67 bp were observed.
  • RNA was fractionated on a 0.8% denaturating agarose gel, transferred to Hybond N + nitrocellulose membrane filters and hybridized with a [a- 32 P]dCTP-labeled 213-bp rat lung mrpl probe and a 1-kb HindiII/AvalI fragment of cmoat m hybridization solution (11) for 20 hours at 65 C Filters were washed 4x30 mm 0 2x SSC/0 1% SDS at 65°C and autoradiographed A 32 P- labeled 1 2-kb PstI fragment of the rat glyceraldehyde-3- phosphate dehydrogenase cDNA [Ph Forth et al , Nucleic Acid Res 13, 1431 (1985)] was used to estimate variations in RNA loading
  • Membranes containing 50 ⁇ g of protein were fractionated by 7 5% SDS polyacrylamide gel electrophoresis, electrophoretically transterred to nitrocellulose filters blocked for at least 2h in PBS/M/T (phosphate-buffered saline containing 1% BSA and 1% milk powder and 0 05% Tween-20), and incubated with the monoclonal antibody (M2 III-5 hyb ⁇ doma culture medium diluted eightfold with PBS/M/T) for 2h Immunoreactivity was visualized with peroxidase-conjugated rabbit anti-mouse lmmunoglobulms and subsequent staining with 3,3'- diaminobenzidine and 4-chloro-l-naphthol substrate P- glycoprote s were detected using the monoclonal antibody C219 and peroxidase-conjugated rabbit anti-mouse IgG Immune complexes were visualize
  • the human homolog of the rat cMOAT cDNA was isolated using a 4 kb fragment of the rat cMOAT cDNA The fragment was labelled as described for the rat cMOAT cDNA. The labelled probe was then used to screen a human lambda gtll liver cDNA library. Three clones with inserts hybridizing with the rat cMOAT cDNA sequence were isolated and designated clone 12,7 and 20. Clone 12 contained an insert of 2716 nucleotides comprising coding sequence 130-2846
  • Clone 7 contained an insert of 2000 nucleotides comprising coding sequence 2517-3185.
  • Clone 20 contained an insert of 2231 nucleotides comprising the coding sequence 3069-5300 Missing nucleotides 1-130 encompassing the translation initiation site were obtained from the Wash ⁇ -Merck EST library, clone 1243479. Furthermore noncoding 3' sequences were found to be present in additional EST clones and were used to complete the full coding sequence of the human cMOAT cDNA. Clones 193244 and 199655 were used for this purpose and completed the full length sequence from 5300 to 5582 nucleotides .
  • the rat cMOAT cDNA was cloned into the mammalian expression vector pSVK3 (Pharmacia).
  • pSVK3-rat-cMOAT and ⁇ SVK3 with rat-cMOAT in the reverse orientation (pSVK3-rat-cMOAT/Rev) relative to the promotor were transfected into COS-7 cells grown in 75 cm 2 tissue culture flasks.Three days after transfection, the cells were used for GS-DNP transport experiments and analyzed for cMOAT protein expression using anti-cMOAT antibodies.
  • For transport measurements the cells were washed with Hanks buffer and loaded with Hanks/l 4 C-CDNB at 15'C. Samples were taken after various time points.
  • COS-7 cells transfected as described above were also used to isolate membrane vesicles and perform transport experiments
  • cell homogenates were prepared from transfected COS-7 cells and were centrifuged over a discontinous gradient of 19, 38 and 56 % sucrose The 38-19 % interface was collected and revesiculated and total protein content was determined using the Lowry method
  • the vesicle suspensions were incubated wuth 3 H-GS-DNP at 37 "C in the presence of an ATP regenerating system
  • vesicles isolated from cells expressing rat-cMOAT exhibited GS-DNP transport above the level of transport observed with vesicles isolated from pSVK3-rat-cMOAT/Rev transfected COS-7 cells. This transport was completely dependent on the presence of ATP characteristic for a member of the ABC transporter superfamily (figure 6).
  • the human LLC-PK1 cell line was also transfected with the same human cMOAT DNA construct and 90 clones were screened for expression None of these clones expressed the human cMOAT protein as detected with antibody M2III-6. In contrast, expression of human cMOAT in yeast was also studied and was high after the translational core sequences of the human cMOAT cDNA were converted to yeast consensus sequences
  • Phenix cells were transfected with pCMV- neo-human-cMOAT DNA using a commercially available calcium phosphate transfection kit (Gibco/BRL) . After 16h at 5% C0 2
  • MDCKII MDCK cell line strain II
  • DOTAP Transfection Reagent
  • Stably infected cells were selected for 2-3 weeks in medium with G418 at 200 mg/ml . Thirty clones were picked and analyzed for the presence of hcMOAT protein. Western blot analysis of crude membrane fractions of these clones revealed that several clones contained a substantial, but between individual clones variable, amount of human cMOAT. Two of these clones are shown in Figure 7. A weak signal was observed in wild- type MDCKII cells with a slightly higher molecular weight than hcMOAT after prolonged exposure. This might either represent canine cMOAT or another protein to which this mAt cross-reacts.
  • Antibody binding was detected with a FITC-labeled sheep anti-mouse IgG (1:50; Boehringer Mannheim, Germany). Filters were mounted with Vectashield (Vector Laboratories, Burlinga e, CA) containing propidiumiodide (1 mg/ml) for counterstaining of nucleic acids. Cells were examined with a MRC-600 confocal microscope (Bio Rad, Hertfordshire, UK) . Expression of hcMOAT protein was visualized by indirect immunolocalization using confocal laser scanning microscopy (CLSM) .
  • CSM confocal laser scanning microscopy
  • Example 5 A mutation in the human cMOA T gene causes the Dubin-Johnson syndrome.
  • the human Dubm-Johnson syndrome is an autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubmemia Patients have impaired hepatobiliary transport of non-bile salt organic anions a phenotype similar as has been described for the TR " rat, which has a defective cmoat.
  • TR " rats and Dubm-Johnson patients we have tested whether a mutation in the human cMOAT gene also underlies the transport defect in Dubm-Johnson syndrome.
  • the human homologue of rat cMOAT, human cMOAT also subject of this invention is deficient in a patient with Dubm-Johnson syndrome.
  • liver fibroblasts from the patient and a normal control were obtained by skin biopsy and cultered in Ham F-10 (Life Technologies), supplemented with 10 % fetal bovine serum and antibiotics, at 37 C.
  • Paraffin-embedded liver sections of Dubin-Johnson and control liver were examined for the presence and localization of the cMOAT protein, using monoclonal antibody M 2 III-6.
  • formaldehyde-fixed paraffin- embedded liver sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activicy was blocked with 0.3 % (v/v) H2O 2 in methanol for 30 min. Before staining, the sections were pretreated with 0.01 M citric acid (pH
  • the M2IH-6 antibody was produced against a bacterial fusion protein containing the 202-amino acid COOH-terminus of rat cMOAT; it cross-reacts with human cMOAT, but not with human MRPl.
  • human control liver like in rat control liver, the antibody stained the canalicular membrane of the hepatocyte.
  • PCR reactions were carried out in a Perkin Elmer GeneAmp PCR system 2400, in Ix Taq polymerase buffer (Life Technologies), 1.5 mM of MgCl2, 0.5 mM of dNTPs, 400 nM of each primer, and 0.5 units of Taq polymerase.
  • the PCR products were obtained after application of the "touch down" PCR protocol; the reactions were denatured at 96 C for 5 min, followed by five times 2 cycles with annealing temperatures of 72, 70, 68, 66, and 65 C respectively, and subsequent 30 cycles with an annealing temperature of 64°C. Each cycle started with 20 s at 94°C, 30s at the indicated annealing temperature, and 90s at 72°C.
  • PCR reaction was terminated after an extension step at 72°C for 10 min.
  • PCR fragments obtained from fibroblasts were excised from agarose gel, purified, ligated into the TA-cloning plasmid pCRTMII (Invitrogen, Leek, The Netherlands), and transformed into INVaF' competent cells (Invitrogen). White colonies were picked, grown overnight, and plasmid DNA was isolated using the alkaline lysis method. Nucleotide sequences of 5-8 pooled clones were determined by the dideoxynucleotide chain method.
  • the identi ication of a mutation in human cMOAT in a patient with the DJS confirms the hypothesis that the TR " rat is an animal model for Dubin Johnson syndrome and provides additional evidence that the cMOAT gene encodes the major transporter for organic anions in the liver canalicular membrane.Our demonstration of a low, but detectable expression of the cMOAT gene in fibroblasts in addition to a nucleic acid based diagnostic assay for Dubm- Johnson syndrome, allows a simple identification of this inherited disorder, without the need for liver biopsy.
  • Example 6 Identification and isolation of other members of the family of anorganic anion transporters .
  • All cells were grown in DMEM or RPMI medium (Gibco, BRL), supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (50 units/ml) and streptomycin (50 ⁇ g/ml) . All cells were free of Mycoplasma as tested by the use of the Gene-Probe rapid Mycoplasma detection system (Gen-Probe, San Diego, USA).
  • the drug sensitivity of cells was determined in clonogenic survival assays in the continuous presence of drugs. Five hundred cells per well were seeded in 24-well plates and incubated for 24 hrs at 37 C. Drugs, of which concentrations were varied in 2-fold steps, were added and cells were o incubated for 5-6 days at 37 C. After this the cells were stained with 0.2% crystal violet in 3.7% glutaraldehyde and colonies containing more then 50 cells were counted. The relative resistance was calculated as the ratio of IC50
  • human cDNA clones were obtained from the I.M.A.G.E. consortium (64). Additional MRP3 cDNA clones were isolated by screening a human liver 5' stretch plus cDNA library, oligo(dT) and random primed (Clontech, Palo Alto, USA), using a 1 kb EcoRI - SacI fragment of a human cDNA clone (no. 84966, Stratagene liver cDNA library #937224) as probe. Several overlapping cDNAs were isolated and sequenced. For MRP4 the insert of a human cDNA clone (no.
  • MRP5 cDNA clones were isolated by screening a fetal brain cDNA library (Clontech, Palo Alto, USA), using the insert of human cDNA clone (no. 50857, Soares infant brain 1NIB cDNA library) as probe (J. Wijnholds, C. Mol, and P. B., unpublished results) .
  • Several overlapping cDNAs were isolated and sequenced. For sequencing the ABI 377 Automatic Sequencer was used. Sequence analysis was done using the GCG package of the Wisconsin University (20). All the sequences have been deposited with GenBank ( MRP3 accession number U83659; MRP4 accession number U83660; MRP5 accession number U83661) .
  • Cytoplasmic RNA from cell lines was isolated by a Nonidet P- 40 lysis procedure (24) .
  • Total cellular RNA from tissue samples obtained during surgery or at autopsy was isolated by acid guanidium isothiocyanate-phenol-chloroform extraction (65).
  • nts nucleotides 4136-4376 (49; GenBank accession number U49248) was generated.
  • the primers used for amplification were 5' -CTGCCTCTTCAGAATCTTAG-3 ' (forward primer) and 5 ' -CCCAAGTTGCAGGCTGGCC-3 ' (reverse primer).
  • MRP3, MRP4 , and MRP5 RNA detection were generated by PCR amplification: (i) for MRP3 a 262 bp fragment was generated using the primers 5' -GATACGCTCGCCACAGTCC-3 ' (forward primer) and 5 ' -CAGTTGGCCGTGATGTGGCTG-3 ' (reverse primer); (ii) for MRP4 a 239 bp fragment was generated using the primers 5'- CCATTGAAGATCTTCCTGG-3 ' (forward primer) and 5'- GGTGTTCAATCTGTGTGC-3 ' (reverse primer); (iii) for MRP5 a 381 bp fragment was generated using the primers 5 ' -GGATAACTTCTCAGTGGG-3 ' (forward primer) and 5' -GGAATGGCAATGCTCTAAAG-3 ' (reverse primer).
  • RNA transcripts were transcribed from Wotl-linearized DNA of hcMOAT-241 and MRP3-262, using T7 RNA polymerase, or from Ncol -linearized DNA from MRP4-239 and MRP5-381, using Sp6 RNA polymerase.
  • RNA detection For MDRl RNA detection, a 301 bp MDRl cDNA fragment was used (nt positions 3500-3801 (66)), and for MRPl RNA detection a 244 bp MRPi cDNA fragment was used (nt positions 239-483 (7)) .
  • RNase protections were carried out according to Zinn et al . (67), modified by Baas et al . (58) . Protected probes were visualized by electrophoresis through a denaturing 6% acrylamide gel, followed by autoradiography. In all experiments a probe for actin (68) was included as control for RNA input.
  • the amount of MDRl , MRPl , cMOAT, MRP3 , MRP4 , or MRP5 RNA relative to the amount of actin was calculated using a phosphorimager (Fuji BAS 2000, TINA 2.08b) .
  • Total cell lysates were made by lysing harvested cells in 10 mM KC1/1.5 mM MgCl /10mM Tris-HCl, pH 7.4/0.5% (wt/vol) SDS supplemented with 1 mM phenylmethylsulfonyl fluoride, leupeptin (2 ⁇ g/ml) , pepstatin (1 ⁇ g/ml), and aprotinin (2 ⁇ g/ml). DNA was sheared by sonication and samples containing 40 ⁇ g of protein were fractionated by SDS/7.5% PAGE and then transferred onto a nitrocellulose filter by electroblotting .
  • cMOAT protein was detected with mouse monoclonal antibodies M2lH"5 or M2III-6, generated against a bacterial fusion protein containing the 202 amino acid COOH-terminus of rat cmoat (48) .
  • Immunoreactivity was visualized with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dako, Denmark) followed by enhanced chemiluminescence detection (Amersham, U.K. ) .
  • fusion proteins were made of the Escherichia col i maltose-binding protein with COOH-terminal ends of human cMOAT, MRP3, and MRP5, respectively, using the plasmid vector pMal-c (69) .
  • the expression plasmids encoded, respectively, for cMOAT the 202-ammo acid COOH-terminal end, for MRP3 the 190-am ⁇ no acid COOH-terminal end, and for MRP5 the 169-ammo acid COOH-terminal end.
  • the fusion proteins were produced in E . coli DH5a and purified by amylose resin affinity chromatography (69).
  • MRP3, MRP4, and MRP5 For the chromosome localization of MRP3, MRP4, and MRP5, radiation hybrid mapping was performed with MRP3, MRP4 , and MRP5 specific primers and two different cell panels, Stanford G3 (StG3; 71) and Genebridge 4RH (Gb4RH; 72) .
  • the primers used for amplification were: (i) for MRP 3 5 ' -CTCAATGTGGCAGACATCGG-3 ' and 5 ' -GGGAGCTCACAAACGTGTGC-3 ' ; (ii) for MRP 4 5 ' -CCATTGAAGATCTTCCTGG-3 ' and 5'- GGTGTTCAATCTGTGTGC-3 ' ; (iii) for MRP 5 5'-
  • MRP homologs other than MRPl and cMOAT. Alignment and comparison of EST sequences with homology specific to the 3 '-terminal ends of MRPl and cMOAT, including the coding sequence for the second ATP-binding domain, revealed that there are at least 4 more MRP homologs expressed in humans.
  • One of these homologs is the human sulfonylurea receptor ( SUR) gene (73). The other three MRP homologs had not been identified before, and were designated MRP3, MRP4, and MRP5.
  • MRP3 and MRP5 Additional cDNA clones for MRP3 and MRP5 were isolated from a human liver and a fetal brain cDNA library, respectively.
  • MRP3 and MRP5 cDNA clones were sequenced as well as the MRP4 cDNA clone obtained from the I.M.A.G.E. consortium.
  • Both MRP3 and MRP5 encode four domain proteins, i.e. proteins with two ATP-binding domains and two domains with transmembrane regions (M.K. and J. Wijnholds, unpublished results). More sequence data will determine whether this is also the case for MRP4 .
  • Figure 14 shows the protein alignment for the COOH-terminal ends of the various members of the human MRP family and human SUR.
  • the alignment includes the Walker A and B motifs and the signature sequence of the second ATP-binding domain.
  • the percentages of homology for the COOH-terminal 124 amino acids are shown in table 1. The highest homology is found between MRPl and MRP3 (86% similarity) and the lowest between SUR and any of the MRPs ( ⁇ 69% similarity) .
  • MRP3 , MRP4 , and MRP5 Chromosome localization of MRP3, MRP4 , and MRP5
  • the MRPl gene has been mapped to chromosome 16 at band pl3.13-13.12 (5) and recently the cMOAT gene to chromosome 10, band q24 (52, 74) .
  • MRP3 , MRP4 , and MRP5 are located on chromosomes 17, 13, and 3, respectively.
  • cMOAT and the three new MRP homologs are believed to encode transporter proteins involved in drug resistance.
  • High MDRl overexpression was detected only in two sublines of the human non-small-cell lung cancer cell line SW1573/S1, both selected for high level doxorubicin resistance (2R160 and 1R500) .
  • the low level of MDRl RNA in the other cell lines is not remarkable as most of the cell lines selected for our panel were known to have a non-Pgp MDR phenotype. Low MDRl overexpression was found in the
  • MRPl RNA is highly overexpressed in the four non-Pgp MDR cell lines GLC 4 /ADR, MOR/R, COR-L23/R, and HL60/ADR, all selected for high level doxorubicin resistance (7, 75, 76).
  • the doxorubicin selected cell lines derived from the SW1573/S1 cell line, showed no or only a minor increase in MRPl RNA, as reported before (7, 77). In the cell lines, selected for cisplatin resistance, we detected no major changes in MRPl RNA.
  • cMOAT varied greatly between the cell lines Most parental cell lines did not express cMOAT or at very low levels Only the MOR/P and the KB-3-1 parental cell lines showed substantial cMOAT RNA levels Overexpression of cMOAT was found in several doxorubicin-resistant sublines of SW1573/S1 (30 3M, lR50b, 2R120, 2R160, and 1R500), and some cisplatin selected cell lines (2008/C13*5 25, 2008/A, A2780/DDP, and HCT8/DDP)
  • MRP4 is expressed only at low or very low levels in the cell lines we analyzed and no overexpression of MRP4 was detected in resistant sublines
  • MRP5 is expressed in every cell line we analyzed, with the highest levels in MOR/P and 2008, but in none of the resistant sublines MRP5 is highly overexpressed Only in three cisplatin resistant cell lines, T24/DDP10, HCT8/DDP, and n the KCP-4(-), a minor increase in MRP5 RNA was detected
  • MRP5 and MRP5 RNA is raised in the KCP-4(-) cells, but a similar result was obtained with Mab M2III-6 which does not cross-react with MRP5.
  • MRP3 , MRP4 , and MRP5 are not alternative splice products of MRPl oi cMOAT Klugbauei and Hofmann (78) recently cloned another ABC transporter ( ABC-C) , located in the same chromosomal band as MRPl , but this is not a MRP homolog, because the identity between these two proteins is only 18%.
  • ABC-C ABC transporter
  • liver canaliculi and erythrocytes contain two different ATP- dependent transport activities for organic anions (81-841 cMOAT is localized in the canalicular membranes of hepatocytes and the absence of th s protein in the TR " rats as well as in a patient with the Dubin-Johnson svndrome shows a role for the cmoat/cMOAT proteins in the transport of non-bile acidic organic compounds from liver to bile (48 49, 55)
  • the other ATP-dependent transport activity in liver canaliculi responsible for transport of bile acids from liver to bile is not attributable to cMOAT, because studies with TR " rats and Dubin-Johnson patients showed that
  • BRIC Benign Recurrent Intrahepatic Chclestasis
  • PFIC Progressive Familial Intrahepatic Cholestasis
  • MRP3 may be the bile salt transporter Since none of the human ABC transporter genes identified thus far maps to chromosome 18 (79, this study) it is unlikely that BRIC/PFIC is caused by a defect in a readily recognizable ABC transporter gene
  • GS-X activity has also been found in erythrocytes
  • human and rat erythrocytes contain a low- and a high-affinity S- (2 , 4-dinitrophenyl) - glutathione (DNP-SG) transporter (84, 91, 92) .
  • DNP-SG transporter is most likely MRPl, since the presence of this protein and its binding to LTC4 have been shown for erythrocytes (93, 94).
  • the other transporter with low affinity for DNP-SG but high affinity for glucuronides and mercapturates (84) is not cMOAT or the bile salt transporter, because (i) no major alterations in DNP-SG transport in erythrocytes from TR " rats and Dubin-Johnson patients were detected (IB), and (ii) erythrocytes transport DNP-SG and GSSG but no bile salts (83) .
  • This second transporter may be encoded by one of the other MRP homologs.
  • MRP4 was not overexpressed in any of the lines .
  • MRP3 RNA was only found to be elevated in the cisplatin resistant HCT8/DDP cell line and several SW1573/S1 sublines selected for doxorubicin resistance. However, overexpression did not correlate with the level of doxorubicin resistance.
  • MRP5 low overexpression was found in three cell lines selected for cisplatin resistance (T24/DDP10, HCT8/DDP, and KCP-4(-); Table 4), but many other cisplatin selected cell lines showed no overexpression.
  • Table 3 shows that the classical non-Pgp cell lines selected for high doxorubicin resistance and known to highly overexpress the MRPJ gene, do not significantly overexpress other members of the MRP family. This is compatible with the interpretation that MRPl is the transporter responsible for MDR in these cell lines. In the non-Pgp derivatives of the SW1573/S1 cell line presented in Table 3 a more complex situation is found and the contribution of MRPl, cMOAT, MRP3, and the major vault protein, also present at increased levels in some of these cell lines (95), remains to be sorted out . 6.3.4 The involvement of organic anion transporters in cisplatin resistance
  • organic anion transporters such as MRPl and cMOAT have been speculatively linked to resistance to oxyanions (arsenite, antimonite) and cisplatin
  • oxyanions arsenite, antimonite
  • cisplatin organic anion transporters
  • These compounds can form complexes with GSH and there is now considerable evidence that these complexes are substrates for organic anion transporters Resistance caused by increased export of these complexes is bound to be complex as pointed out by Ishikawa (80) and by us (6b, 19, 95) Increased levels of pump or GSH, increased GSH synthesis, oi a combination may be required depending on the rate limiting step in drug export
  • Some other cisplatin-resistant lines contain increased levels of cMOAT as well, notably 2008/C13*5.25, 2008/A, A2780/DDP, and HCT8/DDP (Fig. 3, Table 4 and 5).
  • the combination of cisplatin with doxorubicin resistance n resistant cell lines has been reported before (100, 103) and is also present in twj other platin selected lines, studied here, PXN94/tetR and KCP-4(-) (Table 5) . All other platin-selected lines in Table 5 are doxorubicin sensitive, however.
  • Figure la cDNA sequence of human cMOAT Figure lb. cDNA sequence of rat cMOAT Figure 2.
  • A Northern blot analysis of 2 ⁇ q poly(A) + RNA from Wistar rat tissues hybridized to a 1-kb Hmdl ll/Avall cDNA fragment of cmoa t RNA was analyzed as described in (the experimental part Prolonged exposure of the film revealed no detectable expression m other tissues then kidney, duodenum, and ileum
  • B Northern blot analysis of 2 ⁇ g of poly(A) "r RNA from Wistar and TR " rat liver and hepatocytes hybridized with the same probe as described m
  • A The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal is shown at the bottom Molecular size standards are indicated at the right
  • FIG. 3 Immunoblot analysis of cmoat and P-glycoprotem in canalicular and basolateral membrane fractions of Wistar and TR " rat hepatocytes Lane 1, Wistar basolateral membranes, Lane 2, Wistar canalicular membranes, Lane 3 TR “ basolateral membranes, Lane 4, TR " canalicular membranes Upper panel the blot was incubated with the monoclonal antibody M2 III-5 directed to cmoat (27) This antibody did not crossreact with the hMRPl protein as tested in total lysates from the MRP-overexpress g cell line GLC4/ADR (20) Lower panel.
  • Figure 4 Deduced amino acid sequence of the rat cMOAT and alignment with the deduced 70 am o acid sequence of the translated 213-bp putative rat mrpl cDNA
  • FIG. 1 Transport of GS-DNP in COS-7 cells transiently transfected with rat cMOAT expression constructs
  • Closed circles represent cells transfected with pSVK3-rat cMOAT
  • Open circles represent cells that have been transfected with a pSVK3 construct with the rat cMOAT cDNA in the reverse orientation and serves as a negative control
  • the results depicted are the mean of three measurements .
  • Figure 6 Transport of GS-DNP in membrane vesicles prepared from COS-7 cells transiently transfected with rat cMOAT expression constructs in the presence or absence of an ATP regenerating system Closed squares represent cells transfected with pSVK3-rat cMOAT Open squares represent cells that have been transfected with a pSVK3 construct with the rat cMOAT cDNA in the reverse orientation and serves as a negative control The results depicted are the mean of three measurements.
  • Figure 7 Human cMOAT expression in crude lysates from MDCKII derived transfectants 2 or 20 mg of total protein was size fractionated in a 7 5% polyacrylamide gel containing 0.1% (wt/vol) SDS.
  • MDCKII-217 cells Nucleic acids were detected using propidiumiodide (red signal). Top view of the cell layer is shown. B. Optical section perpendicular to the plane of the cell layer.
  • Figure 10 Deduced amino acid sequence of human cMOAT . Predicted transmembrane regions are underlined. Walker A, B, and signature sequence are doubly underlined. Predicted N- glycosylation sites conserved in other cMOAT proteins (rat, rabbit) and MRPl proteins (human, mouse) are indicated with triple asterisks. The triangle indicates the location (amino acid 1066) at which a stop codon is introduced by a C to T transition in DJS cMOAT .
  • Figure 11 Immunohistochemical detection of the cMOAT protein in human and rat liver using monoclonal antibody 2III-6. Sections of a normal human liver (A) and normal rat liver ( B) , which demonstrate the exclusive canalicular localization of the protein.
  • MRP homologs and human SUR The alignment was performed with the PILEUP program of GCG (48) .
  • GenBank accession numbers for the proteins used n this comparison are the following MRPl - L05628, CM0AT/MRP2 - U49248, MRP3 - U83659, MRP4 -U83660, MRP5 - U83661, SUR - L78207
  • the nucleotide binding domain specific signature sequence and the Walker A and B motifs are shown in bold Asterisks above the alignment indicate identical amino acids in at least four of the five MRP proteins
  • Figure 15 RNase protection assays of RNA transcript levels of MDRl MRPl cMOAT ( MRP2 ) MRP3, MRP4 , and MRP5 m the human non- small-cell lung cancer cell line SW1573/S1 and its doxorubicin selected subiine 30 3M 10 ⁇ g total cytoplasmic RNA from each cell line was used per probe The positions oi the protected fragments
  • RNA levels were determined as m Figure 2. The relative expression level is indicated by filled circles, very low expression by _, and undetectable RNA levels by open circles .
  • Table 4 Characteristics of cell lines selected for resistance to cisplatin, tetraplatin or CdCl2- Resistant cell lines were selected by chronically exposing them to the concentrations of drugs as shown Only A2780/DDP and HCT8/DDP were selected by challenging them 1 h weekly with 50 ⁇ M cisplatm RNA levels were determined as in Figure 2.
  • the relative expression level is indicated by filled circles, very low expression by _, and undetectable RNA levels by open circles .
  • Data for total intracellular glutathione concentrations were obtained from three independently isolated cell extracts assayed in three independent experiments using the recycling method of Tietze (56) and presented as the mean GSH ⁇ SD.
  • IC5 Q values and relative resistance factors (RF) of the cell lines analyzed for cisplatin and doxorubicin were obtained from clonogenic survival assays with continuous exposure to drugs.
  • the relative resistance factor was determined by dividing the IC50 °f each resistant cell line by the IC 5 0 of the corresponding parental cell line. Also shown are the levels of RNA transcripts of MRPl and cMOAT , taken from Table 3 and 4.
  • the human multidrug resistance- associated protein MRP is a plasma membrane drug-efflux pump. Proc. Natl. Acad. Sci. , USA 91 : 8882-8826, 1994.
  • amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
  • yeast cadmium factor protein (YCF1) is a vacuolar glutathione 5-conjugate pump. J. Biol. Chem. 271 : 6509-6517, 1996. 34. T. Ishikawa and F. Ali-Osman, Glutathione-associated czs-diamm ⁇ nedichloroplatinum(II) metabolism and ATP- dependent efflux from leukemia cells. J. Biol. Chem. 268 : 20116-20125, 1993. 35. T. Ishikawa et al .
  • GS-X pump is functionally overexpressed m cis-diamm ⁇ nedichloroplatmum(II ) - resistant human leukemia HL60 cells and down-regulated by cell differentiation. J. Biol. Chem. 269 : 29085- 29093, 1994.
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