WO1998029437A2 - Mammalian equilibrative nucleoside transporters - Google Patents

Mammalian equilibrative nucleoside transporters Download PDF

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WO1998029437A2
WO1998029437A2 PCT/IB1997/001657 IB9701657W WO9829437A2 WO 1998029437 A2 WO1998029437 A2 WO 1998029437A2 IB 9701657 W IB9701657 W IB 9701657W WO 9829437 A2 WO9829437 A2 WO 9829437A2
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seq
polypeptide
polynucleotide
group
nucleoside transporter
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PCT/IB1997/001657
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French (fr)
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WO1998029437A3 (en
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James D. Young
Carol E. Cass
Stephen A. Baldwin
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Governors Of The University Of Alberta
The University Of Leeds
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Priority to AU57756/98A priority Critical patent/AU5775698A/en
Publication of WO1998029437A2 publication Critical patent/WO1998029437A2/en
Publication of WO1998029437A3 publication Critical patent/WO1998029437A3/en

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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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This application relates generally to nucleoside transporters, and more specifically to a polynucleotide which encodes a nucleoside transporter that may be useful for the development of new nucleoside drugs.
  • Adenosine for example, functions as a local hormone in regulation of lipolysis. neurotransmitter release, platelet aggregation, coronary vasodilation. and cardiac contractility (Belardinelli, L., et al., 1989, Prog. Cardiovasc. Dis. 32:73-97; Jacobson, .A.. et al. (eds), 1990, Purines in Cellular Signaling: Targets for New Drugs, Springer-Verlag,
  • Nucleoside antimetabolites have therapeutic applications in human neoplastic and viral diseases, including leukemias and AIDS (Perigaud et al. 1992. Nucleosides & Nucleotides 11, 903-945; Handshumacher and Cheng, 1993, in: Cancer Metabolism (Holland, E., Frei, E., Bast, R.C., Kufe, D.W., Morton, D.L., and Weichselbaum, R.R. eds)pp. 712-732, Lea & Febiger, Philadelphia.). Nucleoside transport processes also play an important role in adenosine-mediated regulation of coronary vasodilation. renal vasoconstriction, neurotransmission, platelet aggregation and lipolysis (Belardinelli et al., 1989; Jacobson et al., 1990).
  • nucleoside drugs act intracellularly, after anabolic phosphorylation, by interfering either directly or indirectly with DNA synthesis.
  • mediated nucleoside transport systems NT processes
  • NT processes mediated nucleoside transport systems
  • the activity of NT processes can be an important determinant of pharmacologic action of cytotoxic nucleoside drugs.
  • cultured cells made incapable of transporting nucleosides by genetic mutations or treatment with NT inhibitors exhibit low levels of uptake of adenosine and other endogenous nucleosides and are resistant to a variety of nucleoside analogs with anticancer activity.
  • the permeant selectivities and mechanisms regulating distribution and expression of NT processes are important factors to be considered in the design of nucleoside analogs as therapeutic agents in human diseases.
  • the transport of nucleosides across the plasma membranes of mammalian cells occurs both by equilibrative (facilitated diffusion, sodium independent) processes and by concentrative (sodium-dependent) processes (Griffith, D.A. and Jarvis, S.M., 1996, Biochim. Biophys. Acta 1286, 153-181 ; Cass, C.E., 1995, in "Drug transport in antimicrobial and anticancer chemotherapy", Georgopapadakou, N.H. (ed.), Marcel Dekker, New York, pp. 403-451 ).
  • NBMPR 6-thiopurine ribonucleoside nitrobenzylthioinosine
  • ei- type transporters are generally less susceptible to the coronary vasodilators, dipyridamole and dilazep, and to lidoflazine analogues, than are the es-t ⁇ oe transporters from the same species (Griffith and Jarvis, supra). Both classes of transporter have a similar broad substrate specificity for purine and pyrimidine nucleosides, although some e -type transporters have been reported to have lower affinities for nucleosides than es-type transporters from the same animal species (Griffith and Jarvis, supra) or even from the same cell type (Boleti, H., et al, 1997, Neuropharmacol. 36, 1167-1 179). Although some cell types express only es or e -type transporters, many express both
  • the es and ei nucleoside transporters are under independent genetic control and are produced either singly or together in different cell types and tissues (Belt, J.A. and Noel, L.D., 1988, J. Biol. Chem. 263, 13819- 13822; Cass, C. E., et al.. 1974, Biochim. Biophys. Acta 345: 1 -10; Jarvis, S.M., and Young, J.D.. 1980, Biochem. J. 190:377-383; Jarvis, S.M., et al., 1980, Biochim. Biophys. Acta 597: 183-188; Jarvis, S.M., and Young, J.D., 1986, J. Membr. Biol. 93: 1-10; Belt, J.A., and Noel, L.D.. 1985, Biochem.
  • Transporters of the e.v-type are widely distributed in different cell types (Paterson et al., 1991 ; Kwong, F.Y.P, et al., 1993, J. Biol. Chem. 268:22127-22134) and tissues, bind NBMPR reversibly (Cass, C.E., 1995, in: Drug Transport in Antimicrobial and Anticancer chemotherapy, Georopapadakou, N.H., ed., Marcel Dekker, New York, pp. 403-4 1 ), and can be covalently radiolabeled with [ 3 H]NBMPR by exposure of the transporter-ligand complex to UV light (Wu. J.-S. R.. et al., 1983, J.
  • an es-type nucleoside transporter is the erythrocyte transporter, an integral membrane glycoprotein of apparent M r 55,000 (Wu et al. 1983), has been purified to apparent homogeneity by a combination of ion-exchange and immunoaffinity chromatography (Kwong et al.,1988, Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography, Biochem. J. 255, 243-249). The erythrocyte transporter is inhibited by nanomolar concentrations of NBMPR (Young, J. D. & Jarvis, S. M.. 1983, Nucleoside transport in animal cells. Biosci. Rep.
  • the invention is based on the discovery of equilibrative nucleoside transporters (ENT) that transport a variety of purines and pyrimidines, including, but not limited to adenosine, uridine, guanosine, inosine. formycin B, tubercidin, and thymidine.
  • ENTs are bidirectional; they transport a suitable permeant both into and out of cells.
  • the ENT can be used as a tool for the development of new nucleoside drugs. Two human and two rat genes encoding ENT have been isolated.
  • the invention provides substantially purified equilibrative nucleoside transporter polypeptide.
  • Isolated polypeptides having an amino acid sequence which are at least 85% identical to a polypeptide sequence including SEQ ID NO:2, SEQ ID NO:
  • SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 are also provided.
  • SEQ ID NO:6, and SEQ ID NO:8 are further disclosed.
  • Polynucleotides encoding the polypeptides of the invention are further provided.
  • Antibodies which bind equilibrative nucleoside transporter polypeptide and formulations for administration of these antibodies are also disclosed.
  • a method for identifying a compound which affects an equilibrative nucleoside transporter is provided.
  • a method for determining if a compound utilizes a equilibrative nucleoside transporter is also further provided.
  • a kit for the detecting the presence of an equilibrative nucleoside transporter is provided.
  • a kit useful for the detection of target nucleic acid sequences indicative of the ability to transport nucleoside using an equilibrative nucleoside transporter is also disclosed.
  • a method of assessing a therapeutic intervention for a subject, where the effectiveness of the therapeutic intervention is correlated with the presence or absence of an equilibrative nucleoside transporter is provided.
  • a method of treating a subject having or at risk of having a disorder associated with an equilibrative nucleoside transporter is also disclosed.
  • transgenic animals having a transgene encoding a equilibrative nucleoside transporter are provided.
  • Fig. 1 is the cDNA nucleotide sequence (SEQ ID NO: l ) and deduced amino acid sequence (SEQ ID NO:2) of human equilibrative nucleoside transporter (hENTl).
  • Fig. 2 is the cDNA nucleotide sequence of the hENTl (SEQ ID NO: l )
  • Fig. 3 is the deduced amino acid sequence (SEQ ID NO:2)of human equilibrative nucleoside transporter (hENTl).
  • Fig. 4 shows a topographical model of hENTl .
  • Potential membrane-spanning a- helices are numbered and the putative N-glycosylation site at Asn 8 is indicated.
  • the positions of basic (Arg, Lys. His), acidic (Asp, Glu) and polar but uncharged residues (Ser, Thr, Gin, Asn) are indicated by + , - and darkened circles respectively.
  • Fig. 5 is an alignment of the predicted amino acid sequence of hE ⁇ Tl as compared with the amino acid sequnece of the yeast protein FU ⁇ 26 (SEQ ID NO:9) (Ouellette et al.. 1993, Sequencing of chromosome I from Saccharomyces cerevisiae: analysis of a 32 kb region between the LTE1 and SP07 genes. Genome 36, 32-42), the nematode proteins ZK809.4 (SEQ ID NO: 10) and F16H 1 1.3 (SEQ ID NO: l 1), and the mouse (mHNP36)
  • Fig. 6A shows the time-course of uridine uptake into Xenopus oocytes.
  • Oocytes were injected with water (o) or with water containing in vitro transcribed R ⁇ A encoding hE ⁇ Tl
  • Fig. 6B illustrates the substrate specificity of hE ⁇ Tl -mediated uridine influx for physiological nucleosides and chemotherapeutic nucleoside analogues. The hatched column corresponds to uptake measured in the absence of sodium.
  • Fig. 6C shows the concentration- dependence of hE ⁇ Tl -mediated uridine influx. Oocytes were injected with water (o) or water containing in vitro transcribed R ⁇ A encoding hE ⁇ Tl (•). The inset shows influx of uridine in R ⁇ A-injected oocytes minus that in water-injected oocytes.
  • Fig. 6D shows the inhibition of hENTl -mediated uridine influx by NBMPR, dilazep and dipyridamole. The inset shows the dose-response curve for NBMPR inhibition of uridine influx.
  • Fig. 7 A shows the time course of adenosine uptake into Xenopus oocytes. Oocytes were injected with water alone (o) or with water containing in vitro transcribed RNA encoding hENTl (•).
  • Fig. 7B shows the inhibition of hENTl -mediated adenosine influx by uridine. NBMPR, dilazep and dipyridamole.
  • Fig. 8 is the cDNA nucleotide sequence (SEQ ID NO: 3) of the rat equilibrative nucleoside transporter (rENTl).
  • Fig. 9 is the deduced amino acid sequence (SEQ ID NO: 4) of rat equilibrative nucleoside transporter (rENTl ).
  • Fig. 10 shows the functional expression of rENTl Xenopus oocytes.
  • Influx of [ N C]-labelled uridine and adenosine ( 10 ⁇ M, 20°C) was measured in oocytes injected with water or with water containing RNA transcript. Values are means (+ SEM) of 10 oocytes.
  • Fig. 1 1 illustrates the substrate specificity of rENTl -mediated uridine influx. Influx of uridine (10 ⁇ M) in oocytes containing rENTl transporter was measured in the absence
  • Fig. 12 shows NBMPR and dipyridamole inhibition of hENTl and rENTl .
  • Uridine influx (10 ⁇ M) in oocytes containing either rENTl (m) or hENTl (1) transporter was measured in the presence of increasing concentrations of NBMPR (A) and dipyridamole (B) and expressed as a percentage of the control flux in the absence of inhibitor.
  • Fig. 13 shows a comparison of the predicted amino acid sequence of hENTl (SEQ ID NO:2) with that of rENTl (SEQ ID NO:6). Identical amino acids are marked in bold.
  • Fig. 14 is an alignment of the predicted amino acid sequences of rENTl (SEQ ID NO:2)
  • rENT2 SEQ ID NO: 6
  • Amino acid sequences of rENTl and rENT2 were deduced from the nucleotide open reading frames of clones pAN2 and pAN3.
  • the sequence of hENTl is from reference 25.
  • the arrow shows the position of the residue previously assigned as the amino-terminus of the two HNP36 proteins (Williams, J. B., and Lanahan, A.
  • Fig. 15A shows the hydropathy profiles for the predicted amino acid sequences of rENTl (SEQ ID NO:4) and rENT2 (SEQ ED NO:6) were determined by the method of Eisenberg et al. ( ⁇ isenberg, D., et al., 1984, J. Mol. Biol. 179, 125-142).
  • Fig. 15B shows the potential membrane -spanning -helices in the topographical model are numbered and putative ⁇ ' -linked glycosylation sites in rENTl and rENT2 are indicated solid and open stars, respectively. Residues identical in the two proteins are shown as darkened circles. Residues corresponding to insertions in the sequences of rENTl or rENT2 are indicated by circles containing "+" and "-" signs, respectively.
  • Fig. 16 illustrates the nucleoside uptake by recombinant rENTl and rENT2 expressed in Xenopus oocytes.
  • Oocytes injected with either 10 nl of water alone or 10 nl of water containing 10 ng of rENTl (Fig. 16A) or rENT2 (Fig. 16B) RNA transcript were incubated for 3 days at 18°C in MBM. Fluxes of uridine and adenosine (10 ⁇ M. 20 °C) were determined in transport buffer containing 100 mM NaCl. Each value is the mean ⁇ SEM of 10-12 oocytes.
  • Fig. 17 illustrates the nucleoside specificity of rENTl and rENT2.
  • Uridine influx (10 ⁇ M. 20 C C. 1 min flux) in transport buffer containing 100 mM NaCl (open columns) or 100 mM choline chloride (hatched columns) was measured in rENTl (Fig. 17A) and rENT2 (Fig. 17B) RNA transcript-injected oocytes in the absence (control) or presence of 2 mM nonradioactive physiological nucleosides (T. thymidine; C, cytidine; G, guanosine; I, inosine: A. adenosine; U, uridine) or uracil.
  • T. thymidine C
  • C cytidine
  • G guanosine
  • I inosine: A. adenosine
  • U uridine
  • Nonradioactive nucleosides and uracil were added to oocytes at the same time as [ M C]uridine. H 0, water-injected oocytes. Each value is the mean + SEM of 10-12 oocytes.
  • Fig. 18 show the inhibition of rENTl , hENTl and rENT2 by NBMPR. dipyridamole and dilazep. Dose-response curves for NBMPR (Fig. 18 A), dipyridamole (Fig. 18B) and dilazep (Fig.
  • IG 0 values (+ SE) determined by linear regression analysis of indirect Hill plot transformations of the dose- response curves (Enzfitter, Elsevier-Biosoft), are presented in the text.
  • rENT2 (D) NBMPR, dipyridamole (Dp) and dilazep (Dz) were present at a concentration of 1 ⁇ M.
  • Fig. 19 demonstrates the concentration dependence of uridine influx by rENTl and rENT2.
  • Oocvtes were injected with water alone (o) or water containing RNA transcript (•) for rENTl (Fig. 19A) and rENT2 (Fig. 19B).
  • Influx of uridine (10 ⁇ M, 20°C, 1 min) was measured in NaCl transport buffer. Each value is the mean + SEM of 10-12 oocytes.
  • the insets show v versus vis plots of the transporter-mediated fluxes, calculated as influx in RNA transcript-injected oocytes minus influx in water-injected cells).
  • Apparent K,_ and V ⁇ values (+ SE) were determined by non-linear regression analysis (Enzfitter, Elsevier-Biosoft).
  • Fig. 20 shows alignment of the predicted amino acid sequence of hENT2 (SEQ ED NO:8) with that of the human NBMPR-sensitive nucleoside transporter hENTl (SEQ ID NO:2).
  • the amino acid sequence of hENT2 was deduced from the nucleotide sequence of the open reading frame of the insert in clone pHNPc7.
  • the underlined region of the hENT2 sequence is missing from the published sequence of human HNP36, and an arrow shows the position of the residue previously assigned as the amino-terrninus in that protein.
  • the positions of putative transmembrane regions are shown as open rectangles and residues identical in both sequences are indicated by black boxes.
  • Potential N-linked glycosylation sites in the hydrophilic segment linking putative TMs 1 and 2 of the proteins are shown in lower case C"n ') and their positions highlighted by an asterisk above the aligned sequences.
  • Fig. 21 Nucleoside uptake by recombinant hENTl and hENT2 expressed Xenopus oocytes. Oocytes injected with either 10 nl water alone or 10 nl water containing 10 ng hENTl or hENT2 RNA transcript were incubated for 3 days at 18°C. Fluxes of 10 ⁇ M uridine (open bars) and adenosine (hatched bars) at 20 °C were then determined as described (see Examples below).
  • Fig. 22 shows the concentration dependence of hENT2-mediated uridine influx.
  • Oocytes were injected with water alone (O) or with water containing RNA transcript for hENT2 (•). After incubation for 3 days, the uptake of uridine was measured over the concentration range indicated, as described in Materials and Methods.
  • the inset shows a v versus v/s plot of the transporter-mediated flux, calculated as influx in RNA transcript-injected oocytes minus influx in water-injected cells. Apparent ⁇ and V mx . values, determined by nonlinear regression analysis, are presented in the text.
  • Fig. 23 shows the inhibition of hENTl and hENT2 by NBMPR.
  • Dose response curves for the inhibition of uridine influx by NBMPR were produced in oocytes injected with RNA transcripts encoding hENTl (•) or hENT2 (O). Fluxes were corrected for the small contribution of endogenous transport activity seen in water-injected oocytes, and are expressed as a percentage of the uninhibited hENTl and hENT2 values, which were 0.318 ⁇ 0.023 and 0.224 ⁇ 0.023 pmol/oocyte.min "1 respectively.
  • the invention provides substantially purified equilibrative nucleoside transporter (ENT) polypeptides and polynucleotide sequences encoding the polypeptides.
  • ENT equilibrative nucleoside transporter
  • "Equilibrative nucleoside transporter” refers to a transport protein which is permeable to a variety of purines and pyrimidines and analogs.
  • an ENT is able to transport three or more of the following nucleosides or analogs: adenosine, uridine, guanosine, inosine, formycin B, tubercidin, and thymidine.
  • ENTs include, but are not limited to, equilibration sensitive (es) transporters.
  • the "equilibration sensitive ' ' transporters are Na+ independent nucleoside transporters which reversibly bind NBMPR with high affinities (Y ⁇ values 0.1-10 nM), can be covalently radiolabelled with [3H]NBMR by exposure of the transporter-ligand complex to UV light (Williams, J.B., and Lanahan, A.A., 1995, Biochem. Biophys. Res. Comm. 213:325-333) and are widely distributed in different cell types and tissues.
  • the equilibrative-insensitive (ei) transporters are Na + -independent nucleoside transporters that are unaffected by micromolar concentrations of NBMR. ENT POLYPEPTIDES AND POLYNUCLEOTIDES
  • ENTs are "bi-directional," that is, they transport a suitable permeant both into and out of a cell.
  • a "permeant” as used herein is any molecule which can be transported by an ENT.
  • a human equilibrative nucleoside transporter has an amino acid sequence set forth in SEQ ID NO:2 (hENTl ) or SEQ ID NO:8 (hENT2)
  • a rat equilibrative nucleoside transporter has an amino acid sequence as set forth in and SEQ ID NO:4 (rENTl) or SEQ ID NO:6 (rENT2).
  • substantially purified refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify human or rat ENT using standard techniques for protein purification.
  • the substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
  • the purity of the ENT polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • ENT polypeptide includes functional fragments of the polypeptide. as long as the ENT activity remains. Smaller peptides containing the biological activity of ENT are included in the invention. Included in the invention are polypeptides having an amino acid sequence which is at least 65% identical to
  • SEQ ID NO:2 more preferably 80% identical to SEQ ID NO:2, and most preferably 98% identical to SEQ ED NO:2; polypeptides having an amino acid sequence which is at least 65% identical to SEQ ID NO:4, more preferably 80% identical to SEQ ID NO:4. and most preferably 98%) identical to SEQ ID NO:4; polypeptides having an amino acid sequence which is at least 65% identical to SEQ ID NO:6. more preferably 80% identical to SEQ ID NO:6.
  • hENTl, rENTl, hENT2, or rENT2 primary amino acid sequences may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of the ENT still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity.
  • the ENT polypeptides of the invention also include dominant negative forms of the polypeptides which do not have the biological activity of the ENTs.
  • a "dominant negative form" of an ENT is a polypeptide that is structurally similar to an ENT of the invention which does not have normal ENT function: it interferes with normal ENT function by binding to, or otherwise sequestering, regulating agents, such as upstream or downstream components, that normally interact functionally with the ENT polypeptide.
  • the equilibrative nucleoside transporters of the invention can be inhibited by one or more of the following inhibitors: NBMPR, dipyridamole, or dilazep.
  • inhibitor means a decrease in the ability to transport purines and pyrimidines or analogs. In general, an inhibitor of transport will decrease, to some extent, transport of purines. pyrimidines. or analogs relative to the transport in the absence of inhibitor.
  • the invention also provides polynucleotides encoding ENTs, specifically polynucleotides encoding hENTl , hENT2, rENTl, and rENT2. These polynucleotides include
  • DNA DNA.
  • cDNA and RNA sequences which encode ENT are also included herein, as long as they encode a polypeptide with ENT activity.
  • Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
  • ENT polynucleotide may be subjected to site-directed mutagenesis.
  • the ENT also includes antisense sequences and sequences encoding the dominant negative forms.
  • the polynucleotides of the invention include sequences that are degenerate nucleotide sequences of the ENTs of the invention that occur as a result of the degeneracy of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the ENT polypeptide (e.g., hENTl, rENTl, hENTs, or rENT2) encoded by the nucleotide sequence is functionally unchanged.
  • polynucleotide sequences that encode the hENTl , rENTl . hENT2. and rENT2 polypeptides.
  • An exemplary hENTl polynucleotide sequence is SEQ ID NO: 1
  • an exemplary rENTl polynucleotide sequence is SEQ ED NO:3.
  • an exemplary rENT2 polynucleotide sequence is SEQ ID NO:5.
  • an exemplary hENT2 polynucleotide sequence is SEQ ID NO:7.
  • polynucleotide or “nucleic acid sequence” refers to a polymeric form a nucleotides at least 10 bases in length.
  • isolated polynucleotide is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus: or into the genomic DNA of a prokaryote or eukaryote. or which exists as a separate molecule (e.g. a cDNA) independent of other sequences.
  • the nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single and double forms of DNA.
  • a polynucleotide encoding hENTl includes SEQ ED NO: 1, as well as nucleic acid sequences complementary to SEQ ID NO: 1.
  • a polynucleotide encoding rENTl includes SEQ ID NO:3, as well as nucleic acid sequences complementary to SEQ ID NO:3.
  • a polynucleotide encoding rENT2 includes SEQ ID NO:5. as well as nucleic acid sequences complementary to SEQ ID NO:5.
  • a polynucleotide encoding hENT2 includes SEQ ID NO:7, as well as nucleic acid sequences complementary to SEQ ED NO:7.
  • a complementary sequence may include an antisense nucleic acid sequence.
  • the sequence is RNA
  • the deoxynucleotides A, G, C, and T of SEQ ED NO: l, SEQ ED NO:3, SEQ ED NO:5, and SEQ ED NO:7 are replaced by ribonucleotides A, G, C, and U, respectively.
  • fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein of SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, or SEQ ED NO:8 or selectively hybridize to a structurally related ENT.
  • selective hybridize refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences (e.g., Sambrook, J., et al, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized (see Sambrook et al.. supra). For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1 % SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1%) SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g.. high stringency conditions, or each of the conditions can be used. e.g.. for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • the polynucleotide sequences encoding the hENTl, hENT2, rENTl, and rENT2 polypeptides of the invention include the disclosed sequences and conservative variations thereof.
  • the term "conservative variation” as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine. leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like.
  • the term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
  • DNA sequences encoding an ENT can be expressed in vitro by DNA transfer into a suitable host cell.
  • ENT e.g., hENTl, rENTl, and rENT2
  • hENTl, rENTl, and rENT2 are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term
  • host cell is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the ENT polynucleotide sequences may be inserted into an expression vector.
  • expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the ENT genetic sequences.
  • Polynucleotide sequence which encode an ENT e.g. hENTl , rENTl , hENT2, or rENT2
  • ENT e.g. hENTl , rENTl , hENT2, or rENT2
  • “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence went the expression control sequences control and regulate the transcription and. as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, as start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns.
  • control sequences is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • promoter is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter- dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents: such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al.. 1987, Methods in Enzymology 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viaises (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.
  • the polynucleotide encoding an ENT e.g. hENTl , rENTl . hENT2.
  • rENT2 may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al., 1987, Gene, 56:125), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans. 1988, J. Biol. Chem. 263:3521) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g.. T7, metallothionein I. or polyhedrin promoters).
  • a promoter e.g.. T7, metallothionein I. or polyhedrin promoters.
  • Polynucleotide sequences encoding an ENT can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • transformation is meant a genetic change induced in a cell following incorporation of new DNA (i.e. DNA exogenous to the cell).
  • new DNA i.e. DNA exogenous to the cell
  • the pgenetic change is generally achieved by introduction of the DNA into the genome of the cell (i.e. stable).
  • transformed cell is meant a cell into which (or into an ancestor of which has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding an ENT. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E.
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl method using procedures well known in the art. Alternatively. MgCl, or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the ENT (e.g. hENTl , rENTl , hENT2, or rENT2) of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, 1982, Cold Spring Harbor Laboratory, Gluzman ed.).
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Isolation and purification of microbial expressed polypeptides. or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • the ENT polypeptides of the invention can be used to produce antibodies which are immunoreactive or bind to epitopes of the ENT polypeptides.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • the preparation of polyclonal antibodies is well-known to those skilled in the art (e.g.,
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas. cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose. size-exclusion chromatography. and ion-exchange chromatography. See, e.g., Coligan et al., 1992, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3, supra; Barnes et al., 1992, "Purification of Immunoglobulin G (IgG)" in: Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press).
  • Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
  • Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
  • Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
  • Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g.. syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
  • antibodies of the present invention may also be derived from subhuman primate antibody.
  • General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., 1991, International PCT Patent Publication WO 91/1 1465 and Losman et al. 1990. Int. J. Cancer 46:310, which are hereby incorporated by reference.
  • a therapeutically useful anti-ENT antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al.. 1989. Proc. Nat'l Acad. Sci. USA 86:3833. which is hereby incorporated in its entirety by reference.
  • Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., 1991 , in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 1 19; Winter et al., 1994, Ann. Rev. Immunol. 12:433, which are hereby incorporated by reference.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
  • antibodies of the present invention may be derived from a human monoclonal antibody.
  • Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al..
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab'),, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • ( 1 ) Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain: two Fab' fragments are obtained per antibody molecule;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab' ⁇ .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al., 1972, Proc. Nat'l Acad. Sci. USA 69:2659.
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu. supra.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are described, for example, by Whitlow et al., 1991, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97;
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., 1991 , Methods: a Companion to Methods in Enzymology, Vol. 2, page 106.
  • Antibodies which bind to an ENT polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.
  • adenosine and adenine nucleotides have been shown to be powerful regulators of cellular function in a variety of physiological systems through their specific interactions with purinergic receptors.
  • Purinergic receptors have important roles in many different process including platelet function, regulation of blood flow in vascular beds, inflammation, neuro transmitter release, angiogenesis. lipogenesis, atrial contractility and AV conduction. Because the cellular release and reuptake of adenosine occurs primarily by meditated transport processes, nucleotide inhibitors are potential modulators of interaction of adenosine with its receptors and thus have potential applications in many different diseases.
  • the classic inhibitors of ENT-mediated transport have been shown in model systems to be capable of potentiating the purinergic activity of adenosine.
  • drug discovery programs are directed towards the identification of nucleoside transporter inhibitors that are used to modulate the vasodilatory action of adenosine in the treatment of coronary or cerebrovascular anoxia.
  • the pharmaceutical applications of inhibitors of nucleoside transporters extend into ADDS and cancer treatment by providing an opportunity to manipulate intracellular levels of chemotherapeutic nucleoside drugs.
  • the invention provides a method for identifying a compound which can modulate ENT activity. The method includes incubating compounds and a sample under conditions sufficient to allow the components to interact, and measuring the permeability of the sample to a nucleoside using a permeant (which may also be a nucleoside), and then comparing the permeability of the sample, incubated with the compound, to the permeant with the permeability of a control sample not incubated with the compound.
  • the compounds which affect ENT include peptides. polypeptides.
  • Antiviral and chemotherapeutic compounds can be tested using the method of the invention.
  • “Incubating” includes conditions which allow contact between the test compound and the ENT.
  • Contacting includes in solution and solid phase.
  • the test compound may also be a combinatorial library for screening a plurality of compounds.
  • Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced. and the like, either in solution of after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence, such as PCR.
  • oligomer restriction (Saiki et al..l985, Bio/Technology, 3: 1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,1983, Proc. Natl. Acad. Sci. USA, 80:278), oligonucleotide ligation assays (OLAs) (Landegren et al, 1988, Science, 241 : 1077), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., 1988. Science, 242:229-237).
  • a compound can affect an equilibrative nucleoside transporter by either stimulating or inhibiting transport of a nucleoside.
  • a compound "inhibits " ' an equilibrative nculeoside transporter if the ability to transport purines and pyrimidines or analogs is decreased.
  • a compound "stimulates " an equilibrative nucleoside transporter if the ability to transport purines and pyrimidines or analogs is increased.
  • the sample can be any sample of interest.
  • the sample may be a cell sample or a membrane sample prepared from a cell sample.
  • Suitable cells include any host cells containing a recombinant ENT vector of the invention.
  • the cells functionally express the ENT polypeptide. but preferably have no other nucleoside transporters or have low background nucleoside transporters.
  • Permeability is defined as the ability of a permeant to move from one side of a membrane to another. Permeability can be an efflux (movement of the permeant across a membrane from the inside of a cell or intracellular organelle to the outside of a cell or intracellular organelle) or an influx (movement of the permeant across a membrane from the outside of a cells or an intracellular organelle into the inside of a cell or intracellular organelle).
  • the permeant can be any molecule known to be transported via an ENT.
  • the permeant can be detectably labeled. Most preferably, the permeant is radiolabeled; alternatively the label can be a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.
  • the invention further provides a method for determining if a compound utilizes an equilibrative nucleoside transporter to enter or leave a cell.
  • the method includes incubating the compound with a sample of interest under conditions sufficient to allow the compound to interact with the sample in the presence of an equilibrative nucleoside transporter inhibitor, detecting the permeability of the sample of interest to the compound, and comparing the permeability of the sample incubated in the presence of both the compound and the inhibitor to a control sample incubated with the compound but without inhibitor.
  • Compounds of use with the invention include, but are not limited to, anticancer and antiviral agents.
  • the sample can be any sample of interest.
  • the sample may be a cell sample or a membrane sample prepared from a cell sample.
  • Suitable cells include any host cells containing a recombinant ENT vector of the invention.
  • the cells functionally express the ENT polypeptide. but preferably have no other nucleoside transporters or have low background nucleoside transports.
  • kits may comprise a carrier means containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • One of the container means may comprise a probe which is or can be detectably labeled.
  • probe may be an antibody or nucleic acid sequence specific for an ENT, or fragments thereof.
  • oligonucleotide probes of the present invention can be included in a kit and used for examining the presence of hENTl, hENT2, rENTl, or rENT2, in a sample, as well as the quantitative (relative) degree of binding of the probe for determining the occurrence of specific strongly binding (hybridizing) sequences.
  • the kit may also contain a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin. bound to a reporter molecule, such as an enzymatic, fluorescent, or radionucleotide label to identify the detectably labeled oligonucleotide probe.
  • a reporter-means such as a biotin-binding protein, such as avidin or streptavidin.
  • a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the detectably labeled oligonucleotide probe.
  • the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence.
  • nucleotide(s) for amplification of the target nucleic acid sequence.
  • the kit may also contain a container containing antibodies which bind to hENTl, hENT2. rENTl or rENT2, or fragments thereof. Such antibodies can be used to distinguish the presence of an ENT or the level of expression of ENT in a specimen. Where the kit utilizes antibodies to detect the ENT, these antibodies may be directly labeled.
  • the kit may also contain a container containing a reporter means, such as avidin or steptavidin. bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the directly labeled antibody.
  • the kit can utilizes antibodies that bind hENTl, hENT2, rENTl, or rENT2 that are unlabeled.
  • the kit may then also contain a container containing a second antibody which binds to the antibody specific for the ENT of interest.
  • the second antibody can be directly labeled.
  • the kit may also a container containing a reporter means, such as avidin or steptavidin, bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the directly labeled second antibody.
  • the level of expression of the transporter or transporters, and their functional properties in the neoplastic cells is important.
  • drugs that are nucleoside analogs or function like nucleoside analogs must be able to enter the cell to be effective. If the neoplastic cells express high levels of a particular ENT, then the therapy can include drugs that are permeants for that particular ENT. If the neoplastic cells are deficient in the expression of a particular ENT, permeants that are more suitable for other transporters may be useful.
  • the invention provides a method for assessing the effectiveness of a therapeutic intervention for a subject which includes obtaining a sample from the subject, determining the presence or absence of the expression of an ENT in the sample, and then correlating the presence or absence of the ENT with the effectiveness of the therapeutic intervention.
  • the invention also provides a method for assessing if a therapeutic intervention will produce side effects in a subject, and a means to determine if ENT inhibitors may lessen side effects.
  • a sample of tissue not affected by the disease process is obtained from a subject, and the presence or absence of the expression of the ENT is determined. If a particular therapeutic intervention can be transported through a particular ENT, and this ENT is identified in the sample taken from the tissue not affected by the disease process, then side effects can occur. If protection of the tissue not affected by the disease process from the effects of the theapeutic interention is desired, then protection of normal tissues from the therapeutic intervention may be possible by using a specific ENT inhibitor (see U.S. patent 5,236,902).
  • the inhibitor may be classical pharmaceutical agents such as NBMPR, dipyridamole, or dilazep, or it may be an antibody that binds an ENT of the invention, so long as the ability to transport purines and pyrimidines or analogs is decreased.
  • an antibody or nucleic acid probe specific for an ENT may be used to detect the ENT polypeptide (using antibody) or polynucleotide (using nucleic acid probe) in subject samples such as biological fluids, cells, tissues, or nucleic acid. Any specimen containing a detectable amount of antigen or polynucleotide can be used.
  • biological fluids of use with the invention are blood, serum, plasma, urine, mucous, and saliva. Tissue or cell samples can also be used with the subject invention. The samples can be obtained by many methods such as cellular aspiration, or by surgical removal of a biopsy sample.
  • the invention provides a method for detecting an ENT, for example, which comprises contacting an anti-ENT antibody or nucleic acid probe with a cell suspected of expressing the ENT and detecting binding to the antibody or nucleic acid probe.
  • the antibody reactive with the ENT or the nucleic acid probe is preferably labeled with a compound which allows detection of binding to the ENT of interest.
  • a preferred sample of this invention is neoplastic tissue or tissue affected by HEV (as in tissue takent from AEDS patients).
  • the level of the ENT in the subject cell can be compared with the level in a cell not affected by the disease process.
  • the subject is human.
  • PCR polymerase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleic acid sequence-based amplification
  • the antibodies of the invention can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy.
  • the antibodies of the invention are suited for use. for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimrnunoassay (RIA) and the sandwich (immunometric) assay.
  • RIA radioimrnunoassay
  • sandwich immunometric
  • Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • the antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the polypeptide of the invention.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran. nylon, amylases. natural and modified celluloses, polyacrylamides. agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
  • labels and methods of labeling known to those of ordinary skill in the art.
  • examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.
  • Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl.
  • the present invention identifies a polynucleotide sequence that can be expressed in an altered manner as compared to expression in a normal cell, therefore it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence.
  • the antibodies and polynucleotides of the invention can be used to detect or to treat an equilibratvie nucleoside transporter-associated disorder.
  • equilibratvie nucleoside transporter-associated disorder denotes malignant as well as non-malignant disorders, where tire cells involved in the disorder differ from the surrounding tissue or from unaffected cells in their expression of an equilibrative nucleoside transporter.
  • Detection of elevated levels of ENT expression is accomplished by hybridization of nucleic acids isolated from a cell of interest with an ENT polynucleotide of the invention. Analysis, such as Northern Blot analysis, are utilized to quantitate expression of the ENT. Other standard nucleic acid detection techniques will be known to those of skill in the art.
  • Treatment can include modulation of ENT gene expression and ENT activity by administration of a therapeutically effective amount of a reagent that modulates the ENT.
  • modulate envisions the suppression of expression of a particular ENT when it is over- expressed, or augmentation of the expression of a particular ENT when it is under-expressed.
  • nucliec acid sequences that encode that ENT can be used.
  • nucleic acid sequences that interfere with the expression of the ENT at the translational level can be used.
  • This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific ENT mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • disorders include cancer, for example.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American. 262:40). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target ENT-producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art
  • triplex strategy Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al.. 1991, Antisense Res. and Dev.. 1(3):227; Helene, C, 1991, Anticancer Drug Design. 6(6):569).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs. it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J.Amer.Med. Assn., 260:3030). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, 1988. Nature, 334:585) and "hammerhead"-type. Tetrahymena-typs ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 1 1-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • the present invention also provides gene therapy for the treatment of disorders which are associated with an ENT protein. Such therapy would achieve its therapeutic effect by introduction of a therapeutic polynucleotide into cells having the disorder.
  • the "therapeutic polynucleotide” may be polynucleotide sequences encoding an ENT of the invention, or antisense polynucleotide specific for an ENT of the invention, designed to treat an ENT- associated disorder. Delivery of the therapeutic polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Especially preferred for therapeutic delivery of antisense sequences, or ENT polynucleotides is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to:
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • GaLV gibbon ape leukemia virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting an ENT sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific.
  • Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • a sugar for example, a sugar, a glycolipid, or a protein.
  • Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • Those of skill in the art will know of. or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ENT polynucleotide.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • Another targeted delivery system for the therapeutic polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres. beads, and lipid-based systems including oil-in-water emulsions. micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al..1981, Trends
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al.,1988, Biotechniques. 6:682).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids. especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds. such as phosphatidylglycerol. phosphatidylcholine, phosphatidylserine, phosphatidyletha- nolamine. sphingolipids, cerebrosides, and gangliosides.
  • d-iacylphosphatidylglycerols where the lipid moiety contains from 14-18 carbon atoms. particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active.
  • Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • This invention involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier.
  • administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • subject is meant any mammal, preferably a human.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • Tire administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • the pharmaceutical compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle. Suitable solid or liquid pharmaceutical preparation forms are.
  • compositions for example, granules, powders, tablets, coated tablets, (micro)capsules. suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, 1990, Science, 249:1527-1533, which is incorporated herein by reference.
  • compositions according to the invention may be administered locally or systemically.
  • therapeutically effective dose is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disorder and its complications. Amounts effective for this use will, of course. depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
  • the identification of an ENT provides a useful tool for diagnosis, prognosis and therapeutic strategies associated with expression of a particular ENT. Measurement of levels of an ENT using anti-ENT antibodies is a useful diagnostic for determining therapeutic strategies. TRANSGENIC ANIMALS
  • the present invention relates to transgenic animals having cells that express an ENT.
  • Such transgenic animals represent a model system for the study of ENT related disorders and the or the study of therapeutics targeted to an ENT.
  • the term "animal” here denotes all mammalian species except human. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Farm animals (pigs, goats, sheep, cows, horses, rabbits and the like), rodents (such as mice), and domestic pets (for example, cats and dogs) are included within the scope of the present invention.
  • a “transgenic” animal is any animal containing cells that bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with recombinant vims.
  • Transgenic in the present context does not encompass classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule. Although it is highly preferred that this molecule be integrated within the animal's chromosomes, the present invention also contemplates the use of extrachromosomally replicating DNA sequences, such as might be engineered into yeast artificial chromosomes.
  • transgenic animal also includes a "germ cell line” transgenic animal.
  • a germ cell line transgenic animal is a transgenic animal in which the genetic information has been taken up and incorporated into a germ line cell, therefore conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too. are transgenic animals.
  • the transgenic animals of the present invention be produced by introducing into single cell embryos DNA encoding hENTl, hENT2, rENTl. or rENT2. in a manner such that the polynucleotides are stably integrated into the DNA of germ line cells of the mature animal and inherited in normal Mendelian fashion.
  • Advances in technologies for embryo micromanipulation now permit introduction of heterologous DNA into fertilized mammalian ova.
  • totipotent or pluripotent stem cells can be transformed by microinjection. calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal.
  • developing embryos are infected with
  • _o a retrovirus containing the desired DNA. and transgenic animals produced from the infected embryo.
  • the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.
  • mammalian (mouse, pig, rabbit, sheep, goat, cow) fertilized ova include: Hogan et al., 1986. Manipulating the Mouse Embryo, Cold Spring Harbor Press; Krimpenfort et al.. 1991. Bio/Technology 9:86; Palmiter et al. 1985, Cell 41 :343; Kraemer et al., 1985, Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Laboratory Press; Hammer et al.. 1985, Nature
  • the cDNA that encodes the ENT of interest can be fused in proper reading frame under the transcriptional and translational control of a vector to produce a genetic construct that is then amplified, for example, by preparation in a bacterial vector, according to conventional methods (e.g., Sambrook et al., 1989, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, the contents of which are incorporated by reference).
  • the amplified construct is thereafter excised from the vector and purified for use in producing transgenic animals.
  • transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout.
  • transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination.
  • transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or knocked out.
  • the transgene to be used in the practice of the subject invention is a DNA sequence comprising a modified ENT coding sequence.
  • the ENT gene is disrupted by homologous targeting in embryonic stem cells.
  • the entire ENT gene may be deleted.
  • the ENT disruption or deletion may be accompanied by insertion of or replacement with other DNA sequences, such as a non-functional ENT sequence.
  • the transgene comprises DNA antisense to the coding sequence for an ENT.
  • the transgene comprises DNA encoding an antibody which is able to bind to an ENT.
  • DNA sequences that encode proteins having ENT activity but differ in nucleic acid sequence due to the degeneracy of the genetic code may also be used herein, as may truncated forms, allelic variants and interspecies homologues.
  • a non-degenerate DNA sequence encoding residues 8 to 15 of this sequence was PCR- amplified using Taq polymerase from an oligo(dT)-primed human placental cDNA library constructed, using BstXl linkers, in the plasmid vector pEF-BOS (Mizushima. S.
  • the 5' primer used for the amplification was an 8, 192-fold degenerate oligonucleotide corresponding to residues 1 - 7 plus an additional N-terminal methionine, while the 3' primer was a 512-fold degenerate primer corresponding to residues 16 - 21. EcoRI and Hind ⁇ I sites respectively were incorporated at the 5' ends of the primers to facilitate subsequent cloning.
  • a PCR product of the expected size (84 bp) was gel-purified, digested with EcoRI and sacred! and then ligated into the corresponding cloning sites of pBluescript II KS (+). Sequencing of the resultant clone (80A) showed that it encoded the amino acids expected for positions 8 to 15 in the TV-terminal sequence of the transporter, and identified position 14 as tryptophan. Database searching showed the presence of an identical nucleotide sequence in a 265 bp EST (GenBank accession no. T25352) cloned from a human Burkitt's lymphoma cDNA library. This information was exploited to amplify the 3' portion of the hENTl cDNA by a nested PCR approach. Initial amplification of the placental library by touchdown PCR using
  • Vent polymerase employed a 5', non-degenerate primer based on the clone 80A sequence shown to encode residues 8 - 15, and a 3 ' primer (pEF-BOS 2) corresponding to a region downstream of the BstXl sites in pEF-BOS.
  • the resultant products were subjected to a second amplification using Vent polymerase and a nested 5' primer corresponding to bases 219 - 240 of the EST sequence. This yielded a single product of size 1.9 kb, which contained the complete coding sequence of the C-terminal region of the transporter, plus 616 bp of 3' non-translated region.
  • the 5' end of the transporter cDNA sequence was obtained by PCR amplification of the placental library using as 3' primer an oligonucleotide corresponding to nucleotides 1553-1572 of the sequence shown above and a 5' primer (pEF-BOS 1 ) corresponding to a region of the pEF-BOS vector upstream of the BstXl sites.
  • the resultant 1.6 kb product which contained the complete coding sequence of the transporter plus 178 bp of untranslated 5' sequence, was ligated into the Smal site of pBluescript II KS (+) to yield clone 17.1 , which was used for the expression experiments described in Figs. 6 and 7.
  • Rat Equilibrative Nucleoside Transporter rENTl cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of total RNA extracted from mucosal scrapings of Sprague-Dawlev rat jejunum (Huang, Q.Q., et al.. 1993, J. Biol. Chem. 268:20613-20619) using primers (Al . A2) flanking the hENTl open reading frame.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • First strand cDNA synthesis was performed on 5 mg RNA as template using the Superscript Pre-amplification System (GibcoBRL) and oligo-dT as primer.
  • the PCR reaction 50 ml contained 10 ng template 1st strand cDNA, 5 units TaqfDeep Vent DNA polymerase (100: 1) and 10 pmol each of primers Al and A2.
  • Primer Al corresponded to hENTl nucleotide positions 179-203 (sense,
  • primer A2 corresponded to hENTl nucleotide positions 2108-2127 (antisense, 5--TGAAGGCACCTGGTTTCTGTC-3' herein referred to as SEQ ID NO: 16) and was identical in sequence to nucleotides 39-59 of a 353 bp expressed sequence tag (EST) cloned from a rat PC- 12 cell cDNA library (GeneBank accession no. H31422).
  • EST expressed sequence tag
  • Amplification for one cycle at 94° C for 5 min, 55° C for 1 min 20 s and 72°C for 2 min and 34 cycles at 94° C for 1 min, 55°C for 1 min 20 s and 72° C for 2 min generated a ⁇ 1.8 kb product that was ligated into the PCR vector pGEM-T (Promega) to generate the plasmid prENTl.
  • the prENTl insert was sequenced in both directions by Taq DyeDeoxy terminator cycle sequencing using an automated model 373A DNA Sequencer (Applied Biosystems. Inc.).
  • the 1766 bp insert had an open reading frame (Fig. 8) that encoded a 456-residue protein (designated rENTl , Fig. 9) that was 78% identical in amino acid sequence to hENTl (Fig. 13).
  • rENTl was shown to transport uridine and adenosine (Fig. 10) and to have a broad permeant selectivity for both purine and pyrimidine nucleosides (Fig. 1 1). rENTl was inhibited by NBMPR but, unlike its human homolog hENTl, was unaffected by dipyridamole (Fig. 12).
  • Example 3 Expression of transporter in cells Xenopus expression and nucleoside uptake assays. Plasmid DNA for hENTl was linearised with Notl and transcribed with T3 polymerase in the presence ofTM 7 GpppG cap using the MEGASCRIPT (AMBIO ⁇ ) transcription system. Remaining template was removed by digestion with R ⁇ ase-free D ⁇ ase 1. Oocytes were treated with collagenase to remove follicular layers (Huang. Q.Q., et al., 1993. J. Biol. Chem. 268:20613-20619) and then injected with 10 ng hE ⁇ Tl R ⁇ A transcript in 10 nl of water or 10 nl of water alone.
  • the transport buffer contained 1 mM deoxycoformycin to inhibit adenosine deaminase activity. At this concentration, deoxy- coformycin had no effect on uridine influx. Except where otherwise indicated the incubation period was 5 min and the permeant concentration was 10 mM. Competing unlabelled nucleosides or uracil were used at a concentration of 2 mM.
  • oocytes were treated for 1 h with inhibitor (1 mM unless otherwise indicated) before the addition of permeant. At the end of the incubation, extracellular label was removed by six rapid washes with ice-cold transport buffer. Individual oocytes were dissolved in 5% SDS for quantification of radioactivity by liquid scintillation counting. Each of the values shown represents the mean ⁇ SEM of 8 - 10 oocytes. Fluxes shown in Figs. 6B, 6D and 7B were corrected for endogenous nucleoside uptake activity by subtraction of fluxes seen in water-injected oocytes. This flux was not affected by the presence of competing nucleosides or inhibitors in the transport buffer.
  • the cDNA encoding rENT2 was obtained by first amplifying rENT2 partial cDNA from oligo-dT primed rat jejunal cDNA using primers (A3, A4) for conserved regions of mouse/human HNP36. corresponding to human HNP36 nucleotide positions 265-286 (sense, 5"-AACAACTGGGTGACACTGCTG-3') (SEQ ID NO: 17) and 1217-1238 (antisense. 5'-
  • a cDNA encoding full-length rat HNP36 was then amplified from oligo-dT primed rat jejunal cDNA using a pair of primers (A7, A8) corresponding to nucleotide positions 157- 136 bp upstream of start codon (A7 sense, 5'-TTACCCAACCTGCACCCTCTC-3', SEQ ID NO:21 ) and positions 138-150 bp downstream the stop codon (A8 antisense, 5'-
  • the resulting - 1.7 kb product which contained the complete coding sequence of rat HNP36. designated here as rENT2. was ligated into the pGEM-T vector to generate the plasmid pAN3 and sequenced in both directions.
  • the 1766-bp rENT2 cDNA had an open reading frame encoding a 456-residue protein flanked by 157 bp of untranslated 5'-nucleotide sequence and 150 bp of untranslated 3'- sequence.
  • rENTl - Sequence homology between rodent and human es transporter proteins was suggested by the identification, in the GenBankTM /EMBL EST database (dbEST), of ESTs encoding partial sequences of rat and mouse homologs of hENTl (Griffiths, M., et al.. 1997. Nature Medicine 3:89-93).
  • the additional residue was a cysteine. located just before TM 8.
  • TMs are connected by short ( ⁇ 16 residue) hydrophilic regions, with the exception of large loops linking TMs 1 and 2 (extracellular) and TMs 6 and 7 (intracellular) which contain 41 and 66 residues, respectively (Fig. 15B).
  • the intracellular amino-terminus and TMs 1 -8 and 10 and 11 were highly conserved between the two proteins.
  • the nucleotide sequence upstream of the assigned start codon of the human HNP36 cDNA includes an open reading frame that is 49% identical in predicted amino acid sequence to the amino-terminal region (TMs 1 and 2) of hENTl .
  • the cellular location of the HNP36 protein determined by immunocytochemical staining of murine BALB/c fibroblasts, F9 embryonal carcinoma cells and rat intestinal epithelial cells, was reported as nucleolar (Williams, J.B., and Lanahan, A.A., 1995, supra). Based upon the strong sequence similarity between HNP36 and rENTl/hENTl.
  • HNP36 might be a plasma membrane nucleoside transporter, perhaps with NBMPR-insensitive (e -type) transport activity.
  • pAN3 encoded a rat jejunal protein with the same number of residues as hENTl (456 amino acids, M ⁇ 50,232). The predicted start codon lies in a reasonably good Kozak consensus sequence and is preceded by an in-frame stop codon. There was 88%o and 95% identity (93% and 98%o similarity) to the incomplete amino acid sequences of human and mouse HNP36 (Fig. 14), establishing that the protein, designated here as rENT2, corresponds to full-length rat
  • HNP36 The sequence of rENT2 was 49% identical (68%o similar) to rENTl and 50% identical (69%o similar) to hENTl (Fig. 14).
  • a search of the GenBank /EMBL dbEST revealed that the human homolog of rENT2, human HNP36, is expressed in adult human ovary and ovarian tumours, and in fetal heart and brain.
  • Hydropathy analysis of rENT2 (Fig. 15 A) predicted the same membrane topology as rENTl hENTl . Homology between rENT2 and rENTl/hENTl was greatest within the putative transmembrane helices and there was little homology in either of the two large loops.
  • the predicted extracellular loop of rENT2 between TMs 1 and 2 contained 14 fewer amino acids than rENTl/hENTl, while the corresponding intracellular loop linking TMs 6 and 7 had 14 more residues (Fig. 15B).
  • the putative N-linked glycosylation site at Asn48 of rE ⁇ Tl and hE ⁇ Tl was conserved in rE ⁇ T2 at Asn47, allowing for the presence of a 1 residue insertion before this point in the ENT1 sequences, and was similarly present in the human HNP36 sequence (Figs. 14 and 15B).
  • a second putative glycosylation site was present at Asn56 in rENT2 and at a corresponding location in human HNP36 (Figs. 14 & 15B).
  • Plasmids pAN2 (rENTl) and pAN3 (rENT2) were linearized with Sphl and transcribed with SP6 polymerase in the presence ofTM 7 GpppG cap (Ambion, Austin, TX) using the MEGAscriptTM (Ambion) transcription system. Plasmid cDNA encoding hENTl (Griffiths. M., et al., 1997, supra) in the vector pBluescript II KS (+) was linearized with Not ⁇ and transcribed with T3 polymerase. Healthy stage VI oocytes of Xenopus laevis. treated with collagenase to remove follicular layers (Huang, Q.Q., et al., 1993, J. Biol. Chem.
  • MBM Barth's medium
  • the transport buffer also contained 1 ⁇ M deoxycoformycin to inhibit adenosine deaminase activity. Unless otherwise indicated, the incubation period was 1 min and the permeant concentration was 10 ⁇ M. Competing unlabelled nucleosides and uracil were used at a concentration of 2 mM.
  • oocytes were pretreated with inhibitor (1 ⁇ M unless otherwise indicated) for 1 h before addition of permeant. At the end of the incubation, extracellular radioactivity was removed by six rapid washes in ice-cold transport buffer.
  • oocytes were dissolved in 5% (w/v) SDS for quantitation of radioactivity by liquid scintillation counting.
  • Uridine is only slowly metabolised in oocytes (Huang, Q.Q., et al., supra), while adenosine is efficiently trapped as the 5'-triphosphate ester (10). Results for uptake experiments are given as means ⁇ standard errors
  • uptake in rENTl RNA- injected oocytes was 1.13 ⁇ 0.06 pmol/oocyte, compared with 0.004 + 0.002 pmol/oocyte in water-injected oocytes (Fig. 16A). Similar transport data were obtained in oocytes injected with rENT2 RNA transcript.
  • uptake of uridine (10 ⁇ M. 20°C, 1 min flux) by rENT2 RNA-injected oocytes was 0.47 ⁇ 0.009 pmol/oocyte, compared with 0.01 + 0.003 pmol/oocyte in water-injected cells.
  • Neither protein was dependent upon the presence of sodium ions in the external medium (Figs. 17A & B). Therefore. rENTl and rENT2 were both confirmed to be functional equilibrative nucleoside transporters.
  • rENTl The characteristic that distinguishes es-type and e -type nucleoside transport processes is inhibition by NBMPR.
  • Fig. 18A recombinant rENTl and hENTl exhibited comparable NBMPR inhibition profiles, with I 0 values of 4.6 ⁇ 1.4 and 3.6 ⁇ 1.4 nM, respectively.
  • rENT2 was unaffected by 1 ⁇ M NBMPR (Fig. 18D). rENTl is therefore NBMPR-sensitive (es-type), while rENT2 is NBMPR-insensitive (e -type).
  • rENTl was, as expected, highly sensitive to inhibition by dipyridamole and dilazep, with IQ 0 values of 140 ⁇ 2 and 60 ⁇ 2 nM. respectively.
  • concentrations of dipyridamole or dilazep up to 1 ⁇ M had no effect on rENTl transport activity, demonstrating insensitivity of the rat transporter to both compounds.
  • rENT2 was also relatively unaffected by 1 ⁇ M dipyridamole or dilazep (Fig. 18D).
  • the present invention shows the cloning and expression of cDNAs encoding two nucleoside transporter proteins from rat tissues.
  • rENTl and rENT2 were NBMPR-sensitive and NBMPR-insensitive, respectively, and thus correspond functionally to the two major equilibrative nucleoside transporter subtypes (es and ei) found in mammalian cells.
  • Neither rat protein was inhibited by the coronary vasoactive drugs dipyridamole or dilazep. Since rENTl and hENTl are close structural homologs, it is likely that small changes in the molecular structure within, or adjacent to.
  • nucleoside substrate binding site lead to differences in vasoactive drug binding without affecting the affinity of the site for either NBMPR or nucleoside substrates. That site may include structural domains located within the amino- terminal half of the transporter.
  • Example 7 Cloning of hENT2 The C-terminal two thirds of the hENTl sequence shows strong similarity (approx. 44% identity) to the 36 kDa mouse and human HNP36 proteins, which are delayed-early proliferative response gene products (Williams, J.B. and Lanahan. A.A., 1995. supra). These HNP36 proteins were reported to have a nucleolar location and were predicted to have only 8 membrane-spanning segments (Williams. J.B. and Lanahan, A.A.,1995, supra).
  • nucleotide sequence upstream of the assigned start codon in the human HNP36 cDNA included an open reading frame 49% identical in predicted amino acid sequence to the N-terminal region (transmembrane helices 1-2) of hE ⁇ Tl .
  • the cD ⁇ A might have been artefactually truncated during its preparation, and that a full length cD ⁇ A might encode a second mammalian passive nucleoside transporter isoform (Griffiths, M., et al., 1997, supra).
  • cDNA encoding hENT2 was amplified from an oligo(dT)-primed human placental cDNA library (Griffiths, M., et al., 1997, supra) using a PCR approach.
  • the nucleotide sequence has been submitted to the GenBankTM / ⁇ MBL Data Bank with accession number AF029358.
  • the cDNA amplified from a human placental cDNA library with primers derived from the HNP36 nucleotide sequence contained a 1 ,368 bp open reading frame that encodes a 456-residue protein of ⁇ -( 50,173 (Fig. 20).
  • h ⁇ NT2 The cDNA amplified from a human placental cDNA library with primers derived from the HNP36 nucleotide sequence contained a 1 ,368 bp open reading frame that encodes a 456-residue protein of ⁇ -( 50,173 (Fig. 20).
  • TM hydrophobic transmembrane
  • hENT2 is predicted to be a glycoprotein and contains two potential sites of N-linked glycosylation in the extramembranous loop connecting TMs 1 and 2. one of which. Asq 8 , is conserved in hE ⁇ Tl (Fig. 20).
  • Plasmid DNA (pHNPc7 encoding hENT2 and clone 17.1 encoding hENTl (Griffiths, M.. et al.. 1997, supra) was linearised with Notl and transcribed with T3 R ⁇ A polymerase in the presence of m7 GpppG cap using the MEGASCRIPT (AMBION) transcription system.
  • Xenopus oocytes were injected with 10 ng cRNA in 10 nl of water or 10 nl of water alone, and transport experiments were subsequently performed using radiolabelled uridine and adenosine and a transport buffer containing 100 mM NaCl.
  • the transport buffer also contained 1 ⁇ M deoxycoformycin to inhibit adenosine deaminase activity. An uptake period of 1 min and a permeant concentration of 10 ⁇ M were used unless otherwise indicated.
  • oocytes were treated for 1 h with inhibitor before the addition of permeant.
  • hENTl is a classic es-type transporter, its mediated influx of uridine (10 ⁇ M) being inhibited by NBMPR with a calculated ⁇ J value of 2 nM (Fig. 4, Table 1 and Griffiths, M., et al., 1997, supra).
  • hENT2 -mediated influx of uridine into Xenopus oocytes was inhibited by less than 15% in the presence of 1 ⁇ M NBMPR and by only 70% in the presence of 10 ⁇ M NBMPR (Fig.
  • h_ENT2-mediated adenosine transport exhibited a similar resistance to inhibition by NBMPR.
  • h_ENT2 is therefore an example of an NBMPR- insensitive, e.-type of nucleoside transporter.
  • ei transporters are reported to have a lower affinity for substrates, including uridine, than the corresponding es transporters (Griffith, D.A. and Jarvis, S.M.. 1996, supra: Boleti, H., et al., 1997. supra) no such differences were observed between hENTl and hENT2.
  • hENT2 human HNP36
  • hENT2 human HNP36
  • the physiological role(s) of hENT2 in these tissues is presently unclear.
  • adenosine is thought to play an important part in the regulation of myocardial supply-demand balance (Mubagwa, K., et al., 1996, Cardiovasc. Res. 32:797-813), and there is considerable evidence that adenosine also exerts beneficial, cardioprotective effects in the ischaemic/reperfused myocardium (Lasley, R.D.
  • nucleoside transport inhibitors as therapeutic agents in the heart (Dennis, D.M., et al, 1996, Circulation 94:2551-2559).
  • infusion of the lidoflazine analogue R-75231 before coronary artery occlusion enhances local adenosine concentrations and postischaemic recovery of function, while reducing infarct size in the pig (Martin, B.J., et al., 1997, Am. J. Physiol. 41 :H1857-H1865).
  • HNP36 e -type transporter
  • hENT2 a cDNA encoding an e -type transporter
  • the initial identification of HNP36 as a delayed- early proliferative response gene product may reflect the enhanced need for nucleotide precursors during cell proliferation.
  • es and ei transporters may represent two forms of the same protein (Aronow, B.. et al., 1985, J. Biol. Chem. 260:6226-6233)
  • our results establish that these two classes of transporters are the products of separate genes.
  • Transgenic mice that can produce an ENT of the invention are made according to methods well known in the art. For example, young female mice are injected with hormones to induce superovulation, and are mated. The one-celled embryos are collected, and the pronuclei injected with a purified DNA solution containing the constructs which encode an ENT of the invention. The injected eggs are cultured briefly, and re-implanted into pseudopregnant female mice. Genomic DNA is prepared from each progeny, and analyzed by PCR or Southern blot to determine the mouse's genotype. Mice who carry the transgene are subsequently mated in order to produce a line of mice.
  • mice which contain transgenes encoding an ENT.
  • transgenic mice which carry genes encoding the ENT under the control of specific promoters. Examples of such constructs are genes encoding SEQ ED NO:2 under the control of the rat whey acidic protein regulatory sequences (Yarus, S., et al., 1997, "The carboxy-terminal domain of human surfactant protein B is not required for secretion in milk of transgenic mice," Front. Biosci.
  • mice 2:A1-A8 mouse mammary tumor virus (MMTV) LTR
  • MMTV mouse mammary tumor virus
  • SEQ ED NO:4 under control of the immunoglobulin promoter

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Abstract

The invention provides substantially purified equilibrative nucleoside transporter polypeptide, and polynucleotides encoding ENTs. Antibodies which bind equilibrative nucleoside transporter polypeptide and formulations for administration of these antibodies are also disclosed. Methods for identifying a compound which affects an equilibrative nucleoside transporter, and for determining if a compound utilizes an equilibrative nucleoside transporter are provided. A method of assessing a therapeutic intervention for a subject, where the effectiveness of the therapeutic intervention is correlated with the presence or absence of an equilibrative nucleoside transporter, is provided. A method of treating a subject having or at risk of having a disorder associated with an equilibrative nucleoside transporter is also disclosed. Kits for detecting the presence of an equilibrative nucleoside transporter are provided. Transgenic animals having a transgene encoding an equilibrative nucleoside transporter are described.

Description

MAMMALIAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application Serial No. 60/034,083, filed December 30, 1996, and U.S. Provisional Application Serial No. 60/064,004, filed November 3, 1997.
FIELD OF THE INVENTION
This application relates generally to nucleoside transporters, and more specifically to a polynucleotide which encodes a nucleoside transporter that may be useful for the development of new nucleoside drugs.
BACKGROUND OF THE INVENTION
Natural and synthetic nucleosides have important physiologic and pharmacologic activities in humans. Adenosine, for example, functions as a local hormone in regulation of lipolysis. neurotransmitter release, platelet aggregation, coronary vasodilation. and cardiac contractility (Belardinelli, L., et al., 1989, Prog. Cardiovasc. Dis. 32:73-97; Jacobson, .A.. et al. (eds), 1990, Purines in Cellular Signaling: Targets for New Drugs, Springer-Verlag,
New York). Nucleoside antimetabolites have therapeutic applications in human neoplastic and viral diseases, including leukemias and AIDS (Perigaud et al. 1992. Nucleosides & Nucleotides 11, 903-945; Handshumacher and Cheng, 1993, in: Cancer Metabolism (Holland, E., Frei, E., Bast, R.C., Kufe, D.W., Morton, D.L., and Weichselbaum, R.R. eds)pp. 712-732, Lea & Febiger, Philadelphia.). Nucleoside transport processes also play an important role in adenosine-mediated regulation of coronary vasodilation. renal vasoconstriction, neurotransmission, platelet aggregation and lipolysis (Belardinelli et al., 1989; Jacobson et al., 1990).
Most nucleoside drugs act intracellularly, after anabolic phosphorylation, by interfering either directly or indirectly with DNA synthesis. For those nucleosides that are hydrophilic, mediated nucleoside transport systems (NT processes) are required for passage across the plasma membrane. In experimental systems, there is evidence that the activity of NT processes can be an important determinant of pharmacologic action of cytotoxic nucleoside drugs. For example, cultured cells made incapable of transporting nucleosides by genetic mutations or treatment with NT inhibitors exhibit low levels of uptake of adenosine and other endogenous nucleosides and are resistant to a variety of nucleoside analogs with anticancer activity. The permeant selectivities and mechanisms regulating distribution and expression of NT processes are important factors to be considered in the design of nucleoside analogs as therapeutic agents in human diseases. The transport of nucleosides across the plasma membranes of mammalian cells occurs both by equilibrative (facilitated diffusion, sodium independent) processes and by concentrative (sodium-dependent) processes (Griffith, D.A. and Jarvis, S.M., 1996, Biochim. Biophys. Acta 1286, 153-181 ; Cass, C.E., 1995, in "Drug transport in antimicrobial and anticancer chemotherapy", Georgopapadakou, N.H. (ed.), Marcel Dekker, New York, pp. 403-451 ). The latter are thought to be restricted to specialized cells such as intestinal and renal epithelia and liver, whilst equilibrative, sodium- independent processes are more widely distributed. These can be divided into two types on the basis of their sensitivity to inhibition by the 6-thiopurine ribonucleoside nitrobenzylthioinosine (NBMPR) (Griffith and Jarvis, supra; Cass, supra). Transporters of the es type (equilibrative sensitive) are inhibited by NBMPR, which binds with high affinities (Kd 0.1 - 10 nM). In contrast, transporters of the ez-type (equilibrative insensitive) are little affected by concentrations of NBMPR below 1 μM. Similarly, ei- type transporters are generally less susceptible to the coronary vasodilators, dipyridamole and dilazep, and to lidoflazine analogues, than are the es-tγoe transporters from the same species (Griffith and Jarvis, supra). Both classes of transporter have a similar broad substrate specificity for purine and pyrimidine nucleosides, although some e -type transporters have been reported to have lower affinities for nucleosides than es-type transporters from the same animal species (Griffith and Jarvis, supra) or even from the same cell type (Boleti, H., et al, 1997, Neuropharmacol. 36, 1167-1 179). Although some cell types express only es or e -type transporters, many express both
(Griffith and Jarvis, supra). The es and ei nucleoside transporters are under independent genetic control and are produced either singly or together in different cell types and tissues (Belt, J.A. and Noel, L.D., 1988, J. Biol. Chem. 263, 13819- 13822; Cass, C. E., et al.. 1974, Biochim. Biophys. Acta 345: 1 -10; Jarvis, S.M., and Young, J.D.. 1980, Biochem. J. 190:377-383; Jarvis, S.M., et al., 1980, Biochim. Biophys. Acta 597: 183-188; Jarvis, S.M., and Young, J.D., 1986, J. Membr. Biol. 93: 1-10; Belt, J.A., and Noel, L.D.. 1985, Biochem.
J. 232:681-688; Gati, W.P., et al., 1986, Biochem. J. 236:665-670; Dagnino, L.. and Paterson. A.R.P., 1990, Cancer Res. 50:6549-6553).
Transporters of the e.v-type are widely distributed in different cell types (Paterson et al., 1991 ; Kwong, F.Y.P, et al., 1993, J. Biol. Chem. 268:22127-22134) and tissues, bind NBMPR reversibly (Cass, C.E., 1995, in: Drug Transport in Antimicrobial and Anticancer chemotherapy, Georopapadakou, N.H., ed., Marcel Dekker, New York, pp. 403-4 1 ), and can be covalently radiolabeled with [3H]NBMPR by exposure of the transporter-ligand complex to UV light (Wu. J.-S. R.. et al., 1983, J. Biol. Chem. 258: 13745- 13751 ). These transporters have a broad substrate selectivity for purine and pyrimidine nucleosides, are essential for nucleotide synthesis by salvage pathways in haemopoietic and other cells that lack de novo pathways, and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy (Cass, 1995, supra). They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs such as dipyridamole and dilazep (Belardinelli, L.,1989, supra; Jacobson, K. A., et al.,
1990, supra).
One example of an es-type nucleoside transporter is the erythrocyte transporter, an integral membrane glycoprotein of apparent Mr 55,000 (Wu et al. 1983), has been purified to apparent homogeneity by a combination of ion-exchange and immunoaffinity chromatography (Kwong et al.,1988, Purification of the human erythrocyte nucleoside transporter by immunoaffinity chromatography, Biochem. J. 255, 243-249). The erythrocyte transporter is inhibited by nanomolar concentrations of NBMPR (Young, J. D. & Jarvis, S. M.. 1983, Nucleoside transport in animal cells. Biosci. Rep. 3, 309-322; Paterson, A.R.P. et al. (1983) in Regulatory Functions of Adenosine, Berne, R.M., et al.(eds), Martinus Nijhoff. The Hague pp. 203-220). SUMMARY OF THE INVENTION
The invention is based on the discovery of equilibrative nucleoside transporters (ENT) that transport a variety of purines and pyrimidines, including, but not limited to adenosine, uridine, guanosine, inosine. formycin B, tubercidin, and thymidine. The ENTs are bidirectional; they transport a suitable permeant both into and out of cells. The ENT can be used as a tool for the development of new nucleoside drugs. Two human and two rat genes encoding ENT have been isolated.
In a first embodiment, the invention provides substantially purified equilibrative nucleoside transporter polypeptide. Isolated polypeptides having an amino acid sequence which are at least 85% identical to a polypeptide sequence including SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, and SEQ ID NO:8 are also provided. Isolated polypeptides which are fragments or analogs of SEQ ID NO:2. SEQ ID NO:4. SEQ ID NO:6, and SEQ ID NO:8 are further disclosed. Polynucleotides encoding the polypeptides of the invention are further provided. Antibodies which bind equilibrative nucleoside transporter polypeptide and formulations for administration of these antibodies are also disclosed.
In another embodiment, a method for identifying a compound which affects an equilibrative nucleoside transporter is provided. A method for determining if a compound utilizes a equilibrative nucleoside transporter is also further provided. In an additional embodiment, a kit for the detecting the presence of an equilibrative nucleoside transporter is provided. A kit useful for the detection of target nucleic acid sequences indicative of the ability to transport nucleoside using an equilibrative nucleoside transporter is also disclosed.
A method of assessing a therapeutic intervention for a subject, where the effectiveness of the therapeutic intervention is correlated with the presence or absence of an equilibrative nucleoside transporter, is provided. A method of treating a subject having or at risk of having a disorder associated with an equilibrative nucleoside transporter is also disclosed.
In a further embodiment, transgenic animals having a transgene encoding a equilibrative nucleoside transporter are provided. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is the cDNA nucleotide sequence (SEQ ID NO: l ) and deduced amino acid sequence (SEQ ID NO:2) of human equilibrative nucleoside transporter (hENTl). Fig. 2 is the cDNA nucleotide sequence of the hENTl (SEQ ID NO: l ) Fig. 3 is the deduced amino acid sequence (SEQ ID NO:2)of human equilibrative nucleoside transporter (hENTl).
Fig. 4 shows a topographical model of hENTl . Potential membrane-spanning a- helices are numbered and the putative N-glycosylation site at Asn 8 is indicated. The positions of basic (Arg, Lys. His), acidic (Asp, Glu) and polar but uncharged residues (Ser, Thr, Gin, Asn) are indicated by + , - and darkened circles respectively.
Fig. 5 is an alignment of the predicted amino acid sequence of hEΝTl as compared with the amino acid sequnece of the yeast protein FUΝ26 (SEQ ID NO:9) (Ouellette et al.. 1993, Sequencing of chromosome I from Saccharomyces cerevisiae: analysis of a 32 kb region between the LTE1 and SP07 genes. Genome 36, 32-42), the nematode proteins ZK809.4 (SEQ ID NO: 10) and F16H 1 1.3 (SEQ ID NO: l 1), and the mouse (mHNP36)
(SEQ ID NO: 12) and human (hHNP36) (SEQ ID NO: 13) delayed early response gene proteins (Williams. J.B., and Lanahan, A.A.. 1995, Biochem. Biophys. Res. Commun. 213 : 325-333). The positions of putative transmembrane regions are shown as open rectangles, and residues identical in four or more of the sequences are indicated by black boxes. The arrow shows the position of the residue previously assigned (Williams & Lanahan, 1995. supra) as the N-terminus for the two HΝP36 proteins, and the translated open reading frame upstream of this location in the human protein is underlined. Asterisks indicate potential N- linked glycosylation sites in hEΝTl .
Fig. 6A shows the time-course of uridine uptake into Xenopus oocytes. Oocytes were injected with water (o) or with water containing in vitro transcribed RΝA encoding hEΝTl
(•). Fig. 6B illustrates the substrate specificity of hEΝTl -mediated uridine influx for physiological nucleosides and chemotherapeutic nucleoside analogues. The hatched column corresponds to uptake measured in the absence of sodium. Fig. 6C shows the concentration- dependence of hEΝTl -mediated uridine influx. Oocytes were injected with water (o) or water containing in vitro transcribed RΝA encoding hEΝTl (•). The inset shows influx of uridine in RΝA-injected oocytes minus that in water-injected oocytes. Fig. 6D shows the inhibition of hENTl -mediated uridine influx by NBMPR, dilazep and dipyridamole. The inset shows the dose-response curve for NBMPR inhibition of uridine influx.
Fig. 7 A shows the time course of adenosine uptake into Xenopus oocytes. Oocytes were injected with water alone (o) or with water containing in vitro transcribed RNA encoding hENTl (•). Fig. 7B shows the inhibition of hENTl -mediated adenosine influx by uridine. NBMPR, dilazep and dipyridamole.
Fig. 8 is the cDNA nucleotide sequence (SEQ ID NO: 3) of the rat equilibrative nucleoside transporter (rENTl).
Fig. 9 is the deduced amino acid sequence (SEQ ID NO: 4) of rat equilibrative nucleoside transporter (rENTl ).
Fig. 10 shows the functional expression of rENTl Xenopus oocytes. Influx of [NC]-labelled uridine and adenosine ( 10 μM, 20°C) was measured in oocytes injected with water or with water containing RNA transcript. Values are means (+ SEM) of 10 oocytes.
Fig. 1 1 illustrates the substrate specificity of rENTl -mediated uridine influx. Influx of uridine (10 μM) in oocytes containing rENTl transporter was measured in the absence
(Hatched column) or presence of competing nonradioactive nucleosides (+T, Thymidine; +A, adenosine; +G,guanosine; +U, Uridine; +1, inosine;+C, cytidine; +DA. deoxyadenosine ) or uracil.
Fig. 12 shows NBMPR and dipyridamole inhibition of hENTl and rENTl . Uridine influx (10 μM) in oocytes containing either rENTl (m) or hENTl (1) transporter was measured in the presence of increasing concentrations of NBMPR (A) and dipyridamole (B) and expressed as a percentage of the control flux in the absence of inhibitor.
Fig. 13 shows a comparison of the predicted amino acid sequence of hENTl (SEQ ID NO:2) with that of rENTl (SEQ ID NO:6). Identical amino acids are marked in bold. Fig. 14 is an alignment of the predicted amino acid sequences of rENTl (SEQ ID
NO:4) and rENT2 (SEQ ID NO: 6) as compared with the amino acid sequences of the human NBMPR-sensitive nucleoside transporter hENTl (SEQ ED NO: 2) and the human (hHNP36) (SEQ ID NO: 13) and mouse (mHNP36) (SEQ ID NO: 12) delayed-early response gene products. Amino acid sequences of rENTl and rENT2 were deduced from the nucleotide open reading frames of clones pAN2 and pAN3. The sequence of hENTl is from reference 25. The arrow shows the position of the residue previously assigned as the amino-terminus of the two HNP36 proteins (Williams, J. B., and Lanahan, A. A., 1995, supra), and the translated open reading frame upstream of this location in the human protein is underlined. The positions of putative transmembrane regions are shown as open rectangles, and residues identical in three or more of the sequences are indicated by black boxes. Potential N-linked glycosylation sites in the hydrophilic segment linking putative TMs 1 and 2 of hEΝTl, rEΝTl, rEΝT2, mHNP36 and hHNP36 are shown in lower case ("n") and their positions highlighted by an asterisk above the aligned sequences.
Fig. 15A shows the hydropathy profiles for the predicted amino acid sequences of rENTl (SEQ ID NO:4) and rENT2 (SEQ ED NO:6) were determined by the method of Eisenberg et al. (Εisenberg, D., et al., 1984, J. Mol. Biol. 179, 125-142). Fig. 15B shows the potential membrane -spanning -helices in the topographical model are numbered and putative Λ'-linked glycosylation sites in rENTl and rENT2 are indicated
Figure imgf000009_0001
solid and open stars, respectively. Residues identical in the two proteins are shown as darkened circles. Residues corresponding to insertions in the sequences of rENTl or rENT2 are indicated by circles containing "+" and "-" signs, respectively.
Fig. 16 illustrates the nucleoside uptake by recombinant rENTl and rENT2 expressed in Xenopus oocytes. Oocytes injected with either 10 nl of water alone or 10 nl of water containing 10 ng of rENTl (Fig. 16A) or rENT2 (Fig. 16B) RNA transcript were incubated for 3 days at 18°C in MBM. Fluxes of uridine and adenosine (10 μM. 20 °C) were determined in transport buffer containing 100 mM NaCl. Each value is the mean ± SEM of 10-12 oocytes. In A: •, oocytes injected with RNA: o, oocytes injected with water.
Fig. 17 illustrates the nucleoside specificity of rENTl and rENT2. Uridine influx (10 μM. 20 CC. 1 min flux) in transport buffer containing 100 mM NaCl (open columns) or 100 mM choline chloride (hatched columns) was measured in rENTl (Fig. 17A) and rENT2 (Fig. 17B) RNA transcript-injected oocytes in the absence (control) or presence of 2 mM nonradioactive physiological nucleosides (T. thymidine; C, cytidine; G, guanosine; I, inosine: A. adenosine; U, uridine) or uracil. Nonradioactive nucleosides and uracil were added to oocytes at the same time as [MC]uridine. H 0, water-injected oocytes. Each value is the mean + SEM of 10-12 oocytes. Fig. 18 show the inhibition of rENTl , hENTl and rENT2 by NBMPR. dipyridamole and dilazep. Dose-response curves for NBMPR (Fig. 18 A), dipyridamole (Fig. 18B) and dilazep (Fig. 18C) inhibition of uridine influx (10 μM, 20 °C, 1 min flux) were measured in NaCl transport buffer in oocytes injected with RNA transcript for rENTl (o) and hENTl (•). Cells were preincubated with inhibitor for 1 h before addition of permeant. Each value is the mean + SEM of 10-12 oocytes. Fluxes were not corrected for the small contribution of endogenous transport activity and are expressed as a percentage of the uninhibited rENTl and hENTl values, which were 0.98 + 0.03 and 0.82 + 0.04 pmol/oocyte, respectively, in A and C, and 0.66 + 0.02 and 0.61 + 0.02 pmol/oocyte, respectively, in B. IG0 values (+ SE), determined by linear regression analysis of indirect Hill plot transformations of the dose- response curves (Enzfitter, Elsevier-Biosoft), are presented in the text. For rENT2 (D), NBMPR, dipyridamole (Dp) and dilazep (Dz) were present at a concentration of 1 μM.
Fig. 19 demonstrates the concentration dependence of uridine influx by rENTl and rENT2. Oocvtes were injected with water alone (o) or water containing RNA transcript (•) for rENTl (Fig. 19A) and rENT2 (Fig. 19B). Influx of uridine (10 μM, 20°C, 1 min) was measured in NaCl transport buffer. Each value is the mean + SEM of 10-12 oocytes. The insets show v versus vis plots of the transporter-mediated fluxes, calculated as influx in RNA transcript-injected oocytes minus influx in water-injected cells). Apparent K,_ and V^ values (+ SE) were determined by non-linear regression analysis (Enzfitter, Elsevier-Biosoft).
Fig. 20 shows alignment of the predicted amino acid sequence of hENT2 (SEQ ED NO:8) with that of the human NBMPR-sensitive nucleoside transporter hENTl (SEQ ID NO:2). The amino acid sequence of hENT2 was deduced from the nucleotide sequence of the open reading frame of the insert in clone pHNPc7. The underlined region of the hENT2 sequence is missing from the published sequence of human HNP36, and an arrow shows the position of the residue previously assigned as the amino-terrninus in that protein. The positions of putative transmembrane regions are shown as open rectangles and residues identical in both sequences are indicated by black boxes. Potential N-linked glycosylation sites in the hydrophilic segment linking putative TMs 1 and 2 of the proteins are shown in lower case C"n ') and their positions highlighted by an asterisk above the aligned sequences.
Fig. 21. Nucleoside uptake by recombinant hENTl and hENT2 expressed Xenopus oocytes. Oocytes injected with either 10 nl water alone or 10 nl water containing 10 ng hENTl or hENT2 RNA transcript were incubated for 3 days at 18°C. Fluxes of 10 μM uridine (open bars) and adenosine (hatched bars) at 20 °C were then determined as described (see Examples below).
Fig. 22 shows the concentration dependence of hENT2-mediated uridine influx. Oocytes were injected with water alone (O) or with water containing RNA transcript for hENT2 (•). After incubation for 3 days, the uptake of uridine was measured over the concentration range indicated, as described in Materials and Methods. The inset shows a v versus v/s plot of the transporter-mediated flux, calculated as influx in RNA transcript-injected oocytes minus influx in water-injected cells. Apparent Λ^ and Vmx. values, determined by nonlinear regression analysis, are presented in the text. Fig. 23 shows the inhibition of hENTl and hENT2 by NBMPR. Dose response curves for the inhibition of uridine influx by NBMPR were produced in oocytes injected with RNA transcripts encoding hENTl (•) or hENT2 (O). Fluxes were corrected for the small contribution of endogenous transport activity seen in water-injected oocytes, and are expressed as a percentage of the uninhibited hENTl and hENT2 values, which were 0.318 ± 0.023 and 0.224 ± 0.023 pmol/oocyte.min"1 respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention provides substantially purified equilibrative nucleoside transporter (ENT) polypeptides and polynucleotide sequences encoding the polypeptides. "Equilibrative nucleoside transporter" refers to a transport protein which is permeable to a variety of purines and pyrimidines and analogs. In general, an ENT is able to transport three or more of the following nucleosides or analogs: adenosine, uridine, guanosine, inosine, formycin B, tubercidin, and thymidine. ENTs include, but are not limited to, equilibration sensitive (es) transporters. The "equilibration sensitive'' transporters are Na+ independent nucleoside transporters which reversibly bind NBMPR with high affinities (Y^ values 0.1-10 nM), can be covalently radiolabelled with [3H]NBMR by exposure of the transporter-ligand complex to UV light (Williams, J.B., and Lanahan, A.A., 1995, Biochem. Biophys. Res. Comm. 213:325-333) and are widely distributed in different cell types and tissues. The equilibrative-insensitive (ei) transporters are Na+ -independent nucleoside transporters that are unaffected by micromolar concentrations of NBMR. ENT POLYPEPTIDES AND POLYNUCLEOTIDES
ENTs are "bi-directional," that is, they transport a suitable permeant both into and out of a cell. A "permeant" as used herein is any molecule which can be transported by an ENT. Preferably, a human equilibrative nucleoside transporter has an amino acid sequence set forth in SEQ ID NO:2 (hENTl ) or SEQ ID NO:8 (hENT2), and a rat equilibrative nucleoside transporter has an amino acid sequence as set forth in and SEQ ID NO:4 (rENTl) or SEQ ID NO:6 (rENT2). The term "substantially purified" as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify human or rat ENT using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the ENT polypeptide can also be determined by amino-terminal amino acid sequence analysis. ENT polypeptide includes functional fragments of the polypeptide. as long as the ENT activity remains. Smaller peptides containing the biological activity of ENT are included in the invention. Included in the invention are polypeptides having an amino acid sequence which is at least 65% identical to
SEQ ID NO:2. more preferably 80% identical to SEQ ID NO:2, and most preferably 98% identical to SEQ ED NO:2; polypeptides having an amino acid sequence which is at least 65% identical to SEQ ID NO:4, more preferably 80% identical to SEQ ID NO:4. and most preferably 98%) identical to SEQ ID NO:4; polypeptides having an amino acid sequence which is at least 65% identical to SEQ ID NO:6. more preferably 80% identical to SEQ ID NO:6. and most preferably 98%> identical to SEQ ID NO:6; and polypeptides having an amino acid sequence which is at least 65% identical to SEQ ED NO:8, more preferably 80%> identical to SEQ ID NO:8, and most preferably 98%> identical to SEQ ID NO:8.
Minor modifications of the hENTl, rENTl, hENT2, or rENT2 primary amino acid sequences may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of the ENT still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity.
This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which are not required for ENT biological activity.
The ENT polypeptides of the invention also include dominant negative forms of the polypeptides which do not have the biological activity of the ENTs. A "dominant negative form" of an ENT is a polypeptide that is structurally similar to an ENT of the invention which does not have normal ENT function: it interferes with normal ENT function by binding to, or otherwise sequestering, regulating agents, such as upstream or downstream components, that normally interact functionally with the ENT polypeptide.
The equilibrative nucleoside transporters of the invention can be inhibited by one or more of the following inhibitors: NBMPR, dipyridamole, or dilazep. The term "inhibit" as used herein, means a decrease in the ability to transport purines and pyrimidines or analogs. In general, an inhibitor of transport will decrease, to some extent, transport of purines. pyrimidines. or analogs relative to the transport in the absence of inhibitor.
The invention also provides polynucleotides encoding ENTs, specifically polynucleotides encoding hENTl , hENT2, rENTl, and rENT2. These polynucleotides include
DNA. cDNA and RNA sequences which encode ENT. It is understood that all polynucleotides encoding all or a portion of hENTl, hENT2, rENTl, and rENT2 are also included herein, as long as they encode a polypeptide with ENT activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, ENT polynucleotide may be subjected to site-directed mutagenesis. The polynucleotide sequence for
ENT also includes antisense sequences and sequences encoding the dominant negative forms. The polynucleotides of the invention include sequences that are degenerate nucleotide sequences of the ENTs of the invention that occur as a result of the degeneracy of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the ENT polypeptide (e.g., hENTl, rENTl, hENTs, or rENT2) encoded by the nucleotide sequence is functionally unchanged.
Specifically disclosed herein are isolated polynucleotide sequences that encode the hENTl , rENTl . hENT2. and rENT2 polypeptides. An exemplary hENTl polynucleotide sequence is SEQ ID NO: 1 , an exemplary rENTl polynucleotide sequence is SEQ ED NO:3. an exemplary rENT2 polynucleotide sequence is SEQ ID NO:5. and an exemplary hENT2 polynucleotide sequence is SEQ ID NO:7. The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form a nucleotides at least 10 bases in length. By "isolated polynucleotide" is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus: or into the genomic DNA of a prokaryote or eukaryote. or which exists as a separate molecule (e.g. a cDNA) independent of other sequences. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.
A polynucleotide encoding hENTl includes SEQ ED NO: 1, as well as nucleic acid sequences complementary to SEQ ID NO: 1. A polynucleotide encoding rENTl includes SEQ ID NO:3, as well as nucleic acid sequences complementary to SEQ ID NO:3. A polynucleotide encoding rENT2 includes SEQ ID NO:5. as well as nucleic acid sequences complementary to SEQ ID NO:5. A polynucleotide encoding hENT2 includes SEQ ID NO:7, as well as nucleic acid sequences complementary to SEQ ED NO:7. A complementary sequence may include an antisense nucleic acid sequence. When the sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ED NO: l, SEQ ED NO:3, SEQ ED NO:5, and SEQ ED NO:7 are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the invention are fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein of SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, or SEQ ED NO:8 or selectively hybridize to a structurally related ENT. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions which excludes non-related nucleotide sequences (e.g., Sambrook, J., et al, 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, New York).
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized (see Sambrook et al.. supra). For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1 % SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1%) SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g.. high stringency conditions, or each of the conditions can be used. e.g.. for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
The polynucleotide sequences encoding the hENTl, hENT2, rENTl, and rENT2 polypeptides of the invention include the disclosed sequences and conservative variations thereof. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine. leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
DNA sequences encoding an ENT (e.g., hENTl, rENTl, and rENT2) can be expressed in vitro by DNA transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term
"host cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
In the present invention, the ENT polynucleotide sequences (e.g. hENTl. rENTl. hENT2, or rENT2) may be inserted into an expression vector. The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the ENT genetic sequences. Polynucleotide sequence which encode an ENT (e.g. hENTl , rENTl , hENT2, or rENT2) can be operatively linked to expression control sequences. "Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence went the expression control sequences control and regulate the transcription and. as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, as start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns. maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to included, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
By "promoter" is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter- dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents: such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al.. 1987, Methods in Enzymology 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viaises (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention. In the present invention, the polynucleotide encoding an ENT (e.g. hENTl , rENTl . hENT2. or rENT2) may be inserted into an expression vector which contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al., 1987, Gene, 56:125), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans. 1988, J. Biol. Chem. 263:3521) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g.. T7, metallothionein I. or polyhedrin promoters). Polynucleotide sequences encoding an ENT can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
By "transformation" is meant a genetic change induced in a cell following incorporation of new DNA (i.e. DNA exogenous to the cell). Where the cell is a mammalian cell, the pgenetic change is generally achieved by introduction of the DNA into the genome of the cell (i.e. stable). By "transformed cell" is meant a cell into which (or into an ancestor of which has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding an ENT. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl method using procedures well known in the art. Alternatively. MgCl, or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection. electroporation. insertion of a plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with DNA sequences encoding the ENT (e.g. hENTl , rENTl , hENT2, or rENT2) of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, 1982, Cold Spring Harbor Laboratory, Gluzman ed.).
Isolation and purification of microbial expressed polypeptides. or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
ANTIBODIES
The ENT polypeptides of the invention can be used to produce antibodies which are immunoreactive or bind to epitopes of the ENT polypeptides. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. The preparation of polyclonal antibodies is well-known to those skilled in the art (e.g.,
Green et al., 1992, "Production of Polyclonal Antisera" in: Immunochemical Protocols, Manson, ed., pages 1-5, Humana Press: Coligan et al., 1992, "Production of Polyclonal Antisera in Rabbits. Rats. Mice and Hamsters" in: Current Protocols in Immunology, section 2.4.1 , which are hereby incorporated by reference. The preparation of monoclonal antibodies likewise is conventional (e.g.. Kohler &
Milstein, 1975, Nature 256:495; Coligan et al., 1992, sections 2.5.1-2.6.7, supra; and Harlow et al., 1988, in: Antibodies: a Laboratory Manual, page 726, Cold Spring Harbor Publications, which are hereby incorporated by reference). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas. cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose. size-exclusion chromatography. and ion-exchange chromatography. See, e.g., Coligan et al., 1992, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3, supra; Barnes et al., 1992, "Purification of Immunoglobulin G (IgG)" in: Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press).
Methods of in vitro and in vivo multiplication of monoclonal antibodies are well known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g.. syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
Therapeutic applications for antibodies disclosed herein are also part of the present invention. For example, antibodies of the present invention may also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., 1991, International PCT Patent Publication WO 91/1 1465 and Losman et al. 1990. Int. J. Cancer 46:310, which are hereby incorporated by reference.
Alternatively, a therapeutically useful anti-ENT antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al.. 1989. Proc. Nat'l Acad. Sci. USA 86:3833. which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., 1986, Nature 321 :522; Riechmann et al., 1988, Nature 332:323; Verhoeyen et al., 1988, Science 239: 1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89:4285; Sandhu. 1992. Crit. Rev. Biotech. 12:437; and Singer et al., 1993, J. Immunol. 150:2844, which are hereby incorporated by reference.
Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., 1991 , in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 1 19; Winter et al., 1994, Ann. Rev. Immunol. 12:433, which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al..
1994, Nature Genet. 7: 13; Lonberg et al.. 1994, Nature 368:856; and Taylor et al., 1994, Int. Immunol. 6:579, which are hereby incorporated by reference.
The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab'),, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
( 1 ) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain: two Fab' fragments are obtained per antibody molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab' is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) Single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of making these fragments are known in the art (e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, incorporated herein by reference). As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab'^ . This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. patents No. 4,036.945 and No. 4.331.647, and references contained therein. These patents are hereby incorporated in their entireties by reference. See also Nisonhoff et al, 1960, Arch. Biochem. Biophys. 89:230, Porter, 1959, Biochem. J. 73 : 1 19; Edelman et al, 1967, Methods in Enzymology, Vol. 1 , page 422. Academic Press; and Coligan et al., at sections 2.8.1-2.8.10 and 2.10.1-2.10.4. supra. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al., 1972, Proc. Nat'l Acad. Sci. USA 69:2659. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu. supra. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., 1991, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97;
Bird et al., 1988, Science 242:423-426; Ladner et al., U.S. patent No. 4,946,778; Pack et al., 1993, Bio/Technology 11 :1271-77; and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., 1991 , Methods: a Companion to Methods in Enzymology, Vol. 2, page 106.
Antibodies which bind to an ENT polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies. as well as monoclonal antibodies (See for example, Coligan. et al., 1991. Unit 9, Current
Protocols in Immunology, Wiley Interscience, incorporated by reference).
It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.
METHOD FOR IDENTIFYING COMPOUNDS WHICH AFFECT OR USE EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS
In the past two decades, adenosine and adenine nucleotides have been shown to be powerful regulators of cellular function in a variety of physiological systems through their specific interactions with purinergic receptors. Purinergic receptors have important roles in many different process including platelet function, regulation of blood flow in vascular beds, inflammation, neuro transmitter release, angiogenesis. lipogenesis, atrial contractility and AV conduction. Because the cellular release and reuptake of adenosine occurs primarily by meditated transport processes, nucleotide inhibitors are potential modulators of interaction of adenosine with its receptors and thus have potential applications in many different diseases.
The classic inhibitors of ENT-mediated transport (e.g., NPMPR, dipyridamole) have been shown in model systems to be capable of potentiating the purinergic activity of adenosine. Currently drug discovery programs are directed towards the identification of nucleoside transporter inhibitors that are used to modulate the vasodilatory action of adenosine in the treatment of coronary or cerebrovascular anoxia. The pharmaceutical applications of inhibitors of nucleoside transporters extend into ADDS and cancer treatment by providing an opportunity to manipulate intracellular levels of chemotherapeutic nucleoside drugs. The availability of recombinant ENTs, which can be reproducibly produced in a variety of expression systems, offers the possibility of rapid identification of ENT inhibitors that could be used in developing new approaches to combination chemotherapy with antiviral or anticancer drugs. The invention provides a method for identifying a compound which can modulate ENT activity. The method includes incubating compounds and a sample under conditions sufficient to allow the components to interact, and measuring the permeability of the sample to a nucleoside using a permeant (which may also be a nucleoside), and then comparing the permeability of the sample, incubated with the compound, to the permeant with the permeability of a control sample not incubated with the compound. The compounds which affect ENT include peptides. polypeptides. chemical compounds and biological agents. Antiviral and chemotherapeutic compounds can be tested using the method of the invention. "Incubating" includes conditions which allow contact between the test compound and the ENT. "Contacting" includes in solution and solid phase. The test compound may also be a combinatorial library for screening a plurality of compounds. Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced. and the like, either in solution of after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence, such as PCR. oligomer restriction (Saiki et al..l985, Bio/Technology, 3: 1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,1983, Proc. Natl. Acad. Sci. USA, 80:278), oligonucleotide ligation assays (OLAs) (Landegren et al, 1988, Science, 241 : 1077), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., 1988. Science, 242:229-237).
A compound can affect an equilibrative nucleoside transporter by either stimulating or inhibiting transport of a nucleoside. A compound "inhibits"' an equilibrative nculeoside transporter if the ability to transport purines and pyrimidines or analogs is decreased. A compound "stimulates" an equilibrative nucleoside transporter if the ability to transport purines and pyrimidines or analogs is increased.
The sample can be any sample of interest. The sample may be a cell sample or a membrane sample prepared from a cell sample. Suitable cells include any host cells containing a recombinant ENT vector of the invention. The cells functionally express the ENT polypeptide. but preferably have no other nucleoside transporters or have low background nucleoside transporters.
The permeability of the sample is measured in order to determine if the compound can effect an ENT. "Permeability" is defined as the ability of a permeant to move from one side of a membrane to another. Permeability can be an efflux (movement of the permeant across a membrane from the inside of a cell or intracellular organelle to the outside of a cell or intracellular organelle) or an influx (movement of the permeant across a membrane from the outside of a cells or an intracellular organelle into the inside of a cell or intracellular organelle). The permeant can be any molecule known to be transported via an ENT. The permeant can be detectably labeled. Most preferably, the permeant is radiolabeled; alternatively the label can be a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.
Most candidate ENT permeants are not available in a labeled form. Such compounds can be screened in a cis-inhibition assay for their ability to compete for influx with a know radiolabelled permeant. Alternatively, they can be tested in a trans-acceleration assay for their ability to stimulate efflux of a radiolabelled permeant. Trans-acceleration assays have the important advantage over cis-inhibition tests for distinguishing between compounds which bind to the transporter but are not true permeants (details of efflux and influx experiments can be found in Cass et al., supra) The invention further provides a method for determining if a compound utilizes an equilibrative nucleoside transporter to enter or leave a cell. The method includes incubating the compound with a sample of interest under conditions sufficient to allow the compound to interact with the sample in the presence of an equilibrative nucleoside transporter inhibitor, detecting the permeability of the sample of interest to the compound, and comparing the permeability of the sample incubated in the presence of both the compound and the inhibitor to a control sample incubated with the compound but without inhibitor. Compounds of use with the invention include, but are not limited to, anticancer and antiviral agents.
The sample can be any sample of interest. The sample may be a cell sample or a membrane sample prepared from a cell sample. Suitable cells include any host cells containing a recombinant ENT vector of the invention. The cells functionally express the ENT polypeptide. but preferably have no other nucleoside transporters or have low background nucleoside transports.
KITS
The materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. One of the container means may comprise a probe which is or can be detectably labeled. Such probe may be an antibody or nucleic acid sequence specific for an ENT, or fragments thereof. For example, oligonucleotide probes of the present invention can be included in a kit and used for examining the presence of hENTl, hENT2, rENTl, or rENT2, in a sample, as well as the quantitative (relative) degree of binding of the probe for determining the occurrence of specific strongly binding (hybridizing) sequences.
The kit may also contain a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin. bound to a reporter molecule, such as an enzymatic, fluorescent, or radionucleotide label to identify the detectably labeled oligonucleotide probe.
Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. When it is desirable to amplify the hENTl, hENT2, rENTl or rENT2 target sequence, this can be accomplished using oligonucleotide(s) that are specific primers for amplification.
The kit may also contain a container containing antibodies which bind to hENTl, hENT2. rENTl or rENT2, or fragments thereof. Such antibodies can be used to distinguish the presence of an ENT or the level of expression of ENT in a specimen. Where the kit utilizes antibodies to detect the ENT, these antibodies may be directly labeled. The kit may also contain a container containing a reporter means, such as avidin or steptavidin. bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the directly labeled antibody. Alternatively, the kit can utilizes antibodies that bind hENTl, hENT2, rENTl, or rENT2 that are unlabeled. The kit may then also contain a container containing a second antibody which binds to the antibody specific for the ENT of interest. The second antibody can be directly labeled. The kit may also a container containing a reporter means, such as avidin or steptavidin, bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the directly labeled second antibody. THERAPEUTIC INTERVENTION
For treatment of cancer, knowing the type of transporter or transporters, the level of expression of the transporter or transporters, and their functional properties in the neoplastic cells is important. In cancer chemotherapy, drugs that are nucleoside analogs or function like nucleoside analogs must be able to enter the cell to be effective. If the neoplastic cells express high levels of a particular ENT, then the therapy can include drugs that are permeants for that particular ENT. If the neoplastic cells are deficient in the expression of a particular ENT, permeants that are more suitable for other transporters may be useful.
The invention provides a method for assessing the effectiveness of a therapeutic intervention for a subject which includes obtaining a sample from the subject, determining the presence or absence of the expression of an ENT in the sample, and then correlating the presence or absence of the ENT with the effectiveness of the therapeutic intervention.
The invention also provides a method for assessing if a therapeutic intervention will produce side effects in a subject, and a means to determine if ENT inhibitors may lessen side effects. A sample of tissue not affected by the disease process is obtained from a subject, and the presence or absence of the expression of the ENT is determined. If a particular therapeutic intervention can be transported through a particular ENT, and this ENT is identified in the sample taken from the tissue not affected by the disease process, then side effects can occur. If protection of the tissue not affected by the disease process from the effects of the theapeutic interention is desired, then protection of normal tissues from the therapeutic intervention may be possible by using a specific ENT inhibitor (see U.S. patent 5,236,902). The inhibitor may be classical pharmaceutical agents such as NBMPR, dipyridamole, or dilazep, or it may be an antibody that binds an ENT of the invention, so long as the ability to transport purines and pyrimidines or analogs is decreased. For purposes of the invention, an antibody or nucleic acid probe specific for an ENT may be used to detect the ENT polypeptide (using antibody) or polynucleotide (using nucleic acid probe) in subject samples such as biological fluids, cells, tissues, or nucleic acid. Any specimen containing a detectable amount of antigen or polynucleotide can be used. Examples of biological fluids of use with the invention are blood, serum, plasma, urine, mucous, and saliva. Tissue or cell samples can also be used with the subject invention. The samples can be obtained by many methods such as cellular aspiration, or by surgical removal of a biopsy sample.
The invention provides a method for detecting an ENT, for example, which comprises contacting an anti-ENT antibody or nucleic acid probe with a cell suspected of expressing the ENT and detecting binding to the antibody or nucleic acid probe. The antibody reactive with the ENT or the nucleic acid probe is preferably labeled with a compound which allows detection of binding to the ENT of interest. A preferred sample of this invention is neoplastic tissue or tissue affected by HEV (as in tissue takent from AEDS patients). The level of the ENT in the subject cell can be compared with the level in a cell not affected by the disease process. Preferably the subject is human.
When the cell component is nucleic acid, it may be necessary to amplify the nucleic acid prior to binding with the ENT specific probe. Preferably, polymerase chain reaction (PCR) is used, however, other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
The antibodies of the invention can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy. The antibodies of the invention are suited for use. for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimrnunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
The antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the polypeptide of the invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran. nylon, amylases. natural and modified celluloses, polyacrylamides. agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation. Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl. puridoxal, and fluorescein. which can react with specific antihapten antibodies. The present invention identifies a polynucleotide sequence that can be expressed in an altered manner as compared to expression in a normal cell, therefore it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence. The antibodies and polynucleotides of the invention can be used to detect or to treat an equilibratvie nucleoside transporter-associated disorder. The term "equilibratvie nucleoside transporter-associated disorder" denotes malignant as well as non-malignant disorders, where tire cells involved in the disorder differ from the surrounding tissue or from unaffected cells in their expression of an equilibrative nucleoside transporter.
Detection of elevated levels of ENT expression is accomplished by hybridization of nucleic acids isolated from a cell of interest with an ENT polynucleotide of the invention. Analysis, such as Northern Blot analysis, are utilized to quantitate expression of the ENT. Other standard nucleic acid detection techniques will be known to those of skill in the art.
Treatment can include modulation of ENT gene expression and ENT activity by administration of a therapeutically effective amount of a reagent that modulates the ENT. The term "modulate" envisions the suppression of expression of a particular ENT when it is over- expressed, or augmentation of the expression of a particular ENT when it is under-expressed. Where a disorder is associated with the decreased expression of an ENT, nucliec acid sequences that encode that ENT can be used. Where a disorder is associated with the increased expression of an ENT, nucleic acid sequences that interfere with the expression of the ENT at the translational level can be used. This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific ENT mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme. Such disorders include cancer, for example.
Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American. 262:40). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target ENT-producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art
(Marcus-Sakura, 1988, Anal.Biochem., 172:289).
Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al.. 1991, Antisense Res. and Dev.. 1(3):227; Helene, C, 1991, Anticancer Drug Design. 6(6):569).
Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs. it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J.Amer.Med. Assn., 260:3030). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, 1988. Nature, 334:585) and "hammerhead"-type. Tetrahymena-typs ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 1 1-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
The present invention also provides gene therapy for the treatment of disorders which are associated with an ENT protein. Such therapy would achieve its therapeutic effect by introduction of a therapeutic polynucleotide into cells having the disorder. The "therapeutic polynucleotide" may be polynucleotide sequences encoding an ENT of the invention, or antisense polynucleotide specific for an ENT of the invention, designed to treat an ENT- associated disorder. Delivery of the therapeutic polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Especially preferred for therapeutic delivery of antisense sequences, or ENT polynucleotides, is the use of targeted liposomes.
Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus. herpes virus, vaccinia, or. preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to:
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) is utilized. A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting an ENT sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of. or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ENT polynucleotide.
Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium. Another targeted delivery system for the therapeutic polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres. beads, and lipid-based systems including oil-in-water emulsions. micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al..1981, Trends
Biochem. Sci.. 6:77,). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al.,1988, Biotechniques. 6:682).
The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids. especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds. such as phosphatidylglycerol. phosphatidylcholine, phosphatidylserine, phosphatidyletha- nolamine. sphingolipids, cerebrosides, and gangliosides. Particularly useful are d-iacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms. particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.
This invention involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier. "Administering" the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. By
"subject" is meant any mammal, preferably a human.
The pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a patient. depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate. Tire administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals. The pharmaceutical compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle. Suitable solid or liquid pharmaceutical preparation forms are. for example, granules, powders, tablets, coated tablets, (micro)capsules. suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, 1990, Science, 249:1527-1533, which is incorporated herein by reference.
The pharmaceutical compositions according to the invention may be administered locally or systemically. By "therapeutically effective dose" is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disorder and its complications. Amounts effective for this use will, of course. depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al., eds., 1990, Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press: and Remington's Pharmaceutical Sciences, 1990,
17th ed., Mack Publishing Co., Easton. Pa., each of which is herein incorporated by reference.
The identification of an ENT provides a useful tool for diagnosis, prognosis and therapeutic strategies associated with expression of a particular ENT. Measurement of levels of an ENT using anti-ENT antibodies is a useful diagnostic for determining therapeutic strategies. TRANSGENIC ANIMALS
In another embodiment, the present invention relates to transgenic animals having cells that express an ENT. Such transgenic animals represent a model system for the study of ENT related disorders and the or the study of therapeutics targeted to an ENT. The term "animal" here denotes all mammalian species except human. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Farm animals (pigs, goats, sheep, cows, horses, rabbits and the like), rodents (such as mice), and domestic pets (for example, cats and dogs) are included within the scope of the present invention. A "transgenic" animal is any animal containing cells that bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with recombinant vims. "Transgenic" in the present context does not encompass classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule. Although it is highly preferred that this molecule be integrated within the animal's chromosomes, the present invention also contemplates the use of extrachromosomally replicating DNA sequences, such as might be engineered into yeast artificial chromosomes.
The term "transgenic animal" also includes a "germ cell line" transgenic animal. A germ cell line transgenic animal is a transgenic animal in which the genetic information has been taken up and incorporated into a germ line cell, therefore conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too. are transgenic animals.
It is highly preferred that the transgenic animals of the present invention be produced by introducing into single cell embryos DNA encoding hENTl, hENT2, rENTl. or rENT2. in a manner such that the polynucleotides are stably integrated into the DNA of germ line cells of the mature animal and inherited in normal Mendelian fashion. Advances in technologies for embryo micromanipulation now permit introduction of heterologous DNA into fertilized mammalian ova. For instance, totipotent or pluripotent stem cells can be transformed by microinjection. calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal. In a preferred method, developing embryos are infected with
_o a retrovirus containing the desired DNA. and transgenic animals produced from the infected embryo.
In a most preferred method the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals. These techniques are well known. For instance, reviews of standard laboratory procedures for microinjection of heterologous DNAs into mammalian (mouse, pig, rabbit, sheep, goat, cow) fertilized ova include: Hogan et al., 1986. Manipulating the Mouse Embryo, Cold Spring Harbor Press; Krimpenfort et al.. 1991. Bio/Technology 9:86; Palmiter et al. 1985, Cell 41 :343; Kraemer et al., 1985, Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Laboratory Press; Hammer et al.. 1985, Nature
315:680; Purcel et al.. 1986. Science. 244: 1281 ; Wagner et al., U.S. patent No. 5.175.385; Krimpenfort et al.. U.S. patent No. 5,175.384. the respective contents of which are incorporated by reference.
The cDNA that encodes the ENT of interest can be fused in proper reading frame under the transcriptional and translational control of a vector to produce a genetic construct that is then amplified, for example, by preparation in a bacterial vector, according to conventional methods (e.g., Sambrook et al., 1989, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, the contents of which are incorporated by reference). The amplified construct is thereafter excised from the vector and purified for use in producing transgenic animals. The term "transgenic" as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout. The term "gene knockout" as used herein, refers to the targeted disruption of a gene in vivo with complete loss of function that has been achieved by any transgenic technology familiar to those in the art. In one embodiment, transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination. As used herein, the term "transgenic" includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or knocked out. The transgene to be used in the practice of the subject invention is a DNA sequence comprising a modified ENT coding sequence. In a preferred embodiment, the ENT gene is disrupted by homologous targeting in embryonic stem cells. For example, the entire ENT gene may be deleted. Optionally, the ENT disruption or deletion may be accompanied by insertion of or replacement with other DNA sequences, such as a non-functional ENT sequence. In other embodiments, the transgene comprises DNA antisense to the coding sequence for an ENT. In another embodiment, the transgene comprises DNA encoding an antibody which is able to bind to an ENT. Where appropriate, DNA sequences that encode proteins having ENT activity but differ in nucleic acid sequence due to the degeneracy of the genetic code may also be used herein, as may truncated forms, allelic variants and interspecies homologues.
EXAMPLES The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
Example 1 hENTl cDNA Cloning and Analysis
The sequence of the N-terminal 21 residues of the human erythrocyte nucleoside transporter, purified as previously described (Kwong et al., 1988, supra), was determined by gas-phase sequencing using an Applied Biosystems Model 477A sequencer to be TTSHQPQDRYKAV7LIFFMLG (SEQ ID NO: 14) where "?" indicates an unidentified residue (Xaa). A non-degenerate DNA sequence encoding residues 8 to 15 of this sequence was PCR- amplified using Taq polymerase from an oligo(dT)-primed human placental cDNA library constructed, using BstXl linkers, in the plasmid vector pEF-BOS (Mizushima. S. & Nagata, S., 1990, pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322). The 5' primer used for the amplification was an 8, 192-fold degenerate oligonucleotide corresponding to residues 1 - 7 plus an additional N-terminal methionine, while the 3' primer was a 512-fold degenerate primer corresponding to residues 16 - 21. EcoRI and HindΩI sites respectively were incorporated at the 5' ends of the primers to facilitate subsequent cloning.
A PCR product of the expected size (84 bp) was gel-purified, digested with EcoRI and Hindu! and then ligated into the corresponding cloning sites of pBluescript II KS (+). Sequencing of the resultant clone (80A) showed that it encoded the amino acids expected for positions 8 to 15 in the TV-terminal sequence of the transporter, and identified position 14 as tryptophan. Database searching showed the presence of an identical nucleotide sequence in a 265 bp EST (GenBank accession no. T25352) cloned from a human Burkitt's lymphoma cDNA library. This information was exploited to amplify the 3' portion of the hENTl cDNA by a nested PCR approach. Initial amplification of the placental library by touchdown PCR using
Vent polymerase employed a 5', non-degenerate primer based on the clone 80A sequence shown to encode residues 8 - 15, and a 3 ' primer (pEF-BOS 2) corresponding to a region downstream of the BstXl sites in pEF-BOS.
The resultant products were subjected to a second amplification using Vent polymerase and a nested 5' primer corresponding to bases 219 - 240 of the EST sequence. This yielded a single product of size 1.9 kb, which contained the complete coding sequence of the C-terminal region of the transporter, plus 616 bp of 3' non-translated region. The 5' end of the transporter cDNA sequence was obtained by PCR amplification of the placental library using as 3' primer an oligonucleotide corresponding to nucleotides 1553-1572 of the sequence shown above and a 5' primer (pEF-BOS 1 ) corresponding to a region of the pEF-BOS vector upstream of the BstXl sites. The resultant 1.6 kb product, which contained the complete coding sequence of the transporter plus 178 bp of untranslated 5' sequence, was ligated into the Smal site of pBluescript II KS (+) to yield clone 17.1 , which was used for the expression experiments described in Figs. 6 and 7. At least two independent clones of each PCR product were sequenced and revealed no differences. All products were sequenced at least once on both strands by Taq DyeDeoxy terminator cycle sequencing using an Applied Biosystems model 373A DNA sequencer. Assignment of the translation initiation site of the ORF in hENTl cDNA was based on its resemblance to the consensus sequence described by Kozak (Kozak. M., 1989, The scanning model for translation: an update. J Cell Biol. 108, 229-241) and exact correspondence between the deduced N-terminal sequence and that determined for the erythrocyte protein, except that the latter lacks the initial methionine residue. Analysis of the protein sequence for the presence of putative membrane-spanning segments (Figs. 4 and 5) was performed by the method of Hofmann and Stoffel (Hofmann. K. & Stoffel, W., 1993, TMbase - A database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler 347, 166). Example 2 Rat Equilibrative Nucleoside Transporter rENTl cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of total RNA extracted from mucosal scrapings of Sprague-Dawlev rat jejunum (Huang, Q.Q., et al.. 1993, J. Biol. Chem. 268:20613-20619) using primers (Al . A2) flanking the hENTl open reading frame. First strand cDNA synthesis was performed on 5 mg RNA as template using the Superscript Pre-amplification System (GibcoBRL) and oligo-dT as primer. The PCR reaction (50 ml) contained 10 ng template 1st strand cDNA, 5 units TaqfDeep Vent DNA polymerase (100: 1) and 10 pmol each of primers Al and A2. Primer Al corresponded to hENTl nucleotide positions 179-203 (sense,
5"-CACCATGACAACCAGTCACCAGCCT-3' herein referred to as SEQ ID NO: 15): primer A2 corresponded to hENTl nucleotide positions 2108-2127 (antisense, 5--TGAAGGCACCTGGTTTCTGTC-3' herein referred to as SEQ ID NO: 16) and was identical in sequence to nucleotides 39-59 of a 353 bp expressed sequence tag (EST) cloned from a rat PC- 12 cell cDNA library (GeneBank accession no. H31422). Amplification for one cycle at 94° C for 5 min, 55° C for 1 min 20 s and 72°C for 2 min and 34 cycles at 94° C for 1 min, 55°C for 1 min 20 s and 72° C for 2 min generated a ~ 1.8 kb product that was ligated into the PCR vector pGEM-T (Promega) to generate the plasmid prENTl.
The prENTl insert was sequenced in both directions by Taq DyeDeoxy terminator cycle sequencing using an automated model 373A DNA Sequencer (Applied Biosystems. Inc.).
The 1766 bp insert had an open reading frame (Fig. 8) that encoded a 456-residue protein (designated rENTl , Fig. 9) that was 78% identical in amino acid sequence to hENTl (Fig. 13).
To investigate the functional properties of rENTl, the recombinant protein was expressed in Xenopus oocytes. Plasmid DNA was linearized with Sph and transcribed with SP6 polymerase in the presence of™7 GpppG cap. Subsequent procedures and experimental conditions were the same as those described below for hENTl . rENTl was shown to transport uridine and adenosine (Fig. 10) and to have a broad permeant selectivity for both purine and pyrimidine nucleosides (Fig. 1 1). rENTl was inhibited by NBMPR but, unlike its human homolog hENTl, was unaffected by dipyridamole (Fig. 12). Example 3 Expression of transporter in cells Xenopus expression and nucleoside uptake assays. Plasmid DNA for hENTl was linearised with Notl and transcribed with T3 polymerase in the presence of™7 GpppG cap using the MEGASCRIPT (AMBIOΝ) transcription system. Remaining template was removed by digestion with RΝase-free DΝase 1. Oocytes were treated with collagenase to remove follicular layers (Huang. Q.Q., et al., 1993. J. Biol. Chem. 268:20613-20619) and then injected with 10 ng hEΝTl RΝA transcript in 10 nl of water or 10 nl of water alone.
After 3 d, uptake of [5, 6-3 H] uridine (2 Ci/mmol, Moravek Biochemicals, HPLC- purified before use) (Fig. 6) or f14 Cjadenosine (Amersham Life Sciences) (Fig. 7) was performed at 20°C on groups of 10-12 oocytes in transport buffer (0.2 ml) containing 100 mM ΝaCl or 100 mM choline chloride and 2 mM KCl, 1 mM CaCl . 1 mM MgCl . 10 mM HEPES. pH 7.5. In adenosine uptake and competition experiments, the transport buffer contained 1 mM deoxycoformycin to inhibit adenosine deaminase activity. At this concentration, deoxy- coformycin had no effect on uridine influx. Except where otherwise indicated the incubation period was 5 min and the permeant concentration was 10 mM. Competing unlabelled nucleosides or uracil were used at a concentration of 2 mM.
For experiments involving ΝBMPR. dilazep and dipyridamole. oocytes were treated for 1 h with inhibitor (1 mM unless otherwise indicated) before the addition of permeant. At the end of the incubation, extracellular label was removed by six rapid washes with ice-cold transport buffer. Individual oocytes were dissolved in 5% SDS for quantification of radioactivity by liquid scintillation counting. Each of the values shown represents the mean ± SEM of 8 - 10 oocytes. Fluxes shown in Figs. 6B, 6D and 7B were corrected for endogenous nucleoside uptake activity by subtraction of fluxes seen in water-injected oocytes. This flux was not affected by the presence of competing nucleosides or inhibitors in the transport buffer.
Apparent K^ (0.24 ± 0.03 mM) and V^ (18 ± 1 pmol oocyte.5 mirf ) values for uridine influx (Fig. 6Q were determined by non-linear regression analysis (Enzfitter, Elsevier-Biosoft). Linear regression analysis of an indirect Hill plot transformation of the dose-response curve for ΝBMPR inhibition of hEΝTl -mediated uridine influx (Fig. 6D) gave a Hill coefficient of 0.98 ± 0.03 (indicating interaction of ΝBMPR with a single population of binding sites) and an I , value of 3.4 ± 0.03 nM. Correction of the latter value for inhibitor depletion caused by partitioning into oocyte lipids (determined using HPLC-purified [3 HJNBMPR (Moravek Biochemicals) to be 26%) gave an apparent K, value for NBMPR inhibition of uridine influx of 2.4 nM (calculated using competitive inhibition (Cass, C.E., 1995, in: Drug Transport in Antimicrobial and Anticancer chemotherapy, Georopapadakou, N.H., ed.. Marcel Dekker. New York. pp. 403-451 ; Young & Jarvis, 1983) and a uridine , of 0.24 mM). Gemcitabine was provided by Eli Lilly Company.
There are many Molecular Biology procedures and approaches described herein and the details of the techniques are known to those skilled in the art or described in standard molecular biology references including but not limited to: Ausubel, F.M. ,1996, "Current Protocols in Molecular Biology CD-ROM", John Wiley & Sons Inc., and references therein. There are several additional methods to investigate the function of nucleoside transport including methods described by Cass, C. E., 1995, Nucleoside Transport. :Drιιg Transport in Antimicrobial and Anticancer Chemotherapy. Georgopapadakou. N.H.. ed.. Marcel Dekker, New York, pp. 403- 451 , and references therein.
Example 4
Cloning of rENT2
The cDNA encoding rENT2 was obtained by first amplifying rENT2 partial cDNA from oligo-dT primed rat jejunal cDNA using primers (A3, A4) for conserved regions of mouse/human HNP36. corresponding to human HNP36 nucleotide positions 265-286 (sense, 5"-AACAACTGGGTGACACTGCTG-3') (SEQ ID NO: 17) and 1217-1238 (antisense. 5'-
TGGTGAGAGACACCAAGTAGCC-3') (SEQ ID NO: 18) (Williams, J.B., and Lanahan, A.A., 1995, Biochem. Biophys. Res. Commun. 213: 325-333) (GenBank™ /EMBL accession no. X86681). PCR amplification as described above for rENTl , but with a shorter extension time of 1 min, generated an approximately 1.0 kb product that was 84% and 99%) identical in nucleotide sequence to human and mouse HNP36, respectively. This fragment, labelled with j2P
Figure imgf000041_0001
kit, Pharmacia Biotech, Uppsala, Sweden), was used to screen a directional rat jejunal cDNA library using the Stratagene vector Uni-ZAP™ XAR. A clone of- 1.6 kb was obtained that contained -600 bp of rat HNP36 coding sequence and -700 bp of untranslated 3' sequence. Two rat-specific primers A5 (antisense, 5'-TAGAAGGCATGGTACCCAAC-3'. SEQ ID NO: 19) and A6 (antisense, 5'-TCAAATCCACCTTCACCAGC-3', SEQ ID NO:20) were then constructed to amplify the missing 5' portion of rat HNP36 by a nested approach. Initial amplification of the Uni-Zap™XAR rat jejunal cDNA library by the protocol described above used A5 as the 3' primer and a T3 oligonucleotide corresponding to a region of the Uni- Zap™XAR insertion vector upstream of the EcoRl cloning site as the 5' primer. A second round of amplification using the nested 3" primer A6 yielded a - 500bp product that contained
157 bp of 5' -untranslated sequence as well as the initial 5' portion of the rat HNP36 coding sequence. A cDNA encoding full-length rat HNP36 was then amplified from oligo-dT primed rat jejunal cDNA using a pair of primers (A7, A8) corresponding to nucleotide positions 157- 136 bp upstream of start codon (A7 sense, 5'-TTACCCAACCTGCACCCTCTC-3', SEQ ID NO:21 ) and positions 138-150 bp downstream the stop codon (A8 antisense, 5'-
TCACCATATGCAATGTGGCTAC-3', SEQ ID NO:22). The resulting - 1.7 kb product, which contained the complete coding sequence of rat HNP36. designated here as rENT2. was ligated into the pGEM-T vector to generate the plasmid pAN3 and sequenced in both directions. The 1766-bp rENT2 cDNA had an open reading frame encoding a 456-residue protein flanked by 157 bp of untranslated 5'-nucleotide sequence and 150 bp of untranslated 3'- sequence.
Example 5 Homology Comparisons
Electrophoretic analyses of NBMPR-photolabelled proteins in membrane preparations from various cell types have produced evidence of considerable species and/or tissue-related variations in the size of es transporters (Cass, 1995. supra; Griffith, D.A., and Jarvis. S.M.. 1996, Biochem. Biophys. Acta 1286: 153-181). raising questions about the extent of structural homology among ENT proteins. In some instances the apparent heterogeneity has been shown to be due to differences in glycosylation state (Jarvis, S.M.. and Young, J.D.. 1986, J. Membr. Biol. 93: 1-10; Hogue, D.L., et al., 1990. Biochem. Cell Biol. 68: 199-209; Crawford. C.R.. et al.,
1990, Biochim. Biophys. Acta 1024:289-297; Kwong, F.Y.P, et al., 1993, J. Biol. Chem. 268:22127-22134; Boleti, H. et al., 1997, NeuropharmacoL, in press). Peptide mapping experiments have established structural conservation among several mammalian es transporter proteins (Kwong, F.Y.P.. et al., supra), and it has been shown that the locations of NBMPR covalent labelling, carbohydrate attachment and trypsin cleavage are similar in erythrocytes. liver and/or lung from human, rat, guinea pig and pig. The apparent structural similarity between rat and human es transporters is of particular interest because nucleoside transport in rodent species has a reduced sensitivity to inhibition by vasoactive drugs (Cass. C.E., 1995, supra; Griffith. D.A.,and Jarvis, S.M., supra). Although nothing was known about the molecular properties of e -type nucleoside transporter proteins, the functional similarities between es and e/'-mediated processes suggested an underlying structural homology. rENTl - Sequence homology between rodent and human es transporter proteins was suggested by the identification, in the GenBank™ /EMBL EST database (dbEST), of ESTs encoding partial sequences of rat and mouse homologs of hENTl (Griffiths, M., et al.. 1997. Nature Medicine 3:89-93). The insert of plasmid pAN2 encoded a rat jejunal protein, designated here as rENTl, that contained one more amino acid than hENTl (457 residues. .1/ 49,984) and was 78% identical (88% similar) to hENTl in amino acid sequence (Fig. 14). Interestingly, the additional residue was a cysteine. located just before TM 8. This residue was not present in an otherwise similar dbEST sequence corresponding to mouse ENT1 (GenBank™/EMBL accession no. AA270375). Message for rENTl was also found in rat liver and lung, tissues used previously in ligand binding (32-34), photoaffinity labelling (Kwong, F.Y.P., et al.. supra; Shi, M.M., et al. 1984, Biochem. Biophys. Res. Commun. 1 18:584-600; Wu, J.S.R.. and Young, J.D.. 1984, Biochem. J. 220:499-506) and immunologic (Kwong, F.Y.P., et al., 1992, J. Biol. Chem. 267:21954-21960) and peptide mapping studies (Kwong, F. Y. P, et al., 1993, supra) of native rat e..-type nucleoside transporter proteins. Hydropathy analysis (Fig. 15 A) predicted a topology for rENTl similar to that of hENTl . In this model. 1 1 TMs are connected by short (< 16 residue) hydrophilic regions, with the exception of large loops linking TMs 1 and 2 (extracellular) and TMs 6 and 7 (intracellular) which contain 41 and 66 residues, respectively (Fig. 15B). The intracellular amino-terminus and TMs 1 -8 and 10 and 11 were highly conserved between the two proteins.
A single putative N-linked glycosylation site at Asn48 of the extracellular domain between TMs 1 and 2 of hEΝTl was present in rEΝTl, which also contained two additional N- linked glycosylation sites in the same loop at Asn44 and Asn54 (Figs. 14 and 15B). Differences in the glycosylation states of rEΝTl and hEΝTl are consistent with photoaffinity labelling studies of native rat and human transporters from various tissues (Jarvis. S.M..and
Young, J.D., 1986. supra; Kwong, F.Y.P., et al, 1993, supra; Shi, M.M.. et al.. 1984. supra). which have found that rat es migrates on SDS-polyacrylamide gels with an apparent M value of 62,000, compared with 55,000 for human es. This difference in apparent molecular weight was abolished by digestion with endoglycosidase-F (Kwong, F.Y.P., et al., 1993, supra; Jarvis, S.M., and Young, J.D.. 1986, supra). Since membrane proteins frequently exhibit increased mobilities relative to soluble protein standards on SDS-polyacrylamide gels (Mueckler,
M..et al, 1985. Science 229:941-945), the value of 47,000 for deglycosylated rat and human es is consistent with the calculated sizes of rENTl and hENTl (A-/ 49,984 and 50.249, respectively).
Peptide mapping experiments with native human erythrocyte and rat lung/liver es transporters have identified a common trypsin cleavage site situated in an intracellular domain in the approximate centre of both proteins (Kwong, F.Y.P., et al., 1993, supra). The site of N- linked glycosylation was located close to one end of the human protein, and the site of NBMPR photolabelling to within 16 kDa of that site (Kwong. F.Y.P., et al., 1993. supra). Tire ENT topographical model presented in Fig. 15B predicts that (i) the site of trypsin cleavage is within the central cytoplasmic loop between TMs 6 and 7, and (ii) the site of NBMPR attachment is within the amino-terminal half of the protein. The deduced amino acid sequences of rENTl and hENTl predict a number of potential trypsin cleavage sites within the central cytoplasmic loop.
rENT2 - The carboxy-terminal two-thirds of the hENTl amino acid sequence showed similarity (44%) identity) to the 36-kDa mouse fibroblast and human heart HNP36 proteins
(Williams. J.B., and Lanahan, A.A., 1995, supra). The latter are delayed-early proliferative response gene products, predicted to be integral membrane proteins with eight membrane- spanning segments that correspond to TMs 4-11 of hENTl . However, as we have previously noted (Griffiths, M, et al., 1997, supra), the 198 nucleotides upstream of the assigned start codon in the mouse HNP36 cDNA are in frame with the rest of the hENTl coding sequence and could therefore encode an amino acid sequence with 65% identity to the region of hENTl containing TMs 2 and 3. Similarly, the nucleotide sequence upstream of the assigned start codon of the human HNP36 cDNA includes an open reading frame that is 49% identical in predicted amino acid sequence to the amino-terminal region (TMs 1 and 2) of hENTl . The cellular location of the HNP36 protein, determined by immunocytochemical staining of murine BALB/c fibroblasts, F9 embryonal carcinoma cells and rat intestinal epithelial cells, was reported as nucleolar (Williams, J.B., and Lanahan, A.A., 1995, supra). Based upon the strong sequence similarity between HNP36 and rENTl/hENTl. we hypothesized that HNP36 might be a plasma membrane nucleoside transporter, perhaps with NBMPR-insensitive (e -type) transport activity. pAN3 encoded a rat jejunal protein with the same number of residues as hENTl (456 amino acids, Mτ 50,232). The predicted start codon lies in a reasonably good Kozak consensus sequence and is preceded by an in-frame stop codon. There was 88%o and 95% identity (93% and 98%o similarity) to the incomplete amino acid sequences of human and mouse HNP36 (Fig. 14), establishing that the protein, designated here as rENT2, corresponds to full-length rat
HNP36. The sequence of rENT2 was 49% identical (68%o similar) to rENTl and 50% identical (69%o similar) to hENTl (Fig. 14). A search of the GenBank /EMBL dbEST revealed that the human homolog of rENT2, human HNP36, is expressed in adult human ovary and ovarian tumours, and in fetal heart and brain. Hydropathy analysis of rENT2 (Fig. 15 A) predicted the same membrane topology as rENTl hENTl . Homology between rENT2 and rENTl/hENTl was greatest within the putative transmembrane helices and there was little homology in either of the two large loops. The predicted extracellular loop of rENT2 between TMs 1 and 2 contained 14 fewer amino acids than rENTl/hENTl, while the corresponding intracellular loop linking TMs 6 and 7 had 14 more residues (Fig. 15B). The putative N-linked glycosylation site at Asn48 of rEΝTl and hEΝTl was conserved in rEΝT2 at Asn47, allowing for the presence of a 1 residue insertion before this point in the ENT1 sequences, and was similarly present in the human HNP36 sequence (Figs. 14 and 15B). In addition, a second putative glycosylation site was present at Asn56 in rENT2 and at a corresponding location in human HNP36 (Figs. 14 & 15B).
Example 6
Expression of rENT2 Methods
Plasmids pAN2 (rENTl) and pAN3 (rENT2) were linearized with Sphl and transcribed with SP6 polymerase in the presence of™7 GpppG cap (Ambion, Austin, TX) using the MEGAscript™ (Ambion) transcription system. Plasmid cDNA encoding hENTl (Griffiths. M., et al., 1997, supra) in the vector pBluescript II KS (+) was linearized with Not\ and transcribed with T3 polymerase. Healthy stage VI oocytes of Xenopus laevis. treated with collagenase to remove follicular layers (Huang, Q.Q., et al., 1993, J. Biol. Chem. 268:20613-20619), were maintained at 18°C in modified Barth's medium (MBM) for 24 h until injection with 10 nl water or 10 nl water containing rENTl, rENT2 or hENTl RNA transcript (1 ng/nl). Injected oocytes were incubated for 3 days at 18°C with a daily change of MBM before the assay of transport activity.
Uptake of uridine and adenosine by oocytes was traced with the respective14 C-labelled nucleoside (Amersham, Arlington Heights, IL) (1 μCi ml). Assays were performed at 20 °C on groups of 10-12 oocytes in transport buffer (0.2 ml) containing either 100 mM NaCl or 100 mM choline chloride and 2 mM KCl, 1 mM CaCL , 1 mM MgCl , 10 mM HEPES, pH 7.5 (Griffiths et al., supra). In adenosine uptake and inhibition experiments, the transport buffer also contained 1 μM deoxycoformycin to inhibit adenosine deaminase activity. Unless otherwise indicated, the incubation period was 1 min and the permeant concentration was 10 μM. Competing unlabelled nucleosides and uracil were used at a concentration of 2 mM. For experiments involving NBMPR, dipyridamole and dilazep, oocytes were pretreated with inhibitor (1 μM unless otherwise indicated) for 1 h before addition of permeant. At the end of the incubation, extracellular radioactivity was removed by six rapid washes in ice-cold transport buffer. Individual oocytes were dissolved in 5% (w/v) SDS for quantitation of radioactivity by liquid scintillation counting. We have determined previously that oocytes lack endogenous nucleoside transport processes and have a low basal permeability to uridine and adenosine (Huan. Q.Q., et al., 1994. J. Biol Chem. 269: 17757-17760; Yao. S.Y.M., et al, 1996, Mol. Pharmacol. 50:1529-1535; Huang, Q.Q.. et al., 1993, supra). Uridine is only slowly metabolised in oocytes (Huang, Q.Q., et al., supra), while adenosine is efficiently trapped as the 5'-triphosphate ester (10). Results for uptake experiments are given as means ± standard errors
(SE) for 10-12 individual oocytes. Kinetic constants (apparent i^_ and Vmm ) and IQ values were determined using programs of the ENZFITTER software package (Elsevier-Biosoft. Cambridge, UK). Each experiment was performed at least twice on different batches of oocytes.
Functional expression and NBMPR-sensitivity of recombinant rENTl and rENT2 To investigate the functional characteristics of rENTl and rENT2, the recombinant proteins were expressed in Xenopus oocytes. As shown in Fig. 16A, uptake of the pyrimidine nucleoside [, C]uridine (10 μM, 20° C) was substantially greater in oocytes injected with rENTl RNA transcript than in control oocytes injected with water alone. Uptake was essentially linear for the first minute of incubation, and in subsequent experiments this incubation period was used to approximate initial rates of transport. At this time interval, uptake in rENTl RNA- injected oocytes was 1.13 ± 0.06 pmol/oocyte, compared with 0.004 + 0.002 pmol/oocyte in water-injected oocytes (Fig. 16A). Similar transport data were obtained in oocytes injected with rENT2 RNA transcript. In the experiment shown in Fig. 16B, uptake of uridine (10 μM. 20°C, 1 min flux) by rENT2 RNA-injected oocytes was 0.47 ± 0.009 pmol/oocyte, compared with 0.01 + 0.003 pmol/oocyte in water-injected cells. Neither protein was dependent upon the presence of sodium ions in the external medium (Figs. 17A & B). Therefore. rENTl and rENT2 were both confirmed to be functional equilibrative nucleoside transporters.
The characteristic that distinguishes es-type and e -type nucleoside transport processes is inhibition by NBMPR. As shown in Fig. 18A. recombinant rENTl and hENTl exhibited comparable NBMPR inhibition profiles, with I 0 values of 4.6 ± 1.4 and 3.6 ± 1.4 nM, respectively. In contrast. rENT2 was unaffected by 1 μM NBMPR (Fig. 18D). rENTl is therefore NBMPR-sensitive (es-type), while rENT2 is NBMPR-insensitive (e -type).
Kinetic properties and substrate selectivity of recombinant rENTl and rENT2 Mediated influx of uridine by the two transporters, defined as the difference in uptake between RNA-injected and water-injected oocytes, was saturable (Figs. 19A & B) and conformed to simple Michaelis-Menten kinetics. The calculated apparent K^ value of 0.15 + 0.02 mM for rENTl was within the range expected for uridine transport by mammalian es-type nucleoside transporters (Cass, C.E., 1995. supra; Griffith, D.A., and Jarvis, S.M., 1996, supra), including hENTl (Griffiths. M.. et al.. 1997, supra), and was significantly lower than the value of 0.30 + 0.04 mM determined for rENT2 under the same experimental conditions. A number of studies have reported differences in affinity between native es-type and e/-type nucleoside transporters (Jarvis, S.M., and Young. J.D., 1986, J. Membr. Biol. 93: 1-10; Boleti, H.. et al.. 1997, supra; Lee. C.W.. and Jarvis. S.M.. 1988, Neurochem. Int. 12:483-492: Hammond, J.R., 1992, Biochem. J. 287:431 -436). Vmax estimates for rENTl and rENT2 in Figs. 19A and 19B were 18 + 1 and 14 + 1 pmol/oocyte.miri' , respectively.
Transport of uridine by rENTl was unaffected by the presence of a high concentration of the nucleobase uracil (2 mM), but was strongly inhibited by both pyrimidine (thymidine. cytidine and uridine) and purine nucleosides (adenosine. guanosine. inosine) (Fig. 17A). This result is consistent with the broad specificity typical of e. -type nucleoside transporters (Cass. C.E., 1995. supra: Griffith. D ., and Jarvis. S.M., 1996. supra), and has been found also for recombinant hENTl (Griffiths, M., et al.. supra). Greatest inhibition was observed with adenosine. suggesting that, like native es transporters (Cass, C.E., 1995, supra; Griffith, D .. and Jarvis, S.M., 1996, supra), heterologously-expressed rENTl has a higher affinity for this purine nucleoside than for other substrates. Direct measurements of f4 Cjadenosine (10 μM) transport confirmed that adenosine was an rENTl permeant (inset to Fig. 16A). The rate of mediated influx of adenosine was greater than for uridine.
Similar transport characteristics were obtained for rENT2 (Figs. 16B & 17B), except that cytidine was a relatively poor inhibitor of rENT2-mediated uridine influx. The kinetic characteristics of cytidine transport by native e.-type nucleoside transport processes have not been investigated (Cass, C.E., 1995, supra; Griffith, D.A.. and Jarvis, S.M., 1996, supra).
Interaction of rENTl and rENT2 with dipyridamole and dilazep - Nucleoside transporters of the e..-type are pharmacologic targets of the coronary vasodilators dipyridamole and dilazep. which compete with permeant (and NBMPR) for the substrate binding site (Cass.
C.E., 1995, supra; Griffith, D.A., and Jarvis, S.M., 1996. supra; Jarvis. S.M.. et al., 1982, J. Physiol. (Lond.) 324:31-46; Jarvis, S. M., 1986, Mol. Pharmacol. 30:659-665). These agents, therefore, block adenosine influx across the plasma membrane, thereby potentiating of the interaction of extracellular adenosine with purinoreceptors (Belardinelli, L., et al., 1989, Prog. Cardiovasc. Dis. 32:73-97; Jacobson, K.A., Daly, J.W., and Manganiello. V., eds, 1990, Purines in Cellular Signalling: Targets for New Drugs, Springer Verlag, New York). In most species, including humans, dipyridamole and dilazep inhibit es transport with potencies in the nanomolar concentration range (Cass, C.E.. 1995, supra; Griffith, D.A., and Jarvis, S.M., 1996, supra), whereas they inhibit ei transport with micromolar apparent K, values. In contrast, es and ei transporters of rat origin are generally both resistant to dipyridamole and dilazep inhibition. Figs. 18B and 18C compare dose-response curves for dipyridamole and dilazep inhibition of uridine transport by recombinant rENTl and hENTl . hENTl was, as expected, highly sensitive to inhibition by dipyridamole and dilazep, with IQ0 values of 140 ± 2 and 60 ± 2 nM. respectively. In contrast, concentrations of dipyridamole or dilazep up to 1 μM had no effect on rENTl transport activity, demonstrating insensitivity of the rat transporter to both compounds. rENT2 was also relatively unaffected by 1 μM dipyridamole or dilazep (Fig. 18D). Since recombinant rENTl, rENT2 and hENTl proteins were expressed in the same membrane environment, it can be concluded that the observed differences in sensitivity of the three transporters to inhibition by vasoactive drugs and NBMPR reflect differences in their amino acid sequences. Differences in vasoactive drug sensitivities are unlikely to be a consequence of glycosylation state because the interaction of dipyridamole with the native rat and human es transporters is unaffected by carbohydrate removal (Kwong, F.Y.P., 1993, supra).
Conclusions The present invention shows the cloning and expression of cDNAs encoding two nucleoside transporter proteins from rat tissues. rENTl and rENT2 were NBMPR-sensitive and NBMPR-insensitive, respectively, and thus correspond functionally to the two major equilibrative nucleoside transporter subtypes (es and ei) found in mammalian cells. Neither rat protein was inhibited by the coronary vasoactive drugs dipyridamole or dilazep. Since rENTl and hENTl are close structural homologs, it is likely that small changes in the molecular structure within, or adjacent to. the nucleoside substrate binding site lead to differences in vasoactive drug binding without affecting the affinity of the site for either NBMPR or nucleoside substrates. That site may include structural domains located within the amino- terminal half of the transporter. The cloning and functional expression of rENTl and rENT2, identified previously as the delayed-early proliferative response gene product HNP36, establishes that es and ei transport activities are mediated by separate, but homologous, proteins. Despite close topographical and kinetic similarities, rENTl and rENT2 are only moderately similar in amino acid sequence. Residues conserved between the two proteins, which may have structural and/or functional significance, were located largely within putative TMs. Both rENTl and rENT2 were expressed in jejunum, where, like their concentrative counterparts rCNTl and rCNT2, they may have a physiological role in the absoφtion of dietary nucleosides.
Example 7 Cloning of hENT2 The C-terminal two thirds of the hENTl sequence shows strong similarity (approx. 44% identity) to the 36 kDa mouse and human HNP36 proteins, which are delayed-early proliferative response gene products (Williams, J.B. and Lanahan. A.A., 1995. supra). These HNP36 proteins were reported to have a nucleolar location and were predicted to have only 8 membrane-spanning segments (Williams. J.B. and Lanahan, A.A.,1995, supra). However, we noted that the nucleotide sequence upstream of the assigned start codon in the human HNP36 cDNA included an open reading frame 49% identical in predicted amino acid sequence to the N-terminal region (transmembrane helices 1-2) of hEΝTl . We therefore suggested that the cDΝA might have been artefactually truncated during its preparation, and that a full length cDΝA might encode a second mammalian passive nucleoside transporter isoform (Griffiths, M., et al., 1997, supra).
This hypothesis was tested by the PCR amplification of a cDΝA containing the full- length coding region of human I ΝP36. Production of the encoded protein, which we designate hENT2. in Xenopus oocytes has revealed that it is indeed a functional transporter but, in contrast to hENTl , exhibits NBMPR-insensitive, e/'-type nucleoside transport activity. Although hENT2 shares with hENTl the ability to transport adenosine and uridine. it is much less sensitive to inhibition both by the coronary vasodilator drugs, dipyridamole and dilazep, and by the lidoflazine analogue, draflazine. It should now be possible to exploit the marked differences in activity between hENTl and hENT2 to explore the structural features responsible for solute recognition in these physiologically important transport proteins. The cDNA encoding hENT2 was amplified from an oligo(dT)-primed human placental cDNA library (Griffiths, M., et al., 1997, supra) using a PCR approach. Initial amplification of the placental library by touchdown PCR using Pwo polymerase (Boehringer Mannheim) employed primers corresponding to human HNP36 nucleotide positions 61 - 80 (sense, 5'- GCCATGGCCCGAGGAGACGC-3', SEQ ID NO: 22)and 1551 - 1570 (antisense, 5'- TCCCAATCTTACTGGCCACC-3',SEQ ID NO: 23) (Williams, J.B., and Lanahan. A.A.. 1995. supra) (GenBank™ /EMBL accession no. X86681 ). A product from this amplification of size -1.6 kb was gel purified and re-amplified using the same primers. The resultant - 1.6 kb product, which contained the complete coding region of hENT2, was ligated into the EcoRV site of pBluescript II KS (+) to yield construct pHNPc7. This was sequenced on both strands by Taq DyeDeoxy terminator cycle sequencing using an Applied Biosystems model 373A
DNA sequencer. The nucleotide sequence has been submitted to the GenBank™ /ΕMBL Data Bank with accession number AF029358.
The cDNA amplified from a human placental cDNA library with primers derived from the HNP36 nucleotide sequence contained a 1 ,368 bp open reading frame that encodes a 456-residue protein of Λ-( 50,173 (Fig. 20). We have designated this protein hΕNT2
(human equilibrative nucleoside transporter 2). The nucleotide sequence was identical to the human HNP36 sequence, except that it contained an additional segment of 68 bp immediately following nucleotide position 337 in the HNP36 sequence. hENT2 is 46%) identical in amino acid sequence to hENTl and hydropathic analysis (not shown) of the protein sequence predicts a similar transmembrane topology of 1 1 hydrophobic transmembrane (TM) α-helices connected by hydrophilic loops (Fig. 20). Most of the latter are predicted to be short, except for the loops connecting TMs 1 and 2 (28 residues) and TMs 6 and 7 (80 residues). The former (loop 1) is 13 residues shorter and the latter (loop 6) is 14 residues longer than the corresponding regions of hENTl. hENT2 is predicted to be a glycoprotein and contains two potential sites of N-linked glycosylation in the extramembranous loop connecting TMs 1 and 2. one of which. Asq8 , is conserved in hEΝTl (Fig. 20).
Example 8 Expression Analysis of hEΝT2
Plasmid DNA (pHNPc7 encoding hENT2 and clone 17.1 encoding hENTl (Griffiths, M.. et al.. 1997, supra) was linearised with Notl and transcribed with T3 RΝA polymerase in the presence of m7GpppG cap using the MEGASCRIPT (AMBION) transcription system. Xenopus oocytes were injected with 10 ng cRNA in 10 nl of water or 10 nl of water alone, and transport experiments were subsequently performed using radiolabelled uridine and adenosine and a transport buffer containing 100 mM NaCl. 2 mM KCl, 1 mM CaCl , 1 mM MgCl , 10 mM FIEPES, pH 7.5 exactly as previously described for hENTl (Griffiths. M.. et al.. 1997, supra). In adenosine uptake experiments, the transport buffer also contained 1 μM deoxycoformycin to inhibit adenosine deaminase activity. An uptake period of 1 min and a permeant concentration of 10 μM were used unless otherwise indicated. For experiments involving NBMPR, dilazep, dipyridamole and draflazine, oocytes were treated for 1 h with inhibitor before the addition of permeant. Each of the values shown for uptake experiments represents the mean ± standard error for 10 - 12 oocytes. Apparent K^ and J^_. values for uridine influx were determined by non-linear regression analysis using the ENZFITTER software package (Elsevier-Biosoft, Cambridge, UK). Each experiment was performed at least twice on different batches of oocytes, yielding closely similar results. To investigate the functional characteristics of hENT2 in comparison with those of hENTl, the recombinant proteins were expressed in Xenopus oocytes. As shown in Fig. 21. uptake of both the pvrimidine nucleoside [" Cluridine (10 μM, 20°C) and the purine nucleoside [l C]adenosine ( 10 μM, 20°C) was substantially greater in oocytes injected with the hENT2 transcript than in control oocytes injected with water alone. Uptake of both nucleosides was approximately half that seen in oocytes injected with the hENTl transcript, although the significance of this observation with respect to the intrinsic kinetic properties of the two transporters remains unclear in the absence of any knowledge about their respective cell surface concentrations. Both hENTl and hENT2 RNA-injected oocytes exhibited flaxes of adenosine that were about 2.5-fold greater than the corresponding flux for uridine. Confirmation that hENT2 is a functional nucleoside transporter was provided by the demonstration that mediated influx of uridine, defined as the difference in uptake between RNA-injected and water-injected oocytes, was saturable and conformed to simple Michaelis- Menten kinetics (Fig. 22). The calculated apparent _ζ- value of 0.20 ± 0.03 mM for hENT2 was essentially identical to the value of 0.24 mM that we have previously reported for hENTl (Griffiths. M., et al, 1997, supra). The Vmm estimate for hENT2 was 6.43 ± 0.21 pmol/oocyte.min"1.
As previously discussed, mammalian equilibrative nucleoside transport processes can be classified into es- and e -types by virtue of their sensitivity or resistance to inhibition by NBMPR. hENTl is a classic es-type transporter, its mediated influx of uridine (10 μM) being inhibited by NBMPR with a calculated ΛJ value of 2 nM (Fig. 4, Table 1 and Griffiths, M., et al., 1997, supra). In contrast. hENT2 -mediated influx of uridine into Xenopus oocytes was inhibited by less than 15% in the presence of 1 μM NBMPR and by only 70% in the presence of 10 μM NBMPR (Fig. 23 and Table 1 ). h_ENT2-mediated adenosine transport exhibited a similar resistance to inhibition by NBMPR. h_ENT2 is therefore an example of an NBMPR- insensitive, e.-type of nucleoside transporter. Although in some cell types ei transporters are reported to have a lower affinity for substrates, including uridine, than the corresponding es transporters (Griffith, D.A. and Jarvis, S.M.. 1996, supra: Boleti, H., et al., 1997. supra) no such differences were observed between hENTl and hENT2. However, another reported characteristic of e -type transport processes that distinguishes them from e..-type transport processes in the same animal species is their relative insensitivity to inhibition by dipyridamole, dilazep and lidoflazine analogues such as draflazine (Griffith, D.A. and Jarvis. S.M.. 1996, supra). Table 1 shows that while uridine (10 μM) fluxes mediated by the ei'-type transporter hENTl are potently inhibited by all three compounds (__ 94% inhibition by 1 μM inhibitor). hENT2-mediated uridine fluxes were poorly inhibited (_; 71% inhibition by 10 μM inhibitor). A search of the GenBank EST database (dbEST) showed that, in addition to placenta. hENT2 mRNA is expressed in adult ovary and ovarian tumours and in fetal brain and heart.
The truncated cDNA of hENT2 (human HNP36) previously reported (Williams. J.B. and Lanahan, A. A., 1995, supra) was isolated from an adult heart cDNA library. The physiological role(s) of hENT2 in these tissues is presently unclear. In the heart adenosine is thought to play an important part in the regulation of myocardial supply-demand balance (Mubagwa, K., et al., 1996, Cardiovasc. Res. 32:797-813), and there is considerable evidence that adenosine also exerts beneficial, cardioprotective effects in the ischaemic/reperfused myocardium (Lasley, R.D. and Mentzer, R.M., 1996, Drug Develop. Res. 39:314-318). Over the last few years there has been increasing interest in the potential use of nucleoside transport inhibitors as therapeutic agents in the heart (Dennis, D.M., et al, 1996, Circulation 94:2551-2559). For example, infusion of the lidoflazine analogue R-75231 before coronary artery occlusion enhances local adenosine concentrations and postischaemic recovery of function, while reducing infarct size in the pig (Martin, B.J., et al., 1997, Am. J. Physiol. 41 :H1857-H1865). Preliminary trials of draflazine, an enantiomer of R75321, as a potential cardioprotective agent in humans have already been performed (Rongen, G.A., et al, 1995, J. Clin. Invest. 95:658-668). While these agents appear to increase and prolong the effects of adenosine by inhibiting its uptake into metabolising cells during ischaemia and delaying its washout during reperfusion (Griffith. D.A. and Jarvis, S.M., 1996. supra; Van Belle, H, 1993, Drug Develop. Res. 28:344-348), the precise roles of the different cell types in adenosine fluxes in the heart remain unclear. Our discovery that the heart expresses mRNA encoding not only the es-type transporter, hENTl (Griffiths, M., et al.. 1997, supra), but also an e.-type transporter, hENT2. which is relatively insensitive to draflazine, is therefore of considerable interest. In particular, it will now be possible, using specific antibody and DNA probes for these two isoforms, to assess their distributions and individual contributions to the regulation of extracellular adenosine concentration in cardiac and other tissues.
Thus, a cDNA encoding an e -type transporter, hENT2, has been cloned from human tissues, and expressed m Xenopus oocytes. The initial identification of HNP36 as a delayed- early proliferative response gene product (Williams, J.B. and Lanahan, A.A., 1995, supra) may reflect the enhanced need for nucleotide precursors during cell proliferation. Although some workers have previously suggested that es and ei transporters may represent two forms of the same protein (Aronow, B.. et al., 1985, J. Biol. Chem. 260:6226-6233), our results establish that these two classes of transporters are the products of separate genes. Comparison of the sequences of hENTl and hENT2 (Fig. 20) shows the presence of conserved residues both within the putative transmembrane helices and in the hydrophilic loops connecting them, although the two large loops connecting TMs 1 and 2 and TMs 6 and 7 are poorly conserved. The conserved residues are likely to have structural and functional significance. Determination of the residues that are responsible for the profound differences in the affinities of hENTl and hENT2 for substrate analogues and inhibitors can be achieved through the production of chimaeras and other sequence modifications. Such knowledge should be of value in the rational design of novel nucleoside drugs with better selectivity for target tissues.
Table 1 Inhibition of hENTl- and hENT2-mediated uridine fluxes by NBMPR, dipyridamole, dilazep and draflazine. Uridine influx (10 μM. 20°C, 1 min flux) was measured 3 days after injection of oocytes with hENTl or hENT2 RNA transcripts, as described in Materials and Methods. Oocytes were pre-incubated with inhibitor for lh before addition of permeant. Fluxes were corrected for the small contribution of endogenous transport activity and are expressed as a percentage of the uninhibited value. N.D. = not determined.
Figure imgf000055_0001
Example 9 Transgenic Mice Expressing ENT
Transgenic mice that can produce an ENT of the invention are made according to methods well known in the art. For example, young female mice are injected with hormones to induce superovulation, and are mated. The one-celled embryos are collected, and the pronuclei injected with a purified DNA solution containing the constructs which encode an ENT of the invention. The injected eggs are cultured briefly, and re-implanted into pseudopregnant female mice. Genomic DNA is prepared from each progeny, and analyzed by PCR or Southern blot to determine the mouse's genotype. Mice who carry the transgene are subsequently mated in order to produce a line of mice.
Using these methods mice are produced which contain transgenes encoding an ENT. In order to target the expression to specific cell types, transgenic mice are produced which carry genes encoding the ENT under the control of specific promoters. Examples of such constructs are genes encoding SEQ ED NO:2 under the control of the rat whey acidic protein regulatory sequences (Yarus, S., et al., 1997, "The carboxy-terminal domain of human surfactant protein B is not required for secretion in milk of transgenic mice," Front. Biosci. 2:A1-A8) , or the mouse mammary tumor virus (MMTV) LTR (Mediavilla, M.D., et al., 1997, "Effects of melatonin on mammary gland lesions in transgenic mice overexpressing N-ras proto-oncogenes," J Pineal Res. 22:86-94), to specifically target the expression of hENT2 to breast tissue, or genes encoding SEQ ED NO:4 under control of the immunoglobulin promoter (A vital. N., and
Calame, K., 1996, A 125 bp region of the Ig VH1 promoter is sufficient to confer lymphocyte- specific expression in transgenic mice. Int. Immunol. 8(9): 1359-1366) to specifically targets the expression of rENTl to the B cells.
Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:
1. Substantially purified equilibrative nucleoside transporter polypeptide.
2. The polypeptide of claim 1, wherein said polypeptide is inhibited by dipryridamole.
3. The polypeptide of claim 1, wherein said polypeptide is inhibited by dialzep.
4. The polypeptide of claim 1, wherein said polypeptide is inhibited by nitrobenzylthioinosine (6-[(4-rύtorber_zyl)thiol]-9-β-D-bribo-furanosylpurine.
5. The polypeptide of claim 1, having an amino acid sequenceselected from the group consisting of SEQ ED NO:2, SEQ ID NO:4, SEQ ED NO:6, and SEQ ED NO:8.
6. The polypeptide of claim 5, having the amino acid sequence selected from the group consisting of SEQ ED NO:2 and SEQ ED NO:4.
7. The polypeptide of claim 5, having the amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ED NO:8.
8. An isolated polypeptide having an amino acid sequence which is at least 85% identical to a polypeptide sequence selected from the group consisting of SEQ ED NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
9. The isolated polypeptide of claim 8, wherein said polypeptide is at least 85%o identical to a polypeptide sequence selected from the group consisting of SEQ ED NO:2 and SEQ _D NO:4.
10. The isolated polypeptide of claim 8, wherein said polypeptide is at least 85% identical
? to a polypeptide sequence selected form the group consisting of SEQ ID N0:6 and SEQ ID NO:8.
4 11. An isolated polypeptide which is a fragment or analog of an amino acid sequence
5 selected from the group consisting of SEQ ED NO:2, SEQ ID NO:4, and SEQ ID NO:6.
1 12. The isolated polypeptide of claim 1 1 , wherein said polypeptide is a fragment or analog
2 of a polypeptide sequence selected form the group consisting of SEQ ID NO:2 and SEQ
3 ID NO:4.
u. The isolated polypeptide of claim 11, wherein said polypeptide is a fragment or analog of a polypeptide sequence selected from the group consisting of SEQ ID NO:6 and SEQ
ED NO:8.
1 14. An isolated polynucleotide encoding an equilibrative nucleoside transporter of claim 1.
1 15. The polynucleotide of claim 14, wherein said equilibrative nucleoside transporter is a
2 mammalian equilibrative nucleoside transporter.
1 16. The polynucleotide of claim 15, wherein said equilibrative nucleoside transporter is a
? human equilibrative nucleoside transporter.
1 17. The polynucleotide of claim 15, wherein said equilibrative nucleoside transporter is a
2 rat equilibrative nucleoside transporter.
1 18. The polynucleotide of claim 1. wherein said polynucleotide encodes an amino acid
2 sequence selected from the group consisting of SEQ ED NO:2 and conservative variants
3 thereof, SEQ ID NO:4 and conservative variants thereof, SEQ ED NO:6 and
4 conservative variants thereof, SEQ ED NO: 8 and conservative variants thereof.
19. The polynucleotide of claim 18, wherein said polynucleotide encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2 and conservative variants thereof, and SEQ ID NO:4 and conservative variants thereof.
20. The polynucleotide of claim 18, wherein said polynucleotide encodes an amino acid sequence selected from the group consisting of SEQ D NO:6 and conservative variants thereof and SEQ ID NO:8 and conservative variants thereof.
21. The polynucleotide of claim 14, wherein said polynucleotide is selected from the group consisting of SEQ ID NO: 1 and degenerate variants thereof, SEQ ID NO:3 and degenerative variants thereof, SEQ ID NO:5 and degenerate variants thereof, SEQ ID NO:7 and conseivative variants thereof.
22. The polynucleotide of claim 21. wherein said polynucleotide is selected from the group consisting of SEQ ED NO: 1 and degenerate variants thereof, and SEQ ID NO:3 and degenerative variants thereof.
23. The polynucleotide of claim 21, wherein said polynucleotide is selected from the group consisting of SEQ EN NO:5 and degenerate variants thereof, and SEQ ID NO:7 and degenerate variants thereof.
24. A Ann isolated polynucleotide selected from the group consisting of:
(a) SEQ ED NO: 1 , where T can also be a U;
( ) nucleic acid sequences complementary to SEQ ED NOT;
(c) fragments of a) or b) that are at least 15 bases in length and will hybridize to DNA which encodes a polypeptide as set forth in SEQ ED NO:2;
(d) SEQ ID NO:3, where T can also be a U;
(e) nucleic acid sequences complementary to SEQ ED NO:3; and
( ) fragments of d) or e) that are at least 15 bases in length and will hybridize to DNA which encodes a polypeptide as set forth in SEQ ED NO:4.
25. A Ann iissoollated polynucleotide selected from the group consisting of:
(g) SEQ ID NO:5, where T can also be a U;
(h) nucleic acid sequences complementary to SEQ ED NO:5;
(i) fragments of g) or h) that are at least 15 bases in length and will hybridize to DNA which encodes a polypeptide as set forth in SEQ ID NO:6. ϋ) SEQ ID NO:7, where T can also be a U;
00 nucleic acid sequences complementary to SEQ ED NO:7; and
(I) fragments of j) or k) that are at least 15 bases in length and will hybridize to DNA which encodes a polypeptide as set forth in SEQ ID NO:8.
26. An isolated polynucleotide having 50% or greater homology with a member selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5. and SEQ ID NO:7.
27. The isolated polynucleotide of claim 26, wherein said polynucleotide has 50%) homology with a member selected from the group consisting of SEQ ED NO: 1 and SEQ ID NO:3.
28. The isolated polynucleotide of claim 26, wherein said polynucleotide has 50%> homology with a member selected form the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7.
29. The polynucleotide of claim 14, further comprising an operatively linked expression control sequence.
30. The polynucleotide of claim 29, wherein the expression control sequence is a promoter.
31. The polynucleotide of claim 30, wherein the promoter is tissue specific.
32. An expression vector containing the polynucleotide of claim 1.
33. The vector of claim 32, wherein the vector is a plasmid.
34. The vector of claim 32, wherein the vector is a viral vector.
35. The vector of claim 34, wherein the viral vector is a retroviral vector.
36. A host cell containing the vector of claim 32.
37. The host cell of claim 36, wherein the cell is a eukaryotic cell.
38. The host cell of claim 36, wherein said host cell is a prokaryotic cell.
39. An antibody which binds to an equilibrative nucleoside transporter polypeptide.
40. The antibody of claim 41 , wherein the antibody is monoclonal.
41. The antibody of claim 42, wherein the monoclonal antibody is a humanized monoclonal antibody.
42. A method for identifying a compound which affects an equilibrative nucleoside transporter, comprising: (a) incubating the compound and a sample of interest under conditions sufficient to allow the compound to interact with the sample, (b) detecting the permeability of the sample of interest to a nucleoside using a permeant, (c) comparing the permeability of said sample incubated with said compound to said permeant with the permeability of a control sample not incubated with said compound.
43. The method of claim 42. wherein said sample is a cell.
44. The method of claim 42, wherein said sample is a membrane sample.
45. The method of claim 42, wherein the efflux of a permeant is detected.
46. The method of claim 42, wherein the influx of a permeant is detected.
47. The method of claim 42. wherein the permeant is detectably labeled.
48. The method of claim 42, wherein the detectable label is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, and a chemiluminescent compound.
49. The method of claim 42. wherein said compound inhibits transport of the nucleoside.
50. The method of claim 42, wherein said compound stimulates transport of the nucleoside.
51. A method for determining if a compound utilizes an equilibrative nucleoside transporter, to enter or leave a cell, comprising: (a) incubating the compound and a sample of interest under conditions sufficient to allow the compound to interact with the sample in the presence of an equilibrative nucleoside transporter inhibitor; (b) detecting the permeability of the sample of interest to the compound; (c) comparing the permeability of said sample incubated with said compound in the presence of said inhibitor to the permeability of a control sample incubated with said compound in the absence of said inhibitor.
52. The method of claim 51 , wherein said sample is a cell.
53. The method of claim 51 , wherein said compound is an anticancer agent.
54. The method of claim 51 , wherein said compound is an antiviral agent.
55. A kit useful for detecting the presence of an equilibrative nucleoside transporter in a sample, the kit comprising: carrier means being compartmentalized to receive in close confinement therein one or more containers containing an antibody which specifically binds to the equilibrative nucleoside transporter.
56. The kit of claim 55, wherein said equilibrative nucleoside transporter is selected from the group consisting of SEQ ID NO:2. SEQ ID NO:4, SEQ ID NO:6. and SEQ ID NO:8.
57. A kit useful for the detection of a target nucleic acid sequence in a sample, wherein the presence of the target nucleic acid is indicative of the ability to transport nucleoside using an equilibrative nucleoside transporter, the kit comprising: carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a container containing oligonucleotides which hybridize to an equilibrative nucleoside transporter.
58. The kit of claim 57. wherein said oligonucleotides are selected from the group consisting of SEQ ID NO: 15, the complement of SEQ ID NO: 15, SEQ ID NO: 16, the complement of SEQ ED NO: 16. SEQ ID NO: 17, the complement of SEQ ED NO: 17. SEQ ED NO: 18, the complement of SEQ ED NO: 18, SEQ ED NO: 19. the complement of SEQ ED NO: 19, SEQ ED NO:20, the complement of SEQ ID NO:20. SEQ ED NO:21. the complement of SEQ ID NO:21, SEQ ID NO:22, and the complement of SEQ ED NO:22.
59. A method for assessing the effectiveness of a therapeutic intervention for a subject. comprising: obtaining a sample from said subject; determining the presence of absence of the expression of an equilibrative nucleoside transporter in said sample; correlating the presence or absence of the expression of the equilibrative ' nucleoside transporter in said sample with the effectiveness of the therapeutic intervention.
60. A method of treating a subject having or at risk of having a disorder associated with an equilibrative nucleoside transporter comprising administering to the subject a therapeutically effective amount of a reagent that enhances a function of the equilibrative nucleoside transporter.
61. The method of claim 60, wherein said function is the ability to transport anticancer nucleoside-based drugs.
62. The method of claim 60, wherein said function is the ability to transport antiviral drugs.
63. A transgenic nonhuman animal characterized by expression of an equilibrative nucleoside transporter polypeptide otherwise not naturally occurring in the animal, the expression being conferred by a transgene contained in the somatic and germ cells of the animal, the transgene comprising a nucleic acid sequence which encodes an equilibrative nucleoside transporter polypeptide.
64. The transgenic nonhuman animal of claim 64, wherein the animal is a mouse.
65. The transgenic nonhuman animal of claim 64, wherein the equilibrative nucleoside transporter polypeptide is selected from the group consisting of SEQ ED NO:2, SEQ ED NO:4. SEQ ED NO:6, and SEQ ID NO: 8.
PCT/IB1997/001657 1996-12-30 1997-12-30 Mammalian equilibrative nucleoside transporters WO1998029437A2 (en)

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