Title: Molecular Identification and Characterization of Novel Human and Mouse Concentrative Na+-Nucleoside Cotransporter Proteins (hCNT3 and mCNT3) Broadly Selective for Purine and Pyrimidine Nucleosides (System cib)
Abstract: The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (systemcit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na+-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na+-dependentcib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na+concentration indicated a Na+:uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostrate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element. The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (systemcit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na+-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na+-dependentcib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na+concentration indicated a Na+:uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostrate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element. nucleoside transporter 3′-azido-3′-deoxythymidine bacterial artificial chromosome concentrative nucleoside transporter base pair(s) equilibrative nucleoside transporter kilobase(s) nitrobenzylthioinosine (6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine) N-methyl-d-glucamine polymerase chain reaction reverse transcriptase-PCR putative transmembrane helix expressed sequence tag group of overlapping clones Most nucleosides, including those with antineoplastic and/or antiviral activities (1Periguad C. Gosselin G. Imbach J.L. Nucleosides Nucleotides. 1992; 11: 903-945Crossref Scopus (306) Google Scholar, 2Handschumacher R.E. Cheng C.Y. Holland E. Frei E. Bast R.C. Kufe D.W. Morton D.L. Weichselbaum R.R. Cancer Metabolism. Lea & Febiger, Philadelphia, PA1993: 712-732Google Scholar), are hydrophilic, and specialized plasma membrane nucleoside transporter (NT)1 proteins are required for uptake into or release from cells (3Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, New York1995: 404-451Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). NT-mediated transport is therefore a critical determinant of metabolism and, for nucleoside drugs, their pharmacologic actions (5Mackey J.R. Baldwin S.A. Young J.D. Cass C.E. Drug Resistance Updates. 1998; 1: 310-324Crossref PubMed Scopus (138) Google Scholar). NTs also regulate adenosine concentrations in the vicinity of cell surface receptors and have profound effects on neurotransmission, vascular tone, and other processes (6Fredholm B.B. Curr. Med. Chem. 1997; 4: 35-66Google Scholar, 7Shryock J.C. Belardinelli L. Am. J. Cardiol. 1997; 79: 2-10Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Seven nucleoside transport processes 2The abbreviations used in transporter acronyms are: c, concentrative; e, equilibrative;s and i, sensitive and insensitive to inhibition by NBMPR, respectively; f, formycin B (nonmetabolized purine nucleoside); t, thymidine; g, guanosine;b, broad selectivity. that differ in their cation dependence, permeant selectivities and inhibitor sensitivities have been observed in human and other mammalian cells and tissues. The major concentrative systems (cit,cif, and cib) are inwardly directed Na+-dependent processes and have been primarily described in specialized epithelia such as intestine, kidney, liver, and choroid plexus, in other regions of the brain, and in splenocytes, macrophages, and leukemic cells (3Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, New York1995: 404-451Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Concentrative NT transcripts have also been found in heart, skeletal muscle, placenta, and pancreas. The equilibrative (bidirectional) transport processes (esand ei) have generally lower substrate affinities and occur in most, possibly all, cell types (3Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, New York1995: 404-451Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Epithelia (e.g.intestine and kidney) and some nonpolarized cells (e.g.leukemic cells) coexpress both concentrative and equilibrative NTs, whereas other nonpolarized cells (e.g. erythrocytes) exhibit only equilibrative NTs (3Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, New York1995: 404-451Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Systems cit andcif are generally pyrimidine nucleoside selective and purine nucleoside selective, respectively, whereas systems cib,es, and ei transport both pyrimidine and purine nucleosides. System ei also transports nucleobases. Molecular cloning studies have isolated cDNAs encoding the human and rat proteins responsible for four of these NT processes (cit, cif, es, and ei) (8Huang Q.Q. Yao S.Y.M. Ritzel M.W.L. Paterson A.R.P. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar, 9Che M. Ortiz D.F. Arias I.M. J. Biol. Chem. 1995; 270: 13596-13599Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Yao S.Y.M. Ng A.M.L. Ritzel M.W.L. Gati W.P. Cass C.E. Young J.D. Mol. Pharmacol. 1996; 50: 1529-1535PubMed Google Scholar, 11Ritzel M.W.L. Yao S.Y.M. Huang M.Y. Elliot J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar, 12Wang J. Su S.F. Dresser M.J. Schaner M.E. Washington C.B. Giacomini K.M. Am. J. Physiol. 1997; 273: F1058-F1065PubMed Google Scholar, 13Ritzel M.W.L. Yao S.Y.M. Ng A.M.L. Mackey J.R. Cass C.E. Young J.D. Mol. Membr. Biol. 1998; 15: 203-211Crossref PubMed Scopus (177) Google Scholar, 14Griffiths M. Beaumont N. Yao S.Y.M. Sundaram M. Bouman C.E. Davies A. Kwong F.Y.P. Coe I.R. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (358) Google Scholar, 15Yao S.Y.M. Ng A.M.L. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 16Griffiths M. Yao S.Y.M. Abidi F. Phillips S.E.V. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (226) Google Scholar, 17Crawford C.R. Patel D.H. Naeve C. Belt J.A. J. Biol. Chem. 1998; 273: 5288-5293Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These proteins and their homologs in other mammalian species comprise two previously unrecognized families of integral membrane proteins (CNT and ENT) with quite different predicted architectural designs (3Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Dekker, New York1995: 404-451Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (302) Google Scholar). The relationships of these NT proteins to the processes defined by functional studies are: CNT1 (cit), CNT2 (cif), ENT1 (es), and ENT2 (ei). Although the NT protein(s) responsible for mammalian cibhave remained elusive, we have recently identified a CNT protein withcib-type transport activity from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti) (18Loewen S.K. Ng A.M.L. Yao S.Y.M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1999; 274: 24475-24484Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 3S. Y. M. Yao, A. M. L. Ng, S. K. Loewen, C. E. Cass, and J. D. Young, manuscript in preparation. The CNT family also includes the Escherichia coli proton/nucleoside symporter NupC (19Craig J.E. Zhang Y. Gallagher M.P. Mol. Microbiol. 1994; 11: 1159-1168Crossref PubMed Scopus (77) Google Scholar). Human and rat CNT1 (650 and 648 residues, 71 kDa), designated hCNT1 and rCNT1, respectively, are 83% identical in amino acid sequence (8Huang Q.Q. Yao S.Y.M. Ritzel M.W.L. Paterson A.R.P. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar, 11Ritzel M.W.L. Yao S.Y.M. Huang M.Y. Elliot J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar) and contain 13 putative TMs with an exofacial glycosylated tail at the carboxyl terminus (18Loewen S.K. Ng A.M.L. Yao S.Y.M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1999; 274: 24475-24484Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 4S. R. Hamilton, S. Y. M. Yao, M. P. Gallagher, P. J. F. Henderson, C. E. Cass, J. D. Young, and S. A. Baldwin, manuscript in preparation. hCNT2 (658 residues) (12Wang J. Su S.F. Dresser M.J. Schaner M.E. Washington C.B. Giacomini K.M. Am. J. Physiol. 1997; 273: F1058-F1065PubMed Google Scholar, 13Ritzel M.W.L. Yao S.Y.M. Ng A.M.L. Mackey J.R. Cass C.E. Young J.D. Mol. Membr. Biol. 1998; 15: 203-211Crossref PubMed Scopus (177) Google Scholar) is 83% identical to rCNT2 (659 residues) (9Che M. Ortiz D.F. Arias I.M. J. Biol. Chem. 1995; 270: 13596-13599Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Yao S.Y.M. Ng A.M.L. Ritzel M.W.L. Gati W.P. Cass C.E. Young J.D. Mol. Pharmacol. 1996; 50: 1529-1535PubMed Google Scholar) and 72% identical to hCNT1 (11Ritzel M.W.L. Yao S.Y.M. Huang M.Y. Elliot J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar). The hagfish transporter hfCNT (683 residues) (18Loewen S.K. Ng A.M.L. Yao S.Y.M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1999; 274: 24475-24484Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) is 50–52% identical to h/rCNT1/2 and has a similar predicted membrane topology. NupC (19Craig J.E. Zhang Y. Gallagher M.P. Mol. Microbiol. 1994; 11: 1159-1168Crossref PubMed Scopus (77) Google Scholar), in contrast, is a smaller protein with 27% identity to mammalian CNTs, with the major difference being the absence of the equivalents of TM 1–3 and the amino- and carboxyl-terminal regions of the other proteins. In structure/function studies, the characteristics of hCNT1/2 chimeras and sequence comparisons between h/rCNTs and hfCNT have identified two sets of adjacent residues in TMs 7 and 8 of hCNT1 that, when converted to the corresponding residues in hCNT2, changed the specificity of the transporter from cit to cif (18Loewen S.K. Ng A.M.L. Yao S.Y.M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1999; 274: 24475-24484Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Mutation of the two residues in TM 7 alone produced a protein with intermediate,cib-like activity. In this cit/cibconversion, mutation of hCNT1 Ser319 to Gly was sufficient to enable transport of purine nucleosides, whereas mutation of the adjacent residue Gln320 to Met (which had no effect on its own) augmented this transport. TMs 7 and 8 have also been identified as potential determinants of substrate selectivity in rCNT1/2 (21Wang J. Giacomini K.M. J. Biol. Chem. 1997; 272: 28845-28848Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and mutation of rCNT1 Ser318 (the rat counterpart of hCNT1 Ser319) resulted in a cib-type phenotype similar to that seen with the hCNT1 Ser319 mutation (22Wang J. Giacomini K.M. J. Biol. Chem. 1999; 274: 2298-2302Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Although an earlier study had identified a member of the SGLT glucose transporter family, SNST1, as a candidate cib-type transporter (23Pajor A.M. Wright E.M. J. Biol. Chem. 1992; 267: 3557-3560Abstract Full Text PDF PubMed Google Scholar), its nucleoside-transport activity is very low, and we hypothesized that the missing mammalian concentrative NT was more likely to be a CNT transporter. Following a search for additional mammalian CNT isoforms, we now report the cDNA cloning of new human and mouse members of the CNT transporter family. The encoded proteins, designated hCNT3 and mCNT3, respectively, exhibit strongcib-type functional activity when expressed inXenopus oocytes and have primary structures that place them together with hfCNT in a CNT subfamily separate from h/rCNT1/2. BLAST searches of CNT sequences in the GenBankTM data base identified overlapping human ESTs from mammary gland (AI905993) and colon adenocarcinoma (AW083022) different from established members of the CNT transporter family. Together, they formed a composite cDNA fragment 807 bp in length with an open reading frame of 245 residues followed by 69 bp of 3′-untranslated sequence. The cDNA was 62% identical in nucleotide sequence to corresponding regions of the hCNT1 (U62968) and hCNT2 (AF036109) cDNAs and 68% identical to the hfCNT (AF132298) cDNA. The encoded amino acid sequence was 79% identical to the carboxyl terminus of hfCNT and 58 and 62% identical, respectively, to hCNT1 and hCNT2. These indications of a novel human CNT distinct from hCNT1 and hCNT2 were tested by RT-PCR in a panel of total RNA samples from human mammary gland, small intestine, kidney (CLONTECH, Palo Alto, CA), and liver (13Ritzel M.W.L. Yao S.Y.M. Ng A.M.L. Mackey J.R. Cass C.E. Young J.D. Mol. Membr. Biol. 1998; 15: 203-211Crossref PubMed Scopus (177) Google Scholar). Because the close sequence similarity between the EST composite sequence and hfCNT suggested that the new CNT might correspond to system cib, we also performed RT-PCR on differentiated human myeloid HL-60 cells, a source of functionalcib-type transport activity (see below). First strand cDNA was synthesized using the Superscript Preamplification system (Life Technologies, Inc.) and oligo(dT) as primer. The PCR reaction (30 μl) contained 50 ng of template first-strand cDNA, 2.5 units of Taq-DeepVent DNA polymerase (100:1) and 10 pmol each of the 5′- and 3′-oligonucleotide primers 5′-GAAACATGTTTGACTACCCACAG-3′ and 5′-GTGGAGTTGAAGGCATTCTCTAAAACGT-3′. Amplification for one cycle at 94 °C for 55 s, 54 °C for 55 s, and 72 °C for 70 s, two cycles at 94 °C for 55 s, 55 °C for 55 s, and 72 °C for 70 s, and 30 cycles at 94 °C for 55 s, 58 °C for 55 s, and 72 °C for 70 s (Robocycler™ 40 Temperature Cycler, Stratagene, La Jolla, CA) generated visible PCR products of the predicted size (480 bp) from four of the samples (differentiated HL-60 cells, mammary gland, small intestine, and liver). We extended the partial EST cDNA sequence by 5′-rapid amplification of cDNA ends amplification of mRNA from differentiated HL-60 cells using the FirstChoice RLM-RACE kit (Ambion, Austin, TX). Poly(A)+-selected RNA was treated with calf intestinal phosphatase to degrade 5′-truncated transcripts, followed by tobacco acid pyrophosphatase to remove cap from the remaining full-length mRNAs. A synthetic RNA adaptor from the kit was then ligated to the full-length 5′-monophosphate transcript population using T4 RNA ligase, followed by first strand cDNA synthesis with oligo(dT) as primer. For the initial PCR, the 5′-primer was the outer adaptor primer provided by the kit and the gene-specific 3′-primer was 5′-GATATATATTGCTGCACACCGTTTACAA-3′. Amplification byTaq-DeepVent DNA polymerase (100:1) was for 40 cycles at 94 °C for 55 s, 65 °C for 55 s, and 72 °C for 3 min and 1 cycle at 72 °C for 10 min, the reaction mixture being heated to 94 °C for 1 min before addition of the Taq-DeepVent DNA polymerase mixture. The PCR reaction mixture was resolved on a 1% agarose gel, and faint bands between 1.5 and 2.0 kb in size were isolated and purified (QIAEX II Gel Extraction kit; Qiagen Inc.). This product was then reamplified by nested PCR (35 cycles at 94 °C for 55 s, 65 °C for 55 s, and 72 °C for 3 min and 1 cycle at 72 °C for 10 min) using an inner 5′-primer from the kit and the gene-specific 3′-primer 5′-TTAGCTCAAAACTCAGCTGTGGGTAGTC-3′. A defined band of ∼1.7 kb was isolated, cloned into pGEM-T (Promega, Madison, WI), and sequenced by Taq DyeDeoxyterminator cycle sequencing using an automated model 373A DNA Sequencer (Applied Biosystems, Foster City, CA). The inset overlapped the 807-bp EST sequence by 114 bp and generated an additional 1633 bp of upstream sequence. The new composite 2440-bp sequence was 66% identical to the hfCNT cDNA and contained an open reading frame of 691 amino acids. cDNAs containing the complete coding sequence were then obtained by RT-PCR from differentiated HL-60 cells and mammary gland, as described previously, using 5′- and 3′-primers flanking the open reading frame (5′-CTAAATGAAGAGCGCTTGGGACCT-3′ and 5′-AGCATCTGTACTTCAGAGTTCCACTGG-3′). The resulting ∼2.2-kb products were ligated into pGEM-T and sequenced in both directions to give identical 691-residue open reading frames flanked by 92 bp of 5′-untranslated nucleotide sequence and 41 bp of 3′-untranslated sequence. As expected from their identical nucleotide and predicted amino acid sequences, there was no difference in hCNT3 transport function between cDNA clones isolated from HL-60 cells or mammary gland. Radioisotope transport studies reported in this paper were performed with the HL-60 clone in pGEM-T. BLAST searches of mouse ESTs in the GenBankTM data base identified 630- and 635-bp sequences from two mammary gland IMAGE clones with 73 and 83% sequence identity to parts of the hCNT3 cDNA sequence. IMAGE clone 1514965 aligned with the 5′-coding region, whereas 1515408 ended 54 bp short of the predicted stop codon. Both clones were obtained from the IMAGE Consortium through the American Type Culture Collection (Manassas, VA). PCR showed that they were incomplete, and sequencing of 1515408 gave an additional 85 bp of sequence to complete the 3′-end of the open reading frame. A cDNA with the complete coding sequence was then obtained by RT-PCR from mouse liver RNA (Jackson Laboratories, Bar Harbor, ME) with 5′-primer 5′-AGGATGTCCAGGGCAGACCCGGGAAAGA-3′ and 3′-primer 5′-AGATCACAATTTATTAGGGATCCAATTG-3′. First strand cDNA was synthesized using the Thermoscript RT-PCR System (Life Technologies, Inc.), and amplification by Taq-DeepVent DNA polymerase (100:1) was for 2 cycles at 94 °C for 2 min, 64 °C for 1 min, and 72 °C for 2.5 min, 2 cycles at 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 2.5 min, 30 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2.5 min, and one final extension cycle for 10 min at 72 °C. The resulting ∼2.0-kb product was ligated into pGEM-T and subcloned into the enhanced Xenopusexpression vector pGEM-HE (24Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (983) Google Scholar). Each was sequenced in both directions, giving identical 703-amino acid residue open reading frames flanked by short 3-bp regions of 5′-untranslated or 3′-nucleotide sequence. By providing additional 5′- and 3′-untranslated sequences from aXenopus β-globin gene, the pGEM-HE construct gave greater functional activity and was used in subsequent transport characterization of the mouse protein. pGEM-HE was also used for electrophysiological studies of hCNT3. hCNT3 and mCNT3 plasmid DNAs were linearized withNotI (pGEM-T) or NheI (pGEM-HE) and transcribed with T7 polymerase mMESSAGE mMACHINETM (Ambion). Stage VI oocytes of Xenopus laevis (8Huang Q.Q. Yao S.Y.M. Ritzel M.W.L. Paterson A.R.P. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar) were microinjected with 20 nl of water or 20 nl of water containing capped RNA transcripts (20 ng) and incubated in modified Barth's medium (changed daily) at 18 °C for 72 h prior to the assay of transport activity. Transport was traced using the appropriate 14C/3H-labeled nucleoside, nucleoside drug, or nucleobase (Moravek Biochemicals, Brea, CA or Amersham Pharmacia Biotech) at a concentration of 1 and 2 μCi/ml for 14C-labeled and 3H-labeled compounds, respectively. [3H]Gemcitabine (2′,3′-difluorodeoxycytidine) was a gift from Eli Lilly Inc. (Indianapolis, IN). Radiochemicals were 98–99% pure (see HL-60 transport studies). Flux measurements were performed at room temperature (20 °C) as described previously (8Huang Q.Q. Yao S.Y.M. Ritzel M.W.L. Paterson A.R.P. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar, 11Ritzel M.W.L. Yao S.Y.M. Huang M.Y. Elliot J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar) on groups of 12 oocytes in 200 μl of transport medium containing 100 mmNaCl, 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.5. Except where otherwise indicated, the nucleoside concentration was 20 μm. At the end of the incubation period, extracellular label was removed by six rapid washes in ice-cold transport medium, and individual oocytes were dissolved in 5% (w/v) SDS for quantitation of oocyte-associated radioactivity by liquid scintillation counting (LS 6000 IC; Beckman). Initial rates of transport (influx) were determined using an incubation period of 5 min (8Huang Q.Q. Yao S.Y.M. Ritzel M.W.L. Paterson A.R.P. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar). Choline replaced sodium in Na+ dependence experiments, and the transport medium for adenosine uptake contained 1 μm deoxycoformycin to inhibit adenosine deaminase activity. The flux values shown are the means ± S.E. of 10–12 oocytes, and each experiment was performed at least twice on different batches of cells. Kinetic (K m and V max) and Na+ activation parameters (K 50 and Hill coefficient) ± S.E. were determined using ENZFITTER (Elsevier-Biosoft, Cambridge, UK) and SigmaPlot (SPSS Inc., Chicago, IL) software, respectively. Oocytes were voltage clamped using the two-electrode voltage clamp. Membrane currents were measured at room temperature by use of a GeneClamp 500B oocyte clamp (Axon Instruments, Foster City, CA). The microelectrodes were filled with 3 m KCl and had resistances that ranged from 1–2.5 mΩ. The GeneClamp 500B was interfaced to a computer via a Digidata 1200 A/D convertor and controlled by Axoscope software (Axon Instruments, Foster City, CA). Current signals were filtered at 20 Hz (four-pole Bessel filter) and sampled at intervals of 20 ms. For data presentation, the signals were further filtered at 0.5 Hz by use of pCLAMP software (Axon Instruments). Cells were not used if resting membrane potentials were unstable or less than −30 mV. For measurements of hCNT3-generated currents, oocyte membrane potentials were clamped at −50 mV. Oocytes were perfused with the same medium used for radioisotope flux studies, and transport assays were initiated by changing the substrate-free solution to one containing nucleoside (200 μm). In experiments examining Na+ dependence, sodium in the medium was replaced by choline. The human promyelocytic cell line, HL-60, obtained from the American Type Culture Collection, was propagated as suspension cultures in RPMI 1640 medium, supplemented with 10% fetal calf serum using reagents purchased from Life Technologies, Inc. Stock cultures were maintained in 5% CO2 without antibiotics at 37 °C, subcultured every 3–4 days and demonstrated to be mycoplasma-free. Cell numbers were determined using a Coulter Counter model Z2 (Coulter Electronics Inc., Luton, UK). To induce differentiation, HL-60 cells (3 × 106) growing in logarithmic phase were placed in 10-cm Falcon Primaria tissue culture plates (Becton Dickinson) in the presence of phorbol 12-myristate 13-acetate (200 ng/ml) (Sigma) freshly dissolved in acetone. After 48 h, the plates were washed once with transport buffer (see below) to remove nonadherent cells and then incubated for 15 min in the presence or absence of 100 μm dilazep. Transport assays were performed on the remaining adherent cells. Total RNA and mRNA were prepared from exponentially growing parent and adherent HL-60 cells using the RNeasy Mini Protocol (Qiagen) and Fast Track 2.0 Isolation kit (Invitrogen, Carlsbad, CA), respectively. Nucleoside uptake by differentiated HL-60 cells was measured as described previously (25Graham K.A. Leithoff J. Coe I.R. Mowles D. Mackey J.R. Young J.D. Cass C.E. Nucleosides Nucleotides Nucleic Acids. 2000; 19: 415-434Crossref PubMed Scopus (49) Google Scholar) by exposing replicate cultures at room temperature to3H-labeled permeant (10 μm, 1 μCi/ml) in sodium or sodium-free transport medium (130 mm NaCl or 130 mm NMDG/HCl and 3 mmK2HPO4, 2 mm CaCl2, 1 mm MgCl2, 20 mm Tris/HCl, and 5 mm glucose, pH 7.4). Radiochemicals (Moravek Biochemicals) were 98–99% pure as assessed by high performance liquid chromatography using water-methanol gradients on a C18 reverse phase column, and transport for timed intervals of 1–6 min was terminated by immersion of the culture dish in an excess volume of ice-cold transport solution. Assays to detect concentrative transport were performed in the presence of 100 μm dilazep (a gift from Hoffman La Roche & Co., Basel, Switzerland) to block equilibrative transport of the test nucleoside. Transport by nonadherent parental HL-60 cells was performed as described previously (26Boleti H. Coe I.R. Baldwin S.A. Young J.D. Cass C.E. Neuropharmacology. 1997; 38: 1167-1179Crossref Scopus (75) Google Scholar) using the inhibitor oil stop method. Values are presented as the means of triplicate measurements ± S.D. A human multiple tissue expression (MTETM) RNA array (CLONTECH) and dot blots of mRNA (0.5 μg) from parent and differentiated HL-60 cells on BrightStar-Plus nylon transfer membrane (Ambion) were incubated with a cDNA probe corresponding to hCNT3 amino acid residues 359–549 labeled with32P using the T7QuickPrime kit (Amersham Pharmacia Biotech). Hybridization at high stringency (68 °C) was performed using ExpressHyb hybridization solution (CLONTECH) and 100 μg/ml of sheared herring sperm DNA. Wash conditions were as described in the CLONTECH ExpressHyb user manual. Signals on exposed blots were converted to a high resolution tiff image (Hewlett Packard ScanJet 4C) and quantified using the public domain NIH Image program, version 1.60. For Northern analysis, 5-μg samples of mRNA from human pancreas, bone marrow, trachea, intestine, liver, brain, heart, and kidney (CLONTECH) were separated on a 0.8% formaldehyde-agarose gel, blotted on to BrightStar-Plus nylon transfer membrane, and hybridized with the same hCNT3 probe (residues 359–549) under identical high stringency conditions. Possible cross-hybridization between CNT family members was tested on dot blots of dilutions (0.5 μg-5 ng RNA) of hCNT1, hCNT2, and hCNT3in vitro transcripts. Three identical series of blots were incubated either with hCNT3 probe or with equivalent probes for hCNT1 or hCNT2. The hCNT3 probe, which was 63 and 58% identical in nucleotide sequence to the corresponding regions of hCNT1 and hCNT2, respectively, showed no cross-hybridization with hCNT1 or hCNT2 transcripts. Similarly, there was no cross-reactivity between the hCNT1 and hCNT2 probes and hCNT3 RNA. Some cross-hybridization was seen between the hCNT1 and hCNT2 probes (73% nucleotide sequence identity) and their respective transcripts at RNA loadings ≥50 ng. Under the conditions of high stringency used in our experiments, the hCNT3 probe was therefore specific. In TaqManTMquantitative RT-PCR (Applied Biosystems), an oligonucleotide probe, labeled with a fluorescent tag at the 5′-end and a quenching molecule at the 3′-end, is located between two PCR primers. The 5′-