Oncogene (2003) 22, 7524–7536 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

Nucleoside anticancer drugs: the role of transporters in resistance to cancer chemotherapyw

Vijaya L Damaraju1,2,4, Sambasivarao Damaraju2,4, James D Young1,3,6, Stephen A Baldwin5, John Mackey2,4, MichaelB Sawyer 2,4 and CarolE Cass* ,1,2,4,7

1Membrane Protein Research Group, University of Alberta, Canada; 2Department of Oncology, University of Alberta, Canada; 3Department of Physiology, University of Alberta, Canada; 4Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2; and 5School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

The clinical efficacy of anticancer nucleoside drugs extracellular milieu for the production of for depends on a complex interplay of transporters mediating use in RNA and DNA synthesis. In addition to their entry of nucleoside drugs into cells, efflux mechanisms role as intermediates for many essential cellular biosyn- that remove drugs from intracellular compartments and thetic pathways, nucleotides play key roles in neuro- cellular metabolism to active metabolites. Nucleoside transmission (Baldwin et al., 1999) and regulation of transporters (NTs) are important determinants for salvage cardiovascular activity (Shryock and Belardinelli, 1997) of preformed and mediated uptake of anti- and as signaling molecules (Schachter et al., 1995). NTs metabolite nucleoside drugs into target cells. The focus of play a role in the maintenance of extracellular concen- this review is the two families of human nucleoside trations of available to bind to receptors to transporters (hENTs, hCNTs) and their role in transport modulate a variety of physiological processes. Plasma of cytotoxic chemotherapeutic nucleoside drugs. Resis- membrane transport of nucleosides is a critical determi- tance to anticancer nucleoside drugs is a major clinical nant in the salvage and release of preformed nucleosides problem in which NTs have been implicated. Single in many cell types (Cass, 1995; Griffith and Jarvis, 1996; polymorphisms (SNPs) in drug transporters Baldwin et al., 1999; Young et al., 2001). Polar may contribute to interindividual variation in response to hydrophilic nucleoside analogs used in cancer che- nucleoside drugs. In this review, we give an overview of the motherapy require functional NT proteins to enter cells. functional and molecular characteristics of human NTs Phosphorylation of nucleosides to nucleotides is and their potential role in resistance to nucleoside drugs mediated by nucleoside kinases present in cells. The and discuss the potential use of genetic polymorphism interaction between nucleoside drugs and intracellular analyses for NTs to address drug resistance. (e.g., kinases, deaminases and nucleotidases) Oncogene (2003) 22, 7524–7536. doi:10.1038/sj.onc.1206952 may regulate the rate and extent of metabolism of nucleoside drugs to cytotoxic derivatives. This review Keywords: nucleoside transporters; anticancer nucleo- gives an overview of the functional and molecular sides; polymorphisms; SNPs; drug resistance characteristics of NTs present in human tissues with a focus on localization and regulatory mechanisms. Important structuraldeterminants for recognition of nucleosides by membrane transporters and clinically used nucleoside drugs and resulting resistance mechan- Human nucleoside transporters isms are reviewed. The reader is referred to recently published review articles on the molecular structure and Nucleoside transporters (NTs) mediate the uptake of function of NT proteins and their role in drug resistance physiologic nucleosides as well as both anticancer and (Mackey et al., 1998a; Baldwin et al., 1999; Vickers et al., antiviral nucleoside drugs (Baldwin et al., 1999). 2000; Cabrita et al., 2002; Clarke et al., 2002). Specialized cells, including enterocytes, bone marrow cells and certain brain cells (Murray, 1971) lack de novo synthesis pathways and salvage nucleosides from the Functional characterization *Correspondence: CE Cass, Department of Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G The two major classes of NTs in human cells and tissues 1Z2; E-mail: [email protected] consist of equilibrative nucleoside transporters (ENTs) wSupported by the Alberta Cancer Foundation, the Canadian and concentrative nucleoside transporters (CNTs). In Institutes of Health Research and the National Cancer Institute of the human ENT family, two proteins (hENT1 and Canada 6JDY is a Heritage Scientist of the Alberta Heritage Foundation for hENT2) are known to mediate the transport of MedicalResearch and nucleosides down their concentration 7CEC holds the Canada Research Chair in Oncology gradients. hENTs exhibit broad permeant selectivities Transporters and resistance VL Damaraju et al 7525 and are subdivided based on their sensitivities (hENT1) brane domains and a single extracellular site for or resistance (hENT2) to inhibition by nanomolar glycosylation (Griffiths et al., 1997a, b; Yao et al., concentrations of nitrobenzylmercaptopurine ribonu- 1997; Kiss et al., 2000; Handa et al., 2001). hENT1 is cleoside (NBMPR). hENT1 and hENT2 mediate the known to be a heterogeneously glycosylated protein (45– well-characterized equilibrative-sensitive (es) and equili- 65 kDa) (Kwong et al., 1993) and the predicted 11 brative-insensitive (ei) processes. Although both hENT1 transmembrane topology has been experimentally ver- and hENT2 have similar selectivities for nucleosides, ified by glycosylation scanning mutagenesis and use of hENT2 also recognizes and transports antibodies (Sundaram et al., 2001a). hENT1 and hENT2 and other purine nucleobases (Osses et al., 1996; share 49% amino-acid identity and 69% similarity and Baldwin et al., 1999; Yao et al., 2002). The role of transport purine and pyrimidine nucleosides with differ- hENT3 and hENT4 in nucleoside transport processes ent affinities (Griffiths et al., 1997a, b; Ward et al., 2000). has not yet been established (Baldwin et al., 2003). hCNT1, which is responsible for cit activity in Six functionalsubtypes of CNT processes (Gutierrez humans, was identified in the human kidney by et al., 1992; Belt et al., 1993; Gutierrez and Giacomini, hybridization/RT–PCR cloning and functional expres- 1993, 1994; Paterson et al., 1993; Cass, 1995; Griffith and sion in Xenopus laevis oocytes of two closely related Jarvis, 1996; Flanagan and Meckling-Gill, 1997; Ritzel cDNAs (Ritzel et al., 1997). Minor differences between et al., 1997, 1998, 2001a b; Cass et al., 1999) have been these two cDNAs are felt to be due to genetic described in human cells or tissues. The three best polymorphisms and/or errors during PCR amplification characterized CNTs are Na þ -nucleoside cotransporters and both encode a functionalhCNT1 protein. hCNT1 is and are termed cit, cif and cib to indicate that they are predicted to have 13 transmembrane domains. Human concentrative, insensitive to NBMPR and accept as cDNAs encoding hCNT2 (also termed hSPNT1), which permeants, respectively, pyrimidine nucleosides, purine has cif activity, were isolated from the kidney and nucleosides or both pyrimidine and purine nucleosides. intestine (Wang et al., 1997; Ritzel et al., 1998) by The transporter proteins that are responsible for these hybridization/RT–PCR cloning. The human homologs activities are cit (hCNT1), cif (hCNT2) and cib (hCNT3). were almost identical (658 amino acids), with the However, the proteins responsible for cs (concentrative exception of a single amino-acid substitution at position and sensitive to NBMPR) (Paterson et al., 1993), csg 75, which is arginine in hCNT2 (Ritzel et al., 1998) and (concentrative, sensitive to NBMPR and accepts guano- serine in hCNT2/hSPNT1 (Wang et al., 1997). Mole- sine as permeant) (Flanagan and Meckling-Gill, 1997) cular cloning of cDNAs from differentiated HL-60 cells and cit with specificity towards have not been and human mammary gland resulted in identification of identified (Gutierrez et al., 1992; Gutierrez and Giacomi- sequences encoding a third concentrative transporter ni, 1993, 1994) and are not discussed further in this (hCNT3, 691 amino acids) that exhibits a cib-like review. The mammalian transporter responsible for the activity (Ritzel et al., 2001b). There is an 85% amino- cit process (now known to be CNT1) was first described acid identity between the CNT in transmembrane in freshly isolated mouse intestinal cells (Vijayalakshmi domains 7–9 (Loewen et al., 1999). hCNT2 is 72% and Belt, 1988) and exhibited a preference for pyrimidine identicalto hCNT1 (Ritzel et al., 1997) and hCNT3 is 48 nucleosides. Although inhibited by low concentrations of and 47% identicalto hCNT1 and hCNT2, respectively adenosine, CNT1 does not transport adenosine well. The (Ritzel et al., 2001a). cif process (i.e., CNT2) first described in mouse enter- ocytes (Vijayalakshmi and Belt, 1988) transports purine nucleosides including formycin-B (C-nucleoside analog of Chromosomal localization of transporter genes ) in addition to . Uridine appears to be a universalpermeant for allNTs. The hCNT1 and hCNT2 The genes for hENT1 and hENT2, respectively, have proteins exhibit 1 : 1 Na þ –uridine coupling ratios. been localized to chromosome 6p21.1–p21.2 (Coe et al., The cib process (i.e., hCNT3) is broadly selective and 1997) and to chromosome 11 (GenBankt accession transports both purine and pyrimidine nucleosides in a NT_009379; InternationalHuman Genome Project). concentrative manner into cells. This process was first The genes for hCNT1 (GenBankt accession AF036109) described in freshly isolated leukemic blasts (Belt et al., and hCNT2 (GenBankt accession U62966) map, 1992) and human colon cancer Caco-2 cells (Belt et al., respectively, to chromosome 15q25–26 (Ritzel et al., 1993). The hCNT3 protein exhibits a 2 : 1 stiochiometry 1997) and chromosome 15q15 (15q13–14) (Wang et al., for cotransport of Na þ and uridine. 1997; Ritzel et al., 1998). The hCNT3 gene (GenBankt accession AF305210) has an upstream phorbol-respon- sive element and is localized on chromosome 9q22.2 (Ritzel et al., 2001b) (Table 1). Molecular identification

The isolation of cDNAs encoding hENT1 (Griffiths et al., 1997a) and hENT2 (Griffiths et al., 1997b; Tissue distribution of transporters Crawford et al., 1998) permitted detailed molecular and functionalcharacterization of these transporters. hENT1 appears to be a ubiquitous protein, since its The hENT1 cDNA encodes a protein with 11 transmem- mRNA has been found in a diverse array of human

Oncogene Transporters and resistance VL Damaraju et al 7526 Table 1 Characteristics of functionally characterized human NTs Transporter Chromosomal Tissue distribution Amino-acid MW TMsa location of gene residues (kDa)

hENT1 6p21.1–21.2 Ubiquitous 465 52 11 hENT2 11q13 Predominantly in skeletal muscle, 465 52 11 brain, less in other tissues hCNT1 5q25–26 Kidney, liver, intestine, placenta, 650 71 13 brain, fetalkidney hCNT2 15q15 (15q13–14) Kidney, liver, intestine, placenta, brain, 658 72 13 lung, skeletal muscle, pancreas, heart hCNT3 9q22.2 Bone marrow, intestine, pancreas, tracheas, mammary gland, 691 77 13 prostate, testis, liver, lung kidney, placenta

aTMs: Transmembrane spanning domains

tissues (Pennycooke et al., 2001). The broad tissue Structure-activity studies distribution of hENT1 suggests that this NT is likely to be involved in the uptake of chemotherapeutic nucleo- Uridine transport in hENT1-producing X. laevis oocytes side analogs as well as in the uptake of the physiologic is inhibited by physiological purine and pyrimidine nucleosides. High-level expression of hENT1 mRNA nucleosides and by the nucleoside drugs , was observed in adrenaltissue (Pennycooke et al., 2001) , fludarabine and as well as by although its significance is not yet clear. Expression of NBMPR, , dilazep and draflazine (Grif- hENT1 mRNA in other endocrine tissues suggests the fiths et al., 1997b). Studies of the properties of chimeras possible regulation of hENT1 by steroid hormones between human and rat ENTs have established that (Fideu and Miras-Portugal, 1992, 1993). hENT2 mRNA transmembrane domains 3–6 contain residues respon- has been observed in a variety of tissues, including the sible for sensitivity or resistance to coronary vasodila- brain, heart, lung, thymus, prostate, pancreas, with the tors (Sundaram et al., 1998) and to NBMPR (Sundaram highest levels occurring in skeletal muscle (Crawford et al., 2001b). Transmembrane domains 1–6 of ENT2 et al., 1998). The predominant distribution of hENT2 appear to be responsible for transport of 30-deoxynu- mRNA in skeletal muscle (Pennycooke et al., 2001) cleosides (Yao et al., 2001) while transmembrane suggests its role as a transporter in skeletal muscle (Cass domains 5–6 appear to be involved in its transport of et al., 1998; Crawford et al., 1998). The hENT2 protein nucleobases (Yao et al., 2002). Using site-directed has also been shown to be widely present in the brain mutagenesis, binding of NBMPR to recombinant (Jennings et al., 2001). hENT1 produced in Saccharomyces cerevisiae has been Mammalian CNT families of transporters are found investigated. A mutant in which the methionine at in highly differentiated tissues such as the epithelial position 33 of hENT1 was modified to isoleucine lining of the intestine and kidney (Cass et al., 1999), (generated using a random mutagenesis strategy) although they appear to be present at low levels in other exhibited reduced inhibition of uridine transport by tissues. The high abundance of CNTs in liver and renal dipyridamole (Visser et al., 2002). Modification of tissues may contribute to the renaland hepatic toxicities glycine to serine at position 154 of hENT1 resulted in observed when patients are treated with nucleoside loss of NBMPR binding (Hyde et al., 2001). Uridine analog drugs (Loewen et al., 1999). In the intestine, transport and inhibition by NBMPR were diminished CNTs were localized by functional studies to brush when the highly conserved glycine at position 179 was border membranes (Chandrasena et al., 1997; Patiland changed to alanine, and uridine transport was abolished Unadkat, 1997; Ngo et al., 2001), whereas ENTs were when glycine was changed to leucine, cysteine or valine localized to basolateral membranes (Chandrasena et al., (SenGupta et al., 2002). Glycine at position 184 was 1997; Lum et al., 2000). There is a wide variation in the shown to be important in either targeting of hENT1 to expression of hCNT1 mRNA in normalkidney in the plasma membrane or in correct folding (SenGupta different individuals (Pennycooke et al., 2001) and et al., 2002) using GFP-tagged hENT1 protein in yeast relatively low levels of expression of hCNT1 mRNA in expression system. Interaction of hENT1 with analogs tumor samples (Pennycooke et al., 2001). The expression of uridine and containing base or of hCNT2 mRNA was detected in a variety of human modifications has been described using recombinant tissues, with highest expression in tissues of the digestive transporters produced in S. cerevisiae (Vickers et al., system and kidney, followed by the prostate, cervix and 2002). Decreased recognition of cytidine analogs by lung (Pennycooke et al., 2001). High expression of hENT1 was demonstrated using 20-deoxycytidine and hCNT2 mRNA was seen in samples of tumor tissue cytarabine (araC). Both the 20- and 30-hydroxyls are derived from the lung, ovary, uterus and prostate. important determinants for interaction of cytidine with hCNT3 transcripts have been observed in differentiated hENT1. Addition of bulky substituents at position 5 of HL-60 cells, mammary gland, pancreas, bone marrow, the base resulted in lowered affinity of the analogs for trachea, liver, prostate, brain, heart and intestine (Ritzel hENT1, whereas the 30-hydroxylgroup appeared to be et al., 2001b). absolutely essential for interaction with the transporter

Oncogene Transporters and resistance VL Damaraju et al 7527 (Zimmerman et al., 1987; Vickers et al., 2002). Antiviral of the hydroxylgroup at the 2 0-position did not affect nucleoside drugs that lack the 30-hydroxylgroup on the the interaction of uridine with hCNT2. 5-Fluorouridine ribose ring are not or are very weakly transported by and 5-fluoro-20-deoxyuridine were transported by hENT1 (Yao et al., 2001). hCNT2 with high affinity, suggesting that hCNT2 may Studies with recombinant hENT2 produced in oocytes also be involved in the uptake of these drugs into cells (Griffiths et al., 1997b) revealed complete inhibition of (Lang et al., 2001). Transport activity of halogenated uridine transport by adenosine, inosine or thymidine, analogs of adenosine by hCNT2 was lower compared partialinhibition by guanosine, cytidine or hypoxanthine with halogenated uridine analogs (Lang et al., 2001). and no inhibition by or . hENT2 mediated Loewen et al. (1999) identified amino-acid residues in significant transport of antiviraldrugs, zidovudine, transmembrane domains 7 (Ser 319/Gln 320) and 8 (Ser zalcitabine and didanosine (Yao et al., 2001). Site- 353/Leu 354) that, when converted to the corresponding directed mutagenesis of isoleucine at position 33 to residues in hCNT2, converted the permeant selectivity methionine in hENT2 resulted in the expression of a of CNT1 to a CNT2-like selectivity. protein with higher sensitivity to dipyridamole (Visser Functionalstudies of recombinant hCNT3 in X. laevis et al., 2002). A notable difference between hENT1 and oocytes resulted in the expression of a transport activity hENT2 is the lower affinity of hENT2 to cytidine; kinetic (cib) with broad permeant selectivity. Permeants include studies of fluxes of cytidine in X. laevis oocytes gave Km the physiological purine and pyrimidine nucleosides, as values of 0.56 and 45mm for hENT1 and hENT2, well as anticancer and antiviral nucleoside analogs respectively (Young et al., 2001). Modifications in the 5 including 5-fluorouridine45-fluoro-20-deoxyuridine4 position of the pyrimidine moiety of uridine were better cladribine4zebularine4gemcitabine4fludarabine4 tolerated by hENT2 than by hENT1 and modification in zidovudine42030-dideoxyinosine42030-dideoxycytosine 0 the 3 -hydroxylposition of the ribose moiety of uridine (Ritzel et al., 2001b). Apparent Km values for pyrimidine abolished interaction with hENT2 (Vickers et al., 2002). and purine nucleosides were between 15 and 53 mm and A higher affinity for inosine by hENT2 than by hENT1 hCNT3 does not transport nucleobases (Ritzel et al., has been shown and is consistent with a role for hENT2 2001b). Using site-directed mutagenesis, Loewen et al. in the efflux and reuptake of inosine and hypoxanthine (1999) demonstrated cib-like transport activity by generated from adenosine metabolism during and after mutation of the two residues in transmembrane domain strenuous physicalexercise (Crawford et al., 1998; Ward 7 of hCNT1 (Ser319 to Gly and mutation of Gln320 et al., 2000). to Met). Recombinant hCNT1 produced in X. laevis oocytes transports uridine, zidovudine, zalcitabine, gemcitabine and is inhibited by adenosine, deoxycytidine, thymidine, cytidine and uridine, but not by guanosine or inosine Regulation of NTs (Ritzel et al., 1997; Mackey et al., 1999; Graham et al., 2000). Although adenosine binds with high affinity to The abundance of NT proteins in cells and tissues hCNT1, it is transported very poorly compared to depends in part on the pathways that stimulate uridine. Recombinant hCNT1, when expressed in X. proliferation and differentiation of cells. A variety of laevis oocytes, mediates the transport of uridine (Km mechanisms are thought to contribute to the regulation 42 mm) and gemcitabine (Km 24 mm) with high affinity. of NTs in different cell types. When HL-60 cells were Amino acids 319 (serine) and 320 (glutamine) in induced to differentiate using chemicalinducing agents, transmembrane domain 7 of hCNT1 were shown to be changes in es and ei transport activities, now known to necessary for the pyrimidine nucleoside specificity of be mediated by the hENT1 and hENT2 proteins, were hCNT1-mediated transport (Loewen et al., 1999). observed (Chen et al., 1986; Delicado et al., 1991; Permeant selectivity of hCNT2 was examined using X. Lee et al., 1991; Sokoloski et al., 1991; Lee, 1994). laevis oocyte expression system (Ritzel et al., 1998) and NBMPR-binding sites decreased while the dissociation 0 hCNT2 accepted inosine, adenosine, uridine, 2 -deox- constant (Kd) remained unchanged and hENT1 trans- yadenosine, guanosine and didanosine as permeants. port activity decreased upon induction of differentiation The importance of 30- and 50-hydroxylgroups of the in HL-60 cells (Chen et al., 1986). hENT2 transport ribosylmoiety of uridine for permeant recognition by activity was observed in differentiated human neuro- hCNT2 was demonstrated in the stably transfected blastoma cells but not in undifferentiated cells (Jones CEM-ARAC (transport deficient clone) leukemic cell et al., 1994). line (Lang et al., 2001). The purine nucleoside selectivity Lipopolysaccharides and phorbol esters, which acti- of hCNT2 is demonstrated by its lower Km for vate B cells, upregulate CNTs and downregulate ENTs adenosine (8 mm) than for uridine (40 mm). hCNT1 and in lymphocytes from the bone marrow and in a human B hCNT2 differ in permeant selectivity in that hCNT1 lymphoblast cell line (Soler et al., 1998; Soler et al., transports zidovudine and zalcitabine whereas hCNT2 2000; Soler et al., 2001a, b). These effects were shown to transports didanosine. The importance of 30- and 50- result from differential regulation of NTs by agents like hydroxylgroups of the ribosylmoiety of uridine for protein kinase C (PKC) and tumor necrosis factor-a. permeant recognition by hCNT2 was demonstrated in a Nitric oxide may also be involved in the regulation of stably transfected CEM-ARAC (transport-deficient NTs in activated human B lymphocytes (Soler et al., clone) leukemic cell line (Lang et al., 2001). Removal 2000). Acute stimulation of d and e isoforms of PKC

Oncogene Transporters and resistance VL Damaraju et al 7528 resulted in an increase in transport activity in cultured plasmic 50-nucleotidase. Cytotoxicity of cytarabine MCF-7 breast cancer cells and HeLa cells, whereas results from inhibition of DNA polymerase a (Furth downregulation resulted in a decrease in transport and Cohen, 1968; Heinemann et al., 1988) and from activity (Coe et al., 2002). Transcriptionalregulation incorporation of araCTP into DNA in place of of hENT1 could be due to the presence of regulatory deoxycytidine triphosphate (dCTP). Furthermore when consensus sites in the promoter region of hENT1 and a araCMP is incorporated into DNA, its incorporation possible link to PKC signaling pathways (Sankar et al., prevents DNA chain elongation (Kufe et al., 1980; Fram 2002). et al., 1983) resulting in a blockade of DNA synthesis. Protein and mRNA levels for hENT1 were reduced Cytarabine is used predominately in hematological and transport activity was inhibited in human endothe- malignancies, such as acute myelogenous leukemia and lial cells with exposure to d-glucose, a condition that non-Hodgkin’s lymphoma. When used as a single agent, leads to activation of purinoceptors (Parodi et al., 2002). cytarabine produced remissions in about 30% of Selective regulation of NTs was shown to be critical for patients (Ellison et al., 1968). Combination chemother- proliferation and activation of murine bone marrow- apy that includes cytarabine with other derived macrophages (Soler et al., 2001a). In addition, agents is used in the treatment of chronic myelogenous upregulation of NBMPR-binding sites was described in leukemia, multiple myeloma, Hodgkin’s lymphoma and certain tumors and rapidly dividing cells (Goh et al., non-Hodgkin’s lymphomas. 1995). Inhibitors of protein kinase inhibited ENT1- Gemcitabine (dFdC, 20,20-difluorodeoxycytidine, mediated uptake of uridine and thymidine in human Gemzars) is an analog of cytarabine, which was erythroleukemia K562 cells, independent of kinase modified at the 20-position of the ribose ring by inhibition. These results suggest the involvement of substitution of two fluorine atoms (Baker et al., 1991) ENTs as possible targets for protein kinase inhibitors in to give gemcitabine. hENT1, hENT2, hCNT1 and humans (Huang et al., 2003). hCNT3 mediate uptake of gemcitabine into cells Regulation of CNT processes by lipopolysaccharide, (Mackey et al., 1999) (Zhang J, personalcommunica- phorbolesters and tumor necrosis factor- a was de- tion). hENT1 and hCNT1 appear to be most efficient in scribed in cultured human B and T lymphocytes (Soler the transport of gemcitabine into cells. After entry into et al., 1998; Kichenin et al., 2000; Ritzel et al., 2001b). cells, gemcitabine is phosphorylated to its monopho- Correlation between extracellular metabolism of nucleo- sphate (dFd-CMP) by dCK (Abbruzzese et al., 1991) tides/nucleosides and cADPR-mediated regulation of and to its active triphosphate (dFd-CTP) by pyrimidine intracellular homeostasis was explored to nucleotide kinases (Plunkett et al., 1995). Inactivation of identify specific equilibrative and concentrative NTs gemcitabine or its 50-monophosphate can occur, respec- responsible for cADPR translocation into cells (Guida tively, by deamination by cytidine deaminase or depho- et al., 2002). sphorylation by 50-nucleotidase (Plunkett et al., 1995; Neff and Blau, 1996). Incorporation of dFd-CTP into the growing DNA strand is followed by addition of a naturalnucleoside, thereby preventing DNA repair and Nucleoside analogs currently used in cancer chemotherapy causing masked chain termination (Huang and Plunkett, 1995). Prolonged accumulation of gemcitabine com- NTs are important for cellular uptake of anticancer pared to cytarabine is believed to be due to inhibition of pyrimidine and purine nucleoside analog drugs. NTs are ribonucleotide reductase and deoxycytidine monopho- expected to play a role in drug sensitivity, sphate deaminase (Heinemann et al., 1992). Gemcita- by mediating the uptake of anticancer nucleoside drugs, bine diphosphate inhibits ribonucleotide reductase and in drug resistance, by mediating the salvage of (Huang and Plunkett, 1995) thereby decreasing deox- normalnucleosidesto overcome toxicity. Both pyrimi- ynucleotide triphosphate levels; decreased levels of dine and purine nucleoside analogs are currently used dCTP inhibit deoxycytidine monophosphate deaminase, clinically as antimetabolite drugs. One of the first which increases gemcitabine monophosphate. The high- pyrimidine nucleoside analogs to be developed was er retention of active metabolites of gemcitabine, cytarabine, an analog of deoxycytidine (1-b-d-arabino- compared to cytarabine, in cancer cells is due to a furanosylcytosine, araC, Cytosar-Us) (Ellison et al., combination of different factors: gemcitabine is a better 1967). Intracellular accumulation of cytarabine depends substrate for NTs, is phosphorylated more rapidly and on the plasma concentrations of the drug and enters is eliminated from cells more slowly. Gemcitabine’s cells primarily by hENT1-mediated processes (Wiley unique self-potentiating mechanisms contribute to et al., 1983; Galmarini et al., 2002). Once cytarabine has increased accumulation of gemcitabine and its metabo- entered cells, it is phosphorylated by deoxycytidine lites and are believed to be the basis of its unusual broad kinase (dCK) to the 50-derivative araCMP and subse- spectrum of activity among nucleoside analogs. Gemci- quently by nucleotide kinases to araCTP (Hande and tabine was originally approved to treat symptoms of Chabner, 1978; Owens et al., 1992). Loss of pharmaco- pancreatic cancer (Burris et al., 1997). Subsequent logically active drug occurs through deamination of studies showed that gemcitabine has activity against cytarabine by cytidine deaminase, which results in the metastatic bladder cancer (Stadler et al., 1997) and production of a nontoxic metabolite arabinosyluridine gemcitabine combined with is now the standard and through dephosphorylation of araCMP by cyto- treatment for metastatic bladder cancer (von der Maase

Oncogene Transporters and resistance VL Damaraju et al 7529 et al., 2000). Gemcitabine is also active against non- and ecto-50-nucleotidase to fludarabine (F-araA), which small-lung cancer (Schiller et al., 2002) and breast, is transported into cells by NTs present on plasma ovarian and head and neck cancers (Possinger, 1995; membranes. enters cells mainly by hENT1 Kaye, 1998). and hCNT3 (Gati et al., 1998; Ritzel et al., 2001b). Like (50-deoxy-5-N-[(pentoxy) carbonyl]-cyti- other nucleoside analogs, fludarabine is initially phos- dine, Xelodas) is the most recent pyrimidine nucleoside phorylated by dCK to its monophosphate (fludara, F- to be introduced into clinical practice. Capecitabine was araAMP) form after which it is further phosphorylated developed to overcome the low bioavailability of to F-araATP (Sirotnak et al., 1983). F-AraATP inhibits 5-fluorouracil(Lamont and Schilsky,1999) and, in several enzymes involved in nucleoside synthesis and addition, offers a convenient oraladministration option. DNA replication: (i) DNA polymerase (Parker et al., Capecitabine is a prodrug that is metabolized by 1988; Huang et al., 1990), (ii) DNA primase (Catapano carboxylesterase to 50-deoxy-5-fluorocytidine after oral et al., 1991), (iii) ribonucleotide reductase (Parker et al., administration. 50-Deoxy-5-fluorocytidine is deaminated 1988) and (iv) DNA ligase I (Yang et al., 1992). In by cytidine deaminase to 50-deoxy-5-fluorouridine. nonreplicating cells, cytotoxicity of fludarabine (F- hENT1 mediates the uptake of 50-deoxy-5-fluorouridine araA) is due to inhibition of cellular DNA repair (Mackey et al., 2002). A metabolite of capecitabine, 50- processes (Sandoval et al., 1996). Other mechanisms of deoxy-5-fluorouridine monophosphate, inhibits thymi- action of F-araATP in noncycling cells include incor- dylate synthase and the 50-deoxy-5-fluorouridine tripho- poration into RNA leading to premature chain termina- sphate is incorporated into DNA (Schmoll et al., 1999). tion of RNA transcript and impairing cellular protein The last activation step is catalysed by thymidine synthesis (Huang and Plunkett, 1991). Clinically, phosphorylase, which converts 50-deoxy-5-fluoruridine Fludara is used to treat low-grade lymphomas and into 5-fluorouracil(Lamont and Schilsky, 1999). Thy- chronic lymphocytic leukemia (Chun et al., 1991) midine phosphorylase is highly expressed in tumor (Table 2). tissues and is associated with resistance to conventional Cladribine (2-CdA, 2-chloro-20-deoxyadenosine, 5-fluorouraciltreatment in severalgastrointestinaltu- Leustatins) is closely related to fludarabine. Cladribine mors, in particular colon cancer (Ishikawa et al., 1998). differs from fludarabine in that it has a chlorine Capecitabine has shown activity in metastatic colorectal substitution instead of fluorine at the 2 position of the cancer that is comparable to that of 5-fluorouracil adenine moiety. Cladribine, like fludarabine, is resistant combined with leucovorin (Hoff et al., 2001). Capecita- to deamination by . Cladribine bine has activity against metastatic breast cancer that enters cells via hENT1, hENT2 and hCNT3 (King, has progressed after or (Blum 1994; Ritzel et al., 2001b) and is converted into the et al., 1999; Talbot et al., 2002). active form 2-CdATP by the combined action of dCK Two purine nucleoside antimetabolite drugs, fludar- and cellular nucleotide kinases. The affinity of dCK for abine (9-b-d-arabinofuranosyl-2-fluoroadenine), which cladribine is 10-fold higher than that for fludarabine. is administered as the 50-monophosphate (F-araAMP, Cladribine has a similar mechanism of action to Fludaras), and cladribine (2-chloro-20-deoxyadenosine, fludarabine in that the 50-triphosphate inhibits DNA CdA, Leustatins) are cytotoxic to both dividing and replication and repair as well as ribonucleotide reduc- resting cells. One of the first purine nucleoside analogs, tase thereby reducing deoxyribonucleotide synthesis. 9-b-d-arabinofuranosyladenine (araA), was abandoned Exposure to cladribine is cytotoxic to dividing cells due to its poor solubility and rapid deamination by because of the inhibition of replicative DNA synthesis adenosine deaminase. Addition of a fluorine atom to the (Lassota et al., 1994) and to resting cells because of the adenine moiety to create fludarabine increased resis- inhibition of DNA repair processes (Pettitt et al., tance to adenosine deaminase and addition of a 1999a, b) and alteration of mitochondrial function or phosphate group improved the analog’s solubility and integrity (Genini et al., 2000). Cladribine has been gave rise to Fludara (F-araAMP) (Brockman et al., shown to be active in low-grade lymphomas (Dimopou- 1977; Danhauser et al., 1986). Before entering cells, los et al., 1994), chronic lymphocytic leukemia and hairy fludara is dephosphorylated by plasma phosphatases cell leukemia (Seymour et al., 1994).

Table 2 Nucleoside anticancer drug selectivity of NTs Anticancer nucleosides Transporter(s) Disease

Cytarabine hENT1 Acute myelogenous leukemia, non-Hodgkin’s lymphoma Gemcitabine hENT1/2 and hCNT1/3 Pancreatic cancer, metastatic bladder cancer, non-small-cell lung cancer 50-Deoxy-5-fluorouridinea hENT1 Metastatic colorectal cancer, metastatic breast cancer Fludarabine hENT1/hCNT3 Low-grade lymphoma, chronic lymphocytic leukemia Cladribine hENT1/2 and hCNT3 Low-grade lymphoma, hairy cell leukemia, chronic lymphocytic leukemia Troxacitabine Passive diffusion Acute myelogenous leukemia, pancreatic cancer hENT1/2 and hCNT2, hCNT3? Pediatric refractory or relapsed acute myelogenous leukemia, acute lymphocytic leukemia aMetabolite of capecitabine

Oncogene Transporters and resistance VL Damaraju et al 7530 Nucleoside analogs in development hCNT1 mRNA expression in tumors was more limited with expression observed only in tumors of kidney, Troxacitabine (Troxatyl; BCH-4556; ( )-20-deoxy-30- À uterine, lung and small intestine. Ovarian cancer oxacytidine) has unique properties in terms of its expressed more hCNT1 mRNA than normalovarian structure. An analog of deoxycytidine, troxacitabine tissue, whereas all other tumors had less hCNT1 mRNA exists as an l-enantiomer in contrast to the physiologic expression than the corresponding normaltissues. nucleosides and most nucleoside drugs, which are d- hCNT2 mRNA was expressed in kidney, breast, enantiomers. Cellular uptake of troxacitabine is mainly prostate, uterine, ovarian, colon, lung, stomach and due to passive diffusion (Gourdeau et al., 2001) and the rectalcancers and the majority of breast, prostate, presence or absence of NTs in tumor cells is unlikely to uterine, ovarian and lung cancers expressed more influence sensitivity to troxacitabine. Troxacitabine is hCNT2 mRNA than the corresponding normaltissues. phosphorylated by dCK (Grove et al., 1995). In contrast to most other cytidine analog drugs, troxacitabine is resistant to deoxycytidine deaminase (Grove and Cheng, 1996). The 50-triphosphate of troxacitabine is a good NTs and sensitivity to nucleoside analogs substrate for DNA polymerases but lacks the necessary hydroxyl group to allow chain elongation (Grove et al., In vitro studies have demonstrated that nucleoside 1995). Troxacitabine metabolites do not inhibit ribonu- transport is necessary for many nucleoside analogs to cleotide reductase, in contrast to gemcitabine metabo- enter cells. Mackey et al. (1998b) showed that gemcita- lites (Grove and Cheng, 1996). In phase I studies, bine required nucleoside transport to cause cytotoxicity troxacitabine had activity against renalcellcancer and in a study that compared gemcitabine cytotoxicity in cell primary unknown cancer (Belanger et al., 2002; lines with or without NT activity. Gemcitabine’s IC Townsley et al., 2003). Giles et al. (2001, 2003) observed 50 values (concentrations that inhibited proliferation by the activity of troxacitabine in patients with refractory 50%) were 118–3000-fold greater in cell lines that lacked leukemia. Troxacitabine is being tested as a potential nucleoside transport activity. A major role for hENT1 treatment for acute myeloid leukemia, pancreatic cancer in gemcitabine toxicity was demonstrated by addition of and solid tumors (Ecker, 2002). 0 0 dipyridamole, a potent inhibitor of hENT1 activity, to Clofarabine (Cl-FaraA, 2-chloro-9-(2 -deoxy-2 - culture media, which conferred resistance to gemcitabine fluoro-b-d-arabinofuranosyl) adenine, Clofarex) has by reducing the cellular uptake of gemcitabine. Mata activity against both epithelial and hematologic malig- et al. (2001) showed that hCNT1 was important for nancies. Like cladribine and fludarabine, substitution at cytotoxicity to capecitabine. 50-Deoxy-5-fluorouridine, position 2 of the base with a halogen confers resistance the active metabolite of capecitabine, is a permeant for to deamination (Xie and Plunkett, 1996). Substitution of hCNT1 and the presence of hCNT1 conferred sensitivity fluorine on the arabinosylsugar prevents degradation by of CHO-K1 cells to 50-deoxy-5-fluoruridine. Similarly, bacterial phosphorylase and allows oral administration Lang et al. (2001) showed that the presence of hCNT2 (Carson et al., 1992). Clofarabine enters cells via activity conferred sensitivity to fluoropyrimidine nucleo- hENT1, hENT2, hCNT2 and possibly also hCNT3 sides in CEM cells transfected with cDNA encoding (King et al., 2003). Clofarabine is metabolized to its hCNT2. mono-, di- and triphosphates by dCK and nucleotide Lu et al. (2002) performed a systematic analysis of NT kinases (Parker et al., 1999). Cl-F-araATP is incorpo- mRNA expression and cytotoxicity to common anti- rated into replicating DNA, which terminates chain metabolites such as gemcitabine, cytarabine, cladribine elongation (Parker et al., 1991), and inhibits ribonucleo- and fludarabine and did not find any significant tide reductase, which decreases intracellular deoxynu- relationship between cytotoxicity and NT mRNA cleotide pools (Parker et al., 1991). Clofarabine is expression. They did find relationships between NT undergoing phase II clinical trials for the treatment of mRNA expression and cytotoxicity of the analogs O6- pediatric refractory/relapsed acute myeloid and lym- methylguanine and hCNT1, tiazofurin and hCNT2, phocytic leukemia (Lindemalm et al., 2003). cyclopentenylcytosine and hydroxyurea and hENT2. Several factors may have complicated their analysis of the effects of NT mRNA expression on cytotoxicity: (i) Distribution of NTs in tumor tissues other enzymes such as 5-nucleotidase and cytidine deaminase are known to affect sensitivity to nucleoside Pennycooke et al. (2001) examined the expression of analogs and were not measured, (ii) many nucleoside hENT1 mRNA in tumors and found expression in the analogs are transported by more than one NT and (iii) kidney, breast, prostate, uterus, ovary, cervix, colon, there may not be a direct relationship between mRNA lung, stomach and rectum. Although many tumors expression and protein levels. expressed lower levels of hENT1 mRNA than the Galmarini et al. (2002) studied the effects of hENT1 corresponding normaltissues, breast, lung, stomach mRNA expression on efficacy of cytarabine treatment of and rectal cancers generally expressed higher levels of acute myelogenous leukemia. They retrospectively hENT1 mRNA than normaltissues. hENT2 mRNA studied the expression of dCK, 50-nucleotidase, DNA was expressed in the kidney, breast, prostate, colon and polymerase, topoisomerase I and topoisomerase II, and stomach cancers at higher levels than in normal tissues. hENT1 in blast cells of 123 patients who were treated

Oncogene Transporters and resistance VL Damaraju et al 7531 with cytarabine. Decreased expression of hENT1 was Drug resistance through gene redundancy associated with an increased risk of early relapse. Stam et al. (2003) studied 18 infants and 24 children with Multidrug resistance (MDR) proteins confer a resistant acute lymphoblastic leukemia to determine why infants phenotype to a broad spectrum of drugs used in cancer were sensitive to cytarabine. Leukemic blasts from chemotherapy (Wijnholds et al., 2000). These transpor- infants were threefold more sensitive to cytarabine than ters are broadly classified as ATP-binding cassette blasts from children (P ¼ 0.007). When mRNA levels of proteins (ABC) and constitute a superfamily of proteins dCK, cytidine deaminase, pyrimidine nucleotidase I, (Goldman, 2002). Methyl transferases, organic anion deoxycytidylate deaminase and hENT1 were measured, and cation transporters (OAT and OCT), oxidoreduc- decreased levels of dCK (P ¼ 0.026) but increased levels tase flavoproteins and NTs are major players in drug of hENT1 (P ¼ 0.001) were observed in infants whereas transport and metabolism in cells and may impart drug there were no differences in any of the other cytarabine- resistance phenotypes to cancer cells. Resistance to metabolizing enzymes between infants and children. It nucleoside analogs in tumors is thought to be a major was concluded that increased hENT1 abundance was problem in the clinic and several hypotheses have been responsible for increased sensitivity of infant acute proposed to account for altered NT activities. However, lymphocytic leukemia to cytarabine. regulation through germ line polymorphisms of ENT and CNT genes has not been studied. Redundancy at the level of substrate selectivity or specificity is common among the families of drug Genetic basis of drug resistance metabolism enzymes and transporters. Extensive gene duplication events may be responsible for the evolution Much of the current molecular understanding of the role of these gene families conferring a survival advantage to of transport in the efficacy of drugs has come from the species. As a result, conventional gene knockout or experiments using cell lines and animal models. How- transgenic approaches have limited value in deciphering ever, clinicians and patients are faced with uncertainties the role of these drug transporters in health and disease. that an administered drug may not produce the desired For instance, MDR1 knockout mice showed normal effects. This lack of predictability in the context of development, an indication that this gene product may therapeutic outcome has largely been attributed to serve a detoxifying role only when challenged with genetic variations in patient population. The human xenobiotic compounds (Illmer et al., 2002). It is there- genome across populations is largely identical (up to fore not surprising that transporters that are otherwise 99.9%) and much of the observed variability may lie in well characterized through cloning and expression the remaining genome, prompting large-scale attempts studies are yet to be defined for their role in disease to characterize these variations (Sachidanandam et al., etiology. The near ubiquitous expression of multiple 2001; Strohman, 2002). Over 90% of these variations transporters in various tissues further complicates reside in single nucleotide polymorphism (SNPs) and the genotype–phenotype correlations. Such complex and remaining may be insertions, deletions and short tandem multigene associations are commonly described for repeats. Insertion and deletion polymorphisms as well as polygenic diseases such as cancer, diabetes and cardiac SNPs in transporter proteins when present in coding or abnormalities (Sachidanandam et al., 2001). The im- regulatory regions may produce phenotypes that influ- plication of several transporters in conferring drug ence drug uptake. The mere abundance of SNPs resistance is analogous to the implication of several covering the entire genome over other classes of candidate genes contributing to a phenotype in poly- mutations described above are usefulfor fine mapping genic diseases. Therefore, drug resistance mechanisms the genome as these occur at a frequency of 500–1000 bp must be analysed in the context of the broad permeant (Brookes, 1999; Cargill et al., 1999; Damaraju et al., selectivities of various transporters. For instance, 2002). nucleoside analogs are transported not only by the While somatic mutations in transporters may explain ENT and CNT families but also by the OAT and OCT some of the variability in response to drugs between families (Chen and Nelson, 2000). MRP5, a member of individuals, the genetic basis of drug resistance can only the MDR protein family, has been shown to transport be understood by studying germ line mutations across nucleotide analogs. Inhibitors of OAT also inhibited the populations. Unlike somatic mutations, germ line MDR5 protein function (Wijnholds et al., 2000). variants are stable and heritable (Brookes, 1999). Huge efforts are underway to identify and characterize germ line mutations (most notably SNPs) in different ethnic populations. We provide here a summary of genetic Polymorphism analysis for drug transporters polymorphism analyses for NTs, a rapidly emerging area, and discuss membrane proteins that are potentially Extensive sequencing of drug transport genes in healthy implicated in the transport of nucleosides to address Japanese populations identified germ line variations that drug resistance in tumors. The drug resistance pheno- could potentially explain differences in drug efficacies in type may arise as a result of over- or under-representa- the context of this ethnic group (Iida et al., 2001; tion of transporters, through tissue specific regulation of Hamajima et al., 2002). Detailed experimental evidence expression (Goldman, 2002). linking these polymorphisms to differential drug

Oncogene Transporters and resistance VL Damaraju et al 7532 therapies is awaited. This group has deposited over 350 linking hENT loci to any disease phenotype. The SNPs in genes encoding OAT 1, 2 and 3 and in the genes complexity of the observed expression of NTs in several of the ABC family of transporters in the databases. Over tissues has been ascribed to splice variants of the major 90% of these SNPs were novel, compared to existing transporters, in particular hENT2 (Sankar et al., 2002). SNPs in the dbSNP database (NCBI, NationalCenter It remains to be established if alternative splicing for Biotechnology Information at http://www.ncbi.nlm. of hENT transcripts is due to exposure of cryptic nih.gov/SNP/index.html). SNPs deposited in databases, splice sites as a result of polymorphisms in genomic often from sequence alignments with the expressed DNA. sequence tags (EST) database or from sequencing a NT gene regulation at the transcriptional and post- small sample of chromosomes, may require further transcriptional level is much less studied due to lack of validation by extensive sequencing. Also, a significant information about promoter and enhancer elements as number of chromosomes across different racialgroups well as 30 noncoding regions. Recent work that defined need to be sequenced to identify true polymorphisms or these regions will stimulate further studies on the to detect and correct sequencing errors inherent in the regulation of NTs (Sankar et al., 2002). NT gene initialscreens for SNPs. These qualitycontrolmeasures regulation studies would corroborate earlier findings are essentialprior to routine monitoring of SNPs in on the differentialtissue abundance of NTs in normal affected individuals for comparisons of therapeutic and tumor tissues (Clarke et al., 2002). Tissue-specific efficacy. The OAT and ABC family SNPs were expression of CNT and ENT proteins may also be predominantly in intronic and regulatory regions, and modulated by p53 (Acimovic and Coe, 2002). A simple those that were exonic showed amino-acid changes (Iida approach to correlate the expressed NT proteins to drug et al., 2001; Hamajima et al., 2002). The high occurrence sensitivity or resistance in human cell lines was of SNPs in intronic and regulatory regions needs to be inconclusive, suggesting that NT expression and activity correlated with alternatively spliced transcripts and/or are influenced by other genes in a tissue-specific manner with the levels of expression in tissues to imply a role in (Acimovic and Coe, 2002). disease. Studies on MDR1 gene polymorphisms on the expression of P-glycoprotein and effects of therapy outcome in acute myeloid leukemia patients were reported recently (Illmer et al., 2002). Coding region Polymorphisms in NT genes SNPs in MDR1 gene in exons 12, 21 and 26 were correlated with decreased or increased levels of protein The Human Membrane Transporter Database (HMTD, expression in homozygous wild-type and the hetero- http://128.218.208.23/transporter/trans.html) has infor- zygous variants, providing a clear example of genotype– mation on polymorphisms and tissue-specific expression phenotype associations. Evidence was presented for of severaltransporters. Tissue-specific expression of linkage disequilibrium of the SNPs in the 12, 21 and 26 hENT and hCNT proteins was found in this database, exons, indicating that combined polymorphisms at these whereas data on polymorphisms for these genes were sites could affect the regulation of MDR1 expression missing. A search for SNPs for these genes in the NCBI (Illmer et al., 2002). database revealed SNPs for hENT1 and hENT2 on chromosomes 6 and 11, respectively; a total of 24 SNPs were found, 12 in the mRNA-untranslated region and the remaining in introns. These putative hENT SNPs Comparative genomic studies on NTs need to be validated by sequencing methodology using a large number of chromosomes. Recently, two coding A recent comprehensive comparative genetic analysis of region variants in hENT1 gene were identified in an NT family members has led to the identification of the ethnically diverse DNA samples, and functional char- precise chromosomallocationsfor the genes encoding acterization of these variants in S. cerevisiae indicated hENT1, hENT2 and hENT3 on chromosomes 6, 11 and similar kinetics of uptake of nucleosides and nucleoside 10, respectively (Coe et al., 1997; Sankar et al., 2002). analogs as compared to the native hENT1 (Osato et al., The promoter and other regulatory regions have 2003). Site-directed mutagenesis of the conserved glycine recently been defined for these genes (Sankar et al., in transmembrane domain 5 of hENT1 abolished 2002). hENT1 and hENT2 are believed to have transport and NBMPR-binding activities without af- originated through gene duplications, based on homol- fecting membrane targeting and assembly of the hENT1 ogy and structuralorganization of the genes, although protein (SenGupta et al., 2002). Comparative genomic they are located on two different chromosomes. On the analysis of ENT proteins across species also indicated other hand, the hENT3 gene is structurally very distinct that transmembrane domains 3–6 (exons 4–6) are highly from hENT1 and hENT2 (Sankar et al., 2002). Through conserved and are involved in binding and transport of comparative genomic studies, hENT4 has been identi- nucleosides (Sankar et al., 2002). Experimentalstudies fied on chromosome 7 and is believed to be evolutiona- with chimeras from ENTs of rats and humans have rily distinct and more ancient compared to hENTs 1–3 demonstrated the involvement of transmembrane do- (Acimovic and Coe, 2002). However, the OMIM (On- mains 3–6 in interactions with inhibitors and/or line Mendelian Inheritance in Man, http:// permeants (Sundaram et al., 1998, 2001b; Yao et al., www.ncbi.nlm.nih.gov/Omim/) database has no entries 2001, 2002).

Oncogene Transporters and resistance VL Damaraju et al 7533 A search for hCNT gene polymorphisms in the NCBI Future directions database indicated 61 SNPs in hCNT1, 2, and 3 genes in intronic and untranslated regions. Recently, Gray et al. SNP discovery combined with functionalcharacteriza- (2003) reported the first systematic and functional tion of hENT and hCNT gene families should help analysis of CNT1 gene polymorphisms. These authors address tissue-specific expression and inhibitor sensitiv- used denaturing HPLC and sequencing to identify ities in various tumor groups. Technologies are in place coding region SNPs in 247 DNA samples from now for investigating NT promoter sequence variants ethnically diverse populations. Functional analysis using high-throughput reporter gene expression. Appli- (binding and transport) of these variants was carried cation of genomic technologies by way of association out in X. laevis oocytes (Gray et al., 2003) and several studies to otherwise well-characterized NTs should for variants affecting CNT-mediated functions were identi- the first time offer clues on the role of these proteins in fied. These observations lend credence to the popularly health and disease. It would be interesting to see if one held belief that genetic variations may explain drug or more NT or other drug transporter gene polymorph- resistance mechanisms and could in principle account isms contribute to drug uptake and therapeutic efficien- for the clinically observed therapeutic efficiency of cies in an additive manner, as one would suppose for a nucleoside drugs in the affected populations. polygenic effect in a typical association study.

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