Nucleoside transport and its significance for anticancer drug resistance

Nucleoside transport and its significance for anticancer drug resistance

John R. Mackey,' Stephen A. Baldwin, 2 James D. Youngfl Carol E. Cass ~

'Department of Oncology, University of Alberta, and Cross Cancer Institute, Edmonton,Alberta, Canada;2School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK; 3Department of Physiology, University of Alberta, Edmonton, Canada

AbstractThis article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides.This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and non- nucleoside antimetabolite drugs are also presented.

importance of nucleoside anticancer therapy has :teased recently as a result of the introduction of

o_¢eral new nucleoside drugs into clinical use and the expansion of indications for anticancer nucleoside therapy into the arena of solid tumors. In parallel with these developments, there has been rapid progress in the understanding of nucleoside transport (NT) processes, leading to the cloning and functional expression of cDNAs encoding the four major NT proteins of mammalian cells. In this review, recent developments are highlighted in the molecular biology of NTs and the growing tmderstanding of the relationship of NT to the problem of resistance to nucleoside drugs. Evidence is summarized linking membrane transport processes to the cytotoxic selectivity of nucleoside drugs and the concept of the 'nucleoside transport profile' as a determinant of anticancer nucleoside activity is introduced. Strategies for increasing the therapeutic index of nucleoside anticancer drugs by manipulation of NTs processes to achieve "pharmacologic resistance" of dose-limiting normal tissues are outlined, and the preclinical and clinical experience with modulation of nucleoside salvage by inhibition of NT processes during antimetabolite therapy is reviewed.

NUCLEOSIDE TRANSPORT PROCESSES OF HUMAN CELLS

Seven functionally distinct NT processes have been described in human cells, of which four have been defined in molecular terms, through isolation and functional expression of cDNAs encoding the transporter proteins.l-6 The classification of NT activities is based on functional and pharmacological characteristics, including transport mechanisms (either

equilibrative or concentrative), permeant selectivities and sensitivity to nanomolar concentrations of nucleoside and non-nucleoside inhibitors. 7-1~ The concentrative NTs are inwardly directed sodium/nucleoside symporters, which are capable of moving nucleosides against the concentration gradient through coupled movement of sodium down. its transmembran.e electrochemical gradient. The equilibrative (bidirectional) transporters accept both purine and pyrimidine nucleosides as permeants and are found in most, possi- bly all, cell types, whereas the concentrative transporters have relatively narrow selectivities for nucleoside permeants and are limited to specialized cell types. Equllibrative transporters have been subdivided on the basis of sensitivit 3, to nitrobenzylthioinosine (NBMPR: nitrobenzylmercaptopurine ribonucleoside; 6-[(4 nitrobenzyl)thio]-9-B-D-ribufuranosyl purine); one subtype, termed 'equilibrative sensitive' (es) is inhibited by nanomolar concentrations of NBMPR, as a result of high-affinity (K d < 5 nM) binding of NBMPR at or near the permeant-binding site, whereas the other subtype, termed 'equllibrative insensitive' (et) is unaffected by low concentrations (< 1 btM) of NBMPR. Concentrative NTs comprise several functional subtypes that differ in their substrate selectivities and tissue distributions and, with some excep- tions, are unaffected by high concentrations (< 10 btM) of NBMPR. The chemical structure of NBMPR, together with those of the other classic inhibitors of equilibrative NT processes (dilazep, dipyridamole, draflazine), are shown in Figure 1.

Understanding of relationships among NTs has been greatly advanced by the recent isolation and functional expression of cDNAs encoding rat (r) and human (h) nucleoside transporter (NT) proteins.These NT proteins comprise two structurally unrelated protein families that are designated ENT and CNT, depending on whether they mediate, respectively, equilibrative (E) or concentrative (C) NT processes. The two protein families differ substantially in their architectural design, based on predictions of topologi- cal orientation and organization of the transporter proteins in membranes (Fig. 2). The mammalian ENT proteins identified thus far have approximately 450 amino acid residues 1.3,4,6,~3 and 11 predicted transmembrane domains, whereas the mammalian CNT proteins have approximately 650 amino acid residues and 14 predicted transmembrane domainsfl.5.1416 Members of the ENT family with functional characteristics of es-mediated transport processes are designated ENT1 and those with functional characteristics of el-

mediated transport processes are designated ENT2. Members of the CNT family with preference for pyrimidine nucleosides are designated CNT1 and those with preference for purine nucleosides are designated CNT2. In this nomencla- ture, the numbers represent the chronological order of dis- covery within a transporter subfamily, and a lower case letter precedes the transporter designation (e.g. hENT1, rENT1) to indicate the origin of the transporter protein (e.g. human, rat).

The e s t r a n s p o r t e r (hENT1) The first member of the ENT membrane protein superfamily, the human e quilibrative n_ucleoside transporter 0aENT1), was isolated from a human placental cDNA library by using

Drug Resistance Updates (I 998) I, 310-324 © 1998 Harcourt Brace & Co. Lid

Inhibitors of equilibrative nucleoside transport

o2 -iH2

HO~,~ H 0 H NBMPR

• ~N. N C CH2CIuI20H

N~ y "7" ~.~o.~o. HOCHxCH2% l [ l [

HOCH2Cn: ) Nt '~N 4 J ' ~ N

Dipytidamo/e

CH3Ox ~ OCH~

c H 3 ° ~ c ° ° ( c n 9 3 ~N,~_~N -'(CH2)~ OOc - @ O C H 3

CHfl>" ~.OCH3 Dil~p

H2 O I ~

C

Drntlazine

~ 2

"Z CONH(CH2)sCONH(CH2)2S~ H2 I

Pyrimidine anticancer nucleoside analogs NH2

O H O

SAENTA-tluorescein

NH2 Purine anticancer nucleoside analogs

HO O

H H

Cytarabine Cladribine

NH2

o:+ O

H F

Gemcitabine oH v

NH2

0 H HO

Fludarabine

Chemical structures of transport inhibitors and nucleoside analogs.

© 1998 Harcourt Brace & Co. Ltd Drug Resistance Updates (I 998) I, 310-324

CNT Family

N H 2 ~ C O O H

ENT Family

~ COOH

Fig. 2 The concentrative nucleoside transporter and equilibrative nucleoside transporter protein families.

amino acid sequence information obtained from N-terminal sequencing of the purified human erythrocyte es transporter. 1 The hENT1 cDNA encodes a 50-kDa protein, com- prised of 456 amino acids wi th 11 predicted t ransmembrane domains and three potential glycosylation sites of which one site lies in the predicted extracellular loop be tween transmembrane domains 1 and 2. The native human erythrocyte es transporter is known to be glycosylated near one of its ends. .7 hENT1 possesses a number of potential phosphorylation consensus sites, although it has not been determined if hENT1 is phosphorylated in vivo.The hENT1 gene has been localized to chromosome 6p21.1-p21.2) 8 The rat homologue (rENT1), which has 457 amino acids, also has 11 predicted transmembrane domains but contains three potential glycosylation sites in the loop be tween transmembrane domains 1 and 2. ~3 hENT1 and rENT1 are highly conserved, wi th 78% identity and 88% similarity at the amino acid level.

Recombinant hENT1 has been produced in Xenopus laevis oocytes m and shown to mediate transport of uridine (Km

value, 0.24 raM) (Table 1). Uridine transport in hENTl-containing oocytes is inhibited by physiological purine and pyrimidine nucleosides (but not by uracil) and by cladribine, cTtarabine, fludarabine and gemcitabine (for drug structures, see Fig. 1). Uridine transport is also inhibited by NBMPR (ICs0 value, 3.6 nM) and by the vasoactive drugs, dipyridamole, dilazep and draflazine (for structures, see Fig. 1). Chimeric studies in which various t ransmembrane domains of hENT1 and rENT1 have been interchanged have established that dipyridamole and dilazep (and thus, probably, also NBMPR) bind to a region of hENT1 be tween transmembrane domains 3 and 6. ~9 The tissue distribution of hENT1 is broad, suggesting that the transporter may be ubiquitous; hENT1 transcripts have been observed in several human cancer cell lines probed with the bENT1 cDNA 2° and expressed sequence tags wi th identity to hENT1 from many different normal and neoplastic human tissues have been registered in the genomic data bases.

The e i transporter (hENT2) A second human equilibrative nucleoside transporter (hENT2) with ei-type activity has been identified by isolation

and functional expression in Xenopus oocytes of a hENT1- related cDNA from human placenta. ~ hENT2, a 50-kDa protein, consists of 456 amino acids and is 49% identical and 69% similar to hENT1, hENT2 also has 11 predicted transmembrane domains.Two of its three potential glycosylation sites are on the predicted extracellular loop be tween transmembrane domains 1 and 2. An identical transporter was identified in cultured HeLa ceils by functional expression cloning of a HeLa cDNA in a nucleoside-transport deficient leukemia cell line. 3 A related rat homolog (rENT2), which was isolated from a rat jejunal cDNA library, 13 has 456 amino acids, 11 predicted transmembrane domains and two potential glycosylation sites in the extracellular loop be tween transmembrane domains 1 and 2. bENT2 and rENT2 are 93% similar and 88% identical at the amino acid level.

Recombinant hENT2 has been functionally characterized in Xenopus oocytes 4 and in a NT-defective line of cultured human leukemia cells. 3 The recombinant protein 's insensitiv- ity to inhibition by NBMPR and relatively broad permeant selectivity led to the functional classification of bENT2 as an ei-type transporter. In oocytes, bENT2 exhibits a K m value for uridine transport of 0.20 mM (Table 1). Studies undertaken to identify potential permeants and/or inhibitors, in which 1 mM test compound was assessed for its ability to block inward transport of 10 btM 3H-uridine,3 demonstrated complete inhibition of transport wi th adenosine, inosine, or thymidine, partial inhibition wi th guanosine, cytidine or hypoxanthine, and no inhibition wi th adenine or uracil. Recombinant hENT2 is inhibited by dipyridamole, dilazep and draflazine at 1 btM? ,4 hENT2-encoded mRNA transcripts have been observed in a variety of tissues, including brain, heart, lung, thymus, prostate, pancreas and skeletal muscle. 3 The level of expression of bENT2 mRNA is greatest in skeletal muscle, raising the possibility of differential expression of related isoforms?

Table I Kinetic parameters of nucleoside influx by recombinant nucleoside transporters

NT Substrate K m~p Vm~ × (pmol/ Vrnax:Kmapp Reference

(~M) oocyte.min -~)

hENT I Uridine 240 3.6 0.015 I

rENTI Uridine 150 18 0.120 13

hENT2 Uridine 200 6.4 0.032 4

rENT2 Uridine 300 14 0.047 13

hCNT I Uridine 45 26 0.58 2

rCNT I Uridine 37 21 0.57 16

Adenosine 26 0.07 0.003 14

hCNT2 Uridine 40 0.74 0.02 6

Adenosine 8 0.49 0.06 6

hSPNTI Uridine 80 0.53 0.0007 5

Inosine 5 0.19 0.04 5

rCNT2 Uridine 31 0.86 0.03 6

Adenosine 14 0.90 0.06 6

SPNT Adenosine 6 0.46 0.08 15

Drug Resistance Updates (I 998) I, 310-324 © 1998 Harcourt Brace & Co. Ltd

T h e cit t r a n s p o r t e r (hCNT1) The NT process designated as 'cit' was first described in freshly isolated mouse intestinal epithelial cells as a process that is -concentrative, _insensitive to inhibition by NBMPR and capable of transporting _thymidine. 2' NT processes wi th cit- type activity exhibit a preference for pyrimidine nucleosides and, because they are inhibited by low concentrat ions of adenosine, were originally believed to also mediate transport of adenosine. TM The proteins responsible for the cit process were identified w h e n cDNA encoding a pyrimidine nucleoside-selective, sodium-dependent (i.e. concentrative) trans- por ter was isolated from rat jejunum by functional expression cloning in Xenopus oocytes. 1622 The human homologue of rCNT1 was subsequently identified by hybridization/RT-PCR (reverse transcriptase polymerase chain reaction) cloning and functional expression of two almost identical cDNAs from human kidney.2The two human cDNAs encode the same protein (of 649 and 650 amino acids) te rmed hCNT1 that exhibits cit-type NT activity w h e n produced in Xenopus oocytes, hCNT1 has 83% identity wi th rCNT1 in amino ~tcid sequence, encoding a 71-kDa protein that is predicted to possess 14 t ransmembrane domains.The hCNT1 gene maps to chromosome 15q25-26. Recombinant hCNT1 produced in Xenopus oocytes exhibits cit-type activity, has a K m for uridine uptake of 42 btM (Table 1), mediates uptake of zidovudine (AZT) and zalcitabine (ddC), and is inhibited by adenosine, thymidine, cytidine and uridine, but not by guanosine or inosine. Direct measurements of the kinetics of adenosine uptake by recombinant hCNT1 in Xenopus oocytes have shown that adenosine binds wi th high affinity to the transporter, presmnably at the permeant- binding site, yet is t ransported at very low rates (Table 1). Since physiologic adenosine concentrat ions rarely exceed 5-10 ~tM, it is possible that adenosine regulates hCNT1 activity in vivo, acting to inhibit transport of pyrimidine nucleosides (e.g. uridine).

T h e c / f t r a n s p o r t e r (hCNT2) The NT process designated as ' c / f was first described in freshly isolated mouse intestinal epithelial cells 21 as a process that is sodium-dependent, insensitive to inhibition by NBMPR and capable of transporting formycin B (the C-nucleoside analog of inosine). NT processes wi th c/fltype activity were subsequently shown to be concentrat ive and capable of transporting a variety of purine nucleosides and uridine. TM

cDNAs encoding c ~ t y p e transporters were first isolated from rat liver by functional expression cloning in Xenopus oocytes 's and subsequently by RT-PCR from rat jejunum. ~4 The open reading frames of the liver and intestinal cDNAs predicted an identical 72-kDa protein, te rmed SPNT is and rCNT2.'4 The transporter protein, hereafter te rmed rCNT2, is predicted to have 659 amino acids wi th 14 t ransmembrane domains and is 64% identical to rCNT1, wi th the greatest divergence in the N- and C-terminal portions, cDNAs encoding a human homolog (hSPNT1 or hCNT2) were isolated from kidney s and intestine 6 by hybridization cloning/RT-PCR amplification. The human homolog, hereafter te rmed hCNT2, has 658 amino acids and, like the CNT1 proteins, is predicted to possess 14 t ransmembrane domains. Northern analyses suggest expression in a variety of human tissues,

including heart, liver, skeletal muscle, kidney, intestine, pancreas, placenta, brain and lung.The hCNT2 (or hSPNT1) gene is located on chromosome 15. 5,6

Recombinant hCNT2 in Xenopus oocytes exhibits K m values of 5, 8 and 40 J, tM, respectively, for inward transport of inosine, adenosine and uridine (Table 1). Other hCNT2 permeants include 2 '-deoxyadenosine (adenosine > 2'- deoxyadenosine), guanosine and didanosine (ddD, but not thymidine, cytidine, uracil, zidovudine or zalcitabine. A c / f type NT process has been described in the acute promyelocytic NB4 leukemia cell line; 23 it mediates adenosine and uridine uptake with K m values, respectively, of 10 ~tM and 30 gM and thymidine is not accepted as a permeant.

O t h e r n u c l e o s i d e t r a n s p o r t ac t iv i t i es i n h u m a n ce l l s A N T process te rmed 'cib' that is concentrative, insensitive to NBMPR, and possessing _road permeant selectivity for both purine and pyrimidine nucleosides has been described in freshly isolated human leukemic blasts 24 and human colon cancer CaCo-2 cells. 25 The technical difficulties in functionally distinguishing be tween the various concentrat ive trans- por ter types w h e n multiple types are present in single tissues or cells, together wi th the low levels of cib-type activity, have limited the characterization of cib transport processes in human cells.The cib transporter protein has not been identified.

A sodium-dependent (i.e. concentrative) NBMPR-_sensitive NT process wi th selectivity for guanosine and thus terned 'csg' has recently been repor ted in NB4 acute promyelocytic leukemia cells. 26 Although 2'-deoxyguanosine inhibits the uptake of radiolabelled guanosine by approximately 50%, it is unknown whe the r csg mediates transport of both guanosine and 2'-deoxyguanosine, or whe the r 2 '-deoxyguanosine is a compet i t ive inhibitor of guanosine transport. Further study is required to define the relevance of the csg transport process to cancer chemotherapy, as this transporter has only been described in a single cell line, and its selectivity for anticancer nucleosides is not known.The protein responsible for mediating the csg process has not been identified.

A second sodium-dependent (i.e. concentrat ive) NBMPR- _sensitive NT process, te rmed 'cs', has been described in freshly isolated chronic lymphocytic leukemia cells and acute myelogenous leukemia (AML) cells.The cs transporter mediates cellular uptake of cladribine and fludarabine 27 and thus differs from the csg process, which does not accept adenosine (nor, presumably, adenosine analogs). The permeant selectivity of the cs process has not been fully characterized and the protein responsible has not been identified.

ROLE OF NUCLEOSIDE TRANSPORT IN CYTOTOXICITY AND ANTICANCER NUCLEOSIDE RESISTANCE

The cellular targets of nucleoside chemotherapy have been the focus of intense study, and mechanisms of action of the anticancer nucleosides include incorporat ion into DNA and RNA, DNA strand breakage and perturbat ion of intracellular nucleot ide pools. 28 Because the pharmacologic targets of most nucleoside analogs are intracellular, permeat ion through the plasma membrane is an obligatory first step in

© 1998 Harcourt Brace & Co. Ltd Drug Resistance Updates (I 998) I, 310-324 !

the manifestation of CytOtOXiCity. 7 However, as nucleoside analogs are generally hydrophllic, diffusion through the plasma membrane is slow, and transporter-mediated uptake is the major route of drug influx. Inefficient cellular uptake is, therefore, a potential mechanism of resistance to anticancer nucleosides. 25

In the discussion that follows, we have interpreted earlier studies of NT processes in the context of current knowledge of the molecular biology of NT proteins.Thus far, the molecular identities of the NT proteins responsible for mediating the es, el, cit and c/f processes in various cell types have proven to be members of the ENT or CNT protein families. In the examples given be low for which the molecular identity of the NT process has not yet been defined, the process is designated by its trivial name (e.g. es or et3 linked wi th the name of the presumptive NT protein (e.g. hENT1 or rCNT1). Thus, the NBMPR-sensitive equilibrative process of murine leukemia cells is te rmed ' es /mENTl ' and is a predicted protein, whose existence remains to be established. In addition, the transporter families (lENT vs CNT) to which the cib, cs

and csg proteins belong is unknown.

Transport-related resistance to nucleoside analogs: cultured cancer ce l l l i ne s The first NT-deficient cell line was the AE1 clone, generated from $49 murine T-cell lymphoma cells by chemical mutage- nization and subsequent selection in media containing toxic concentrat ions of adenosine. 29 31 AE1 cells exhibit greatly reduced uptake of physiological nucleosides and high-level resistance to cytotoxic nucleosides, including cytarabine, 5- fluoro-2'-deoxyaaridine, 5-fluorouridine 31 and gemcitabine. 32 AE1 cells lack es/mENTl-mediated transport activity and NBMPR-binding sites, suggesting a mutational loss of functional transporter protein. The first human transport-related resistant cell line, ARAC-SC, was obtained from a hypoxanthine-guanine phosphoribosyltransferase deficient clonal derivative of the human T-lymphoblast CCRF-CEM cell line. 33,34 The parental CCRF-CEM cells exhibit primarily hENTl-mediated transport activity. After chemical mutagenesis, cells were selected for cytarabine resistance.The result- ing ARAC-SC cells exhibit cross-resistance to a spect rum of cytotoxic nucleosides due to reduced uptake. 33'34 ARAC-8C cells, like AEI cells, appear to be resistant by virtue of an absence of functional hENT1 protein since they completely lack NBMPR-sensitive NT activity and high-affinity NBMPR- binding sites.

Transport-related resistance has also been observed in the absence of chemical mutagenesis. 33 Exposure of murine erythroleukemia cells to increasing concentrat ions of periodate- oxidized adenosine yielded cells wi th genetically stable high-level resistance and a 20-fold decrease in NBMPR-binding sites, suggesting a loss of the es/mENT1 transport process. In human HCT-8 colon cancer cells, 36 700-fold resistance to increasing concentrat ions of 5-fluoro-2'-deoxyuridine (FdUrd) was obtained and there was no measurable uptake of FdUrd or binding of NBMPR to FdUrd-resistant cells. Each of these studies demonstrates that the absence of the es process, presumably due to altered ENT1 product ion and/or function, confers high level-resistance to cytotoxic nucleosides.

Transport-related resistance to nucleoside analogs: c l i n i c a l evidence In vitro studies have demonstrated that NT-deficient cells are highly resistant to cytotoxic nucleosides. However, the importance of NT deficiency in clinical drug resistance is uncertain, in large part due to the difficulty of performing transport studies on malignant cells derived from clinical specimens, and the problems associated wi th quantifying NT proteins in malignant clones admixed with normal cells. Nonetheless, there is compell ing evidence that clinical resistance to cytarabine can be mediated by NT deficiency (reviewed below), although NT-based clinical resistance to o ther anticancer nucleosides has yet to be proven.

Transport of anticancer nucleoside drugs The anticancer nucleosides in routine clinical use (Fig. 1) include cytarabine (ara-C), cladribine (2-CdA), fludarabine (F- ara-A) and gemcitabine (dFdC). Deoxycoformycin has largely been supplanted by the arrival of the newer purine nucleoside analogs, and will not be discussed further in this review.

Cytarabine Cytarabine (ara-C, 1-[~-D-arabinofuranosyl cytosine) has a major role in the curative therapy of acute leukemia. Given as a single agent at standard doses, cytarabine induces hematological remissions in about 30% of patients withAML?7 A 7- day infusion of cytarabine in combinat ion with three daily doses of an anthracycline (doxorubicin or idarubicin) is the standard induction therapy for AML, and cytarabine is also a componen t of the consolidation, maintenance and intensifi- cation regimens. Combinations including cytarabine are also employed for the blastic phase of chronic myelogenous leukemia (CML) and for non-Hodgkin's lymphomas? 8

The incorporat ion of cytarabine into DNA is closely linked to its cytotoxic effect on leukemic cells. 39 DNA incorporat ion requires intracellular accumulation of cytarabine 5'- t r iphosphate (ara-CTP), which is the net result of several processes, including membrane transport, anabolism to ara- CTP and degradation of ara-CTP to inactive metaboli tes.The relationship be tween ara-CTP accumulation and treatment efficacy has been clearly demonstrated. The magnitude of radiolabelled cytarabine retention by bone mar row mononu- d e a r cells derived from AML patients correlates positively wi th clinical response to cytarabine-based induct ion chemotherapy. 4° In a study wi th AML patients treated with cytarabine and anthracycline, 41 freshly harvested myeloblasts were classified as 'high' o r ' l ow ' retent ion based on intracellular quantities of ara-CTP; the median duration of clinical remission, once attained, was two-fold greater in the high- retent ion group. Mediated inward transport of cytarabine is the major determinant of ara-CTP accumulation at low cytarahine concentrat ions (< 1 ~tM), whereas phosphorylation capacity predominates at concentrat ions above 10 ~tM;42 44 cytarahlne given during low or standard dose treatment regimens (e.g, 100-200 mg/m2/day) produces plasma levels be low 1 gM. 4~

The efficiency of cytarabine uptake by leukemic blast cells has been related to clinical ou tcome in AML patients receiving standard dose cytarabine. Nueleoside transport parameters were measured in blasts from 15 AML patients


Table 2 Kinetic parameters of nucleoside drug uptake

Drug Nucleoside Species transport process

Cell type Krn (~tM)

Vm~x Reference

Cytarabine es/hENT I Human

Fludarabine cif/mCNT2 Mouse Cladribine es/hENT I Human

Gemcitabine

AML blasts

LI210 CCRF-CEM

ei/hENT2 Human K562

c//TmCNT2 Murine MA27

255_+ I I

13.4 5.1

10.3

4.9

es/hENT I Human CCRF-CEM 329 _+ 91

T-cell lymphoblasts

ei/hENT2 Human HeLa + 832 _+ 204

100 nM NBMPR

cit/hCNT I Human hCNT I - 18.3 _+ 7.2 transfected HeLa cells

cif/hCNT2 Human hCNT2-transfected No measurable transport

0.84 _+ 0.44 49 (pmol/s/I 0 ~ cells)

57 1.43 62

(pmol/uL cell water/s)

0.50 62 (pmol/uL cell water/s) 0.29 62 (pmol/uL cell water/s) 17.0 + 4.3 32 (pmol/s/I 0' cells) 4.2 _+ 1.3 32 (pmol/s/I 06 cells) 0.94 _+ 0.22 32 (pmol/s/106 cells) N.A. 32

cib Human HeLa cells CaCo2 colon Rate insufficient Rate insufficient cancer cells + for accurate for accurate dilazep kinetics kinetics

32

treated with standard induction therapy containing cytarabine? 6 Of the seven patients who failed therapy, three exhibited the lowest rates of cytarabine uptake and NBMPR- binding-site numbers. Another report 47 described a patient with T-cell acute lymphoblastic leukemia in first relapse, whose blasts prior to therapy had (i) rapid eytarabine uptake and ara-CTP accumulation, and (ii) high numbers of NBMPR- binding sites. Mthough this patient's leukemia initially responded to standard doses of cytarabine, the NBMPR-binding-site number and ara-CTP accumulation rate had decreased by approximately 75% at the time of disease relapse, suggesting that cytarabine chemotherapy had selected for variant cells with reduced es/hENT1 activity.

In oilier studies of isolated leukemic blasts, ",48 the mean K and V values of cytarabine influx were approximately 260 ~tM, while the Vma ~ values of cytarabine influx varied 80-fold (Table 2). Approximately 80% of cytarabine entered leukemic blasts by the es/hENTl-mediated process over a wide range of cytarabine concentrations, and a positive correlation was shown between NBMPR-binding site numbers and cytarabine influx in both normal and leukemic leukocytes? ~ The abtm- dance of es/hENT1 in plasma membranes of leukemic blasts has also been determined by binding of SAENTA-fluorescein, an impermeant fluorescent analog of NBMPR, and is correlated with in vitro sensitivity to cytarabine. 49,5°

High-dose cytarabine regimens (e.g. 3 g/m2/day) produce remissions in some patients refractory to conventional-dose cytarabine and generate plasma levels above 50 ~tM, 51 thereby overcoming the rate-limiting effect of es/hENTl-mediated transport on formation of ara-CTP Two randomized studies have shown benefit to high-dose cytarabine-based induction by demonstrating that, although remission rates are similar to standard-dose therapy, superior disease-free survival rates are seen in the high-dose groups. 52,53

In summary, cytarabine uptake in human leukemic cells is mediated by the es/hENT1 process, and its abundance in plasma membranes is a determinant of clinical efficacy of standard dose cytarabine therapy in adult AML. Studies are in progress to more precisely determine cytarabine transportability by the other NT processes of human cells. Cytarabine appears to be a poor permeant for recombinant hCNT1 (the pyrimidine-nucleoside selective concentrative transporter) based on the high concentrations required to inhibit uridine transport in transiently transfected cultured cells (Coe I et al, unpublished results).

Fludarabine Fludarabine (F-ara-A, 9---D-arabinosyl-2-fluoroadenine, Fludara®) is the most active agent in the treatment of chronic lymphocytic leukemia, and also has activity in

© 1998 Harcourt Brace & Co. Ltd Drug Resistance Updates (1998) 1,310-324

indolent non-Hodgkin's lymphoma, prolymphocyt ic leukemia, and Waldenstr6m's macroglobulinemia. Fludara- bine has little solid tumor activity. 28 Myelosuppression is the dose-limiting toxicity and profound cell-mediated immuno- deficiency characterized by reduced CD4+ counts commonly results in opportunistic infections¢ 4 Following intravenous administration, fludarabine 5 '-monophosphate is rapidly dephosphorylated extracellularly to fludarabine. Fludarabine is transported into cells and is rephosphorylated by deoxycytidine kinase and thence to fludarabine 5'-triphosphate, which induces toxicity through incorporation into DNA and/or RNA and inhibition of ribonucleotide reductase, DNA polymerase-o~, DNA primase, and/or DNA ligase I. 28,5s

Cellular differences in mediated transport of fludarabine have been proposed as a mechanism of selectivity of fludarabine therapy, since there is less in vivo accumulation of fludarabine 5'-triphosphate in murine gastrointestinal mucosa and bone marrow compared to that in P388 murine leukemia cells, s~ Evidence support ing a role for differential uptake of fludarabine in its tissue selectivity was obtained in the L1210 murine leukemia cell line, 56 in which fludarabine accumulation was eight-fold more rapid than that observed in mouse intestinal crypt epithelial cells, and the influx step, rather than the subsequent phosphorylat ion step, was rate- limiting for routine gastrointestinal epithelial cells, s6

A kinetic study of fludarabine uptake by L1210 cells 57 found evidence for transport via two separate processes, wi th K m values, respectively, of 190 btM and 13 gM. However, because subsequent studies 58,59 revealed that L1210 cells possess three distinct NT processes (es, ei, cOO , it is likely that the repor ted low-affinity transport of fludarabine in L1210 cells was due to the combined operat ion of the es/mENT1

and ei/mENT2 processes and the high-affinity transport rep- resented the operat ion of the clf/mCNT26° transport process (Table 2). In lymphoblasts harvested from acute lymphoblastic leukemia (ALL) patients, fludarabine sensitivity in vitro correlated wi th es/hENT1 abundance in the plasma membrane, as determined by 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA) - f luorescein binding, s° In summary, fludarabine appears to be a substrate for es/hENT1, ei/hENT2 c/f/hCNT2 and cs ~7 and its tissue selectivity may be related to differences in abundance of functional NTs.

Cladribine Cladribine (CdA, 2-chloro-2'-deoxyadenosine, Leustatin®) is a purine nucleoside analog that is resistant to degradation by adenosine deaminase. Cladribine achieves durable complete remissions in the majority of patients wi th hairy cell leukemia, and short4ived remissions in chronic lymphocytic leukemia and non-Hodgkin's lymphoma; cladribine has minimal solid tumor activity. The major toxicity of standard doses is myelosuppression and cell-mediated immunosuppression. Cladribine, after permeat ion across the plasma membrane, is conver ted to the 5 ' -monophosphate by deoxycytidine kinase. Inhibition of DNA synthesis and repair, inhibition of r ibonuclotide reductase, and imbalances in deoxyribonu- cleotide pools contr ibute to cytotoxicity. 28

Cladribine uptake is evidently mediated by both es/hENT1 and ei/hENT2-mediated processes, as shown by

NBMPR and dipyridamole protect ion studies in cladribine- exposed human hematological cancer cell lines. 61 Transport kinetics of radiolabelled cladribine have been determined for es/hENT1, ei/hENT2, and ci f /mCNT2 in several leukemic cell lines, and the degree of cladribine cytotoxicity was directly correlated wi th the efficiency of transport 6~ (Table 2). In freshly harvested ALL lymphoblasts, in vitro sensitivity to cladribine correlated with es/hENT1 abundance in plasma membranes determined by SAENTA - f luorescein binding. 5°

Uptake of cladribine by the recombinant c~ type transporter of rats (rCNT2) has been demonstrated in the Xenopus oocyte system. TM Additionally, cladribine inhibited inosine uptake (IC50 13 btM) by recombinant rCNT2 in a HeLa cell expression system, t° However, the transportability of cladribine via recombinant hCNT2 has not yet been determined.

In summary, cladribine uptake in human ceils is mediated by es/hENT1, ei/hENT2 and cs, ~7 and inward transport of cladribine appears to be an important determinant of in vitro cytotoxicity. Studies to directly determine the transportability of cladribine by the recombinant human NT proteins are in progress.

Gemcitabine Gemcitabine (dFdC, 2',2'-dlfluorodeoxycytidine, Gemzar®) is a pyrimidine analog of deoxycytidine in which two fluorine atoms are present at the 2'-position of the deoxyribose ring (Fig. 1). The dosedimiting toxicity is myelosuppression. Gemcitabine is unique among nucleoside drugs in that it has considerable activity against solid tumors, including non-small cell lung, breast, bladder, ovarian, head and neck cancers2 8

Gemcitabine is first conver ted intracellularly to dFdCMP by deoxycytidine kinase, and subsequently phosphorylated to the 5'-diphosphate and 5'-triphosphate derivatives by pyrimidine monophospha te and diphosphate kinases. 63 dFdCDP inhibits r ibonucleotide reductase, while dFdCTP is incorporated into DNA and RNA. 64 Although the relative contributions of these effects to cytotoxicity are not known, gemcitabine exhibits the proper ty of self-potention in that dFdCTP inhibits deoxycytidine monophospha te deaminase, thereby decreasing its o w n catabolic degradation. 63 Plasma membrane transport of gemcitabine was initially studied in Chinese hamster ovary cells, where the rates of inward transport of gemcitabine and cytarabine were similar and not responsible for the observed differences in cytotoxicity of gemcitabine and cytarabine; rather, the differences were attributed to gemcitabine's higher affinity for deoxycytidine kinase and the longer intracellular retention of dFdCTP than ara-CTR 65

The transportability of gemcitabine, and its importance in gemcitabine cytotoxicity, has been examined in detail in studies wi th both native and recombinant NTs.32 A deficiency in NT, produced either pharmacologically or genetically, confers two- to three-log protect ion of cultured cells from gemcitabine cytotoxicity. Kinetic studies wi th a panel of murine and human cell lines wi th defined NT processes demonstrated that gemcitabine uptake is mediated by the es, el, cit and cib processes, but not by the c/f process (Table 2); in these studies, the molecular identity of the responsible trans- por ter proteins (i.e. hENT1, hENT2, hCNT1, hCNT2) was known for all but the cib-mediated process. The most

W Drug Resistance Updates (I 998) I, 310-324 © 1998 Harcourt Brace & Co. Ltd

efficient processes were those mediated by hENT1 and hCNT1, although the rates of gemcitabine transport were approximately 10-fold slower than those of uridine. The hCNT2/cif transporter did not accept gemcitabine as a permeant.

Strategies fo r m o d u l a t i o n o f nuc leos ide t r a n s p o r t p rocesses to e n h a n c e the rapeu t i c eff icacy Improvement in the therapeutic index of anticancer nucleosides, to achieve maximal ant±cancer activity with minimal toxicity to the patient, may be possible through manipula- tions based on understanding of NT processes. Clinical NT inhibitors are required for several of the strategies outlined below.At present, however, no ideal NT inhibitor is available for clinical use. NBMPR, the classic inhibitor of es/hENT1- mediated processes, produces significant hypotension when administered as the soluble prodrug NBMPR 5'-monophosphate (Venner PM, et al, unpublished results), and all pub- lished clinical trials attempting to exploit NT inhibitors have used dipyridamole. 6~-73 Dipyridamole, although having the advantage of inhibiting both es/hENTl and ei/hENT2 processes, has the disadvantage of non-specificity (inhibition of phosphodiesterase, stimulation of prostacyclin biosynthesis), and binding to plasma o~-acid glycoprotein and albumin, 74 which limits achievable free serum concentrations 75 even when given at maximally tolerated doses by continuous cen- tral intravenous infusion. 69 Draflazine appears to be well tolerated in the setting of acute ischemia for which it is being studied as a cardioprotective agent 76 and currently appears to be the most promising es/hENT1 transport inhibitor for further in vivo study. Like dipyridamole, draflazine is a less effec- tive inhibitor of ei/hENT2 than es/hENT1.4

Pharmacologic resistance by protection of normal tissues In the 'protection' strategy, transport inhibitors are used to induce pharmacologic resistance by reducing cellular uptake of nucleoside drugs by dose-limiting normal tissues, thereby increasing the maximum tolerated drug dosage. Proof of principle has been demonstrated in studies with rodents in which NBMPR 5'-monophosphate (NBMPR-P) reduced the toxicity of tubercidin (7-deazaadenosine, 4-

77 amino-7-(~-D-ribofuranosyl)pyrrolo [2,3-d] pyrimidine) and nebularine (9-[3-D-ribofuranosylpurine).V7.7a In L1 2 1 0 leukemia-bearing mice, NBMPR-P increased the therapeutic index of both nebularine 79 and flndarabine. 8° BIBW22BS, (4- [N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis(cis -2,6-dimethyl-morpholino)-6-phenylpteridine), which is a potent inhibitor of equilibrative NT processes, did not impair gemcitabine-induced growth inhibition of human tumor xenografts grown subcutaneously in nude mice, but slightly reduced therapy associated weight loss. 8. The efficacy of such protection strategies may be due, in part, to higher levels of es/ENT1 activity in proliferating malignant cells relative to their normal counterparts (Table 3). Human breast, liver, stomach and colorectal tumor tissues have more NBMPR-binding sites than the adjacent normal tissues, 82. and mitotic rates correlate with NBMPR-binding site numbers in human thymocytes 83 and human myeloblasts, s4 Thus, the transport inhibitors appear to reduce normal tissue drug influx below a threshold for cytotoxicity, while leaving

Table 3 Numbers of NBMPR-binding sites on representative human cells

Cell type Binding sites/cell Reference

Erythrocyte II 000 122 Bone marrow 21 000 85

mononuclear cells Peripheral blood I 123 _+ 553 83

lymphomcytes I 120 ± 610 46 Unstimulated peripheral 2203 83

blood lymphocytes Mitogen stimulated 68 024 83

peripheral blood

lymphocytes Normal CD5+/CD 19+ 553 _+ 178 100

lymphocytes Chronic lymphocytic 485 _+ 425 100

leukemia cells 1430 _+ 730 84 Bone marrow plasma 997 _+ 1096 92

cells from normal

donors Normal blood 2270 _+ 690 46

polymorphonuclear cells Leukemic blasts 7490 + 8080 123 Acute lymphocytic 2300-7400 46

leukemic blasts 3100 _+ 940 48

Acute myelogenous 3800-24 200 84 leukmic blasts

Thymocytes 26 068 ± 8776 83 Bone marrow plasma 1777 _+ 2181 92

cells from patients with myeloma

CI 80-13S 232 000 -+ 70 800 124 undifferentiated ovarian cancer

RC2a 100 780 ± 19 700 124

myelomonocytic leukemic blasts

HL-60 promyelocytic 170 000 85 leukemia

CCRF-CEM T 330 000 85 lymphoblast leukemia

N84 promyelocytic 535 000 23

leukemia BeWo choriocarcinoma 2 7000 000 125 MCF-7 breast cancer 245 000 126 HeLa cervical 150 000 127

carcinoma 410 000 125

malignant cells with sufficient transport capability to achieve cytotoxic intracellular drug concentrations.

Pharmacologic resistance to cytotoxic nucleosides has been achieved with human hematopoietic progenitors by treatment with transport inhibitors, suggesting that combinations of equilibrative NT inhibitors and ant±cancer nucleosides may be capable of selectively targeting malignant cells possessing inhibitor-insensitive NT activities, while reducing myelotoxicity. Progenitor cells derived from normal human

© 1998 Harcourt Brace & Co. Ltd Drug Resistance Updates (1998) 1,310-324

bone marrow were spared from in vitro tubercidin toxicity by pretreatment with transport-inhibitory concentrations of NBMPR, dilazep or dipyridamole and tolerated in vitro exposures to relatively high concentrations of the transport inhibitors (5-1 0 ~tM) without loss of viability. 85 Other studies have confirmed that hematopoietic progenitor cells possess predominantly es/hENT 1 activity. 86

In summary, inhibitors of equllibrative NT processes, most likely mediated by the ENT1 and ENT2 proteins, are capable of providing pharmacologic resistance to some normal tissues while permitting cytotoxicity to malignant cells. Pursuit of such strategies in clinical trials will require effec- tive and well-tolerated transport inhibitors.

Nucleoside trapping Cells that possess both equJlibrative and concentrative NT processes may be rendered more sensitive, rather than resistant, to nucleoside drugs by the loss of equllibrative transport capability. For example, dipyridamole enhances the cytotoxicity of ara-A in L1210/C2 c e l l s , 87 which have both eqnilibrative NT processes and the purine-selective concentrative process. 59 Although dipyridamole reduces initial rates of uptake of ara-A, the subsequent intracellular accumulation of ara-A is enhanced because of the almost complete inhibition of ara-A efflux (via es/mENT1 and ei/mENT2) while inward transport of ara-A via the sodium-dependent concentrative transporter continues. Ara-A cytotoxicity is signifi- candy enhanced in cells so treated. Nucleoside 'trapping' by this strategy could be exploited to increase the therapeutic index of anticancer nucleoside drugs in human cancers if (i) the malignant cells possess either cit/hCNT1 (sensitization to pyrimidine nucleoside analogs) or c/f/hCNT2 (sensitization to purine nucleoside analogs) transporters, and (ii) the dose-limiting normal tissues (in which the CNT proteins are presumed to be absent or non-functional) can be rendered pharmacologically resistant. The trapping strategy failed in a recent in vitro study with gemcitabine; 32 dipyridamole treatment of cultured human CaCo-2 cells, which possess both equllibrative and concentrative NT processes, had no effect on gemcitabine sensitivity.

A trapping strategy that requires only equilibrative transporters has also been explored, ss After short exposures to cytarabine, cultured ovarian carcinoma and promyelocytic leukemia cells that were subsequently incubated continu- ously with dipyridamole, which blocked outward transport via the equilibrative processes, experienced a lO-fold augmentation of cytotoxicity. Cladrihine may also be amenable to trapping strategies. Levels of cladribine phosphates present in human leukemic cells declined rapidly when the cells were in cladribine-free mediumfl 9 and in freshly harvested chronic lymphocytic leukemia (CLL) cells, NBMPR significantly decreased the rate of cladribine efflux, thereby increasing the drug content of NBMPR-treated cells over untreated control cells? °

The nuc leos ide t r a n s p o r t prof 'de and drug schedule The 'nucleoside transport profile' is the description of the numbers and types of functional NT proteins present in the plasma membrane of the cells or tissues of interest.There are marked differences in the kinetics of transport of anticancer

nucleoside analogs by the human NTs (Table 2), suggesting that it may be possible to rationally design chemotherapeutic schedules that take advantage of these differences. Were a given malignancy found to exhibit predominantly CNT proteins, one might target malignant cells by low-dose long-duration exposure to a toxic nucleoside for which the transporter in question has high selectivity. Variation in the tissue distribution of NT proteins may contribute to the differing toxicity profiles of continuous versus bolus infusions of gemcitabine, 91 cladribine and fludarabine, z8

Selective increases in nucleoside transport in cancer cells Mthough regulation of equilibrative NT processes is not well understood, it is clear that the number of es/ENT1 proteins (as determined by NBMPR-binding studies) varies with proliferation rate, and in response to cytotoxic and mitogenic stimuli. High cellular proliferation rates are associated with high levels of es/hENTl-mediated activity in human thymocytes, 83 leukemic blasts ~4 and myeloma cells? 2 These observations suggest that proliferative stimulation of malignant cells will increase nucleoside drug uptake, thereby sensitizing oth- erwise resistant cells. Exposure of routine S 1 macrophages to colony stimulating factor-1 (CSF-1) stimulated adenosine uptake by increasing es/mENTl-mediated transport at the time of entry of the cells into the S phase of the cell cyclefl 3 while treatment of freshly isolated human myeloid leukemic blasts with granulocyte-macrophage CSF (GM-CSF) resulted in a four-fold increase in es/hENT1 abundance. 94 Unfor- tunately, the strategy of GM-CSF priming was shown to be ineffective in three randomized trials of treatment of AML with cytarabinc 95-97 as were attempts to use granulocyte CSF (G-CSF) to increase leukemia cell proliferation, thereby increasing cell sensitivity to cytarabine. 98,99

Nucleoside analog therapy itself may increase the number of NBMPR-binding sites, thereby acting in a self-potentiating fashion.Treatment of freshly isolated CLL ceils with fludarabine or cladribine led to a five-fold increase in the binding of SAENTA-fluorescein as well as increases in proliferation and apoptotic rates. ~°8 Additionaily, induction of sodium-dependent NT processes has been demonstrated by agents that promote cellular differentiation, For example, in the human promyelocytic leukemia HL-60 line, chemical induction of differentiation ~°~-1°4 leads to increased rates of sodium-dependent nucleoside transport and decreased rates of NBMPR- sensitive nucleoside transport as well as decreased numbers of high-affinity NBMPR - binding sites.

Gene therapy strategies The application of gene therapy to the treatment of cancer has been limited by several technical hurdles, including the limited transfection efficiencies achievable in vivo.To circum- vent this problem, those strategies that have a significant 'bystander effect' are preferred. 1°5 For example, introduction of viral thymidine kinase followed by gancyclovir therapy leads to cytoxocity in transfected cells, I°6 and the release of activated (i.e. phosphorylated) gancyclovir during cell lysis is toxic to neighboring non-transfected cells. Increased accumulation of gancyclovir within a transfected cell would therefore augment the bystander effect. Since gancyclovir requires


mediated uptake (both by nucleoside and nucleobase transporters1°7), introduction of a gene encoding a transporter wi th high affinity for gancyclovir together wi th the viral thymidine kinase gene might increase cytotoxicity.

Relationship between multidrug-resistant phenotypes and nucleoside transport Recent studies suggest that multidrug-resistant cell lines have increased nucleoside uptake capabilities. MCF-7 human breast cancer cells with different levels of P-glycoprotein-mediated multidrug resistance also exhibited different apparent K m and V a ~ values of adenosine transport, and the increase inVm= correlated with an increase in NBMPR binding, a°8 The influx of cytarabine, and its subsequent accumulation as intracellular ara-CTE, was higher in a P-glycoprotein-containing subllne than in the parental P388 murine leukemia cells, due to an increase in NBMPR-insensitive uptake in the multidrug-resistant cells. 1°9 P-glycoprotein-containing and multidrug resistance protein (MRP)-containing cell lines, although resistant to P-glycoprotein and MRP substrates, are significantly more sensitive to gemcitabine than their parental cell linesY ° Although the mechanism(s) tmderlying these observations are unknown, these studies suggest that cytotoxic nucleosides may selectively target multidrug-resistant cells.

ROLE OF NUCLEOSIDE TRANSPORT IN RESISTANCE TO NON-NUCLEOSIDE ANTIMETABOLITES

Nucleoside salvage pathways are an important means of circumventing antimetabolite drug action. 11~-113 Although pur ine and pyrimidine nucleotides are essential for numer- ous biological processes, they can be synthesized endoge- nously via de novo synthetic pathways. However, some tissues lack these pathways and require salvage of exogenous nucleosides to mee t their metabolic requirements. The relative contributions of de novo and salvage pathways vary be tween different tissues, and in some normal tissues (e.g. hematopoiet ic and intestinal epithelial cells), the rate of salvage synthesis exceeds the rate of de novo synthesis.

The non-nucleoside antimetabolite drugs in clinical anticancer therapy inhibit de novo synthesis of purine and/or pyrimidine nucleotides. Because salvage of nucleosides and nucleobases is enhanced in many human tumors, the salvage pathways can be utilized by cancer cells to replenish intracellular nucleotide pools, thereby overcoming antimetabolite cytotoxicity. TM Cells therefore become resistant to the cytotoxici ty of the antimetabolite drug in quest ion if they have access to substrates of the salvage synthesis pathways (nucleobases such as hypoxanthine or nucleosides), or to components of the de novo pathway distal to the inhibitory block.

Nucleic acid precursors are present in plasma and extracellular fluid, and participate in salvage synthesis pathways by means of functional nucleoside and nucleobase transporters, lnhihitors of equilibrative NT processes have been explored in both preclinical models and clinical trials as modulators of resistance to antimetabolite drugs. Dipyridamole has been most extensively studied, because of its ability to inhibit es/ENT1 and ei/ENT2-mediated processes, as well as nueleobase transport, ~1~,'6 although

other po ten t inhibitors of equilibrative transport (e.g. NBMPR, dilazep, draflazine) also have potential applications as modulators of salvage-dependent resistance to antimetabolites.

5 -F luo ro t t r ac i l Dipyridamole modulat ion of 5-fluorouracil (5-FU) cytotoxicity has been previously reviewed. 117 5-FU treatment leads to inhibition of de novo product ion of dTMP and cytotoxicity by interference wi th DNA synthesis and repair. However, NT- mediated uptake of preformed thymidine can bypass dTMP depletion, thereby conferring resistance to 5-FU. Such resistance can be c i rcumvented in vitro by blocking salvage of exogenous thymidine - e.g. cancer cell lines exposed to 5-FU and inhibitors of eqnilibrative NTs (dipyridamole and NBMPR) show increased 5-fluoro-2'-deoxyuridine 5'- monophospha te (FdUMP) synthesis, decreased FdUrd efflux, prolonged intracelltflar half-life of FdUMP, and greater cytotoxicity compared to controls exposed to 5-FU alone. "7,1~s

Two clinical trials in which 5-FU and dipyridamole were given by concurrent continuous intravenous infusion failed to modulate the clinical toxicity of 5-FU, and clinical responses were rare. 66,~7 Although the strategy of dipyridamole modulat ion of 5-FU toxicity has been abandoned (in part due to the previously discussed pharmacologic limita~ tions of dipyridamole), clinical inhibitors of ENT proteins that are bet ter tolerated and more poten t might warrant further investigation. One such candidate inhibitor, the dipyridamole derivative BIBW22BS, increased the antiproliferative effects of 5-FU two- to six-fold in colon cancer cell lines, but did not affect 5-FU-dependent growth inhibition in human tumor xenografts grown subcutaneously in nude mice. 81

N-phosphonacetyl-L-aspartate N-phosphonacetyl-L-aspartate (PALA), a transition state inhibitor of aspartate transcarbamylase, blocks de novo pyrimidine biosynthesis. ~19 Despite promising preclinical activity, PALA has generally demonstrated little efficacy as a single agent in human cancer.Trials of PALA combined with oral 72 or intravenous dipyridanlole 73 failed to improve anticancer efficacy, although gastrointestinal toxicity was higher than with PALA monotherapy. Further study of NT modulation of PALA toxicity does not seem warranted.

The antifolate drugs Methotrexate and tr imetrexate inhibit dihydrofolate reductase and lead to impaired de novo synthesis of purines and pyrimidine nucleotides. Doseqimiting toxicities are myelo- suppresion and mucositis. Resistance of cancer cells to antifolate cytotoxicity can arise through increased capacity for salvage of nucleosides and/or nucleobases; such resistance can be c i rcumvented by inhibition of cellular uptake. For example, dipyridamole potentiates the growth-inhibitory effects of methotrexate against HCT 1 16 human colon cancer cells through inhibition of thymidine transport.lz° Similar results are seen in routine sarcoma cells 1~1 and in human embryo fibroblast cells, 112 where dipyridamole increases sensitivity to methotrexate by approximately 10-fold (an effect which can be reversed by excess hypoxanthine plus thymidine).

© 1998 Harcourt Brace & Co. Ltd Drug Resistance Updates (I 998) I, 310-324

Unfortunately, dipyridamole also increases methotrcxate toxicity to normal cells. 7° Methotrexate toxicity (moderate to severe myelosuppression and/or mucositis) was greater in patients treated with a bolus injection of methotrexate 24 h after initiation of a high-dose dipyridamole infusion than in patients treated wi th methotrexate alone. Pharmacokinetic studies showed no change in methotrexate elimination. Dipyridamole potentiat ion of methotrexate toxicity appears to require high-dose dipyridamole (given by continuous infusion), since modulat ion of methotrexate toxicity was not observed wi th oral dipyridamole and methotrexate therapy. 71

The myelosuppression induced by antifolates is largely due to toxicity to cells in late stages of hematopoietic develop- ment, while early myeloid progenitor ceils are relatively resistant. In a study of the role of nucleoside salvage in protecting hematopoiet ic stem cells from the cytotoxicity of trimetrexate, 86 human progenitor cells were relatively resistant to trimetrexate toxicity because of their ability to salvage thymidine and hypoxanthine from serum. Exposure to NBMPR-P greatly increased trimetrexate toxicity to progenitors and stem cells, suggesting that severe myelotoxicity could limit the use of inhibitors of the ENT proteins in combination with trimetrexate. In contrast, the interaction of lometrexol (a novel antifolate which inhibits de novo purine biosynthesis) and dipyridamole is more complex. Hematologic cell lines are res- cued from lometrexol cytotoxicity by hypoxanthine, even in the presence of dipyridamole, unlike some human carcinoma cell lines (HeLa,A549)/16 Thus, the combination of lometrexol, dipyridamole and hypoxanthine may be selectively toxic to some malignant cells while sparing bone marrow.

CONCLUSIONS

Nucleoside transport plays a vital physiological role in nucleoside salvage, and is a major determinant of cytotoxicity for anticancer nucleoside analogs. Understanding of NT processes has been greatly aided by the recent cloning of cDNAs encoding four human NT proteins. Acquired resistance to nucleoside analogs can occur as a result of inefficient nucleoside uptake, both in vitro and in vivo, although the contribution of transport deficiency in clinical resistance to nucleosides requires further study.There are several strategies wi th the potential to increase the therapeutic index of nucleoside analog drugs, by way of normal tissue protection, nucleoside trapping, selectively promot ing drug uptake in malignant cells, rational drug scheduling to exploit a tumor 's NT profile, and gene therapy. Strategies have also been con- sidered of modulat ion of antimetabolite chemotherapy by inhibition of nucleoside salvage.

F ~ DIRECTIONS

Although there is strong evidence for a role of membrane transport in the pharmacologic action of nucleoside anticancer drugs, this information has yet to be exploited to improve their efficacy. With the recent cloning of four human NTs, molecular and immunologic probes are becom- ing available to determine the tissue and tumor distribution of these proteins, and will al low a greater understanding of their role in normal physiology and in their relationship to

malignant progression. Such probes may allow characterization of the NT profile of a given cell or tumor, which may in turn allow rational drug selection or predict ion of response to nucleoside anticancer therapy. Furthermore, a molecular understanding of human NTs might allow the design of specific inhibitors, as well as more tumor-specific nucleoside analogs. These approaches have the potential to overcome clinical nucleoside drug resistance and to increase the therapeutic indices of nucleoside anticancer drugs.

Acknowledgments

We thank Joan Thibault, Denise Brooks, Linda Harris mad Xiao Fang for assistance in preparing this manuscript.This work was supported by the Alberta Cancer Board, the Medical Research Council of Canada, the National Cancer Institute of Canada, Medical Research Council and the Wellcome Trust, UK. CEC is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada and JDY is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research.

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Nucleoside transport and its significance for anticancer drug resistance

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