A Comparisonof the Abilitiesof Nitrobenzylthioinosine,Dilazep,And

(CANCERRESEARCH52,5879-5886, November1,19921 A Comparisonofthe Abilitiesof Nitrobenzylthioinosine,Dilazep,and Dipyridamole to Protect Human Hematopoietic Cells from 7-Deazaadenosine (Tubercidin)'

Carol E. Cass,2 Karen M. King, Jencet T. Montaflo, and Anna Janowska-Wieczorek Departrnents ofBiochernistry [C. E. C., K. M. Kj and Medicine (A. J-W.J, University ofAlberta, and the Canadian Red Cross Blood Transfusion Service (A. J-W., I. T.M.J,Edmonton,Alberta,Canada

ABSTRACT substrate specificities, sensitivies to tight-binding inhibitors, and dependence on sodium gradients (for reviews, see Refs. Nltrobenzylthioinosine, dilazep, and dipyridamole are potent inhibi 1â€”4).There are two classes of nucleoside transport systems, tornof equllibrativetransportof nucleosidesthat may have pharmaco logical applications In mOdulatingthe therapeutic index of nucleoside each with at least two functionally distinct subclasses. The antimetabolites used in cancer chemotherapy. We have compared the equiibrative transporters (es, ei)3 exhibit broad substrate spec relative abilities of these inhibitors to reduce the toxicity of in vitro ificities, and because they differ in sensitivity to inhibition by exposures to tubercidin against clonogenic progenitor cells of normal low concentrations (<10 n@i)of NBMPR,4 they can be func human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two tionally distinguished by analysis of concentration-effect rela leukemic human cell lines (HL-60/Cl, CCRF-CEM) that differ in their tionships for inhibition of nucleoside fluxes. Inhibition of es expression of transporter subtypes. Short (1-h) exposures to 1 @iMhi transporters by NBMPR arises because of specific interactions bercidin alone inhibited colonyformation (a) of normal human hemato with high-affinity (Kd â€˜1 nM) sites on the extracellular aspect poletic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and of transporter elements. Dilazep and dipyridamole are also in (b) of HL-60/Ci andCCRF-CEM cellsby >90%. Pretreatment(30 hibitors of nucleoside transport, with activity against both es mm) with nitrobenzylthioinoslne, dilazep, or dipyridaniole followed by and ci transporters (7â€”11);the potency of dipyridamole appar simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoleticprogenitors ently differs widely among cell types (12). The es and ci trans and cell lines. Greater reductions oftoxicity were consistently seen with porters exhibit similar, relatively broad substrate specifIcities bone marrow progenitors and CCRF-CEM cells than with HL-60/Ci (8) and are thought to be separate gene products (13, 14). cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in There are several types of sodium-dependent, concentrative tubercidin toxicity of 50-100% were achieved at these concentrations: nucleoside transporters, based on the analysis of permeant 0giM (nitrobenzylthloinosine) @0.i @tM(dilazep) and 3.0 @tM(dipy specificities (5, 15). These transporters accept adenosine and ridamole). Pretreatment (30 mlii) followed by simultaneous treatment uridine as substrates, but they vary with respect to other purine (1 h) with any of the transportinhibitors(@0.i MM)and0.1 MM13H1- and pyrimidine nucleosides. The concentrative transporters tubercidin blocked the uptake of radioactivity completely in CCRF identified thus far are not inhibited by NBMPR, dilazep, or CEM cells and only partially in IIL-60/Ci cells These effects, which dipyridamole. Concentrative transporters have been described were consistent with the nucleoside transport phenotypes of CCRF CEM cells (inhlbitor-senslth'e)and HL-60/C1 cells (inhibitor-sensitive in epithelial tissues (5, 15â€”17),rodentsplenocytes (18, 19), and and inhibitor-resistant),suggestedthat protectionwas due to the inhi macrophages (20, 21) and in cultured IEC-6 intestinal cells bition of tubercidin uptake via equllibrative nucleoside transport sys (22), L1210 leukemia cells (1 1, 23), and HL-60 leukemia cells tern(s).Ught-density mononuclearcells from humanbone marrow,of (24). which the clonogenic progenitors represented only a minor (<0.01%) The relative levels of expression of nucleoside transporter subpopulation, possessed far fewer nitrobenzylthloinosine-binding sites subclasses vary greatly in different cell types, ranging from the (2 x 10@sites/cell,Kd= 0.7 ni@i)thaneither HL-60/Cl cells (1.7 x 10@ expression ofa single transporter, as in human erythrocytes (4), sites/cell, Kd 0.9 ni@i)orCCRF-CEM cells (3.3 x 10@sites/cell, Kd = to the expression of three transporters, as in Li210 leukemia 0.5 ns4 Initial rates of uptake of 1 @iMI3Hladenosine(0-6 s, 20Â°C)by cells (25). Changes in the relative activities of transporter sub human bone marrow mononuclearcells werereduced partially by 0.1 @iM types have been shown in HL-60 cells during the induction of inhibitor (nitrobenzylthioinosine > dipyridarnole > dilazep) and corn pletely by 10 MMinhibitor.We concludefrom these results that perme myeloid differentiation (24, 26), in IEC-6 cells as they become ation ofadenosine antirnetaboiltes in normal hone marrow progenitors is confluent (22), and in rat-2 fibroblasts after the activation of a mediated, most likely by Inhibitor-sensitive route(s), and may thus be a transforming protein tyrosine kinase (27). pharmacological target for the modulation of dose-limiting hematopol There have been many demonstrations of modulation of etic toxicities associated with their use as anticancer agents. the cellular content of nucleosides and nucleoside drugs by NBMPR and dipyridamole (1-4). In one strategy, transport INTRODUCTION inhibitors have been used to reduce the cellular uptake of nu cleoside drugs by dose-limiting normal tissues, thereby increas Transport of nucleosides into mammalian cells is mediated ing the maximum tolerated drug dosage. This â€œhostprotectionâ€• by multiple systems that can be distinguished by differences in

3 In one terminology (5), the equiibrative transporters are called â€œequilibrative ReceivedI 1/27/91;accepted8/24/92. sensitiveâ€•(es) and â€œequilibrativeinsensitiveâ€•(ci). The latter transporter is also The costs ofpublication ofthis article were defrayed in part by the payment of sometimes called â€œnitrobenzylthioinosine-resistantâ€•since,in some cell types, inhi page charges. This article must therefore be hereby marked advertisement in accord bition by NBMPR is seen,althoughonly at high concentrations(l MM)(6). mice with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviationsand trivial names used are: NBMPR, nitrobenzylthioinosine I Supported by the National Cancer Institute of Canada (C. E. C.) and the 6[(4-altrobenzyl)thioJ-9-@i-o-ribofuranosylpurine;tubercidin,7.deazaadenosine; AlbertaHeritageSavingsTrust Fund-AppliedResearchCancer(A. J-W.).C. E. C. dipyridamole, 2,6-bis(diethanolamino)-4,8-dipetidinopythnido-(5,4-djpytimidine; holds a career award from the National Cancer Institute ofCanada, and K. M. K. CFU-GM, colony-formingunit(s)-granulocyte,macrophage;BFU-E, burst-form holds a studentship from the Medical Research Council of Canada. ing unit(s)-erythroid CFU-GEMM, colony-forming unit(s)-granulocyte, erythroid, 2 To whom requests for reprints should be addressed, at Department of Bio macrophage,megakaryocyte;IMDM,Iscove'smodifiedDulbecco'smedium;IC@, chemistry,474 MedicalSciencesBuilding,Universityof Alberta,Edmonton, Al that concentrationofdrugthat inhibitedtreatedculturesby50%relativeto un berta, Canada T6G 2H7. treated cultures. 5879

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. PROTEC1ION FROM TUBERCIDIN TOXICITY BY TRANSPORT INHIBITORS strategy has been successfully used in studies with rodents in determinationofdrugconcentrationsthatinhibitedproliferationrates which the 5' monophosphate of NBMPR, a prodrug of high in static suspension cultures by 50% (ICso values) as described previ solubility, enhanced the therapeutic index of tubercidin in mice ously (33). with leukemia (28, 29) or schistosomiasis (30) by selectively Uptake, Transport, and Binding Assays. Measurements of cellular reducing the uptake of 7-deazaadenosine (tubercidin) by pan uptake of 0.1 LMI3Hltubercidinby HL-60/C1 and CCRF-CEM cells creas, liver, and kidney (31). during1-hexposureswereconductedat37T inâ€œtransportIMDMâ€•[10 mM 4-(2-hydroxyethyl)-1-piperazinecthanesuffonic acid in NaHCO3- Toxicity to the hematopoietic system is a major limitation of free IMDM]. Assays were conducted in triplicate, and cells were col most nucleoside antimetabolites used in antiviral and antican lected for analysis of their radioactive content using the â€œoil-stopâ€• cer therapy. In this study, we have compared the abilities of procedure described by Harley et al. (34). NBMPR, dilazep, and dipyridamole to reduce in vitro toxicity Measurements of initial rates of cellular uptake of 1 @LM[3H)adeno of tubercidin against normal multipotential progenitor cells sine by light-density mononuclear cells were conducted at 22C in (CFU-GEMM) and committed progenitor cells (CFU-GM, â€œtransportbufferâ€•(3mMK2HPO4, 1.8 mr@iCaCl2,1 mr@tMgCl2,144 BFU-E) with their abilities to reduce toxicity against cell lines mM NaCl, 20 mM Tris) using the â€œcolddilazep stopâ€• procedure de (HL-60/Cl, CCRF- CEM) that were grown under similar cul scribed by Hogue a a!. (35). With this procedure, cellular accumulation ture conditions. The cell lines were used as models of cell types of radioactivitywasinhibitedâ€œinstantaneouslyâ€•bythe additionof ice with inhibitor-sensitive nucleoside transport (CCRF-CEM) or a cold (4CC)transport buffer containing 100 @Mdilazep.Time courses of mixture of inhibitor-sensitive and inhibitor-insensitive nucleo uptake were linear during the first 8â€”10s,and initial rates (transport) side transport (HL-60/Ci). To determine ifthe â€œprotectabilityâ€•weredefinedbymeasuringuptakeat 2-sintervalsfrom0 to 6 s. Assays of cells was due to inhibitor blockade of nucleoside transport, were conducted in triplicate. Mass-lawanalysis ofbinding 0fI3HINBMPR was conducted at 22C we examined the effects of the inhibitors (a) on the uptake of as described previously (36). Cultured cells, harvested from actively 3H-labeled tubercidin by the cultured cell lines under the con proliferating cultures, were incubated under equilibrium binding con ditions of short-term toxicity experiments and (b) on transport ditions in NaHCO3-RPMI that contained 10 mr@t4-(2-hydroxyethyl)- (zero-trans influx) of [3H]adenosine in freshly isolated mono -l-piperazineethanesulfonic acid and graded concentrations (1â€”30nM) nuclear cells from human bone marrow. Our results indicated of [3H]NBMPRwithor without 10 @LMnonradioactiveNBMPR(to the presence of inhibitor-sensitive nucleoside transport activity determine nonspecific binding). Freshly isolated mononuclear bone both in clonogenic hematopoietic progenitors and in freshly marrow cells were assayed under similar conditions, except that binding isolated bone marrow cells, suggesting that it may sometimes be reactions were conducted in transport buffer. possible to reduce the toxicities of adenosine antimetabolites Materials. Cell culture materials were from Grand Jsland Biological against the hematopoietic system with nucleoside transport Co. (Burlington,Ontario,Canada)unless otherwisespecified. Percoll inhibitors. was purchased from Pharmacia Canada, Inc. (Bale D'Urfe, QuÃ©bec, Canada), methylcellulose was from Fluka AG Chemische Fabrischen (Basel, Switzerland), and erythropoietin was from the Terry Fox Lab MATERIALS AND METHODS oratory (Vancouver,British Columbia, Canada). [3H(G)JTubercidin(19 Ci/mmol), [2,8-3Hjadenosine(50 Ci/mmol), and [3H(G)JNBMPR (23 Bone Marrow Cells. Normal bone marrow was obtained, with the Ci/mmol) were from Moravek Biochemicals (Brea, CA) and were re donors' informedconsent,from the sternumsof patientsundergoing purified by high-pressureliquid chromatographyon a Partisil 10 cardiac surgery. Cells from the buffy coats were collected by centrifu ODS-3 column(Whatman,Inc.,Clifton,NJ) withmethanol-watergra gation (400 x g for 10 mm) and then resuspended in IMDM for the dients. NBMPR was prepared (37) in the laboratory ofDr. A. Paterson collection of light-density mononuclear cells by density centrifugation (University ofAlberta, Edmonton, Alberta, Canada) or was purchased, on 60%Percoll.For assayof drugtoxicities,the light-densitymarrow alongwithtubercidinanddipyridamole,fromSigmaChemicalCo.(St. cells (6 x l05/culture) were incubated at 37T (a) for 30 mm in IMDM Louis, MO). Dilazep dihydrochloride was a generous gift of Hoff alone (control) or with NBMPR, dilazep, or dypridamole and (b) then man-La Roche and Co. (Basel, Switzerland). for 1 h in IMDM alone (control) or with graded concentrations of tubercidin with or without transport inhibitor. Drug exposures were terminated by centrifugation, and the cells were washed twice with RESULTS drug-free IMDM and then assayed as previously described (32) for formation of colonies by pluripotent hematopoietic progenitors Toxicity of Tubercidin. Tubercidin is an analogue of adeno (CFU-GEMM), granulocyte-macrophage progenitors (CFU-GM), and sine and, consequently, has multiple biochemical sites of action erythroid progenitors (BFU-E). Uptake and binding studies were con (38â€”40).In an earlier study (40), short (2-h) exposures of cul ducted with light-density mononuclear cells. tured L1210 leukemia cells were highly toxic, with an IC50 Cell Lines. The origin and growth characteristicsof human HL value ofO. 1 @iMforinhibition ofcolony formation. In this work, 60/Cl promyelocyticleukemiccells havebeen described(33). Human CCRF-CEMlymphoblasticcells were obtainedfrom Dr. W. T. Beck tubercidin toxicity against normal hematopoietic progenitors (St. Jude Children'sResearchHospital, Memphis,TN). Cells were and two leukemic cell lines was assessed by subjecting suspen maintained in stationary suspension cultures in RPMI 1640 supple sion cultures to short (1-h) drug exposures, followed by assay of mented with I5% (HL-60/Cl) or 10% (CCRF-CEM) fetal calf serum colony formation in the absence of drug. The protocol (32) for and wereroutinelyrestartedafter 20â€”30subculturegenerationsfrom the detection ofclonogenic human hematopoietic cells was also Mycoplasma-free stocks preserved in liquid nitrogen and, when actively used for the cell lines, so that the in vitro culture conditions for proliferating, exhibited population doubling times of 20â€”24h. Cell cultured cells and progenitors were almost identical. The results numbers were determined with an electronic particle counter. The ef of a large series of experiments are summarized in Table 1, fects ofshort (1-h) drug exposures on HL-60/Cl and CCRF-CEM cells where it is evident that the hematopoietic progenitors (CFU wereassessed using the same procedures as describedabovefor hemato poietic progenitors (32), except that the colony-forming assay was per GM, BFU-E, CFU-GEMM) and the leukemic cell lines formed without erythropoietin; the relationships between cell number (CCRF-CEM,HL-60) exhibitedsimilar high levelsof sensitiv and colony formation were determined in the absence of drugs and ity to tubercidin. For all cell types, clonogenicity was reduced foundto belinearforbothcelllines.Theeffectsofcontinuous(48to 52 partially (20â€”40%)by exposures to 0.1 tM tubercidin and al h) drug exposures on HL-60/C1 and CCRF-CEM cells were assessed by most completely (93â€”100%)by exposures to 1 @LMtubercidin. 5880

tubercidinâ€•TubercidinTable 1 Inhibition ofcolonyformation by short exposures to evident that substantial protection could be achieved with each of the inhibitors. There were no differences among the three (@zM)Colony formation (percentage survival) progenitor populations in â€œprotectabilityâ€•sincethe reductions CFU-GEMM0.01 CFU-GM BFU-E in tubercidin toxicity achieved with the transport inhibitors 18(5) 100Â±25(5) 82Â±28(3) were essentially the same for pluripotent (CFU-GEMM), my 0.1 64Â±14(8) 71Â±14(6) 60Â±22(5) 1.0 0 (11) 0 (10) 0 (6) eloid (CFU-GM), and erythroid (BFU-E) progenitor cells. 10.094Â± (7)CCRF-CEM0 (12) 0 (II) 0 The transport inhibitors were not equivalent in their ability to HL-60/Cl0.1 reduce tubercidin toxicity, with NBMPR and dilazep being (5) 62Â±11 (4) more potent than dipyridamole. Treatment with NBMPR at 0.5 20Â±24 (3) 2Â±2 (4) 0.1, 0.3, and 1 @LMresultedin similar reductions in tubercidin 1.0 7Â±5 (6) 0 (4) toxicity, and a further reduction was seen for treatment with 3 5.078Â±14 0 (3) 0 (4) @LM.For dilazep, similar reductions in toxicity were seen for a Cells were incubated for 1 h in the presence or absence of tubercidin at the concentrationsindicatedand then assayedfor colonyformation in the absenceof treatment with 0. 1 and 1 tM and further reductions for treat tubercidinas describedin â€œMaterialsandMethods.â€•Therewereat least two cul ment with 3 and 10 @LM.Fordipyridamole, tubercidin toxicity tures per conditionin eachexperiment,and the numberofexperimentsis indicated in parentheses. Results are presented as percentages of colonies formed in drug was unaffected by treatment with 0.1 @i;the reductions in treated cultures, relative to colonies formed in untreated (control) cultures. Values toxicity seen with 3 and 10 @iMweresimilar to those achieved are means Â±SE. with NBMPR and dilazep at these concentrations. The increase in protectability seen at the highest inhibitor concentrations The results of the experiments of Table 1 suggested that suggested the presence of multiple pharmacological targets. HL-60/Cl cells were somewhat more sensitiveto tubercidin When the data of Fig. 1 were replotted (not shown) as percent than CCRF-CEM cells. This difference in sensitivity was con age survival versus concentration of transport inhibitor at each firmed by assessing the effects of continuous (2-day) exposures concentration of tubercidin, many of the concentration-effect to tubercidin on the proliferation ofcells maintained in suspen relationships were biphasic, a further indication that the pro sion cultures. The IC50 values in two separate experiments (data tective effect involved multiple processes. not shown) for the inhibition of proliferation rates during ex Effects of Transport Inhibitors on the Toxicity of Tubercidin posures of 48â€”52hwere 0.06 and 0.06 LMforHL-60/C1 cells against HL-60/C1 and CCRF-CEM Cells. The experiments of and 0. 13 and 0. 15 @tMforCCRF-CEM cells. Fig. 2 compared the relative abilities of several concentrations Toxicity of Transport Inhibitors. Before assessing the abil of NBMPR, dilazep, and dipyridamole to reduce tubercidin ity of the transport inhibitors to protect cells from tubercidin, toxicity against clonogenic cells of the lines HL-60/C1 and their toxicities, when administered as single agents, against CCRF-CEM cells during short (1-h) exposuresto tubercidin, human bone marrow progenitors and the leukemic cell lines such as those described above for bone marrow progenitors. were determined (data not shown). Short (1.5-h) exposures Although the transport inhibitors protected the two cell lines to NBMPR, dilazep, or dipyridamole at concentrations as high equally well at the lower concentrations of tubercidin (e.g., 0.5 as 10 @LMhad no effect on colony formation by any of the @LM), they were consistently more effective in protecting CCRF progenitor populations or the cell lines. In these experiments, CEM cells than HL-60/C1 cells at the higher concentrations of suspension cultures were exposed for 1.5 h at 37Â°Ctograded tubercidin (e.g., S @tM). concentrations (0.001â€”10 @zM)ofeach of the transport inhibi The relative abilities of the transport inhibitors to protect tors and then assayed for colony formation in the absence of HL-60/Cl and CCRF-CEM cells from tubercidin toxicity were drug as described above. The absence of inhibitor toxicity was then examined under more stringent conditions by subjecting confirmed in experiments in which the effects of continuous cells to continuous (2-day) exposures. Proliferation rates were (2-day) exposures to graded concentrations (0.0001â€”10 @tM)of determined for suspension cultures that were incubated in the NBMPR, dilazep, or dipyridamole on the proliferation of presence of a growth-inhibitory concentration (0.2 tiM)of tu HL-60/C1 and CCRF-CEM cells was determined in suspen bercidin (a) alone or (b) together with graded concentrations of sion cultures. Proliferation rates were (a) unaffected by the each of the transport inhibitors. Results from a single represen @ presence of the inhibitors at concentrations 1 @tMand(b) only tative experiment with NBMPR are presented in Fig. 3, where slightly reduced (by 15â€”30%)at S @iM(dipyridamole) and 10 tM it is apparent that CCRF-CEM cells were more readily pro (dilazep). These results indicated that both normal and leuke tected than were HL-60/Cl cells. Several such experiments, mic cells could tolerate in vitro exposures to relatively high involvingeach ofthe three transport inhibitors, are summarized concentrations of the transport inhibitors without loss of in Fig. 4. Although there were individual differences in potency, viability. the addition of the transport inhibitors to tubercidin-treated Effects of Transport Inhibitors on Tubercidin Toxicity cultures consistently increased proliferation rates to a greater against Normal Hematopoietic Progenitors. The effects of extent in CCRF-CEM cells than in HL-60/C1 cells. At the NBMPR, dilazep, and dipyridamole on the toxicity of tuberci lower concentrations (@0.Ol @LM),dilazepwas more effective din against hematopoietic progenitor cells were examined in than either NBMPR or dipyridamole in protecting both cell experiments that involved short (1-h) exposures to tubercidin. types. At the higher concentrations (@1MM),eachof the inhib Suspension cultures of mononuclear cells from freshly isolated itors provided virtually complete protection of CCRF-CEM human bone marrow were first exposed to transport inhibitor cells and only partial protection of HL-60/C1 cells from tuber alone for 30 mm and then to transport inhibitor plus tubercidin cidin toxicity. for 1 h, after which colony-forming ability was determined in High-Affinity Binding of NBMPR. The assumption in the the absence of drugs. The 30-mm pretreatment was included to experiments described above was that toxicity was reduced be ensure equilibration of inhibitor molecules with transport-in cause of the inhibition of the inward transport of tubercidin, at hibitory sites, if present, on the cells. The results of a large least in part, via the NBMPR-sensitive (es) transporter. For series of such experiments are summarized in Fig. 1, where it is erythrocytes of a variety of mammalian species, es transporter 5881

elements have been identified by mass-law analysis of high affinity binding of [3H]NBMPR, and there is a correlation be 100 CD tween the cellular abundance of NBMPR-binding sites and transport capacity (4). In this work, site-specific binding of Li. -@-0 [3HJNBMPR was determined under equilibrium conditions for to > (a) the cultured cell lines and (b) light-density mononuclear cells from normal human bone marrow. The latter are a heter U) ogeneous population of which the hematopoietic progenitors detected in the clonogenic assays were only a small fraction (<0.01%). The Scatchard plots (not shown) were linear, and

Cl) although the Kd values for mononuclear cells (0.7 nM) were to C similar to those for cultured cells (HL-60/C1, 0.9 ni@i;CCRF 0 CEM, 0.5 nM), the binding-site densities of mononuclear cells @ 100 (2.1 x 1O@sites/cell) were an order ofmagnitude less than those w C;, of the cultured cells (HL-60/C1, 1.7 x 1O@sites/cell; CCRF LI. CEM, 3.3 x 1O@sites/cell). These results suggested a substan 0 tially greater capacity for transport of nucleosides via es-medi ated transport processes in the cell lines than in mononuclear cells. Tubercidin(@zM) Effects of Transport Inhibitors on Tubercidin Uptake. Tu bercidin is accepted as a permeant by es transporters, with kinetics of influx that are identical to those of adenosine (34). Although tubercidin is also a substrate for ei transporters (13), :@ LI. it does not appear to be a good substrate for the â€œpurine-spe 0 cificâ€•concentrativenucleoside transporters (5, 25). If the pro > tection from tubercidin cytotoxicity described above was due to

U) inhibition of nucleoside transport processes, exposure of cells to NBMPR, dilazep, or dipyridamole under conditions of the 0' â€˜-LI. short-term toxicity experiments would be expected to reduce the cellular uptake of tubercidin. The experiments of Fig. 5 demonstrated that each of the transport inhibitors reduced the C 0 uptake of [3H]tubercidin by HL-60/C1 and CCRF-CEM cells 0 0 during short (1-h) exposures to a growth-inhibitory concentra w tion (0. 1 MM)of drug. CD CCRF-CEM cells express primarily es transport activity (41), Li. whereas HL-60/C1 cells express a mixture ofes and â€œNBMPR 0 resistantâ€•transport activities (24, 26). This difference in trans porter phenotypes is consistent with the differences observed in Tubercidin (MM) Fig. 5 in the inhibitor-induced reductions in tubercidin uptake, which were consistently less in HL-60/C1 cells than in CCRF CEM cells. There were also differences in the potency of the CD inhibitors for HL-60 cells (NBMPR > dilazep > dipyridamole) Li. and CCRF-CEM cells (NBMPR > dilazep = dipyridamole). 0 to These differences in inhibitor potencies are consistent with > those observed in the protection experiments (Figs. 1 and 2) in

U) which cells were subjected to short (1-h) exposures to tuberci towC din. The lack of correspondence with inhibitor potencies in the protection experiments (Fig. 5) was probably due to the much greater duration (>48 h) of drug exposure. Effects of Transport Inhibitors on Adenosine Uptake by Mononuclear Bone Marrow Cells. Nucleoside transport in human bone marrow cells has not been studied by direct measurement of permeant fluxes. Since the NBMPR-binding site densities of light-density mononuclear cells suggested the presence of NBMPR-sensitive transport activity, initial rates ofuptake of 1 @tM[3H]adenosineby freshly isolated mono

@ I 0.1 1.0 nuclear cells were determined in the presence and absence of Tubercidin (MM) each of the transport inhibitors (Table 2). Linear time courses Fig. 1. The ability of different concentrations of NBMPR, dilazep, and dipy (data not shown) were consistently obtained using a rapid-assay ridamole to protect colony-forming hematopoietic progenitors (CFU-GM, BFU-E, CFU-GEMM) during short (1-h) exposuresto tubercidin.Human bone marrowcellswereexposedto drugsand assayedforcolonyformationas described in â€œMaterialsand Methods.â€•Assays were conducted in duplicate, and results 57.4 for CFU-GM, 4.5 to 25.4 for BFU-E, and 1.2 to 51.9 for CFU-GEMM) have from 12separateexperimentshavebeencombined;most data points representthe been omitted for clarity of presentation. The concentrations of transport inhibi mean of 4 to 7 separate experiments, and SE values(whichranged from 1.9 to torswere(1CM):0,0@,â€¢,0.1;@,0.3;A, 1.0 , 3.0 0, 10.0. 5882

0 Co > 0 (9 -I Cl) I C a, 0

Cl) a, w C 0 0 0 LL 0 0 0

Tubercidin (NM) Fig. 2. Ability of NBMPR, dilazep, and dipyridamole to protect colony-forming HL-60/Cl and CCRF-CEM cells during short (I-h) exposures to different concentrations of tubercidin. Cells were exposed to drugs and assayed for colony formation as describedin â€œMaterialsandMethods.â€•Assayswere conducted in duplicate, and results (mean values Â±SE) from a series of four experiments have been combined. SE values are presented only where the error bars extend beyond the @ data symbols.The concentrations of transport inhibitors were (jiM):0, 0@, 0.1; E@,1.0;â€¢,3;A, 10.

procedure described previously (35) that allows the measure GM, BFU-E) of normal bone marrow were compared with ment of permeant uptake at very short (2-s) intervals. In the those oftwo cultured leukemic cell lines that exhibited different absence of transport inhibitors, mononuclear cells exhibited nucleoside transport phenotypes. The cell lines were selected as considerable adenosine uptake activity (0.7 pmol/iil cell water/ examples ofcell types with (a) primarily NBMPR-sensitive (es) s), with rates that were similar to those seen in other cell types under comparable assay conditions (1, 2). The reduction in

initial uptake rates seen in the presence of the inhibitors (NB 7 NBMPR, MM MPR > dilazep > dipyridamole) indicated that adenosine per 6 HL-60/C1 @0 - meation in mononuclear cells occurred primarily by mediated 5 processes. Since a large (about 85%) inhibition was seen in the 4 presence of a concentration (0. 1 MM)of NBMPR that fully saturated the high-affinity binding sites, permeation via the es 3 â€¢10 transporter was evidently the major route of entry under the assay conditions of Table 2. For all three inhibitors, complete 2 (100%) inhibition of adenosine fluxes was seen at the highest concentration of inhibitor tested.

DISCUSSION to 0 0.01 @ I- 0.001 Protection of cultured cells from cytotoxic nucleosides by x 0 treatment with an inhibitor of nucleoside transport was first demonstrated by Warnick et a!. (42). There have since been E 8 7 10,0.1 numerous reports of such protection by NBMPR, and while a, 6 inhibition of transport has generally been assumed to be the 0 5 mechanism, reduced cellular uptake of the cytotoxic nucleoside 4 has been demonstrated in only a few instances (for examples, 0.01 see Refs. 43 and 44). Protection from cytotoxic nucleosides by 3 dipyridamole has been shown in only a few instances (45, 46), although there are many examples of potentiation by dipy 2 0.001 0.0001 ridamole of the toxicity of inhibitors of de novo synthesis of 0 purines or pyrimidines by blocking the salvage of physiological nucleosides (reviewed in Ref. 47). There are no reports of pro tection by dilazep. I I I I This study was undertaken to determine if human hemato 0 24 48 72 poietic cells of normal and leukemic origin could be protected by transport inhibitors from toxicity by tubercidin, an analogue Time (hr) of adenosine previously used to demonstrate the feasibility of Fig. 3. Protection of HL-60/Cl and CCRF-CEM cells during continuous (2- the â€œhostprotectionâ€•strategy in animal models (28â€”31).The day) exposures to tubercidin by different concentrations of NBMPR. Cells were grown in media without drugs (0) or media that contained 0.2 @iMtubercidin(â€¢) effects of NBMPR, dilazep, and dipyridamole on tubercidin together with NBMPR at the concentrationsspecified.Cellconcentrations(three toxicity against clonogenic progenitors (CFU-GEMM, CFU cultures/condition) were determined as described in â€œMaterialsand Methods.â€• 5883

sures, we found (a) that each was capable ofprotecting hemato poietic progenitors and (b) that CFU-GEMM, BFU-E, and CFU-GM cells exhibited the same behavior for all of the expo sure conditions tested. These observations suggested that tuber cidin uptake by progenitors is a mediated process. There were differences in the abilities of the three inhibitors to protect progenitors. When protection during exposures to 1.0 MM a, tubercidin was assessed at 0.1 MM inhibitor, dipyridamole Co was ineffective, whereas NBMPR and dilazep each provided C 0

a, 0 Q. C a, U) CO a, C 0 a, C 0 a, C 0. a, e a, .@ Q.a, Co 0.

C @0 0 a, .0 I-.

0 0.0001 0.001 0.01 0.1 1 10 TransportInhibitor(MM) Fig. 4. Effects of different concentrations of NBMPR, dilazep, and dipy ridamole on the inhibition of proliferation of HL-60/CI and CCRF-CEM cells during continuous (2-day) exposures to 0.2 @iMtubercidin. Cell concentrations were determined at intervals of 22â€”26hin cultures subjected to drug exposures as describedin â€œMaterialsandMethods,â€•andproliferation rates (numberof popu 0 0.0001 0.001 0.01 0.1 lation doublings in 46â€”50h) were calculated for each condition (three cultures! condition). The data are presented as the percentage increase in proliferation rates TransportInhibitor(MM) ofcultures treated with NBMPR (â€¢),dilazep(0), or dipyridamole (A) relative to Fig. 5. Inhibition ofl3Hjtubercidin uptake by cultured HL-60/Cl and CCRF proliferation rates of cultures treated with 0.2 @Mtubercidin;for each cell line, CEM cells during short (1-h)exposuresby NBMPR, dilazep, and dipyridamole. data are from a single representative experiment of a series of four that gave Cells (2 x 105/assay) were first incubated, with gentle mixing, at 37C for 30 similar results. Proliferation rates(number ofpopulation doublings in 48 h) in the mm in modified RPMI 1640 without additives or with graded concentrations presenceof0.2 @LMtubercidinwere0.13 and 0.20 for HL-60/Cl and CCRF-CEM (0.001â€”10@LM)oftransportinhibitor and then for 1 h in modified RPM! 1640 that cells, respectively. contained 0.1 @LMl3Hltubercidinalone or with transport inhibitor. Assays were conducted in triplicate and uptake values for cultures exposed to NBMPR (â€¢), dilazep(0), and dipyridamole(A)are presentedas a percentageofvalues obtained nucleoside transport processes (CCRF-CEM; see Refs. 41 and for cultures in the absence of transport inhibitors. The uptake of tubercidin (per 48) and (b) a mixture of es and â€œNBMPR-insensitiveâ€•nucleo 10@cells)in the absence of inhibitors was 0.192 X l0@cpm (15.5 fmol) for HL-60/Cl cells and 0.169 x l0@cpm(14.3 fmol) for CCRF-CEM cells. side transport processes (HL/60/C1; see Refs. 24, 26, and 48). In proliferating HL-60/Cl cells, such as were used in this study, the NBMPR-insensitive transport activity was probably medi ated by an equilibrative transporter, since significant levels of Table 2 Inhibition ofadenosine transport in light-density mononuclear cells by NBMPR, diazep, and dipyridamolea sodium-dependent transport activity appear in HL-60 cells only after the induction of differentiation (24). InhibitorTransportratesAgent@LMpmol4il/s%None During short (1-h) exposures, tubercidin exhibited similar toxicities against normal and leukemic clonogenic cells, with Â±0.24 NBMPR 0.11Â±0.08 16Â±11 reductions in colony formation of >90% at concentrations of 1 NBMPRb 10.0 0.03 0 MM. During long (2-day) exposures, proliferating HL-60/C1 Dilazep 0.1 0.53 Â±0.26 76 Â±37 cells were twice as sensitive to tubercidin as were CCRF-CEM Dilazepâ€• 10.0 â€”0.03 0 Dipyridamole 0.1 0.25 Â±0.13 36 Â±19 cells. The effects of long-term exposures to tubercidin on bone Dipyridamoleb0.1 10.00.70 â€”0.10100Â±34 0 marrow progenitors were not determined in this study; IC90 aInitialratesoftransportof1LMI3Hladenosineweredeterminedat20'Cas values of 8.6 nM and 71.2 nr@i,respectively, were reported described in â€œMaterialsandMethodsâ€•and are expressed as pmol/s4il cell water. (49, 50) for BFU-E and CFU-GM cells cultured for 2 weeks in The extent of inhibitionis expressedas the percentageof fluxesin the presenceof inhibitor, relative to those in the absence of inhibitor. Except where noted, values the presence of tubercidin. represent the mean Â±SD of three independent determinations using preparations When we examined the ability of the transport inhibitors to of light-density mononuclear cells from bone marrow obtained from different in dividuals (transport assays were conducted in triplicate). reduce tubercidin toxicity against clonogenic progenitor cells b Values represent the average of three determinations of transport rates on a (CFU-GEMM, BFU-E, CFU-GM) during short (1-h) expo singlepreparationof mononuclearcells. 5884

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. PROTECFION FROM TUBERCIDIN TOXICITY BY TRANSPORT INHIBITORS considerable protection. At 3.0 MMinhibitor, dipyridamole pro nucleobasetransport in animal cells.Biochim.Biophys.Acts, 947:405-443, 1988. vided protection equivalent to that observed with NBMPR and 4. Gati, W. P., and Paterson, A. R. P. Nucleoside transport. [email protected] and J. dilazep. C.Parker(eds.),TheRedCellMembrane:Structure,Function,andClinical CCRF-CEM cells were more readily protected from tuberci Implications, pp. 635â€”661.New York: Marcel Dekker, 1989. 5. Vijayalakshmi,D., and Belt,J. A. Sodium-dependentnucleosidetransport in din toxicity by the transport inhibitors than were HL-60/Cl mouse intestinal epithelialcells.J. Biol.Chem., 263: 19419â€”19423,1988. cells, and when clonogenicity was determined after 1-h expo 6. Plagemann, P. G. W., and Wohiltueter,R. M. Nitrobenzylthioinosine-sen sures to tubercidin, the extent of protection resembled that sitive and -resistant nucleoside transport in normal and transformed rat cells. Biochim.Biophys.Acta, 816: 387â€”395,1985. observed for progenitor cells. The differences in protectability 7. Paterson, A. R. P., Lau, E. Y., Dahlig, E., and Cass, C. E. A common basis were most evident at the higher concentrations of tubercidin; for inhibition of nucleoside transport by dipyridamole and nitrobenzylthioi toxicity against CCRF-CEM or progenitor cells was almost nosine? Mol. Pharmacol., 18: 40â€”44,1980. 8. Belt, J. A., and Noel, L. D. Nucleoside transport in Walker 256 rat carcino completely eliminated, whereas toxicity against HL-60/C1 cells sarcoma and S49 mouse lymphomacells. Biochem.J., 232: 681â€”688,1985. was only partially eliminated. The biochemical basis of protec 9. Jarvis, S. M. Nitrobenzylthioinosine-sensitive nucleoside transport system: mechanism of inhibition by dipyridamole. Mol. Pharmacol., 30: 659- 665, tion for the cell lines was a blockade of cellular uptake of 1986. tubercidin. The differences between HL-60/C1 and CCRF 10. Gati, W. P., and Paterson, A. R. P. Interaction of[3Hldilazep at nucleoside CEM cells in intrinsic sensitivity of inhibition of tubercidin transporter-associated binding sites on 549 mouse lymphoma cells. Mol. Pharmacol., 36: 134â€”141,1989. uptake were consistent with the transporter phenotypes of these 11. Dagnino, L., and Paterson, A. R. P. Sodium-dependentand equilibrative cell lines (24, 26, 41, 48). For example, at high concentrations nucleosidetransport systemsin L1210 mouseleukemiacells:effectof inhib (0.1, 1, and 10 MM),the inhibitors almost completely blocked itors of equilibrativesystemson the concentration and retention of nucleo sides. Cancer Res., SO:6549â€”6553,1990. the uptake of radioactivity by CCRF-CEM cells and only par 12. Plagemann,P. G. W., and Woffendin,C. Speciesdifferencesin sensitivityof tially blocked the uptake by HL-60/C1 cells. It was not techni nucleosidetransport in erythrocytesand cultured cells to inhibition by ni trobenzylthioinosine,dipyridamole, dilazep and lidoflazine. Biochim. Bio cally feasible to determine tubercidin uptake in clonogenic phys. Acts, 969: 1â€”8,1988. progenitor cells. However, the similarity in protectability of 13. Belt, J. A., and Noel, D. L. Isolation and characterization of a mutant of CCRF-CEM cells and progenitors cells suggests that tubercidin L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nude oside transport. J. Biol. Chem., 263: 13819â€”13822,1988. influx in progenitor cells, like that in CCRF-CEM cells, occurs 14. Ullman, B. Mutational analysis of nucleoside and nucleobase transport. In.@ primarily by an es-mediated process. This conclusion is sup D. Kessel(ed.), Resistanceto AntineoplasticDrugs, pp. 293-315. Boca Ra ported by the demonstration of NBMPR-sensitive adenosine ton, FL: CRC Press, 1989. 15. Williams, T. C., and Jarvis, S. M. Multiple sodium-dependent nucleoside fluxes in the more mature myeloid elements (low-density mono transport systems in bovine renal brush-border membrane vesicles. Biochem. nuclear cells) of human bone marrow. J., 274: 27â€”33,1991. 16. LeHir, M., and Dubach, U. C. Sodium gradient-energizedconcentrative In summary, we have shown that normal hematopoietic pro transport of adenosine in renal brush border vesicles.PflÃ¼gersArch.Eur. J. genitor cells were protected from an adenosine antimetabolite Physiol., 401: 58â€”63,1984. (tubercidin) by three different inhibitors of equiibrative trans 17. LeHir, M., and Dubach,U. C. Uphill transport ofpyrimidine nucleosidesin renal brush border vesicles.Pflugers Arch. Eur. J. Physiol., 404: 238â€”243, port of nucleosides (NBMPR, dilazep, and dipyridamole). To 1985. determine the basis of this protection, we conducted parallel 18. Darnowski, J. W., Holdridge, C., and Handshumacher, R. E. Concentrative studies with model cell lines whose transporter phenotypes are uridine transport by murine splenocytes: kinetics, substrate specificity, and sodium dependency. Cancer Res., 47: 2614â€”2619,1987. known, to relate inhibitor protection from toxicity of the anti 19. Plagemann,P. G. W., Man, J. M., and Woffendin,C. Na@-dependent,active metabolite with inhibitor blockade of uptake of the antimetab and Na@-independent,facilitatedtransport of formycin B in mouse spleen lymphocytes.Biochim.Biophys.Acts, 1022:93â€”102,1990. olite. Because the protection of hematopoietic progenitors re 20. Baer, H. P., and Moorji, A. Sodium-dependent and inhibitor-insensitive up sembled that seen with CCRF-CEM cells, we concluded that takeofadenosine bymouseperitonealexudatecells.Biochim.Biophys.Acta, tubercidin influx in progenitor cells occurs primarily by inhib 1026:241â€”247,1990. 21. Plagemann,P. G. W., and Aran, J. M. Characterization of NaF@dependent, itor-sensitive, and thus mediated, mechanisms. Since the trans active nucleoside transport in rat and mouse peritoneal macrophages, a port inhibitors by themselves were not toxic, it may be possible mouse macrophage cell line and normal rat kidney cells. Biochim. Biophys. to protect normal hematopoietic cells from myelosuppression Acta, 1028:289â€”298,1990. 22. Jakobs,E. W.,VanOs-Corby,D.J., and Paterson,A.R. P. Expressionof by adenosine antimetabolites through pretreatment or simulta sodium-linked nucleoside transport activity in monolayer cultures of IEC-6 neous treatment with transport inhibitors. Because of the con intestinal epithelial cells. J. Biol. Chem., 265: 22210â€”22216, 1990. 23. Dagnino, L., Bennett, L. L., Jr., and Paterson, A. R. P. Sodium-dependent siderable heterogeneity of expression of nucleoside transport nucleoside transport in mouse leukemia Ll210 cells. J. Biol. Chem., 266: systems among different cell types, defmition ofthe â€œtransport 6308â€”6311,1991. abilityâ€•of adenosine antimetabolites by normal hematopoietic 24. Lee, C. W., Sokoloski,J. A., Sartorelli, A. C., and Handschumacher, R. E. Induction of the differentiationof HL-60 cells by phorbol 12-myristate 13- cells and target tumor cells will be needed to defme circum acetate activates a Na@-dependenturidine-transport system. Biochem. J., stances in which the host protection tactic might be useful. 274: 85â€”90,1991. 25. Crawford, C. R., Ng, C. Y. C., Noel, D., and Belt, J. A. Nucleoside transport in Ll210 murine leukemia cells. J. Biol. Chem., 265: 9732â€”9736,1990. ACKNOWLEDGMENTS 26. Chen, S-F., Cleaveland,J. S., Hollmann, A. B., Wiemann, M. 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31. Kolassa, N., Jakobs, E. S., Buzzell, G. R., and Paterson, A. R. P. Manipu reconstitution of the nucleoside transporter of CEM human leukemin cells. lation of toxicity and tissue distribution of tubercidin in mice by nitroben Biochim. Biophys. Acts, 1024: 289â€”297,1989. zylthioinosine 5'-monophosphate. Biochem. Pharmacol., 31: 1863â€”1874, 42. Warnick, C. 1., Muzik, H., and Paterson, A. R. P. Interferencewith nude 1982. oside transport in mouse lymphoma cells proliferating in culture. Cancer 32. Janowska-Wieczorek, A., Mannoni, P., Turner, A. R., McGann, L. E., Shaw, Res., 32: 2017â€”2022,1972. A. R. E., and Turc, J-M. Monoclonal antibody specific for granulocytic 43. Paterson, A. R. P., Yang, S-E., Lau, E. Y., and Can, C. E. Low specificity of lineage cells and reactive with human pluripotent and committed haemato the nucleosidetransport mechanismof RPMI 6410 cells. MoL PharmacoL, poietic progenitor cells. Br. J. Haematol., 58: 159â€”168,1984. 16:900â€”908,1979. 33. Ferguson, P. J., and Cass, C. E. Differential cellular retention of vincristine 44. Pros, K. L, Averett, D. R., and Zimmerman, T. P. Transport and metabolism and vinblastineby cultured human promyelocyticleukemiaHL-60/Cl cells: of 9-@9-t-arabinofuranosylguaninein a human T-lymphoblastoid cell line: the basis of differential toxicity. Cancer Res., 45: 5480â€”5488,1985. nitrobenzylthioinosine-sensitive and -insensitive influx. Cancer Res., 50: 34. Harley, E. R., Paterson, A. R. P., and Cass, C. E. Initial rate kinetics of the 1817â€”1821,1990. transport of adenosine and 4-amino-7-(jl-t-ribofuranosyl)-pyrrolo[2,3-d]py 45. King, M. E., Naporn, A., Young, B., and Howell, S. B. Modulation of rimidine (tubercidin) in cultured cells. Cancer Res., 42: 1289â€”1295,1982. 35. Hogue, D. L., Hodgson, K. C., and Cass, C. E. Effects of inhibition of cytarabine uptake and toxicity by dipyridamole. Cancer Treat. Rep., 68: 361â€”366,1984. N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of Ll2lO leukemia cells. Biochem. Cell Biol., 68: 199â€”209,1990. 46. Kubota, M., Takimoto, T., Kitoh, T., Tanizawa, A., Kiriyama, Y., Akiyama, 36. Can, C. E., Kolassa, N., Uehara, Y., Dahlig-Harley, E., Harley, E. R., and Y., and Mikawa,H. Preventionof [email protected] Paterson, A. R. P. Absence of binding sites for the transport inhibitor ni 4-nitrobenzyl-6-thioinosine or dipyridamole in human leukemia cell lines. trobenzylthioinosine to nucleoside transport-deficient mouse lymphoma AnticancerRes., 8: 339â€”342,1988. cells. Biochim. Biophys. Acta, 649: 769â€”777,1981. 47. Grem, J. L., and Fischer, P. H. Enhancement of 5-fluorouracil'santicancer 37. Paul, B., Chen, M. F., and Paterson, A. R. P. Inhibitors of nucleoside trans activity by dipyridamole. Pharmacol. Ther., 40: 349â€”371,1989. port. A structure-activity study using human erythrocytes J. Med. Chem., 18: 48. Cass, C. E., Peteya, D. J., Lynch, M. A., Tupas, J., Janmohamed, N., and 968â€”973,1975. Janowska-Wieczorek, A. Differential sensitivity ofleukemic cell lines to m 38. Suhadolnik, R. M. Nucleoside Antibiotics. New York: Intersciences, 1970. trobenzylthioinosine.Bone Marrow Transplant., 2: 124, 1987. 39. Suhadolnik, R. M. Nucleosides as Biological Probes. New York: John Wiley 49. Kaplinsky, C., Yeger, H., Estrov, Z., Barankiewicz, J., Pawlin, G., Freedman, and Sons, 1979. M. H., and Cohen, A. Selectiveprotection oftubercidin toxicityby nitroben 40. Cass, C. E., Selner, M., Tan, 1. H., Muhs, W. H., and Robins, M. J. Com zylthioinosineinnormaltissuesbutnotin humanneuroblastomacells.Can parison of the effects on cultured L1210 leukemia cells of the ribosyl, 2'- cer Chemother. Pharmacol., 17: 264â€”268,1986. deoxyribosyl,and xylosylhomologsof tubercidinand adenosinealone or in 50. el Kouni, M. H., Diop, D., O'Shea, P., Carlisle, R., and Sommadossi, J-P. combination with 2'-deoxycoformycin. Cancer Treat. Rep., 66: 317â€”326, Prevention of tubercidin host toxicity by nitrobenzylthioinosine 5-mono 1982. phosphate for the treatment of schistosomiasis. Antimicrob. Agents 41. Crawford, C. R., Ng, C. Y. C., Ullman, B., and Belt, J. A. Identification and Chemother., 33: 824â€”827,1989.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. A Comparison of the Abilities of Nitrobenzylthioinosine, Dilazep, and Dipyridamole to Protect Human Hematopoietic Cells from 7-Deazaadenosine (Tubercidin)

Carol E. Cass, Karen M. King, Jencet T. Montaño, et al.

Cancer Res 1992;52:5879-5886.

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