Effects of Nucleoside Transport Inhibitors on the Salvage and Toxicity of Adenosine and Deoxyadenosine in L1210 and P388 Mouse Leukemia Cells1

[CANCER RESEARCH 45, 6418-6424, December 1985]

PeterG. W.Plagemann2andRobertM.Wohlhueter

Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT concentrative nucleoside transporter (1, 7, 8), which, however, may exist in the plasma membrane in two forms (9-11); one Incubation of deoxycoformycin-treated L1210 leukemia cells form is inhibited by nmolar concentrations of NBTI (nitrobenzyl with dipyridamole or nitrobenzylthioinosine, inhibitors of nuclec- thioinosine) which correlates with the binding of NBTI to high side transport, enhanced the long-term incorporation of 2'-de- affinity sites on the cell (K,, ~0.5 nw), whereas the other form oxyadenosine and adenosine into the nucleotide pool and the lacks these binding sites and is inhibited only at i/M concentra toxicity of 2'-deoxyadenosine for the cells. In contrast, 2'-deoxy- tions of NBTI. The system, regardless of in which configuration, adenosine uptake in deoxycoformycin-treated P388 leukemia transports all natural pyrimidine and purine ribo- and deoxyribo- cells, which was about 10 times greater than that in L1210 cells, nucleosides with similar, though not equal, efficiency (1, 7, 8). was inhibited by dipyridamole and nitrobenzylthioinosine, and 2'- However, with respect to purine ribo- and deoxyribonucleosides, deoxyadenosine toxicity was not significantly affected by the this conclusion is largely based on the finding that they inhibit transport inhibitors. P388 cells also were about 6 times more the transport of Urd and dThd in various types of cultured cells resistant to 2'-deoxyadenosine than were L1210 cells, in spite and human erythrocytes (1, 8,12-14). Urd and dThd have been of the greater uptake of the nucleoside. We found that purine the favorite nucleosides for transport measurements because nucleoside transport in L1210 and P388 cells exhibited similar human RBC lack Urd and dThd kinases (12), appropriate variants kinetic properties and sensitivity to dipyridamole and nitroben lacking these enzymes are available for a considerable number zylthioinosine (both influx and efflux) and that the stimulation of of cell lines (1), and cultured cells can be sufficiently depleted of 2'-deoxyadenosine uptake by the inhibitors in L1210 cells is not ATP to practically completely block the phosphorylation of Urd mediated at the level of its transport into the cells but rather and dThd (1,15). These cells also lack Urd and dThd phospho- reflects an enhanced intracellular net accumulation of deoxy- rylases or their levels are so low as not to interfere with transport adenosine nucleotides. measurements. Thus, it is possible to measure the equilibration of these nucleosides across the membrane in metabolically inert INTRODUCTION cells. Direct studies of Ado and dAdo transport, on the other hand, Nucleoside salvage pathways in animal cells in their simplest have been more problematic. Their deamination can be inhibited form consist of two steps: the carrier-mediated transport of the effectively by treatment with dCF, but it has been difficult to nucleoside across the membrane, coupled with a simple, irrever block their conversion to nucleotides either by genetic or bio sible Michaelian phosphorylation within the cells (1-3). In the chemical approaches. Several studies have determined the ki case of Ado3 and 2'-dAdo, an additional route consists of their netics of uptake4 of Ado in human RBC and cultured cells (13, deamination to Ino and dlno, respectively, phosphorolysis of the 16-25), but in many studies two kinetic components in uptake latter, and salvage of the resulting Hyp. The non-mediated per were reported, and it was not clear whether the reported kinetic meation of natural nucleosides is so slow, though it varies parameters pertained to Ado transport, a step in Ado metabo somewhat with their lipid solubility, that the effective salvage of lism, or a combination thereof. Even in cells in which Ado a nucleoside depends on the functioning of the transport system. deamination and phosphorylation were greatly inhibited, Ado For example, a transport deficient variant of S49 mouse lym- uptake appeared to be concentrative due either to non-specific phoma cells (4) fails to grow in medium containing aminopterin, binding or to residual conversion to nucleotides by direct phos Hyp, and dThd, since it cannot salvage dThd. phorylation or via adenine, presumably resulting from the action The brush borders of epithelial cells of the kidney and intestine of methylthioadenosine phosphorylase (26). Similar problems in possess a Na+-dependent, concentrative nucleoside transport interpretation apply to three recent reports which showed that system (5, 6), but such a system has not been detected in the uptake of 2'-dAdo by Ado deaminase-deficient or inhibited erythrocytes, various tumor cells, hepatocytes, and numerous human lymphoma cells (27) or mouse L1210 lymphocytic leuke untransformed and transformed mammalian cell culture lines (1). mia cells (28, 29) was stimulated or little affected, rather than The latter types of cells probably express only a single non- inhibited, by NBTI and dipyridamole, another potent inhibitor of nucleoside transport (1,7). The results raised the possibility that Received4/22/85;revised8/27/85;accepted9/3/85. 1This work was supported by USPHS Research Grants GM 24468 and AM dAdo is transported by an alternate NBTI- and dipyridamole- 35211. resistant transporter (27) or that the accumulation of dAdo 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: Ado, adenosine; Ino, inosine; Hyp, hypoxanthine; '"Uptake" denotes the total intracellular accumulation of radioactivity from dThd, thymidine; Urd, uridine; Ade, adenine; dCF, 2-deoxycoformycin; NBTI, 6([4- exogenous labeled substrate regardless of metabolic conversions. "Transport" nitrobenzyl]thio)-9-0-D-ribofuranosylpurine; ICÂ«,,concentration causing 50% inhibi denotes solely the transfer of unmodified substrate across the cell membrane as tion. mediated by a saturable, selective carrier.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6418

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. SALVAGE OF Ado AND dAdO IN P388 AND L1210 CELLS nucleosides was stimulated by the transport inhibitors, perhaps Kmfixed at 100 /tM and the slope at f=0 was taken as the initial velocity by preferentially inhibiting the efflux of dAdo (29). To distinguish (vf2; for details see Refs. 1 and 13). For efflux measurements, a suspension of 1-2 x 108 cells/ml was between these possibilities, we have determined the effect of dipyridamole and NBTI on the long term uptake of dAdo and equilibrated with a specified concentration of radiolabeled substrate. Ado by dCF-treated L1210 and P388 cells, as well as on the Samples of the suspension were mixed using the dual syringe procedure at short time intervals in a ratio of 1:7.3 (opposite to that in influx influx and efflux of these purine nucleosides as measured by measurements) with basal medium containing, where indicated, specified rapid kinetic techniques in ATP-depleted cells. concentrations of transport inhibitors. The ceHs were separated from the medium by centrifugation through oil and analyzed for radioactivity. Materials. [2,8-3H]Ado, [2,8-3H]2'-dAdo, [5-3H]Urd, and [5'-3H]5'- MATERIALS AND METHODS dAdo were purchased from Moravek Bkachemicals, Brea, CA. Unlabeted Cell Cultures. Mouse L1210 and P388 cells were propagated in nucleosides were obtained from Sigma Chemical Co., St. Louis, MO; suspension culture in Eagle's minimum essential medium for suspension unlabeled 5'-dAdo was from Dr. H. Hogenkamp, and unlabeled NBTI cultures supplemented with non-essential amino acids, sera, and extra was from Calbiochem. San Diego, CA. Dipyridamole (Persantin) and dCF D-glucose as described previously (30). For measuring the inhibition of (Pentostatin) were gifts from Geigy Pharmaceuticals, Yonkers, NY and cell growth by 2'-dAdo, the cells were seeded into 24-well tissue culture Parke-Davis and Co., Morris Plains, NJ, respectively. plates (about 1 x 10s cells/well) and incubated in a CO2 incubator at 37Â°Cfor 3 days. The cells were then enumerated in a Coulter Counter. RESULTS AND DISCUSSION The cell lines were examined periodically for Mycoplasma contamination by the Urd/uracil incorporation method (31). No contamination was 2'-dAdo Toxicity of L1210 and P388 Cells. We have com detected. pared the effect of various concentrations of dipyridamole and Measurements of Uptake of Radiolabeled Substrates by Cells. 2'-dAdo on the growth of dCF-treated L1210 and P388 cells. Cells were collected from exponential phase cultures and suspended to 2-3 x 107 cells/ml in basal medium 42 (32). The suspension was P388 cells were chosen for this comparison because they are supplemented with 25 /JM dCF and after 5 min of incubation at 37Â°C; also mouse tumor cells of lymphoidal origin and have a similar samples thereof were mixed with radiolabeled substrate as indicated in size, growth properties, and nucleoside transport characteristics the appropriate experiments. The suspensions were incubated at 37Â°C, as L1210 cells (10). In this comparison, it was further of impor and at specified times the cells from samples of suspension were tance to rule out that any observed growth inhibition was due to separated from the culture medium by centrifugation through an oil the treatment with dipyridamole per se, since the latter is toxic, mixture as described previously (33). The cell pellets were analyzed for at higher concentrations, to L1210 and P388 cells (see Chart radioactivity. The radioactivity values were corrected for substrate 1A, zero 2'-dAdo concentration) and other types of cultured trapped in the extracellular space of cell pellets, as estimated by use of mammalian cells. This toxicity is unrelated to an inhibition of [14C]inulin, and converted to pmol of substrate taken up//il cell water on nucleoside transport.5 The results in Chart ~\A show that the the basis of cell water values, determined experimentally using 3H2O (33), or were expressed on the basis of 107 cells (about 8 Â¿Â¿Icellwater/ presence of concentrations of dipyridamole that exhibited little, 107 cells; Ref. 10). Where indicated, replicate samples of cell suspension if any, toxicity by themselves (1,4, and 10 Â¿/M)greatlypotentiated were analyzed for radioactivity in acid-insoluble material. In experiments the inhibition of growth of dCF-treated L1210 cells by various concentrations of 2'-dAdo. The results confirm those reported in which we analyzed the metabolism of the substrate, additional samples of cells were collected by centrifugation through oil directly into an acid by Kang and Kimball (29). On the other hand, dipyridamole layer, which immediately stops further metabolism (20, 30). The acid showed little synergism with 2'-dAdo in inhibiting the growth of extract was further processed and analyzed by ascending paper chro- P388 cells (Chart 18). Furthermore, P388 cells were significantly matography with a solvent composed of 30 ml of 1 M ammonium acetate, more resistant to 2'-dAdo than were L1210 cells (ICso about 7 pH 5, and 70 ml 95% ethanol (solvent 28) as described previously (20, versus 40 UM 2'-dAdo). 30). For ascertaining the effectiveness of the dCF treatment, the cell- The presence of 0.5 Â»MNBTI also potentiated the toxicity of free medium was removed immediately upon centrifugation of the cell 2'-dAdo for L1210 but not for P388 cells (data not shown). The suspension samples, acidified, and chromatographed with a solvent composed of 79 ml saturated ammonium sulfate, 19 ml 0.05 M phosphate effect was about equivalent to that observed with 2 ^M dipyri buffer, pH 6, and 2 ml isopropanol (solvent 9) for the separation of Ado, damole. NBTI by itself did not affect the growth of either cell line. Effects of Dipyridamole and NBTI on the Uptake of 2'-dAdo Ino, and Hyp or of dAdo, dlno, and Hyp (20, 30). Measurement of Purine Nucleoside Transport. Transport was mea and Ado in L1210 and P388 Cells. Chart 2, A and B, illustrates sured in cells blocked in metabolism as indicated in appropriate experi time courses of uptake of [3H]2'-dAdo into total cell material by ments. Time courses (comprising 15 time points) of transmembrane dCF-treated P388 and L1210 cells, respectively, in the presence equilibration of radiolabeled substrate were determined under zero-frans of 2 and 10 ^M dipyridamole and 500 HM NBTI. First, the results conditions by rapid kinetic techniques as described previously (1, 13, show that control P388 cells took up about 100 times more 2'- 20). For influx measurements, samples of suspensions of 1-3 x 107 dAdo than did L1210 cells in 2 hr of incubation of 37Â°C.Second, cells/ml were mixed with a solution of radiolabeled substrate at short, whereas in P388 cells, as one might expect, dipyridamole and timed intervals with a dual syringe apparatus (5-ml and 1-ml syringes) in NBTI inhibited the uptake of 2'-dAdo, both stimulated its uptake a ratio of 7.3:1. The cells were separated from the medium by centrif in L1210 cells. NBTI had relatively little effect on 2'-dAdo uptake ugation through oil and analyzed for radioactivity. Data were evaluated by fitting an integrated rate equation, based on the simple carrier with in P388 cells when compared to dipyridamole, but this is not directional symmetry and equal mobility of empty and nucleoside-loaded unexpected, since only about 80% of Urd transport of P388 cells carrier (1,34). In experiments to determine Michaelis-Menten parameters, is inhibitable by NBTI at concentrations up to 1 MM (9, 10), six to eight substrate concentrations were used, and the parameters whereas dipyridamole at 20 UM inhibits transport >95% (10). were extracted by least squares regression. In other experiments, where However, the same is true for Urd transport in L1210 cells (9, only initial velocities at a single permeant concentration (500 JJM)were of interest, the integrated rate equation for zero-frans entry was fitted with 5 P. G. W. Plagemann and R. M. Wonlhueter, unpublished data.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6419

IOO-

Chart 1. Effects of 2'-dAdo in combination 80-A with dipyndamoleon the proliferation of L1210 W and P388 (B) cells. 24-well cultured plates were seeded with about 1 x 1tf cells/well in 1 ml of growth medium supplemented with the 60- indicated concentrations of dipyndamole (DIP) and 2 -dAdo plus 5 ^M dCF. The plates were incubated at 37Â°Cfor 3 days, and the cells in each well were enumerated in a Coulter Coun 40- ter. Control cultures of L1210 and P388 cells incubated with dCF alone contained 1.0 x 10" and 1.4 x 10* cells/well, respectively. 20-

20 40 80 0 ' 2 IO IO 20 40 80 2'-DEOXYADENOSINE (pM)

A. P388 B. LI2IO C. ACID-INSOLUBLE 30 Chart 2. Effects of dipyndamole and NBTI CO on the uptake of [2,8-3H]2'-dAdo by P388 and O-CONTROL L1210 cells (A and B, respectively) and its in Â«-+2pM DIP corporation into acid-insoluble material (C). Samples of suspensions of about 1 x 107/ A-HOuM DIP BP388 or L1210 cells/ml of BM428 were sup NBTI plemented with 20 Â¡MdCF and 5 min later as Ã´ 20 indicated with 2 or 10 MMdipyndamole(DIP)or P388 500 nm NBTI plus 40 >M [2,8-3H]2'-

0 I 2 O I 2 TIME(HR)

10), and this fact therefore is unrelated to their different effects Kimball (29). Chart 3 also shows that Ado uptake by L1210 cells on 2'-dAdo uptake in the two types of cells. was slightly stimulated by dipyridamole and NBT!, but far less The inhibition of 2'-dAdo uptake by the transport inhibitors in than was 2'-dAdo uptake. The lesser effect, however, may P388 cells was reflected by a similar inhibition of 2'-dAdo incor simply be related to the fact that Ado uptake in untreated cells poration into acid-insoluble material (Chart 2C). The stimulation was already about 10 times greater than was 2'-dAdo uptake. of dAdo uptake in L1210 cells had little effect on its incorporation In fact, Ado uptake ceased after about 2 hr of incubation (Chart into acid-insoluble material, but incorporation was very low (Chart 3/4), because the medium had become practically depleted of 2C), and in another experiment (Chart 3B), the amount of 2'- substrate. dAdo incorporated into acid-insoluble material was transiently Effects of Dipyridamole and NBTI on Furine Nucleoside increased by the dipyridamole treatment. The reason for this Transport in L1210 and P388 Cells. We have measured the difference is not known; the stimulation of 2'-dAdo incorporation zero-trans influx of Ado and 2'-dAdo in ATP-deleted, dCF-treated into cell material by dipyridamole and NBTI was about the same cells at a substrate concentration of 500 /Â¿M.Underthese con in the two experiments, which demonstrated the reproducibility ditions < 10% of the intracellular substrate was phosphorylated of the effect (compare Charts 2B and 3A, right frame). Further at 3 min of incubation, allowing a reasonable approximation of more, Chromatographie analyses of the acid-soluble pool of 2'- the rate of transmembrane equilibration of unmodified substrate dAdo labeled L1210 cells showed clearly that the transport by the use of rapid kinetic techniques. Representative time inhibitors stimulated the accumulation of 2'-dATP (Table 1). courses of transmembrane equilibration of 2'-dAdo by L1210 These results are in agreement with those reported by Kang and cells in the absence and presence of two concentrations of

CANCER RESEARCH VOL. 45 DECEMBER 1985 6420

previously for untreated cells (data not shown). 5'-dAdo is not a substrate for either Ado deaminase or Ado kinase (35), and it is also not phosphorolyzed in L1210 or P388 O-CONTROL cells (26), since both these cell types lack methylthioadenosine

â€¢-+2PMDIP phosphorylase (36). Thus its transport can be measured in these

*- tlOuM DIP cells without complications due to metabolic conversion and in the absence of prior treatment with dCF or of ATP-depletion T+500 nM NBTI 0 (35). We have compared the kinetics of zero-trans influx of 5'- dAdo in L1210 and P388 cells and have found them to be comparable; the Michaelis-Menten constants (K) and maximum velocities (V) fell between 500 and 900 U.Mand 25 and 45 pmol/ u.\ cell water-s, respectively, and only a single saturable, non- concentrative transport system was detectable in the substrate concentration range from 10 to 1280 MM. Our results overall indicate that the nucleoside transporters of L1210 and P388 cells possess similar kinetic properties and sensitivities to dipyridamole and NBTI, in agreement with pre vious results based on experiments with Urd as substrate (K, ~250 MM;V, -20 pmol/Ml cell water-s; Ref. 10). Furthermore, no Chart 3. Comparison of the effects of dipyridamole and NBTI on the uptake of second dipyridamole- and NBTI-resistant purine nucleoside Ado and 2'-dAdo (A) and its incorporation into acid-insoluble material (3) by L1210 cells. Samples of a suspension of about 1 x 107 cells/ml of BM42B were supple transport system specific for L1210 cells was detected. Our mented with 25 iiU dCF and 5 min later as indicated with 2 or 20 UMdipyridamole results contradict the suggestion that Ado and 2'-dAdo are (DIP) or 500 nM NBTI plus 40 nM [2,8-3H]Ado (11 cpm/pmol) or 40 pu [2,8-3H]2'- dAdo (8.8 cpm/pmol). At the indicated times of incubation at 37Â°C,the cells from transported by carriers that differ in NBTI sensitivity (27). The 0.5-ml samples of suspension were collected by centrifugation through an oil layer results are also inconsistent with the suggestion that dipyrida and were analyzed for radioactivity (total in cell) or were analyzed for radioactivity mole may inhibit to a greater extent the efflux than the influx of in acid-insoluble material. All points are averages of duplicate samples. The broken 2'-dAdo in L1210 cells (29). This hypothesis seemed unlikely on lines indicate the intracellular concentration of substrate equivalent to that initially in the medium. the basis of the directional symmetry of the nucleoside carrier in these cells (1) and the rapid equilibration of this lypophilic sub Table 1 stance across membranes. It has also been ruled out experi Stimulation of dATP accumulation from exogenous dAdo in dCF-treated L1210 mentally; dipyridamole inhibited about equally the efflux of 500 cells by dipyridamole and NBTI MM2'-dAdo from L1210 and P388 cells whether (data not shown) The details of the experiment are described in the legend to Chart 3. After 2.5 h of incubation with 40 utt [3H]Ado or [3H]2'-dAdo, the cells were extracted, and or not (Chart 6) they were ATP-depleted. The same was the the acid extracts were chromatographed with solvent 28. The amounts of nucleo- case for NBTI (Chart 6). It follows that the enhancement of long side triphosphate (XTP) accumulated were calculated on the basis of the Chromat term 2'-dAdo uptake by dipyridamole and NBTI in L1210 cells is ographie separations and the amounts incorporated into total cell material (see Chart 3). not mediated at the transport step. Instead it seems to involve a stimulation of the net formation of dAdo nucleotides. This is also Amounts of XTP accumulated indicated by the finding that transport is not a limiting factor in (nmol/107 cells) the uptake of 2'-dAdo by L1210 cells. Another experiment illustrates the shift from inhibition of 2'- Treatment [3H]Ado [3H]2'-dAdo None 29.0 0.88 dAdo transport to stimulation of uptake by dipyridamole in the +10 fjM dipyridamole 29.2 2.62 overall uptake of 2'-dAdo in dCF-treated L1210 cells (Chart 7). +0.5 MMNBTI 32.6 1.63 Uptake during the first minute was inhibited by NBTI and dipyri damole; uptake during this interval reflects mostly transport. But beginning at about 2 min of incubation, 2'-dAdo uptake became dipyridamole and NBTI are illustrated in Chart 4. Initial transport velocities (vf2 were computed as the zero time slopes of the stimulated greatly in the NBTI- and dipyridamole-treated cells. uptake curves. The viÂ£values from this and other experiments Since the rate of accumulation of dAdo nucleotides in L1210 for Ado and 2'-dAdo zero-frans entry in both types of cells as a cells was very low compared to the transport capacity of the cells, the inhibition of 2'-dAdo transport by dipyridamole or NBTI, function of dipyridamole and NBTI concentration are illustrated in Chart 5. The zero-trans influx of Ado and 2'-dAdo were which was >90% in the case of dipyridamole, was inconsequen inhibited similarly in both L1210 and P388 cells. We found, tial as far as its conversion to nucleotides was concerned. This however, that the transport of Ado and 2'-dAdo in both the ATP- was not the case for P388 cells, which exhibited a much higher rate of conversion of 2'-dAdo to nucleotides. Inhibition of trans depleted P388 and L1210 cells was considerably less inhibited port slowed 2'-dAdo phosphorylation, although not to the same by dipyridamole (IC50about 5 MM)than was reported previously for zero-frans influx of Urd (IC50about 300 nM) in these cells (10). extent (compare Charts 2 and 7), since the capacity even of these cells to transport 40 MM2'-dAdo exceeded considerably We have no explanation for this difference at the molecular level. However, it seems to be related to the iodoacetate-KCN treat their capacity to phosphorylate this substrate. That 2'-dAdo ment used to deplete the cells of ATP, since in a direct compar uptake in human lymphoma cells (27) is less sensitive to inhibition ison of the effect of dipyridamole and NBTI on Ado, 2'-dAdo, by dipyridamole and NBTI than is Ado uptake may be partially and Urd influx in the same population of ATP-depleted P388 and explained on the same basis, since Ado uptake was about 10 L1210 cells, Urd influx was also inhibited less than was reported times higher than was 2'-dAdo uptake by these cells.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6421

Chart 4. Effect of dipyridamote (DIP) (A) and - NBTI (B) on the zero-frans influx of 2'-dAdo in Â°,300 . ATP-depteted, dCF-treated L1210 cells. The cells were depleted of ATP by incubation in

5 mM KCN, 5 mw rodoacetate, and 25 MMdCF (about 2 x 107 cells/ml) at 37Â°Cfor about 10 i 200- mm. Then samples of the suspension were E supplemented with various concentrations of â€” dipyridamole or NBTI (as indicated in Chart 5), ^ and after at least 5 min of incubation at 25Â°C, z the influx of 500 UM[2,8-3H]2 '-dAdo or [2,8-3H]- tu Ado (about 1 cpm/pmol) was measured by rapid 4 IOO- kinetic techniques as described in "Materials *~ and Methods." Only representative time â€¢Â§ courses of substrate equilibration are shown. ^ Initial zero-frans entry velocities (vf2) were esti mated by integrated rate analysis. ISO

A. DIP B. NBTI IOO-

8O o

Charts. Zero-frans influx of 500 Â¡M 2'- i dAdo and Ado as a function of dipyridamole (A) 60 or NBTI (B) concentration in ATP-depleted o L1210 and P388 cells. The experiments were ** conducted as described in the legend to Chart 4. vfj values for untreated, control cells all fell 40 between 10 and 16 pmol//jl cell water -s.

20-

10 100 1000 10000 IO 100 [DIP](nM) [NBTlJ(nM)

Although our results indicate clearly that it is the net formation [3H]2'-dAdo in the presence of 5 UM dipyridamole at 37Â°Cfor 1 of intracellular dAdo nucleotides rather than 2'-dAdo transport h. Under these conditions the two types of cells took up similar into the cell that is stimulated by dipyridamole and NBTI in L1210 amounts of 2'-dAdo (see Chart 2), and >80% of the radioactivity cells, further work is required to elucidate the mechanisms in their acid-soluble pools was associated with dATP (Chart 8ÃŸ). involved at the molecular level. The low net uptake of 2'-dAdo Then the cells were incubated in fresh medium without dAdo could reflect rapid and continuous degradation of deoxyadenine and monitored for cell-associated radioactivity. There was only nucleotides in these cells, and its stimulation by NBTI and a slow loss of radioactivity from the cells during 2 h of incubation dipyridamole could reflect the inhibition of such activity. Carson at 37Â°C,which was, however, comparable in L1210 and P388 et al. (37) reported that the sensitivities of 21 human lymphoid cells (Chart 8/1). In both cultures, the radioactivity lost from the cell lines to 2'-dAdo toxicity were inversely related to their soluble cells was recovered as 2'-dAdo (50-60%), dlno plus Hyp (30- deoxynucleotidase activities. Such a relationship, however, can 40%), and an unidentified component (about 4%) in the medium not explain the difference in 2'-dAdo sensitivity between L1210 (data not shown). There was also a slow chase of radioactivity and P388 cells, since P388 cells are much more resistant to a from the acid-soluble pool to acid-insoluble material, which was 2'-dAdo than L1210 cells, in spite of their much greater capacity comparable in both types of cells (Chart 8A). The only difference to take up the nucleoside. Furthermore, we could not detect any we observed between L1210 and P388 cells was that the difference in the rate of turnover of dATP in L1210 and P388 proportion of radioactivity in the soluble pool recovered in fraction cells (Chart 8). We labeled the dATP pool of both types of cells III containing dAdo (but also Ado, dlno, Ino, and Hyp) was greater by treating them with dCF and then incubating them with 1 Ã•IM in L1210 than P388 cells, both after the labeling period and the

CANCER RESEARCH VOL. 45 DECEMBER 1985 6422

Chart6. Inhibition of 2'-dAdo efflux by di- pyridamele and NBTI in L1210 W and P388 (B) cells. Suspensions of about 1.3 x 10* cells/ml of medium were treated with 50 pM dCF and then equilibrated with 500 ,,M [2,8-3H]2'-dAdo (4.5 cpm/pmol) at 37Â°C.The zero-frans exit of radiolaoeled substrate into balanced salt solu tion or balanced salt solution containing, where indicated, 5 or 20 Â¡Mdipyndamole (DIP) or 1 nu NBTI was then measured at 25Â°C by rapid kinetic techniques as described in "Materials and Methods.*

TIME (SEC)

0-TIME -i

ICO

â€¢g _ 80- tu TOTAL IN CELL S2 S Q. F U o 60h O- A- LI2IO OO 2HR Â» A- P388

20 ACID-INSOLUBLE 60 IZO 240 360 48O TIME (SEC) Chart 7. Comparison of the effects of dipyndamole and NBTI on the influx and phosphorylation of 2'-dAdo in intact L1210 cells at 25Â°C.Samples of a suspension I 0 5 IO I5 of 3 x 10" dCF-treated (25 tat) L1210 cells/ml of BM42B were supplemented TIME (HR) FRACTION NUMBER where indicated with 20 MMdipyridamote (DIP) or 0.5 Â¡MNBTI and then the uptake of 40 MM[2,8-3HJ2'-dAdo (10 cpm/pmol) at 25Â°Cwas measured by the rapid kinetic technique (2-60 s). Another portion of each of the suspensions was mixed with Chart 8. Turnover of dATP in L1210 and P388 cells. Suspensions of about 4 x 10' cells/ml of BM42B were supplemented with 20 Ã•/MdCFand 5 ,/M dipyndamole radiolabeled substrate and manually sampled in a comparable manner over longer and 5 min later with 1 (/M [2,8-3H]2'-dAdo (800 cpm/pmol). After 1 h of incubation intervals (1-8 min). The broken line indicates the intracellular concentration of at 37Â°C, the cells were collected by centrifugatkjn, resuspended to the same substrate equivalent to that initially in the medium. density in fresh medium (0 time) and, at various times of further incubation at 37Â°C, the cells from 0.5-ml samples of suspension were collected by centrifugation through oil and were analyzed for radioactivity. Other 0.5-ml samples of suspension chase (Chart 8, B and C, respectively). This observation has were analyzed for radioactivity in acid-insoluble material. All values in A are averages been confirmed in a second simitar experiment, but its signifi of duplicate samples and are expressed as a percentage of radioactivity associated with the cells at 0 time (about 4 x 106 cpm/0.5 ml of suspension). The acid-soluble cance in relation to dATP synthesis and turnover in these cells pools were extracted from additional samples of cells and chromatographed with is unclear. solvent 28. Representative chromatograms are shown in B and C. Fraction I = In view of the results in Chart 8, it seems more likely that the dATP, dADP; II = dAMP; III = dAdo (Ado, Hyp, Ino, diÃ±o). stimulation of 2'-dAdo uptake in L1210 cells by dipyndamole is at the level of intracellular phosphorylation of 2'-dAdo. For with two different inhibitors of nudeoside transport, one of which example, the low uptake of 2'-dAdo by L1210 cells and its seems to be highly specific for the nucleoside transporter (NBTI), stimulation by NBTI and dipyndamole could be due to some kind it seems likely that the effect is indirect and mediated through of inhibition of 2'-dAdo kinase activity which is somehow relieved an inhibition of nucleoside transport. What mechanism is in by the treatment with the transport inhibitors. Regardless, since volved, however, is unclear at present. the stimulation of 2'-dAdo nucleotide accumulation is observed The results with L1210 cells have additional interest in that

CANCER RESEARCH VOL. 45 DECEMBER 1985 6423

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. SALVAGE OF Ado AND dAdo IN P388 AND L1210 CELLS they represent another example of a synergistic cytotoxic effect with wide nucleoside specificity. J. Cell. Physiol., 89:1-18,1976. 16. Chelto, P. L., Sirotnak, F. M., Dorick, D. M., Yang, C. H., and Montgomery, J. of a combination of substances, each of which alone exhibits A. Initial rate kinetics and evidence for duality of mediated transport of relatively low or no cytotoxicity. It is also of interest that, in this adenosine, related purine nucleosides, and nucleoside analogues in L1210 case, the synergism seems to be relatively specific for a lym- cells. Cancer Res., 43: 97-103,1983. 17. Green, R.D. Adenosine transport by a variant of C1300 murine neuroblastoma phoidal tumor lacking T- or B-cell surface antigens which has cells deficient in adenosine kinase. Biochim. Biophys. Acta, 598: 366-374, been used as a laboratory model for acute lymphocytic leukemia 1980. 18. Hariey, E. R., Paterson, A. R. P., and Cass, C. E. Initial rate kinetics of transport of childhood. of adenosine and tubercidin in cultured cells. Cancer Res., 42: 1289-1295, 1982. 19. Kolassa, N., Plank, B., and Tumheim, K. pH and temperature dependence of ACKNOWLEDGMENTS adenosine uptake by human erythrocytes. Eur. J. Pharmacol., 52: 345-351, 1978. We thank Laurie Erickson and John Erbe for excellent technical assistance and 20. Lum, C. T., Marz, R., Plagemann, P. G. W., and Wohlhueter, R. M. Adenosine Yvonne Guptill for valuable secretarial work. transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes. J. Cell. Physiol., 707:173-200,1979. 21. Paterson, A. R. P., Babb, L. R., Paran, J. H., and Cass, C. E. Inhibition by nitrobenzylthioinosine of adenosine uptake by asynchronous HeLa cells. Mol. REFERENCES Pharmacol., 73: 1147-1158,1977. 22. Pofit, J. T. and Strauss, P. R. Membrane transport by macrophages in 1. Plagemann, P. G. W. and Wohlhueter, R. M. Permeation of nucleosides, nucleic suspension and adherent to glass. J. Cell. Physiol., 92: 249-256,1977. acid bases, and nucleotides in animal cells. Curr. Top. Membr. Transp., 74: 23. Roos, H., and Pfleger, K. Kinetics of adenosine uptake by erythrocytes, and 225-233, 1980. influence of dipyridamole. Mol. Pharmacol., 8: 417-425,1972. 2. Plagemann, P. G. W. and Wohlhueter, R. M. Nucleoside transport in mamma 24. Schrader, J., Beme, R. M., and Rubio, R. Uptake and metabolism of adenosine lian cells and interaction with intracellular metabolism. In: R. M. Berne, R. W., by human erythrocyte ghosts. Am. J. Physiol., 223:159-166,1972. Rail, and R. Rubio, (eds.), Regulatory Function of Adenosine, pp. 179-200. 25. Thampy, K. G. and Barnes, E. M., Jr. Adenosine transport by cultured glial Boston: Martinus Nijhoff Publishers, 1983. cells from chick embryo brain. Arch. Biochem. Biophys., 220: 340-346,1983. 3. Wohlhueter, R. M. and Plagemann, P. G. W. The roles of transport and 26. Plagemann, P. G. W. and Wohlhueter, R. M. 5'-Deoxyadenosine metabolism phosphorylation in nutrient uptake in cultured animal cells. Int. Rev. Cytol., 64: in various mammalian cells. Biochem. Pharmacol., 32: 1433-1440,1983. 171-240,1980. 27. Itzasa, T., Kubota, M., and Carson, D. A. Modulation of adenine nucleoside 4. Cohen, A., Ulman, B., and Martin, D. W., Jr. Characterization of a mutant excretion and incorporation in adenosine deaminase-deficient human lym mouse lymphoma cells with deficient transport of purine and pyrimidine nu phoma cells. Biochem. Biophys. Res. Commun., 727: 514-520,1984. cleosides. J. Biol. Chem., 254: 112-116,1979. 28. Cass, C. E., Seiner, M., Tan, T. H., Muhs, W. H., and Robins, M. J. Comparison 5. LeHir, M. and Dubach, V. C. Sodium gradient-energized concentrative trans of the effects of 2'-deoxyribosyl, and xylolyl homologs of tubercidin and port of adenosine in renal brush border vesicles. PflÃ¼gersArch., 407: 58-63, adenosine alone or in combination with 2'-deoxycoformycin. Cancer Treat. 1984. Rep., 66: 317-326,1982. 6. Schwenk, M., Hegazy, E., and Lopez del Pino, V. Undine uptake by isolated 29. Kang, G. and Kimball, A. P. Dipyridamole enhancement of toxicity to L1210 intestinal epithelial cells of guinea pig. Biochim. Biophys. Acta, 805: 370-374, cells by deoxyadenosine and deoxycoformycin combinations in vitro. Cancer 1984. Res., 44:461-466,1984. 7. Paterson, A. R. P., Kolassa, N., and Cass, C. E. Transport of nucleoside drugs 30. Plagemann, P. G. W. and Wohlhueter, R. M. Adenosine metabolism in wild- in animal cells. Pharmacol. Ther., 72: 515-536, 1981. type and enzyme-deficient variants of Chinese hamster ovary and Novikoff rat 8. Young, J. D. and Jarvis, S. M. Nucleoside transport in animal cells. Biosci. hepatoma cells. J. Cell. Physiol., 776: 236-246,1983. Rep., 3:309-332,1983. 31. Schneider, E. L., Stanbridge, E. F., and Epstein, C. J. Incorporation of 9. Belt, J. A. Heterogeneity of nucleoside transport in mammalian cells. Two [3H]uridine and [3H]uracil into RNA. A simple technique for the detection of types of transport activity in L1210 and other cultured neoplastic cells. Mol. mycoplasma contamination of cultured cells. Exp. Cell. Res., 84: 311-318, Pharmacol., 24: 479-484, 1983. 1974. 10. Plagemann, P. G. W. and Wohlhueter, R. M. Nucleoside transport in cultured 32. Plagemann, P. G. W. Nucleotide pools of Novikoff rat hepatoma cells growing mammalian cells. Multiple forms with different sensitivity to inhibition by nitro- in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide benzylthioinosine. Biochim. Biophys. Acta, 773: 39-52, 1984. pools and pool sizes during growth cycle. J. Cell. Physiol., 77: 213-240,1971. 11. Plagemann, P. G. W. and Wohlhueter, R. M. Nitrobenzylthioinosine-sensitive 33. Wohlhueter, R. M., Marz, R., Graff, J. C., and Plagemann, P. G. W. A rapid- and resistant nucleoside transport in normal and transformed rat cells. Biochim. mixing technique to measure transport in suspended animal cells: application Biophys. Acta, 876: 387-395. to nucleoside transport in rat hepatoma cells. Methods Cell Biol., 20: 211- 12. Cass, C. E. and Paterson, A. R. P. Mediated transport of nucleosides by 236,1978. human erythrocytes. Specificity toward purine nucleosides as permeants. 34. Wohlhueter, R. M. and Plagemann, P. G. W. On the functional symmetry of Biochim. Biophys. Acta, 297: 734-746,1973. nucleoside transport in mammalian cells. Biochim. Biophys. Acta, 689: 249- 13. Plagemann, P. G. W. and Wohlhueter, R. M. Adenosine and tubercidin binding 260, 1982. 35. Kessel, D. Transport of a nonphosphorylated nucleoside, 5'-deoxyadenosine, and transport in Chinese hamster ovary and Novikoff rat hepatoma cells. J. Cell. Physiol., 776: 247-255, 1983. by murine leukemia L1210 cells. J. Biol. Chem., 253: 400-403, 1978. 14. Plagemann, P. G. W., Wohlhueter, R. M., and Erbe, J. Nucleoside transport in 36. Toohey, J. I. Methylthioadenosine nucleoside phosphorylase deficiency in human erythrocytes. A simple carrier with directional symmetry and differential methylthio dependent cancer cells. Biochem. Biophys. Res. Commun., 83:27- mobility of loaded and empty carrier. J. Biol. Chem., 257:12069-12074,1982. 35,1978. 15. Plagemann, P. G. W., Marz, R., and Erbe, J. Transport and countertransport 37. Carson, D. A., Kaye, J., and Wassen, D. B. The potential importance of soluble of thymidine in ATP-depleted and thymidine kinase-deficient Novikoff rat deoxynucleotidase activity in mediating deoxyadenosine toxicity in human hepatoma and mouse L-cells: evidence for a high Kmfacilitated diffusion system lymphoblasts. J. Immunol., 726: 48-352,1981.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6424

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. Effects of Nucleoside Transport Inhibitors on the Salvage and Toxicity of Adenosine and Deoxyadenosine in L1210 and P388 Mouse Leukemia Cells

Peter G. W. Plagemann and Robert M. Wohlhueter

Cancer Res 1985;45:6418-6424.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/12_Part_1/6418

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/45/12_Part_1/6418. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.