Proc. Nati. Acad. Sci. USA Vol. 83, pp. 986-990, February 1986 Cell Biology Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells (hydroxyurea/nucleoside incorporation/metabolic flux) VERA BIANCHI, ELISABET PONTIS, AND PETER REICHARD* Medical Nobel Institute, Department of Biochemistry I, Karolinska Institutet, Box 60400, S104 01 Stockholm, Sweden Contributed by Peter Reichard, September 23, 1985

ABSTRACT Degradation of pyrimidine deoxyribonucleo- but also resulted in a continued uptake and net phosphoryl- side triphosphates plays a major role in the regulation of their ation of deoxyuridine. This effect was secondary to a large pool sizes in 3T6 cells. During normal growth, these cells drop in the intracellular dUMP pool. We propose that excrete deoxyribonucleosides (mostly deoxyuridine) into the deoxyuridine and dUMP form a substrate cycle and that the medium. When DNA strand elongation is inhibited, de novo operative direction of this cycle is regulated by the intracel- synthesis of dCTP and dTTP continues, followed by degrada- lular concentration of dUMP. tion of the deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degra- AND dation of deoxyribonucleotides and leads to an influx of MATERIALS METHODS deoxyuridine from the medium. This effect appears to be Materials. Deoxy[6-3H]uridine (21 Ci/mmol; l Ci = 37 caused by a large drop in the size of the intracellular dUMP GBq) was from New England Nuclear Dupont, and [5- pool. We propose that substrate cycles, involving phosphoryl- 3H]cytidine (25-30 Ci/mmol) and [3H]dNTPs and [a-32P]- ation of deoxyribonucleosides by kinases and dephosphoryla- dNTPs were from Amersham. [5-3H]Cytidine was purified by tion of deoxyribonucleoside 5'-phosphates by a nucleotidase, HPLC before use to remove radiolysis products. Hydroxy- participate in the regulation of the size of pyrimidine deoxy- urea was from Sigma. ribonucleoside triphosphate pools by directing the flow of Incorporation of Labeled Nucleosides into Cells. Parallel deoxyribonucleosides across the cell membrane. While kinases cultures of 3T6 cells were set up and grown as described (8, are regulated mainly by allosteric effects, the activity of the 10). Also the general conditions ofthe experiments, including nucleotidase appears to be regulated by substrate concentra- medium changes, various additions to the media, and the tion. preparation of cells and media for analyses of isotope incorporation into nucleosides, nucleotides, and DNA have The de novo synthesis of the four deoxyribonucleoside been described. Changing the earlier protocol, however, we triphosphates (dNTPs), substrates for the replication of extracted acid-soluble pools from cells still attached to dishes DNA, occurs by reduction of ribonucleoside diphosphates with 2 ml of 60% methanol for 60 min at 0°C. The methanolic (1-3). dNTPs can also be formed by a salvage pathway via solution was poured offcarefully and processed as described phosphorylation of the corresponding deoxyribonucleosides (8). The remaining cells were dissolved in 2 ml of 0.3 M when these are available (4). Key enzymes of both de novo NaOH, kept at room temperature overnight, and used to and salvage pathways are subject to complicated allosteric measure isotope incorporation into DNA. controls. Maintenance of the proper balance between dNTP The size and the specific activity of dNTP pools were pools is apparently important for normal cell function, and its determined enzymatically (11-14). Isotope incorporation into perturbation may increase the mutability of cells and have dUMP and dTMP was measured after separation of the other genotoxic consequences (5, 6). Enzymes involved in nucleotides by HPLC on Nucleosil C18 columns by isocratic the degradation ofdNTPs also participate in the regulation of elution with 0.1 M triethylamine phosphate (pH 3.0). HPLC pools. During normal growth, mouse 3T6 fibroblasts in S was used also for the separation and analyses of deoxyribo- phase utilize for DNA synthesis only 75% of the dCDP nucleosides in the media (7). The in situ activity ofthymidyl- formed by reduction of CDP and excrete the remaining 25% ate synthase was measured by the release of 3H20 into the into the medium as deoxyribonucleosides, mostly as deoxy- medium from cells incubated with 5-3H-labeled nucleosides uridine (7). When DNA replication is blocked with (8, 15, 16). aphidicolin, dNTP turnover continues and all deoxyribonu- All experiments were made when the cells had reached a cleotides synthesized de novo are excreted as deoxyribonu- density of 0.5-1.0 x 106 cells per 5-cm dish, with 60-70% of cleosides (7). In contrast, hydroxyurea (8) and azidocytidine them being in S phase. All results are normalized for 106 cells. (9) inhibitors of ribonucleotide reduction almost completely abolish pyrimidine dNTP turnover. These results suggested RESULTS the existence of a mechanism regulating the degradation of General Plan of Experiments. Isotope was introduced into pyrimidine dNTPs in relation to ongoing dNTP synthesis and the metabolism of pyrimidine dNTPs from either [5- DNA replication. Substrate cycles between deoxyribonucle- 3H]cytidine (Fig. LA) or deoxy[6-3H]uridine (Fig. 1B). In the osides and deoxyribonucleotides were suggested to be in- former case, the dCTP pool attained a constant specific volved in such a mechanism (7). activity after 60 min. From there on, the amount of isotope Here we explore the possible effects of hydroxyurea on entering dCTP by reduction of CDP equaled the amount substrate cycles in S-phase 3T6 cells. Treatment with the leaving the pool to be incorporated into DNA as dCMP plus drug not only blocked the degradation of pyrimidine dNTPs the amount released into the medium as deoxycytidine,

The publication costs of this article were defrayed in part by page charge Abbreviations: dNTP, deoxyribonucleoside triphosphate; DP, dipy- payment. This article must therefore be hereby marked "advertisement" ridamole. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom correspondence should be addressed. 986 Downloaded by guest on October 1, 2021 Cell Biology: Bianchi et al. Proc. Natl. Acad. Sci. USA 83 (1986) 987

[5-3HjCytidine In a second experiment we determined the effect of + HU __0-[ hydroxyurea on the outflow of isotope from dCTP into the A CDP dCDP....dCMP ..,,dUMP.4 dTMP various end products indicated in Fig. LA. Cells were prela- beled for 60 min with cytidine, hydroxyurea was added, and, IdCydi j 10 min after addition of the drug, the labeled medium was replaced with conditioned medium (10) containing cold cy- Deoxy[6-3H]uridine tidine and hydroxyurea. During 60 min of continued incuba- B tion, we determined the accumulation of isotope in DNA and dUMP , dTMP DNA in the medium as well as the loss of isotope from dCTP. Fig. 3 Left shows the results from the control (no hydroxyurea). Because ofthe presence ofa large intracellular prelabeled pool of cytidine phosphates, isotopic dCTP was FIG. 1. Incorporation of isotope from [5-3H]cytidine (A) or regenerated continuously also when labeled cytidine was no deoxy[6-3H]uridine (B) into various end products of pyrimidine longer present in the medium. The specific activity of the dNTP metabolism. Hydroxyurea (HU) inhibits the incorporation of dCTP pool decreased linearly during the 60-min period, with cytidine by blocking the reduction of CDP. an average value of 245 cpm/pmol. We used this value to transform the cpm recorded in Fig. 3 into pmol. Thus, a total deoxyuridine, or 3H20. 3H20 arises during the methylation of of 235,000 cpm was incorporated into DNA during 60 min, [5-3H]dUMP and represents the total amount of dTMP corresponding to the incorporation of 235,000/245 x 60 = 16 formed from dCMP, irrespective of whether dTMP is incor- pmol of dCMP per min into DNA. By similar calculations we porated into DNA or degraded to thymidine and excreted into find that dCTP was degraded to deoxycytidine at a rate of 1.4 the medium. With deoxy[6-3H]uridine, isotope equilibrium pmol/min and to deoxyuridine at a rate of 5.0 pmol/min. was attained more rapidly, the specific activity ofdTTP being Finally, dTMP (= 3H20) was formed from dCMP at a rate of stable after 15 min. During the steady state, the isotope that 10 pmol/min. An estimated 10% was degraded (8) and left the dTTP pool accumulated as dTMP in DNA and as excreted into the medium as thymidine, while the rest was thymidine in the medium (Fig. 1B). incorporated into DNA. Addition of hydroxyurea blocks ribonucleotide reductase Fig. 3 Right shows the flow of isotope from dCTP into the (17-19) and results in an almost immediate cessation of DNA various end products in the presence of hydroxyurea. In this replication. In our experiments we investigated the effect of case the reduction ofCDP was blocked, and isotope no longer the drug on the flow of isotope through pyrimidine dNTPs was incorporated into dCTP. The average specific activity of according to the schemes shown in Fig. 1. the dCTP pool during the 60 min in cold medium was 220 Experiments with [5-3H]Cytidine. In a first experiment (Fig. cpm/pmol. From the total radioactivity incorporated into the 2), we investigated the effects of hydroxyurea on the size of various deoxyribonucleosides in the medium, we calculated the four dNTP pools and on the turnover ofdCTP. During the that dCTP was degraded to deoxycytidine at a rate of 0.6 60 min of the experiment, both the dATP and dGTP pools pmol/min, to deoxyuridine at a rate of 0.3 pmol/min, and to decreased continuously in size, while the size of the dCTP thymidine at a rate of 1.0 pmol/min. In the calculation of the pool was halved during the first 10 min and then did not last value we assumed that all of the dTMP (3H20) was change further (Fig. 2 Left). The dTTP pool expanded and degraded to thymidine, since hydroxyurea had stopped DNA more than doubled. The specific activity of the dCTP pool synthesis completely as judged from the insignificant incor- and its total radioactivity decreased also when the size of the poration of dCMP into DNA (data not shown). pool no longer changed (Fig. 2 Right). This indicates a slow outflow of isotope from the pool, balanced by resynthesis from a nonlabeled source, possibly deoxycytidine in the 400. medium. x 300 x E i c. C. IC s ^200 200 2 l:w c 0: 1 0 *-I ._ 150

-o 0 -0 CZ

ceO. 100 CZ *-x - °Cem ' -N =X 50 7; 100. 1 0.x 0 'oCZ u 0 Ce Ha. f0 'H u aC 20 40 70 30 70 0 A 1T0 z 4\A%~~~ Time, minA

FIG. 3. Effects of hydroxyurea on the flow of isotope from 10 30 60 10 30 60 prelabeled dCTP into metabolic end products. Two parallel sets of Time, min cultures were labeled from 0.3 AM [5-3H]cytidine during 60 min. One set (Left) served as a control, while the other (Right) received 3 mM FIG. 2. Effects of hydroxyurea on the size of the 4 dNTP pools hydroxyurea at time 0. After 10 min (arrowheads), the media of both (Left) and the turnover of the dCTP pool (Right). The cells were sets were replaced with conditioned medium containing 0.3 ,uM cold labeled for 60 min with 0.3 uM [5-3H]cytidine before addition of 3 cytidine and, in the set represented by Right, 3 mM hydroxyurea. mM hydroxyurea at time 0. At that time, 106 cells contained 88 pmol During the following 60 min, we determined the flow of isotope from ofdTTP (o), 100 pmol ofdCTP (e), 20 pmol ofdGTP (A), and 18 pmol dCTP into the various metabolic end products shown in boxes in Fig. of dATP (A). (Left) Percentage changes in pool size during 60 min 1A. The appearance of 3H in water measures the methylation of after addition of hydroxyurea. (Right) Specific activity of dCTP (-o-) dUMP formed from dCTP. Note the difference in scale for total and the total radioactivity present in the dCTP pool (e) during the radioactivity between the two panels. e, DNA; o, dCTP; v, 3H20; same time period. o, dUrd; *, dCyd. Downloaded by guest on October 1, 2021 988 Cell Biology: Bianchi et al. Proc. Natl. Acad. Sci. USA 83 (1986)

Results concerning the effect of hydroxyurea on dCTP 150 degradation are summarized in the last three columns of Table 1. The drug decreased the formation ofdeoxyuridine to ° 100 O 5% and that of deoxycytidine to 46% but did not change x x the values indicate that dCTP thymidine excretion. Together, E 50 degradation was decreased to 25%. c) C.;^ Experiments with Deoxy[63H]uridine. The experiments de- +- scribed in the previous section focused on the effect of C.) *.) hydroxyurea on the catabolism of dCTP and led to the Ce Ce conclusion that the drug strongly inhibited the excretion of 0 "0 deoxyuridine, the major breakdown product in normal cells. Cect In this section we describe the effect of hydroxyurea on the - 4- anabolism of deoxyuridine. od -4 Deoxy[6-3H]uridine was added to four parallel sets of cultures. Two served as controls, and two received hydroxy- urea together with deoxyuridine. One control and one hydroxyurea set contained in addition dipyridamole (DP), an Time, min inhibitor of nucleoside transport (20), at a concentration that inhibited the incorporation of 0.3 AM thymidine into the dTTP pool by about 90% without affecting cell growth (data O 0 not shown). All cultures had received fresh medium before '-4q addition of isotope to remove nonlabeled deoxyribonucleo- x x sides accumulated in the medium during previous growth. Incorporation ofisotope into DNA, intracellular nucleotides, .) and extracellular thymidine was measured during a 60-min C.) period and is shown in Fig. 4, with Fig. 4 Upper representing -0 Cd Ce the two controls; Fig. 4 Lower, the two hydroxyurea sets; 0 0 "0 Fig. 4 Left, data without DP addition; Fig. 4 Right, data with Ceu 5-0 DP addition. - In the controls, a steady state was rapidly attained, with the o0 specific activity of the dTTP pools reaching values of 1300 F- cpm/pmol (without DP) and 150 cpm/pmol (with DP). Since the specific activity of deoxyuridine in the medium was 10 30 60 known, it was possible to calculate the contribution of the Time, mmn deoxyuridine phosphorylation pathway to total dTTP syn- thesis simply by dividing the specific activity of the dTTP FIG. 4. Effects of hydroxyurea on the metabolism of deoxy[6- pool with that of deoxyuridine (17,000 cpm/pmol). We then 3H]uridine. Four parallel sets of cultures were shifted to fresh that the cells under conditions synthesized medium containing dialyzed horse serum 90 min before addition of found steady-state isotope. Sixty minutes later two sets (Right) received 5 ,uM 7.6% (without DP) or 1.4% (with DP) of their dTTP from dipyridamole, an inhibitor of nucleoside transport (20), and two sets deoxyuridine. (Left) did not. After an additional 30 min (at time 0) all four sets Also the dUMP and dTMP pools rapidly attained isotopic received 0.3 AM deoxy[6_3H]uridine with (Lower) and without equilibrium, and we assumed that their specific activities (Upper) 3 mM hydroxyurea. During the following 60-min period, we under steady-state conditions were identical to that of the determined incorporation of 3H into DNA, into intracellular dTTP, dTTP pool. When we calculated the size of these pools from dTMP, and dUMP, and into extracellular thymidine. The amount of their total radioactivities as described above, the following isotope in extracellular thymidine was too low to be measurable in values for the 30-min time point were found: dUMP, 15 pmol the presence ofdipyridamole. Note the difference in scales in the four (without DP) or 32 pmol (with DP); dTMP, 8 pmol (without panels. ---, dTTP specific activity; -, total radioactivity; *, DNA; DP) or 9 pmol (with DP). o and *, dTTP; o, dThd; v, dUMP; A, dTMP. The major part ofdTTP turnover was accounted for by the incorporation of dTMP into DNA (18.2 pmol/min, irrespec- deoxyuridine, the nucleoside was phosphorylated at a rate of tive of the presence of DP). A small amount (0.7 pmol/min) 1.4 pmol/min under steady-state conditions. However, the was degraded to thymidine. Thus, the total turnover ofdTTP phosphorylation rate during the first 10 min was faster. was 18.9 pmol/min. Since 7.6% of dTTP originated from During this time, before isotopic equilibrium had been es- tablished, the cells incorporated 214,000 cpm into DNA, 160,000 cpm into dTTP and 12,000 cpm into dTMP and dUMP Table 1. Summary of the effects of hydroxyurea (HU) on pool (Fig. 4 Upper Left). By dividing the sum of these values with sizes and excretion of deoxyribonucleosides 17,000 (the specific activity of deoxyuridine), a phosphoryl- Pool size,* pmol Excretion, pmol/min ation rate of 2.3 pmol/min was obtained. Addition per 106 cells per 106 cells Hydroxyurea prevented the attainment of a steady state, to dUMPt dThd dUrdt both in the absence (Fig. 4 Lower Left) and presence (Fig. 4 medium dTTPt dTMPt dCydt Lower Right) of DP. The specific activity of the dTTP pool None 106 8 15 0.7t 5.0 1.3 increased continuously during the 60 min of the experiment 1.0* and never reached a plateau value. At all time points, HU 199 10 0.3 0.5t 0.3 0.6 hydroxyurea increased the total amount of radioactivity 1.0t present as dTTP. This effect was particularly pronounced DP 109 9 32 - with DP when the pool, after a 60-min incubation with HU/DP 148 8 0.08 hydroxyurea, contained about 8 times more isotope than that *From 30-min values. of the control shown in Fig. 4 Upper Right. A small amount tFrom incorporation of deoxy[6_3H]uridine. of isotope (12,000 cpm) was incorporated into DNA in the tFrom incorporation of [5-3H]cytidine. absence of DP during the first 10 min, but from there on Downloaded by guest on October 1, 2021 Cell Biology: Bianchi et al. Proc. Natl. Acad. Sci. USA 83 (1986) 989 incorporation was negligible (data not shown). In the pres- continued its increase up to 60 min. A likely explanation for ence of hydroxyurea, the rate of deoxyuridine phosphoryl- this behavior is that the increase in pool size largely depended ation during the first 10 min was 1.9 pmol/min as compared on the phosphorylation of exogenous deoxyuridine and that to the value of2.3 pmol/min calculated above for the control. DP slowed down the internalization of the deoxyribonucleo- However, with hydroxyurea all isotope was incorporated side. The difference between the two curves reflects on the into nucleotide pools and not into DNA. Thus, it appears that rate of deoxyuridine phosphorylation and gives a rate of 4.7 the drug caused isotope to accumulate in an expanding dTTP pmol/min during the first 10 min. This value is higher than the pool instead of flowing through a pool of constant size on its rate calculated above from isotope data. way to DNA. Calculation of the size of the dTMP and dUMP pools and ofthe rate ofthymidine excretion was complicated by the fact DISCUSSION that the cells never reached a steady state in the presence of 3T6 cells obtain their supply ofdCTP and dTTP mainly by the hydroxyurea. Although it was probable that both dTMP and reduction of CDP (7, 8). Somewhat surprising, we found that thymidine rapidly equilibrated their isotope with dTTP and the synthesis of dNTPs during S phase was larger than their therefore had the same specific activity as dTTP, a similar demand for DNA synthesis and that the cells degraded the situation hardly applied to dUMP. In the controls during the surplus material to deoxyribonucleosides (7). When DNA steady state, isotope incorporated into dUMP from deoxy- replication was inhibited, dNTP synthesis continued and all uridine was constantly diluted by de novo synthesis of the material was degraded and excreted (7). The present work nucleotide and further channeled into dTTP. Since both illustrates the reverse situation: when de novo synthesis is dTTP and dUMP turned over rapidly, the specific activity of inhibited, degradation of dNTPs decreases greatly and the two pools soon became identical. The situation changed deoxyribonucleosides are imported into the cell from the drastically on addition of hydroxyurea. De novo synthesis of medium. dUMP ceased, and the dUMP pool soon attained the specific To explain these phenomena, we propose that substrate activity of deoxyuridine. The dTTP pool was not drained for cycles between deoxyribonucleosides and their 5' phos- DNA synthesis but expanded, and it never equilibrated with phates regulate the degradation of pyrimidine dNTPs and the dUMP. Therefore, in our calculations we assumed that the direction of flow of deoxyribonucleosides across the cell specific activities ofdTMP and thymidine were identical with membrane. In general, substrate cycles provide a sensitive that of dTTP but that the specific activity of dUMP equaled mechanism for the control of metabolic pathways. Their that of deoxyuridine in the medium. In this way we obtained participation in various aspects of carbohydrate metabolism the following values for the 30-min time point in the presence is well established, but substrate cycles also appear to of hydroxyurea: thymidine excretion, 0.5 pmol/min; dUMP function in the control ofmany other metabolic pathways (21, pool, 0.3 pmol (without DP) and 0.08 pmol (with DP); and 22), and the recent demonstration of an dTMP pool, 10 pmol (without DP) and 8 pmol (with DP). adenosine/AMP The results concerning the effect of hydroxyurea on the cycle in hepatocytes (23) is of particular interest in connec- size of nucleotide pools are summarized in the first three tion with our work. columns of Table 1. The most pronounced effect of the drug A model for the participation of a substrate cycle in the was a large decrease in the size of the dUMP pool. We might regulation of pyrimidine dNTP metabolism is presented in point out that, with the different assumption that dUMP had Fig. 6. A deoxyribonucleoside diphosphate synthesized de the same specific activity as dTTP, the size ofthe dUMP pool novo by reduction of the ribonucleotide equilibrates rapidly would have been 2.3 pmol in the presence of hydroxyurea, a with both its mono- and triphosphate. In rapidly growing and decrease of more than 83% compared to the control. It is evident that, irrespective ofour assumptions, the drug caused a large decrease in dUMP pool size. The kinetics of the expansion of the dTTP pool caused by addition ofhydroxyurea were strongly influenced by DP (Fig. DNA 5). Without DP, the size of the dTTP pool reached a plateau 1 1 value after 10 min and decreased slightly between 30 and 60 dNTP min. In the presence of DP, the pool expanded slowly and rNDP dNDP

~200 Ic dNMP ADP 44 t I " Pi dPyd ATP , ; 150

n_ 100 dPyd (extracellular) FIG. 6. A model for the involvement of a substrate cycle between a pyrimidine deoxyribonucleoside (dPyd) and its monophosphate in 10 30 60 the regulation of a dNTP pool. De novo synthesis of deoxyribonu- Time, min cleotides occurs at the diphosphate level by reduction of ribonucleotides. If synthesis exceeds demands, a situation that is FIG. 5. Effect ofdipyridamole on the expansion ofthe dTTP pool normal in growing 3T6 cells and becomes pronounced when DNA induced by hydroxyurea. Two parallel sets of cultures were shifted replication is blocked, the dNMP pool increases and the cycle leads to fresh medium containing dialyzed horse serum 90 min before to excretion of the deoxyribonucleoside. If de novo synthesis is addition of 0.3 ,M deoxy[6-3H]uridine and 3 mM hydroxyurea (time insufficient and deoxyribonucleotide pools decrease, the cycle op- 0). One set (o) had received 5 ,M dipyridamole 30 min before erates in the opposite direction and pulls available deoxyribonucle- addition of isotope, while the other (o) served as control. osides into the cell. Downloaded by guest on October 1, 2021 990 Cell Biology: Bianchi et al. Proc. Natl. Acad. Sci. USA 83 (1986) metabolizing cells, the triphosphate accumulates as the but its high Km value for dNMPs would make the enzyme dominating form, with deoxyuridine phosphates forming an exquisitely sensitive to changes in substrate concentration. exception. Because of the presence of a powerful dUTP Our experiments concern pyrimidine dNTPs. A control of nucleotidase (24, 25), only the monophosphate dUMP is the size of purine dNTP pools by catabolic enzymes some normally found in cells. A substrate cycle can either drain time ago was illuminated dramatically by the finding that material from the deoxyribonucleotide pool by dephospho- certain immune deficiency diseases in man are caused by the rylation of the monophosphate or add nucleotide by phos- genetic loss of adenosine deaminase or purine nucleoside phorylation of the deoxyribonucleoside. In either case the phosphorylase (34). It is possible that substrate cycles also extracellular deoxyribonucleoside is in rapid equilibrium may be involved in the regulation of purine dNTPs. with the cycle through a transmembrane carrier system (26, 27). Intracellular degradation leads to export of deoxyribo- This work was supported by grants from the Swedish Medical nucleosides, whereas intracellular phosphorylation leads to Research Council and the Wallenberg Foundation. V.B. is the import of deoxyribonucleosides. recipient of a Research Fellowship from the European Science Previous work (7, 8) provided the following data relevant Foundation, on leave from the University of Padova, Italy. to our model. (i) During normal growth, 3T6 cells exported 1. Thelander, L. & Reichard, P. (1979) Annu. Rev. Biochem. 48, quite large amounts of pyrimidine deoxyribonucleosides, 133-158. roughly two-thirds in the form of deoxyuridine, while the rest 2. Holmgren, A. (1981) Curr. Top. Cell. Regul. 19, 47-76. consisted of approximately equal amounts of deoxycytidine 3. Lammers, M. & Follmann, H. (1983) Struct. Bonding (Berlin) and thymidine. (ii) Inhibition of DNA strand elongation by 54, 27-91. aphidicolin did not stop de novo pyrimidine dNTP synthesis. 4. Kornberg, A. (1980) DNA Replication (Freeman, San Fran- Both dCTP and dTTP turned over with close-to-normal rates, cisco). resulting in an increased export of deoxyribonucleosides, 5. Kunz, B. A. (1982) Environ. Mutagen. 4, 695-725. mainly as thymidine and deoxycytidine. (iii) Inhibition of de 6. Meuth, M. (1984) Mutat. Res. 126, 107-112. 7. Nicander, B. & Reichard, P. (1985) J. Biol. Chem. 260, 9216- novo synthesis ofdNTPs with hydroxyurea greatly decreased 9222. the turnover ofpyrimidine dNTP pools. The dTTP pool more 8. Nicander, B. & Reichard, P. (1985) J. Biol. Chem. 260, 5376- than doubled. This paradox suggests synthesis of dTTP by 5381. salvage of preformed deoxyribonucleosides. 9. Akerblom, L. & Reichard, P. (1985) J. Biol. Chem. 260, 9197- Our experiments with hydroxyurea provide some insight 9202. into the regulation of substrate cycles, in particular the one 10. Nicander, B. & Reichard, P. (1983) Proc. NatI. Acad. Sci. involving deoxyuridine/dUMP. (i) Hydroxyurea blocks the USA 80, 1347-1351. excretion of deoxyuridine, while deoxycytidine and thymi- 11. Skoog, L. (1970) Eur. J. Biochem. 17, 202-208. dine are less affected. Since deoxyuridine is the major 12. Lindberg, U. & Skoog, L. (1970) Anal. Biochem. 34, 152-160. 13. Stimac, E., Housman, D. & Huberman, J. A. (1977) J. Mol. excretion product, the net effect of hydroxyurea is a depres- Biol. 115, 485-511. sion of dNTP degradation. (ii) Hydroxyurea increases the 14. Hellgren, D., Nilsson, S. & Reichard, P. (1979) Biochem. intracellular dTTP pool, largely at the expense of extracel- Biophys. Res. Commun. 88, 16-22. lular deoxyuridine. This effect is not caused by an increased 15. Tomich, P. K., Chiu, C.-S., Wovcha, M. G. & Greenberg, phosphorylation of deoxyuridine but by a decreased dephos- G. R. (1974) J. Biol. Chem. 249, 7613-7622. phorylation of dUMP. The expanding dTTP pool actually 16. Rode, W., Scanlon, K. J., Moroson, B. A. & Bertino, J. R. causes a feedback-inhibition ofthymidine kinase, the enzyme (1980) J. Biol. Chem. 255, 1305-1311. responsible for the phosphorylation of deoxyuridine. The 17. Young, C. W. & Hodas, S. (1964) Science 146, 1172-1174. with DP show that the system is not 18. Turner, M. K., Abrams, R. & Lieberman, I. (1966) J. Biol. experiments permeation Chem. 241, 5777-5780. affected by hydroxyurea but that it is required for the 19. Krakoff, I. H., Brown, N. C. & Reichard, P. (1968) Cancer expansion of the dTTP pool-i.e., dTTP is synthesized from Res. 28, 1559-1565. extracellular deoxyuridine. (iii) The decreased dephospho- 20. Scholtissek, C. (1968) Biochim. Biophys. Acta 158, 435-447. rylation of dUMP results from a large decrease in the size of 21. Newsholme, E. A., Challiss, R. A. J. & Crabtree, B. (1984) the dUMP pool. Trends Biochem. Sci. 9, 277-280. Two kinds of enzyme participate in the substrate cycle 22. Koshland, D. E., Jr. (1984) Trends Biochem. Sci. 9, 155-159. 23. Bontemps, F., Berghe, G. D. V. & Hers, H.-G. (1983) Proc. illustrated in Fig. 6: a kinase and a nucleotidase. Pyrimidine Natl. Acad. Sci. USA 80, 2829-2833. deoxyribonucleoside kinases are well-known enzymes (4, 24. Bertani, L. H., Haggmark, A. & Reichard, P. (1963) J. Biol. 28-31). Their activity is regulated allosterically. Less is Chem. 238, 3407-3413. known about the nature of the nucleotidase. A major con- 25. Williams, M. V. & Cheng, Y. (1979) J. Biol. Chem. 254, clusion from the present work is that the in situ ofthe 2897-2901. activity 26. Plagemann, P. G. W. & Wohlhueter, R. M. (1980) Curr. Top. enzyme degrading dUMP is regulated by substrate concen- Membr. Transp. 14, 225-330. tration. Also the decreased excretion of deoxycytidine after 27. Ullman, B., Kaur, K. & Watts, T. (1983) Mol. Cell. Biol. 3, addition of hydroxyurea can be explained by a parallel 1187-1196. decrease in nucleotide concentration. 28. Okazaki, R. & Kornberg, A. (1964) J. Biol. Chem. 239, a 269-275. Mammalian cells contain a cytosolic nucleotidase with 29. Maley, F. & Maley, G. F. (1962) Biochemistry 1, 847-851. preference for deoxyribonucleotides, which has been de- 30. Durham, J. P. & Ives, D. H. (1970) J. Biol. Chem. 245, scribed and studied in detail by Fritzon (32). This enzyme has 2276-2284. Km values for deoxyribonucleotides in the 10-3 M range. It 31. Hurley, M. C., Palella, I. D. & Fox, I. H. (1983) J. Biol. is present in 3T6 cells (33) and appears to be an excellent Chem. 258, 15021-15027. 32. Fritzon, P. (1978) Adv. Enzyme Regul. 16, 43-61. candidate for the nucleotidase activity ofthe substrate cycle. 33. Magnusson, G. (1971) Eur. J. Biochem. 20, 225-230. No evidence was obtained for an allosteric control of this 34. Martin, D. W., Jr., & Gelfand, E. W. (1981) Annu. Rev. enzyme by dNTPs (L. Hoglund and P.R., unpublished data), Biochem. 50, 845-877. Downloaded by guest on October 1, 2021