Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

130

Biochimica et Biophysica Acta 928 (1987) 130-136 Elsevier

BBA12015

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells Jean-Louis P6rignon, Dominique M. Bories, Anne-Marie Houllier, Laure Thuillier and Pierre H. Cartier Laboratoire de Biochimie, INSERM U75, Facult~ de M~decine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15 (France)

(Received 30 July 1986) (Revised manuscript received 16 December 1986)

Key words: Pyrimidine metabofism; Uridine phosphorylase; Thymidine phosphorylase; (Human lymphoid cell)

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidinenucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides. Introduction The fact that the intermediary metabolism of nucleosides and nucleotides is essential for the Correspondence: J.-L. P6rignon, Laboratoire de Biochimie, INSERM U75, Facult6 de M&tecine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.

maturation and functions of lymphocytes is illustrated by the severe immunodeficiencies associated with adenosine deaminase (EC 3.5.4.4) and purine-nucleoside phosphorylase (EC 2.4.2.1) deficiencies [1,2]. This prompted us to study the intermediary metabolism of pyrimidine nucleosides in human lymphocytes, lymphoblasts and lymphoblastoid cell lines. In previous studies, we

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

131 demonstrated the existence of marked deficiency of cytidine deaminase (EC 3.5.4.5) activity in the lymphoblasts of most patients with acute lymphoblastic leukemia [3] and a total deficiency of the same activity in cell lines derived from Burkitt's lymphoma and in Epstein-Barr virus infected B lymphoblastoid cell lines [4]. In the present work, we investigated the anabolism of pyrimidine bases in intact human lymphoid cells. Since mammalian cells have little, if any, ability to metabolize uracil into UMP by phosphoribosyltransfer [5], we focused our study on the synthesis of pyrimidine nucleosides either by the action of phosphorylases on pyrimidines bases, or by ribosyl or deoxyribosyl transfer between purine and pyrimidine nucleosides. We conducted this study in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in Raji cells (lymphoblasts derived from a Burkitt's lymphoma). The ability of the cells to synthesize uridine from various bases and nucleosides was estimated from their ability to grow on these compounds in the presence of an inhibitor of pyrimidine 'de novo' synthesis (pyrazofurin); their ability to synthesize deoxyuridine and thymidine was estimated from the inhibition of the incorporation of radiolabelled thymidine in the presence of unlabelled metabolic precursors. Our results indicate that the two types of cell have a different, but generally low, capacity to synthesize uridine, deoxynridine and thymidine from combinations of bases and nucleosides, and suggest that the pyrimidine-nucleoside phosphorylases have, physiologically, a catabolic rather than anabolic role. Materials and Methods Materials. Pyrazofurin (3,fl-D-ribofuranosyl,4hydroxypyrazole-5-carboxamide) was from Calbiochem (Los Angeles, CA). The pyrimidine and purine bases, ribo- and 2'-deoxyribonucleosides, ribose 1-phosphate and deoxyribose 1-phosphate were obtained from Boehringer (Mannheim, F.R.G.) or Sigma (St. Louis, MO). [6-3H]Thymidine (1 Ci/mmol), [2-14C]cytidine (43 m C i / mmol), [2-14C]uracil and [8-14C]hypoxanthine were purchased from CEA (Gif-sur-Yvette, France). 3MM and DE 81 chromatographic paper was obtained from Whatman (Maidstone, U.K.) and

TLC plastic sheets coated with PEI-cellulose were from Merck (Darmstadt, F.R.G.). Raji cells and fetal calf serum were purchased from Flow Laboratories (Irvine, U.K.), RPMI 1640 medium was from Gibco (Grand Island, NY) and phytohemagglutinin P was from Difco Laboratories (Detroit, MI). All the enzyme activities were determined by radioisotopic assays. Thymidine phosphorylase activity (EC 2.4.2.4) was assayed by the conversion of thymidine to thymine; the incubation medium consisted of 100 mM Tris-acetate (pH 6.0), 12.5 mM potassium phosphate (pH 6.0), 0.75 mM [63H]thymidine and cell extract in a final vol. of 200 /~1. The reaction was run at 37°C. At various times, 50-ttl portions were taken and the reaction was stopped by deproteinization at 96°C for 2 rain. After centrifugation, 20 #1 of the supernatant were co-chromatographed with unlabelled thymidine and thymine on PEI-cellulose plastic sheets with 0.2 M NaC1/1% NH4OH as the eluent. The spots corresponding to each compound were visualized under ultraviolet light, cut out and counted in toluene/PPO (4 g/1)/POPOP (200 mg/1) in a Packard Tri-Carb liquid scintillation spectrometer. Uridine phosphorylase activity (EC 2.4.2.3) was assayed by the conversion of uracil to uridine; the incubation medium consisted of 55 mM Tris-HC1 (pH 7.4), 6 mM ribose 1-phosphate, 0.2 mM [2-14C]uracil and cell extract in a final vol. of 200 #1. The reaction was run and stopped as described above. Uridine and uracil were separated by descending chromatography on Whatman 1 paper with 5 M ammonium acetate (pH 9.0), saturated sodium tetraborate, ethanol and 5 mM EDTA (10 : 40 : 90 : 50, v/v), and counted as described above. The assay of deoxyuridine phosphorylase activity was the same, except that the reaction mixture contained 6 mM deoxyribose 1phosphate instead of ribose 1-phosphate. Purinenucleoside phosphorylase was assayed as described previously [6], except that hypoxanthine and inosine were separated by descending chromatography on Whatman 3MM paper, eluted with 0.05 mM EDTA/0.5 M NH4OH , according to Uitendaal et al. [7]. The assay of cytidine deaminase activity has been previously described [3]. Human lymphocytes were separated from

132 heparinized venous blood by Ficoll-Hypaque gradient centrifugation and washed twice in Hanks balanced salts solution. The cells were cultured in a medium consisting of RPMI 1640 medium supplemented with 33 mM Hepes, 300 mg/1 Lglutamine, 10% fetal calf serum, penicillin (100 U / m l ) and streptomycin (100 gg/ml). For studies of thymidine incorporation, the cellular suspension of lymphocytes was adjusted to 1.10 6 cells/ml, and 0.2 ml aliquots were distributed in triplicate into 96-well microtiter plates (3040 Falcon Labware, Oxnard, CA). Phytohemagglutinin (final dilution 1/700) and the other effectors were added at culture initiation. The cellular suspension of Raji cells was adjusted to 1-105 cells/m l, and similarly distributed in triplicate into microtiter plates. After 48-72 h of incubation at 37°C in a humidified atmosphere of 5% CO in air, cultures were pulsed for 6 h with 1 gCi/well of [6-3 H]thymidine, and the radioactivity incorporated into DNA was quantitated as previously described [8] (preliminary experiments had shown that, under these conditions, the incorporation of thymidine was linear with time up to 18 h). The culture conditions for the study of cellular proiiferation of Raji cells were the same as above; 5-10 ml of the cellular suspension (1 • 105 cells/ mi) were distributed in 50-ml Falcon flasks, and after 72 h of incubation, aliquots were taken from

each culture to determine the cell number by optic microscopy and cellular viability by the trypan blue exclusion method. The pathways of the intermediary metabolism of nucleosides in cultured cells were estimated either by an isotopic dilution method or by their ability to restore the growth of cells in the presence of pyrazofurin, an inhibitor of pyrimidine synthesis de novo. The isotopic dilution method is an adaptation of the 'deoxyuridine-suppression test' [9], widely used in the investigation of megaloblastic anemias. In their critical study of the mechanism of this test, Pelliniemi and Beck [10] who worked, like ourselves, on phytohemagglutinin-stimulated human peripheral blood lymphocytes, demonstrated that the inhibition by unlabelled deoxyuridine of the incorporation of labelled thymidine subsequently added in the culture medium was due only in part to an inhibition of thymidine transport, and implicated also the expansion of intracellular pools of unlabelled thymidine deoxyribonucleotides and the inhibition of thymidine kinase by thymidine triphosphate and deoxyuridine. We thus hypothesized that the intracellular synthesis of unlabelled thymidine and deoxyuridine from the corresponding bases and deoxyribose 1-phosphate should similarly lead to an inhibition of the incor~ poration of labelled thymidine by the latter mech-

TABLE I ENZYME ACTIVITIESINVOLVEDIN THE SYNTHESISOF PYRIMIDINERIBO- AND DEOXYRIBONUCLEOSIDESBY RIBO- AND DEOXYRIBO-TRANSFERFROM INOSINEOR DEOXYINOSINE purine-nucleosidephosphorylase (1)

dIno Hyp~

dR1P)

(pi

V..

(Thy

~dThd

thymidine phosphorylase purine-nucleosidephosphorylase

(3)

dIn~y/

Pi

2:~dUrd

deoxyuridinephosphorylase

Hyp)

(RIP)

QUra

Purine-nucleosidephosphorylase

Ino (4)

Pi

Ino )

(dUrd

dIno

~kUrd

Urd

uridine phosphorylase purine-nucleosidephosphorylase deoxyuridinephosphorylase uridine phosphorylase

Abbreviations: Thy, thymine; Ura, uracil; dThd, thymidine; Urd, uridine; dUrd, deoxyuridine; Hyp, hypoxanthine; Ino, inosine; dIno, deoxyinosine;RIP, ribose 1-phosphate; dRIP, deoxyribose1-phosphate; Pi, inorganic phosphate.

133 anisms. Since intact cells are not permeable to phosphorylated sugars, the synthesis of pyrimidine deoxyribonucleosides from thymine and uracil was tested with a purine deoxyribonucleoside (deoxyinosine) as a donor of deoxyribose 1-phosphate. The synthesis of cytidine and deoxycytidine from cytosine was not investigated because of the absence of cytidine/deoxycytidine phosphorylase(s) in mammalian cells [11]. The reactions of deoxyribosyl transfer tested, as well as the corresponding enzymatic activities, are mentioned in Table 1. Although a direct transfer of ribose and deoxyribose between nucleosides has been reported for human cells [12], we mention in this table only the indirect mechanism of deoxyribosyl transfer which has recently been well documented for the thymidine phosphorylase from mouse liver [13]. When pyrimidine de novo synthesis is inhibited by pyrazofurin, the cell proliferation depends entirely on the synthesis of U M P from uridine [8]. It is thus possible to estimate the ability of the cells to synthesize uridine either from uracil, with inosine as a donor of ribose 1-phosphate, or by an exchange of bases between deoxyuridine and inosine as mentioned in Table I.

Results

Metabolism of exogenously added pyrimidine bases and nucleosides The incubations with thymidine alone (Table II) show that 10 -4 M thymidine inhibits the incorporation of the radiolabelled nucleoside in phytohemagglutinin-stimulated lymphocytes by only 50%, versus 91% in Raji cells; the inhibition is almost total with 10 -3 M thymidine in both types of cell. This indicates that lymphocytes are less able to accumulate thymidine triphosphate than are Raji cells. Similarly (Table I1), the suppression by unlabelled deoxyuridine of the incorporation of radiolabelled thymidine is much lower in lymphocytes (where 10 -4 M deoxyuridine is not significantly efficient) than in Raji cells. This indicates that lymphocytes are less able to synthesize a n d / o r to accumulate thymine deoxyribonucleotides from deoxyuridine than are Raji cells. The incubations with uracil in the presence of pyrazofurin (Table III) show that neither type of cell is able to synthesize U M P from uracil, which indicates that these cells have no efficient uracil-phosphoribosyltransferase activity. In contrast, in both cases the cell growth was restored by

TABLE II SYNTHESIS OF DEOXYURIDINEAND THYMIDINE FROM URACIL OR THYMINE AND DEOXYINOSINE The effectors were added, at the indicated concentrations, at the beginning of the culture. After 48 or 72 h, cultures were pulsed with [6-3H]thymidine; the decrease of the incorporation of labelled thymidine correlates with the synthesis of unlabelled deoxyuridine or thymidine from the precursors. The results are expressed as the percentage of radioactivityincorporated by the cells in the absence of effectors. All the determinations were made in triplicate; data represent mean+ S.D. for 4-6 independent experiments, as indicated. The incorporation of thymidine ranged from 31970+3270 to 57572+6323 for the lymphocytes and from 30724+3991 to 52949-i-7928 for Raji cells. The abbreviations are the same as those in Table I. Cells

Lymphocytes (n = 5)

Concentration of effectors 10-4 M

Percentof [6-3H]thymidineincorporation in control cultures Ura

dUrd

dlno

Ura + dlno

97.5+16.9 83.2+17.2 125.7+19.8 116.7+16.8

Lymphocytes 10-3M (n = 6)

107.9+16.4 29.4+ 8.2

143.2+18.6

Raji (n = 5)

10-4M

103.5+ 4.4

132.5+28.8 122.0-t-24.4

Raji (n = 4)

10-3M

89.0+11.6

48.3+ 7.4 9.5+ 0.5

95.4+ 5.3

Thy

dThd

64.6+15.0 50.6+4.2

58.2+11.7 124.7+20.7 76.3+22.2

114.5-t-10.2 113.2+14.7

1.7+0.6 8.6+2.2

Thy + dlno 64.2+13.8 22.0+ 6.5 131.4+21.3

0.5+0.1 104.0-1-8.5

134 T A B L E III SYNTHESIS O F U R I D I N E F R O M U R A C I L A N D I N O S I N E A N D F R O M D E O X Y U R I D I N E A N D I N O S I N E Raji cells and phytohemagglutinin-stimulated h u m a n lymphocytes were cultured in the presence of 5.10 -5 M pyrazofurin (PF), an inhibitor of pyrimidine de novo synthesis. The effectors were added at the indicated concentrations, at the beginning of the culture. After 48 or 72 h, the cell growth was estimated either by the incorporation of [6-3 H]thymidine (first four lines) or by counting viable cells (last line). The results are expressed as the percentage of growth of cells cultured in the absence of pyrazofurin or effectors. Data represent mean 4-S.D. of 3 - 6 independent experiments, as indicated. The incorporation of thymidine ranged from 31970± 3 270 to 57 572 4- 6 323 for the lymphocytes and from 30 724 + 3 991 to 52 949 4- 7 928 for Raji cells. The abbreviations are the same as those in Table I. Cells

Concentration of effectors

Percent of cell growth in control cultures PF 5.10- 5 M

PF + Urd

PF + Ura

PF + dUrd

PF + Ino

PF + Ino + Ura

PF + Ino + dUrd

Lymphocytes (n = 6)

10 - 4 M

27.9 _+11.9

77.2 4-11.6

29.0 -+ 14.5

16.6 + 8.2

30.0 -+ 14.4

31.1 -+ 13.6

16.9 + 10.9

Lymphocytes ( n = 3)

10 3 M

12.1 + 3.1

80.9 _+37.7

14.1 + 3.6

3.7 +_ 1.5

11.1 4- 2.0

30.1 _+8.1

12.5 4-11.8

Raji (n = 5)

10 4 M

17.5 4- 5.7

101.6 + 17.5

17.6 + 2.7

6.6 4- 2.2

16.6 + 7.5

22.0 + 7.2

9.0 4- 5.8

Raji (n=4)

10 -3 M

15.1 4-3.4

62.1 4-10.4

17.5 4-4.2

1.3 4-0.5

12.2 +3.6

44.3 4-5.0

1.2 4-0.2

Raji * (n=5)

10 -3 M

25.9 4-8.5

94.9 -+12.2

28.1 4-6.9

22.1 -+3.7

26.0 -+6.6

32.1 4-10.5

24.0 4-7.3

* Cellular viability was determined by the trypan blue exclusion method; the cells were counted by optic microscopy.

the addition of uridine, which indicates an efficient salvage of uridine into UMP by uridine kinase activity. The fact that 10-3 M uridine had an inhibitory effect on the incorporation of thymidine in Raji cells (Table III, fourth line), whereas it restored almost completely the cell growth (Table III, fifth line) is indicative of an effect of uridine on thymidine transport a n d / o r of isotopic dilution by unlabelled uridine due to the synthesis of thymine deoxyribonucleotides from UMP via ribonucleoside-diphosphate reductase (EC 1.17.4.1) and thymidylate synthase (EC 2.1.1.b).

radiolabelled thymidine in lymphocytes, whereas it was ineffective in Raji cells (Table II). This indicates that lymphocytes can salvage thymine to thymidine by deoxyribosyl transfer (involving thymidine phosphorylase and purine-nucleoside phosphorylase activities), contrary to Raji cells. The ability of the lymphocytes to salvage uracil to deoxyuridine by deoxyribosyl transfer is less important than by thymine (at a concentration of 10 -3 M effectors, the incorporation of labelled thymidine was inhibited by 42%); also in this case, Raji cells were unable to synthesize the deoxyribonucleoside from the base with deoxyinosine as a donor of deoxyribose 1-phosphate.

Synthesis of thymidine and deoxyuridine Thymine, uracil and deoxyinosine alone did not inhibit the incorporation of labelled thymidine into DNA, indicating that these compounds did not inhibit the uptake of thymidine. The association of thymine and deoxyinosine at final concentrations of 10 -4 M and 10 -3 M inhibited by 36% and 78%, respectively, the incorporation of

Synthesis of uridine Neither the association of uracil and inosine, nor that of deoxyuridine and inosine, could restore the growth of lymphocytes or of Raji cells, as estimated from the incorporation of radiolabelled thymidine or by cell counting (Table III). This indicates that the synthesis of uridine from

135 TABLE IV ACTIVITIES OF P Y R I M I D I N E A N D P U R I N E - N U C L E O S I D E PHOSPHORYLASES IN H U M A N PERIPHERAL BLOOD LYMPHOCYTES A N D RAJI CELLS Data represent mean ___S.D. and [range]. (nmol- min :- 10 - 6 cells) thymidine phosphorylase

deoxyuridine phosphorylase

uridine phosphorylase

purine-nucleoside phosphorylase

Lymphocytes

1.06 + 0.71 [0.236-2.67] (n = 42)

1.23 _+0.54 [0.77-2.03] (n = 5)

0.081 + 0.033 [0.028-0.130] (n = 24)

16.4 + 7.5 [7.0-41.0] (n = 29)

Raji

0.012 + 0.003 (n = 7)

0.009 + 0.003 (n =11)

0.021 _+0.008 (n =18)

30.2 _+11.2 (n =10)

uracil by uridine phosphorylase, or by the association of uridine phosphorylase and deoxyuridine phosphorylase, is inefficient in either type of cell. Purine and pyrimidine phosphorylases actioities in cell extracts

Since ribo- and deoxyriboribosyl transfer reactions depend on the activities of purine and pyrimidine-nucleoside phosphorylases, these were assayed in cell extracts of lymphocytes and Raji cells (Table IV). Purine-nucleoside phosphorylase activity is high in both types of cell, and higher in Raji cells. On the other hand, Raji cells decreased uridine phosphorylase activity (26% of lymphocytes) and are markedly deficient in thymidine and deoxyuridine phosphorylase activities (1.1 and 0.7% of the lymphocyte activities, respectively). It should be pointed out that deoxyuridine has been reported to be a substrate for both uridine phosphorylase and thymidine phosphorylase [14]; the 'deoxyuridine phosphorylase' activity that we measured may, thus, be due to either of these two enzymes (or both). However, the fact that, in Raji cells as well as in lymphocytes, its activity is quite similar to that of thymidine phosphorylase, strongly suggests that, in human lymphoid cells, the deoxyuridine phosphorylase activity is due to the thymidine phosphorylase eniyme. Discussion

The purpose of this study was to investigate the ability of cultured human lymphoid cells to syn-

thesize pyrimidine nucleosides from uracil a n d thymine. When de novo pyrimidine synthesis was inhibited by pyrazofurin, the addition to the culture medium of uracil and of a donor of ribose 1-phosphate could not support the proliferation of phytohemagglutinin-stimulated lymphocytes nor that of Raji cells, despite the presence of uridine phosphorylase and purine-nucleoside phosphorylase activities in cell extracts. Simil~ results have been obtained by Scott Mc Ivor et al. [15] in Novikoff hepatoma cells; the discrepancy between the measurements of enzyme activities in cell extracts, and the results obtained with cultured cells, was attributed, by these authors, to the high Km of uridine phosphorylase with respect to uracil, and to the presence of relatively high concentrations of inorganic phosphate (5-10 mM) in cultured cells, which could act as an inhibitor of uridine synthesis by uridine phosphorylase. Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases and have a low (uridine phosphorylase) or very low (thymidine phosphorylase/ deoxyuridine phosphorylase) pyrimidine-nucleoside phosphorylase activity. Lymphocytes have a higher pyrimidine-nucleoside phosphorylase activity, they can synthesize deoxyuridine and thymidine from uracil and thymine (with deoxyinosine as a donor of deoxyribose 1-phosphate), but only at high and non-physiological concentrations of precursors; they metabolize deoxyuridine and thymidine into nucleotides less efficiently than do Raji cells. Taken together, these results suggest

136 that proliferating l y m p h o i d cells efficiently salvage pyrimidine nucleosides, but do n o t salvage pyrmidine bases, and that the pyrimidine-nucleoside phosphorylases when they are present may, rather, have a catabolic role. In this respect, the m a r k e d decrease of t h y m i d i n e / d e o x y u r i d i n e phosphorylase activity in Raji cells should be c o m p a r e d to the decrease of thymidine phosphorylase in acute lymphoblastic leukemia [16] and in m a n y neoplastic tissues [17]. This finding is also similar to our previous description of cytidine deaminase deficiency in acute lymphoblastic leukemia and in Raji cells [3,4]. Such a decrease of enzymes catalyzing the catabolism of pyrimidine nucleosides in h u m a n lymphoblasts (blasts of acute lymphoblastic leukemia and Raji cells), could be interpreted as a means for rapidly proliferating cells to spare pyrimidine compounds, as suggested b y W e b e r [18]. The sparing of pyrimidine deoxyribonucleosides is also quite consistent with the hypothesis of the regulation of pyrimidine deoxyribonucleoside triphosphate pools by substrate cycles between deoxyribonucleosides and deoxyribonucleotides, as proposed by Reichard and co-workers [19].

Acknowledgement This work was supported b y a grant from the Facult6 de M&iecine Necker-Enfants Malades.

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