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Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.

Previous studies indicated that uridine is essentially cleared in a single pass through a rat liver and replaced in a highly regulated manner by uridine formed presumably by de novo synthesis. We report a cellular basis for the catabolic component of this apparent paradox by dissociation of the liver with collagenase into two cell fractions, hepatocytes and a nonparenchymal cell population. Suspensions of the nonparenchymal cells rapidly cleave uridine to uracil, whereas in hepatocytes this activity was <5% of that in nonparenchymal cells. Conversely, hepatocytes cause extensive degradation of uracil to -alanine. These differences correlate with the uridine phosphorylase and dihydrouracil dehydrogenase activity in cell-free extracts of each cell type. We have documented the existence of a Na+-dependent, nitrobenzylthioinosine-insensitive transport system for uridine in the parenchymal cells (Michaelis constant 46 +/- 5 microM) that achieves a three- to fourfold concentration gradient in hepatocytes. A similar system is present in the nonparenchymal cell population. In addition, a highly specific and active Na+-dependent transport system for beta-alanine, the primary catabolic metabolite of uracil, has been demonstrated in hepatocytes.

Purpose: To correlate changes in uridine transport and colony morphology with differentiation of human breast cancer cells by tamoxifen and related agents., Materials and Methods: Cultures of MCF-7 human breast cancer were treated with estradiol or the antiestrogen derivatives tamoxifen, hydroxytamoxifen, and ICI 164, 384. Initial rates of uridine transport and equilibrium concentrations were determined and morphological characteristics of the cultures evaluated., Results: Tamoxifen causes an early induction of a Na+ -dependent transport of uridine characteristic of normal epithelial cells but absent in normal MCF-7 cultures and most human neoplasms examined. The pure antiestrogen ICI 164,384 and the more potent 4-hydroxytamoxifen also induced concentrative uridine transport; estradiol could prevent the expression of this transporter. Associated with induction of transport was a dramatic increase in dome formation in the cultures, a measure of unidirectional inorganic ion transport characteristic of the differentiated state., Conclusions: The induction of a concentrative transport of uridine is a concomitant of cellular differentiation of breast tumor cells. These findings give added weight to evidence that uridine may play a regulatory role in the transition to the neoplastic state. The absence of the transporter and low intracellular uridine concentrations in the undifferentiated state may relate to 5-FU sensitivity of breast tumors. Induction of the transporter by tamoxifen and the consequent major increase in intracellular concentrations of free uridine suggests a potentially negative effect of tamoxifen on regimens containing 5-FU.

Uridine transport in undifferentiated HL-60 cells occurs primarily by facilitated diffusion, but a limited Na(+)-dependent process can be demonstrated (Km = 44 +/- 4.4 microM, Vmax = 0.13 +/- 0.01 microM/s). This latter transport system was inhibited by adenosine and inosine (Ki = 110 and 260 microM, respectively), whereas guanosine and thymidine were less effective (Ki = 1600 and 1200 microM, respectively). Dimethylsulfoxide (DMSO) caused a concentration-dependent decrease in facilitated uridine transport. This change was attributable to a decrease in the number of transporter molecules as determined by the binding of [3H]nitrobenzylthioinosine to cell membranes. Moreover, the Na(+)-dependent transport of uridine was enhanced by DMSO at a concentration of the polar solvent as low as 0.4%. When HL-60 cells were exposed to 1.0% DMSO, a marked increase in Na(+)-dependent uridine transport occurred within 72 hr, a time preceding maximum granulocytic differentiation. This change was attributable to an increase in transport affinity (Km = 1.54 +/- 0.65 microM), with no change in Vmax (Vmax = 0.13 +/- 0.02 microM/s). The consequence of these changes was the generation of a 3- to 4-fold increase in the intracellular concentration of uridine relative to the medium at a physiological concentration of 5 microM uridine. Similar increases in transport affinity were observed for adenosine, inosine, guanosine and thymidine in DMSO-differentiated HL-60 cells (Km values of 2 to 5 microM). These results complement our previous studies with phorbol 12-myristate 13-acetate, in which differentiation to a monocytic phenotype was also associated with enhanced Na(+)-dependent nucleoside transport.

Modulation of pyrimidine metabolism or the metabolic fate of 5-fluorouracil by a number of different agents has permitted a significant increase in the response rate to this agent, particularly for colorectal cancers. Brequinar, a noncompetitive inhibitor of mitochondrial dihydroorotate dehydrogenase has been shown to achieve a tumor-specific modulation of the therapeutic effect of 5-fluorouracil. A selective decrease of uridine nucleotide pools in Colon tumor 38 compared to normal tissues of C57/BL6 mice was observed after Brequinar administration. This effect was achieved with very low nontherapeutic doses of Brequinar (8 to 27% of the maximum tolerated dose in this model). Pretreatment with Brequinar 4 and 24 h prior to administration of [3H]fluorouracil significantly increased incorporation of the fluoropyrimidine into Colon 38 tumor RNA, while minimal effects were seen in normal tissues of C57/BL6 mice. Brequinar (15, 30, and 50 mg/kg) was administered 4 h prior to fluorouracil (85 mg/kg) on a weekly basis in Colon 38-bearing mice. All combinations potentiated 5-fluorouracil antitumor activity and the lowest dose of Brequinar (15 mg/kg) showed a reduced toxicity (weight loss) compared to the same dose of 5-fluorouracil as a single agent. When Brequinar preceded fluorouracil by 24 h, greater toxicity and less antitumor activity were observed. A comparison of the optimal Brequinar-fluorouracil regimen with a previously optimized N-(phosphonoacetyl)-L-aspartic acid-fluorouracil combination in Colon 38 tumor indicated that Brequinar-fluorouracil was more effective and less toxic.

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).

The concentration of uridine (Urd) in murine tissues appears to be controlled by Urd catabolism, concentrative Urd transport, and the non-concentrative, facilitated diffusion of Urd. Previous reports document the tissue-specific disruption of these processes, and subsequently intracellular pools of free Urd in mice, by the administration of exogenous Urd (250 mg/kg) or the Urd phosphorylase (EC 2.4.2.3; uracil:ribose-1-phosphate phosphotransferase) inhibitor 5-benzylacyclouridine (BAU) (240 mg/kg). We now report the effect of combinations of BAU (120 mg/kg, p.o.), the nucleoside transport inhibitor dipyridamole (DP) (25 mg/kg, i.p.), and exogenous Urd (250 mg/kg, i.v.) on Urd pools in mice. This dose of BAU increased Urd pools 2- to 6-fold, in a tissue-specific manner, for up to 5 hr. DP increased Urd pools 3-fold in spleen, over a 4-hr period, but did not affect other tissues. Administration of BAU 1 hr prior to exogenous Urd resulted in a 50- to 100-fold expansion of tissue normal after 6 hr. Administration of DP 1 hr prior to exogenous Urd caused a tissue-specific 40- to 100-fold increase in Urd pools which, except in spleen, returned to normal within 2 hr. The marked additive effects of these combinations were in contrast to those obtained following the administration of BAU 1 hr prior to DP. This regimen increased Urd pools from 4- to 9-fold, in a tissue-specific manner. In addition, Urd pools remained elevated for up to 9 hr, except in spleen where the Urd concentration was elevated for up to 15 hr. Analysis of enzyme activities indicated that DP does not enhance the inhibitory effect of BAU against murine liver Urd phosphorylase. However, DP did inhibit plasma clearance of BAU, and this effect may partially explain the apparent synergistic effect of this combination. In spite of the prolonged and dramatic expansion of tissue Urd pools produced by BAU + DP, the total Ura nucleotide content in spleen, gut and colon tumor 38 (CT38) increased by less than 70% over a 12-hr period following administration of this combination. These findings are discussed in light of their biochemical and therapeutic implications.

The Na(+)-dependent transport and facilitated diffusion of uridine were measured after differentiation of HL-60 leukaemia cells along the monocytic pathway by phorbol 12-myristate 13-acetate (PMA). PMA (200 ng/ml) caused a marked increase in Na(+)-dependent uridine transport within 48 h of exposure that was attributable to an increase in transport affinity (apparent Km values of 1.15 +/- 0.22 and 44 +/- 4.4 microM for PMA-induced and uninduced cells respectively), with no change in Vmax. (0.15 +/- 0.02 and 0.13 +/- 0.01 pmol/s per microliter of cell water for PMA-induced and uninduced cells respectively). A corresponding rapid decrease in both the rate of facilitated diffusion and the formation of uracil nucleotides occurred in PMA-induced cells. As a consequence of these changes, intracellular pools of uridine 3-4-fold greater than those in the medium were generated. A similar increase in Na(+)-dependent transport of adenosine, inosine, guanosine, thymidine and cytidine (Km values of 1-4 microM) was observed. The effects of PMA on the activation of the Na(+)-dependent uridine transporter were inhibited by staurosporine, suggesting the involvement of protein kinase C. The findings indicate that a change in the balance of the cellular mechanisms employed for nucleoside transport occurs during the monocytic differentiation of HL-60 leukaemia cells.

HL-60 leukemia cells, induced to differentiate, activate a Na(+)-dependent nucleoside transport system, concomitant with a reduction in the nitrobenzylthioinosine (NBMPR)-sensitive facilitated transport of nucleosides. The consequence of these changes lead to the formation of intracellular pools of uridine. To examine the possible role of accumulated uridine in the commitment of HL-60 leukemia cells to undergo maturation, the effects of uridine on the growth and differentiation of HL-60 cells were monitored. Uridine at millimolar levels caused a concentration-dependent inhibition of cellular growth, resulting in the accumulation of cells in the G2/M phases of the cell cycle, phenomena that preceded the formation of differentiated cells. These effects of uridine were reduced by 10 microM NBMPR, an inhibitor of the facilitated transport of nucleosides. The effects of 24 mM uridine on growth and differentiation of HL-60 cells were also prevented by 5 mM inosine, and partially prevented by either 2 mM hypoxanthine or 20 microM adenosine. Pretreatment of HL-60 cells with 24 mM uridine for 6 days, followed by a 2 h exposure to TPA, resulted in the rapid attachment of cells to the tissue culture dish, and the extension of long processes. Although the concentrations of uridine required for the above effects are greater than those achieved during differentiation, these observations suggest that uridine may play a role in regulating the maturation process.

A previous report from this laboratory indicated that the concentration of free uridine (Urd) in many normal murine tissues greatly exceeds that in plasma. We now report that Urd uptake by isolated murine splenocytes is concentrative, and that the rate of uptake from medium containing 10 to 500 microM [3H]Urd conforms to a process that is saturable with a Km of 38.0 +/- 4.1 (SE) microM and Vmax of 2.70 +/- 0.27 pmol/s/microliter cell water. Other ribosyl and deoxyribosyl pyrimidine nucleosides or their analogues were not concentrated by splenocytes; however, ribosyl and deoxyribosyl purine nucleosides and, to a lesser extent, deoxyuridine did inhibit Urd uptake. In this system Urd uptake was not inhibited by 1 microM nitrobenzylthioinosine or 10 microM dipyridamole but was significantly inhibited by 5 mM NaN3 or 250 microM KCN. Transport of Urd involves Na+ cotransport as evidenced by complete inhibition when Na+ is replaced by Li+ in the incubation medium, and it is also inhibited by 3 mM ouabain. Active Urd transport coexists with the nonspecific, carrier mediated, facilitated diffusion of nucleosides as demonstrated by the inhibition of Urd efflux and thymidine influx in splenocytes by nitrobenzylthioinosine and dipyridamole. Under identical conditions, Urd entry into L1210 leukemia cells was nonconcentrative and non-Na+ dependent but inhibited by nitrobenzylthionosine. That nucleosides enter most cultured neoplastic cell lines by facilitated diffusion and not the active transport mechanism for Urd confirms earlier findings and may represent an exploitable target for chemotherapy.

Evidence is presented that the liver effects an essentially complete degradation of plasma uridine in a single pass and replaces it largely from hepatic pools of acid-soluble uridine nucleotides. The concentration of uridine in the hepatic vein of the rat was essentially the same as that in the arterial circulation and portal vein. However, the isolated perfused rat liver degraded more than 90 percent of infused [5-3H]uridine in a single passage. Similar results were found in vivo when tracer amounts of [3H]uridine and [14C]uridine were infused into the portal vein of an intact rat. Furthermore, less than 2 percent of the infused uridine entered the acid-soluble nucleotide pools of the liver after 30 minutes of infusion. Intraperitoneal injection of [3H]orotate allowed selective labeling of liver (and kidney) pyrimidines. After 3 hours, the specific activity of uridine in the hepatic vein was more than three times that in the arterial circulation. This unusual exchange, which is not saturated even at uridine concentrations as high as 50 microM, contributes to the rapid turnover of plasma uridine and explains its inefficient utilization in peripheral tissues.

Evidence for transport systems that actively concentrate uridine in normal tissues provides a previously unexploited opportunity for manipulation to therapeutic advantage. The ability to expand these pools in a tissue-specific manner by administration of exogenous uridine, inhibition of uridine phosphorylase with BAU or blockade of the facilitated transport of nucleosides with dipyridamole is established. If the apparent defect in the active transport mechanism for uridine in neoplastic cells in culture as well as several model tumors reflect the properties of human neoplasms, a new exploitable therapeutic difference may exist. These approaches may, in the near future, increase the therapeutic effectiveness not only of fluorouracil and the other fluoropyrimidines but also of other agents which disrupt uridine metabolism such as PALA and pyrazofurin.

Pools of free uridine, ranging from 7.3 to 38.0 nmol/g wet weight, have been detected in a variety of freeze-clamped murine tissues. These concentrations average 10-fold greater than that detected in plasma. The kinetics of these pools after an i.v. tracer dose of [3H]uridine suggest that the initial rapid disappearance of [3H]uridine from plasma (t1/2 = 2 min) reflects distribution into tissues as well as catabolism by the liver. Subsequently, the tissue uridine pools turn over with half-lives of 13 to 18 h. Analyses of the activity of the proximal enzymes in uridine metabolism (uridine phosphorylase and uridine kinase) suggest that the phosphorylase correlates with the size of tissue uridine pools. Further evidence for this is seen in the sustained 5- to 15-fold increase in both tissue and plasma uridine concentrations after treatment with benzylacyclouridine, a potent uridine phosphorylase inhibitor. In contrast, a nonphysiological dose of exogenous uridine (250 mg/kg) briefly increases the plasma concentration of uridine to over 1 mM but it returns to below 10 microM within 1 h. Under these conditions as well, tissue concentrations of uridine increase 5- to 10-fold in most tissues, 20-fold in spleen, and 70-fold in kidney. High cellular concentrations of free uridine relative to medium are also observed in dispersed murine splenocytes. Furthermore, splenocytes incubated in 5 microM [3H]uridine achieved a 2-fold higher intracellular concentration of [3H]uridine in less than 1 min independent of phosphorylation. Thymidine was not concentrated in this system nor did nitrobenzylthioinosine inhibit [3H]uridine uptake. These findings suggest that in normal tissues and explanted cells, pools of uridine are sustained by a concentrative transport mechanism and constitute a previously unrecognized reservoir of pyrimidine nucleosides in tissues.

Benzylacyclouridine (BAU), a potent inhibitor of uridine phosphorylase, delays the disappearance of uridine from plasma, affects the utilization of uridine by selected tissues, and enhances the therapeutic effects of 5-fluorouracil (FUra) in female C57BL/6 mice. A single 30-mg/kg i.v. injection of BAU lengthens the plasma half-life of both a tracer dose of [3H]uridine (3 micrograms/kg) and a pharmacological dose of uridine (250 mg/kg) by 250 and 83%, respectively. This dose of BAU also increases the normal plasma concentration of uridine about 4-fold to 9 microM and sustains these levels for 4 h. Four injections of BAU at 30 mg/kg over 6 h or a single injection at 240 mg/kg increases the plasma concentration of uridine over 10-fold to approximately 50 microM. In addition to affecting the pharmacokinetics of uridine, a 30-mg/kg dose of BAU selectively increases up to 4-fold the ability of normal host tissues to salvage a tracer dose of [3H]uridine for nucleic acid biosynthesis, the uracil nucleotide pool size, and the incorporation of uridine into nucleic acids. However, uridine salvage from plasma by colon tumor 38 is increased only slightly by BAU, while the uracil nucleotide pool size and uridine incorporation into tumor nucleic acids are actually decreased by 15 and 37%. The selective effect of BAU on uridine utilization is reflected in the ability of BAU to modify FUra-induced host toxicity. The dose of FUra required to kill 50% of the treated normal mice (350 mg/kg) is modestly increased by "rescue" regimens consisting of the subsequent administration of repeated injections of either BAU alone (30 mg/kg/injection) or uridine alone (250 mg/kg/injection). However, an increase of 54% is achieved when repeated injections of the combination of BAU and uridine are administered. In C57BL/6 mice bearing advanced transplants of colon tumor 38, the period of tumor growth inhibition resulting from multiple courses of FUra-containing drug regimens can be increased by the delayed administration of BAU alone or BAU combined with uridine.