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The Journal of Immunology, 1998, 160: 846-853.
Copyright © 1998 by The American Association of Immunologists

In Vitro and In Vivo Mechanisms of Action of the Antiproliferative and Immunosuppressive Agent, Brequinar Sodium1

Xiulong Xu*, James W. Williams*, Jikun Shen*, Haihua Gong*, Deng-Ping Yin*, Leonard Blinder*, Robert T. Elder*, Howard Sankary*, Alison Finnegan{dagger},{ddagger} and Anita S.-F. Chong2,*,{dagger}

Departments of * General Surgery and {dagger} Immunology/Microbiology and {ddagger} Section of Rheumatology, Department of Internal Medicine, Rush Medical College, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (<=65 µM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (>=65 µM). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Brequinar sodium (BQR)3 (NSC 368390; DuP785) [6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt] was originally developed as an antiproliferative agent for the treatment of cancer and subsequently as an immunosuppressive agent for the control of allograft and xenograft rejection (1, 2, 3, 4, 5, 6, 7, 8). BQR inhibits the enzymatic activity of dihydroorotate dehydrogenase (DHO-DHase), the fourth enzyme of the de novo pyrimidine biosynthetic pathway (9, 10, 11). Inhibition of this enzyme prevents the formation of pyrimidine nucleotides (PyN) necessary for the synthesis of RNA and DNA, thus blocking cell proliferation (4). In vivo, BQR suppresses the growth of tumors, the induction of contact sensitivity, the generation of cytotoxic T cells, and the production of Abs (4). Because clonal expansion of lymphocytes is central to the induction of both humoral and cellular immune responses, it has become widely accepted that immunosuppression by BQR is due to the in vivo inhibition of pyrimidine synthesis (reviewed in Refs. 7 and 8).

At least two sets of data suggest that this hypothesis should be reexamined. First, in vitro experiments indicate that addition of exogenous uridine antagonizes the antiproliferative activity of BQR (10, 12, 13). Because human and rodent plasmas contain uridine (5–10 µM), it seems unlikely that BQR could block in vivo the proliferation of cells that are able to salvage and use uridine to maintain pyrimidine nucleotide triphosphate (PyNTP) levels (14, 15, 16). Second, the symptoms common to all patients with a genetic defect in de novo pyrimidine synthesis, hereditary orotic aciduria, are megaloblastic anemia and orotic acid crystalluria (reviewed in 17 . In contrast, the majority of patients of hereditary orotic aciduria do not have undue susceptibility to infection, indicating that they are not significantly immunosuppressed. In vitro cellular immune defects have been reported in some of these patients; however, these immune defects may be due to lymphopenia arising from a generalized defect in hemopoiesis, rather than to an intrinsic inability of the T cells to proliferate in response to Ag stimulation (18, 19).

We have reexamined the mode of action of BQR in two in vivo models, induction of anemia and control of transplant rejection. We have quantified the effect of BQR on the nucleotide triphosphate (NTP) levels of bone marrow and spleen cells and determined the effect of uridine on the antiproliferative and immunosuppressive activity of BQR in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

BQR was a gift from DuPont Merck Pharmaceuticals (Wilmington, DE), while uridine was purchased from Sigma (St. Louis, MO). These compounds were dissolved in 0.9% NaCl for i.p. injections and in distilled water for in vitro studies. Anti-phosphotyrosine mAb, 4G10, and rabbit anti-p59fyn and anti-p56lck antisera were purchased from UBI (Lake Placid, NY). Histone 2B was purchased from Calbiochem (San Diego, CA).

In vivo treatments

Transplantation of BALB/c hearts into C3H mice was performed as previously described (20). BQR was administered once daily by i.p. injection, while uridine was administered twice daily. Mice were bled through the orbital vein using a microhematocrit capillary tube (Baxter, Deerfield, IL), and the blood was centrifuged for 10 min at 550 x g. The percentage of packed cell volumes was determined with a microhematocrit capillary tube reader (Critocaps, Oxford Labware, St. Louis). All mice were killed 4 h after receiving their last dose of BQR or uridine.

In vitro stimulation

Lymphocytes were isolated from BALB/c spleen by centrifugation through a Ficol gradient and were depleted of macrophages and B cells by adherence to goat anti-mouse IgG-coated plates. The nonadherent cells (>=80% T cells) were harvested and cultured at 4 to 5 x 105/ml in RPMI 1640 supplemented with 10% FBS. Stimulation was performed with 5 µg/ml Con A (Sigma) in the presence of indicated concentrations of BQR or uridine. After 40 to 66 h, the cells were harvested and the levels of cell proliferation or purine NTP and PyNTP were quantified.

Quantitation of lymphocyte proliferation by flow cytometry

The flow cytometric cell proliferation assay was performed as previously described (21). Spleen cells from BALB/c mice were incubated with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) for 10 min at 37°C. The cells were washed three times in cold RPMI 1640 containing 10% FBS before use in in vitro stimulation assays. After 65 h, the proliferating cells were identified as those with sequential halving of fluorescence intensity, using a flow cytometer (Ortho Cytron Absolute, Ortho Diagnostic Systems, Raritan, NJ).

Extraction and quantitation of NTP and uridine

NTP from spleen, bone marrow, or splenic T cells from in vitro cultures were extracted with 0.4 M trichloric acid and neutralized with an equal volume of 0.5 M tri-n-octylamine in Freon 113 as previously described (22). Nucleotides were separated using a Whatman anion exchange column (Particil 10 SAX; Alltech, Deerfield, IL) and a linear gradient elution of potassium phosphate buffer, pH 4.5 (10–500 mM). The corresponding peaks of four nucleotides were detected by HPLC (Waters, Milford, MA) and the concentrations were calculated based on a standard curve of nucleotides (Sigma). Serum sample was diluted twofold in 0.9% NaCl, and uridine was extracted by addition of an equal volume of 0.8 M trichloric acid and then neutralized with an equal volume of 0.5 M tri-n-octylamine in Freon. Serum uridine was detected by HPLC using a Waters HPLC Symmetry C18 column and an elution solution (5 mM KH2PO4, pH 3.8) at a flow of 1 ml/min. The uridine peak was identified by its retention time and spectrum compared with a uridine standard (Sigma), and uridine concentrations were calculated based on a standard curve.

Western blotting and protein-tyrosine phosphorylation

T cells (5 x 106/sample) isolated from BALB/c lymph nodes were preincubated with various concentrations of BQR for 2 h, stimulated with 2 µg of anti-CD3 (2C11-1456) for 2 min, and cross-linked with 8 µg of goat anti-hamster IgG for another 2 min. Cell lysates were separated on an SDS-polyacrylamide gel and then transferred onto a nitrocellulose membrane. Protein-tyrosine phosphorylation was detected by Western blot using enhanced chemiluminescence as previously described (23, 24). Relative intensities of tyrosine phosphorylation were determined on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) using IP Lab Gel software (Signal Analytics Corp., Vienna, VA). The relative intensities were plotted, and IC50 values were determined as 50% reduction in tyrosine phosphorylation.

In vitro tyrosine kinase assay

Immunoprecipitated p59fyn or p56lck from CTLL-4 cells or LSTRA cells (5 x 106) was preincubated with various concentrations of BQR in the PTK buffer (50 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM MnCl2) on ice for 10 min. Exogenous substrate, histone 2B (2 µg), was added and, after 10 min, the reaction was initiated by addition of 10 µCi [{gamma}-32P]ATP. After incubation at 20°C for 10 min, the reaction mixture was subjected to electrophoresis in a 12.5% SDS-polyacrylamide gel. Phosphorylation of the kinase and the exogenous substrate was analyzed by autoradiography as previously described (23, 24). Quantitation of exposed films was performed as described above.

Histology and immunohistochemistry

Spleens were collected and embedded in OCT compound and then snap-frozen in liquid nitrogen. The 5-µm sections of spleen were fixed in 10% formalin and then stained in hematoxylin and eosin solutions. To confirm the presence of megakaryocytes, spleen sections were stained with rabbit anti-human von Willebrand factor Ab (Dako, Carpenteria, CA) using standard avidin-biotin-horseradish peroxidase methods. Megakaryocyte counts of 100 fields/spleen were performed, in which a field was defined by an ocular grid (x400, ~1 µm2).

Statistical analyses

All analyses to determine significant differences between treatment and control groups were performed by ANOVA using the SuperANOVA program for Macintosh (Abacus Concepts Inc., Berkeley, CA). A post hoc Tukey’s compromise test was used to identify the treatments that were statistically different (p <= 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of BQR on the bone marrow

The myelotoxic effects of BQR that result in anemia and leukopenia have been extensively documented (7, 8). We treated normal BALB/c mice with BQR (10–20 mg/kg/day) and observed that the hematocrits, reported as percentage of packed cell volume, were significantly reduced after 4 wk of therapy (Fig. 1Goa; ANOVA and Tukey’s test, p = 0.001). BQR-treated (10–20 mg/kg/day) mice had a 31% reduction in percentage of packed cell volume compared with untreated BALB/c mice (Fig. 1Goa). After treatment with BQR, the UTP and CTP levels in bone marrow cells were reduced by 30 and 25%, respectively (Table IGo). There was no significant decrease in the ATP or CTP levels in the BQR-treated mice.



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  		FIGURE 1. Anemia is induced by BQR treatment. BALB/c mice were treated with 20 or 10 mg/kg/day of BQR, either alone (a) or in combination with uridine (1000 or 2000 mg/kg/day) (b). Blood samples were obtained at 3, 4, and 6 wk, and at ~24 h after treatment with BQR and 12 h after treatment with uridine (a). The percentage of packed cell volume was determined after 3, 4, and 6 wk of treatment (a) or wk 4 (b) as described in Materials and Methods. The results represent the means ± SEM from three to four mice in each group, and the experiment was performed twice (in some groups the SEM bars are too small to be visible). Statistically significant differences were determined, by ANOVA and Tukey’s tests (p <= 0.0001), between BQR-treated and untreated or uridine monotherapy controls (*); between BQR-treated and uridine monotherapy groups (#); and between BQR-treated and BQR-plus-uridine-treated groups (**).

 

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  		Table I. Depletion of intracellular nucleotides in vivo by BQRa

 
Coadministration of BQR (10–20 mg/kg/day) with uridine (1000–2000 mg/kg/day) prevented anemia, and the hematocrits remained at levels (61–63%) comparable with those of untreated controls (68–71%, see Fig. 1Gob). The UTP and CTP levels in the bone marrow remained depressed by 25% in mice treated with BQR (10 mg/kg/day) plus 1000 mg/kg/day of uridine, but approached those of normal mice in the BQR- and uridine-treated (2000 mg/kg) mice (Table IGo). These observations confirm our ability to detect the consequence of inhibiting DHO-DHase activity in vivo, and support the conclusion that induction of anemia by BQR is mediated through inhibition of DHO-DHase activity and de novo pyrimidine synthesis.

Effects of BQR on the spleen

When BQR monotherapy was continued for 6 wk, a spontaneous reversal in anemia was unexpectedly observed (Fig. 1Goa). The spleens from these BQR-treated mice were significantly enlarged (Fig. 2Goa; ANOVA and Tukey’s test, p < 0.05) and histologic examination revealed a marked increase in the number of megakaryocytes (Fig. 2Go, b and e), a characteristic of extramedullary hemopoiesis. The number of megakaryocytes was significantly reduced in mice treated with the combination of BQR and uridine (Fig. 2Go, b and f). While it is possible that this observation may be due to toxicity of uridine for megakaryocytes, we did not observe infiltrating neutrophils or macrophages or other signs of cell death in the spleen. The modest decrease in the weights of spleen in the uridine-treated groups was not statistically significant. Therefore, we interpret these data as suggesting that extramedullary hemopoiesis is responsible for the recovery from BQR-induced anemia, and that myeloid cell proliferation can occur in the spleen even when it is inhibited in the bone marrow.



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  		FIGURE 2. Extramedullary hemopoiesis in BALB/c mice treated with BQR for 6 wk. Weights of spleens (a), and the numbers of megakaryocytes in 100 high-power fields in the spleens (b), of normal mice or mice treated with BQR (10 mg/kg/day for 6 wk) or BQR plus uridine (1000 or 2000 mg/kg/day). All mice were killed 4 h after the last dose of BQR or uridine was administered. The results represent the mean ± SEM from three to five mice/group, and each experiment was performed twice (in some groups the SEM bars are too small to be visible). Hematoxylin and eosin-stained sections of spleens from normal mice (c), or mice treated with uridine (2000 mg/kg/day) (d), BQR (10 mg/kg/day) (e), or BQR (10 mg/kg/day) plus uridine (2000 mg/kg/day) (f), illustrate the presence of megakaryocytes. The inset in c confirms the identity of megakaryocytes that stain positively for rabbit anti-human von Willebrand-factor Ab. Statistically significant differences were determined, by ANOVA and Tukey’s tests (p <= 0.0001), between BQR-treated and untreated or uridine monotherapy controls (*), and between BQR-treated and BQR-plus-uridine-treated groups (**).

 
Next, we quantified the PyNTP levels in the spleens of mice treated for 6 wk with either BQR or uridine, or both. The PyNTP levels in mice treated with 10 mg/kg of BQR for 6 wk were comparable with those observed in normal mice. In contrast, administration of uridine alone (2000 mg/kg for 6 wk) significantly elevated PyNTP levels (Table IGo). The PyNTP levels were also elevated by uridine in the BQR-treated mice, although these increases were not statistically significant (Table IGo). These observations led us to hypothesize that uridine can be salvaged from the serum to maintain normal intracellular PyNTP levels, despite inhibition of de novo pyrimidine synthesis by BQR. Furthermore, because hemopoiesis in the presence of BQR can occur in the spleen but not in the bone marrow, it is likely that the rate-limiting factor for cell proliferation in vivo is the availability of uridine for salvage in the bone marrow.

Immunosuppressive activity of BQR is unaffected by uridine coadministration

The observation of extramedullary hemopoiesis and normal PyNTP levels in the spleen prompted us to test whether the ability of BQR to inhibit T cell immune responses in vivo is due to inhibition of de novo pyrimidine synthesis. We reasoned that if immunosuppression by BQR was solely through inhibition of de novo pyrimidine synthesis, then coadministration of uridine at doses that reversed anemia in BQR-treated mice should reduce its immunosuppressive activity. We monitored the immunosuppressive activity of BQR in a BALB/c into C3H cardiac allograft model. The untreated allograft survived for a mean of 9.2 ± 0.83 days (Table IIGo). Recipients were treated with either 10 or 2 mg/kg/day of BQR as monotherapy or in combination with 1000 mg/kg/day of uridine. This dose of uridine was chosen because it effectively prevented anemia and elicited fewer toxic effects than the higher 2000-mg/kg dose. The higher dose of BQR was toxic, and the recipients died 3 to 8 days post-transplant (n = 4), while treatment with 2 mg/kg/day of BQR extended allograft survival to 47.17 ± 16.4 days. When uridine was coadministered with either 10 or 2 mg/kg/day of BQR, the allografts survived 51.0 ± 16.1 and 51.8 ± 16.5 days, respectively. This experiment simultaneously demonstrates that uridine can control the toxicity associated with BQR administration, and that uridine has no significant effect on the immunosuppressive activity of BQR. These data further suggest that BQR effects immunosuppression by an unidentified mechanism that is independent of inhibition of de novo pyrimidine synthesis. The alternative explanation is that lymphocytes are unable to salvage sufficient uridine to normalize intracellular pyrimidine stores and to undergo clonal expansion. The subsequent in vitro experiments address the latter possibility.


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  		Table II. Uridine does not antagonize the immunosuppressive effect of BQRa

 
Effect of serum concentrations of uridine on the activity of BQR in vitro

Our in vivo studies led us to speculate that serum is a significant source of salvageable uridine. We determined that the levels of serum uridine were similar in untreated (5.26 ± 2.13 µM uridine; n = 4) and BQR-treated mice (8.55 ± 1.52 µM; n = 4). We next quantified the in vitro ability of splenic T cells to salvage uridine and reverse the depression of intracellular PyNTP levels by BQR. Lymphocytes stimulated with 5 µg/ml Con A for 40 h increased both their UTP and CTP levels by approximately fourfold, while more moderate increases in ATP and GTP levels of two- to threefold were observed (Table IIIGo). In a dose-dependent manner, BQR effectively prevented the increase in PyNTP levels with an IC50 of 0.26 µM. Higher concentrations of BQR (>0.5 µM) also prevented the increase in purine NTP levels, possibly due to a generalized block of T cell activation (Table IIIGo). Uridine, in a dose-dependent manner, was able to restore PyNTP levels in lymphocytes treated with 2.6 µM of BQR (Table IIIGo).


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  		Table III. Effect of BQR and uridine on the nucleotide levels in Con A-activated T cellsa

 
Next, we tested whether these concentrations of uridine could reverse the inhibition of cell proliferation by BQR. Spleen cells were prestained with a intracellular fluorescent label, CFSE, that is divided equally between daughter cells upon cell division (21). Flow cytometry was used to identify successive generations of proliferating cells as those with sequential halving of fluorescence intensity (Fig. 3Goa). We chose this assay to measure cell proliferation over the more conventional radiolabeled thymidine uptake assay because depletion of PyN can also result in a reduction of intracellular thymidine and a decrease in the ratio of intracellular unlabeled and labeled thymidine. This would result in underestimation of the potency of BQR and of exogenous uridine. Using the CFSE assay, we determined that BQR effectively inhibited cell proliferation with an IC50 of 0.26 µM. Uridine (200 µM) completely reversed the ability of <=27 µM of BQR to inhibit cell proliferation, confirming that the primary mechanism of action of low doses of BQR was solely due to inhibition of de novo pyrimidine synthesis (Fig. 3Gob). In a second experiment, we examined the effects of serum concentrations (1.25–20 µM) of uridine on the antiproliferative effects of BQR (Fig. 3Goc). We observed that 15, 5, and 1.25 µM uridine reversed the antiproliferative activity by 100, 50, and 30%, respectively. The ability of physiologic concentrations of uridine to significantly reverse the effects of low doses of BQR demonstrates the ability of T cells to salvage uridine at a rate that can compensate for an inhibition of de novo PyN.



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  		FIGURE 3. Effect of uridine on the antiproliferative activity of BQR in Con A-activated T cells. T cells enriched from BALB/c spleen were prestained with an intracellular fluorescent label, CFSE. Upon cell division, CFSE is divided equally between daughter cells. Thus, cells that have undergone successive generations of cell proliferation will have sequential halving of fluorescence intensity. a, Flow cytometry was used to identify cells that had undergone proliferation. Five and six-tenths percentage of cells proliferated in the unstimulated cultures, while 53.9% of cells proliferated in Con A-stimulated cultures after 68 h in vitro. Uridine (200 µM) partially reversed the antiproliferative activities of 67 µM of BQR, but completely reversed the antiproliferative activities of 2.6 µM of BQR. b, BQR was able to inhibit cell proliferation in a dose-dependent manner, both in the absence (open squares) or presence of 200 µM (filled squares) uridine. c, Physiologic concentrations of uridine countered the antiproliferative activities of 2.6 µM of BQR. The data in b and c are presented as percentage of proliferating cells; each data point represents the mean of triplicate cultures from one representative experiment ± SEM (in some groups the SEM bars are too small to be visible). Each experiment was repeated four times.

 
It is notable that at concentrations of >=65 µM of BQR, uridine was unable to reverse the inhibitory effects of BQR on cell proliferation (Fig. 3Gob, filled squares). In the presence of 200 µM of uridine, the IC50 for inhibition of T cell proliferation was 65 µM BQR. These data prompted us to test the possibility that at high concentrations of BQR, other activities may inhibit lymphocyte proliferation.

Ability of BQR to inhibit tyrosine phosphorylation

Another pharmacologic agent that has profound immunosuppressive activity and the ability to inhibit the enzymatic activity of DHO-DHase is leflunomide (25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Because leflunomide is also an inhibitor of tyrosine phosphorylation (23, 24), we tested whether this property is shared by BQR. We first tested whether BQR could inhibit the in vitro kinase activity of purified p56lck and p59fyn (Fig. 4Go, a and b). BQR inhibited autophosphorylation of p56lck with an IC50 of 70 µM; inhibition was 39, 41, and 60% for 25, 50, and 100 µM BQR, respectively. BQR also inhibited the phosphorylation by p56lck of the exogenous substrate, histone 2B, with an IC50 of 70 µM; inhibition was 10, 43, 59, and 86% for 25, 50, 100, and 200 µM BQR, respectively. BQR inhibited autophosphorylation of p59fyn with an IC50 of 105 µM; inhibition was 0, 17, 48, and 65% for 25, 50, 100, and 200 µM BQR, respectively. BQR also inhibited the phosphorylation by p59fyn of histone 2B with an IC50 of 20 µM; inhibition was 26, 54, 79, 83, and 84% for 10, 25, 50, 100, and 200 µM BQR, respectively.



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  		FIGURE 4. Inhibition of p59fyn and p56lck activity by BQR. p59fyn from 1 x 107 CTLL-4 cells (a) or p56lck from 5 x 106 LSTRA cells (b) was immunoprecipitated with 5 µl of anti-p59fyn or anti-p56lck antiserum, respectively, and aliquoted with histone 2B into each tube containing reagents for an in vitro protein-tyrosine kinase assay. The in vitro kinase assays were performed with the indicated concentrations of BQR as described in Materials and Methods. This experiment was performed three times, and one representative experiment is presented. c, Inhibition of protein-tyrosine phosphorylation in CD3-stimulated T cells by BQR. T cells isolated from lymph nodes of BALB/c mice were preincubated with BQR for 2 h and then stimulated with anti-CD3 mAb plus goat anti-hamster IgG for a total of 4 min. Cell lysates were prepared, separated on a 10% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane as described in Materials and Methods. Protein-tyrosine phosphorylation was detected with anti-phosphotyrosine mAb 4G10 and enhanced chemiluminescence as described in Materials and Methods.

 
We also tested the ability of BQR to inhibit tyrosine phosphorylation in normal murine T cells stimulated with anti-CD3 mAbs. As shown in Figure 4Goc, BQR was able to inhibit anti-CD3-stimulated tyrosine phosphorylation. Scanning densitometry of seven proteins of 42, 60, 70, 90, 95, 120, and 135 kDa, indicated that the IC50 of BQR for inhibition of anti-CD3-stimulated tyrosine phosphorylation ranged from 10 to 50 µM. BQR also inhibited the constitutively phosphorylated 60-kDa protein in murine T cells. Thus BQR, like leflunomide, has at least two biochemically independent activities that can be defined in vitro: inhibition of pyrimidine synthesis and inhibition of tyrosine phosphorylation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cellular requirements for PyN can be met either by de novo synthesis or by reutilization of preformed pyrimidine nucleoside, such as uridine, from extracellular sources. The importance of the pyrimidine synthetic pathway to T cell proliferation under defined conditions has been demonstrated by Fairbanks et al. (35). They reported that the metabolic requirements for pyrimidines were met by salvaging uridine in resting T lymphocytes, but the synthetic pathway is critical for the eightfold expansion of PyN pools in T cells activated in culture conditions in which exogenous uridine is limiting. Nanomolar concentrations of BQR inhibit the enzymatic activity of DHO-DHase, resulting in the inhibition of PyN synthesis and of cell proliferation (9, 10, 11). These observations suggest a hypothesis that the immunosuppressive activity of BQR is mediated through inhibition of DHO-DHase activity that ultimately prevents clonal expansion of activated T cells.

We provide experimental evidence that anemia induced by BQR correlates with depression of PyN in bone marrow cells, and that exogenous uridine is able to prevent this induction of anemia. Thus, the ability of BQR to inhibit cell proliferation and hemopoiesis in the bone marrow is largely due to inhibition of DHO-DHase. In contrast, similar concentrations of uridine are unable to significantly reduce the immunosuppressive activity of BQR. The ability of uridine to reverse the effects of BQR on bone marrow, but not on secondary lymphoid tissues, can be explained by differences in the ability of bone marrow cells and T cells to use salvaged uridine, or by differences in the availability of uridine in the bone marrow and the secondary lymphoid tissue. Our observation of hemopoiesis in the spleen, but not in the bone marrow, of mice treated for 6 wk with BQR supports the latter possibility.

Extramedullary hemopoiesis has been observed in a number of experimental models of sustained anemia or suppression of bone marrow hemopoiesis, and a cause-and-effect relationship has been suggested (36, 37, 38, 39). We speculate that prolonged depression of hemopoiesis in the bone marrow of BQR-treated mice triggers extramedullary hemopoiesis in their spleens. The ability of BQR to inhibit hemopoiesis in the bone marrow, but not in the spleen, correlated with a depression of PyNTP levels in the bone marrow, but not the spleen. The administration of uridine restores hemopoiesis in the bone marrow and prevents the induction of extramedullary hemopoiesis in the spleen. These observations collectively support the hypothesis that the availability of serum uridine for salvage in the bone marrow, rather than the inability to salvage uridine, limits the ability of hemopoietic stem cells to counter the effects of BQR.

The inability of uridine to significantly antagonize the immunosuppressive activity of BQR can be explained by two possibilities. First, the mode of immunosuppression may not be related to the depression of intracellular PyN; or second, lymphocytes may be unable to effectively salvage uridine to compensate for the inhibition of de novo pyrimidine synthesis. Our inability to detect reduction of intracellular PyN levels in the spleen of mice treated with BQR for up to 6 wk supports the former possibility. In addition, in vitro studies clearly demonstrate the ability of lymphocytes treated with <=2.6 µM of BQR to salvage plasma concentrations of uridine (2.5–15 µM) (14) and to convert salvaged uridine into intracellular PyNTP pools. These concentrations of uridine (2.5–15 µM) also antagonize the ability of BQR (2.6 µM) to inhibit splenocyte proliferation. These observations are consistent with the report that plasma concentrations of uridine can reverse the effects of inhibition of de novo pyrimidine synthesis in cultured L1210 cells (40). Based on our in vitro observations, we conclude that T lymphocytes can salvage uridine from physiologic extracellular concentrations at a rate that counters the inhibition of de novo pyrimidine synthesis and cell proliferation by BQR.

An intriguing observation was the inability of uridine to completely reverse the antiproliferative activity at high doses of BQR (>=67 µM). This suggested a second activity at high concentrations of BQR that is independent of attenuation of PyN levels. The similar in vivo activities of BQR to leflunomide (25, 26, 27, 28, 29), a compound with the ability to inhibit DHO-DHase and tyrosine kinase activity (23, 24, 31, 32, 34), prompted us to test whether BQR might also have the ability to inhibit the activity of T cell-associated tyrosine kinases. Indeed, tyrosine phosphorylation induced in murine T cells by cross-linking with anti-CD3 mAbs was significantly inhibited by BQR (10–50 µM). In addition, the in vitro kinase activities of two src family kinases were also inhibited with 20 to 105 µM of BQR. While these concentrations are significantly higher than the IC50 for inhibition of DHO-DHase (10–50 nM) and for reduction of PyN levels (0.26 µM), it should be noted that tissue and serum BQR concentrations can range from a peak of 200 to 500 µM to a 24-h trough level of 20 to 50 µM in mice treated with a single i.v. dose of 50 mg/kg of BQR (41). Mean levels of BQR in the sera of rats treated with an immunosuppressive dose of 3 mg/kg/day is ~40 µM with a 24-h trough level of <=10 µM (42). Thus, the data from pharmacologic monitoring do not exclude the possibility that inhibition of tyrosine phosphorylation may contribute to the immunosuppressive activities of BQR in vivo.

In conclusion, we report that uridine completely reverses the inhibition by BQR of de novo pyrimidine synthesis and proliferation in lymphocytes. However, uridine is unable to reverse the antiproliferative effects of high concentrations of BQR in vitro. We observed that uridine can reverse anemia, but not the immunosuppressive activity of BQR, in vivo. Thus, anemia is due to inhibition of de novo pyrimidine synthesis by BQR, while the mechanism of immunosuppression is more complex and may involve the newly identified activity of BQR as an inhibitor of tyrosine phosphorylation. Finally, our observations collectively suggest that uridine may be used to increase the therapeutic index of BQR by preventing the toxicities associated with inhibition of PyN synthesis and unmasking the true immunosuppressive potential of BQR.


   Acknowledgments
 
We thank Drs. Ian Boussy, Jeffery Short, and Howard Gebel for helpful discussions and comments on the manuscript, and Dr. Ian Boussy for assistance with the statistical analyses. We also thank Dr. Christopher Parish for introducing and assisting us in the development of the CFSE proliferation assay. We gratefully acknowledge the gift of BQR from DuPont Merck Pharmaceuticals (Wilmington, DE), and LSTRA cells from Dr. Tamara Hurly (Salk Institute, San Diego, CA).


   Footnotes
 
1 This work is funded in part by a grant from the National Institutes of Health (AI34061). Back

2 Address correspondence and reprint requests to Dr. Anita Chong, Department of General Surgery, Rush Presbyterian St. Luke’s Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612. Back

3 Abbreviations used in this paper: BQR, brequinar sodium; CFSE, carboxyfluorescein diacetate succinimidyl ester; DHO-DH, dihydroorotate dehydrogenase; PyN, pyrimidine nucleotides; NTP, nucleotide triphosphate; PyNTP, pyrimidine nucleotide triphosphate; IC50, 50% inhibitory concentration. Back

Received for publication July 18, 1997. Accepted for publication October 1, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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