Fung, JJ

Lemster, B

Starzl, TE

Thomson, AW

Todo, S

Woo, J

English

PubMed

Name not available

Potentiation of the antiproliferative activity of brequinar sodium for murine lymphocytes by exogenous cytidine

D-Scholarship@Pitt

. , i I r1 • Brequinar Potentiation of the Antiproliferative Activity of Brequinar Sodium for Murine Lymphocytes by Exogenous Cytidine A.W. Thomson, J. Woo, B. Lemster, S. Todo, J.J. Fung, and T.E. Starzl THE antimetabolite brequinar sodium (BQR) is a novel quinoline carboxylic acid analogue with broad anti-tumor activity in mice, including inhibition of the growth of tumor xenografts. I The mechanism of action of BQR in tumor cells is believed to involve inhibition of dihydrooro-tate dehydrogenase, a step in de novo pyrimidine biosyn-thesis,2.3 resulting in depletion of precursors required for RNA and DNA synthesis. There is recent evidence that BQR is very effective either alone or in combination with FK 506 or cyclosporine (Cy A) in the prolongation of experimental allograft or xenograft survival.4 To obtain more information on the influence of BQR on T - and B-cell activation and proliferation, we have investigated its ef-fects on the responses of murine spleen cells to various stimulants, including concanavalin A (ConA), phorbol myristate acetate (PMA) ± ionomycin, anti-CD3, and anti-Igs. Since BQR inhibits pyrimidine biosynthesis. we have also determined the effects of exogenous uridine and cytidine on the antilymphocytic activities of the drug in vitro. MATERIALS AND METHODS Reagents BQR (DuPont Merck Pharmaceutical Co. Wilmington. Dell was kindly provided by Dr L. Makowka. Cedars-Sinai Medical Center (Los Angeles. Calif). It was dissolved fresh before each experi-ment in sterile saline (1 mg/mL) and diluted in RPMI-I640 (Gibco. Grand-Island, NY). ConA, PMA. ionomycin, uridine, cytidine. and goat anti-mouse Igs were from Sigma Chemical Co (St Louis. Mo). Antimurine CD3 monoclonal antibody (MAb; clone KT3) was from Serotec (Oxford, England, UK). Lymphocyte Culture Nucleated spleen cells isolated from spleens of 8- to 12-week-old. male C57BUIO SnJ mice (Jackson Laboratory, Bar Harbor. Me) were prepared as described previously~ in RPMI-I640 with 10% vol/vol fetal bovine serum (FBS) and 5 x 105 cells added to triplicate round-bottom wells of 96-well microtiter plates (Corn-ing. NY). Cells were stimulated with ConA <0.5 to 5 J,Lg/mL). PMA (1 ng/mL). ionomycin (0.5 J,LmoIlL). anti-CD3 (I of 100). or anti-Ig (I of 50) for 24 to 120 hours. For estimation of DNA synthesis. 1 J,LCi 3H-thymidine was added 24 hours before harvest and incor-poration of radioactivity into DNA estimated by liquid scintilla-tion counting. Quantitation of Cytokine Production and Cytokine Gene Expression Interleukin-2 (lL-2) and IL-4 levels in supernatants of cultures of 50 x 106 spleen cells stimulated for 20 hours with ConA (5 JLg/mL) in Falcon tissue culture tubes were estimated using enzyme-linked immunosorbent assay (ELISA) kits for murine IL-2 (Collabora-tive Research Inc. Bedford. Mass) or IL-4 (Endogen Inc. Boston. Mass). Production of mRNA for IL-2 and IL-4 in ConA-stimu-lated cells was determined by semiquantitative reverse transcrip-tion polymerase chain reaction as described elsewhere.6 RESULTS AND DISCUSSION In 72-hour cultures, addition of BQR (5 JLg/mL to 0.1 JLg/mL) at 0 hour caused dose-dependent inhibition of the strong proliferative responses induced by both ConA (5 JLg/mL) (Fig ta) and PMA + ionomycin (data not shown). In contrast, no impairment oflow concentration ConA (0.5 From the Transplant Institute, University of Pittsburgh, Health Science Center, Pittsburgh, Pennsylvania. Supported by NIH grant OK 29961-09. Address reprint requests to A.W. Thomson, PhD, University of Pittsburgh. Biomedical Science Tower. 15th floor. Terrace and Lothrop Sts. Pittsburgh, PA 15213. © 1993 by Appleton & Lange 0041-1345/931$3.00/+0 704 Transplantation Proceedings, Vol 25, No 1 (February), 1993: pp 704-705 EXOGENOUS CYTIDINE 200000 150000 ~ 100000 U 50000 30000 20000 10000 30000 ::& 20000 Q. U 10000 .001 (a) B:JI BOR+CYfIDINE (0.1 mM) BOR+URIDINE (0.1 mMj .01 .1 1 10 100 BaR CONC. lug/mil (b) -0-- OORONLY ~ eOR+CYf (O.lmM) .01 .1 1 10 100 BaR CONC. lug/mil (e) .01 .1 1 10 BaR CONC. lug/mil -0-- OORON..Y ~ eOR+CYf(O.lmM) 100 Fig 1. Effects of exogenous nucleosides on the anti proliferative activity of BaR. (8) The inhibitory effect of BaR on ConA (5 ILQImL)-stimulated DNA synthesis was reversed by undine but potentiated by cytidine. (b) Inhibition of weaker anti-CD3-induced DNA synthesis by BaR + cytidine but not by BaR alone. (c) Inhibition of anti-Ig-induced DNA synthesis by BaR + cytidine but not by BaR alone. 705 or 0.1 ~g/miFK anti-CD3. or anti-Ig responses was ob-served. Addition of BQR to ConA (5 ~g/miF cultures as late as 24 hours gave 70% inhibition of DNA synthesis. The capacity of synthetic nucleosides to reverse the inhibitory effect of BQR was examined by addition of either uridine (0.1 mmollL) or cytidine (0.1 mmollL) together with BQR at the start of ConA-. anti-CD3-. or anti-Ig-stimulated cultures. Uridine almost totally reversed the inhibitory effect of BQR. whilst in contrast. cytidine potentiated its action (Fig la~FK Cytidine alone had a modest inhibitory effect on ConA-stimulated DNA synthesis. These obser-vations are consistent with inhibition by BQR of a step in de novo pyrimidine nucleotide biosynthesis. Moreover. they suggest that ConA-stimulated murine lymphocytes may lack cytidine deaminase. which permits formation of uridine or its anabolites from cytidine (salvage pathway). ConA (5 ~g/miF-stimulated lymphocytes cultured for 24 hours in the presence of BQR (5 ~g/miF showed no inhibition of production of mRNA for IL-2 or IL-4 or of cytokine production. Combination of BQR with cytidine inhibited IL-2 and IL-4 production but not mRNA expression (data not shown). The observed antimetabolic effects of BQR com-plement the cytokine synthesis inhibitory action of FK 506 or Cy A and suggest that this new agent may prove useful as adjunctive immunosuppressive therapy in combination with either FK 506 or CyA. Moreover. combination of BQR and cytidine may offer a further possibility for inhibition of T - and B-ceU proliferation. REFERENCES I. Loveless SE. Neubauer RH: Proc Am Assoc Cancer Res 27:276. 1986 2. Peters GJ. Sharma SL, Laurensse G, et al: Invest New Drugs 5:235, 1987 3. Peters OJ. Schwartsmann G. Nadal JC. et al: Cancer Res 50:4644. 1990 4. Makowka L, Cramer DV: Transplant Sci 2:50,1992 5. Woo J. Ross CSK, Milton JI, et al: Clin Exp Immunol 79:109. 1990 6. Lemster B, Woo J. Strednak J, et al: Transplant Proc 24:2845, 1992 

Potentiation of the antiproliferative activity of brequinar sodium for murine lymphocytes by exogenous cytidine.

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Potentiation of the antiproliferative activity of brequinar sodium for murine lymphocytes by exogenous cytidine.

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