Potentiation of the Antitumor Activity of 5-Fluorouracil in Colon Carcinoma Cells by the Combination of Interferon and Deoxyribonucleosides Results from Complementary Effects on Thymidine Phosphorylase1

a-Interferon ill Nu) potentiates the cytotoxicity of 5-fluorouracil ¡Il raÃ-in vitro, and the combination has clinical efficacy in advanced colorectal cancer. We have reported previously an IFNa-mediated eleva tion in cellular FdUMP levels accompanied by the stimulation of thymidine phosphorylase (TP) activity in extracts from HT-29 human colon carcinoma cells treated with IFNa. We have now found that this effect of IFNa can be measured in vivo as an increase in thymine incorporation in intact cells. The increase was only 3-fold, however, compared to the 12-fold increase seen in TP activity in cell extracts. This suggested that the cosubstrate for TP, deoxyribose-1-phosphate, was rate limiting in the cells. Since the synthetic pathway of TP can also proceed via a transferase reaction, natural and modified deoxyribonucleosides were tested as deoxyribosyl donors. TP activity was measurable in cell extracts using deoxyinosine as cosubstrate with either thymine or FUra, although activity was only 10% ofthat measured with deoxyribose-1-phosphate. The pyrimidine analogue 5-propynyloxy-2'-deoxyuridine (PO-dUrd) had 15% of the maximal TP activity in cell extracts and also increased thymine incor poration in intact cells 10-fold. Both 2'-deoxyinosine and PO-dUrd poten tiated the cytotoxicity of FUra by 8-11-fold. IFNa potentiated the cyto toxicity of FUra by 1.8-fold, and the combination of IFNa and PO-dUrd produced a 25-fold increase in the cytotoxicity of FUra. Neither the cor responding analogue riboside, 5-propynyloxyuridine, nor the analogue base, 5-propynyloxyuracil, had any effect on FUra cytotoxicity. There was a significant correlation between the ability of a nucleoside and/or IFNa combination to increase thymine incorporation and to reduce the 50% inhibitory concentration for FUra. IFNa and PO-dUrd also potentiated the inhibition by FUra of thymidylate synthase activity. These findings suggest that the use of a deoxyribonucleoside to provide the rate limiting cosubstrate would complement the stimulation of TP by IFNa, and to gether they should further enhance the antitumor activity of FUra. INTRODUCTION IFNa3 potentiates the antitumor activity of FUra in colon carci noma in vitro, in animal models, and clinically (1-9). The enhance ment of cytotoxicity by IFNa seen in vitro was accompanied by an increased conversion of FUra to its active metabolite, FdUMP (1011). The elevated metabolic activation of FUra appears to be due to an IFNa-induced increase in TP activity, the first enzyme in the pathway for the direct conversion of FUra to deoxyribonucleotides (10). TP (thymidine orthophosphate deoxyribosyltransferase; EC 2.4.2.4) catalyzes the reversible synthesis of thymidine from thymine using Received 6/24/93; accepted 1/17/94. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA54422 and CA36517, by Cancer Center Support Grant CA13330 from the National Cancer Institute, and by a grant from the Wendy Will Case Cancer Fund. 2 To whom requests for reprints should be sent, at Department of Oncology, Albert Einstein Cancer Center, 111 East 210th Street, Bronx, NY 10467. 1 The abbreviations used are: IFNa, a-interferon; dlno, 2'-deoxyinosine; FUra, 5-flu orouracil; dR-l-P, deoxyribose-1-phosphate; TS, thymidylate synthase; PO-dUrd, 5-(2propynyloxy)-2'-deoxyuridine; PO-Urd, 5-(2-propynyloxy)-uridine; PO-Ura, 5-(2-propynyloxy)-uracil; TP, thymidine phosphorylase; NMR, nuclear magnetic resonance; bs, broad singlet; d, doublet; t, triplet; PBS, phosphate-buffered saline; PCA, perchloric acid; FBS, fetal bovine serum; IC.so, 50% inhibitory concentration. dR-l-P as a cosubstrate (12); alternatively, the enzyme can transfer a deoxyribose moiety from one deoxynucleoside to a pyrimidine base to form a second deoxynucleoside (13-15). The deoxyribosyl transferase activity has been proposed to proceed by both direct and indirect mechanisms; the latter involves an "enzyme-bound" dR-l-P interme diate (14,16,17). Kinetic studies with purified TP have shown that the initial velocity of the synthetic reaction is higher than that of the phosphorolytic reaction (17). The gene for human TP has recently been shown to be identical to that for platelet-derived endothelial cell growth factor, an angiogenic factor that stimulates endothelial cell growth and chemotaxis (18, 19). Expression of TP was found to vary up to 15-fold in different human tissues and was elevated up to 10-fold in biopsies from carcinomas of the stomach, colon, and ovary when compared to normal parenchymal tissue of these organs (20, 21). Despite the presence of this enzyme in most cells, however, the utilization of thymine by cells is low (com pared to thymidine incorporation) due to the lack of dR-l-P or de oxyribose donors (22). The addition of deoxyribonucleosides (both purine and pyrimidines) to intact cells greatly increases thymine in corporation. Since FUra can readily serve as a substrate for TP, it was anticipated that its conversion to FdUMP would also be suboptimal in IFNa-treated cells, despite the activation of the enzyme activity by IFNa. FUra is thought to be cytotoxic to tumor cells by three potential mechanisms: inhibition of thymidylate synthase by FdUMP; incorpo ration of FdUTP into DNA; and incorporation of FUTP into RNA (reviewed in Ref. 23). The mechanism by which IFNa potentiates the actions of FUra has been examined, and studies have implicated both DNA-mediated events and indirect or secondary effects on thymidy late synthase as potential targets (3, 4). Other IFNa-mediated activi ties for which there is experimental evidence include effects on DNA repair and the transcriptional upregulation of the TS gene (4, 24). These biochemical events would all potentially be further enhanced by increased cellular formation of FdUMP. We tested the hypothesis, therefore, that deoxyribonucleoside supplementation would lead to increased FUra cytotoxicity when used in combination with IFNa due to complementary effects on TP, which presumably cause a further increase in the formation of fluoropyrimidine-nucleotides, including FdUMP, and a greater inhibition of TS activity. MATERIALS AND METHODS Cell Culture. HT-29 human colon carcinoma cells (25) were maintained in RPMI 1640 with 10% FBS (GIBCO) in 5% CO2 and were free of Mycoplasma contamination. Synthesis of the 5-(2-Propynyloxy)pyrimidines. PO-dUrd and PO-Urd were prepared as described previously (Refs. 26 and 27, respectively). Essen tially, the same approach was used to synthesize the hitherto unreported POUra, i.e., treating an aqueous solution of the monosodium salt of 5-hydroxyuracil with an excess of 3-bromopropyne. The resulting PO-Ura was isolated by preparative high performance liquid chromatography on a C-18 reversed phase silica gel column with 20% methanol as the eluting solvent; m.p. > 250°C (darkens and shrinks above 200°C), 'H NMR (methyl sulfoxide-d6), 8 11.27 (IH, bs, N3-H), 10.57 (IH, bs, Nl-H), 7.21 (IH, d, H-6, J6,NH = 3.3 Hz), 4.60