(CANCER RESEARCH 52, 4922-4928, September 15. 1992| Elevation of Thymidylate Synthase following 5-Fluorouracil Treatment Is Prevented by the Addition of Leucovorin in Murine Colon Tumors1 Clasina L. Van der Wilt, Herbert M. Pinedo, Kees Smid, and Godefridus J. Peters2 Department of Oncology, Free University Hospital, 1007 MB Amsterdam, The Netherlands ABSTRACT The inhibition of thymidylate synthase (TS) by the 5-fluorouraciI (5FU) metabolite 5-fluorodeoxyuridine monophosphate can be biochem ically modulated by leucovorin (LV). LV administration increases the level of reduced folates in tissues, which promotes the inhibition of TS. We have studied the antitumor effect, free 5-fluorodeoxyuridine mono- phosphate levels, and inhibition of TS in two murine colon tumors at several time points after weekly 5FU or LV and 5FU administration. The antitumor effect of 5FU alone could be potentiated by LV in both tumors. 5-Fluorodeoxyuridine monophosphate levels (212 and 46 pmol/g wet wt. after 2 h for Colon 26 and Colon 38, respectively) were sufficient to mediate TS inhibition, but the levels were not related to antitumor activity. Untreated controls of the 5M -sensitive tumor Colon 38 had 3 times lower TS levels than did those of the more resistant tumor Colon 26. One course of treatment resulted in a comparable extent and retention of TS inhibition for 511 and LV/5FU therapy in both tumors. After 1 week there was complete recovery of TS inhibition, but the TS levels in tumors from 5FU-treated mice tended to be higher than the controls, which was more pronounced after three courses of therapy. A 4-fold increase of TS levels was seen in Colon 26 after SFU therapy. The elevation of TS in this tumor affected the extent of TS inhibition. Tumors treated with 5FU and LV also showed an increase of TS, but to a lesser extent, while the absolute effect on TS inhibition remained the same. This might be related to the potentiating effect of LV on 5FU antitumor activity in vivo in these tumors. INTRODUCTION The enzyme TS3 is considered to be a major target of 5FU, which is commonly used in the treatment of advanced colorec- tal cancer. TS catalyzes the reductive methylation of dUMP, which is the final reaction in the de novo synthesis of dTMP. Inhibition of TS leads to depletion of dTMP and, thereby, to cessation of DNA biosynthesis. The inhibition of TS is medi ated by the formation of a ternary complex consisting of TS, FdUMP, and the cofactor CH2-THF (1). The availability of CH2-THF, especially in its polyglutamated form, determines the stability of the ternary complex (2-4), and limiting amounts might affect the action of 5FU on TS. This is the rationale for the combined treatment with 5FU and LV, a precursor of CH2- THF. In vitro (5-7) and in animal models (8-10), LV potentiated the effect of 5FU. In patients, it has also been demonstrated that LV can enhance the response rate of single-agent 5FU, in advanced colorectal cancer, from <20% to 30-40% (11). An Received 12/20/91; accepted 7/3/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds Grant IKA-VU 88-20) and Lederle Cyanamid, The Nether lands. 2 To whom requests for reprints should be addressed, at Department of Oncol ogy, Free University Hospital, P.O. Box 7057, 1007 MB Amsterdam, The Neth erlands. 3 The abbreviations used are: TS, thymidylate synthase; CH2-THF, 5,10-meth- ylenetetrahydrofolate; 5FU. 5-fluorouracil; LV, leucovorin; FdUMP, 5-fluorode oxyuridine monophosphate. effective schedule now used clinically (12) is comparable to the most effective schedule in mice, consisting of pretreatment with LV followed by simultaneous administration of LV and 5FU (10). It is generally believed that this potentiation is due to enhancement of TS inhibition and that the degree and duration of TS inhibition induced by 5FU treatment might influence the success of treatment. Measurements of TS before and after exposure to 5FU have been performed in vitro (13, 14). TS levels and TS inhibition after treatment have also been measured in tumor tissue of animal models (15-17) and patients (18-22). Most studies con centrated on the effect of a single dose of 5FU on TS levels, which was evaluated for a maximum of 72 h (16), while enzyme assays were usually limited to the ligand binding assay with FdUMP. We studied the long term effects of 5FU and LV/5FU treat ment in two murine colon tumors, Colon 26 and Colon 38. These tumors have been characterized extensively with respect to 5FU metabolism and response to 5FU-containing regimens (10, 23-25). Colon 38 is more sensitive to 5FU than is Colon 26. Potentiation of the antitumor effect of 5FU by LV appeared to be schedule dependent, and the combination was more effec tive when LV was administered before and together with 5FU (10). Recently, a purified stereoisomer of LV, /-LV, has become available. This is thought to be the biologically active form of LV and might, therefore, have a better effect than the racemic mixture of d- and /-LV. The clinical effects of/-LV are currently being studied (26, 27). TS inhibition after 5FU treatment has been studied for short periods, but little is known about long term effects. Several mechanisms of resistance related to TS (19) might, however, be present in the tumor and could be activated or develop during therapy. This study concentrates on the effects of 5FU treat ment on TS after more than one treatment course, since pa tients usually receive more than one course of 5FU administra tion. The present results demonstrate that prolonged treatment with 5FU can induce an increase in TS levels, which can, how ever, be prevented by concomitant LV therapy. MATERIALS AND METHODS Chemicals. 5FU was obtained from Hoffman LaRoche (Basel, Swit zerland). Leucovorin and purified /-leucovorin were kindly provided by Lederle Cyanamid (Etten-Leur, The Netherlands). [6-3H]-FdUMP (specific activity, 20 Ci/mmol) was purchased from Moravek Bio- chemicals Inc. (Brea, CA) and [5-3H]-dUMP (specific activity, 10.9 Ci/mmol) from Amersham International (Buckinghamshire, England). Lactobacillus casei thymidylate synthase (purified enzyme) was ob tained from BIOPURE (Boston, MA). ¿/-Tetrahydrofolate (Sigma Chemical Co., St Louis, MO) was converted into CH2-THF by addition of formaldehyde (15, 28). All other chemicals were of analytical grade and were commercially available. In Vivo Studies. Maintainance of the tumors in mice and treatment with 5FU and LV have been described in detail previously (9, 10). The most effective schedule for the combined treatment with LV and 5FU was found to be 50 mg/kg LV, followed, after 1 h, by 50 mg/kg LV and 4922 on June 22, 2015. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from