Chen T L, Erlichman C
Department of Pharmacology and Medicine, University of Toronto, Canada.
Cancer Chemother Pharmacol. 1992;30(5):370-6. doi: 10.1007/BF00689965.
Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical anti-tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/- leucovorin (LV) +/- brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 microM hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).
低剂量的从头嘧啶生物合成抑制剂与5-氟尿嘧啶（FU）联合使用，被认为可增强FU的抗肿瘤活性。布雷喹那就是这样一种抑制剂，单独使用时临床抗肿瘤效果甚微。我们测定了用FU ± 亚叶酸（LV） ± 布雷喹那处理的MGH-U1细胞的克隆形成存活率，并研究了这些处理对胸苷酸合成酶（TS）的影响。暴露24小时后，布雷喹那、FU和FU + LV（100微摩尔）导致50%细胞生长抑制（IC50）的浓度分别为0.4、20和10微摩尔。用布雷喹那的IC10（0.1微摩尔）进行24小时预处理和48小时连续处理，均可增加FU的细胞毒性，但不会增强FU + LV的细胞毒性。同时暴露于0.1微摩尔布雷喹那和FU ± LV 24小时，不会增加FU ± LV的细胞毒性。在单独用0.1微摩尔布雷喹那预处理24小时或随后再暴露于FU（25微摩尔） ± LV（100微摩尔）2小时的细胞中，测定细胞内三磷酸胞苷（CTP）和三磷酸尿苷（UTP）池、游离TS结合位点以及游离氟脱氧尿苷一磷酸（FdUMP）和脱氧尿苷一磷酸（dUMP）的水平。在用布雷喹那处理的细胞中，CTP和UTP池分别相当于对照值的68%和46%。在用FU处理的细胞中，剩余的游离TS结合位点相当于对照值的70%，而在用FU + 布雷喹那处理的细胞中，仅为对照水平的9%。与仅用FU处理的细胞相比，用布雷喹那预处理的细胞中游离FdUMP水平增加了5倍。同时暴露于100微摩尔次黄嘌呤和25微摩尔FU可抑制FdUMP形成的增加。细胞内dUMP水平不受布雷喹那影响。我们得出结论，当在FU处理之前暴露于布雷喹那时，低剂量的布雷喹那可增加FU的细胞毒性，但不会增强FU + LV的细胞毒性。这种增强作用似乎是由共底物磷酸核糖焦磷酸（PRPP）增加导致FdUMP形成增加所介导的。
