Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluorodUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human .8-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) catalyzes the conversion of 2'-deoxyuridine 5'-monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu, where H4PteGlu is tetrahydropteroylglutamic acid) to thymidine monophosphate (dTMP) and dihydrofolate (H2PteGlu) (1). This enzymatic reaction provides the sole intracellular de novo source of dTMP, and because of its central role in the synthesis of DNA precursors, TS remains an important target enzyme in cancer chemotherapy (2).Both the cDNA and corresponding mRNA clones for mouse (3) and human (4) TS have been isolated and sequenced, and these probes have facilitated the analysis of TS structure and expression and the study of the molecular basis of TS regulation. This enzyme has been purified and well characterized from various sources, including bacteria, bacteriophage, yeast, viruses, parasites, and mammals (5-9). TS is a dimeric protein with identical subunits, each =35 kDa, and comparison of the predicted primary amino acid sequence ofthe protein from eight different sources reveals that it is one of the most highly conserved proteins.Previous studies examining regulation of TS expression have concentrated on cell-cycle-directed events. Various investigators have shown that maximal TS activity occurs during periods of active DNA synthesis (10-12). Moreover, this increase in TS enzyme levels that arises as cells enter S phase appears to be regulated at both the transcriptional and posttranscriptional levels (13-15). Takeishi et al. (4) also suggested the possibility of translational regulation of TS expression given the theoretical potential of three interconvertible secondary structures, each containing a stem-loop structure in the 5' untranslated region (5' U...

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The expression of TS is an important independent prognosticator of disease-free survival and survival in patients with rectal cancer. Adjuvant fluorouracil (5-FU)-based chemotherapy demonstrated significant improvement in disease-free and overall survival for patients with high TS levels. Prospective studies measuring TS levels will be needed to understand further the role of TS as a prognosticator of survival and chemotherapeutic benefit.

We report the enhanced inhibitory potency of methotrexate (MTX) polyglutamates and dihydrofolate pentaglutamate on the catalytic activity of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase purified from MCF-7 human breast cancer cells. In the present work, MTX (4-amino-10-methylpteroylglutamic acid) and dihydrofolate, both monoglutamates, were found to be weak competitive inhibitors of AICAR transformylase with Kis of 143 and 63 ,uM, respectively, and their inhibitory capacity was largely unaffected by the glutamated state of the folate cosubstrate. In contrast, MTX polyglutamates were found to be potent competitive inhibitors, with an =10-fold increase in inhibitory potency with the addition of each glutamate group up to four (i.e., the pentaglutamate derivative). MTX (2), and the polyglutamated forms of MTX have been detected in a number of neoplastic and, to a lesser extent, normal tissues following MTX exposure (3-9). While retaining a potent inhibitory effect on H2PteGlu reductase, the polyglutamated forms of MTX differ from parent MTX in that they possess a more prolonged intracellular half-life resulting from a slower efflux rate from cells (10). The selective retention of the MTX polyglutamates results in prolonged antimetabolic effects after the removal of extracellular drug.We have investigated the possibility that polyglutamation of MTX may increase its ability to inhibit other folatedependent enzymes, particularly those that have a higher affinity for polyglutamated folate substrates. This report describes the 2500-fold enhanced capacity of MTX polyglutamate to inhibit 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase [5-amino-4-imidazolecarboxamide ribotide (AICAR) transformylase, EC 2.1.2.3; AICAR TFase], a folate-requiring enzyme that catalyzes the reaction: 10-formyl-tetrahydrofolate (10-formyl-H4PteGlu) + AICAR, yielding 5'-phosphoribosyl-5-formamido-4-imidazole-carboxamide (formyl-AICAR), an intermediate in the de novo purine biosynthetic pathway, and tetrahydrofolic acid (H4PteGlu). We also report that H2PteGlu5, which increases in the cell following inhibition of H2PteGlu reductase (11) (Glu-Glu), and H2PteGlu were purchased from Sigma, and H2PteGlu5 was reduced from PteGlu5 as described (13,14) and purified by recrystallization. Affi-Gel Blue was purchased from Bio-Rad. All other chemicals were of reagent grade and purchased from Sigma.Preparation of Reduced Folates. Pure, biologically active l-L-10-formyl-H4PteGlu and -Glu5 were prepared by enzymatic reduction of H2PteGlu or -Glu5 to H4PteGlu or -Glu5 (15). For the enzymatic reduction, H2PteGlu or -Glu5 (50 mg) and NADPH (125 mg) in 20 ml 0.05 M Tris HCl buffer (pH 7.4) were incubated at 370C with partially purified Lactobacterium casei H2PteGlu reductase (New England Enzyme Center, Boston). The reaction was followed spectrophotometrically at 340 nm until no additional NADPH was metabolized. The H4PteGlu and -Glu5 thus formed were purified by elution from a DEAE-cel...