Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2-deoxyuridine 5-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N 5 ,N 10 -methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH 2 H 4 PteGlu, generating dTMP and dihydrofolate (for a review, see Ref. 1). Because the enzyme is indispensable in the de novo synthesis of dTMP, it plays an important role in DNA replication in actively dividing cells and has been an attractive target at which anti-neoplastic agents are directed. Fluoropyrimidines (e.g. 5-fluorouracil and FdUrd) and, more recently, anti-folates (e.g. AG337, ZD1694, BW1843U89) have been useful in the clinical management of tumors of the breast, colon, stomach, and head and neck (2-5). Fluoropyrimidines exert their effects through formation of the nucleotide analog FdUMP, which inhibits TS via formation of a covalent complex containing the analog CH 2 H 4 PteGlu and the enzyme (1). This complex, which is termed the inhibitory ternary complex, is quite stable and leads to prolonged inhibition of the enzyme, depletion of dTMP pools, and thymineless death.A number of studies with cultured cell lines, tumor models, and clinical specimens have shown that TS inhibitors induce enzyme levels by about 2-4-fold (6 -8). Because response to TS-directed chemotherapy is dependent upon the enzyme concentration, such induction has been viewed as a potential barrier to successful therapeutic outcomes. As a result, there has been a great deal of interest in the mechanism of the induction and in strategies to ameliorate its effects. The increases in TS levels do not involve change...

Thymidylate synthase (EC 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for DNA replication in actively dividing cells. The enzyme is a critical target at which chemotherapeutic agents such as fluoropyrimidines (e.g., 5-fluorouracil and 5-fluoro-2'-deoxyuridine) and folic acid analogues (e.g., raltitrexed, LY231514, ZD9331, and BW1843U89) are directed. These agents exert their effects through the generation of metabolites that bind the active site of TS and inhibit catalytic activity. The binding of ligands to the TS molecule leads to dramatic changes in the conformation of the enzyme, particularly within the C-terminal domain. Stabilization of the enzyme and an increase in its intracellular level are associated with ligand binding and may be important in cellular response to TS-directed drugs. In the present study, we have examined molecular features of the TS molecule that control its degradation. We find that the C-terminal conformational shift is not required for ligand-mediated stabilization of the enzyme. In addition, we demonstrate that the N-terminus of the TS polypeptide, which is extended in the mammalian enzyme and is disordered in crystal structures, is a primary determinant of the enzyme's half-life. Finally, we show that TS turnover is carried out by the 26S proteasome in a ubiquitin-independent manner. These findings provide the basis for a mechanistic understanding of TS degradation and its regulation by antimetabolites.

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrimidines 5-fluorouracil and 5Ј-fluoro-2Ј-deoxyuridine (FdUrd) or the antifolates AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of cancer, particularly cancer of the colon and rectum. Numerous studies have shown that TS gene amplification, leading to mRNA and enzyme overproduction, is a major mechanism of resistance to these inhibitors. In the present work, we have isolated and characterized FdUrd-resistant derivatives of several human colon tumor cell lines. Although gene amplification was commonly observed, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to be caused by expression of a metabolically unstable TS molecule. The reduced half-life of TS in this line was caused by a Pro-to-Leu substitution at residue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resistant line, which had an excess of enzyme relative to mRNA, the TS molecule was more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that the stabilization must be caused by changes in one or more cellular factors that regulate TS degradation. The results indicate that changes in the stability of the TS polypeptide accompany, and even contribute to, acquired resistance to TS inhibitors in colon tumor cells.

Glucocorticoids regulate proliferation of lymphosarcoma P1798 in culture. Treatment with dexamethasone caused a redistribution of cells with respect to the cell cycle. A decrease in cells in S and G2 + M phases was observed. This was accompanied by a corresponding increase in G1 cells. Growth arrest was preceded by a rapid and precipitous decrease in the expression of the cellular c-myc gene. Restriction analysis of the c-myc gene indicated that this locus was neither amplified nor grossly rearranged in P1798 cells. Glucocorticoids caused a decrease in the abundance of c-myc mRNA. After 24 h in 0.1 microM dexamethasone, c-myc mRNA levels declined to less than 5% of control. Fifty percent inhibition occurred within 90 min. The effects of dexamethasone were completely reversible. The amount of c-myc mRNA returned to control levels within 4 h after withdrawal of the hormone. Nuclear run-on transcription analysis indicated that glucocorticoids regulate transcription of the c-myc gene in P1798 cells. Transcription of exons I and II was inhibited to the same extent, suggesting that glucocorticoids inhibit initiation of transcription. Inhibition of transcription may account for decreased expression of c-myc which may, in turn, account for the antiproliferative effects of the hormone.

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