Background 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). G1 phase and hTS is localized in the nuclei during S and G2-M phase, buy 88901-36-4 the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena buy 88901-36-4 occur CD1E in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies. Introduction Human thymidylate synthase of the ThyA family [hTS (EC 18.104.22.168), encoded by the gene TYMS] is a folate-dependent enzyme that converts 2-deoxyuridine-5-monophosphate (dUMP) and N5-N10-methylenetetrahydrofolate (mTHF) to dihydrofolate and 2-deoxythymidine-5-monophosphate (dTMP). buy 88901-36-4 Recent papers demonstrated that hTS is localized not only in the cytoplasm, but also in the nuclei and in the mitochondria. Nuclear hTS is associated with proliferating cell nuclear antigen (PCNA) and other components of the DNA replication machinery, suggesting that thymidylate biosynthesis occurs at replication forks . On the other hand, mitochondrial hTS prevents uracil accumulation in mitochondrial DNA and is essential for mtDNA integrity . Human cells do not possess the flavin-dependent thymidylate synthase ThyX that is found in many free living microbes . Consequently, hTS provides the only pathway for thymidylate synthesis in human cells and represents an essential target enzyme for cancer chemotherapy . Several inhibitors that prevent the catalytic activity of human thymidylate synthase through binding to dUMP and/or mTHF binding pockets have been identified. For instance, the uracil- analog 5-fluorouracil (5-FU), after metabolic conversion to 5-FdUMP, is a well characterized active-site inhibitor of hTS that has been widely used in chemotherapy since 1957 . FdUMP forms a covalent ternary complex with hTS and mTHF, resulting in the irreversible inhibition of the catalytic activity of hTS. Inhibition buy 88901-36-4 of hTS provokes an increase of the intracellular dUMP concentration ,  and causes depletion of deoxythymidine triphosphate (dTTP) . The imbalance of intracellular deoxynucleotide pools disrupts DNA replication and triggers cell death , . In addition to direct inhibition of hTS, the 5-FU metabolites 5-fluorouridine-5-triphosphate (F-UTP) buy 88901-36-4 and 5-fluoro-2-deoxyuridine-5-triphosphate (FdUTP) cause cell death through incorporation into RNA and DNA, respectively (for a review see ). To prevent formation of resistant cell populations and to improve the response rate of treatment, 5-FU is usually given in combination with other drugs in clinical settings. For instance, a combination of 5-FU with irinotecan and oxaliplatin has increased the response rate to treatment for advanced colorectal cancer from 10%C15% to 40%C50% , , and histone deacetylase (HDAC) inhibitors have shown synergistic effects in combination with 5-FU , . Although the reliability of thymidylate synthase expression as a clinical predictor of the response to 5-FU remains controversial , , it should be noted that the nuclear to cytosolic expression ratio of hTS predicts the outcome of 5-FU treatment better than the overall expression level . It is well established that 5-FU administration increases the steady-state expression level of hTS in tissues and cell lines (for a review see ). Different regulatory mechanisms contributing to this phenomenon have been described in distinct human cell lines. For instance, in human gastrointestinal cell lines (Hutu 80, HT-29 and WIDR), as well as in human ovarian carcinoma cell lines (2008 and C13*), the ternary complex 5-FdUMP-MTF-hTS has increased stability as compared with the non-complexed enzyme, thus increasing up to 6-fold the steady-state expression level of hTS , ..