Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.
TAS-106 [1-(3-C-ethynyl-β β ββ-D-ribo-pentofuranosyl)cytosine] is a new anticancer ribo-nucleoside with promising antitumor activity. We have previously presented evidence suggesting that the TAS-106 sensitivity of cells is correlated with intracellular accumulation of the triphosphate of TAS-106, which may be affected both by cellular membrane transport mechanisms and uridine-cytidine kinase (UCK) activity. Since the presence of a UCK family consisting of two members, UCK1 and UCK2, has recently been reported in human cells, we investigated the relation between expression of UCK1 and UCK2 at both the mRNA and protein levels and UCK activity (TAS-106 phosphorylation activity) in a panel of 10 human cancer cell lines. Measurement of UCK activity in these cell lines revealed that it was well correlated with the cells' sensitivity to TAS-106. In addition, the mRNA or protein expression level of UCK2 was closely correlated with UCK activity in these cell lines, but neither the level of expression of UCK1 mRNA nor that of protein was correlated with enzyme activity. We therefore compared the protein expression level of UCK2 in several human tumor tissues and the corresponding normal tissues. Expression of UCK2 protein was barely detectable in 4 of the 5 human tumor tissues, but tended to be high in the pancreatic tumor tissue. It could not be detected at all in any of the normal tissues. Thus, expression of UCK2 appeared to be correlated with cellular sensitivity to TAS-106, and it may contribute to the tumor-selective cytotoxicity of TAS-106.Key words: TAS-106 -Anticancer ribo-nucleoside -Uridine-cytidine kinase 1 -Uridine-cytidine kinase 2 1-(3-C-Ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) is a new ribo-nucleoside analogue of cytidine that has been found to possess significant cytotoxicity and antitumor activity in preclinical therapeutic models.1-3) As shown in Fig. 1, TAS-106 taken up by the cells is rapidly phosphorylated to the monophosphate by uridine-cytidine kinase (UCK, EC 2.7.1.48), and this product is subsequently phosphorylated to the di-and the triphosphate. 4)The triphosphate of TAS-106, ECTP, is an active metabolite and inhibits RNA synthesis by nonspecifically blocking RNA polymerases I, II, and III. [5][6][7][8] Thus, intracellular accumulation of ECTP seems critical for TAS-106 to exert cytotoxic activity. In fact, we have reported that the sensitivity of cells to TAS-106 is correlated with inhibition of cellular RNA synthesis by TAS-106 and that inhibition of cellular RNA synthesis by TAS-106 is correlated with intracellular accumulation of ECTP. We have also shown that the amount of TAS-106 transported into cells and the cells' UCK activity are decreased in TAS-106-resistant cell variants in comparison with their parental cells. 4)These results suggest that both cellular membrane transport by nucleoside transporters (NTs) and initial phosphorylation by UCK may be factors limiting the display of antitumor activity of TAS-106.Most mammalian cells possess NTs that mediate the cellular m...
The mechanism of resistance to 5‐fluorouracil (5‐FU) was studied with NUGC‐3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5‐day exposures to stepwise‐increasing concentrations of 5‐FU in vitro. NUGC‐3/5FU/L was 200‐fold and over 16‐fold resistant to 96‐h and 1‐h exposures to 5‐FU, respectively. NUGC‐3/5FU/L incorporated less 5‐FU into RNA, indicating resistance to the RNA‐directed action of 5‐FU. On the other hand, NUGC‐3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5‐FU. Polymerase chain reaction‐single‐strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5‐FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC‐3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl‐transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC‐3/5FU/L than in the parent NUGC‐3. These results indicated that resistance to 5‐FU in NUGC‐3/5FU/L is due to reduced activities of 5‐FU‐anabolizing enzymes, but not to an alteration of TS. 2′‐Deoxyinosine effectively enhanced TS inhibition by 5‐FU in the resistant cells, thus markedly sensitizing them to 5‐FU.
The small GTPase RhoD, activated by fibroblast growth factor (FGF) signaling, forms actin-based, cytoneme-like, thin and long cellular protrusions through activating mDia3C. These protrusions transmit FGF receptors toward the cell body. They are likely to be responsible for intercellular communication between FGF-producing cells and target cells.
To identify genes differentially expressed in association with resistance to 5-fluorouracil (5FU), an mRNA differential display (DD) analysis was used to compare transcripts from the NUGC-3 human gastric tumor cell line and the NUGC-3/5FU/L line, which had acquired 208-fold resistance as a consequence of repeated exposure to escalating concentrations of 5FU. The 110 cDNA fragments differentially expressed in the DD analysis of either the NUGC-3 or NUGC-3/5FU/L cells were sequenced and subjected to a homology search, and 29 overexpressed and 22 underexpressed genes were identified in NUGC-3/5FU/L as a result. To confirm whether the changes in the gene expression levels in the NUGC-3/5FU/L cells were shared by other 5FU-resistant cells, 35 genes were analyzed by northern hybridization in 3 pairs of parent / 5FU-resistant human gastrointestinal tumor cell lines. The analysis revealed 20 overexpressed and 10 underexpressed genes in at least one of the three 5FU-resistant cells as compared with those in the parent cells. Among them, P-glycoprotein, equilibrative nucleoside transporter 1, and methylenetetrahydrofolate dehydrogenase were highly expressed in two of the three 5FU-resistant cells and cytidine deaminase and integrin α α α α3 were underexpressed. The acquisition of resistance to 5FU by tumor cells may result from multiple changes in cellular functions. Key words:Resistance to 5-fluorouracil -Profiling of gene expression -Human gastrointestinal tumor cells -mRNA differential display -Northern hybridization 5-Fluorouracil (5FU) is widely used in the treatment of solid tumors, but the inherent or acquired resistance of certain tumors to 5FU therapy is a major clinical problem. Various mechanisms of resistance to 5FU have been proposed: overexpression of the target enzyme thymidylate synthase (TS), depletion of folate cofactors, an increase in the level of the competing substrate deoxyuridine monophosphate, altered drug transport/metabolism, and alterations in DNA repair/cell-cycle control pathways. [1][2][3][4][5] Following repeated exposure to escalating concentrations of 5FU, a 5FU-resistant human gastric tumor cell line we established, NUGC-3/5FU/L, developed a 208-fold increase in resistance to 5FU compared with the parent line. Our previous studies 6) showed a significant decrease in the inhibitory effect of 5FU on intracellular TS activity and the incorporation of 5FU into the RNA of NUGC-3/5FU/L cells compared to parent cells. Assays for several enzymes involved in 5FU metabolism or anabolism revealed that the activities of uridine phosphorylase (UP), uridine kinase (UK), and orotate phosphoribosyltransferase (OPRT) were about 70% lower in NUGC-3/ 5FU/L cells than in the parent line. It is possible that other as yet unknown molecular mechanisms are also involved in 5FU resistance, since there appear to be considerable differences between the degree of resistance and the rates of inhibition of these 5FU-activating enzymes.In this study, an mRNA differential display (DD) were performed to screen for genes...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.