Orotate Phosphoribosyltransferase Research Articles

An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound, Acetoin, in Rice Vinegar Production

Gluconacetobacter europaeus, one of the microorganisms most commonly used for vinegar production, produces the unfavorable flavor compound acetoin. Since acetoin reduction is important for rice vinegar production, a genetic approach was attempted to reduce acetoin produced by G. europaeus KGMA0119 using specific gene knockout without introducing exogenous antibiotic resistance genes. A uracil-auxotrophic mutant with deletion of the orotate phosphoribosyltransferase gene (pyrE) was first isolated by positive selection using 5-fluoroorotic acid. The pyrE disruptant designated KGMA0704 (ΔpyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and the pyrE gene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption of aldC or als, which encodes α-acetolactate decarboxylase or α-acetolactate synthase, was attempted in KGMA0704. The disruption of these genes was expected to result in a decrease in acetoin levels. A disruption vector harboring the pyrE marker within the targeted gene was constructed for double-crossover recombination. The cells of KGMA0704 were transformed with the exogenous DNA using electroporation, and genotypic analyses of the transformants revealed the unique occurrence of targeted aldC or als gene disruption. The aldC disruptant KGMA4004 and the als disruptant KGMA5315 were cultivated, and the amount of acetoin was monitored. The acetoin level in KGMA4004 culture was significantly reduced to 0.009% (wt/vol) compared with KGMA0119 (0.042% [wt/vol]), whereas that of KGMA5315 was not affected (0.037% [wt/vol]). This indicates that aldC disruption is critical for acetoin reduction. G. europaeus KGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Substitution of anti-androgens and tegafur-uracil combination therapy for castration-resistant prostate cancer: Results of a multi-center randomized phase II study

We conducted this study to determine whether substitution with anti-androgen (SOA) and tegafur-uracil (a pro‑drug of 5-FU) combination therapy is more effective than SOA alone after relapse from initial hormonal therapy. Patients who were histologically confirmed and relapsed after initial hormonal therapy were included. All patients were randomly allocated into two groups: SOA alone (group A) or SOA combined with tegafur-uracil (group B). The mRNA expression of four enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phospho-ribosyltransferase (OPRT) and thymidine phosphorylase (TP), in prostate cancer cells was analyzed by quantitative reverse-transcription polymerase chain reaction. Fifty-two patients were enrolled in this study. The median age was 77 (range: 47-92) years. The PSA response rate in group B (61.5%) tended to be higher compared to that in group A (34.6%) (p=0.095). Group B (median: 15.9 months) had a significantly longer time to PSA progression (TTP) compared to group A (6.4 months) (p=0.014). In patients with a lower TS expression or a higher OPRT expression, group B demonstrated a higher PSA response rate compared to group A (p=0.019 and p=0.041, respectively). In addition, in the patients with a lower TS expression, group B demonstrated a significantly longer TTP compared to group A (p=0.018). There were no severe adverse events in either treatment group. After relapse from initial hormonal therapy, SOA combined with tegafur-uracil is effective and well tolerated. The TS mRNA expression level may be a predictive factor for this combination therapy.

Construction of an Endogenous Selectable Marker Gene for the Rice Straw Degrading White-Rot BasidiomyceteCyathus stercoreus

Cyathus stercoreus has a superior ability to increase the cellulase saccharification yields of rice straw under biological pretreatment. As a first step to improve the biological pretreatment efficiency of C. stercoreus further, a new gene transformation system was constructed. Uracil auxotrophic mutant uv-180 was generated as host strain by ultraviolet radiation. It was transformed using plasmid pGE containing the orotate phosphoribosyltransferase (CsURA5) and enhanced green fluorescent protein (egfp). Many transformants exhibited strong fluorescence, indicating successful genetic transformation. An intron was needed for the expression of egfp. The EGFP fluorescence intensities of the three transformants did not decay even after subculturing on uracil-containing semisolid medium 5 times, suggesting that the transformed plasmids were stably retained in the absence of selective pressure.

Ultrasound-assisted improvement of drug solubility: a simple and useful method for the formation of salts from 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one

The low aqueous solubility of drug-like compounds has been described as one of the main problems causing the failure of new chemical entities in medicinal chemistry programmes. This paper describes our efforts to overcome lack of solubility and to produce a novel template protocol for forming salts of poorly soluble compounds. We prepared a series of 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one amine salts by using ultrasound irradiation. The poorly water-soluble molecule 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one was recently described as an inhibitor of the Mycobacterium tuberculosis orotate phosphoribosyltransferase enzyme. Salts of this molecule were prepared in 88–98 % yield from a mixture of 4-hydroxy-6-methyl-3-nitropyridin-2(1H)-one with primary and secondary amines (dimethylamine, diethylamine, dipropylamine, ethanolamine, 2-amino-2-methyl-1,3-propanediol, d-threoninol, pyrrolidine and piperidine) after ultrasound irradiation. The main advantages of this method are a significant reduction in reaction times and the use of a renewable solvent as a reaction medium. Thus the compounds are obtained after irradiation for 8 min in ethanol.

Study protocol of the B-CAST study: a multicenter, prospective cohort study investigating the tumor biomarkers in adjuvant chemotherapy for stage III colon cancer

BackgroundAdjuvant chemotherapy for stage III colon cancer is internationally accepted as standard treatment with established efficacy. Several oral fluorouracil (5-FU) derivatives with different properties are available in Japan, but which drug is the most appropriate for each patient has not been established. Although efficacy prediction of 5-FU derivatives using expression of 5-FU activation/metabolism enzymes in tumors has been studied, it has not been clinically applied.Methods/designThe B-CAST study is a multicenter, prospective cohort study aimed to identify the patients who benefit from adjuvant chemotherapy with each 5-FU regimen, through evaluating the relationship between tumor biomarker expression and treatment outcome. The frozen tumor specimens of patients with stage III colon cancer who receives postoperative adjuvant chemotherapy are examined. Protein expression of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) are evaluated using enzyme-linked immunosorbent assay (ELISA). mRNA expression of TP, DPD, thymidylate synthase (TS) and orotate phosphoribosyl transferase (OPRT) are evaluated using reverse transcription polymerase chain reaction (RT-PCR). The patients’ clinical data reviewed are as follow: demographic and pathological characteristics, regimen, drug doses and treatment duration of adjuvant therapy, types and severity of adverse events, disease free survival, relapse free survival and overall survival. Then, relationships among the protein/mRNA expression, clinicopathological characteristics and the treatment outcomes are analyzed for each 5-FU derivative.DiscussionA total of 2,128 patients from the 217 institutions were enrolled between April 2009 and March 2012. The B-CAST study demonstrated that large-scale, multicenter translational research using frozen samples was feasible when the sample shipment and Web-based data collection were well organized. The results of the study will identify the predictors of benefit from each 5-FU derivative, and will contribute to establish the “personalized therapy” in adjuvant chemotherapy for colon cancer.Trial registrationClinicalTrials.gov: NCT00918827, UMIN Clinical Trials Registry (UMIN-CTR) UMIN000002013

Enhancement of 5-fluorouracil-induced cytotoxicity by leucovorin in 5-fluorouracil-resistant gastric cancer cells with upregulated expression of thymidylate synthase

BackgroundElucidation of the mechanisms by which gastric cancer cells acquire resistance to 5-fluorouracil (5FU) may provide important clues to the development of effective chemotherapy for 5FU-resistant gastric cancerMethodsFour 5FU-resistant cell lines (MKN45/5FU, MKN74/5FU, NCI-N87/5FU, and KATOIII/5FU) were established by continuous exposure of the cells to progressively increasing concentrations of 5FU for about 1 year. Then, mRNA expression levels of four genes associated with 5FU metabolism, i.e., thymidylate synthase (TS), dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase, were quantitatively evaluated by real-time reverse transcriptase-polymerase chain reaction. In addition, TS protein expression was measured by Western blot analysis.ResultsAs compared with the parent cell lines, the 5FU-resistant cell lines showed 3.8- to 11.6-fold higher resistance to 5FU, as well as 1.9- to 3.5-fold higher TS mRNA expression and 1.6- to 7.1-fold higher TS protein expression. In contrast, the expressions of other genes did not differ significantly among the cell lines. The cytotoxicity of 5FU was enhanced 2.3- to 2.8 fold by leucovorin (LV) against three of the four 5FU-resistant cell lines.ConclusionsCollectively, LV enhanced the cytotoxicity of 5FU not only against the parent gastric cancer cell lines, but also against the 5FU-resistant cell lines, even those with elevated TS expression levels. These results suggest that clinical studies of a combination of 5FU and LV are warranted in patients who have recurrent gastric cancer after 5FU-based therapy.

Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms

BackgroundAfrican trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.Methodology/Principal FindingsPyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.Conclusions/SignificancePyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.

Predictive value of orotate phosphoribosyltransferase in colorectal cancer patients receiving 5-FU-based chemotherapy

Pretreatment knowledge of chemosensitivity and side-effects of chemotherapy for colorectal cancer (CRC) patients are likely to ensure the best chemotherapeutic outcome. The aim of this study was to identify additional predictive factors of chemosensitivity to the key CRC treatment drug 5-fluorouracil (5-FU). Surgically obtained specimens from 106 patients treated for CRC were immunohistochemically assessed to investigate the correlation between the protein expression of the 5-FU metabolic enzymes orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD), and clinicopathological characteristics as well as the correlation between the protein expression and outcomes of 5-FU-based chemotherapy. A correlation was detected between the high expression of the 5-FU metabolic enzyme OPRT and negative lymph node metastasis (P=0.0496), as well as between DPD and advanced Tumor-Node-Metastasis (TNM) grade cases (IIIA-IVB) and positive lymph node metastases (P=0.0414, respectively). In all 106 patients and in 79 patients undergoing 5-FU-based chemotherapy, survival was improved in those patients with a positive OPRT expression (P=0.0144 and 0.0167, respectively). OPRT expression was higher in the 79 patients with no recurrence (P=0.0179) as well as in patients treated with R0 surgery and 5-FU-based chemotherapy without side-effects (P=0.0126). Disease-free survival (DFS) rate was higher in patients without side-effects, and in patients with a positive OPRT expression without side-effects (P=0.0021 and 0.0031, respectively). Findings of this study demonstrated that OPRT expression positively correlated with fewer side-effects of 5-FU-based chemotherapy and longer patient survival.

OPRT Is a Potential Predictive Factor for the Response to S-1 in Gastric Cancer

Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric cancer patients received S-1 (80 mg/m2/day; days 1-14) and oxaliplatin (130 mg/m2; day 1) every 3 weeks and subsequently received gastrectomy with D1/D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase using quantitative reverse-transcriptase polymerase chain reaction. Results: Between December 2009 and October 2010, thirty-five patients were enrolled in this study. 24 (68.5%) patients had clinical tumor response and 10 (28.6%) patients achieved histological response. Quantitative reverse-transcriptase polymerase chain reaction results showed that orotate phosphoribo-syltransferase (OPRT) mRNA expression was significantly higher in histological responders than non-responders (3.75 vs. 1.81, P = 0.005). Diffuse-type gastric cancer patients demonstrated higher orotate phosphoribosyltransferase (OPRT) expression levels than intestinal-type ones (2.79 vs. 1.60, P = 0.014). Similar results were not found when comparing thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression levels. Conclusion: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy. Mini Abstract: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy.

Backbone 1H-13C-15N NMR Assignments and Ligand Binding Study of OMP Synthase from Saccharomyces Cerevisiae

Catalysis in OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10) is coupled to the unstructured-to-structured transition of a 10-residue peptide loop. OMP synthase from yeast is a small, homodimeric (49 kDa) and highly stable domain-swapped enzyme that catalyzes the formation of the UMP precursor orotidine 5'-monophosphate (OMP) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA). Inter-subunit communication has been proposed to result in half-of-the-sites cooperativity of binding [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342]. High-resolution X-ray structures of the highly homologous Salmonella typhimurium and E. coli enzymes show that major and apparently essential movement of protein (mobile peptide loops and entire core) and substrates accompany catalysis and can result in an occupied enzyme showing asymmetry. A novel unproductive OA analog, citrazinic acid (CA), allows structural exploration of stable Michaelis-like complexes by NMR. Here, we report the backbone 1H-13C-15N NMR assignments of the yeast enzyme in unliganded, OMP and MgPRPP·CA forms. Equilibrium binding of PRPP in the presence of CA shows two independent binding sites present in equal concentrations, but with KD values 19-fold lower and 3-fold higher than the single for PRPP of 6.3 ± 0.6 μM to two sites in the native dimer. Binding and potential structural asymmetry was further investigated by isothermal titration calorimetry and ligand titration by NMR. The implications for cooperativity and the origin of the domain swapped structure are discussed.

Factors predicting the response to oral fluoropyrimidine drugs: A phase II trial on the individualization of postoperative adjuvant chemotherapy using oral fluorinated pyrimidines in stage III colorectal cancer treated by curative resection (ACT-01 Study)

We evaluated the predictive relevance of several biomarkers on the survival of patients with stage III colorectal cancer treated with adjuvant chemotherapy of oral fluoropyrimidines. This was a multicenter phase II trial on adult patients with histologically confirmed resected stage III (Dukes' C) colorectal cancer. Patients received oral doxifluridine (800 mg/m2/day) in 3 divided doses, or oral uracil/tegafur (UFT) (400 mg/m2/day) in 2 divided doses for 5 days, every 7 days for 12 months with a 5-year follow-up. Outcome measures were disease-free survival and tissue markers [thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) protein levels and TP, DPD, thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) mRNA levels in tumor samples and TS tandem-repeat type in blood samples]. There was a significant association between the intratumoral TP/DPD enzyme ratio and disease-free survival when the model included the drug, the parameter and the interactions between them [hazard ratio (HR)=2.76; P=0.00469]. The 5-year disease-free survival rate was statistically significantly higher in patients with high TP/DPD ratios [median ≥2.63: 71.9%; 95% confidence interval (CI) 61.4-80.0] compared to patients with low TP/DPD ratios (<2.63: 57.0%; 95% CI 46.3-66.3) (log-rank P=0.0277) following adjuvant therapy with oral fluoropyrimidines. No significant association was observed between the intratumoral TP/DPD enzyme ratio (cut-off value 2.0) and the disease-free survival rate in the doxifluridine group; primary endpoint (log-rank P=0.6850). The magnitude of the intratumoral TP/DPD enzyme ratio may be a potential indicator for the individualization of postoperative adjuvant chemotherapy with oral fluoropyrimidines for stage III colorectal cancer.

Predictive markers of capecitabine sensitivity identified from the expression profile of pyrimidine nucleoside-metabolizing enzymes

Molecular markers predicting sensitivity to anticancer drugs are important and useful not only for selecting potential responders but also for developing new combinations. In the present study, we analyzed the difference in the sensitivity of xenograft models to capecitabine (Xeloda®), 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine, Furtulon®) and 5-FU by comparing the mRNA levels of 12 pyrimidine nucleoside-metabolizing enzymes. Amounts of mRNA in the tumor tissues of 80 xenograft models were determined by real-time RT-PCR and mutual correlations were examined. A clustering analysis revealed that the 12 enzymes were divided into two groups; one group consisted of 8 enzymes, including orotate phosphoribosyl transferase (OPRT), TMP kinase (TMPK) and UMP kinase (UMPK), and was related to the denovo synthesis pathway for nucleotides, with mRNA expression levels showing significant mutual correlation. In the other group, 4 enzymes, including thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD), were involved in the salvage/degradation pathway of the nucleotides, and the mRNA levels of this group were dispersed more widely than that of the denovo group. Antitumor activity was assessed in 24 xenograft models for each drug. The antitumor activity of capecitabine and 5'-DFUR correlated significantly with the mRNA levels of TP and with the TP/DPD ratio, whereas the activity of 5-FU correlated significantly with OPRT, TMPK, UMPK and CD. In a stepwise regression analysis, TP and DPD were found to be independent predictive factors of sensitivity to capecitabine and 5'-DFUR, and UMPK was predictive of sensitivity to 5-FU. These results indicate that the predictive factors for sensitivity to capecitabine and 5'-DFUR in xenograft models may be different from those for 5-FU, suggesting that these drugs may have different responders in clinical usage.

Better efficacy of S-1 treatment for thymic carcinoma: case report and review of the literature

Primary thymic carcinoma is a rare tumor of the thymus gland. The role of chemotherapy in treating advanced thymic carcinoma is unclear. It has recently been reported that thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) expression may be important factors when predicting the effectiveness of 5-fluorouracil-based chemotherapy (Kaira et al., 74:419–425, 2011). In this report, we describe three cases in which S-1 or S-1 combination treatment was given as second-line therapy for thymic carcinoma, and examine the relationships between the immunohistochemical expression of TS, DPD, and OPRT and treatment effectiveness. There were no deaths due to toxicity. Two patients achieved partial remission (PR), and one patient achieved stable disease (SD). These results suggest that S-1 is a safe and promising regimen for thymic carcinoma. Immunohistochemical staining for OPRT expression tended to be stronger in the two PR cases than in the SD case. Based on the present results, it appears that S-1 may be useful for treating unresectable thymic carcinoma, and that OPRT expression may predict the response to S-1 treatment. Further clinical studies are needed to confirm these findings.

Analysis of the mRNA Expression Levels of Thymidylate Synthase (TS), Dihydropyrimidine Dehydrogenase (DPD) and Orotate Phosphoribosyl Transferase (OPRT) in Liver Metastases from Colorectal Cancer

We measured the mRNA expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT) in liver metastases from colorectal cancer and retrospectively analyzed their association with other clinicopathological factors and the prognosis of these patients. The study involved 55 patients of colorectal cancer with liver metastasis who underwent surgery at our department between February 1988 and April 2003. Analysis of the mRNA expression levels of each of the aforementioned enzymes in the liver metastases revealed no significant differences in the mRNA expression levels of the enzymes depending on the timing of development of the liver metastasis, histological type of the cancer, or the patient gender. In the analysis of the survival period, no association was observed between the survival prognosis and the mRNA expression level of DPD or OPRT in the liver metastases; on the other hand, the prognosis was better in the group showing low TS mRNA expression (median survival period: 3.77 years) than in the group showing high TS mRNA expression (median survival period: 2.39 years) (p=0.0134). These results suggest that the mRNA expression level of TS in the liver metastases in colorectal cancer patients is a useful prognostic indicator.

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.

Suppression of heat shock protein 27 expression promotes 5-fluorouracil sensitivity in colon cancer cells in a xenograft model

The present study investigates whether the expression levels of heat shock protein27 (HSP27) in colon cancer cells are associated with 5-fluorouracil (5-FU) sensitivity in a xenograft model, as well as the mechanism responsible for regulating 5-FU sensitivity. HCT116 cells which have a high expression of HSP27 were stably transfected with specific short hairpin RNA (shRNA) in order to suppress HSP27 expression. The association between HSP27 protein expression levels and 5-FU sensitivity was evaluated in a mouse xenograft model. The mRNA expression of 5-FU metabolic enzymes and cell apoptosis were also analyzed in the transfected cells. The suppression of HSP27 protein expression led to enhanced 5-FU sensitivity. The mRNA expression levels of dihydropyrimidine dehydrogenase and orotate phosphoribosyltransferase, but not those of thymidylate synthase, and the number of apoptotic cells increased in the transfected cells after 5-FU exposure. In conclusion, the suppression of HSP27 expression in colon cancer cells may promote 5-FU sensitivity by inducing apoptosis, despite the acceleration in 5-FU metabolism.

Expression of thymidylate synthase and orotate phosphoribosyltransferase in thymic carcinoma

Thymic carcinoma is a rare thymic epithelial tumor in which chemotherapy for advanced disease has not yet been established. Thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) protein expression levels in thymic carcinoma were evaluated as possible indicators of the anticancer activity of 5-fluorouracil (5-FU) drugs using immunohistochemistry (IHC). A total of 24 samples of thymic carcinoma were used in the present study. The tumor sections were immunohistochemically stained for TS and OPRT. As a comparison with thymic carcinoma, we also assessed the TS and OPRT protein expression levels in 55 lung cancer samples. The TS expression was positive in 12 of 24 thymic carcinoma samples (50%) and OPRT expression was positive in 10 (42%). The association between TS and OPRT expression and Masaoka stages of thymic carcinoma was analyzed. The TS and OPRT expressions in stage IV were significantly higher compared to that in stages I, II or III. We also compared the TS and OPRT expression levels between thymic carcinoma and lung cancer (33 adenocarcinomas and 22 squamous cell carcinomas). TS expression in thymic carcinoma was significantly lower compared with lung squamous cell carcinoma. OPRT expression in thymic carcinoma was significantly higher compared to lung adenocarcinoma. The combination of a relatively low expression of TS and high expression of OPRT suggests an improved antitumor effect of 5-FU drugs in thymic carcinoma compared to in lung carcinoma.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

Loop Residues and Catalysis in OMP Synthase

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.