Orotate Phosphoribosyltransferase Activity Research Articles

Expression and Enzyme Kinetics of Fused Plasmodium falciparum Orotate Phosphoribosyltransferase and Orotidine 5′-monophosphate Decarboxylase in Different Escherichia Coli

Background: Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inverse order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OPRT-OMPDC, has been described in many organisms. Objective: The study aimed to select the optimum host cell and temperature for expressing the recombinant fusion OMPDC-OPRT having the enzymatic activity. Methods: We constructed gene fusions of the human malaria parasite Plasmodium falciparum OMPDC-OPRT (1,836 bp) in the pTrcHisA vector and expressed it as a 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21(DE3), TOP10, Rosetta) at 18°C and 25°C. The recombinant bifunctional protein was partially purified by Ni-nitrilotriacetic acid affinity chromatography and confirmed via Western blot and LC-MS/MS. The enzyme kinetics of OPRT and OMPDC was assessed. Results: Specific enzymatic activities of both OPRT and OMPDC domains expressed in E. coli BL21(DE3) cells were approximately eight-to-nine-fold higher than those in the TOP10 cells at 18°C. However, the specific activities of both domains expressed in the TOP10 cells were twice higher than those of the BL21(DE3) cells at 25°C. Very low and no enzymatic activities were observed when the constructed vector was expressed in the Rosetta cells at both induction temperatures. The bifunctional enzyme had specific activities of the OPRT and OMPDC domains in a ratio of 1:2. Kinetic study values of the OPRT domain in the bifunctional OMPDC-OPRT enzyme were found to be relatively low at µM level and at the perfect catalytic efficiency (kcat/Km). Conclusion: The recombinant fusion of OMPDC-OPRT exhibited a high expression level of E. coli BL21(DE3) at 18°C. The kinetic parameter is greater than 108M-1s-1.

A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

Control of pyrimidine nucleotide formation in Pseudomonas aurantiaca

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

The pyrimidine biosynthetic pathway and its regulation in Pseudomonas jessenii.

The control of the pyrimidine biosynthetic pathway by pyrimidine bases was examined in Pseudomonas jessenii ATCC 700870. The pyrimidine biosynthetic enzymes aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate (OMP) decarboxylase activities were found to be higher in the succinate-grown ATCC 700870 cells than the glucose-grown cells. All the enzyme activities were depressed in uracil-supplemented ATCC 700870 glucose-grown cells relative to the unsupplemented cells which was indicative of possible repression of enzyme synthesis by uracil. In the succinate-grown, ATCC 700870 cells, transcarbamoylase, dihydroorotase and dehydrogenase activities were decreased by uracil and orotate supplementation while decarboxylase activity was decreased following uracil addition. A pyrimidine auxotroph was isolated by conventional chemical mutagenesis and resistance to 5-fluoroorotic acid whose pyrimidine requirement was met by uracil or cytosine. The mutant strain was deficient for orotate phosphoribosyltransferase activity. Pyrimidine limitation of the mutant strain cells for 1 or 2h caused about a two-fold increase in aspartate transcarbamoylase or dihydroorotase activity independent of carbon source relative to excess uracil growth conditions. At the level of enzyme activity, aspartate transcarbamoylase activity in P. jessenii ATCC 700870 was inhibited strongly by pyrophosphate, ATP, UTP, GTP and UMP under saturating substrate concentrations.

Pyrimidine nucleotide synthesis in the emerging pathogen Pseudomonas monteilii.

Regulation of pyrimidine biosynthesis by pyrimidines in the emerging, opportunistic human pathogen Pseudomonas monteilii ATCC 700476 was evident. When wild-type cells were grown on succinate in the presence of uracil or orotic acid, the activities of all 5 pyrimidine biosynthetic enzymes were depressed while the activities of 3 of the enzymes decreased in glucose-grown cells supplemented with uracil or orotic acid compared with unsupplemented cells. Pyrimidine limitation of succinate- or glucose-grown pyrimidine auxotrophic cells lacking orotate phosphoribosyltransferase activity resulted in more than a doubling of the pyrimidine biosynthetic enzyme activities relative to their activities in uracil-grown cells. Independent of carbon source, pyrimidine-limited cells of the pyrimidine auxotrophic cells deficient for dihydroorotase activity generally resulted in a slight elevation or depression of the pyrimidine biosynthetic enzyme activities compared with their activities in cells grown under saturating uracil conditions. Aspartate transcarbamoylase activity in P. monteilii was regulated at the enzyme activity level, since the enzyme was strongly inhibited by CTP, UMP, GMP, GDP, ADP, and UTP. In summary, the regulation of pyrimidine biosynthesis in P. monteilii could be used to control its growth or to differentiate it biochemically from other related species of Pseudomonas.

Impact of 5-fluorouracil metabolizing enzymes on chemotherapy in patients with resectable colorectal cancer.

Although 5-fluorouracil (5-FU) is an important drug for colorectal cancer (CRC) treatment, no useful biomarker is currently available to predict treatment response. Since 5-FU is converted into active or inactive forms by orotate phosphoribosyltransferase (OPRT) or dihydropyrimidine dehydrogenase (DPD), a correlation between these enzymes and response to 5-FU has been suggested. However, such a correlation has not been investigated prospectively. Therefore, in the present study, we aimed to prospectively evaluate whether OPRT and DPD were predictive factors of the response to 5-FU treatment in patients with resectable CRC. The present investigation was designed as a multicenter prospective cohort study. OPRT and DPD activities were assessed in biopsy samples, obtained surgically from patients with resectable CRC. The OPRT/DPD ratio was calculated and the cut-off values for this ratio were determined for 5-year disease-free survival (DFS) and overall survival (OS). Patients were treated with 5-FU/leucovorin (LV) regimens and oral 5-FU. The endpoint of this study was the correlation between the OPRT/DPD ratio and 5-year DFS and OS. The cut-off value for the OPRT/DPD ratio was determined by using the maximum χ2 statistic method against 5-year DFS and OS. Sixty-eight patients were enrolled from July 2003 to May 2005. The median follow-up period was 1925 days. The OPRT/DPD ratio cut-off values for 5-year DFS and OS were 0.015 and 0.013, respectively. During the 5-year DFS and OS periods, patients with higher cut-off values had a better prognosis than those with lower ratios (P=0.03 and 0.02, respectively). In conclusion, our results suggest that the OPRT/DPD ratio could be a predictive factor for response to 5-FU/LV adjuvant chemotherapy.

Abstract 5572: Prognostic significance of TS, TK, OPRT and DPD activities in renal cell carcinoma

Abstract Introduction: Thymidylate synthase (TS) and thymidine kinase (TK) are the key enzymes in the de novo and salvage DNA synthetic process, respectively. Orotate phosphoribosyltransferase (OPRT) plays an important role in the de novo pathway of DNA and RNA synthesis. In contrast, dihydropyrimidine dehydrogenase (DPD) is a key enzyme involved in degradation of DNA and RNA. We examined the activities of TS, TK, OPRT and DPD in 83 renal cell carcinomas and normal kidneys and evaluated the clinical significance. Methods: TS, TK, OPRT and DPD activities in non-fixed fresh frozen renal cell carcinomas and normal kidneys were determined enzymatically by the FdUMP binding assay, the DEAE cellulose disc method, the 5-FU phosphorylation assay and the 5-FU degradation assay, respectively. Results: TS activity was 5-fold higher in renal cell carcinoma compared to normal kidney. TS activity in Stage III/IV renal cell carcinoma was 3-fold higher than that in Stage I/II renal cell carcinoma. In addition, the level of TS activity in Grade 3 renal cell carcinoma was 2-fold higher than that in Grade 1 and Grade 2 carcinomas. TK activity was approximately 4-fold higher in normal kidney compared with renal cell carcinoma. TK activity in Stage IV renal cell carcinoma was 2-fold higher than that in Stage I-III renal cell carcinoma. Furthermore, the level of TK activity in Grade 3 renal cell carcinoma was 2-fold higher than that in Grade 1 and Grade 2 carcinomas. OPRT activity was approximately 7-fold higher in renal cell carcinoma compared to normal kidney. OPRT activity in Stage III/IV renal cell carcinoma was 2-fold higher than that in Stage I/II renal cell carcinoma. The level of OPRT activity in Grade 3 renal cell carcinoma was 3-fold higher than those in Grade 1 and Grade 2 carcinomas. DPD activity was approximately 2-fold higher in normal kidney compared to renal cell carcinoma. The higher stage and grade of renal cell carcinoma was, the lower DPD activity was observed. The level of OPRT activity correlated with those of TS and TK activities. However, DPD activity was not associated with TS activity. High TS, TK, OPRT activities and low DPD activity predicted worse prognosis in patients with renal cell carcinoma.Conclusions: The present study has demonstrated that TS, TK, OPRT and DPD activities in renal cell carcinoma correlated with the stage/grade status of renal cell carcinoma. In addition, the levels of TS, TK, OPRT and DPD activities were prognostic parameters in patients with renal cell carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5572. doi:1538-7445.AM2012-5572

Comment on “Influence of Orotate Phosphoribosyl Transferase Levels on Tumor Prognosis and Response to Chemotherapy”

I read with great interest the recent article by Yasumatsu et al. [1]. Interestingly, recent data suggests that orotate phosphoribosyl transferase (OPRT) may significantly influence tumor prognosis and response to 5-fluorouracil therapy in a number of systemic malignancies besides head and neck carcinomas. For instance, a close association exists between OPRT levels and prognosis in esophageal malignancies. Early stages such as stage I and stage II express higher levels of orotate phosphoribosyl transferase (OPRT) in contrast to late stages [2]. Hence, OPRT may significantly influence prognosis in esophageal carcinomas. Similarly, increased chances of metastasis following surgery for colorectal carcinomas is seen in primary tumors that show accentuated OPRT expression. An increased risk of hematogeneous metastasis is seen in colorectal tumors with elevated OPRT levels [3]. Similarly, OPRT expression enhances the antineoplastic effects of 5-fluorouracil in gastric carcinomas and markedly improves prognosis in these patients [4]. Similar, relationships are seen in pancreatic malignancies. For instance, OPRT negative tumors are associated with a lower postsurgical resection survival rates in comparison to OPRT-positive pancreatic malignancies [5]. Patients with OPRT-positive tumors who are administered chemotherapy with chemotherapeutic agents such as gemcitabine have higher survival rates in contrast to those who do not receive similar adjuvant therapy. Similarly, there is upregulation of OPRT expression in prostate malignancies. In fact, a close association is seen between grading and tumor OPRT mRNA expression. In addition, hormone-refractory prostate cancers demonstrate a higher OPRT/dihydropyrimidine dehydrogenase (DPD) ratio in contrast to hormone-sensitive prostate cancer [6, 7]. This higher OPRT/DPD ratio explains the higher sensitivity of these tumors to 5-fluorouracil therapy. Similarly, bladder carcinomas demonstrate increased OPRT activity in contrast to normal bladder tissue. In fact, low-grade bladder carcinomas express lower levels of OPRT in contrast to high-grade bladder carcinomas. OPRT expression significantly influences the sensitivity of the tumors to 5-fluorouracil and this is an important factor in assessing prognosis in bladder carcinomas [7]. The above examples illustrate the close association between OPRT and treatment outcome and response to 5-fluorouracil in systemic malignancies and the need for further studies in this regard.

Biologic significance of TS, TK, and OPRT activities in renal cell carcinoma.

e15003 Background: Thymidylate synthase (TS), thymidine kinase (TK) and orotate phosphoribosyltransferase (OPRT) are the key enzymes in the de novo and salvage DNA synthetic process. 5-FU is a prod...

Abstract 904: Significance of thymidylate synthase (TS), thymidine kinase (TK) and orotate phosphoribosyltransferase (OPRT) activities as biomarkers in bladder cancer

Abstract Introduction and Objective : Thymidylate synthase (TS) and thymidine kinase (TK) are the key enzymes in the de novo and salvage DNA synthetic process, respectively. Orotate phosphoribosyltransferase (OPRT) plays a key role in the de novo pathway of DNA and RNA synthesis. Little is known about the significance of TS, TK and OPRT activities in various cancers including bladder cancer. We examined TS, TK and OPRT activities in 82 bladder cancers and normal bladders and evaluated the correlation between the level of the enzymatic activities and the stage/grade status of bladder cancer. In addition, the prognostic significance was also examined in patients with bladder cancer. Methods : TS, TK and OPRT activities in non-fixed fresh frozen bladder cancers and normal bladders were measured biochemically by the FdUMP binding assay, the DEAE cellulose disc method and the 5-FU phosphorylation assay, respectively. Results : TS activity was 10-fold higher in bladder cancer compared to normal bladder. The activity of TS in muscle-invasive bladder cancer was 3-fold higher than that in non-muscle-invasive cancer. In addition, the level of TS activity in Grade 3 bladder cancer was 4.5-fold and 3.5-fold higher than those in Grade 1 and Grade 2 cancers, respectively. TK activity was approximately 2-fold higher in bladder cancer compared with normal bladder. TK activity in muscle-invasive bladder cancer was 2-fold higher than that in non-muscle-invasive bladder cancer. Furthermore, the level of TK activity in Grade 3 bladder cancer was 2-fold higher than those in Grade 1 and Grade 2 cancers. The activity of OPRT was approximately 7.5-fold higher in bladder cancer compared to normal bladder. OPRT activity in muscle-invasive bladder cancer was 2-fold higher than that in non-muscle-invasive bladder cancer. The level of OPRT activity in Grade 3 bladder cancer was 6-fold and 2-fold higher than those in Grade 1 and Grade 2 cancers, respectively. The level of TS activity was associated with the level of TK activity. Moreover, the level of OPRT activity correlated with those of TS and TK activities. High TS, TK and OPRT activities predicted worse prognosis in patients with Ta and T1 non-muscle-invasive bladder cancer. OPRT activity had the strongest relationship between prognosis and the level of activity. Conclusions : The current study has shown that TS, TK and OPRT activities in bladder cancer were higher than that in normal bladder, and that TS, TK and OPRT activities positively correlated with the stage/grade status of bladder cancer. In addition, the levels of TS, TK and OPRT activities were prognostic indicators in patients with bladder cancer. The present study has demonstrated that TS, TK and OPRT activities may be biomarkers in bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 904. doi:10.1158/1538-7445.AM2011-904

Effect of carbon source on pyrimidine biosynthesis in Pseudomonas oryzihabitans

The effect of carbon source on the regulation of pyrimidine biosynthesis in the opportunistic human pathogen Pseudomonas oryzihabitans was studied at the level of enzyme synthesis. Although pyrimidine supplementation of glucose-grown Ps. oryzihabitans cells produced a slight but statistically significant effect on the de novo pyrimidine biosynthetic pathway enzyme activities, catabolite repression of the enzyme activities by glucose appeared to be occurring. Pyrimidine limitation experiments undertaken using an orotidine 5'-monophosphate decarboxylase mutant strain grown on glucose indicated that repression of enzyme synthesis by pyrimidines was occurring. Following pyrimidine limitation of the mutant strain cells, dihydroorotase and dihydroorotate dehydrogenase activities were found to about double while aspartate transcarbamoylase and orotate phosphoribosyltransferase activities were slightly elevated compared to their activities in the mutant strain cells grown on excess uracil.

Control of pyrimidine nucleotide formation in Pseudomonas fulva

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 50-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.

Kinetic benefits and thermal stability of orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase enzyme complex in human malaria parasite Plasmodium falciparum

We have previously shown that orotate phosphoribosyltransferase (OPRT) and orotidine 5′-monophosphate decarboxylase (OMPDC) in human malaria parasite Plasmodium falciparum form an enzyme complex, containing two subunits each of OPRT and OMPDC. To enable further characterization, we expressed and purified P. falciparum OPRT–OMPDC enzyme complex in Escherichia coli. The OPRT and OMPDC activities of the enzyme complex co-eluted in the chromatographic columns used during purification. Kinetic parameters (Km, kcat and kcat/Km) of the enzyme complex were 5- to 125-folds higher compared to the monofunctional enzyme. Interestingly, pyrophosphate was a potent inhibitor to the enzyme complex, but had a slightly inhibitory effect for the monofunctional enzyme. The enzyme complex resisted thermal inactivation at higher temperature than the monofunctional OPRT and OMPDC. The result suggests that the OPRT–OMPDC enzyme complex might have kinetic benefits and thermal stability significantly different from the monofunctional enzyme.

Regulation of pyrimidine formation inPseudomonas lundensis

The regulation of pyrimidine formation in the food spoilage agent Pseudomonas lundensis ATCC 49968 by pyrimidines was examined. In P. lundensis cells grown on glucose as a carbon source, the enzymes aspartate transcarbamoylase, dihydroorotase, and orotidine 5'-monophosphate decarboxylase were induced by orotic acid. Pyrimidine auxotrophs containing reduced transcarbamoylase or orotate phosphoribosyltransferase activity were isolated using chemical mutagenesis and selection procedures. Independent of carbon source, the maximum derepression of enzyme activity was observed for orotidine 5'-monophosphate decarboxylase after pyrimidine limitation of either auxotroph. In the glucose-grown cells of the transcarbamoylase mutant strain, orotic acid induced dihydroorotase and decarboxylase activities. Aspartate transcarbamoylase activity in succinate-grown P. lundensis cells was highly regulated by pyrophosphate as well as by pyrimidine and purine ribonucleotides. It was concluded that pyrimidine formation in P. lundensis was controlled both at the level of de novo pyrimidine biosynthetic enzyme synthesis and at the level of transcarbamoylase activity.

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

BckgroundA number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU.MethodsThis study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors.ResultsWhile there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity.ConclusionTS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

Challenge for a better combination with basic evidence

5-Fluorouracil (5-FU) has been the most widely accepted and studied chemotherapeutic agent, and many combination chemotherapeutic regimens have been reported. However, until recently, a standard regimen for metastatic gastric cancer had not been established. The combination of S-1 and cisplatin is a good candidate as a standard first-line regimen for metastatic gastric cancer. On the other hand, interest in biochemical modulation has become wide spread recently. The low level of dihydropyrimidine denhydrogenase (DPD), thymidylate synthase (TS) activities, and a high level of orotate phosphoribosyl-transferase (OPRT) activity enhance the antitumor effect of 5-FU and S-1. Docetaxel is one of the agents that modulate these enzyme expressions and activities. Moreover, the response rate of combination therapy of docetaxel and S-1 for metastatic gastric cancer was 56.3% and median survival time was 14.3 months in a phase II study, showing it to be a good candidate for a new standard regimen for gastric cancer. A phase III collaborative study, START (S-1 and Taxotere for advanced gastric cancer randomized phase III trial), is now under way in Japan and Korea.

Correlation between Clinical Pathologic Factors and Activity of 5-FU-Metabolizing Enzymes in Colorectal Cancer

Orotate phosphoribosyl transferase (OPRT), dihydropyrimidine dehydrogenase (DPD), and thymidylate synthase (TS) are initial key enzymes in the 5-fluorouracil (5-FU) metabolic pathway. The expression levels and activities of these three enzymes play important roles in the response of cancer patients to 5-FU-based chemotherapy. The purpose of this study was to investigate the relationship between the activities of 5-FU metabolic enzymes and clinicopathologic factors in colorectal cancer. We measured the activities of OPRT, DPD, and TS in colorectal cancer tissues. We also investigated the correlations between the activities of these three enzymes and clinicopathologic factors (histological type, depth of tumor invasion, extent of lymph node metastasis, Dukes' stage, lymphatic invasion, and vascular invasion). We examined 100 patients with surgically resected colorectal cancer. Poorly differentiated adenocarcinoma showed significantly higher DPD activities than did moderately differentiated or well-differentiated adenocarcinoma. In patients with lymph-node metastasis, OPRT activity was significantly lower than in patients without lymph-node metastasis. No significant relation was found between TS activity and histological type, depth of tumor invasion, extent of lymph node metastasis, Dukes' stage, lymphatic invasion, or vascular invasion. The response to 5-FU may be poor in patients with lymph-node metastasis, because of low OPRT activity, and in patients with poorly differentiated adenocarcinoma, because of high DPD activity.

Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.

Regulation of pyrimidine formation in Pseudomonas oryzihabitans

The regulation of pyrimidine formation in the opportunistic human pathogen Pseudomonas oryzihabitans was investigated at the level of enzyme synthesis and at the level of activity for the pyrimidine biosynthetic pathway enzyme aspartate transcarbamoylase. Although pyrimidine supplementation of succinate-grown P. oryzihabitans cells produced little effect on the de novo pyrimidine biosynthetic pathway enzyme activities, pyrimidine limitation experiments undertaken using an orotidine 5'-monophosphate decarboxylase mutant strain isolated from P. oryzihabitans ATCC 43272 indicated that repression of enzyme synthesis by pyrimidines was occurring. Following pyrimidine limitation of the succinate-grown decarboxylase mutant strain cells, aspartate transcarbamoylase and dihydroorotase activities were found to increase by about 3-fold while dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities were also observed to increase relative to their activities in the mutant strain cells grown on excess uracil. At the level of enzyme activity, aspartate transcarbamoylase in P. oryzihabitans was strongly inhibited by pyrophosphate, ADP, ATP and GTP in the presence of saturating substrate concentrations.