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PubMed1981

Pyrimidine metabolism of Bdellovibrio bacteriovorus grown intraperiplasmically and axenically.

Bdellovibrio bacteriovorus grown axenically or intraperiplasmically on Escherichia coli has pathways for the interconversion of pyrimidines and the synthesis of pyrimidine nucleoside 5'-triphosphates similar to those found in the enteric bacteria. Minimal differences in enzyme activities were observed for axenically and intraperiplasmically grown cells. As might be expected for an organism which takes up deoxyribonucleoside 5'-monophosphates per se, high levels of enzymes which catalyze the generation of deoxyribonucleoside triphosphates from monophosphates were found. In addition, all enzymes of the thymine salvage pathway, except for thymidine kinase, were directly demonstrated in wild-type strains. It was possible to demonstrate this activity only indirectly owing to an inhibitor in wild-type extracts. Investigations with inhibitors of pyrimidine interconversion reactions showed that essentially all B. bacteriovorus deoxyribonucleic acid not synthesized from units derived from E. coli deoxyribonucleic acid is made from components of the substrate organism's ribonucleic acid. Evidence for de novo pyrimidine synthesis from the amino acid level was not found for B. bacteriovorus grown on E. coli that had a high protein/deoxyribonucleic acid ratio or on normal E. coli. The potential for de novo pyrimidine synthesis by intraperiplasmically grown B. bacteriovorus, however, cannot be totally ruled out on the basis of these investigations.

Bdellovibrio↗

PubMed1989

Multiple control mechanisms for pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12.

Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated over a several-hundredfold range by pyrimidine availability. This regulation occurs, at least in large part, through a UTP-sensitive attenuation control mechanism in which transcriptional termination at the pyrBI attenuator, a rho-independent transcriptional terminator located immediately upstream of the pyrB structural gene, is regulated by the relative rates of transcription and translation within the pyrBI leader region. There is suggestive evidence that an additional, attenuator-independent control mechanism also contributes to this regulation. To measure the level of regulation that occurs through the attenuation and attenuator-independent control mechanisms, we constructed a mutant strain in which a 9-base-pair deletion was introduced into the attenuator of the chromosomal pyrBI operon. This deletion, which removes the run of thymidine residues at the end of the attenuator, completely abolishes rho-independent transcriptional termination activity. When the mutant strain was grown under conditions of pyrimidine excess, the level of operon expression was 51-fold greater than that of an isogenic pyrBI+ strain. Under conditions of pyrimidine limitation, operon expression was increased an additional 6.5-fold in the mutant. These results demonstrate that the attenuation control mechanism is primarily responsible for pyrimidine-mediated regulation but that there is a significant contribution by an attenuator-independent control mechanism.

Aspartate Carbamoyltransferase↗

PubMed1995

Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression.

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J. Turner, Y. Lu, and R.L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.

Bacillus subtilis↗

PubMed2001

The pyrimidine operon pyrRPB-carA from Lactococcus lactis.

The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.

Amino Acid Sequence↗

PubMed1981

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.

DNA Glycosylases↗

PubMed1982

Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.

An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.

Cell-Free System↗

PubMed1998

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point.

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

Amino Acid Sequence↗

PubMed1999

Activation of transcription by metabolic intermediates of the pyrimidine biosynthetic pathway.

Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold. The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p. Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates. We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system. Transcriptional activation by Ppr1p can be observed, however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions. The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein. Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.

Animals↗

PubMed1983

Canine cyclic hematopoiesis is associated with abnormal purine and pyrimidine metabolism.

Canine cyclic hematopoiesis is an autosomal recessive disease characterized by regular 11-13-d cycles of the neutrophil, reticulocyte, and platelet counts caused by a defect in regulation of marrow stem cell proliferation. Treatment with lithium abrogates cycling of the cell counts in these grey collie dogs. Aware of the defective lymphopoiesis associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies, we hypothesized that abnormal purine or pyrimidine metabolism might be present in these dogs. Using high pressure liquid chromatography, we measured erythrocyte purine and pyrimidine nucleotide levels and plasma purine and pyrimidine nucleosides and bases in normal and grey collie dogs before and during lithium treatment. During neutropenic periods in the grey collies, erythrocyte ATP, GTP, and UTP levels were significantly elevated. Normal dogs made neutropenic with cyclophosphamide did not show such elevations. Lithium treatment normalized the levels of erythrocyte ATP, GTP, and UTP in the grey collies and eliminated the differences between normal and grey collie nucleotide levels. Plasma thymine levels were markedly increased during neutropenia in the grey collie but were not increased in cyclophosphamide-treated normal dogs. The finding of abnormal concentrations of purine and pyrimidine metabolites in these dogs suggest that a metabolic derangement in purine or pyrimidine metabolism may be the cause of the defective stem cell proliferation in this disease.

Animals↗

PubMed2004

Purine and pyrimidine metabolism and electrocortical brain activity during hypoxemia in near-term lambs.

Insufficient cerebral O(2) supply leads to brain cell damage and loss of brain cell function. The relationship between the severity of hypoxemic brain cell damage and the loss of electrocortical brain activity (ECBA), as measure of brain cell function, is not yet fully elucidated in near-term newborns. We hypothesized that there is a strong relationship between cerebral purine and pyrimidine metabolism, as measures of brain cell damage, and brain cell function during hypoxemia. Nine near-term lambs (term, 147 d) were delivered at 131 (range, 120-141) d of gestation. After a stabilization period, prolonged hypoxemia (fraction of inspired oxygen, 0.10; duration, 2.5 h) was induced. Mean values of carotid artery blood flow, as a measure of cerebral blood flow, and ECBA were calculated over the last 3 min of hypoxemia. At the end of the hypoxemic period, cerebral arterial and venous blood gases were determined and CSF was obtained. CSF from 11 normoxemic siblings was used for baseline values. HPLC was used to determine purine and pyrimidine metabolites in CSF, as measures of brain cell damage. Concentrations of purine and pyrimidine metabolites were significantly higher in hypoxemic lambs than in their siblings, whereas ECBA was lower in hypoxemic lambs. Significant negative linear relationships were found between purine and pyrimidine metabolite concentrations and, respectively, cerebral O(2) supply, cerebral O(2) consumption, and ECBA. We conclude that brain cell function is related to concentrations of purine and pyrimidine metabolites in the CSF. Reduction of ECBA indeed reflects the measure of brain damage due to hypoxemia in near-term newborn lambs.

Animals↗

PubMed2001

Fluorouracil and the new oral fluorinated pyrimidines.

OBJECTIVE: To briefly review the biotransformation and bioavailability of fluorouracil (5-FU); discuss the effects of dihydropyrimidine dehydrogenase (DpD) on the efficacy and toxicity profiles of 5-FU; and review a new class of drugs known collectively as the oral fluorinated pyrimidines, which inhibit or circumvent DpD activity and, when administered with 5-FU, alter its pharmacokinetic and pharmacodynamic properties. DATA SOURCES: A MEDLINE literature search was conducted (1966-March 1999) using the search terms fluoropyrimidines, fluorouracil, 5-FU, fluorinated pyrimidines, capecitabine, eniluracil, uracil-tegafur, uracil-ftorafur, UFT, S1, BMS-247616, and BOF-A2. Reference lists, bibliographies of pertinent articles, and abstracts from the American Society of Clinical Oncology and the San Antonio Breast Cancer Symposium annual meetings were also identified and reviewed. Both preclinical and clinical literature were reviewed and analyzed. DATA SYNTHESIS: The new oral fluorinated pyrimidines appear to produce antitumor activity equivalent or superior to that of intravenously administered 5-FU by achieving higher intratumoral 5-FU concentrations or sustained 5-FU exposure. These agents are generally associated with manageable and non-life-threatening toxicities. The oral route of administration facilitates ease of administration and may reduce total healthcare costs associated with 5-FU-sensitive tumors. More studies are needed to assess the therapeutic and economic benefits of the oral fluorinated pyrimidines. CONCLUSIONS: The bioavailability, efficacy, and toxicity of 5-FU depend on its catabolic rate-limiting enzyme, The new oral fluorinated pyrimidines inhibit or circumvent DpD activity and, when combined with 5-FU, increase 5-FU's bioavailability and cytotoxic effects and decrease its toxicities. Results of Phase I and II studies in patients with a variety of malignancies suggest positive outcomes, including greater efficacy, less drug-related toxicity, lower costs related to drug administration, and greater patient convenience.

Animals↗

PubMed2004

Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry.

BACKGROUND: The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry. METHODS: Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. RESULTS: All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls. CONCLUSION: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.

Argininosuccinic Aciduria↗

PubMed1985

Enhancement of pyrimidine nucleoside uptake into K562 and YAC-1 cells by cadeguomycin.

Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.

Animals↗

PubMed1999

1H-NMR spectroscopy of body fluids: inborn errors of purine and pyrimidine metabolism.

BACKGROUND: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders. METHODS: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 microL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism. RESULTS: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field. CONCLUSION: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Structure of animal mitochondrial DNA: nucleotide composition, pyrimidine clusters, and methylation character.

The nucleotide composition, relative concentration of pyrimidine clusters, and the degree of methylation of the mitochondrial and nuclear DNA's of various vertebrates and the protozoan Crithidia oncopelti have been studied. With respect to the relative concentration of GC pairs, the mtDNA of animals (bull, rat) does not differ from the corresponding nDNA. The relative concentration of GC pairs in the mtDNA of certain fish and birds is 1.5-2.5 mole% higher than in the respective nDNA. The kinetoplast DNA of the protozoan C. oncopelti (where the relative concentration of the GC pairs is 42.9 mole %) differs very sharply in composition from the nDNA (where the relative concentration of GC pairs is 51.3 mole %). The mtDNA's and kDNA's studied are distinguished from the respective nDNA'S by a lower degree of clustering of pyrimidine nucleotides. The proportion of mono- and dipyrimidine fragments in the mtDNA and kDNA is 30 mole %, while in the nDNA it does not exceed 23 mole %. The relative concentration of long pyrimidine clusters (hexapyrimidine clusters of larger) in the mtDNA is smaller than in the nDNA by a factor of 2-5. The low degree of clustering of the pyrimidine nucleotides is apparently characteristic of all the known mtDNA's and may support the fact that they have a single type of organization and are of a single origin. All the vertebrate mtDNA's studied contain 5-methylcytosine as a minor base (1.5-3.15 mole %), and their level of methylation is 1.5-2 times greater than that in the respective nDNA's. It has been shown that animals display species specificity with respect to the 5-methylcytosine content in the mtDNA. Its distribution among the pyrimidine clusters in the bovine heart mtDNA differs substantially from that in the nDNA. This suggests that the methylation specificities of nuclear and mitochondrial DNA are different. A DNA methylase, which effects the in vitro methylation of cytosine residues both in the homologous mtDNA and in different heterologous DNA's, has been found in rat liver and bovine heart mitochondria. The specificity of the in vitro methylation of the cytosine residues in the same heterologous Escherichia coli B DNA by the nuclear and mitochondrial enzymes is different: The mitochondrial enzyme methylates predominantly in monopyrimidine fragments, and the nuclear enzyme methylates mostly in di- and tripyrimidine fragments. They, therefore, recognize different nucleotide sequences.

Animals↗

PubMed2000

Defects in pyrimidine degradation identified by HPLC-electrospray tandem mass spectrometry of urine specimens or urine-soaked filter paper strips.

BACKGROUND: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. METHODS: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N:-carbamyl-ss-aminoisobutyric acid, and N:-carbamyl-ss-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. RESULTS: All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 micromol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1. 0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. CONCLUSIONS: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.

Chromatography, High Pressure Liquid↗

PubMed2002

Uridine phosphorylase (-/-) murine embryonic stem cells clarify the key role of this enzyme in the regulation of the pyrimidine salvage pathway and in the activation of fluoropyrimidines.

We have reported the elevation of uridine phosphorylase (UPase) in many solid tumors and the presence of a variant phosphorolytic activity in breast cancer tissues (M. Liu et al., Cancer Res., 58: 5418-5424, 1998). To better understand the biological and pharmacological significance of these findings, we have developed an UPase gene knockout embryonic stem (ES) cell model by specific gene targeting techniques. In this cellular model, we establish the critical role of UPase as an important anabolic enzyme in 5-fluorouracil (5-FU) activation and pyrimidine salvage pathway regulation. It has long been known that UPase regulates the plasma concentration of uridine; however, little is known of the role of UPase in the activation and metabolism of 5-FU and its derivatives, mainly because of the lack of an appropriate model system. The experimental data indicate that the disruption of UPase activity in murine ES cells leads to a 10-fold increase in 5-FU IC(50) and a 2-3-fold reduction in its incorporation into nucleic acids, whereas no differences in toxicity is seen with other pyrimidine nucleoside analogues such as 5-fluorouridine, 2'-deoxy-5-fluorouridine, and 1-beta-D-arabinofuranosylcytosine compared with WT (wild-type) ES cells. Benzylacyclouridine can specifically prevent the WT ES cells from the sensitivity of 5-FU. Our data also shows the effect of UPase on the cytotoxicity of 5'-deoxy-5-fluorouridine (5'DFUR), a 5-FU prodrug. The IC(50) is increased almost 16-fold in the knockout cells compared with the wild type cells, demonstrating the role of UPase in catalyzing the conversion of 5'DFUR to 5-FU. These findings additionally elucidate the tumor-specific selectivity of capecitabine, the oral fluoropyrimidine prodrug approved for the treatment of metastatic breast and colorectal cancers. Not only do the knockout cells present a decreased incorporation of 5-FU into nucleic acids but also an increased reliance on the pyrimidine salvage pathway. The reduced dependence of UPase knockout cells on the pyrimidine de novo synthesis is reflected in the apparent resistance to phosphonacetyl-L-aspartic acid, a specific inhibitor of pyrimidine pathway, with a 5-fold elevation in its IC(50) in UPase-nullified cells compared with WT. In summary, we have successfully generated an UPase gene knockout cell model that presents reduced sensitivity to 5-FU, 5'DFUR, and phosphonacetyl-L-aspartic acid, although it does not affect the basic cellular physiology under normal tissue culture conditions. Considering the role of UPase in 5-FU metabolism and the elevated expression of this protein in cancer cells compared with paired normal tissues, additional investigation should be warranted to firmly establish the clinical role of UPase in the tumor selective activation of 5-FU and capecitabine.

Animals↗

PubMed1989

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase.

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.

Animals↗