Thymidine Salvage Research Articles

Transition Metal-Catalyzed Nonoxidative Decarboxylation Reactions

Decarboxylases typically utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates. The recent characterization of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) has revealed that this enzyme adopts a TIM-barrel (beta/alpha)(8) fold and employs a mononuclear transition metal center to decarboxylate the substrate in an oxidant-independent fashion. Thus, ACMSD represents a type of decarboxylation reaction that has been so far uncharacterized in biological systems. Several close homologues of ACMSD were analyzed, including isoorotate decarboxylase (IDCase), 5-carboxyvanillic acid decarboxylase (5-CVD), gamma-resorcylate decarboxylase (gamma-RSD), and 4-oxalomesaconate hydratase (OMAH). These enzymes are involved in the catabolism of tryptophan and vanillate, the biodegradation of hydroxylbenzoates, and the thymidine salvage pathways in certain organisms. They possess the signature sequence motifs of the amidohydrolase superfamily and likely share the same structural and mechanistic characteristics as that of ACMSD. Analysis of the sequence conservation and evolutionary relationship of ACMSD-related proteins suggests an emerging ACMSD protein family that includes ACMSD and ACMSD-like decarboxylases and hydratases with diverse substrate specificities, many of which are poorly understood in regard to their functions and mechanisms. Progress in the biochemical and structural characterization of ACMSD not only sheds light on the active site of this protein family but also promises the elucidation of the detailed catalytic mechanism of these novel transition metal-dependent nonoxidative decarboxylation reactions.

Thymidine Kinase Diversity in Bacteria

Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.

An evaluation of thymidine phosphorylase as a means of preventing thymidine rescue from the thymidylate synthase inhibitor raltitrexed

The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.

Mitochondrial Deoxynucleotide Pools in Quiescent Fibroblasts: A POSSIBLE MODEL FOR MITOCHONDRIAL NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE)

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.

Activation of thymidine salvage pathway measured with FLT PET, as a potential indicator of response to 5FU in colorectal cancer

3572 Background: The commonly used positron emission tomography (PET) tracer is [18F]-Fluoro-2-deoxy-D-glucose (FDG). [18F]-3-fluoro-3-deoxy-L-thymidine (FLT) is a novel tracer, with cellular uptake mechanisms linked to the pyrimidine salvage enzyme, thymidine kinase-1 (TK-1). The colorectal cancer chemotherapeutic agent, 5-Fluorouracil (5FU), inhibits de novo pyrimidine synthesis. Therefore, we investigated changes in FLT and FDG uptake after 5FU treatment, to determine their potential in detecting early evidence of response to 5FU in vitro. Methods: Human colorectal cancer cell cultures (SW480, SW620) were treated with 5FU (50μg/ml, 2h). Cells were washed, incubated with FLT or [18F]-Fluoro-2-deoxy-D-glucose (FDG) for comparison (100kBq, 90 min). Cells were subsequently harvested and uptake was determined using a gamma counter. TK-1 expression was detected by Western blotting. Changes in S-phase fraction (SPF) of treated cell were examined by flow cytometry. Results: Cytotoxic dose of 5FU chosen showed minimal cell killing immediately after treatment (0–10% (p=NS). However, 50% cell killing was achieved at 24 hours. A mean 2.96 fold increase (2.82 ± 0.2, SW480; 3.10± 0.3 SW620) in total FLT uptake (per 10 5 cells) was observed in treated cells vs controls (p<0.01). However, no difference was observed in FDG uptake. The increase in FLT uptake post 5FU treatment correlated with increased TK-1 expression in both cell lines. No significant change in the SPF of 5FU treated cells was observed. Conclusions: An early biological response to 5FU treatment was measured with FLT and not FDG. This effect correlated with an increased expression of thymidine salvage enzyme TK-1, before changes in the cell cycle were observed. FLT PET may potentially be used as an indicator of response to 5FU based chemotherapeutic agents before changes in morphology become apparent. No significant financial relationships to disclose.

Genes of the thymidine salvage pathway: Thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa

Genes for two enzymes in the thymidine salvage pathway, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and iso-orotate decarboxylase (IDCase) have been isolated from fungal sources. THase was isolated from a Rhodotorula glutinis cDNA library using a degenerate oligonucleotide based on the published amino acid sequence. The coding sequence was transferred to an Escherichia coli expression system, from which recombinant THase activity was measured using 14C-labeled thymine. The THase sequence shows an almost complete avoidance of codons ending in A or T: 95.8% GC content is present in the third position of codons. A connection between this codon bias and the role of the thymidine salvage pathway in pyrimidine metabolism is proposed. The THase sequence is similar to Group I Fe+2-dependent, alphaKG-dependent dioxygenases. The R. glutinis THase gene was used to locate the probable THase genes in the sequenced genomes of Neurospora crassa and Aspergillus nidulans. The genes neighboring THase in these two genomes are similar to each other, and are similar to the mammalian 2-amino-3-carboxymuconate-6-semialdhyde decarboxylase (ACMSD), leading to their identification as IDCase genes. The N. crassa version was isolated by PCR of genomic DNA, and IDCase activity was measured in recombinant E. coli carrying this gene. A new family of decarboxylases, using similar substrates, is identified by virtue of the protein sequence similarity.

P14ARF expression increases dihydrofolate reductase degradation and paradoxically results in resistance to folate antagonists in cells with nonfunctional p53.

The p14(ARF) protein, the product of an alternate reading frame of the INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2 to target p53 for proteosomal degradation and causes an increase in steady-state p53 levels, leading to a G(1) and G(2) arrest of cells in the cell cycle. Although much is known about the function of p14(ARF) in the p53 pathway, not as much is known about its function in human tumor growth and chemosensitivity independently of up-regulation of p53 protein levels. To learn more about its effect on cellular proliferation and chemoresistance independent of p53 up-regulation, human HT-1080 fibrosarcoma cells null for p14(ARF) and harboring a defective p53 pathway were stably transfected with p14(ARF) cDNA under the tight control of a doxycycline-inducible promoter. Induction of p14(ARF) caused a decrease in cell proliferation rate and colony formation and a marked decrease in the level of dihydrofolate reductase (DHFR) protein. The effect of p14(ARF) on DHFR protein levels was specific, because thymidylate kinase and thymidylate synthase protein levels were not decreased nor were p53 or p21WAF1 protein levels increased. The decrease in DHFR protein was abolished when the cells were treated with the proteasome inhibitor MG132, demonstrating that p14(ARF) augments proteasomal degradation of the protein. Surprisingly, induction of p14(ARF) increased resistance to the folate antagonists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine in the medium reversed this resistance, indicating that p14(ARF) induction increases the reliance of these cells on thymidine salvage.

Significance of PET for ENT-tumors

Significance for ENT-tumors. Molecular imaging with PET is based on the use of specific radioactive molecules as source of image contrast. In ENT-tumors mostly glucose and occasionally amino acid/protein metabolism were assessed with PET for tumor diagnosis. Thymidine salvage pathway and proliferation as well as tissue hypoxia are tested already in clinical studies and bear considerable potential for modulation of radiation ports and therapeutic response of cytoreductive regimens. This short review summarises actual clinical indications and potential of PET in ENT-tumors. For both nodal staging as well as detection of recurrent disease, sensitivity and specifity of FDG-PET were 80 - 100 %. FDG-PET proved to be superior to conventional imaging in most published studies. In CUP-syndrome the primary could be detected in 25 - 50 % of patients. Acquisition of PET and CT images in a combined scanner allow versatile PET based metabolic imaging in combination with high resolution and anatomical precise CT-based morphological imaging and thus combines advantages of both imaging modalities. Clinical as well as scientific potential of this functional metabolic and high resolution morphological imaging approach is high.

In vivo labeling of fission yeast DNA with thymidine and thymidine analogs

In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies. Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine. Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP. We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk). hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs. We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers.

Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse-chase labelling.

Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools.

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C‐labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐14C]cytidine, [2‐14C]deoxycytidine and [2‐14C]thymidine. The de‐novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce‐cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO2 at all stages of embryo development.

Quantitation of plasma thymidine by high-performance liquid chromatography—atmospheric pressure chemical ionization mass spectrometry and its application to pharmacodynamic studies in cancer patients

A novel method employing high-performance liquid chromatographic–mass spectrometry analysis (LC–MS) has been developed and validated for the quantitation of plasma thymidine (TdR). It involves a plasma clean-up step with strong anion-exchange solid-phase extraction (SAX-SPE) followed by HPLC separation and atmospheric pressure chemical ionization mass spectrometry detection (APCI–MS) in a selected-ion monitoring (SIM) mode. The ionization conditions were optimised in negative ion mode to give the best intensity of the dominant formate adduct [ M+HCOO] − at m/ z 287. Relative retention times were 6.5 and 8.5 min for thymidine and 5-iodo-deoxyuridine, an iodinated analogue internal standard (IS), respectively. Peak area ratios of thymidine to IS were used for regression analysis of the calibration curve. The latter was linear from 1.0 to 100 ng/ml using 1 ml sample volume of plasma. The average recovery was 80.0 and 62.8% for thymidine and 5-iodo-deoxyuridine, respectively. The method has sufficient sensitivity, precision, accuracy and selectivity for routine analysis of human plasma thymidine concentration with a lower limit of quantitation (LOQ) of 1 ng/ml. The method was used to measure plasma thymidine in healthy volunteers and cancer patients as well as laboratory rodents. Preliminary studies in cancer patients treated with the new fluoropyrimidine analogue capecitabine ( N 4-pentoxycarbonyl-5′-5-fluorocytidine) found that plasma thymidine was reduced after 1 week of treatment, consistent with inhibition of thymidylate synthase (TS). Marked differences between plasma thymidine in humans as compared to mice and rats confirms the observation that studies of anticancer drugs targeting TS may be confounded by differences in thymidine salvage between these species. In conclusion, the method developed is highly selective and sensitive and enables the study of thymidine pharmacodynamics in several species.

2-[11C]thymidine positron emission tomography as an indicator of thymidylate synthase inhibition in patients treated with AG337.

Some anticancer drugs inhibit thymidylate synthase (TS), a key enzyme for thymidine nucleotide biosynthesis. Cells can compensate for depleted thymidine levels by taking up extracellular thymidine via a salvage pathway. We investigated the use of 2-[11C]thymidine positron emission tomography (PET) to measure thymidine salvage kinetics in vivo in humans. Five patients with advanced gastrointestinal cancer were PET scanned both before and 1 hour after oral administration of the TS inhibitor AG337 (THYMITAQ [nolatrexed]); seven control patients were scanned twice but not treated with AG337. Thymidine salvage kinetics were measured in vivo using 2-[11C]thymidine PET and spectral analysis to obtain the standardized uptake values (SUV), the area under the time-activity curve (AUC), and the fractional retention of thymidine (FRT). Changes in PET parameters between scans in the AG337-treated and control groups were compared using the Mann-Whitney U test. The relationship between AG337 exposure and AG337-induced changes in tumor FRT and in plasma deoxyuridine levels (a conventional pharmacodynamic systemic measure of TS inhibition) was examined using Spearman's regression analysis. Statistical tests were two-sided. The between-scan change in FRT in patients treated with AG337 (38% increase, 95% confidence interval [CI] = 8% to 68%) was higher than that in control patients (3% increase, 95% CI = -11% to 17%) (P =.028). The level of AG337-induced increase in both 2-[11C]thymidine FRT and plasma deoxyuridine levels was statistically significantly correlated with AG337 exposure (r = 1.00, P =.01 for both). AG337 administration was associated with increased tumor tracer retention that was consistent with tumor cell uptake of exogenous 2-[11C]thymidine as a result of TS inhibition. 2-[11C]Thymidine PET can be used to measure thymidine salvage kinetics directly in the tissue of interest.

The history of folic acid.

The history of folic acid.

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis.

Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.

MNGIE: from nuclear DNA to mitochondrial DNA.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a unique autosomal recessive disorder with mitochondrial DNA alterations. The disease is characterized clinically by ptosis, progressive external ophthalmoparesis, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Muscle biopsies typically reveal mitochondrial abnormalities including ragged-red fibers and focal cytochrome c oxidase deficiency. Analysis of mitochondrial DNA in skeletal muscle shows partial depletion, multiple deletions, or both. To identify the cause of MNGIE, we mapped the disease locus to chromosome 22q13.32-qter. Within this region, we identified the gene encoding thymidine phosphorylase as the MNGIE gene. We have identified homozygous or compound-heterozygous thymidine phosphorylase gene mutations in 35 MNGIE patients (21 families) from diverse ethnic groups, including: Ashkenazi Jewish, Western European, Jamaican, Hispanic, and Japanese. We confirmed pathogenicity of the mutations by a spectrophotometric assay of thymidine phosphorylase activity with peripheral leukocytes of 15 MNGIE patients. Thymidine phosphorylase enzymatic activity was severely reduced, thus enabling us to conclude that the loss-of-function mutations in thymidine phosphorylase gene cause MNGIE. Thymidine phosphorylase catabolizes thymidine to thymine. In agreement with this notion, we noted that plasma thymidine level is increased more than 20-fold in MNGIE patients compared to controls. Therefore, we have hypothesized that increased thymidine causes mitochondrial nucleotide pool imbalance which, in turn, leads to motochondrial DNA alterations, via a mitochondria-specific thymidine salvage pathway. The identification of the MNGIE gene has allowed us to classify MNGIE as a disease of nucleoside dysmetabolism. We may be entering a new era of research on mitochondrial nucleoside metabolism.

Modulation of RTX cytotoxicity by thymidine and dipyridamole in vitro: implications for chemotherapy.

The cytotoxicity of the thymidylate synthase (TS) inhibitor raltitrexed (RTX) is reversed by supraphysiologic thymidine concentrations. Dipyridamole (DP) blocks thymidine salvage (uptake) and potentiates several chemotherapeutic agents in vitro. The purpose of this study was to determine whether DP is capable of potentiating RTX cytotoxicity in the presence of physiologic thymidine concentrations. We examined the effect of DP on RTX cytotoxicity in the presence of various physiologic thymidine concentrations in eight colorectal adenocarcinoma cell lines by colony formation assay, and in p53-defective HL60 S and p53-competent HL60 SN3 promyelocytic leukemia cells by a tetrazolium reduction-based assay. In WiDr colon adenocarcinoma cells the cytotoxicity of 1.0 microM RTX (4 h) varied from 0% to >99% within the reported range of human serum thymidine concentrations from 500 to <50 nM, respectively. DP potentiated RTX cytotoxicity 2- to >93-fold within this range. Free DP concentrations of 10 to 20 nM, achievable with oral dosing, potentiated RTX at thymidine concentrations up to 100 nM. Potentiation at higher physiologic thymidine concentrations of >/=200 nM required DP concentrations of at least 50 nM, or about twice the steady-state DP concentration obtainable from oral and intravenous dosing, but well within that obtainable by intraperitoneal infusion. Maximal potentiation required 72 to 120 h of DP exposure. DP also potentiated RTX in the absence of exogenous thymidine in six of eight colon cell lines and HL60 S and SN3 cells suggesting a second, nonthymidine salvage-dependent mechanism of potentiation. Clinically achievable free DP concentrations potentiated RTX cytotoxicity within the range of physiologic serum thymidine concentrations. RTX/DP or DP analogue-based combination therapy should, therefore, be considered for clinical trial. Serum or tumor thymidine concentration determinations may aid in identification of patients likely to respond to TS and nucleoside salvage inhibitors versus alternate, non-TS-directed therapies.

Radioactivity-Based and Spectrophotometric Assays for Isoorotate Decarboxylase: Identification of the Thymidine Salvage Pathway in Lower Eukaryotes

A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use.Neurospora crassapossesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated14CO2is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement ofKmvalues for both substrates. IDCase activity is found to be significantly higher inN. crassastrains lackinguc-1,a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimatedKivalue that is too low for accurate determination.

Ribose-enhanced Synthesis of UTP, CTP, and GTP from Parent Nucleosides in Cardiac Myocytes

The participation of ribose and its metabolites in some nucleoside salvage reactions is well established. Isolated adult rat cardiac myocytes were used as a model system to determine whether ribose acts as a general stimulant of salvage reactions in cardiac muscle, or whether only certain classes of nucleosides are affected by ribose. Myocytes were incubated with [3H]-adenosine, [3H]-cytidine, [3H]-guanosine, [3H]-thymidine, or [3H]-uridine for 30 or 60 min in the presence or absence of 5 mM ribose. The cells were extracted and the extracts assayed for [3H]-nucleoside and [3H]-nucleotide products. Salvage synthesis of cytosine, guanine and uracil nucleotides from the parent nucleosides was stimulated by ribose. Guanosine and uridine salvage appeared saturated at 50 microM external nucleoside (the dose response of cytidine salvage was not examined). Adenosine salvage was unaffected by ribose addition; the response to increasing external adenosine concentration was non-Michaelis-Menten, showing a peak of activity at 25 microM external nucleoside. Thymidine salvage was also unaffected by ribose, and was saturated at 50 microM external thymidine. These data suggest that adenosine and thymidine are metabolized to their respective nucleotide monophosphates by kinase activity. Cytidine, guanosine, and uridine salvage are stimulated by ribose, and must therefore be metabolized in part by nucleoside phosphorylase and phosphoribosyltransferase activity.