Enzymes Of Pyrimidine Metabolism Research Articles

Decitabine- and 5-azacytidine resistance emerges from adaptive responses of the pyrimidine metabolism network

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients’ bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72–96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

Abstract C58: p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy

Abstract Although several reasons for chemorefractoriness of pancreatic ductal adenocarcinoma (PDAC) response have been proposed, a well-established basis is genetic inactivation of the p53/p16 apoptosis axis. Therefore, there is a need for p53/p16-independent PDAC treatments. The key epigenetic regulator DNA methyltransferase 1 (DNMT1) has been scientifically validated as a molecular target for p53-independent cytoreduction of pancreatic cancer, e.g., by activating terminal epithelial programs. Moreover, the pyrimidine nucleoside analog prodrugs decitabine (Dec) or 5-azacytidine (5Aza) are metabolically processed into a nucleotide analog that depletes DNMT1. Motivated by the scientific data, several clinical trials evaluating Dec, 5Aza, or analogs to treat PDAC have been completed. However, results from these trials have been discouraging and contradict encouraging preclinical results. To model the clinical situation, we evaluated Dec and 5Aza efficacy in gemcitabine-resistant PDAC in vitro and in mice. Gemcitabine-resistant PDAC cells were cross-resistant to Dec but not 5Aza. Measurement of the pyrimidine metabolism enzymes needed for prodrug conversion demonstrated the reason: DCK that activates gemcitabine and Dec was suppressed, but UCK2 that activates 5Aza was preserved and upregulated. Also, the catabolic enzyme cytidine deaminase (CDA), which rapidly catabolizes Dec and 5Aza into inactive uridine counterparts, was upregulated. Importantly, these shifts in pyrimidine metabolism that were observed in established gemcitabine-resistant cells were rapidly recapitulated (within hours/days) upon exposure of naïve PDAC cells to Dec or 5Aza, although 5Aza exposure immediately upregulated DCK, whereas Dec exposure immediately upregulated UCK2. Measurements of nucleotide levels by LC-MS/MS indicated that these contrasting shifts were caused by opposite effects of Dec and 5Aza on dCTP levels. These observations suggested potential solutions to overcome resistance: We evaluated alternating Dec with 5Aza, to exploit their mutual priming effects on DCK and UCK2 expression, and the addition of tetrahydrouridine (THU) to inhibit the catabolism of Dec and 5Aza by CDA. This optimized regimen was very efficacious in a murine xenograft model of gemcitabine-resistant PDAC (median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P<0.00001). Terminal epithelial-differentiation of gemcitabine-resistant PDAC cells was confirmed by a significant increase in pancreatic epithelial markers PTF1A, MIST1, and N-cadherin, while apoptosis markers were not induced. In sum, the pyrimidine metabolism network, which senses and regulates nucleotide amounts, automatically dampens Dec (and gemcitabine) and 5Aza activity. Nevertheless, these reactions can be anticipated and exploited instead to extend and deepen DNMT1 depletion and noncytotoxic tumor cytoreduction. Citation Format: Rita Tohme, Xiaorong Gu, Asmaa El Desoky, Caroline Schuerger, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. p53/p16-independent cytoreduction of chemoresistant pancreatic adenocarcinoma by metabolically optimized epigenetic therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C58.

Feedback Responses of the Pyrimidine Metabolism Network Mediate Resistance to Decitabine and 5-Azacytidine

There is a need to understand and counter mechanisms-of-resistance to decitabine (Dec) and 5-azacytidine (5Aza), the only agents approved to treat all subtypes of myelodysplastic syndromes (MDS). Both Dec and 5Aza are pro-drugs, and here, in vitro, in vivo and patient data show that while processing the pro-drugs, pyrimidine metabolism is itself altered, dampening pro-drug conversion and causing resistance. Anticipation of these adaptive responses of the pyrimidine metabolism network enables their exploitation instead. Dec and 5Aza are processed by pyrimidine metabolism into a nucleotide analog that depletes DNA methyltransferase 1 (DNMT1). We found that DNMT1 protein, although substantially depleted (~50%) in patients' bone marrows at time-of-response, rebounded at time-of-relapse (Fig 1A). The pyrimidine metabolism enzymes DCK and UCK2 rate-limit cellular uptakes of Dec and 5Aza, respectively. At relapse on Dec (n=13), DCK was halved vs patients' baseline bone marrows (Fig 1B). At relapse on 5Aza (n=14), it was UCK2 that was halved (Fig 1B). Simultaneous with DCK or UCK2 suppression, however, the other enzyme (UCK2 or DCK) was upregulated up to 40-fold (Fig 1B). Seesaw DCK and UCK2 expression was observed also in DCK knock-out (KO) and UCK2-KO AML cells (HAP1): DCK-KO cells upregulated UCK2, resisted Dec, but were more sensitive to 5Aza. UCK2-KO cells upregulated DCK, resisted 5Aza, but were more sensitive to Dec (concentrations for 50% growth inhibition [GI50] Dec: HAP1 wildtype 3mM, DCK-KO 12mM, UCK2-KO 0.1mM; 5Aza: wildtype 4mM, DCK-KO 2mM, UCK2-KO 15mM). These compensatory upregulations of UCK2 or DCK were adaptive to DCK- or UCK2-KO-induced decreases in dCTP or dTTP respectively. We thus examined if Dec or 5Aza also perturbed dCTP/dTTP amounts to trigger adaptive metabolic shifts. Dec decreased dTTP and increased dCTP, while 5Aza decreased dCTP, 24 hours after their addition to AML cells (THP1, OCI-AML3, MOLM13, K562)(Fig 1C). A single dose of Dec or 5Aza was also sufficient to upregulate UCK2 or DCK >2-fold, and the catabolic enzyme CDA >4-fold, within 72 hours (Fig 1D). Protein levels tracked the mRNA changes. Exponentially proliferating Dec or 5Aza-resisting AML cells emerged within 35 days in presence of pro-drug (5 AML cell lines) and displayed the same metabolic reconfigurations seen acutely. We then used patient-derived xenotransplant (PDX) models of chemorefractory AML to identify methods to counter this automatic metabolic adaptation or 'auto-resistance'. The Dec and 5Aza doses used (subcutaneous Dec 0.2 mpk or 5Aza 2 mpk) were chosen to deplete DNMT1 from marrow without cytotoxicity, demonstrated by flow cytometry for DNMT1 and gH2AX. We found (i) Dec timed to avoid DCK troughs (D1 & 2 each week) was superior to Dec timed to coincide with DCK troughs (D1 & 4 each week)(median survival 74 vs 62 days [vehicle 40 days], Log-rank p=0.006); (ii) Adding a CDA inhibitor (intraperitoneal tetrahydrouridine 4-10 mpk) to halved doses of Dec or 5Aza (0.1 mpk, 1 mpk respectively, to avoid cytotoxicity) was superior to no CDA inhibitor + Dec 0.2 mpk or 5Aza 2 mpk (median survival 180 vs 115 days [vehicle 50 days], Log-rank p=0.004), and (iii) Alternating Dec and 5Aza timed to peaks of mutual priming (e.g., D1 & 4 each week) was superior to Dec alone or 5Aza alone, or Dec/5Aza alternating every month or Dec/5Aza given together - the regimen incorporating these lessons extended survival by several months vs vehicle (median survival 223 vs 50 days, Log-rank p=0.003). Eventual AML relapse/progression on this therapy was again caused by AML cells in which DNMT1 was not depleted (Fig 1E), because of even larger increases in CDA and upregulation of de novo pyrimidine synthesis. We are thus evaluating higher CDA-inhibitor doses (40 mpk) and incorporation of non-cytotoxic doses of a de novo pyrimidine synthesis inhibitor. In sum, the pyrimidine metabolism network responds automatically to Dec or 5Aza perturbation with adaptive shifts that decrease pro-drug conversion into the DNMT1-depleting nucleotide. These metabolic shifts can be anticipated (Fig 1F) and exploited, using simple, clinically viable treatment modifications. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Saunthararajah:EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties; Novo Nordisk: Consultancy.

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets.

Schistosomes are responsible for the parasitic disease schistosomiasis, an acute and chronic parasitic ailment that affects >240 million people in 70 countries worldwide. It is the second most devastating parasitic disease after malaria. At least 200,000 deaths per year are associated with the disease. In the absence of the availability of vaccines, chemotherapy is the main stay for combating schistosomiasis. The antischistosomal arsenal is currently limited to a single drug, Praziquantel, which is quite effective with a single-day treatment and virtually no host-toxicity. Recently, however, the question of reduced activity of Praziquantel has been raised. Therefore, the search for alternative antischistosomal drugs merits the study of new approaches of chemotherapy. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and host. Pyrimidine metabolism is an excellent target for such studies. Schistosomes, unlike most of the host tissues, require a very active pyrimidine metabolism for the synthesis of DNA and RNA. This is essential for the production of the enormous numbers of eggs deposited daily by the parasite to which the granulomas response precipitates the pathogenesis of schistosomiasis. Furthermore, there are sufficient differences between corresponding enzymes of pyrimidine metabolism from the host and the parasite that can be exploited to design specific inhibitors or "subversive substrates" for the parasitic enzymes. Specificities of pyrimidine transport also diverge significantly between parasites and their mammalian host. This review deals with studies on pyrimidine metabolism in schistosomes and highlights the unique characteristic of this metabolism that could constitute excellent potential targets for the design of safe and effective antischistosomal drugs. In addition, pyrimidine metabolism in schistosomes is compared with that in other parasites where studies on pyrimidine metabolism have been more elaborate, in the hope of providing leads on how to identify likely chemotherapeutic targets which have not been looked at in schistosomes.

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

Enzymology of Pyrimidine Metabolism and Neurodegeneration.

It is well known that disorders of pyrimidine pathways may lead to neurological, hematological, immunological diseases, renal impairments, and association with malignancies. Nucleotide homeostasis depends on the three stages of pyrimidine metabolism: de novo synthesis, catabolism and recycling of these metabolites. Cytidine and uridine, in addition to be used as substrates for pyrimidine nucleotide salvaging, also act as the precursors of cytidine triphosphate used in the biosynthetic pathway of both brain's phosphatidylcholine and phosphatidylethanolamine via the Kennedy cycle. The synthesis in the brain of phosphatidylcholine and other membrane phosphatides can utilize, in addition to glucose, three compounds present in the blood stream: choline, uridine, and a polyunsaturated fatty acids like docosahexaenoic acid. Some authors, using rat models, found that oral administration of two phospholipid precursors such as uridine and omega-3 fatty acids, along with choline from the diet, can increase the amount of synaptic membrane generated by surviving striatal neurons in rats with induced Parkinson's disease. Other authors found that in hypertensive rat fed with uridine and choline, cognitive deficit resulted improved. Uridine has also been recently considered as a neuroactive molecule, because of its involvement in important neurological functions by improving memory, sleep disorders, anti-epileptic effects, as well as neuronal plasticity. Cytidine and uridine are uptaken by the brain via specific receptors and successively salvaged to the corresponding nucleotides. The present review is devoted to the enzymology of pyrimidine pathways whose importance has attracted the attention of several researchers investigating on the mechanisms underlying the physiopathology of brain.

Mechanisms of Resistance to 5-Azacytidine/Decitabine in MDS-AML and Pre-Clinical In Vivo Proof of Principle of Rational Solutions to Extend Response

Only 5-azacytidine (5Aza) and decitabine (Dec), both DNA methyltransferase (DNMT1) depleting drugs, are FDA-approved to treat all myelodysplastic syndrome (MDS) subtypes. Neither agent is curative. What are the mechanisms of resistance? MDS is genetically heterogeneous, thus, this question has been approached by attempting to correlate MDS genetics with response, or by examining genetics at relapse. Unfortunately, results have been contradictory/inconclusive. Another approach is to consider that achievement of the intended molecular pharmacodynamic (PD) effect of DNMT1-depletion is a minimum requirement for response, superceding other biological considerations including genetics/susceptibility to apoptosis or differentiation. As such, a logical first-step is to examine whether relapse is driven by sub-clones thriving despite DNMT1-depletion, or from failure to deplete DNMT1 in the first place. In both murine and clinical studies, relapse was from failure to deplete DNMT1 (Leukemia 2011;25(11):1739-50; Oncotarget 2012;3(10):1137-45; J Clin Invest 2015;125(3):1043-55). Thus, the question can be reframed as: Why is molecular PD not achieved, and can mechanisms be identified to extend response?Achievement of molecular PD by 5Aza/Dec depends on intra-cellular exposure/half-life (t½). Intra-cellular t½ depends in turn on extra-cellular exposure time (plasma t½) and pyrimidine metabolism enzymes that phosphorylate 5Aza/Dec, trapping these drugs in cells. There is controversy regarding the identify of the enzymes that phosphorylate and trap 5Aza: UCK1, UCK2 or both. To answer this question, we correlated UCK1, UCK2 and DCK expression with sensitivity of NCI60 cancer cell lines (n=60) to 5Aza or Dec. 5Aza sensitivity correlated strongly with UCK2 expression but not UCK1 or DCK (corr. coeff.: 5AzaGI50 vs UCK2-0.28, p=0.03; vs UCK1 0.22, p=0.1; vs DCK -0.07, p=0.5). Dec sensitivity correlated with DCK expression but not UCK2 or UCK1 (corr. coeff.: DecGI50 versus DCK-0.27, p=0.04; vs UCK1-0.01, p=0.9; vs UCK2-0.19, p=0.2). There was greater than 2-fold variation of UCK2/DCK expression in primary MDS CD34+ bone marrow cells, not linked with WHO-subtypes or genetics, suggesting flexible use of one or the other enzyme for pyrimidine salvage, although a trend for higher DCK expression was noted in MDS containing mutated TP53 (GSE58831). Does treatment select for sub-clones utilizing salvage enzymes that avoid drug? DCK and UCK2 expression was serially measured by QRT-PCR in bone marrow from MDS patients treated with a Dec regimen rationalized for non-cytotoxic DNMT1-depletion (0.1-0.2 mg/kg SC 1-3X/week, n=12) or with conventional 5Aza (n=6). UCK2 consistently increased (up to 100-fold) and DCK consistently decreased at relapse on Dec, with vice-versa for 5Aza (Fig. 1).Mechanism-based solutions to such resistance were evaluated in xenotransplant models of human MDS-AML that phenocopy human disease with bone marrow infiltration and death by cytopenia. Candidate solutions investigated were (i) Dec + thymidine, or HU, or methotrexate, to increase DCK (by inhibition of ribonucleotide reductase [RNR]); (ii) 5Aza or Dec at lower dose to decrease Cmax and avoid cytotoxicity + tetrahydrouridine (THU) to increase plasma t½/Tmax for DNMT1-depletion (THU inhibits the enzyme cytidine deaminase that rapidly inactivates 5Aza and Dec in vivo); (iii) 5Aza alternating or combined with Dec, since 5Aza-mediated increases in DCK are expected to increase sensitivity to Dec, while Dec-mediated increases in UCK2 are expected to increase sensitivity to 5Aza; Of these interventions, reduced dosage 5Aza or Dec + THU, and alternating THU-5Aza with THU-Dec, but not simultaneous THU-5Aza+THU-Dec, nor the RNR inhibitors, increased response and survival to striking extents (approaching 1 year)(Fig. 2). Bone marrow gH2AX and DNMT1-expression by flow-cytometry and preservation of murine hematopoiesis confirmed the non-cytotoxic, epigenetic mechanism of action (Fig. 2).We show that straightforward shifts in pyrimidine metabolism demonstrably account for much of the resistance to 5Aza or Dec. Fortunately, such resistance is amenable to logical, non-toxic, clinically applicable solutions, validated in pre-clinical in vivo models. Clinical trials of non-cytotoxic THU-Dec (IND#112914) and alternating THU-Dec/THU-5Aza, incorporating companion PD/metabolism biomarkers, are planned. [Display omitted] DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Not applicable: Other: patent applications around decitabine, 5-azacytidine and tetrahydrouridine. Off Label Use: decitabine used by route of administration and dosages other than FDA approved regimens.

A biochemical basis for the myeloid:lymphoid cell ratio, increases in which are associated with poor prognoses in numerous pathological conditions

Background Elevated myeloid:lymphoid cell ratios in peripheral blood, which are indicative of inflammation, have been associated with poor prognoses in a wide variety of disease states. The biochemical and physiological basis of this phenomenon has not previously been investigated. Hypothesis Proliferation of lymphocytes is absolutely dependent on an adequate supply of pyrimidine nucleotides. Increases in the activities of thymidine phosphorylase (TP) and cytidine deaminase (CDA), two key enzymes of pyrimidine metabolism that are found in myeloid cells, are frequently associated with inflammation. Neutrophils and platelets do not proliferate but have high rates of metabolism as well as the capacity to control nucleotide supply by catabolizing essential precursors including pyrimidine nucleosides and glutamine, which is required for de novo nucleotide synthesis. Aim To investigate the relationship between CDA and TP activities and myeloid:lymphoid cell ratios in peripheral blood. Methods Activities of CDA and TP in whole blood samples were simultaneously quantified using a simple HPLC-based assay. Metabolic activity was assessed by resazurin reduction. Results and conclusion Elevated neutrophil:lymphocyte ratios correlated closely with increased CDA and TP activities and increased metabolic flux, supporting the hypothesis that competition for pyrimidine nucleotide precursors is an important influence on the peripheral blood myeloid:lymphoid cell ratio.

DPD is a molecular determinant of capecitabine efficacy in colorectal cancer

Capecitabine is a fluoropyrimidine-based drug that offers physicians a more convenient treatment for advanced colorectal cancer (CRC), with manageable toxicity and antitumor activity comparable to that of continuous-infusion therapies with 5-fluorouracil (5-FU). However, there are no validated and established predictive factors for clinical outcome of capecitabine efficacy in CRC. The gene expressions of the pyrimidine metabolism enzymes dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and thymidylate synthase (TS) have previously been shown to be response determinants of fluoropyrimidine-based drugs in various tumors. Therefore, we investigated whether intratumoral mRNA expression levels of these genes are also associated with the clinical outcome of patients with metastatic CRC treated with first-line capecitabine. Thirty-seven patients with metastatic CRC were enrolled in this study and treated with single agent capecitabine. The intratumoral mRNA levels of DPD, TP and TS were assessed from paraffin-embedded tissue samples using laser-capture-microdissection methods and quantitative real-time PCR. There were 20 women and 17 men with a median age of 61 years (range 49-74). The median progression-free survival was 6.7 months (95% CI, 4.8-11.6 months), with a median follow-up of 14.4 months (range 1.3-18.7 months). Complete response was observed in 1 (3%), partial response in 6 (20%), stable disease in 14 (47%) and progressive disease in 9 (30%) patients (response was inevaluable in 7 patients). Higher gene expression levels of DPD were associated with resistance to capecitabine (P=0.032; Kruskal-Wallis test). Patients with a lower mRNA amount of DPD (<or=0.46) had a longer progression-free survival compared with patients that had a higher mRNA amount (8.0 vs. 3.3 months; adjusted P=0.048; log-rank test). This pilot study suggests that intratumoral gene expression levels of DPD may be useful in predicting the clinical outcome of patients with metastatic CRC with first-line single agent capecitabine treatment. Our data should be validated in larger and prospective clinical trials.

Pyrimidine metabolism during somatic embryo development in white spruce ( Picea glauca)

Summary Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce ( Picea glauca ). The contribution of the de novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6- 14 C]orotic acid, an intermediate of the de novo pathway, and with [2- 14 C]uridine and [2- 14 C]uracil, substrates of the salvage pathways. The de novo pathway was very active throughout embryo development. More than 80 percnt; of [6- 14 C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as 14 CO 2 , probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of the de novo and salvage pathways of pyrimidine nucleotide biosynthesis in vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measured in vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.

Thymidine phosphorylase and dihydropyrimidine dehydrogenase protein expression in colorectal cancer.

It is essential for actively proliferating cells to increase their rate of DNA synthesis to progress through the cell cycle. This is reflected in the increased uracil usage that is a common feature in solid tumours. Thymidine phosphorylase (TP) anabolises formation of pyrimidine nucleosides available for DNA synthesis, whereas dihydropyrimidine dehydrogenase (DPD) catabolises the degradation of pyrimidine bases, thereby reducing levels of uracil and thymine available for DNA synthesis. In addition, tissue levels of TP or DPD have been associated with the clinical efficacy of pyrimidine anti-metabolites commonly used in the treatment of colorectal cancer. There is little information, however, on the relative expression or degree of co-ordinated regulation of either protein in primary or metastatic colorectal cancer. DPD and TP protein levels were measured in 15 primary colorectal carcinomas, 10 colorectal liver metastases and 25 adjacent uninvolved tissues. DPD was reduced in 67% (10/15) of colorectal tumours (mean tumour/normal = 0.52) and in all liver metastases (mean tumour/normal = 0.41) compared with the corresponding normal tissue. In contrast, TP was increased in 80% (12/15) of colorectal tumours (mean tumour/normal = 18.91) and in all metastases (mean tumour/normal = 3.70). TP and DPD protein expression were highly variable in uninvolved and tumour tissues. The ratio of TP:DPD was higher in 87% of colorectal tumours and in all liver metastases compared with the adjacent uninvolved tissues. This suggests the presence of co-ordinated regulation of these pyrimidine metabolic enzymes and offers a strategy for optimising the use of pyrimidine-based chemotherapy.

Purine and pyrimidine nucleoside content of the neuronal extracellular space in rat. An in vivo microdialysis study.

Neurological side-effects of chemotherapeutic agents1 known to act on purine and pyrimidine metabolism enzymes raised a possible role for nucleosides and deoxynucleosides in brain cell survival as it is demonstrated in recent investigations.2–5 Some new data also demonstrated the involvement of nucleotides and nucleosides in various brain functions. Apart from the well-known transmitter function of ATP6 and neuromodulator role of adenosine7 other nucleotides and nucleosides have also been suggested to be neuroactive. Pyrimidine nucleotides have specific pyrimidinoceptors even in cells of brain origin.8 Nucleosides could have their own modulatory actions as indicated by sleep modifying effect of uridine.9 The in vivo measurement of nucleosides and related substances in behaving animals could be the following major contribution to understanding the mechanisms of neurological side-effects of chemotherapeutic agents and the functional roles of nucleosides in the brain. A recently developed method, the in vivo microdialysis technique was used in the present study. Dialysis measures the composition of local extracellular space under various experimental conditions in freely moving animals.10 The main applications of microdialysis technique is measuring synaptic neurotransmitter overflow,10 investigation of transport functions, and pharmacokinetics of various drugs,11 and also gaining information about intracellular metabolic processes.12 Microdialysis has already been applied in human patients13 with subarachnoidal haemorrhage, head trauma, Parkinson’s disease, brain tumors and epilepsy.

Structure and function of purine and pyrimidine metabolic enzymes

Structure and function of purine and pyrimidine metabolic enzymes

Pyrimidine metabolism and secondary product formation; biogenesis of albizziine, 4-hydroxyhomoarginine and 2,3-diaminopropanoic acid

In relation to the biogenesis of pyrimidine-derived secondary products, the relative activities and substrate specificities of the enzymes of pyrimidine metabolism were determined in extracts of seedlings from a number of leguminous species. Carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) were 4 to 6 × more active, and 5′-nucleotidase and uridine hydrolase were 2 to 3 × more active in the secondary metabolite producing species examined. As it was also confirmed that UMP is the most effective nucleotide inhibitor of ATCase activity, these relatively high rates of hydrolysis of UMP to uracil would lessen effective negative feedback control of pyrimidine synthesis in the secondary metabolite producers and further increase the availability of uracil for secondary metabolism. The key enzyme of pyrimidine catabolism, NADPH-dependent dihydrouracil dehydrogenase was shown to use uracil, thymine, lathyrine and 5-aminouracil as substrates and its activity was 2 to 3 × higher in those species not yielding pyrimidine secondary products. This observation is consistent with the diversion, in the secondary product-forming plants examined, of significant amounts of uracil away from catabolism and into secondary product synthesis. The dihydrouracil dehydrogenase of Albizia julibrissin exhibited higher activity with 5-aminouracil as substrate than with uracil or thymine. Dihydropyrimidinase, the next enzyme in the catabolic pathway, was extracted from the various species and shown to use 5,6-dihydrouracil 5,6-dihydrothymine and 5-amino-5,6-dihydrouracil as substrates. Using a NADPH-regenerating system, the combined action of the two enzymes, dihydrouracil dehydrogenase and dihydropyrimidinase, was shown to produce 4-hydroxyhomoarginine from lathyrine. This was identified by co-chromatography and electrophoresis with authentic samples of 4-hydroxy-homoarginine. Preparations of β-ureidopropionase, the third enzyme in the catabolic pathway, were obtained from seedlings of Pisum sativum and shown to hydrolyse N-carbamoyl- β-alanine to 3-aminopropanoic acid, and N-carbamoyl-2-methyl-3-aminopropanoic acid to 3-amino-2-methylpropanoic acid. Additionally, the preparations from P. sativum and A. julibrissin hydrolysed albizziine to 2,3-diaminopropanoic acid, identified by co-chromatography and electrophoresís with authentic samples. It is concluded that there is a greater production and diversion of uracil into secondary product formation in the plants producing these compounds than in those that do not. In the non-producing plants, there is a more active catabolism of uracil. With A. julibrissin, degradation of 5-aminouracil by the pyrimidine catabolic process was shown to yield the non-protein amino acids albizziine and 2,3-diamino-propanoic acid. In Lathyrus tingitanus, this process demonstrably produces 4-hydroxyhomoarginine from lathyrine. The observations described are consistent with the view that production of pyrimidine-derived secondary products is attributable to the operation of detoxication processes for bioactive pyrimidines.

Circadian rhythm of orotate phosphoribosyltransferase, pyrimidine nucleoside phosphorylases and dihydrouracil dehydrogenase in mouse liver: Possible relevance to chemotherapy with 5-fluoropyrimidines

In female mice (30–35 g) maintained in standardized conditions of 12 hr light (0600–1800 hr) alternating with 12 hr darkness (1800–0600 hr), food and water ad lib., there was a 24-hr cycle change (P < 0.0001, Cosinor analysis) in the activity of hepatic orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), uridine phosphorylase (UrdPase; EC 2.4.2.3), and dihydrouracil dehydrogenase (DHUDase; 1.3.1.2) but not thymidine phosphorylase (EC 2.4.2.4). The peaks of both OPRTase and UrdPase activities occurred in the activity span at around 18 and 15 hours after light onset (HALO) and the trough at 6 and 3 HALO, respectively, when samples were taken every 4 hr. Conversely, the peak of DHUDase occurred in the rest span at around 3 HALO and the trough at 15 HALO. The maximal enzyme activities (3146 ± 172, 561 ± 25, and 6.7 ± 0.7 pmol/min/mg protein) was 210, 400 and 560% higher than the minimal activities (1507 ± 172, 139 ± 25, and 1.2 ± 0.7 pmol/min/mg protein), for OPRTase UrdPase, and DHUDase, respectively. A circadian rhythm was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition there was a corresponding shift in UrdPase and DHUDase activities with a period of 1 hr difference in the time of maximum and minimum enzyme activities. OPRTase, on the other hand, showed little change after 4 weeks of adaptation under the reverse light cycle. The circadian rhythm of these key enzymes of pyrimidine metabolism, the interrelationship of their activities, and their role in the regulation of uridine bioavailability could be of particular significance in modulating the therapeutic regimens with 5-fluorinated pyrimidines.

Purine and pyrimidine metabolism in human muscle and cultured muscle cells

Using radiochemical methods, we determined the activities of various enzymes of purine and pyrimidine metabolism in homogenates of human skeletal muscle and of cultured human muscle cells. Results show a large discrepancy between the enzyme activities in muscle and cultured cells. With regard to purine metabolism, adenylate (AMP) deaminase activity was only 1–3% in cultured cells compared to that in muscle, whereas the activity of adenosine deaminase, purine-nucleoside phosphorylase, adenosine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase was 7–15-fold higher in the cultured cells. The enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase, orotidine 5′-monophosphate decarboxylase and uridine kinase showed activity of 100–200-fold higher in cultured cells than in adult muscle. The differences in enzyme activity are probably related to the low differentiation stage and the absence of contractile activity in the cultured muscle cells. Care must be taken when using these cells as a model for studying purine and pyrimidine metabolism of adult myofibers.

Effects of N,N-dimethylformamide and sodium butyrate on enzymes of pyrimidine metabolism in cultured human tumor cells

Effects of a 7-day treatment with the maturational agents DMF and sodium butyrate on enzymes of pyrimidine metabolism, growth rate and cell maturation were assessed in 5 human tumor cell lines, ARH-77 (myeloma), K-562 (chronic myeloid leukemia), KG-1 (myeloid leukemia), HL-60 (promyelocytic leukemia) and RWLy-1 (non-Hodgkin's lymphoma). DMF lengthened the doubling times of all five cell lines while sodium butyrate lengthened only those of K-562, HL-60 and RWLy-1. Full maturation was induced only in HL-60 by either agent and in K-562 by butyrate. Exposure resulted in a decreased activity of the anabolic enzyme orotate phosphoribosyltransferase (EC 2.4.2.10) and increased activities of the catabolic enzymes thymidine phosphorylase (EC 2.4.2.4) and dihydrouracil dehydrogenase (EC 1.3.1.2). Changes in the amphibolic enzyme, uridine phosphorylase (EC 2.4.2.3) did not follow any apparent pattern. This study indicates that the pattern of pyrimidine metabolism differs between the differentiated and slowly growing, and undifferentiated rapidly growing counterpart of several human tumors, suggesting that enzymes of pyrimidine metabolism can be used as markers for cellular growth and/or maturity.

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0°C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37°C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37°C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0°C and at 37°C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.

Growth of a dihydrofolate reductaseless mutant of bacteriophage T4.

A mutant of bacteriophage T4 was isolated which was unable to induce virus-specific dihydrofolate reductase in infected cells. The mutant was able to form several other early enzymes of pyrimidine metabolism. Growth of the mutant in a wild-type host, Escherichia coli B, was compared with that of the parent strain, T4BO(1), and T4td8, a mutant which lacks the ability to induce thymidylate synthetase. Growth studies were carried out in minimal medium, which gave higher growth rates and phage yields than the supplemented media used in previous studies. The reductase mutant formed deoxyribonucleic acid and plaque-forming particles at a rate slightly higher than the synthetase mutant but 1.5-to 2-fold lower than that of the wild-type phage under all conditions studied. The addition of thymine to a culture infected by the mutant increased the growth rate significantly, suggesting that the genetic lesion leads to a partial thymidylate deficiency. Like other viral genes controlling steps in thymidylate metabolism, the dihydrofolate reductase gene appears to be useful but not completely essential for growth.