Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.

The Warburg effect modulates DHODH role in ferroptosis: a review.

Ferroptosis is an iron-dependent regulated cell death that suppresses tumor growth. It is activated by extensive peroxidation of membrane phospholipids caused by oxidative stress. GPX4, an antioxidant enzyme, reduces these peroxidized membrane phospholipids thereby inhibiting ferroptosis. This enzyme has two distinct subcellular localization; the cytosol and mitochondria. Dihydroorotate dehydrogenase (DHODH) complements mitochondrial GPX4 in reducing peroxidized membrane phospholipids. It is the rate-limiting enzyme in de novo pyrimidine nucleotide biosynthesis. Its role in ferroptosis inhibition suggests that DHODH inhibitors could have two complementary mechanisms of action against tumors; inhibiting de novo pyrimidine nucleotide biosynthesis and enhancing ferroptosis. However, the link between mitochondrial function and ferroptosis, and the involvement of DHODH in the ETC suggests that its role in ferroptosis could be modulated by the Warburg effect. Therefore, we reviewed relevant literature to get an insight into the possible effect of this metabolic reprogramming on the role of DHODH in ferroptosis. Furthermore, an emerging link between DHODH and cellular GSH pool has also been highlighted. These insights could contribute to the rational design of ferroptosis-based anticancer drugs. Video Abstract.

ENT1 blockade by CNX-774 overcomes resistance to DHODH inhibition in pancreatic cancer.

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nucleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from inhibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resistance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest pyrimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Control of a pyrimidine ribonucleotide salvage pathway in Pseudomonas oleovorans.

The control of a pyrimidine ribonucleotide salvage pathway in the bacterium Pseudomonas oleovorans ATCC 8062 was studied. This bacterium is important for its ability to synthesize polyesters as well as for its increasing clinical significance in humans. The pyrimidine salvage pathway enzymes pyrimidine nucleotide N-ribosidase and cytosine deaminase were investigated in P. oleovorans ATCC 8062 under selected culture conditions. Initially, the effect of carbon source on the two pyrimidine salvage enzymes in ATCC 8062 cells was examined and it was observed that cell growth on the carbon source succinate generally produced higher enzyme activities than did glucose or glycerol as a carbon source when ammonium sulfate served as the nitrogen source. Using succinate as a carbon source, growth on dihydrouracil as nitrogen source caused a 1.9-fold increase in the pyrimidine nucleotide N-ribosidase activity and a 4.8-fold increase in cytosine deaminase activity compared to the ammonium sulfate-grown cells. Growth of ATCC 8062 cells on cytosine or dihydrothymine as a nitrogen source elevated deaminase activity by more than double that observed for ammonium sulfate-grown cells. The findings indicated a relationship between this pyrimidine salvage pathway and the pyrimidine reductive catabolic pathway since growth on dihydrouracil appeared to increase the degradation of the pyrimidine ribonucleotide monophosphates to uracil. The uracil produced could be degraded by the pyrimidine base reductive catabolic pathway to ß-alanine as a source of nitrogen. This investigation could prove helpful to future work examining the metabolic relationship between pyrimidine salvage pathways and pyrimidine reductive catabolism in pseudomonads.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

Dysregulation of de novo nucleotide biosynthetic pathway enzymes in cancer and targeting opportunities.

Cancer is a disease of uncontrolled cell growth and a major cause of death worldwide. Many molecular events characterize tumor initiation and progression. Global gene expression analyses using next-generation sequencing, proteomics and metabolomics show genomic, epigenetic, and metabolite concentration changes in various tumors. Molecular alterations identified include multiple cancer-driving mutations, gene fusions, amplifications, deletions, and post-translational modifications. Data integration from many high-throughput platforms unraveled dysregulation in many metabolic pathways in cancer. Since cancer cells are fast-growing, their metabolic needs are enhanced, hence the requirement for de novo synthesis of essential metabolites. One critical requirement of fast-growing cells and a historically important pathway in cancer is the nucleotide biosynthetic pathway and its enzymes are valuable targets for small molecule inhibition. Purines and pyrimidines are building blocks of DNA synthesis and due to their excessive growth, cancer cells extensively utilize de novo pathways for nucleotide biosynthesis. Methotrexate, one of the early chemotherapeutic agents, targets dihydrofolate reductase of the folate metabolic pathway that is involved in nucleotide biosynthesis. In this review, we discuss the nucleotide biosynthetic pathways in cancer and targeting opportunities.

Genomic sites hypersensitive to ultraviolet radiation.

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at A2-15TTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers.

Housecleaning of pyrimidine nucleotide pool coordinates metabolic adaptation of nongrowing Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.

Muscle and serum metabolomes are dysregulated in colon-26 tumor-bearing mice despite amelioration of cachexia with activin receptor type 2B ligand blockade.

Cancer-associated cachexia reduces survival, which has been attenuated by blocking the activin receptor type 2B (ACVR2B) ligands in mice. The purpose of this study was to unravel the underlying physiology and novel cachexia biomarkers by use of the colon-26 (C26) carcinoma model of cancer cachexia. Male BALB/c mice were subcutaneously inoculated with C26 cancer cells or vehicle control. Tumor-bearing mice were treated with vehicle (C26+PBS) or soluble ACVR2B either before (C26+sACVR/b) or before and after (C26+sACVR/c) tumor formation. Skeletal muscle and serum metabolomics analysis was conducted by gas chromatography-mass spectrometry. Cancer altered various biologically functional groups representing 1) amino acids, 2) energy sources, and 3) nucleotide-related intermediates. Muscle metabolomics revealed increased content of free phenylalanine in cancer that strongly correlated with the loss of body mass within the last 2 days of the experiment. This correlation was also detected in serum. Decreased ribosomal RNA content and phosphorylation of a marker of pyrimidine synthesis revealed changes in nucleotide metabolism in cancer. Overall, the effect of the experimental C26 cancer predominated over blocking ACVR2B ligands in both muscle and serum. However, the level of methyl phosphate, which was decreased in muscle in cancer, was restored by sACVR2B-Fc treatment. In conclusion, experimental cancer affected muscle and blood metabolomes mostly independently of blocking ACVR2B ligands. Of the affected metabolites, we have identified free phenylalanine as a promising biomarker of muscle atrophy or cachexia. Finally, the decreased capacity for pyrimidine nucleotide and protein synthesis in tumor-bearing mice opens up new avenues in cachexia research.

Structure-Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5'-Nucleotidase (CD73) Inhibitors.

Cluster of differentiation 73 (CD73) converts adenosine 5'-monophosphate to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogues, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3-methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N4-(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5'- O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N4-benzoyl-cytidine (7f), N4-[ O-(4-benzyloxy)]-cytidine (9h), and N4-[ O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) ( Ki values 5-10 nM at human CD73). Selected compounds tested at the two uridine diphosphate-activated P2Y receptor subtypes showed high CD73 selectivity, especially those with large nucleobase substituents. These nucleotide analogues are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

Epitranscriptomic Code and Its Alterations in Human Disease.

Innovations in epitranscriptomics have resulted in the identification of more than 160 RNA modifications to date. These developments, together with the recent discovery of writers, readers, and erasers of modifications occurring across a wide range of RNAs and tissue types, have led to a surge in integrative approaches for transcriptome-wide mapping of modifications and protein-RNA interaction profiles of epitranscriptome players. RNA modification maps and crosstalk between them have begun to elucidate the role of modifications as signaling switches, entertaining the notion of an epitranscriptomic code as a driver of the post-transcriptional fate of RNA. Emerging single-molecule sequencing technologies and development of antibodies specific to various RNA modifications could enable charting of transcript-specific epitranscriptomic marks across cell types and their alterations in disease.

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines.

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.

Dihydroorotate Dehydrogenase as a Target for the Development of Novel Helicobacter pylori-Specific Antimicrobials.

Helicobacter pylori (H. pylori) infection is the world's most common bacterial infection, affecting approximately 50% of the global population. H. pylori is the strongest known risk factor for stomach diseases, including cancer. Hence, treatment for H. pylori infection can help reduce the risk of these diseases. However, the emergence of drug-resistant strains of H. pylori and the occurrence of adverse effects resulting from current therapies have complicated the successful eradication of H. pylori infection. Although various antibiotics that target several bacterial enzymes have been discovered, dihydroorotate dehydrogenase (DHODH) may hold potential for the development of novel anti-H. pylori agents with reduced toxicity and side effects. Here we review the existing literature that has focused on strategies for developing novel therapeutic agents that target the DHODH of H. pylori.

Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding.

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.

Role of sequence evolution and conformational dynamics in the substrate specificity and oligomerization mode of thymidylate kinases.

Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.

Aspartate Rescues S-phase Arrest Caused by Suppression of Glutamine Utilization in KRas-driven Cancer Cells.

During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

Expression and characterization of purinergic receptors in rat middle meningeal artery-potential role in migraine.

The dura mater and its vasculature have for decades been central in the hypothesis of migraine and headache pathophysiology. Although recent studies have questioned the role of the vasculature as the primary cause, dural vessel physiology is still relevant in understanding the complex pathophysiology of migraine. The aim of the present study was to isolate the middle meningeal artery (MMA) from rodents and characterize their purinergic receptors using a sensitive wire myograph method and RT-PCR. The data presented herein suggest that blood flow through the MMA is, at least in part, regulated by purinergic receptors. P2X1 and P2Y6 receptors are the strongest contractile receptors and, surprisingly, ADPßS caused contraction most likely via P2Y1 or P2Y13 receptors, which is not observed in other arteries. Adenosine addition, however, caused relaxation of the MMA. The adenosine relaxation could be inhibited by SCH58261 (A2A receptor antagonist) and caffeine (adenosine receptor antagonist). This gives one putative molecular mechanism for the effect of caffeine, often used as an adjuvant remedy of cranial pain. Semi-quantitative RT-PCR expression data for the receptors correlate well with the functional findings. Together these observations could be used as targets for future understanding of the in vivo role of purinergic receptors in the MMA.