HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport.

Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na electrochemical gradient, while hCNT3 is both Na- and H-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [H] or [C] pyrimidine and purine nucleosides once inside cells.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response.

Fine-mapping of a novel premenopausal breast cancer susceptibility locus at Chr4q31.22 in Caucasian women and validation in African and Chinese women.

We previously identified a novel breast cancer susceptibility variant on chromosome 4q31.22 locus (rs1429142) conferring risk among women of European ancestry. Here, we report replication of findings, validation of the variant in diverse populations and fine-mapping of the associated locus in Caucasian population.

2-Nitroimidazole-Furanoside Derivatives for Hypoxia Imaging-Investigation of Nucleoside Transporter Interaction, F-Labeling and Preclinical PET Imaging.

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives-mimicking nucleosides-have proven their potential with [F]FAZA ([F]fluoro-azomycin--arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios.

Role of cysteine 416 in -ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent -ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent -chloromercuriphenyl sulfonate.

Multitargeted kinase inhibitors imatinib, sorafenib and sunitinib perturb energy metabolism and cause cytotoxicity to cultured C2C12 skeletal muscle derived myotubes.

Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment and prognosis but have also resulted in adverse effects such as fatigue, diarrhea, hypothyroidism, and other toxicities. We investigated TKI effects on skeletal muscle as a possible explanation of TKI induced fatigue. Changes in mitochondrial function due to inhibition of oxidative phosphorylation complexes, generation of superoxides, and inhibition of key transporters involved in uptake of glucose and/or nucleosides may result in alteration of energy metabolism and/or mitochondrial function.

Breast cancer associated germline structural variants harboring small noncoding RNAs impact post-transcriptional gene regulation.

Copy Number Variants (CNVs) are a class of structural variations of DNA. Germline CNVs are known to confer disease susceptibility, but their role in breast cancer warrants further investigations. We hypothesized that breast cancer associated germline CNVs contribute to disease risk through gene dosage or other post-transcriptional regulatory mechanisms, possibly through tissue specific expression of CNV-embedded small-noncoding RNAs (CNV-sncRNAs).

Germline copy number variations are associated with breast cancer risk and prognosis.

Breast cancer is one of the most common cancers among women, and susceptibility is explained by genetic, lifestyle and environmental components. Copy Number Variants (CNVs) are structural DNA variations that contribute to diverse phenotypes via gene-dosage effects or cis-regulation. In this study, we aimed to identify germline CNVs associated with breast cancer susceptibility and their relevance to prognosis.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance.

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na and/or H electrochemical gradients. CNT family member NupC resembles hCNT1 in permeant selectivity but is H-coupled.

Synthesis and preclinical characterization of 1-(6'-deoxy-6'-[F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-6'-[F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia.

Positron emission tomography (PET) using fluorine-18 (F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6'-deoxy-6'-[F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-[F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12±8% (n=10, based on [F]fluoride starting activity) in a total synthesis time of 60min with a specific activity at end of synthesis of 218±58GBq/μmol (n=10).

Selective Inhibition of Human Equilibrative and Concentrative Nucleoside Transporters by BCR-ABL Kinase Inhibitors: IDENTIFICATION OF KEY hENT1 AMINO ACID RESIDUES FOR INTERACTION WITH BCR-ABL KINASE INHIBITORS.

Human nucleoside transporters (hNTs) mediate cellular influx of anticancer nucleoside drugs, including cytarabine, cladribine, and fludarabine. BCR-ABL tyrosine kinase inhibitors (TKIs) imatinib and dasatinib inhibit fludarabine and cytarabine uptake. We assessed interactions of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib with recombinant hNTs (hENT1, 2; hCNT1, -2, and -3) produced individually in yeast Saccharomyces cerevisiae Nilotinib inhibited hENT1-mediated uridine transport most potently (IC50 value, 0.

A Uridine Glucuronosyltransferase 2B7 Polymorphism Predicts Epirubicin Clearance and Outcomes in Early-Stage Breast Cancer.

Background: Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in single nucleotide polymorphisms (SNPs). We studied whether the -161 C > T germline SNP in UGT2B7 was related to epirubicin metabolism and whether differences exist in the toxicity and efficacy of epirubicin-based chemotherapy among patients who were TT homozygotes, CT heterozygotes, and CC homozygotes.

Patients And Methods: A total of 132 women with non-metastatic breast cancer receiving FEC (5-fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), cyclophosphamide 500 mg/m(2)) were prospectively enrolled.

Inhibition of sodium-independent and sodium-dependent nucleobase transport activities by tyrosine kinase inhibitors.

Purpose: Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sodium-dependent nucleobase transport (SNBT) activities were investigated in normal human renal proximal tubule epithelial cells (hRPTECs) and in pig kidney cell line (LLC-PK1).

Methods: Uptake assays were performed by assessing accumulation of radiolabeled nucleobases over time into hRPTECs or LLC-PK1 cell lines which express ENBT and SNBT activities, respectively. Dose-response curves for inhibition of 1 µM [(3)H]adenine or 1 µM [(3)H]hypoxanthine were examined in hRPTECs and in LLC-PK1 cells with varying TKI concentrations (0-100 µM) to calculate the IC50 values (mean ± S.

Designing and Testing of Novel Taxanes to Probe the Highly Complex Mechanisms by Which Taxanes Bind to Microtubules and Cause Cytotoxicity to Cancer Cells.

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of β-tubulin. Since β-tubulin isotypes have tissue-dependent expression--specifically, the βIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects.

Interactions of multitargeted kinase inhibitors and nucleoside drugs: Achilles heel of combination therapy?

Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to treat many solid tumors. Combination trials of TKIs with gemcitabine, a nucleoside anticancer drug, in pancreas, renal, lung, ovarian, and other malignancies resulted in little benefit to patients. TKI interactions with human nucleoside transporters (hNT) were studied by assessing inhibition of [(3)H]uridine uptake in yeast producing recombinant hNTs individually and in cultured human cancer cell lines.

Specimen quality evaluation in Canadian biobanks participating in the COEUR repository.

Human biological specimens are important for translational research programs such as the Canadian Ovarian Experimental Unified Resource (COEUR) funded by the Terry Fox Research Institute. Sample quality is an important consideration, as it directly impacts the quality of ensuing research. The aim of the present study was to determine the quality of tissues collected from different sites contributing to the COEUR cohort.

Breast cancer prediction using genome wide single nucleotide polymorphism data.

Background: This paper introduces and applies a genome wide predictive study to learn a model that predicts whether a new subject will develop breast cancer or not, based on her SNP profile.

Results: We first genotyped 696 female subjects (348 breast cancer cases and 348 apparently healthy controls), predominantly of Caucasian origin from Alberta, Canada using Affymetrix Human SNP 6.0 arrays.

Comparative in vitro evaluation of transportability and toxicity of capecitabine and its metabolites in cells derived from normal human kidney and renal cancers.

The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.

Erlotinib, gefitinib, and vandetanib inhibit human nucleoside transporters and protect cancer cells from gemcitabine cytotoxicity.

Purpose: Combinations of tyrosine kinase inhibitors (TKI) with gemcitabine have been attempted with little added benefit to patients. We hypothesized that TKIs designed to bind to ATP-binding pockets of growth factor receptors also bind to transporter proteins that recognize nucleosides.

Experimental Design: TKI inhibition of uridine transport was studied with recombinant human (h) equilibrative (E) and concentrative (C) nucleoside transporters (hENT, hCNT) produced individually in yeast.

Cellular influx, efflux, and anabolism of 3-carboranyl thymidine analogs: potential boron delivery agents for neutron capture therapy.

3-[5-{2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine (N5-2OH) is a first generation 3-carboranyl thymidine analog (3CTA) that has been intensively studied as a boron-10 ((10)B) delivery agent for neutron capture therapy (NCT). N5-2OH is an excellent substrate of thymidine kinase 1 and its favorable biodistribution profile in rodents led to successful preclinical NCT of rats bearing intracerebral RG2 glioma. The present study explored cellular influx and efflux mechanisms of N5-2OH, as well as its intracellular anabolism beyond the monophosphate level.

Expression of nucleoside transporters and deoxycytidine kinase proteins in muscle invasive urothelial carcinoma of the bladder: correlation with pathological response to neoadjuvant platinum/gemcitabine combination chemotherapy.

Purpose: In pancreatic cancer, deoxycytidine kinase and the human equilibrative nucleoside transporter 1 have been validated as predictive markers for benefit from gemcitabine therapy. Gemcitabine is used with cisplatin or carboplatin as neoadjuvant chemotherapy for muscle invasive urothelial cancer of the bladder before radical cystectomy and patients rendered disease-free at surgery tend to have better outcomes. In this trial we examined if nucleoside transporter or deoxycytidine kinase protein abundance in biopsy specimens before chemotherapy is related to the response to neoadjuvant chemotherapy.

Assessing SNP-SNP interactions among DNA repair, modification and metabolism related pathway genes in breast cancer susceptibility.

Genome-wide association studies (GWASs) have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs) associated with breast cancer susceptibility.

Identification of a breast cancer susceptibility locus at 4q31.22 using a genome-wide association study paradigm.

More than 40 single nucleotide polymorphisms (SNPs) for breast cancer susceptibility were identified by genome-wide association studies (GWASs). However, additional SNPs likely contribute to breast cancer susceptibility and overall genetic risk, prompting this investigation for additional variants. Six putative breast cancer susceptibility SNPs identified in a two-stage GWAS that we reported earlier were replicated in a follow-up stage 3 study using an independent set of breast cancer cases and controls from Canada, with an overall cumulative sample size of 7,219 subjects across all three stages.

The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29.

Nucleoside transport in humans is mediated by members of two unrelated protein families, the SLC28 family of cation-linked concentrative nucleoside transporters (CNTs) and the SLC29 family of energy-independent, equilibrative nucleoside transporters (ENTs). These families contain three and four members, respectively, which differ both in the stoichiometry of cation coupling and in permeant selectivity. Together, they play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis.

Human concentrative nucleoside transporter 3 transfection with ultrasound and microbubbles in nucleoside transport deficient HEK293 cells greatly increases gemcitabine uptake.

Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep.