Monitoring HSVtk suicide gene therapy: the role of [18F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [F-18] FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [F-18] FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [F-18] FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour- bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2- chloroadenosine did not significantly affect the [F-18] FHPG uptake in vitro. Thymidine and uridine significantly decreased [F-18] FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [F-18] FHPG uptake in C6tk and C6 tumours decreased from 3.070.5 to 1.070.2 after infusion of adenine. Thus, in our tumour model, [F-18] FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier

Synergistic activity of troxacitabine (Troxatyl™) and gemcitabine in pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl™) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials.</p> <p>Methods</p> <p>The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For <it>in vivo </it>studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired <it>t </it>test using GraphPad prism software.</p> <p>Results</p> <p>Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. <it>In vivo </it>testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine.</p> <p>Conclusion</p> <p>These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.</p

A case of serendipity*

An account is given of how a sensitive bioassay system for measurement of the neurotransmitter acetylcholine serendipitously led to the identification of adenosine triphosphate (ATP) released in vitro from active skeletal muscle. Subsequent application of the identification procedures to exercising human muscle in vivo, cardiac muscle cells in vitro, and human erythrocytes exposed to hypoxia gave rise to the general concept of ATP as a molecule that could influence cell function from the extracellular direction. Mechanisms of ATP release from cells in terms of “trigger” events such as mechanical distortion of the membrane, depolarization of the membrane, and exposure to hypoxia are discussed. Potential therapeutic uses of extracellular ATP in cancer therapy, radiation therapy, and a possible influence upon aging are discussed. Possible roles (distant and local) of extracellular ATP released from muscle during whole body exercise are discussed

Efficient Visual Search from Synchronized Auditory Signals Requires Transient Audiovisual Events

BACKGROUND: A prevailing view is that audiovisual integration requires temporally coincident signals. However, a recent study failed to find any evidence for audiovisual integration in visual search even when using synchronized audiovisual events. An important question is what information is critical to observe audiovisual integration. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that temporal coincidence (i.e., synchrony) of auditory and visual components can trigger audiovisual interaction in cluttered displays and consequently produce very fast and efficient target identification. In visual search experiments, subjects found a modulating visual target vastly more efficiently when it was paired with a synchronous auditory signal. By manipulating the kind of temporal modulation (sine wave vs. square wave vs. difference wave; harmonic sine-wave synthesis; gradient of onset/offset ramps) we show that abrupt visual events are required for this search efficiency to occur, and that sinusoidal audiovisual modulations do not support efficient search. CONCLUSIONS/SIGNIFICANCE: Thus, audiovisual temporal alignment will only lead to benefits in visual search if the changes in the component signals are both synchronized and transient. We propose that transient signals are necessary in synchrony-driven binding to avoid spurious interactions with unrelated signals when these occur close together in time

Bone Marrow Stromal Cells Modulate Mouse ENT1 Activity and Protect Leukemia Cells from Cytarabine Induced Apoptosis

BACKGROUND: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. METHODS: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of (3)H-adenosine. RESULTS: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. CONCLUSION: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML

Microsporidia::Why Make Nucleotides if You Can Steal Them?

Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions

A quantitative analysis of the effect of cycle length on arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

The clinically established proarrhythmic effect of bradycardia and antiarrhythmic effect of lidocaine (10 μM) were reproduced in hypokalaemic (3.0 mM K+) Langendorff-perfused murine hearts paced over a range (80–180 ms) of baseline cycle lengths (BCLs). Action potential durations (at 90% repolarization, APD90s), transmural conduction times and ventricular effective refractory periods (VERPs) were then determined from monophasic action potential records obtained during a programmed electrical stimulation procedure in which extrasystolic stimuli were interposed following regular stimuli at successively decreasing coupling intervals. A novel graphical analysis of epicardial and endocardial, local and transmural relationships between APD90, corrected for transmural conduction time where appropriate, and VERP yielded predictions in precise agreement with the arrhythmogenic findings obtained over the entire range of BCLs studied. Thus, in normokalaemic (5.2 mM K+) hearts a statistical analysis confirmed that all four relationships were described by straight lines of gradients not significantly (P > 0.05) different from unity that passed through the origin and thus subtended constant critical angles, θ with the abscissa (45.8° ± 0.9°, 46.6° ± 0.5°, 47.6° ± 0.5° and 44.9° ± 0.8°, respectively). Hypokalaemia shifted all points to the left of these reference lines, significantly (P < 0.05) increasing θ at BCLs of 80–120 ms where arrhythmic activity was not observed (∼63°, ∼54°, ∼55° and ∼58°, respectively) and further significantly (P < 0.05) increasing θ at BCLs of 140–180 ms where arrhythmic activity was observed (∼68°, ∼60°, ∼61° and ∼65°, respectively). In contrast, the antiarrhythmic effect of lidocaine treatment was accompanied by a significant (P < 0.05) disruption of this linear relationship and decreases in θ in both normokalaemic (∼40°, ∼33°, ∼39° and ∼41°, respectively) and hypokalaemic (∼40°, ∼44°, ∼50° and ∼48°, respectively) hearts. This extended a previous approach that had correlated alterations in transmural repolarization gradients with arrhythmogenicity in murine models of the congenital long QT syndrome type 3 and hypokalaemia at a single BCL. Thus, the analysis in terms of APD90 and VERP provided a more sensitive indication of the effect of lidocaine than one only considering transmural repolarization gradients and may be particularly applicable in physiological and pharmacological situations in which these parameters diverge

The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=−0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=−0.30; P=0.04), decitabine (rp=−0.29; P=0.04) and gemcitabine (rp=−0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=−0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=−0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=−0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML

Purinergic modulation of microglial cell activation

Microglial cells are resident macrophages in the brain and their activation is an important part of the brain immune response and the pathology of the major CNS diseases. Microglial activation is triggered by pathological signals and is characterized by morphological changes, proliferation, phagocytosis and the secretion of various cytokines and inflammatory mediators, which could be both destructive and protective for the nervous tissue. Purines are one of the most important mediators which regulate different aspects of microglial function. They could be released to the extracellular space from neurons, astrocytes and from the microglia itself, upon physiological neuronal activity and in response to pathological stimuli and cellular damage. Microglial activation is regulated by various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, gene transcription, the production of inflammatory mediators and cell survival. Among them, the role of P2X7 receptors is especially well delineated, but P2X4, various P2Y, A1, A2A and A3 receptors also powerfully participate in the microglial response. The pathological role of microglial purine receptors has also been demonstrated in disease models; e.g., in ischemia, sclerosis multiplex and neuropathic pain. Due to their upregulation and selective activation under pathological conditions, they provide new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses