Biomarker alterations associated with distinct patterns of metastatic spread in colorectal cancer.

Metastatic spread is the most important life-threatening feature of colorectal cancer and is supposed to be mainly driven by alterations in different carcinogenic pathways. The present study compared mutation and expression profiles of distinctive biomarkers in colorectal cancer patients with different clinical metastatic patterns. As for a case-control study, patients were matched according to T category, grading and primary tumour site. Overall, 246 patients with either exclusive lung metastasis (N = 82), exclusive liver metastasis (N = 82) or non-metastatic colorectal cancer (N = 82) were identified. Paraffin-embedded specimens were examined for mutations in the RAS and RAF genes and for the expression of ß-catenin and CD133. Clinical endpoints were presence or absence of distant metastasis, formation of metastasis in lungs versus the liver and survival. MAPK pathway mutations in either the KRAS, NRAS or BRAF gene were associated with the development of lung metastasis (63.4%) compared to the control group (47.6%; p = 0.04). MAPK pathway alterations plus high ß-catenin expression were associated with metastasis to the lungs but not to the liver (28.0% vs. 13.4%; p = 0.02). High CD133 expression correlated with the development of liver metastasis compared to the control group (30.5% vs. 14.6%; p = 0.02). This data indicates that different patterns of distant spread are associated with specific biomarker alterations and may represent different molecular subtypes of colorectal cancer. However, underlying mechanisms of metastasis formation in different anatomic sites remains unclear. Since knowledge of the anticipated site of distant spread would substantially impact clinical management, further research is needed to identify solid biomarkers for different metastatic patterns.

Early reduction of serum TARC levels may predict for success of ABVD as frontline treatment in patients with Hodgkin Lymphoma.

BACKGROUND: Many efforts have been made to predict prognosis of newly diagnosed Hodgkin Lymphoma (HL) patients. Objective of this study was to investigate the association between early reduction of Thymus and Activation-Regulated Chemokine after the first ABVD cycle (TARC-1) and prognosis of HL patients. METHODS: Serum samples of 116 HL patients were collected at baseline, after every ABVD cycle and during follow-up. The 99th centile of TARC distribution in a group of 156 independent healthy subjects (800pg/ml) was considered as cut-off for discriminating between abnormal and normal TARC values. FINDINGS: 101 patients out of 116 had baseline TARC above 800pg/ml (median value 27515pg/ml (IQR, 11001-68139)) and were the object of this analysis. TARC-1 significantly decreased to a median value of 556pg/ml (IQR, 378-977pg/ml). TARC-1 values below 800pg/ml were associated with success of therapy (p=0.0003) and PET-2 negativity (p=0.001). TARC-1≤800pg/ml identified a population with a significantly higher 5-years PFS in the whole cohort (90.1% vs 55.6%; p<0.0001) and in both subgroups of advanced (p=0.003) and early stage patients (p=0.021). At multivariable analysis, TARC-1 was significant independent predictor of PFS (p=0.0035). INTERPRETATION: Early reduction of TARC serum levels can predict success of treatment, being associated with achievement of interim PET-2 negative and favorable long-term outcome in HL patients receiving ABVD as front-line therapy.

Hypoxia determines survival outcomes of bacterial infection through HIF-1alpha dependent re-programming of leukocyte metabolism.

Hypoxia and bacterial infection frequently co-exist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both S. aureus and S. pneumoniae infections rapidly induced progressive neutrophil mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, prior to infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of HIF pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilisation, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes following bacterial infection through suppression of HIF-1α and neutrophil metabolism. The therapeutic implications of this work are that in the context of systemic or tissue hypoxia therapies that target the host response could improve infection associated morbidity and mortality.

The influence of glutamate receptor 2 expression on excitotoxicity in Glur2 null mutant mice.

AMPA receptor (AMPAR)-mediated ionic currents that govern gene expression, synaptic strength, and plasticity also can trigger excitotoxicity. However, native AMPARs exhibit heterogeneous pharmacological, biochemical, and ionic permeability characteristics, which are governed partly by receptor subunit composition. Consequently, the mechanisms governing AMPAR-mediated excitotoxicity have been difficult to elucidate. The GluR2 subunit is of particular interest because it influences AMPAR pharmacology, Ca(2+) permeability, and AMPAR interactions with intracellular proteins. In this paper we used mutant mice lacking the AMPAR subunit GluR2 to study AMPAR-mediated excitotoxicity in cultured cortical neurons and in hippocampal neurons in vivo. We examined the hypothesis that in these mice the level of GluR2 expression governs the vulnerability of neurons to excitotoxicity and further examined the ionic mechanisms that are involved. In cortical neuronal cultures AMPAR-mediated neurotoxicity paralleled the magnitude of kainate-evoked AMPAR-mediated currents, which were increased in neurons lacking GluR2. Ca(2+) permeability, although elevated in GluR2-deficient neurons, did not correlate with excitotoxicity. However, toxicity was reduced by removal of extracellular Na(+), the main charge carrier of AMPAR-mediated currents. In vivo, the vulnerability of CA1 hippocampal neurons to stereotactic kainate injections and of CA3 neurons to intraperitoneal kainate administration was independent of GluR2 level. Neurons lacking the GluR2 subunit did not demonstrate compensatory changes in the distribution, expression, or function of AMPARs or of Ca(2+)-buffering proteins. Thus GluR2 level may influence excitotoxicity by effects additional to those on Ca(2+) permeability, such as effects on agonist potency, ionic currents, and synaptic reorganization.

Endogenous Zn(2+) is required for the induction of long-term potentiation at rat hippocampal mossy fiber-CA3 synapses.

The functional role of the abundant Zn(2+) found in some hippocampal synapses has been an enigma. We show here, using N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide (TSQ) staining, that chelatable-Zn(2+) can be removed from hippocampal synaptic boutons using dietary depletion or with Zn(2+) chelators. A chronic dietary deficiency of bouton Zn(2+) resulted in the impairment of long-term potentiation (LTP) at mossy fiber-CA3 synapses. The averaged normalized fEPSP slope 30 min after tetanus was 209 +/- 28% of baseline value in control (mean +/- SEM, n = 10), and 118 +/- 12% in Zn(2+)-deficient rats (mean +/- SEM, n = 12, P < 0.01). In the deficient rats with Zn(2+) supplements, mossy fiber LTP returned to normal levels. The acute depletion of bouton Zn(2+) in the hippocampal slice with membrane-permeable Zn(2+) chelators, dithizone, or diethyldithiocarbamic acid (DEDTC) blocked the induction of mossy fiber LTP. The mean amplitudes of EPSCs after tetanus were 194 +/- 22% of baseline value in control (n = 5), compared to 108 +/- 14% in dithizone (n = 6) and 101 +/- 12% in DEDTC (n = 5). The averaged value of LTP, at the associational commisural fiber-CA3 synapses, was 193 +/- 20% in the control (n = 6), compared to 182 +/- 21% (n = 6, P > 0.1) in the presence of dithizone. The blockade of mossy fiber LTP by dithizone was reversible after washout. In addition, normal LTP could be induced by tetanus if exogenous Zn(2+) was applied immediately following dithizone. Our results indicate that the endogenous Zn(2+) is specifically required for LTP induction at the mossy fiber input into CA3 neurons.

The Lurcher mutation of an alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor subunit enhances potency of glutamate and converts an antagonist to an agonist.

A point mutation of the GluRdelta2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L(c)) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85- and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.

Selective abolition of the NMDA component of long-term potentiation in mice lacking mGluR5.

The mechanisms underlying the differential expression of long-term potentiation (LTP) by AMPA and NMDA receptors, are unknown, but could involve G-protein-linked metabotropic glutamate receptors. To investigate this hypothesis we created mutant mice that expressed no metabotropic glutamate receptor 5 (mGluR5), but showed normal development. In an earlier study of these mice we analyzed field-excitatory postsynaptic potential (fEPSPs) in CA1 region of the hippocampus and found a small decrease; possibly arising from changes in the NMDAR-mediated component of synaptic transmission. In the present study we used whole-cell patch clamp recordings of evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons to identify the AMPAR- and NMDAR-mediated components of LTP. Recordings from control mice following tetanus, or agonist application (IS, 3R-1-amino-cyclopentane 1,3-dicarboxylic acid) (ACPD), revealed equal enhancement of the AMPA and NMDA receptor-mediated components. In contrast, CA1 neurons from mGluR5-deficient mice showed a complete loss of the NMDA-receptor-mediated component of LTP (LTP(NMDA)), but normal LTP of the AMPA-receptor-mediated component (LTP(AMPA)). This selective loss of LTP(NMDA) was seen in three different genotypic backgrounds and was apparent at all holding potentials (-70 mV to +20 mV). Furthermore, the LTP(NMDA) deficit in mGluR5 mutant mice could be rescued by stimulating protein kinase C (PKC) with 4beta-phorbol-12,13-dibutyrate (PDBu). These results suggest that PKC may couple the postsynaptic mGluR5 to the NMDA-receptor potentiation during LTP, and that this signaling mechanism is distinct from LTP(AMPA). Differential enhancement of AMPAR and NMDA receptors by mGluR5 also supports a postsynaptic locus for LTP.

Enhanced LTP in mice deficient in the AMPA receptor GluR2.

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.

Sensitivity of AMPA receptors to pentobarbital.

The inhibitory effects of pentobarbital on various AMPA receptors expressed (GluR1, GluR3, GluR1/3, GluR1/2, and GluR2/3) in Xenopus oocytes were examined. Combinations of AMPA receptor subunits that included GluR2 demonstrated a much higher sensitivity to blockade by this barbiturate and the apparent co-operativity of the interaction of pentobarbital with the receptor was reduced. This evidence demonstrates that the GluR2 subunit alters the structure of AMPA receptors in such a way as to facilitate any interaction with this barbiturate.

Expression of functional metabotropic and ionotropic glutamate receptors in baculovirus-infected insect cells.

An ionotropic glutamate receptor of the kainate subtype (GluR6) and a G-protein coupled metabotropic glutamate receptor (mGluR1 alpha) were expressed and studied in two insect cell lines: sf9 cells from Spodoptera frugiperda and MG1 cells from Trichoplusia ni. Application of kainate to GluR6-infected MG1 cells produced kainate-activated currents. Glutamate activation of mGluR1 alpha in MG1- and sf9-infected cells caused rapid, transient increases in intracellular calcium levels. This effect was more pronounced in MG1 cells compared to sf9 cells. These results indicate that functional glutamate receptors can be expressed in the baculovirus system, and that MG1 cells may have several advantages over the widely used sf9 cells for studying the functional properties of receptors and channels.

A transmembrane model for an ionotropic glutamate receptor predicted on the basis of the location of asparagine-linked oligosaccharides.

Several different models have been proposed for the transmembrane structure of receptors for the neurotransmitter L-glutamate. In this study, the sites of N-linked oligosaccharides on GluR6, a member of the kainate class of ionotropic glutamate receptors, were examined. Site-directed mutagenesis was utilized to alter the consensus sequence at three potential sites for N-linked glycosylation in the carboxyl-terminal half of the molecule. The presence of a carbohydrate substitution was monitored by shifts in the relative molecular weight of the mutant receptors on immunoblots. Molecular weight shifts were observed for the mutants N515Q and N720Q and for two companion mutants, T517A and T722A, which also eliminate the consensus sequence for N-linked glycosylation. No shift in molecular weight was observed in the the mutant N574Q. These results indicate that asparagines 515 and 720 are glycosylated and thus are likely located extracellularly. In immunocytochemical analyses of GluR6 expressed in baculovirus-infected cells, permeabilization with detergents was required for immunostaining with a carboxyl-terminal antibody, indicating that the carboxyl terminus is located intracellularly. Electrophysiological recordings of the mutant receptors expressed in human embryonic kidney cells demonstrated that the amplitudes of the kainate-activated currents mediated by the N574Q, N720Q, and the T722A mutants were not significantly different from currents mediated by wild type GluR6 receptors, while the currents mediated by the N515Q and T517A mutants were significantly depressed. Based on these findings, we propose a model for the transmembrane topology of GluR6.

Properties of a recombinant kainate receptor expressed in baculovirus-infected insect cells.

GluR6 is a glutamate receptor of the kainate subtype that is expressed in the mammalian central nervous system. The cDNA coding for the rat brain receptor was subcloned into a baculovirus expression vector and the purified recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The pharmacological profile and the status of several post-translational modifications of the GluR6 protein were analyzed. Saturation analyses of [3H]kainic acid binding demonstrated that GluR6 expressed in Sf9 cell membranes bound [3H]kainic acid at a single high affinity site with a dissociation constant of 12 nM. Competition studies indicated the inhibitory potencies of various excitatory amino acids, including the potent neurotoxin domoic acid, were comparable to those observed in mammalian brain tissue. Immunoblots of infected Sf9 cell membranes using an anti-GluR6 antibody revealed two immunoreactive bands. Enzymatic deglycosylation indicated that the higher molecular weight form corresponded to the glycosylated receptor whereas the lower molecular weight form corresponded to the unglycosylated protein. The phosphorylation of GluR6 by cyclic AMP-dependent and cyclic GMP-dependent protein kinase was examined in partially purified preparations of the receptor. GluR6 was phosphorylated by cyclic AMP but not by cyclic GMP-dependent protein kinase in vitro. These results indicate that GluR6 expressed in Sf9 cells has similar pharmacological properties and is processed post-translationally in a manner similar to GluR6 expressed in mammalian cells and tissues. The ease of production and the high levels of expression in baculovirus-infected insect cells relative to other expression systems should facilitate further biochemical and pharmacological characterization of this receptor.

Phosphorylation and modulation of a kainate receptor (GluR6) by cAMP-dependent protein kinase.

Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.