Biomedical subjects

Luigi Varesio

Publications and source records attributed to Luigi Varesio.

15 recordsLinked to original sources

PubMed2007

Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile.

BACKGROUND: The normalization of DNA microarrays allows comparison among samples by adjusting for individual hybridization intensities. The approaches most commonly used are global normalization methods that are based on the expression of all genes on the slide and on the modulation of a small proportion of genes. Alternative approaches must be developed for microarrays where the proportion of modulated genes and their distribution are unknown and they may be biased towards up- or down-modulated trends. RESULTS: The aim of the work is to study the use of spike-in controls to normalize low-density microarrays. Our test-array was designed to analyze gene modulation in response to hypoxia (a condition of low oxygen tension) in a macrophage cell line. RNA was extracted from controls and cells exposed to hypoxia, mixed with spike RNA, labeled and hybridized to our test-array. We used eight bacterial RNAs as source of spikes. The test-array contained the oligonucleotides specific for 178 mouse genes and those specific for the eight spikes. We assessed the quality of the spike signals, the reproducibility of the results and, in general, the nature of the variability. The small values of the coefficients of variation revealed high reproducibility of our platform either in replicated spots or in technical replicates. We demonstrated that the spike-in system was suitable for normalizing our platform and determining the threshold for discriminating the hypoxia modulated genes. We assessed the application of the spike-in normalization method to microarrays in which the distribution of the expression values was symmetric or asymmetric. We found that this system is accurate, reproducible and comparable to other normalization methods when the distribution of the expression values is symmetric. In contrast, we found that the use of the spike-in normalization method is superior and necessary when the distribution of the gene expression is asymmetric and biased towards up-regulated genes. CONCLUSION: We demonstrate that spike-in controls based normalization is a reliable and reproducible method that has the major advantage to be applicable also to biased platform where the distribution of the up- and down-regulated genes is asymmetric as it may occur in diagnostic chips.

Action Potentials↗

PubMed2006

Inhibition of PI3K induces Rac activation and membrane ruffling in proto-Dbl expressing cells.

Proto-Dbl protein, a guanine nucleotide exchange factor (GEF) for Rho GTPases, is tightly regulated by a combination of mechanisms that involve intra- and intermolecular interaction and N- and C-terminal domain-dependent turnover of the protein. Moreover, the interaction of the PH domain of proto-Dbl with phosphoinositides regulates its subcellular localization and biological activity. Here we show that inhibition of the phosphatidylinositol 3-kinase (PI3K) by molecular and pharmacological inhibitors causes a strong inhibition of proto-Dbl-induced cell proliferation and transformation. Conversely, inhibition of PI3K results in the translocation of proto-Dbl to the plasma membrane, Rac activation and increased membrane ruffles and cell motility. Furthermore, we investigated the signaling molecules involved in proto-Dbl-induced cell transformation and motility and observed that inhibition of PI3K in proto-Dbl expressing cells induces an increase in p38 activity and a decrease in ERK phosphorylation. Our results suggest that proto-Dbl activates distinct downstream effectors to induce morphological changes and cell transformation.

Animals↗

PubMed2006

Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages.

Hypoxia, a local decrease in oxygen tension, occurring in many pathological processes, modifies macrophage (Mphi) gene expression and function. Here, we provide the first evidence that hypoxia inhibits transgene expression driven by the Moloney murine leukemia virus-long terminal repeats (MoMLV-LTR) in IFN-gamma-activated Mphi. Hypoxia silenced the expression of several MoMLV-LTR-driven genes, including v-myc, enhanced green fluorescence protein, and env, and was effective in different mouse Mphi cell lines and on distinct MoMLV backbone-based viruses. Down-regulation of MoMLV mRNA occurred at the transcriptional level and was associated with decreased retrovirus production, as determined by titration experiments, suggesting that hypoxia may control MoMLV retroviral spread through the suppression of LTR activity. In contrast, genes driven by the CMV or the SV40 promoter were up-regulated or unchanged by hypoxia, indicating a selective inhibitory activity on the MoMLV promoter. It is interesting that hypoxia was ineffective in suppressing MoMLV-LTR-controlled gene expression in T or fibroblast cell lines, suggesting a Mphi lineage-selective action. Finally, we found that MoMLV-mediated gene expression in Mphi was also inhibited by picolinic acid, a tryptophan catabolite with hypoxia-like activity and Mphi-activating properties, suggesting a pathophysiological role of this molecule in viral resistance and its possible use as an antiviral agent.

Animals↗

PubMed2006

Hypoxia modifies the transcriptome of primary human monocytes: modulation of novel immune-related genes and identification of CC-chemokine ligand 20 as a new hypoxia-inducible gene.

Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O(2), with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.

Cell Hypoxia↗

PubMed2006

Growth arrest-inducing genes are activated in Dbl-transformed mouse fibroblasts.

The Dbl oncogene is a guanine nucleotide exchange factor for Rho GTPases and its activity has been linked to the regulation of gene transcription. Dbl oncogene expression in NIH3T3 cells leads to changes in morphological and proliferative properties of these cells, inducing a highly transformed phenotype. To gain insights into Dbl oncogene-induced transformation we compared gene expression profiles between Dbl oncogene-transformed and parental NIH3T3 cells by cDNA microarray. We found that Dbl oncogene expression is associated with gene expression modulation involving upregulation of 51 genes and downregulation of 49 genes. Five of the overexpressed genes identified are known to exert antiproliferative functions. Our observations suggest that the expression of Dbl oncogene in NIH3T3 may lead to the induction of genes associated with cell cycle arrest, possibly through the activation of stress-induced kinases.

Animals↗

PubMed2005

Constitutively active Cdc42 mutant confers growth disadvantage in cell transformation.

The Rho family small GTPase Cdc42 is critical for diverse cellular functions including the regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell cycle progression and cell transformation. Like other members of the Rho family, Cdc42 cycles between the GTP-bound, active state, and the inactive, GDP-bound state under tight regulation, and it is believed that the GTP bound form of Cdc42 represents the active signaling module in eliciting effector activation and cellular responses. The constitutively active mutant, V12, derived from the analogous mutations found in oncogenic Ras that are GTPase-defective, and a "fast-cycling" self-activating mutant, F28, of Cdc42, have been widely in use to study the cellular effects of Cdc42. Here we report that the constitutively active V12 mutant of Cdc42, when stably expressed in cells, could behave in a dominant negative fashion in inhibiting cell proliferation while the F28 mutant was growth stimulatory. The V12 mutant failed to transform NIH3T3 cells while F28 potently stimulated anchorage-independent growth. The growth inhibitory effect of the V12 mutant correlated with activation of JNK2 and suppression of the cyclin D1 and NF-kappaB expressions that were instead upregulated by the F28 mutant. Furthermore, the V12 mutant could suppress, whereas the F28 mutant potentiated or had no effect on, a wide variety of oncogene-induced cell transformation, including that by the Dbl family GEFs Dbl, Vav and Lbc and the oncogenic Ras, ErbB-2, PDGF B or Raf. These results raise the possibility that over-saturation or constitutive activation of Cdc42 signal may negatively impact on cell proliferation and that both the activation and deactivation steps, or the complete GTPase cycle, of Cdc42 is required for proper function.

Animals↗

PubMed2004

Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy. EXPERIMENTAL DESIGN: Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting. RESULTS: Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN. CONCLUSIONS: These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.

Apoptosis↗

PubMed2004

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium.

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.

Amino Acid Transport Systems↗

PubMed2004

Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages.

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1beta (MIP-1beta), RANTES, IFN-gamma-inducible protein-10, and MIP-2 were not affected, and MIP-1alpha was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-gamma and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.

Animals↗

PubMed2004

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages.

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.

Animals↗

PubMed2003

The signature motif in human glucose-6-phosphate transporter is essential for microsomal transport of glucose-6-phosphate.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Sequence alignments identify a signature motif shared by G6PT and a family of transporters of phosphorylated metabolites. Two null signature motif mutations have been identified in the G6PT gene of GSD-Ib patients. In this study, we characterize the activity of seven additional mutants within the motif. Five mutants lack microsomal G6P uptake activity and one retains residual activity, suggesting that in G6PT the signature motif is a functional element required for microsomal glucose-6-phosphate transport.

Amino Acid Sequence↗

PubMed2003

Macrophage activating properties of the tryptophan catabolite picolinic acid.

Recent studies have suggested a role for aminoacid catabolites as important regulators of macrophage (Mphi) activities. We reported previously that picolinic acid (PA), a tryptophan catabolite produced under inflammatory conditions and a costimulus with IFNgamma of Mphi effector functions, is a selective inducer of the Mphi inflammatory protein-1alpha (MIP-1alpha) and -1beta (MIPs), two CC-chemokines involved in the elicitation of the inflammatory reactions and in the development of the Th1 responses. In this study, we have investigated the effects of IFNgamma on PA-induced MIPs expression and secretion by mouse Mphi as well as the regulation of MIP-1alpha/beta receptor, CCR5, by both stimuli alone or in combination. We demonstrated that IFNgamma inhibited MIPs mRNA stimulation by PA in a dose-and time-dependent fashion, despite its ability to induce other CC- or CXC chemokines. MIPs mRNA down-regulation was associated with decreased intracellular chemokine expression and secretion and was dependent on both mRNA destabilization and gene transcription inhibition. Moreover, IFNgamma inhibitory effects were stimulus-specific because MIPs induction by PA was either unaffected or increased by the anti-inflammatory cytokines, IL-10 and IL-4, or the pro-inflammatory stimulus, LPS, respectively. In contrast, we found that IFNgamma increased CCR5 basal expression, whereas PA down-regulated both constitutive and IFNgamma-induced CCR5 mRNA and protein levels. These results demonstrate that IFNgamma and PA have reciprocal effects on the production of MIPs chemokines and the expression of their receptor. The concerted action of IFNgamma and PA on MIP-1alpha/beta chemokine/receptor system is likely to be of pathophysiological significance and to represent an important regulatory mechanism for leukocyte recruitment and distribution into damaged tissues during inflammatory responses.

Animals↗

PubMed2002

Antifungal activity of macrophages engineered to produce IFNgamma: inducibility by picolinic acid.

Macrophages are important antimicrobial effectors, whose efficacy is greatly enhanced by interferon-gamma (IFNgamma). We recently engineered a mouse macrophage cell line to express the IFNgamma gene in a inducible manner. Such macrophages, Mphi10, include a construct containing the IFNgamma gene under the control of the synthetic promoter HRE3x-Tk. Picolinic acid (PA) is a catabolite of tryptophan, known to exert costimulatory activities on macrophages and expected to act on transcriptional elements within HRE3x-Tk promoter. Since evidence exists on the efficacy of engineered macrophages as carriers of therapeutic genes against tumors, we tested Mphi10, under basal conditions and following exposure to PA, as IFNgamma-producing cells in in vitro models of fungal infection. We found that Mphi10 constitutively exhibited anticryptococcal and anticandidal activity, low but detectable levels of IFNgamma mRNA and undetectable levels of nitric oxide synthase (iNOS) transcripts. Treatment with PA caused time-dependent enhancement of antifungal activity. The phenomenon was associated with the induction of both IFNgamma and iNOS gene expression and was followed by IFNgamma and NO production. The effect of the Mphi10-produced IFNgamma on other cells was also investigated by a transwell co-culture system. A major enhancement of phagocytosis and antifungal activity was observed in BV2 microglial cells that had been co-cultured with Mphi10. Such an increase was only evident when Mphi10 had been pretreated with PA and was abrogated by concomitant addition of anti-IFNgamma antibodies. In conclusion, we show that Mphi10 respond to PA with the production of IFNgamma, which retains the ability to induce antifungal activity in the producing macrophages as well as in other macrophage populations. The potential use of Mphi10 as vectors for therapeutic genes in infectious diseases is discussed.

Animals↗

PubMed2002

Flavopiridol inhibits vascular endothelial growth factor production induced by hypoxia or picolinic acid in human neuroblastoma.

Human neuroblastoma (NB) tumors elaborate angiogenic peptides, and enhanced angiogenesis correlates with their aggressive behavior, metastatic spread and poor clinical outcome. Hence, inhibition of angiogenic factor production may represent a potential therapeutic target for NB treatment. There is currently little information regarding the stimuli that control NB production of angiogenic mediators. In this study, we analyzed the effects of hypoxia, a common feature of solid tumors and a major drive to tumor angiogenesis, and of PA, a tryptophan catabolite produced under inflammatory conditions and endowed with several biologic properties, on the production of the angiogenic activator VEGF by advanced-stage human NB cell lines. We demonstrate that both stimuli are potent inducers of VEGF expression and secretion. VEGF upregulation by PA involved iron chelation because iron sulfate prevented this effect whereas the iron-chelating agent DFX induced VEGF production. Conversely, the CDK inhibitor Flp completely blocked VEGF induction by hypoxia. This effect occurred as early as 3 hr after stimulation and did not require de novo protein synthesis. Moreover, Flp exerted similar inhibitory activity on VEGF induction by PA or DFX, suggesting that this compound targets an essential step in the signaling pathway that leads to VEGF expression. Our findings demonstrate that PA can modulate angiogenic factor production by tumor cells and establish the importance of Flp as an inhibitor of VEGF production by human NB.

Antineoplastic Agents↗

PubMed2002

Antagonistic effect of picolinic acid and interferon-gamma on macrophage inflammatory protein-1alpha/beta production.

The L-tryptophan catabolite, picolinic acid (PA), is an activator of macrophage effector functions and an inducer of macrophage inflammatory protein-1alpha (MIP-1alpha) and -1beta (MIPs). We have investigated the regulation of PA-induced MIPs production in mouse macrophages. We demonstrated a dose- and time-dependent downregulation of MIPs mRNA by the Th1 cytokine, IFN-gamma, that was associated with inhibition of intracellular chemokine production and secretion. This effect was IFN-gamma-specific because MIPs induction was unaffected by the Th2 cytokines, IL-10 and IL-4, or the proinflammatory stimulus, LPS. Moreover, MIPs downregulation by IFN-gamma was dependent on both mRNA destabilization and gene transcription inhibition. These results demonstrate that MIP-1alpha/beta production by macrophages is a tightly regulated process resulting from the interaction between inhibitory stimuli derived from the immune system and stimulatory signals of non-immunologic origin. The antagonistic effect of PA and IFN-gamma on MIPs production may be important for the regulation of the inflammatory responses in vivo.

Animals↗