Ana Fontalba

Imatinib Inhibits Proliferation of Ewing Tumor Cells Mediated by the Stem Cell Factor/KIT Receptor Pathway, and Sensitizes Cells to Vincristine and Doxorubicin-Induced Apoptosis

Purpose and Experimental Design: The stem cell factor/KIT receptor loop may represent a novel target for molecular-based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWS-FLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 μm) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 μm). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 μm) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 μm) was able to increase the antitumor in vitro effect of doxorubicin (DXR) and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.

Deficiency of the NF-κB Inhibitor Caspase Activating and Recruitment Domain 8 in Patients with Rheumatoid Arthritis Is Associated with Disease Severity

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-κB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-κB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-κB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-α and the p65 subunit of NF-κB. Moreover, TNF-α and overexpression of p65 induced the formation of NF-κB-CARD8 promoter complexes. Thus, CARD8 may control NF-κB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-κB transcriptional activity, and abrogates the binding of NF-κB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-κB target genes (cIAP, A1), in response to TNF-α. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-κB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

Case-control study and meta-analysis of low density lipoprotein receptor-related protein gene exon 3 polymorphism in Alzheimer's disease

The low density lipoprotein receptor-related protein (LRP) may influence both the clearance and the production of beta-amyloid peptide and thus plays a role in Alzheimers disease (AD) pathogenesis. Previous studies, although inconsistent, have suggested that the LRP exon 3 CC genotype contributes to the risk of AD. A case-control study utilizing a clinically well-defined group of 305 sporadic AD patients and 304 control subjects was performed to test this association in an ethnically homogeneous population from Spain. In the current study, the LRP CC genotype was not over-represented in AD patients compared to non-demented controls. A meta-analysis of previous studies revealed a weak correlation of LRP CC genotype with AD (odds ratio of 1.35, P=0.01).

NLRP2, an Inhibitor of the NF-κB Pathway, Is Transcriptionally Activated by NF-κB and Exhibits a Nonfunctional Allelic Variant

NLRP2 has been shown to inhibit the NF-κB signaling pathway, and thus may contribute to modulate the inflammatory response, where NF-κB plays a major role. In this study, we report that expression of NLRP2 is induced upon differentiation of CD34+ hemopoietic progenitors into granulocyte or monocyte/macrophages. We also found that NLRP2 was up-regulated following differentiation of mesenchymal stem cells toward adipocytes. Notably, stimulation of HEK293T cells with TNF-α or overexpression of the p65 subunit of NF-κB resulted in up-regulation of NLRP2 and the formation of NF-κB-NLRP2 promoter complexes. Moreover, ectopic expression of p65 but not of other transcriptional regulators induced transactivation of the NLRP2 promoter. Thus, NLRP2 may control NF-κB activation through a regulatory loop. Nucleotide changes within the NACHT domain of other NLRP proteins have been associated with hereditary fever syndromes and chronic inflammatory diseases. We identified five single nucleotide polymorphisms present in the NACHT domain of NLRP2 by sequencing genomic DNA from 319 healthy controls. The frequencies of the rare alleles varied between 0.2 and 10%. Of note, one of these variants, I352S was unable to block the transcriptional activity of NF-κB and the formation of NF-κB-DNA-binding complexes following stimulation with TNF-α. Overall, our findings provide molecular insight into the expression of NLRP2 by NF-κB and suggest that a polymorphism within the NACHT domain of NLRP2 may contribute to the amplification of inflammatory responses due to a reduction of inhibitory signals on the NF-κB pathway.

Poly (ADP-ribose) polymerase-1 (PARP-1) genetic variants are protective against Parkinson's disease

Poly (ADP-ribose) polymerase-1 (PARP-1) is involved in crucial pathogenic events in Parkinsons disease (PD). We studied the effect of promoter variations of PARP-1 gene on the risk for PD in a case-control association study comprising 146 PD patients and 161 controls from Northern Spain. Three polymorphisms from the promoter region of PARP-1 gene were analyzed: -410C/T, -1672G/A, and a (CA)n microsatellite. A protective effect against PD was found for heterozygosity at (-410) (OR 0.44) and (CA)n microsatellite (OR 0.53) polymorphisms, and heterozygosity at (-1672) polymorphism delayed by 4 years on the onset age of PD. Variations in the regulatory region of PARP-1 gene might modify the risk for PD.

Mutation study of Spanish patients with Hereditary Hemorrhagic Telangiectasia

BackgroundHereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes.MethodsHere, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT.ResultsWe identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model.ConclusionOverall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.

Deficiency of CARD8 Is Associated with Increased Alzheimer’s Disease Risk in Women

NF-ĸB, a major transcription factor controlling inflammation, is activated in Alzheimer’s disease (AD) brains. CARD8 protein has been implicated in the suppression of NF-ĸB activity, but a truncating polymorphism (p.C10X, rs2043211) renders a non-functional CARD8 protein that gives rise to a more active NF-ĸB and an amplification of the inflammatory process. Apolipoprotein E (ApoE) Ε4 allele, the major genetic risk factor of AD, is associated with hyperactivation of NF-ĸB and enhanced brain inflammation. In a case-control study in 300 AD patients and 300 healthy controls, we examined whether the CARD8 (p.C10X) polymorphism, independently or in concert with the ApoE Ε4 allele, might predispose to AD. Women, but not men, carrying the CARD8 AA genotype (truncated protein) had a 2.39-fold higher risk of developing AD than subjects with the CARD8 TT genotype (full-length protein). This association with susceptibility to AD was independent of the ApoE Ε4 allele.

Expression and function of toll-like receptors in peripheral blood mononuclear cells of patients with polymyalgia rheumatica and giant cell arteritis

Objective To investigate the expression and function of the Toll-like receptor (TLR) family in peripheral blood mononuclear cells (PBMCs) of patients with polymyalgia rheumatica (PMR) and giant cell arteritis (GCA). Methods The authors analysed 70 patients with PMR, 20 with GCA, and 24 healthy controls (HC). TLR expression was assessed by flow cytometry. TLR function was assessed by stimulating PBMCs with specific ligands. Results A significantly increased expression of TLR7 in PBMCs of patients with active disease compared with HC was found. Despite increased expression of TLR7, circulating monocytes from patients showed a significantly lower in vitro response to TLR7 agonists. No amino acid substitutions predicted to be functionally damaging were found in TLR7. A normal response to specific TLR7 agonists in patients in complete remission eliminated a genetic defect. TLR expression and function were also affected to some degree in other diseases characterised by a strong acute phase response. Conclusion These data suggest activation of TLR7 during the active phase of PMR and GCA which resolves with complete disease remission. Whether this finding is the consequence of the marked inflammatory process in these disorders or activation by natural ligands remains to be explored.

Gene-gene interaction between CARD8 and interleukin-6 reduces Alzheimer's disease risk

Sirs, A chronic inflammatory process with activation of microglial cells contribute to the neurodegeneration associated with Alzheimer’s disease (AD) [9]. NF-jB, a major transcription factor controlling inflammation, is activated in AD brains [5], and genes encoding several different proinflammatory cytokines, such as interleukin-6 (IL-6), are induced by NF-jB in activated microglia surrounding senile plaques [4]. In contrast with the majority of CARD proteins described to date that activate NF-jB, CARD8 potently suppresses NF-jB activation and reduces the inflammatory process [8]. A biallelic (T/A) polymorphism located at the third nucleotide of codon 10 of CARD8 (rs2043211) generates a premature stop codon (allele A) that severely truncates CARD8 protein giving rise to a small N-terminal peptide unable to regulate the NF-jB signaling pathway; in contrast, the full-length protein (allele T) represses the activity of NF-jB and limits the induction of inflammatory mediators [3]. CARD8 (rs2043211) polymorphism has been associated with chronic inflammatory diseases, such as Crohn’s disease [6] and rheumatoid arthritis [3]. A biallelic (G/C) polymorphism in the promoter region (-174, rs1800795) of the IL-6 gene was found to be associated with susceptibility for AD [1, 7]. The postulated common pathway of CARD8 and IL-6 in the cerebral inflammatory response (CARD8 suppressing the NF-jB activity in the context of proinflammatory signals) prompted us to examine the combined contribution of both genes to the susceptibility for AD. The study included 239 AD patients (66% women; mean age at the time of study 75.6 years; SD 7.8; range 50– 97 years; mean age at onset 72.4 years; SD 7.9; range 48– 94 years) who met NINCDS/ADRDA criteria for probable AD. All AD cases were defined as sporadic because their family history did not mention any first-degree relative with dementia; this information was obtained by direct interviews with relatives. Control subjects consisted of 165 unrelated individuals (69% women; mean age 80.8 years; SD 7.1; range 63–98 years) who had complete neurological and medical examinations establishing that they were free of significant illness and all had MMSE scores of 28 or more. Control subjects were randomly selected from a nursing home, and they arose from the same base population as the cases. The AD and control samples were Caucasians originating from a homogeneous population in a limited geographical area in Northern Spain. All patients and control subjects were ascertained to have parents and grandparents born in Northern Spain to ensure ethnicity; consequently, possible confounding effects of the inclusion in the study of members of different ethnic groups have been minimized. Blood samples were taken after written informed consent had been obtained from the subjects or their representatives. The study was approved by the ethical committee of the University Hospital ‘‘Marques de Valdecilla’’. The CARD8 (rs2043211) and IL-6 (-174, rs1800795) polymorphisms were determined as described A. Fontalba O. Gutierrez J. L. Fernandez-Luna Unidad de Genetica Molecular, ‘‘Marques de Valdecilla’’ University Hospital, Santander, Spain

Copy number variations in endoglin locus: mapping of large deletions in Spanish families with hereditary hemorrhagic telangiectasia type 1

BackgroundThe hereditary hemorrhagic telangiectasia syndrome (HHT), also known as the Rendu–Osler-Weber syndrome is a multiorganic vascular disorder inherited as an autosomal dominant trait. Diagnostic clinical criteria include: epistaxis, telangiectases in mucocutaneous and gastrointestinal sites, arteriovenous malformations (AVMs) most commonly found in pulmonary, hepatic and cerebral circulations, and familial inheritance. HHT is transmitted in 90% of the cases as an autosomal dominant condition due to mutations in either endoglin (ENG), or activin receptor-like kinase 1 (ACVRL1/ALK1) genes (HHT type 1 and 2, respectively).MethodsWe have carried out a genetic analysis of four independent Spanish families with HHT clinical criteria, which has permitted the identification of new large deletions in ENG. These mutations were first detected using the MLPA technique and subsequently, the deletion breakpoints were mapped using a customized copy number variation (CNV) microarray. The array was designed to cover the ENG gene and surrounding areas.ResultsAll tested families carried large deletions ranging from 3-kb to 100-kb, involving the ENG gene promoter, several ENG exons, and the two downstream genes FGSH and CDK9. Interestingly, common breakpoints coincident with Alu repetitive sequences were found among these families.ConclusionsThe systematic hybridization of DNA from HHT families, with deletions or duplications, to custom designed microarrays, could allow the mapping of breakpoints, coincident with repetitive Alu sequences that might act as “hot spots” in the development of chromosomal anomalies.