Wnt activation promotes neuronal differentiation of glioblastoma.

One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme (GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of ß-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of ß-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133(+) fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy.

Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.

Characterization of C14orf4, a novel intronless human gene containing a polyglutamine repeat, mapped to the ARVD1 critical region.

Within the ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region, mapped to 14q24.3, we detected an intronless gene of 4859 bp, predominantly expressed in the heart tissue. This gene encodes a 796-amino-acid, proline-rich protein showing polyglutamine and polyalanine tracks with variable length at the N-terminus and a C3HC4 RING finger domain at the C-terminus. CREB and AP-2 binding sites are present in the promoter region. The 5' flanking region contains neither a TATA box nor a CAAT box, but it is high in GC content and includes several Sp1 binding sites. Protein similarity searches revealed a significant match between the C-terminus and a human hypothetical protein, whose gene is located on the chromosome 19 long arm. The predicted protein shows PEST sequences, suggesting its rapid degradation. The novel intronless gene, provisionally named C14orf4 and probably encoding a nuclear protein, was excluded from being the ARVD1 gene.

ARVD4, a new locus for arrhythmogenic right ventricular cardiomyopathy, maps to chromosome 2 long arm.

Autosomal dominant arrhythmogenic right ventricular dysplasia (ARVD; MIM 107970) is a genetically heterogeneous cardiomyopathy, which often causes sudden death in juveniles and athletes. Two disease loci were previously mapped respectively to 14q23-q24 (ARVD1) and to 1q42-q43 (ARVD2). A third possible locus was assigned to 14q12-q22. We report here on a linkage study performed on three independent families with recurrence of ARVD characterized by localized involvement of the left ventricle. In these families the disease appears to be transmitted with three polymorphic DNA markers of the chromosome 2 long arm, showing a maximum lod score of 3.46 at theta = 0 for the marker D2S152. The multipoint linkage analysis suggests that the novel ARVD locus, provisionally named ARVD4, maps to 2q32. 1-q32.3, within the chromosomal region including markers D2S152, D2S103, and D2S389.

Chromosomal localization of the human genes, CPP32, Mch2, Mch3, and Ich-1, involved in cellular apoptosis.

Members of the ICE/CED-3 protease family appear to play an essential role in programmed cell death process. In this paper the chromosomal localization of the human genes CPP32, Mch2, Mch3 and Ich-1 is reported, obtained by Radiation Hybrid Mapping. CPP32 was assigned to chromosome 4q33-q35.1, Mch2 to chromosome 4q25-q26, Mch3 to chromosome 10q25.1-q25.2 and Ich-1 to chromosome 7q35. Ich-1 was found to map very close to the marker WI-9353. The possible overlapping of the two independent locus assignments is considered. The genomic distribution of these genes is discussed, with particular reference to the co-location with some human genetic diseases all characterized by autosomal dominant inheritance and by similar malformative features.

Fine mapping of the human endothelin-converting enzyme gene by fluorescent in situ hybridization and radiation hybrids.

Based on the sequence of the human endothelin-1-converting enzyme (hECE-1) cDNA, we cloned a 1982 bp cDNA fragment and we amplified by PCR a 3' fragment located on the last exon (exon 19). Human metaphase chromosomes were studied by Fluorescent in Situ Hybridization (FISH) using the 1982 digoxigenated hECE-1 fragment as a probe and chromosome 1-specific probes. Twin spot signals on each of the two homologous chromosomes 1 were found by FISH on band p36. The results of monochromosomal hybrids confirmed that hECE-1 is on chromosome 1. Radiation hybrid mapping localized the hECE-1 gene 3.15 cR far from D1S2436 (WI-3177), at about 25 cM from the telomere of the short arm, possibly at the border between 1p36.3 and 1p36.2.