Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia.

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Distinctive gene expression profiles in Balb/3T3 cells exposed to low dose cobalt nanoparticles, microparticles and ions: potential nanotoxicological relevance.

Size-dependent characteristics of novel engineered nanomaterials might result in unforeseen biological responses and toxicity. To address this issue, we used cDNA microarray analysis (13443 genes) coupled with bioinformatics and functional gene annotation studies to investigate the transcriptional profiles of Balb/3T3 cells exposed to a low dose (1 μM) of cobalt nanoparticles (CoNP), microparticles (CoMP) and ions (Co2+). CoNP, CoMP and Co2+ affected 124, 91 and 80 genes, respectively. Hierarchical clustering revealed two main gene clusters, one up-regulated, mainly after Co2+, the other down-regulated, mainly after CoNP and CoMP. The significant Gene Ontology (GO) terms included oxygen binding and transport and hemoglobin binding for Co2+, while the GOs of CoMP and CoNP were related to nucleus and intracellular components. Pathway analysis highlighted: i) mitochondrial dysfunction for Co2+, ii) signaling, activation of innate immunity, and apoptosis for CoNP, and iii) cell metabolism, G1/S cell cycle checkpoint regulation and signaling for CoMP. Unlike ions, particles affected toxicologically-relevant pathways implicated in carcinogenesis and inflammation.

Helicobacter pylori biofilm: a protective environment for bacterial recombination.

AIMS: The aim of this work was to investigate the interaction between two Helicobacter pylori strains in promoting genetic transfer, when grown in the biofilm mode. METHODS AND RESULTS: Biofilms produced by H. pylori 9/10 (A), H. pylori 15/4 (B) and their mixture (C) were studied for biomass production and cell viability. The genetic heterogeneity of 45 clones, coming from mature biofilm of co-cultured H. pylori strains was studied by both RAPD and cagA (EPIYA motifs)/vacA virulence genes analysis. Helicobacter pylori A, B and C developed a well-structured biofilm without significant differences in viability. No significant differences were recorded between A and B biomass measurement, whereas C biofilm expressed a significant (P < 0.001) higher adhesive capability when compared with A and B biofilms. C-clones DNA-fingerprintings showed an high genetic heterogeneity (mean similarity value = 0.528). The 60% of C-clones displayed vacA allelic combination s1i1m1m2 associated with cagA EPIYA motif pattern P1P2P3P3P3. CONCLUSIONS: Biofilms developed by multiple H. pylori strains are more complex than those associated with single strains. Such condition might promote the genetic exchange favouring the generation of more virulent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The 'biofilm niche' represents a successful strategy and a suitable environment for promoting bacterial population persistence by recombination events.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

Gastric adenomas: relationship between clinicopathological findings, Helicobacter pylori infection, APC mutations and COX-2 expression.

Gastric adenomas are rare neoplastic growths characterized by localized polypoid proliferations of dysplastic epithelium that tend to progress to infiltrating adenocarcinoma. Therefore, the identification of molecular markers that could reliably recognize adenomas at risk of progression is advocated in the clinical management. In this study we investigated, in a series of gastric adenoma specimens from an area at high risk of gastric cancer, the relationship between clinicopathological characteristics of adenoma and Helicobacter pylori infection, APC mutational status, and COX-2 and the down-stream enzyme mPGES1 expression. Helicobacter pylori infection, detected in 24%, and 33% by histology and PCR analyses, respectively, did not show any relationship with growth pattern, localization, size, dysplasia grade and presence of synchronous cancer. Pathogenetic mutations of MCR region (codons 1269-1589) of the APC gene were detected only in one case corresponding to a single, small size, low grade, H. pylori-negative adenoma. The expression of COX-2 largely matched that of mPGES(1). Both were overexpressed in 79% of cases showing a relationship with high-grade dysplasia, size >10 mm and presence of a synchronous carcinoma. In conclusion, COX-2 may play a key role in the development and progression of gastric adenoma and could be an attractive target in the management of gastric adenoma at major risk of cancer development.

Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin.

BACKGROUND: K-ras mutations are a key step in colorectal cancer progression. Such mutations have been widely studied in case series from Western countries but there are few data on the rate and spectrum of mutations in tumors from countries where the epidemiological features of the disease are different. PATIENTS AND METHODS: Tumor samples from 182 Iranian colorectal cancer patients (170 sporadic cases and 12 HNPCC cases) were screened for K-ras mutations at codons 12, 13 and 61 by sequencing analysis. The cases were also characterized for microsatellite instability at mononucleotide repeats by PCR and fragment analysis, and classified according to microsatellite instability status. The frequency and the spectrum of K-ras mutations were compared with those observed in a series of colorectal cancer patients from Italy. RESULTS: K-ras mutations were observed in 68/182 (37.4%) cases. Mutation frequencies were similar in HNPCC-associated, sporadic MSI-H and sporadic microsatellite-stable (MSS) tumors. However, the G13D substitution was more frequent in HNPCC (3/4, 75%) and sporadic MSI-H (7/11, 63.6%) tumors compared to sporadic MSS tumors (11/53, 20.4%) (P <0.01). Comparison of mutations in the two series from Iran and Italy showed a significantly higher frequency of G13D among Italian patients. CONCLUSIONS: While the frequency of K-ras mutations could be similar, the mutational spectrum could be differentially influenced by genetic and environmental factors.

Cobalt nano-particles modulate cytokine in vitro release by human mononuclear cells mimicking autoimmune disease.

The use of particles from micro to nanoscale provides benefits to diverse scientific fields, but because a large percentage of their atoms lie on the surface, nanomaterials could be highly reactive and pose potential risks to humans. Due to their wide range of application, Cobalt nano-particles are of great interest both in industry and in life-science. To date, there are few studies on Co nano-particle toxicology. In this respect, this study aims at evaluating in vitro the potential interference of Co nano-particles on the production of several cytokines (IL-2, IL-4, IL-6, IL-10, IFNgamma and TNFalpha) by PBMCs, comparing their effects to those of Co micro-particles and Co solution (CoCl2). Cells were cultured in Opticell flasks with escalating concentrations (10-5, 10-6 and 10-7 M), of Co nano and micro-particles and CoCl2 or without metal. Cytokines were quantified in the supernatants using a human Th1/Th2 cytokine cytometric bead array. Co micro-particles showed a greater inhibitory effect compared to other Co forms. Its inhibitory activity was detected at all concentrations and towards all cytokines, whereas Co solutions selectively inhibited IL-2, IL-10 and TNF-alpha at maximal concentration. Co nano-particles induced an increase of TNF-alpha and IFN-gamma release and an inhibition of IL-10 and IL-2: a cytokine pattern similar to that detected in the experimental and clinical autoimmunity. On the basis of the obtained data, immune endpoints should be sought in the next series of studies both in vitro and in vivo in subjects exposed to cobalt nano-particles.

Origin and gender determination of dried blood on a statue of the Virgin Mary.

In Italy, blood exudation from objects of worship recurs frequently in ancient chronicles and literature, in popular beliefs, and even in modern mass-media reports. This phenomenon, that was associated with epochal or catastrophic events, has roots that reach classical antiquity. In the last few years, several events connected with the detection of bloody "tears" on statues of the Virgin Mary required forensic medicine investigations. In the present report we describe genetic investigations conducted on dried blood of unknown derivation found on a statuette representing the Virgin Mary. To test the human or animal origin of the blood, we amplified Alu-specific sequences from DNAs obtained from the unknown sample and from humans, large apes, various Old and New World monkeys, a prosimian, mouse, common domestic artiodactyls and chicken. This investigation restricted the range of possible origin of the statue blood to humans, apes and Old World monkeys. To test the male or female origin of the blood, we used a multiplex nested polymerase chain reaction method, that allows the simultaneous amplification of the X-specific locus DXZ4 and of the Y-specific locus SRY. Considering the unlikelihood of an origin from simian Old World primates, the exclusive amplification of the X-specific product from the unknown sample and from human female blood controls, compared to the amplification of distinct X- and Y-specific bands from human male blood controls, strongly supports a human female origin of the statue blood.