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A great bulk of recent experimental evidence suggests the key role of the complex crosstalk between the extracellular matrix (ECM) and the cellular component of tissues during morphogenesis and embryogenesis. In particular, remodeling of the ECM and of its physical interactions pattern with surrounding cells represent two crucial processes that might be involved in muscle development. However, little information is available on this topic, especially on invertebrate species. To obtain new insights on how tuning the ECM microenvironment might drive cellular fate during embryonic development, we used the invertebrate medicinal leech Hirudo verbana as a valuable experimental model, due to its simple anatomy and the recapitulation of many aspects of the basic biological processes of vertebrates. Our previous studies on leech post-embryonic development have already shown the pivotal role of ECM changes during the growth of the body wall and the role of Yes-associated protein 1 (YAP1) in mechanotransduction. Here, we suggest that the interactions between stromal cell telocytes and ECM might be crucial in driving the organization of muscle layers during embryogenesis. Furthermore, we propose a possible role of the pleiotropic enzyme HvRNASET2 as a possible modulator of collagen deposition and ECM remodeling not only during regenerative processes (as previously demonstrated) but also in embryogenesis. [ABSTRACT FROM AUTHOR]

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease. [ABSTRACT FROM AUTHOR]

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer. Oncogene (2001) 20, 980–988. [ABSTRACT FROM AUTHOR]

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors. [ABSTRACT FROM AUTHOR]

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence. [ABSTRACT FROM AUTHOR]

OTX homeobox genes have been extensively studied for their role in development, especially in neuroectoderm formation. Recently, their expression has also been reported in adult physiological and pathological tissues, including retina, mammary and pituitary glands, sinonasal mucosa, in several types of cancer, and in response to inflammatory, ischemic, and hypoxic stimuli. Reactivation of OTX genes in adult tissues supports the notion of the evolutionary amplification of functions of genes by varying their temporal expression, with the selection of homeobox genes from the "toolbox" to drive or contribute to different processes at different stages of life. OTX involvement in pathologies points toward these genes as potential diagnostic and/or prognostic markers as well as possible therapeutic targets. [ABSTRACT FROM AUTHOR]

The patient reported here underwent hematopoietic stem cell transplantation (HSCT) due to chronic granulomatous disease (CGD) caused by biallelic mutations of the NCF1 gene. Two years later, he developed AML, which was unexpected and was recognized via sex-mismatched chromosomes as deriving from the donor cells; the patient was male, and the donor was his sister. Donor cell leukemia (DCL) is very rare, and it had never been reported in patients with CGD after HSCT. In the subsequent ten years, the AML relapsed three times and the patient underwent chemotherapy and three further HSCTs; donors were the same sister from the first HSCT, an unrelated donor, and his mother. The patient died during the third relapse. The DCL was characterized since onset by an acquired translocation between chromosomes 9 and 11, with a molecular rearrangement between the MLL and MLLT3 genes—a quite frequent cause of AML. In all of the relapses, the malignant clone had XX sex chromosomes and this rearrangement, thus indicating that it was always the original clone derived from the transplanted sister's cells. It exhibited the ability to remain quiescent in the BM during repeated chemotherapy courses, remission periods and HSCT. The leukemic clone then acquired different additional anomalies during the ten years of follow-up, with cytogenetic results characterized both by anomalies frequent in AML and by different, non-recurrent changes. This type of cytogenetic course is uncommon in AML. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) is a rare myeloproliferative disorder caused by the reciprocal translocation t(9;22)(q34;q11) in hematopoietic stem cells (HSCs). This chromosomal translocation results in the formation of an extra-short chromosome 22, called a Philadelphia chromosome (Ph), containing the BCR-ABL1 fusion gene responsible for the expression of a constitutively active tyrosine kinase that causes uncontrolled growth and replication of leukemic cells. Mechanisms behind the formation of this chromosomal rearrangement are not well known, even if, as observed in tumors, repetitive DNA may be involved as core elements in chromosomal rearrangements. We have participated in the explorative investigations of the PhilosoPhi34 study to evaluate residual Ph+ cells in patients with negative FISH analysis on CD34+/lin- cells with gDNA qPCR. Using targeted next-generation deep sequencing strategies, we analyzed the genomic region around the t(9;22) translocations of 82 CML patients and one CML cell line and assessed the relevance of interspersed repeat elements at breakpoints (BP). We found a statistically higher presence of LINE elements, in particular belonging to the subfamily L1M, in BP cluster regions of both chromosome 22 and 9 compared to the whole human genome. These data suggest that L1M elements could be potential drivers of t(9;22) translocation leading to the generation of the BCR-ABL1 chimeric gene and the expression of the active BCR-ABL1-controlled tyrosine kinase chimeric protein responsible for CML. [ABSTRACT FROM AUTHOR]

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation. [ABSTRACT FROM AUTHOR]

Ovarian cancer represents one of the most malignant gynecological cancers worldwide, with an overall 5-year survival rate, being locked in the 25–30% range in the last decade. Cancer immunotherapy is currently one of the most intensively investigated and promising therapeutic strategy and as such, is expected to provide in the incoming years significant benefits for ovarian cancer treatment as well. Here, we provide a detailed survey on the highly pleiotropic oncosuppressive roles played by the human RNASET2 gene, whose protein product has been consistently reported to establish a functional crosstalk between ovarian cancer cells and key cellular effectors of the innate immune system (the monocyte/macrophages lineage), which is in turn able to promote the recruitment to the cancer tissue of M1-polarized, antitumoral macrophages. This feature, coupled with the ability of T2 ribonucleases to negatively affect several cancer-related parameters in a cell-autonomous manner on a wide range of ovarian cancer experimental models, makes human RNASET2 a very promising candidate to develop a "multitasking" therapeutic approach for innovative future applications for ovarian cancer treatment. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn's disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy‐dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro‐inflammatory genes such as interleukin‐1beta, interleukin‐8 and tumour necrosis factor alpha were upregulated in the second week and anti‐inflammatory genes such as transforming growth factor beta and interleukin‐10. Interestingly, an additional expression peak was found for interleukin‐8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food‐dependent intestinal inflammation. [ABSTRACT FROM AUTHOR]

Introduction. Müller glial cells typically activate to react to hypoxic tissue damage in several retinal diseases. We evaluated the in vitro response to a hypoxia-mimicking stimulus on the expression of a set of genes, known to contribute to eye morphogenesis and cell differentiation. Materials and Methods. A MIO-M1 Müller cell line was cultured in a hypoxia-mimicking environment by the addition of cobalt chloride to the culture medium, followed by a recovery time in which we mimic restoration from the hypoxic insult. The HIF-1α protein and VEGF-A gene expression were quantified to verify the induction of a hypoxia-like state. Results. Among the genes under study, we did not observe any difference in the expression levels of Otx1 and Otx2 during treatment; conversely, Otx1 was overexpressed during recovery steps. The VEGF-A gene was strongly upregulated at both the CoCl2 and recovery time points. The transactivated isoform (TA) of the TP73 gene showed an overexpression in long-term exposure to the hypoxic stimulus with a further increase after recovery. Discussion. Our molecular analysis is able to describe the activation of a set of genes, never before described, that can drive the response to a hypoxia-like status. The improved comprehension of these cellular events will be useful for designing new therapeutical approaches for retinal pathologies. [ABSTRACT FROM AUTHOR]

Background: An isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q), are the most frequent anomalies in the bone marrow of patients with Shwachman-Diamond syndrome, which is caused in most cases by mutations of the SBDS gene. These clonal changes imply milder haematological symptoms and lower risk of myelodysplastic syndromes and acute myeloid leukaemia, thanks to already postulated rescue mechanisms. Results: Bone marrow from fourteen patients exhibiting either the i(7)(q10) or the del(20)(q) and coming from two large cohorts of patients, were subjected to chromosome analyses, Fluorescent In Situ Hybridization with informative probes and array-Comparative Genomic Hybridization. One patient with the i(7)(q10) showed a subsequent clonal rearrangement of the normal chromosome 7 across years. Four patients carrying the del(20)(q) evolved further different del(20)(q) independent clones, within a single bone marrow sample, or across sequential samples. One patient with the del(20)(q), developed a parallel different clone with a duplication of chromosome 3 long arm. Eight patients bore the del(20)(q) as the sole chromosomal abnormality. An overall overview of patients with the del(20)(q), also including cases already reported, confirmed that all the deletions were interstitial. The loss of material varied from 1.7 to 26.9 Mb and resulted in the loss of the EIF6 gene in all patients. Conclusions: Although the i(7)(q) and the del(20)(q) clones are frequent and clinically benign in Shwachman Diamond-syndrome, in the present work we show that they may rearrange, may be lost and then reconstructed de novo, or may evolve with independent clones across years. These findings unravel a striking selective pressure exerted by SBDS deficiency driving to karyotype instability and to specific clonal abnormalities. [ABSTRACT FROM AUTHOR]

Muscle differentiation has been widely described in zebrafish and Xenopus, but nothing is known about this process in amphibian urodeles. Both anatomical features and locomotor activity in urodeles are known to show intermediate features between fish and anurans. Therefore, a better understanding of myogenesis in urodeles could be useful to clarify the evolutionary changes that led to the formation of skeletal muscle in the trunk of land vertebrates. We report here a detailed morphological and molecular investigation on several embryonic stages of Ambystoma mexicanum and show that the first differentiating muscle fibers are the slow ones, originating from a myoblast population initially localized close to the notochord that forms a superficial layer on the somitic surface afterwards. Subsequently, fast fibers differentiation ensues. We also identified and cloned A. mexicanum Myf5 as a muscle-specific transcriptional factor likely involved in urodele muscle differentiation. [ABSTRACT FROM AUTHOR]

It is widely recognized that metal compounds may modify gene expression. In this context, we have searched for genes whose expression may be affected by cadmium and platinum ions within the context of a cell culture system. Cadmium is well known for its carcinogenic potential while platinum is destined to become more and more interesting because of its increasing use in the automotive industries. By applying differential display to cultures of mouse fibroblast, we have identified two transcripts (acute lymphoblastic leukemia-1, All-1, and a novel gene named metal-responsive gene, MERE-1) that were responsive to platinum and cadmium ions. Moreover, further experiments with a panel of metal compounds have shown that MERE-1 was strongly induced also by La(NO3)2 and Cr(NO3)3, and to a lesser extent, by Na2CrO4 and (NH4)2TeCl6. [Copyright &y& Elsevier]

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection. Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization. [ABSTRACT FROM AUTHOR]

Background: Clonal chromosome changes are often found in the bone marrow (BM) of patients with Shwachman-Diamond syndrome (SDS). The most frequent ones include an isochromosome of the long arm of chromosome 7, i (7)(q10), and an interstitial deletion of the long arm of chromosome 20, del (20)(q). These two imbalances are mechanisms of somatic genetic rescue. The literature offers few expression studies on SDS. Results: We report the expression analysis of bone marrow (BM) cells of patients with SDS in relation to normal karyotype or to the presence of clonal chromosome anomalies: del (20)(q) (five cases), i (7)(q10) (one case), and other anomalies (two cases). The study was performed using the microarray technique considering the whole transcriptome (WT) and three gene subsets selected as relevant in BM functions. The expression patterns of nine healthy controls and SDS patients with or without chromosome anomalies in the bone marrow showed clear differences. Conclusions: There is a significant difference between gene expression in the BM of SDS patients and healthy subjects, both at the WT level and in the selected gene sets. The deletion del (20)(q), with the EIF6 gene consistently lost, even in patients with the smallest losses of material, changes the transcription pattern: a low proportion of abnormal cells led to a pattern similar to SDS patients without acquired anomalies, whereas a high proportion yields a pattern similar to healthy subjects. Hence, the benign prognostic value of del (20)(q). The case of i (7)(q10) showed a transcription pattern similar to healthy subjects, paralleling the positive prognostic role of this anomaly as well. [ABSTRACT FROM AUTHOR]

Highlights • A role for human RNASET2 protein in macrophage polarization is proposed. • The RNASET2 protein reduces M2-like in favour of M1-like polarization pattern of human macrophages in vitro under appropiate stimuli. • Tuning of the macrophage polarization pattern by RNASET2 might play a critical role in RNASET2-mediated in vivo tumor suppression. Abstract Macrophages represent key inflammatory cellular effectors of the innate immune response. Despite being widely acknowledged as professional phagocytes, the functional roles played by these cells have been progressively widened over the years to encompass regulation of the adaptive immune system, stimulation or suppression of cancer cell growth and tissue remodeling. These diverse functional features have led to the concept of "macrophage plasticity", i.e. the ability of these cells to express a wide range of phenotypes endowed with different functional roles. Several activation programs have been described for mammalian macrophages, based mainly on their differential transcriptional profiles. Based on established in vitro experimental conditions, many researchers currently refer to the M1 (or M1-like) and M2 (or M2-like) terms to describe the two extremes of a rather broad spectrum of polarization states that macrophages can experience in vivo. In light of the widely recognized opposite roles of M1-like and M2-like macrophages on cancer growth, and our largely incomplete knowledge of the cellular and molecular mechanisms underlying the establishment of the M1-like versus M2-like balance within a tumor mass, we report here results from in vitro assays pointing at the human RNASET2 gene as a potential regulator of the balance between M1-like/M2-like macrophage polarization. Not only do our results confirm previous in vivo data, thus further supporting a role for this pleiotropic protein in the innate immune system, but they also define RNASET2 as a new molecular target with potential applications for in vivo reprogramming of macrophage polarization, an increasingly appraised anticancer strategy. [ABSTRACT FROM AUTHOR]

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68 and HmAIF-1 macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches. [ABSTRACT FROM AUTHOR]

Abstract: Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients'' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted. [Copyright &y& Elsevier]

Background information The ribonucleases (RNases) constitute a heterogeneous group of enzymes, which exert diverse and specific biological functions. Several RNases have been shown to control gene expression and cell differentiation. RNASET2, a novel member of the Rh/T2/S family of RNases, exerts micro-environmental control of malignancy in different experimental models with a general onco-suppressor activity, since it prevents cancer proliferation. Indeed, RNASET2 was found to be downregulated at the transcript level in several primary ovarian tumours or cell lines and in melanoma cell lines. Although recent works shed light on the biological role of RNASET2 in delaying tumour growth, its trafficking within the cell is still poorly understood. RNASET2 seems to play diverse biological roles including turnover of tRNA in yeast as well as rRNA degradation in zebrafish. Results Here, we have studied the intracellular trafficking of RNASET2 in mammalian cells. RNASET2 co-localizes with markers for the trans-Golgi network (TGN), which is the central sorting and processing station of the secretory pathway. Moreover, using the temperature-sensitive vesicular stomatitis glycoprotein, we demonstrate that RNASET2 undergoes delivery to the plasma membrane. In contrast to other RNA-interacting proteins, RNASET2 does not accumulate in stress granules upon metabolic stress in mammalian cells. Surprisingly, RNASET2 shows co-localization with processing bodies (P-bodies), which increases upon metabolic stress. Finally, cells lacking RNASET2 show a reduced numbers of P-bodies. Conclusions In this study, we have identified two distinct cellular pools of RNASET2-containing granules. One pool undergoes membrane delivery using the TGN, and it is released to the extracellular environment. The second pool is recruited into P-bodies, suggesting a possible involvement of RNASET2 in P-body formation in mammalian cells. [ABSTRACT FROM AUTHOR]

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family. [ABSTRACT FROM AUTHOR]

Abstract: The ANKRD1/CARP gene encodes a muscle-specific protein which has been implicated in transcriptional regulation and myofibrillar assembly. Several features at both the mRNA and protein levels define ANKRD1 as a gene whose expression is tightly regulated, and deregulated expression of this protein has been recently associated to human congenital heart disease. It is therefore crucial to define the intracellular pathways that regulate the ANKRD1 protein’s steady-state levels. Here, we show that ANKRD1 is a short-lived protein whose levels are tightly regulated by the 26S proteasome. In addition, a critical role for a putative PEST motif was established, although other degrons within the ANKRD1 protein are likely implicated in the control of its intracellular levels. [Copyright &y& Elsevier]

BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of β cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of β cells suggests that it may play a role in diabetes-associated amyloidogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Ribonucleases are ubiquitous enzymes involved in RNA metabolism and are classified in several families on the basis of their structural, catalytic, and biological properties. Here, we describe characterization of the only human member of the Rh/T2/S family of acid hydrolases so far described, named RNASET2. This protein was previously reported to have an interesting biological function in the control of tumourigenesis and metastatization. We show that RNASET2 is present in multiple forms in human cell lines and mouse tissues, one of which represents the full length, glycosylated and secreted form, while the others are proteolytic products. RNASET2 is endowed with catalytic activity as demonstrated with purified recombinant protein expressed in the Baculovirus Expression Vector System and in a human cell line ectopically expressing various types of constructs. Furthermore, we document for this protein a lysosomal localization as described for other members of the Rh/T2/S family of ribonucleases. The results presented herein represent a further advancement toward the molecular understanding of the tumour suppressive properties of the human RNASET2 protein. [Copyright &y& Elsevier]

Styrene is one of the most important monomers produced worldwide, and it finds major use in the production of polystyrene, acrylonitrile–butadiene–styrene resins and unsaturated polystyrene resins. Epidemiological studies on styrene showed that the malignancies observed most frequently in humans after exposure are related to the lymphatic and haemopoietic system. IARC classified styrene a possible carcinogenic to humans (Group 2B). In this study, we evaluated the effect of styrene on gene expression profiles of human cord blood cells, as well as its activity on the apoptosis and bcl-2 related protein expression. Data demonstrated that, after 24 and 48 h of exposure, styrene (800 μM) induced an increase in the necrosis of mononuclear cord blood cells, whereas it did not cause any increase in the apoptotic process. Western blot analysis revealed a modified expression of Bax, BCl-2, c-Jun, c-Fos and Raf-1 proteins in the human cord blood cells after direct exposure to styrene, whereas p53 expression did not change. Furthermore, Macroarray analysis showed that styrene changed cord blood gene expression, inducing up-regulation of monocyte chemotactic protein 1 (MCP-1), and down-regulation of CC chemokine receptor type 1 (CCR-1) and SLP-76 tyrosine–phosphoprotein. [Copyright &y& Elsevier]

In this report we present the characterization of ovarian neoplasms including benign tumors, borderline tumors, and invasive carcinomas in order to assess whether a sharing of cytogenetic abnormalities is present in all three types of tumors. A cohort of 114 newly diagnosed and untreated ovarian epithelial tumors were analyzed by cytogenetic and molecular cytogenetic approaches with probes specific for chromosome 6. Three groups of chromosome abnormalities were identified: the first group included abnormalities common to all tumor classes (losses of chromosomes 6, 8, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, and X; gains of chromosomes 1, 3, 5, and 12; 6q24∼qter deletions); the second group presented specific abnormalities present in malignant but not in benign tumors (losses of chromosomes 2, 7, 13, and 14; gains of chromosome 4 and chromosome markers); and the last group included abnormalities unique to invasive carcinomas (loss of chromosome 4; gains of chromosomes 2, 7, 8, 9, 10, 16, 17, 18, 19, 20, and 21; 6q16∼q24 deletions; rearrangements of 3p, 3q, 13q, and 21q regions). The presence of shared chromosomal alterations in all three types of ovarian neoplasms investigated in this report seems therefore to suggest a progression model for these types of tumors. [Copyright &y& Elsevier]

Hypoxia is a key component of the tumor microenvironment (TME) and promotes not only tumor growth and metastasis, but also negatively affects infiltrating immune cells by impairing host immunity. Dendritic cells (DCs) are the most potent antigen-presenting cells and their biology is weakened in the TME in many ways, including the modulation of their viability. RNASET2 belongs to the T2 family of extracellular ribonucleases and, besides its nuclease activity, it exerts many additional functions. Indeed, RNASET2 is involved in several human pathologies, including cancer, and it is functionally relevant in the TME. RNASET2 functions are not restricted to cancer cells and its expression could be relevant also in other cell types which are important players in the TME, including DCs. Therefore, this study aimed to unravel the effect of hypoxia (2% O2) on the expression of RNASET2 in DCs. Here, we showed that hypoxia enhanced the expression and secretion of RNASET2 in human monocyte-derived DCs. This paralleled the HIF-1α accumulation and HIF-dependent and -independent signaling, which are associated with DCs' survival/autophagy/apoptosis. RNASET2 expression, under hypoxia, was regulated by the PI3K/AKT pathway and was almost completely abolished by TLR4 ligand, LPS. Taken together, these results highlight how hypoxia- dependent and -independent pathways shape RNASET2 expression in DCs, with new perspectives on its implication for TME and, therefore, in anti-tumor immunity. [ABSTRACT FROM AUTHOR]

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors. [ABSTRACT FROM AUTHOR]

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech's innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health. [ABSTRACT FROM AUTHOR]

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO–S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO–S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth. • RNASET2 possesses an oncosuppressive role. • Human RNASET2 was previously produced in different non-mammalian heterologous systems. • For the first time, we produced human RNASET2 in CHO–S cells, a mammalian system. • Recombinant human RNASET2 was purified in high yield and as a single 38 kDa band. • Recombinant human RNASET2 was active at both enzymatic and biological level. [ABSTRACT FROM AUTHOR]

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme. [ABSTRACT FROM AUTHOR]

Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment of M1-polarized macrophages. Here, we report a murine Rnaset2-based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2-overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2-expressing C51 cells showed strong delayed tumor growth. CD86+ M1 macrophages were massively recruited in Rnaset2-expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206+ M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8+ T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2-expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8+ T cells. [ABSTRACT FROM AUTHOR]

RNASET2 is an extracellular ribonuclease endowed with a marked antitumorigenic role in several carcinomas, independent from its catalytic activity. Besides its antitumorigenic role by the recruitment to the tumor mass of immune cells from the monocyte/macrophage lineage, RNASET2 is induced by cellular stress and involved in actin cytoskeleton remodeling affecting cell interactions with the extracellular matrix (ECM). Here, we aimed to investigate the effects of RNASET2 expression modulation on cell phenotype and behavior in epithelial ovarian cancer (EOC) cellular models. In silico analysis on two publicly available datasets of gene expression from EOC patients (n = 392) indicated that increased RNASET2 transcript levels are associated with longer overall survival. In EOC biopsies (n = 101), analyzed by immunohistochemistry, RNASET2 was found heterogeneously expressed among tumors with different clinical–pathological characteristics and, in some cases, its expression localized to tumor-associated ECM. By characterizing in vitro two models of EOC cells in which RNASET2 was silenced or overexpressed, we report that RNASET2 expression negatively affects growth capability by conferring a peculiar cell phenotype upon the interaction of EOC cells with the ECM, resulting in decreased src activation. Altogether, these data suggest that drugs targeting activated src might represent a therapeutic approach for RNASET2-expressing EOCs. [ABSTRACT FROM AUTHOR]