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Induced hypoxia stress on cervical cancer derived cells (HeLa cells) leads to significant changes in their membrane lipid profiles. The lipidome of HeLa cells was characterized by a joint approach wherein liquid chromatography-mass spectrometry (LC-MS) analysis was followed by high resolution NMR measurements. Multivariate data analysis showed apparent separation between control and hypoxia-treated HeLa cells and thus demonstrated hypoxia effects on lipid metabolism. The most striking finding was that hypoxia stimulation significantly reduced the total amount of cellular phosphoinositols (PI) but caused a prominent increase in the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). The observed decrease of PI amount under hypoxic conditions is probably due to the accumulation of cellular myo-inositol, which is known to play a critical role in de novo synthesis of PI. Moreover, our study suggests that polyunsaturated phospholipid species are stronger biomarkers for discriminating the effect of hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains reveals that UI slightly increases in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by which HeLa cells adapt to hypoxia conditions are also briefly reported. This journal is © 2014 The Royal Society of Chemistry.

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.

SERPINB3 is a serine protease inhibitor with pleiotropic functions. It is involved in several physiological and pathological processes, where it appears to exert antiapoptotic effects. Little is known about its expression on immune system cells, the major players in mechanisms of viral defense and autoimmune disorders. The aim of this study was to characterize the expression of SERPINB3 on the surface of peripheral blood mononuclear cell subsets in both normal subjects and in patients with chronic viral infections and autoimmune diseases. Sixty-two patients were analyzed by flow cytometric analysis, including 45 with hepatitis C virus (HCV)-related chronic liver disease and 17 with systemic lupus erythematosus (SLE). SERPINB3 was expressed on B lymphocytes in 79% of the controls, in 32% of the HCV-infected patients and in none of the SLE patients. Surface localization of SERPINB3 was confirmed by confocal microscopy. SERPINB3 positivity was associated with CD27 reactivity ( r = 0.98), but not to other activation molecules (CD69, CD71, CD86 and CXCR3). SERPINB3 is physiologically expressed on the surface of CD27+ B lymphocytes, but its expression is reduced in HCV viral infection and not detectable in SLE patients. These results may suggest a role for SERPINB3 in B-cell defects typically found in viral infections and autoimmune disorders.

This work presents an improved platform for single-site electroporation and controlled transfectants delivery. The device consists of a gold microelectrode array (MEA) with integrated microfluidics and nanostructured titanium dioxide (ns-TiO2) functionalized electrodes for the improvement of cell adhesion. Human cervical cancer cells (HeLa) have been successfully cultivated on chip surface using traditional protocols. The system has been previously tested by electroporating HeLa cells with Lucifer Yellow (LY) and then, in order to validate the approach and cell viability, with plasmid for the enhanced expression of Green Fluorescence Protein (pEGFP-N1) delivered through microchannels.

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

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

Epithelial–mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over-expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial–mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E-cadherin and an increase of β-catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti-protease activity of the protein, as documented by the results obtained with an active loop-deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti-SERPINB3 antibody in a dose-dependent manner and SERPINB3-transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial–mesenchymal transition, acting at both the autocrine and the paracrine level. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Squamous Cell Carcinoma Antigen-1 (SCCA1) overexpression has been observed in tumours of epithelial origin and in hepatocellular carcinoma. Previous data indicate that this serpin inhibits apoptosis, while its proliferative activity was only recently proposed. The aim of this study was to evaluate the effect of SCCA1 on liver cells in a transgenic mouse model after partial hepatectomy. Twenty-one C57BL/6J mice (11 transgenic for human SCCA1, 10 wild-type) underwent partial hepatectomy and were sacrified after one week. Apoptosis and proliferation markers were determined in the liver at sacrifice, while a cytokine panel was measured in serum. Transgenic mice showed a relative liver weight significantly higher than wild-type mice at sacrifice (mean +/- SD, 5.38+/-0.50% vs 4.84+/-0.29%, p=0.0221), while no difference (p=0.2403) was observed in two untreated control groups (6 transgenic, 6 wild-type mice). Active caspase-3 was significantly lower in transgenic mice than in wild-type mice (p=0.0047). The transgenic mouse group showed overall higher proliferative activity at sacrifice, compared to wild-type mice, with increased proliferation parameters. Cytokine analysis revealed a remarkable and opposite sex-dependent behaviour of interleukin (IL)-6 after hepatectomy. At variance with wild-type mice, a significant IL-6 increase was documented only in transgenic females (p=0.0313), even more relevant than that observed in wild-type males. In conclusion, transgenic mice expressing SCCA1 showed higher liver regenerative potential compared to wild-type mice, supporting the dual role of this serpin as an anti-apoptotic and pro-proliferative stimulus for liver cells in vivo.

SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA1) is a member of the ov-serpins, a serine protease inhibitors family expressed in many cell types including normal epithelium, leukocytes, tumors of epithelial origin and primary liver cancer. Several studies, carried out in vitro and in vivo, have documented an important role of SERPINB3 in the modulation of programmed cell death by different mechanisms, both in inflammatory processes and in cancer. SERPINB3 significantly attenuates apoptosis by contrasting cytochrome c release from the mitochondria and by antichemotactic effect for NK cells. Mechanisms involved in apoptosis induction and regulation play a key role in the balance between cell proliferation and death. Imbalance of this equilibrium may contribute to the development of autoimmune diseases, as defective apoptosis of immune cells leads to deregulated autoreactive cell proliferation. Since defective programmed cell death represents a critical feature of autoimmunity, the involvement of SERPINB3 in this pathological field deserves further studies.

The liver is the main site of HBV replication, however extrahepatic organs, such as the lymphoid system, are an important reservoir of the virus. Viral DNA into different mononuclear cell subsets has been mainly detected in monocytes and B lymphocytes. The attachment site of the virus has been identified in the preS1 encoded protein of the virus envelope, the same involved in hepatocyte infection. The risk of HBV transmission by infected lymphocytes has been clearly documented in the setting of liver transplantation where de novo HBV infection has been found in up to about 80% of liver grafts from HBsAg negative but anti-HBc positive donors. In the hemodialysis setting the percentage of HBV DNA detection in mononuclear cells of HBsAg negative patients has been described in up to 54% of the cases. Vertical transmission studies indicate that HBV-infected mononuclear cells of the mother may result in viral infection of mononuclear cells of the newborns and possible HBV vaccine response failure. HBV can also infect bone marrow cells and in vitro studies demonstrate a block of hematopoiesis by HBV, supporting clinical observations of isolate cases of aplastic anemia associated to the infection.

This work presents an improved platform for single-site electroporation and controlled transfectants’ delivery. The device consists of a gold microelectrode array (MEA) with integrated microfluidics and nanostructured titanium dioxide (ns-TiO2) functionalized electrodes. Human cervical cancer cells (HeLa) have been successfully cultivated on chip surface. The system has been previously tested by electroporating them with Lucifer Yellow (LY) and then, in order to validate the approach and cell viability, with plasmids for the enhanced expression of Green Fluorescence Protein (pEGFP) delivered through microchannels.