Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by 'RTK swapping' by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by 'RTK swapping' by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.

The role of alveolar type II cells in swine leptospirosis

Abstract: This study aimed to investigate a possible relationship between alveolar type II cells and the inflammatory response to infection with Leptospira spp., and thus comprise a further element that can be involved in the pathogenesis of lung injury in naturally infected pigs. The study group consisted of 73 adult pigs that were extensively reared and slaughtered in Teresina, Piauí state, and Timon, Maranhão state, Brazil. The diagnosis of leptospirosis was made using the microscopic agglutination test (MAT) aided by immunohistochemistry and polymerase chain reaction. The MAT registered the occurrence of anti-Leptospira antibodies in 10.96% (8/73) of the pigs. Immunohistochemistry allowed for the visualization of the Leptospira spp. antigen in the lungs of 87.67% (64/73) of the pigs. There was hyperplasia of bronchus-associated lymphoid tissue and circulatory changes, such as congestion of alveolar septa, parenchymal hemorrhage and edema within the alveoli. Lung inflammation was more intense (p = 0.0312) in infected animals, which also showed increased thickening of the alveolar septa (p = 0.0006). Evaluation of alveolar type II (ATII) cells using an anti-TTF-1 (Thyroid Transcription Factor-1) antibody showed that there were more immunostained cells in the non-infected pigs (53.8%) than in the infected animals (46.2%) and that there was an inverse correlation between TTF-1 positive cells and the inflammatory infiltrate. There was no amplification of Leptospira DNA in the lung samples, but leptospiral DNA amplification was observed in the kidneys. The results of this study showed that a relationship exists between a decrease in alveolar type II cells and a leptospire infection. Thus, this work points to the importance of studying the ATII cells as a potential marker of the level of lung innate immune response during leptospirosis in pigs.

Frequency of B cells in normal mice which recognize self proteins

The mechanism whereby the immune system avoids self-aggression is one of the central issues of Immunology. The discovery of natural autoantibodies, mainly of IgM isotype, and of idiotypic interactions between antibodies indicates that elements of the immune system interact with self constituents and with themselves. Results of studies with soluble antibodies have indicated that the pool of circulating IgM represents the end result of a highly selective process of B cells activation and differentiation by self proteins resulting in the formation of a network. The objective of the present work was to determine the frequency of self-reacting B cells in normal mice. We were able to detect B cells that recognize self proteins present in extracts of different organs in normal adult, 2-3-month old, BALB/c and C57BL/6 mice with an ELISA spot assay. About 1% of total IgM-secreting cells among small, LPS-stimulated spleen cells reacted with organ extracts, whereas among large spleen cells the frequency was 5- to 10-fold lower. Immunization induced an increase in the frequency of IgM-secreting cells. The present results provide cellular evidence for the results of studies done at the serological level. The physiological role of these self-recognizing cells, as well as their participation in autoimmune processes, remain to be established

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

p16INK4 expression in precursor lesions of squamous cell cervical cancer related to the presence of HPV-DNA

The purpose of the present study was to identify the expression of p16INK4 in cervical cancer precursor lesions by immunohistochemistry and to correlate it with lesion grade and presence of human papillomavirus (HPV) infection. Cervical specimens from 144 women seen consecutively at the gynecology outpatient clinic of our institution from December 2003 to May 2005 were analyzed by cytopathology, histopathology, polymerase chain reaction for HPV-DNA, and p16INK4 immunostaining. Histologically normal biopsies, HPV-DNA negative by polymerase chain reaction, were used as control. HPV-DNA prevalence, including the control group, was 68.1% and the prevalence of p16INK4 expression was 55.0%. The percentage of cells stained by p16INK4 ranged from 10 to 100%, both in the group consisting of cervical intraepithelial neoplasia (CIN)1/HPV specimens and in the group of CIN2/CIN3 specimens with P value of 0.0001. p16INK4 expression was 48.3% in the CIN1/HPV group, as opposed to 94.3% in the CIN2/CIN3 group (P = 0.001), showing a statistically significant difference between the two groups. The quantitative method used here is simple and less subjective than the different semiquantitative methods described in the literature. In view of the different definitions of a p16INK4-positive case, it is almost impossible to compare the findings reported by different investigators. This study confirms the association between p16INK4 and CIN2 and CIN3 lesions. Moreover, it shows that some low grade lesions expressed high levels of this protein. This may indicate that such low grade lesions may be predisposed to progress to high grade lesions. This means that p16INK4 may be a strong marker for "neoplastic lesions" induced by HPV and not just an infection marker.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Prolonged acceptance of skin grafts induced by B cells places regulatory T cells on the histopathology scene

The participation of regulatory T (Treg) cells in B cell-induced T cell tolerance has been claimed in different models. In skin grafts, naive B cells were shown to induce graft tolerance. However, neither the contribution of Treg cells to B cell-induced skin tolerance nor their contribution to the histopathological diagnosis of graft acceptance has been addressed. Here, using male C57BL/6 naive B cells to tolerize female animals, we show that skin graft tolerance is dependent on CD25+ Treg cell activity and independent of B cell-derived IL-10. In fact, B cells from IL-10-deficient mice were able to induce skin graft tolerance while Treg depletion of the host inhibited 100% graft survival. We questioned how Treg cell-mediated tolerance would impact on histopathology. B cell-tolerized skin grafts showed pathological scores as high as a rejected skin from naive, non-tolerized mice due to loss of skin appendages, reduced keratinization and mononuclear cell infiltrate. However, in tolerized mice, 40% of graft infiltrating CD4+ cells were FoxP3+ Treg cells with a high Treg:Teff (effector T cell) ratio (6:1) as compared to non-tolerized mice where Tregs comprise less than 8% of total infiltrating CD4 cells with a Treg:Teff ratio below 1:1. These results render Treg cells an obligatory target for histopathological studies on tissue rejection that may help to diagnose and predict the outcome of a transplanted organ.

BAFF promotes regulatory T-cell apoptosis and blocks cytokine production by activating B cells in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology. A number of questions regarding its etiology are unclear. CD4+CD25+ regulatory T cells (Tregs) play a critical role in self-tolerance and, for unknown reasons, their relative number is reduced in PBC patients. B-cell-activating factor (BAFF) is a key survival factor during B-cell maturation and its concentration is increased in peripheral blood of PBC patients. It has been reported that activated B cells inhibit Treg cell proliferation and there are no BAFF receptors on Tregs. Therefore, we speculated that excessive BAFF may result in Treg reduction via B cells. To prove our hypothesis, we isolated Tregs and B cells from PBC and healthy donors. BAFF and IgM concentrations were then analyzed by ELISA and CD40, CD80, CD86, IL-10, and TGF-β expression in B cells and Tregs were measured by flow cytometry. BAFF up-regulated CD40, CD80, CD86, and IgM expression in B cells. However, BAFF had no direct effect on Treg cell apoptosis and cytokine secretion. Nonetheless, we observed that BAFF-activated B cells could induce Treg cell apoptosis and reduce IL-10 and TGF-β expression. We also showed that BAFF-activated CD4+ T cells had no effect on Treg apoptosis. Furthermore, we verified that bezafibrate, a hypolipidemic drug, can inhibit BAFF-induced Treg cell apoptosis. In conclusion, BAFF promotes Treg cell apoptosis and inhibits cytokine production by activating B cells in PBC patients. The results of this study suggest that inhibition of BAFF activation is a strategy for PBC treatment.