Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis.     

Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells

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MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2

MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.     

DDX3 acts as a tumor suppressor in colorectal cancer as loss of DDX3 in advanced cancer promotes tumor progression by activating the MAPK pathway

Objective: The treatment and prognosis of patients with advanced colorectal cancer (CRC) remain a difficult problem. Herein, we investigated the role of DEAD (Asp-Glu-Ala-Asp) box helicase 3 (DDX3) in CRC and proposed potential therapeutic targets for advanced CRC.Methods: The expression of DDX3 in CRC and its effect on prognosis were explored by databases and CRC tissue microarrays. Stable DDX3 knockdown and overexpression cell lines were established with lentiviral vectors. The effects of DDX3 on CRC were investigated by functional experiments in vitro and in vivo. The molecular mechanism of DDX3 in CRC was explored by western blotting. Molecular-specific inhibitors were further used to explore potential therapeutic targets for advanced CRC.Results: The expression of DDX3 was decreased in advanced CRC, and patients with low DDX3 expression had a poor prognosis. In vitro and in vivo experiments showed that low DDX3 expression promoted the proliferation, migration and invasion of CRC. DDX3 loss regulated E-cadherin and β-catenin signaling through the mitogen-activated protein kinase (MAPK) pathway as shown by western blotting. In addition, the MEK inhibitor, PD98059, significantly reduced the increased cell proliferation, migration and invasion caused by knockdown of DDX3.Conclusions: DDX3 acts as a tumor suppressor gene in CRC. DDX3 loss in advanced cancer promotes cancer progression by regulating E-cadherin and β-catenin signaling through the MAPK pathway, and targeting the MAPK pathway may be a therapeutic approach for advanced CRC.     

PALI1 promotes tumor growth through competitive recruitment of PRC2 to G9A-target chromatin for dual epigenetic silencing

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Enhancer of zeste homolog 2 activates wnt signaling through downregulating CXXC finger protein 4

Through silencing tumor suppressor genes, epigenetic changes can activate signaling pathways important to cancer development. In this report, we found an epigenetic contribution to the aberrant activation of wnt signaling in human gastric cancer. CXXC4 (CXXC finger protein 4) was identified as a novel target of EZH2 (enhancer of zeste homolog 2), and EZH2 promotes the activation of wnt singaling by downregulating CXXC4 expression. CXXC4 inhibits the growth of gastric cancer cells both in vitro and in vivo through inactivating wnt signaling. In contrast, depletion of CXXC4 activates wnt signaling and promotes the anchorage-independent growth of nontumor gastric epithelial cells. CXXC4 is downregulated in gastric carcinoma tissues and its downregulation is associated with poor outcome of gastric cancer patients (hazard ratio: 5.053, P<0.05). Through its binding to dishevelled (Dvl), CXXC4 stabilizes the destruction complex of β-catenin to inhibit wnt signaling. Two critical amino acid residues in CXXC4, K161 and T162 were found to be important to its binding to Dvl and the growth inhibitory effect of CXXC4. In summary, EZH2 promotes the activation of wnt signaling in gastric carcinogenesis through the downregulation of CXXC4 expression. CXXC4 is a novel potential tumor suppressor directly regulated by EZH2, and its expression is a significant prognosis factor for patients with early stages of gastric cancer.     

RDH10, RALDH2, and CRABP2 are required components of PPARγ-directed ATRA synthesis and signaling in human dendritic cells

All-trans retinoic acid (ATRA) has a key role in dendritic cells (DCs) and affects T cell subtype specification and gut homing. However, the identity of the permissive cell types and the required steps of conversion of vitamin A to biologically active ATRA bringing about retinoic acid receptor-regulated signaling remains elusive. Here we present that only a subset of murine and human DCs express the necessary enzymes, including RDH10, RALDH2, and transporter cellular retinoic acid binding protein (CRABP)2, to produce ATRA and efficient signaling. These permissive cell types include CD103⁺ DCs, granulocyte-macrophage colony-stimulating factor, and interleukin-4-treated bone marrow-derived murine DCs and human monocyte-derived DCs (mo-DCs). Importantly, in addition to RDH10 and RALDH2, CRABP2 also appears to be regulated by the fatty acid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and colocalize in human gut-associated lymphoid tissue DCs. In our model of human mo-DCs, all three proteins (RDH10, RALDH2, and CRABP2) appeared to be required for ATRA production induced by activation of PPARγ and therefore form a linear pathway. This now functionally validated PPARγ-regulated ATRA producing and signaling axis equips the cells with the capacity to convert precursors to active retinoids in response to receptor-activating fatty acids and is potentially amenable to intervention in diseases involving or affecting mucosal immunity.     

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

In situ analysis of epigenetic modifications in the chromatin of Brachypodium distachyon embryos

Epigenetic modifications of the chromatin structure are crucial for many biological processes and act on genes during the development and germination of seeds. The spatial distribution of 3 epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 was investigated in ‘matured,’ ‘dry,’ ‘imbibed” and ‘germinating’ embryos of a model grass, Brachypodium. Our results indicate that the patterns of epigenetic modification differ in the various types of tissues of embryos that were analyzed. Such a tissue-specific manner of these modifications may be linked to the switch of the gene expression profiles in various organs of the developing embryo.     

Heuristic estimation of the α/β ratio for a cohort of Mexican patients with prostate cancer treated with external radiotherapy techniques

BackgroundThe aim of the study was to Estimate and compare the radiobiological ratio α/β with the heuristic method for a cohort of Mexican patients with prostate cancer (PCa) who were treated with external radiotherapy (RT) techniques at three Hospital Institutions in Mexico City. With the Kaplan-Meier technique and the Cox proportional hazards model, the biochemical relapse-free survival (bRFS) is determined and characterized for cohorts of Mexican patients with PCa who received treatment with external RT. Using these clinical outcomes, the radiobiological parameter α/β is determined using the heuristic methodology of Pedicini et. al.Materials and methodsThe α/β is calculated from the survival curves for different treatment schemes implemented at three distinct hospitals. The Pedicini’s techniques allow to determine the parameters α/β, k and N₀ when treatments are not radiobiologically equivalent, therefore, are built up of a set of curved pairs for the biologically effective dose (BED) versus the ratio α/β, where the ratio is given by the intersection for each pair of curves.ResultsSix different values of α/β were found: the first α/β = 2.46 Gy, the second α/β = 3.30 Gy, the third for α/β = 3.25 Gy, the fourth α/β = 3.24 Gy, the fifth α/β = 3.38 Gy and the last α/β = 4.08 Gy. These values can be explained as follows: a) The bRFS of the schemes presents a statistical variation; b) The absorbed doses given to the patient present uncertainties on the physical dosimetry that are not on the modeling; c) Finally, in the model for the bRFS of Eq. (3), there are parameters that have to be considered, such as: the number of clonogenic tumor cells N₀, the overall treatment time (OTT), the kick-off time for tumor repopulation T_k and the repopulation doubling time. Therefore, the mean value to α/β for all schemes has an average value of 3.29 (± 0.52) Gy.ConclusionsThe value of α/β¯=3.29 (±0.52) Gy is determined from cohorts of Mexican patients with PC a treated with external radiotherapy using the time-dependent LQ model, which is a higher value with respect to the “dogma” value of α/β 1.5 Gy obtained with the LQ model without temporal dependence. Therefore, there is a possibility of optimizing treatments radiobiologically and improving the results of bRFS in Mexican patients with PCa treated with external radiotherapy.     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Biology of IL-27 and its Role in the Host Immunity against Mycobacterium Tuberculosis

IL-27, a heterodimeric cytokine of IL-12 family, regulates both innate and adaptive immunity largely via Jak-Stat signaling. IL-27 can induce IFN-γ and inflammatory mediators from T lymphocytes and innate immune cells. IL-27 has unique anti-inflammatory properties via both Tr1 cells dependent and independent mechanisms. Here the role and biology of IL-27 in innate and adaptive immunity are summarized, with special interest with immunity against Mycobacterium tuberculosis.     

The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.     

Cellular prostatic acid phosphatase,a PTEN-functional homologue in prostate epithelia,functions as a prostate-specific tumor suppressor

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis.     

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus

Background

Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.

Results

The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5x10⁵ cfu/ml to 10³ cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.

Conclusions

So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.     

The role of thioredoxin 1 in the mycophenolic acid-induced apoptosis of insulin-producing cells

Mycophenolic acid (MPA) is one of many effective immunosuppressive drugs. However, MPA can induce cellular toxicity and impair cellular function in β-cells. To explore the effects of MPA and the relation between MPA and Trx-1, we used various methods, including an Illumina microarray, to identify the genes regulated during pancreatic β-cell death following MPA treatment. INS-1E cells (a pancreatic β-cell line) and isolated rat islets were treated with MPA for 12, 24, or 36 h, and subsequent microarray analysis showed that (Trx1) gene expression was significantly reduced by MPA. Further, Trx1 overexpression increased the cell viability, decreased the activations of c-jun N-terminal kinase (JNK) and caspase-3 by MPA, and attenuated ROS upregulation by MPA. Furthermore, siRNA knockdown of Trx1 increased MPA-induced cell death and the activations of p-JNK and caspase-3, and MPA significantly provoked the apoptosis of insulin-secreting cells via Trx1 downregulation. Our findings suggest that the prevention of Trx1 downregulation in response to MPA is critical for successful islet transplantation.