Pleural mesothelial cells express both BLT2 and PPARalpha and mount an integrated response to pleural leukotriene B4.

PubMed

Pace, Elisabetta; Ferraro, Maria; Mody, Christopher H; Melis, Mario R; Scafidi, Valeria; Bonanno, Anna; Profita, Mirella; Giarratano, Antonino; Gjomarkaj, Mark

2008-11-15

Leukotriene B(4) (LTB(4)) plays a crucial role in the recruitment of neutrophils into the pleural space. We identified for the first time the mechanisms by which LTB(4) interacts with mesothelial cells and recruits neutrophils in the pleural compartment. Primary pleural mesothelial cells express both the proinflammatory receptor for LTB(4) BLT2, and the anti-inflammatory receptor for LTB(4), PPARalpha. Parapneumonic pleural effusions highly increase BLT2 expression and, via BLT2 activation, increase the adhesion between mesothelial cells and neutrophils and the expression of ICAM-1 on mesothelial cells. The block of PPARalpha further increases both cell adhesion and ICAM-1 expression. BLT2 activation promotes the activation, on mesothelial cells, of STAT-1 but not the activation of NF-kappaB transcription factor. The increase of ICAM-1 expression is achieved via increased tyrosine phosphorylation activity since herbimycin, a tyrosine kinase inhibitor, reduces and since Na orthovanadate, a tyrosine phosphatase inhibitor, further increases ICAM-1 expression. This study demonstrates that pleural mesothelial cells, expressing both proinflammatory and anti-inflammatory LTB(4) receptors, are able to mount an integrated response to LTB(4) with a prevalence of BLT2 activities in the presence of an inflammatory milieu within the pleura.

Tissue Factor promotes breast cancer stem cell activity in vitro.

PubMed

Shaker, Hudhaifah; Harrison, Hannah; Clarke, Robert; Landberg, Goran; Bundred, Nigel J; Versteeg, Henri H; Kirwan, Cliona C

2017-04-18

Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231,T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

Expression patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and activating CD94/NKG2C on decidual CD56bright natural killer cells differ from those on peripheral CD56dim natural killer cells.

PubMed

Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji

2006-06-01

The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.

Regulation of expression of the ligand for CD40 on T helper lymphocytes.

PubMed

Castle, B E; Kishimoto, K; Stearns, C; Brown, M L; Kehry, M R

1993-08-15

Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell-delivered contact signals to B cells and may contribute to the high degree of specificity in B cell responses.

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

PubMed

Bartlett, W C; McCann, J; Shepherd, D M; Roy, M; Noelle, R J

1990-12-15

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

PubMed

Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A

2015-12-01

Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD.

Alterations in receptor expression or agonist concentration change the pathways gastrin-releasing peptide receptor uses to regulate extracellular signal-regulated kinase.

PubMed

Chen, Pei-Wen; Kroog, Glenn S

2004-12-01

G protein-coupled receptors activate extracellular signal-regulated kinases (ERKs) via different pathways in different cell types. In this study, we demonstrate that gastrin-releasing peptide receptor (GRPr) regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration. We examined stably transfected BALB/c 3T3 fibroblasts expressing GRPr constructs at different levels and treated the cells with several concentrations of bombesin (BN, a GRPr agonist) to activate a variable number of GRPr per cell. GRPr induced two waves of ERK activation and one wave of ERK inhibition. One wave of activation required an intact GRPr carboxyl-terminal domain (CTD). It peaked 6 min after addition of high BN concentration ([BN]) in cells with high GRPr expression. Another wave of activation was CTD-independent. It peaked 2 to 4 min after BN addition in cells when [BN] and/or GRPr expression were lower. The early wave of ERK activation was more sensitive than the later one to pretreatment with Bisindolylmaleimide I (GF 109203X) (a protein kinase C inhibitor) or hypertonic sucrose. Because these two waves of activation differ in time course, dose-response curve, requirement for GRPr CTD, and sensitivity to inhibitors, they result from different signaling pathways. A third pathway in these cells inhibited ERK phosphorylation 2 min after addition of high [BN] in cells with high GRPr expression. Furthermore, a GRPr-expressing human duodenal cancer cell line showed differential sensitivity to GF 109203X throughout BN-induced ERK activation, indicating that GRPr may activate ERK via multiple pathways in cells expressing endogenous GRPr.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

PubMed

Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2008-07-16

Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

PubMed Central

Yang, Xiao-Feng; Weber, Georg F.

2014-01-01

Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

Increased mast cell expression of PAR-2 in skin inflammatory diseases and release of IL-8 upon PAR-2 activation.

PubMed

Carvalho, Ricardo Filipe da Silva; Nilsson, Gunnar; Harvima, Ilkka Tapani

2010-02-01

Mast cells are increasingly present in the lesional skin of chronic skin inflammatory diseases including psoriasis and basal cell carcinoma (BCC). It has previously been shown that proteinase-activated receptor (PAR)-2 is expressed by mast cells, and tryptase is a potent activator of this receptor. In this study, skin biopsies from both healthy-looking and lesional skin of patients with psoriasis and superficial spreading BCC were collected and the expression of PAR-2 immunoreactivity in tryptase-positive mast cells was analysed. PAR-2 expression was confirmed in vitro in different mast cell populations. Cord-blood derived mast cells (CBMC) were stimulated with a PAR-2 activating peptide, 2-furoyl-LIGRLO-NH(2). Consequently, IL-8 and histamine production was analysed in the supernatants. We observed a significant increase in the percentage of mast cells expressing PAR-2 in the lesional skin of psoriasis and BCC patients compared with the healthy-looking skin. HMC-1.2, LAD-2 and CBMC mast cells all expressed PAR-2 both intracellularly and on the cell surface. CBMC activation with the PAR-2 activating peptide resulted in an increased secretion of IL-8, but no histamine release was observed. Furthermore, both PAR-2 and IL-8 were co-localized to the same tryptase-positive mast cells in the lesional BCC skin. These results show that mast cells express increased levels of PAR-2 in chronic skin inflammation. Also, mast cells can be activated by a PAR-2 agonist to secrete IL-8, a chemokine which can contribute to the progress of inflammation.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

PubMed

Yoon, Mee Sun; Pham, Chanh Tin; Phan, Minh-Trang Thi; Shin, Dong-Jun; Jang, Youn-Young; Park, Min-Ho; Kim, Sang-Ki; Kim, Seokho; Cho, Duck

2016-12-01

Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R 2  = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Gab1 Mediates Hepatocyte Growth Factor-Stimulated Mitogenicity and Morphogenesis in Multipotent Myeloid Cells

PubMed Central

Felici, Angelina; Giubellino, Alessio; Bottaro, Donald P.

2012-01-01

Hepatocyte growth factor (HGF)-stimulated mitogenesis, motogenesis and morphogenesis in various cell types begins with activation of the Met receptor tyrosine kinase and the recruitment of intracellular adaptors and kinase substrates. The adapter protein Gab1 is a critical effector and substrate of activated Met, mediating morphogenesis, among other activities, in epithelial cells. To define its role downstream of Met in hematopoietic cells, Gab1 was expressed in the HGF-responsive, Gab1-negative murine myeloid cell line 32D. Interestingly, the adhesion and motility of Gab1-expressing cells were significantly greater than parental cells, independent of growth factor treatment. Downstream of activated Met, Gab1 expression was specifically associated with rapid Shp-2 recruitment and activation, increased mitogenic potency, suppression of GATA-1 expression and concomitant upregulation of GATA-2 transcription. In addition to enhanced proliferation, continuous culture of Gab1-expressing 32D cells in HGF resulted in cell attachment, filopodia extension and phenotypic changes suggestive of monocytic differentiation. Our results suggest that in myeloid cells, Gab1 is likely to enhance HGF mitogenicity by coupling Met to Shp-2 and GATA-2 expression, thereby potentially contributing to normal myeloid differentiation as well as oncogenic transformation. PMID:20506405

Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8+ T-cell response

PubMed Central

Fann, Monchou; Godlove, Jason M.; Catalfamo, Marta; Wood, William H.; Chrest, Francis J.; Chun, Nicholas; Granger, Larry; Wersto, Robert; Madara, Karen; Becker, Kevin; Henkart, Pierre A.; Weng, Nan-ping

2006-01-01

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8+ T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8+ T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8+ T-cell response. PMID:16868257

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

Inflammatory cytokines IL-17 and TNF-α up-regulate PD-L1 expression in human prostate and colon cancer cells

PubMed Central

Wang, Xun; Yang, Lingyun; Huang, Feng; Zhang, Qiuyang; Liu, Sen; Ma, Lin; You, Zongbing

2017-01-01

Programmed cell death protein 1 (PD-1) acts on PD-1 ligands (PD-L1 and PD-L2) to suppress activation of cytotoxic T lymphocytes. Interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) are co-expressed by T helper 17 (TH17) cells in many tumors. The purpose of this study was to test if IL-17 and TNF-α may synergistically induce PD-L1 expression in human prostate cancer LNCaP and human colon cancer HCT116 cell lines. We found that IL-17 did not induce PD-L1 mRNA expression, but up-regulated PD-L1 protein expression in HCT116 and LNCaP cells. TNF-α induced PD-L1 mRNA and protein expression in both cell lines. Neither IL-17 nor TNF-α induced PD-L2 mRNA or protein expression. IL-17 and TNF-α acted individually rather than cooperatively in induction of PD-L1 expression. IL-17 and/or TNF-α activated AKT, nuclear factor-κB (NF-κB), and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathways in HCT116 cells, whereas only NF-κB signaling was activated in LNCaP cells. NF-κB inhibitor could diminish PD-L1 protein expression induced by IL-17 and/or TNF-α in both HCT116 and LNCaP cell lines. ERK1/2 inhibitor could also reduce PD-L1 protein expression induced by IL-17 and/or TNF-α in HCT116 cells, while AKT inhibitor could abolish PD-L1 protein expression induced by IL-17 and/or TNF-α in LNCaP cells. These results suggest that IL-17 and TNF-α act individually rather than cooperatively through activation of NF-κB and ERK1/2 signaling to up-regulate PD-L1 expression in HCT116 cells, while the two inflammatory cytokines act through activation of NF-κB signaling, in the presence of AKT activity, to up-regulate PD-L1 expression in LNCaP cells. PMID:28223102

The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

PubMed Central

Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

2009-01-01

Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

Dihydroavenanthramide D inhibits human breast cancer cell invasion through suppression of MMP-9 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Young-Rae; Noh, Eun-Mi; Oh, Hyun Ju

2011-02-25

Research highlights: {yields} MMP-9 plays a pivotal role in the invasion of MCF-7 breast cancer cells. {yields} TPA stimulates MMP-9 expression through activation of MAPK/NF-{kappa}B and MAPK/AP-1 pathways. {yields} Dihydroavenanthramide D suppresses MMP-9 expression via inhibition of TPA-induced MAPK/NF-{kappa}B and MAPK/AP-1 activations. {yields} Dihydroavenanthramide D blocks cell invasion of MCF-7 breast cancer cells. -- Abstract: Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Previous study demonstrates that DHAvD strongly inhibits activation of nuclear factor-kappa B (NF-{kappa}B), which is a major component in cancer cell invasion. The present study investigated whethermore » DHAvD can modulate MMP-9 expression and cell invasion in MCF-7 human breast cancer cells. MMP-9 expression and cell invasion in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased, whereas these inductions were muted by DHAvD. DHAvD also suppressed activation of mitogen-activated protein kinase (MAPK), and MAPK-mediated nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1) activations in TPA-treated MCF-7 cells. The results indicate that DHAvD-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the MAPK/NF-{kappa}B and MAPK/AP-1 pathways in MCF-7 cells. DHAvD may have potential value in breast cancer metastasis.« less

Gab3 is required for human colorectal cancer cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Shihao; Wang, Na; Hui, Pingping

Here, we focused on the potential function of Gab3, an uncommon Gab family protein, in human colorectal cancer (CRC) cells. We found that Gab3 was only expressed in human colon cancer tissues as well as in established (HCT-116 and HT-29 lines) and primary human CRC cells. It was however absent in normal human colon cancer tissues and in FHC colon epithelial cells. Knockdown of Gab3 by targeted-shRNAs inhibited proliferation of the CRC cells. Reversely, exogenous over-expression of Gab3 promoted CRC cell proliferation. At the signaling level, Gab3 co-precipitated with p85 and SHP2 in CRC cells, which was required for subsequentmore » Akt and Erk activation. Gab3 shRNA knockdown inhibited Akt and Erk activation, yet Gab3 over-expression augmented it. In vivo, HCT-116 xenograft tumor growth in severe combined immune deficient (SCID) mice was suppressed following expressing Gab3 shRNAs. Meanwhile, Akt and Erk activation in Gab3 shRNA-expressing tumors was also largely inhibited. Together, our results suggest that Gab3 expression in CRC cells is important for Akt-Erk activation and cell proliferation. - Highlights: • Gab3 is only expressed in colorectal cancer (CRC) cells, but not in colon epithelial cells. • Gab3 shRNA knockdown inhibits CRC cell proliferation. • Exogenous over-expression of Gab3 promotes HCT-116 cell proliferation. • Gab3 co-precipitates with p85 and SHP2 to mediate Akt and Erk activation in CRC cells. • HCT-116 tumor growth in SCID mice is suppressed with expression of Gab3 shRNAs.« less

Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

PubMed

Jiang, Hong Ning; Li, Yuan; Jiang, Wen Yi; Cui, Zong Jie

2018-01-01

Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm -2 ) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a "neglected" IL-2low Bimhigh phenotype, poor CTL function and cell death.

PubMed

Holz, Lauren E; Benseler, Volker; Vo, Michelle; McGuffog, Claire; Van Rooijen, Nico; McCaughan, Geoffrey W; Bowen, David G; Bertolino, Patrick

2012-10-01

The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

PubMed

Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

2016-03-01

T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

Autophagy promotes metastasis and glycolysis by upregulating MCT1 expression and Wnt/β-catenin signaling pathway activation in hepatocellular carcinoma cells.

PubMed

Fan, Qing; Yang, Liang; Zhang, Xiaodong; Ma, Yingbo; Li, Yan; Dong, Lei; Zong, Zhihong; Hua, Xiangdong; Su, Dongming; Li, Hangyu; Liu, Jingang

2018-01-19

Autophagy is a dynamic physiological process that can generate energy and nutrients for cell survival during stress. Autophagy can regulate the migration and invasive ability in cancer cells. However, the connection between autophagy and metabolism is unclear. Monocarboxylate transporter 1 (MCT1) plays an important role in lactic acid transport and H + clearance in cancer cells, and Wnt/β-catenin signaling can increase cancer cell glycolysis. We investigated whether autophagy promotes glycolysis in hepatocellular carcinoma (HCC) cells by activating the Wnt/β-catenin signaling pathway, accompanied by MCT1 upregulation. Autophagic activity was evaluated using western blotting, immunoblotting, and transmission electron microscopy. The underlying mechanisms of autophagy activation on HCC cell glycolysis were studied via western blotting, and Transwell, lactate, and glucose assays. MCT1 expression was detected using quantitative reverse transcription-PCR (real-time PCR), western blotting, and immunostaining of HCC tissues and the paired adjacent tissues. Autophagy promoted HCC cell glycolysis accompanied by MCT1 upregulation. Wnt/β-catenin signaling pathway activation mediated the effect of autophagy on HCC cell glycolysis. β-Catenin downregulation inhibited the autophagy-induced glycolysis in HCC cells, and reduced MCT1 expression in the HCC cells. MCT1 was highly expressed in HCC tissues, and high MCT1 expression correlated positively with the expression of microtubule-associated protein light chain 3 (LC3). Activation of autophagy can promote metastasis and glycolysis in HCC cells, and autophagy induces MCT1 expression by activating Wnt/β-catenin signaling. Our study describes the connection between autophagy and glucose metabolism in HCC cells and may provide a potential therapeutic target for HCC treatment.

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

PubMed

Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

2009-02-01

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells.

PubMed

Park, Song Yi; Shin, Jee-Hye; Kee, Sun-Ho

2017-09-01

β-Catenin is a central player in Wnt signaling, and activation of Wnt signaling is associated with cancer development. E-cadherin in complex with β-catenin mediates cell-cell adhesion, which suppresses β-catenin-dependent Wnt signaling. Recently, a tumor-suppressive role for E-cadherin has been reconsidered, as re-expression of E-cadherin was reported to enhance the metastatic potential of malignant tumors. To explore the role of E-cadherin, we established an E-cadherin-expressing cell line, EC96, from AGS cells that featured undetectable E-cadherin expression and a high level of Wnt signaling. In EC96 cells, E-cadherin re-expression enhanced cell proliferation, although Wnt signaling activity was reduced. Subsequent analysis revealed that nuclear factor-κB (NF-κB) activation and consequent c-myc expression might be involved in E-cadherin expression-mediated cell proliferation. To facilitate rapid proliferation, EC96 cells enhance glucose uptake and produce ATP using both mitochondria oxidative phosphorylation and glycolysis, whereas AGS cells use these mechanisms less efficiently. These events appeared to be mediated by NF-κB activation. Therefore, E-cadherin re-expression and subsequent induction of NF-κB signaling likely enhance energy production and cell proliferation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

PubMed

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-10-17

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells

PubMed Central

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-01-01

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. PMID:29156827

Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Harato, Misako; Feng, Guo-Gang; Ishikawa, Naoshisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression. PMID:24466034

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Ping; Jiang Xiaohong; Arcasoy, Murat O.

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutivelymore » active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.« less

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PubMed

Nickel, Sabrina; Selo, Mohammed Ali; Fallack, Juliane; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

2017-12-01

Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

Colorectal cancer cells suppress CD4+ T cells immunity through canonical Wnt signaling.

PubMed

Sun, Xuan; Liu, Suoning; Wang, Daguang; Zhang, Yang; Li, Wei; Guo, Yuchen; Zhang, Hua; Suo, Jian

2017-02-28

Understanding how colorectal cancer escapes from immunosurveillance and immune attack is important for developing novel immunotherapies for colorectal cancer. In this study we evaluated the role of canonical Wnt signaling in the regulation of T cell function in a mouse colorectal cancer model. We found that colorectal cancer cells expressed abundant Wnt ligands, and intratumoral T cells expressed various Frizzled proteins. Meanwhile, both active β-catenin and total β-catenin were elevated in intratumoral T cells. In vitro study indicated that colorectal cancer cells suppressed IFN-γ expression and increased IL-17a expression in activated CD4+ T cells. However, the cytotoxic activity of CD8+ T cells was not altered by colorectal cancer cells. To further evaluate the importance of Wnt signaling for CD4+ T cell-mediated cancer immunity, β-catenin expression was enforced in CD4+ T cells using lentiviral transduction. In an adoptive transfer model, enforced expression of β-catenin in intratumoral CD4+ T cells increased IL-17a expression, enhanced proliferation and inhibited apoptosis of colorectal cancer cells. Taken together, our study disclosed a new mechanism by which colorectal cancer impairs T cell immunity.

Gastrin regulates ABCG2 to promote the migration, invasion and side populations in pancreatic cancer cells via activation of NF-κB signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Juan; Xin, Beibei; Wang, Hui

Gastrin is absent in most normal adult pancreatic tissues but is highly expressed in pancreatic cancer tissues. Although Gastrin expression was reported to be associated with tumor proliferation in human pancreatic cancer, studies on the relationship between Gastrin and tumor metastasis in pancreatic cancer are rare. In this study, we performed an analysis to determine the effects of Gastrin on modulating the side populations, cell proportion and tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Gastrin and ABCG2 were widely expressed in pancreatic cancer cell lines and overexpressed in cancer tissues.more » Gastrin induced ABCG2 expression, and this effect was mediated by NF-κB activation. Gastrin regulated the SP proportion of BxPC-3 cells via modulating ABCG2 expression. Through the regulation of the functions of NF-κB/ABCG2, Gastrin functionally promoted the migration and invasion in pancreatic cancer cell. The present study indicated that Gastrin induced ABCG2 expression by activating NF-κB and thereby modulated the SP proportion, tumor cell metastatic potential and invasion activity in pancreatic cancer. Gastrin could serve as an effective therapeutic target for the metastasis of pancreatic cancer. - Highlights: • Gastrin induces ABCG2 expression mediated by NF-κB activation. • Gastrin regulates NF-κB's function that binds to the ABCG2 promoter in BxPC-3 cells. • Gastrin promotes the SP proportion in BxPC-3 cells by modulating ABCG2 expression via activation of NF-κB molecule. • Gastrin induces an increase in migration and invasion potential in pancreatic cancer cell by regulating NF-κB/ABCG2 signaling.« less

Relevance of P-glycoprotein on CXCR4+ B cells to organ manifestation in highly active rheumatoid arthritis.

PubMed

Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Kawabe, Akio; Tanaka, Yoshiya

2018-03-01

In rheumatoid arthritis (RA), P-glycoprotein (P-gp) expression on activated B cells is associated with active efflux of intracellular drugs, resulting in drug resistance. CXCR4 is associated with migration of B cells. This study was designed to elucidate the relevance of P-gp expression on CXCR4 + B cells to clinical manifestations in refractory RA. CD19 + B cells were analyzed using flow cytometry and immunohistochemistry. P-gp was highly expressed especially on CXCR4 + CD19 + B cells in RA. The proportion of P-gp-expressing CXCR4 + B cells correlated with disease activity, estimated by Simplified Disease Activity Index (SDAI), and showed marked expansion in RA patients with high SDAI and extra-articular involvement. In highly active RA, massive infiltration of P-gp + CXCR4 + CD19 + B cells was noted in CXCL12-expressing inflammatory lesions of RA synovitis and RA-associated interstitial pneumonitis. In RA patient with active extra-articular involvement, intracellular dexamethasone level (IDL) in lymphocytes diminished with expansion of P-gp + CXCR4 + CD19 + B cells. Adalimumab reduced P-gp + CXCR4 + CD19 + B cells, increased IDL in lymphocytes, and improved the clinical manifestation and allowed tapering of concomitant medications. Expansion of P-gp + CXCR4 + B cells seems to be associated with drug resistance, disease activity and progressive destructive arthritis with extra-articular involvement in RA.

Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling

PubMed Central

Saldanha-Araujo, Felipe; Haddad, Rodrigo; de Farias, Kelen C R Malmegrim; Souza, Alessandra de Paula Alves; Palma, Patrícia V; Araujo, Amélia G; Orellana, Maristela D; Voltarelli, Julio C; Covas, Dimas T; Zago, Marco A; Panepucci, Rodrigo A

2012-01-01

Abstract Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling. PMID:21777379

PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

PubMed

Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

2001-10-11

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

PubMed

Stephen, Tom L; Payne, Kyle K; Chaurio, Ricardo A; Allegrezza, Michael J; Zhu, Hengrui; Perez-Sanz, Jairo; Perales-Puchalt, Alfredo; Nguyen, Jenny M; Vara-Ailor, Ana E; Eruslanov, Evgeniy B; Borowsky, Mark E; Zhang, Rugang; Laufer, Terri M; Conejo-Garcia, Jose R

2017-01-17

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity. Copyright © 2017 Elsevier Inc. All rights reserved.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin, E-mail: Jqin710@vip.sina.com

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-addedmore » active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.« less

Unripe Rubus coreanus Miquel suppresses migration and invasion of human prostate cancer cells by reducing matrix metalloproteinase expression.

PubMed

Kim, Yesl; Lee, Seung Min; Kim, Jung-Hyun

2014-01-01

Rubus coreanus Miquel (RCM) is used to promote prostate health and has been shown to have anti-oxidant and anti-carcinogenic activities. However, the effects and mechanisms of RCM on prostate cancer metastasis remain unclear. PC-3 and DU 145 cells were treated with ethanol or water extract of unripe or ripe RCM and examined for cell invasion, migration, and matrix metalloproteinases (MMPs) activity and expression. Phosphoinositide 3-kinase (PI3K) and Akt activities were examined. Unripe RCM extracts exerted significant inhibitory effects on cell migration, invasion, and MMPs activities. A significant reduction in MMPs activities by unripe RCM ethanol extract treatment (UE) was associated with reduction of MMPs expression and induction of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, PI3K/Akt activity was diminished by UE treatment. In this study, we demonstrated that UE decreased metastatic potential of prostate cancer cells by reducing MMPs expression through the suppression of PI3K/Akt phosphorylation, thereby decreasing MMP activity and enhancing TIMPs expression.

The role of annexin A1 in expression of matrix metalloproteinase-9 and invasion of breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Hyereen; Ko, Jesang; Jang, Sung-Wuk, E-mail: swjang@amc.seoul.kr

2012-06-22

Highlights: Black-Right-Pointing-Pointer We evaluated the effect of ANXA1 on promoting migration and invasion in MDA-MB-231 cells. Black-Right-Pointing-Pointer ANXA1 siRNA inhibits invasion and migration. Black-Right-Pointing-Pointer ANXA1 regulates MMP-9 expression and activity. Black-Right-Pointing-Pointer ANX-1 siRNA inhibits the activation of NF-{kappa}B in MDA-MB-231 cells. -- Abstract: Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. However, the regulatory mechanism of MMP-9 expression and its biological effects on breast cancer development remain obscure. In the current study, we examined the potential role of annexin A1 (ANXA1) in regulating migration and invasion in breast cancer cell lines. Both ANXA1more » mRNA and protein are expressed in the highly invasive, hormone-insensitive human breast cancer cell lines MDA-MB-231 and SKBr3, but not in the hormone-responsive cell lines MCF-7 and T47D. Downregulation of ANXA1 expression with specific small interfering RNAs (ANXA1 siRNA) in MDA-MB-231 cells resulted in decreased cancer cell migration and invasion. Ablation of ANXA1 expression decreases the expression of MMP-9 at both the mRNA and protein levels and also reduces the proteolytic activity of MMP-9 in MDA-MB-231 cells. Moreover, silencing ANXA1 also decreases the transcriptional activity of MMP-9 by the suppression of nuclear factor kappa-B (NF-{kappa}B) activity. Collectively, these results indicate that ANXA1 functions as a positive regulator of MMP-9 expression and invasion of breast cancer cells through specific activation of the NF-{kappa}B signaling pathway.« less

Analysis of immune activation and clinical events in acute infectious mononucleosis.

PubMed

Williams, Hilary; Macsween, Karen; McAulay, Karen; Higgins, Craig; Harrison, Nadine; Swerdlow, Anthony; Britton, Kate; Crawford, Dorothy

2004-07-01

The symptoms of infectious mononucleosis (IM) are thought to be caused by T cell activation and cytokine production. Surface lymphocyte activation marker (SLAM)-associated protein (SAP) regulates lymphocyte activation via signals from cell-surface CD244 (2B4) and SLAM (CD150). We followed T cell activation via this SAP/SLAM/CD244 pathway in IM and analyzed whether the results were associated with clinical severity. At diagnosis, SAP, SLAM, and CD244 were significantly up-regulated on CD4 and CD8 T cells; expression decreased during IM, but CD244 and SLAM levels remained higher on CD8 cells 40 days later. There were significantly more lymphocytes expressing CD8 and CD244/CD8 in patients with severe sore throat. The expression of CD8 alone and CD244 on CD8 cells correlated with increased virus load. We suggest that T cells expressing CD244 and SLAM are responsible for the clinical features of IM but that the control of activation is maintained by parallel increased expression of SAP.

Altering calcium influx for selective destruction of breast tumor.

PubMed

Yu, Han-Gang; McLaughlin, Sarah; Newman, Mackenzie; Brundage, Kathleen; Ammer, Amanda; Martin, Karen; Coad, James

2017-03-04

Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

SVEP1 is a novel marker of activated pre-determined skeletal muscle satellite cells.

PubMed

Shefer, Gabi; Benayahu, Dafna

2010-03-01

In this study we explored the expression pattern of SVEP1, a novel cell adhesion molecule (CAM), in bona fide satellite cells and their immediate progeny. We show that SVEP1 is expressed in activated satellite cells prior to their determination to the myogenic lineage. SVEP1 was also expressed during early phases of myogenic differentiation through the initial stage of myoblast fusion and myotube formation. The expression of SVEP1 was shown by immunostaining two cell culture systems: freshly isolated myofibers and primary myoblasts. Pax7 was used to pinpoint satellite cells situated in their niche on myofibers, and activated satellite cells were determined based on BrdU incorporation (Pax7(+)/BrdU(+)cells). MyoD marked satellite cells fated to undergo myogenesis as well as proliferating and differentiating myoblasts. Differentiating myoblasts and myotubes were identified based on their sarcomeric myosin expression. We showed that SVEP1 was specifically expressed in pre-determined activated satellite cells (Pax7(+)/ BrdU(+) /MyoD(-)) accounting for about 24% of total satellite cells. On the other hand, SVEP1 expression was absent in quiescent satellite cells (Pax7(+)/BrdU(-)/MyoD(-)). Moreover, based on MyoD/sarcomeric myosin co-expression SVEP1 was shown to be expressed throughout the early phases of myogenesis up until myoblast fusion and myotube formation. A decline in SVEP1 expression occurred upon myotube maturation. We suggest SVEP1 as a potential biomarker for pre-fated satellite cells. The impact of this finding is that it may allow scrutinizing signals that affect differentiation commitment. Thus, holds a therapeutic potential for maladies that involve deregulated stem cell fate-decision.

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell–Derived Factor-1α–Mediated Stellate Cell Activation

PubMed Central

Hong, Feng; Tuyama, Ana; Lee, Ting Fang; Loke, Johnny; Agarwal, Ritu; Cheng, Xin; Garg, Anita; Fiel, M. Isabel; Schwartz, Myron; Walewski, Jose; Branch, Andrea; Schecter, Alison D.; Bansal, Meena B.

2010-01-01

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell–derived factor-1α (SDF-1α), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1α/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1α is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1α increases expression of α-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1α/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. Conclusion HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1α is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1α–induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches. PMID:19434726

Dominant expression of interleukin-10 and transforming growth factor-beta genes in activated T-cells of chronic active Epstein-Barr virus infection.

PubMed

Ohga, Shouichi; Nomura, Akihiko; Takada, Hidetoshi; Tanaka, Tamami; Furuno, Kenji; Takahata, Yasushi; Kinukawa, Naoko; Fukushima, Noriyasu; Imai, Shosuke; Hara, Toshiro

2004-11-01

Chronic active Epstein-Barr virus (EBV) infection is a chronic mononucleosis syndrome associated with clonal proliferation of EBV-carrying T-/natural killer (NK)-cells. High levels of circulating EBV and activated T-cells are sustained during the prolonged disease course, whereas it is not clear how ectopic EBV infection in T-/NK-cells has been established and maintained. To assess the biological role of activated T-cells in chronic active EBV infection (CAEBV), EBV DNA and cellular gene expressions in peripheral T-cells were quantified in CAEBV and infectious mononucleosis (IM) patients. In CAEBV, HLA-DR(+) T-cells had higher viral load and larger amounts of IFN gamma, IL-10, transforming growth factor-beta (TGF beta), and cytotoxic T lymphocyte antigen-4 (CTLA4) mRNA than HLA-DR(-)T-cells. HLA-DR(+) T cells of IM patients transcribed more IFN gamma and IL-10 than their HLA-DR(-)T cells. Expression levels of IFN gamma and forkhead box p3 (Foxp3) in CAEBV HLA-DR(+) T-cells were higher than in IM HLA-DR(+) T-cells. The effective variables to discriminate the positivity of HLA-DR were IL-10, IFN gamma, CTLA4, TGF beta, and IL-2 in the order of statistical weight. EBV load in CAEBV T-cells correlated with the expression levels of only IL-10 and TGF beta. These results suggest that CAEBV T-cells are activated to transcribe IFN gamma, IL-10, and TGF beta excessively, and the latter two genes are expressed preferentially in the EBV-infected subsets. The dominant expression of regulatory cytokines in T-cells may imply a viral evasion mechanism in the disease.

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakuci, Enkeleda; Mahner, Sven; DiRenzo, James

2006-10-01

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ER{alpha} signaling. However, many ER{alpha}-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ER{alpha} signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ER{alpha}-negative cells. We previously noticed that both ER{alpha}-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ER{alpha}-negative cell lines even exceeded its over-expression level inmore » ER{alpha}-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ER{alpha}-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.« less

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells.

PubMed Central

Kondoh, N.; Yamada, T.; Kihara-Negishi, F.; Yamamoto, M.; Oikawa, T.

1998-01-01

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9743289

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

PubMed

Detry, C; Lamour, V; Castronovo, V; Bellahcène, A

2008-02-01

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

Identification of an inducible regulator of c-myb expression during T-cell activation.

PubMed Central

Phan, S C; Feeley, B; Withers, D; Boxer, L M

1996-01-01

Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation. PMID:8628306

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

PubMed

Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

2000-04-01

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

PubMed Central

2012-01-01

Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

The bile acid receptor GPBAR1 (TGR5) is expressed in human gastric cancers and promotes epithelial-mesenchymal transition in gastric cancer cell lines

PubMed Central

Cipriani, Sabrina; Marchianò, Silvia; Marino, Elisabetta; Zampella, Angela; Rende, Mario; Mosci, Paolo; Distrutti, Eleonora; Donini, Annibale; Fiorucci, Stefano

2016-01-01

GPBAR1 (also known as TGR5) is a bile acid activated receptor expressed in several adenocarcinomas and its activation by secondary bile acids increases intestinal cell proliferation. Here, we have examined the expression of GPBAR1 in human gastric adenocarcinomas and investigated whether its activation promotes the acquisition of a pro-metastatic phenotype. By immunohistochemistry and RT-PCR analysis we found that expression of GPBAR1 associates with advanced gastric cancers (Stage III-IV). GPBAR1 expression in tumors correlates with the expression of N-cadherin, a markers of epithelial-mesenchymal transition (EMT) (r=0.52; P<0.01). Expression of GPBAR1, mRNA and protein, was detected in cancer cell lines, with MKN 45 having the higher expression. Exposure of MKN45 cells to GPBAR1 ligands, TLCA, oleanolic acid or 6-ECDCA (a dual FXR and GPBAR1 ligand) increased the expression of genes associated with EMT including KDKN2A, HRAS, IGB3, MMP10 and MMP13 and downregulated the expression of CD44 and FAT1 (P<0.01 versus control cells). GPBAR1 activation in MKN45 cells associated with EGF-R and ERK1 phosphorylation. These effects were inhibited by DFN406, a GPBAR1 antagonist, and cetuximab. GPBAR1 ligands increase MKN45 migration, adhesion to peritoneum and wound healing. Pretreating MKN45 cells with TLCA increased propensity toward peritoneal dissemination in vivo. These effects were abrogated by cetuximab. In summary, we report that GPBAR1 is expressed in advanced gastric cancers and its expression correlates with markers of EMT. GPBAR1 activation in MKN45 cells promotes EMT. These data suggest that GPBAR1 antagonist might have utility in the treatment of gastric cancers. PMID:27409173

Inflammatory cytokines IL-17 and TNF-α up-regulate PD-L1 expression in human prostate and colon cancer cells.

PubMed

Wang, Xun; Yang, Lingyun; Huang, Feng; Zhang, Qiuyang; Liu, Sen; Ma, Lin; You, Zongbing

2017-04-01

Programmed cell death protein 1 (PD-1) acts on PD-1 ligands (PD-L1 and PD-L2) to suppress activation of cytotoxic T lymphocytes. Interleukin-17 (IL-17) and tumor necrosis factor-α (TNF-α) are co-expressed by T helper 17 (T H 17) cells in many tumors. The purpose of this study was to test if IL-17 and TNF-α may synergistically induce PD-L1 expression in human prostate cancer LNCaP and human colon cancer HCT116 cell lines. We found that IL-17 did not induce PD-L1 mRNA expression, but up-regulated PD-L1 protein expression in HCT116 and LNCaP cells. TNF-α induced PD-L1 mRNA and protein expression in both cell lines. Neither IL-17 nor TNF-α induced PD-L2 mRNA or protein expression. IL-17 and TNF-α acted individually rather than cooperatively in induction of PD-L1 expression. IL-17 and/or TNF-α activated AKT, nuclear factor-κB (NF-κB), and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathways in HCT116 cells, whereas only NF-κB signaling was activated in LNCaP cells. NF-κB inhibitor could diminish PD-L1 protein expression induced by IL-17 and/or TNF-α in both HCT116 and LNCaP cell lines. ERK1/2 inhibitor could also reduce PD-L1 protein expression induced by IL-17 and/or TNF-α in HCT116 cells, while AKT inhibitor could abolish PD-L1 protein expression induced by IL-17 and/or TNF-α in LNCaP cells. These results suggest that IL-17 and TNF-α act individually rather than cooperatively through activation of NF-κB and ERK1/2 signaling to up-regulate PD-L1 expression in HCT116 cells, while the two inflammatory cytokines act through activation of NF-κB signaling, in the presence of AKT activity, to up-regulate PD-L1 expression in LNCaP cells. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Pro-Inflammatory Activated Kupffer Cells by Lipids Induce Hepatic NKT Cells Deficiency through Activation-Induced Cell Death

PubMed Central

Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Background Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. Aims The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Methods Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. Results High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. Conclusion High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD. PMID:24312613

Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent mu to gamma1 class switch recombination in CD38-activated B cells.

PubMed

Tsukamoto, Yumiko; Uehara, Shoji; Mizoguchi, Chieko; Sato, Atsushi; Horikawa, Keisuke; Takatsu, Kiyoshi

2005-10-21

Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to mu to gamma1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce mu to gamma1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces mu to gamma1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce mu to gamma1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce mu to gamma1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR.

Granulysin-Expressing CD4+ T Cells as Candidate Immune Marker for Tuberculosis during Childhood and Adolescence

PubMed Central

Mueller, Henrik; Faé, Kellen C.; Magdorf, Klaus; Ganoza, Christian A.; Wahn, Ulrich; Guhlich, Ute; Feiterna-Sperling, Cornelia; Kaufmann, Stefan H. E.

2011-01-01

Background Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB. Methods Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4+ CD45RO+ memory T cells. Results CD4+ CD45RO+ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSElowCD4+CD45RO+) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4+ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells. Conclusions Our data suggest granulysin expression by CD4+ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence. PMID:22216262

Role of Bruton’s tyrosine kinase in myeloma cell migration and induction of bone disease

PubMed Central

Bam, Rakesh; Ling, Wen; Khan, Sharmin; Pennisi, Angela; Venkateshaiah, Sathisha Upparahalli; Li, Xin; van Rhee, Frits; Usmani, Saad; Barlogie, Bart; Shaughnessy, John; Epstein, Joshua; Yaccoby, Shmuel

2014-01-01

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton’s tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL) –6– or stroma–dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. PMID:23456977

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression.

PubMed

Borgerding, Andrea; Hasenkamp, Justin; Engelke, Michael; Burkhart, Nina; Trümper, Lorenz; Wienands, Jürgen; Glass, Bertram

2010-03-01

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells. A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)-defined NK cells from human leukocyte antigen (HLA)-typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay. HLA, but not CD20 expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications. KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA. Copyright 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

AMPK inhibits MTDH expression via GSK3β and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

PubMed

Gollavilli, Paradesi Naidu; Kanugula, Anantha Koteswararao; Koyyada, Rajeswari; Karnewar, Santosh; Neeli, Praveen Kumar; Kotamraju, Srigiridhar

2015-10-01

Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3β (GSK3β) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3β and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3β) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3β and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells. © 2015 FEBS.

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

PubMed

Frazao, Alexandra; Colombo, Marina; Fourmentraux-Neves, Emmanuelle; Messaoudene, Meriem; Rusakiewicz, Sylvie; Zitvogel, Laurence; Vivier, Eric; Vély, Frédéric; Faure, Florence; Dréno, Brigitte; Benlalam, Houssem; Bouquet, Fanny; Savina, Ariel; Pasmant, Eric; Toubert, Antoine; Avril, Marie-Françoise; Caignard, Anne

2017-07-01

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF -mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR . ©2017 American Association for Cancer Research.

The role of transient receptor potential vanilloid type-2 ion channels in innate and adaptive immune responses

PubMed Central

Santoni, Giorgio; Farfariello, Valerio; Liberati, Sonia; Morelli, Maria B.; Nabissi, Massimo; Santoni, Matteo; Amantini, Consuelo

2013-01-01

The transient receptor potential vanilloid type-2 (TRPV2), belonging to the transient receptor potential channel family, is a specialized ion channel expressed in human and other mammalian immune cells. This channel has been found to be expressed in CD34+ hematopoietic stem cells, where its cytosolic Ca2+ activity is crucial for stem/progenitor cell cycle progression, growth, and differentiation. In innate immune cells, TRPV2 is expressed in granulocytes, macrophages, and monocytes where it stimulates fMet-Leu-Phe migration, zymosan-, immunoglobulin G-, and complement-mediated phagocytosis, and lipopolysaccharide-induced tumor necrosis factor-alpha and interleukin-6 production. In mast cells, activation of TRPV2 allows intracellular Ca2+ ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56+ natural killer cells. TRPV2 orchestrates Ca2+ signal in T cell activation, proliferation, and effector functions. Moreover, messenger RNA for TRPV2 are expressed in CD4+ and CD8+ T lymphocytes. Finally, TRPV2 is expressed in CD19+ B lymphocytes where it regulates Ca2+ release during B cell development and activation. Overall, the specific expression of TRPV2 in immune cells suggests a role in immune-mediated diseases and offers new potential targets for immunomodulation. PMID:23420671

Peroxisome proliferator-activated receptor (PPAR)-gamma expression in human vascular smooth muscle cells: inhibition of growth, migration, and c-fos expression by the peroxisome proliferator-activated receptor (PPAR)-gamma activator troglitazone.

PubMed

Benson, S; Wu, J; Padmanabhan, S; Kurtz, T W; Pershadsingh, H A

2000-01-01

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells.

PubMed

Liao, Kai-Sheng; Wei, Chia-Li; Chen, Jyh-Cheng; Zheng, Hao-Yu; Chen, Wen-Ching; Wu, Chia-Hung; Wang, Tai-Jing; Peng, Yi-Shuan; Chang, Po-Yuan; Lin, Yun-Wei

2016-11-01

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Costimulation dependent expression of miR-214 increases the ability of T cells to proliferate by targeting Pten

PubMed Central

Jindra, Peter T.; Bagley, Jessamyn; Godwin, Jonathan G.; Iacomini, John

2010-01-01

T cell activation requires signaling through the T cell receptor (TCR) and costimulatory molecules such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally and are also known to be involved in lymphocyte development and function. Here we set out to examine potential roles of miRNAs in T cell activation by using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs up-regulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Up-regulation of miR-214 in T cells inversely correlated with PTEN levels. In vivo, transcripts containing the 3' untranslated region (3' UTR) of Pten including the miR-214 target sequence were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 up-regulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation dependent up-regulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is in part related to its ability to regulate expression of miRNAs that control T cell activation. PMID:20548023

Novel cell-cell signaling by microglial transmembrane TNFα with implications for neuropathic pain

PubMed Central

Zhou, Zhigang; Peng, Xiangmin; Hagshenas, Jafar; Insolera, Ryan; Fink, David J.; Mata, Marina

2010-01-01

Neuropathic pain is accompanied by neuroimmune activation in dorsal horn of spinal cord. We have observed that in animal models this activation is characterized by increased expression of transmembrane tumor necrosis factor α (mTNFα) without release of soluble (sTNFα). Here we report that the pain-related neurotransmitter peptide substance P (SP) increases expression of mTNFα without release of sTNFα from primary microglial cells. We modeled this interaction using an immortalized microglial cell line; exposure of these cells to SP also resulted in increased expression of mTNFα but without any increase in expression of the TNF-cleaving enzyme (TACE) and no release of sTNFα. In order to evaluate the biological function of uncleaved mTNFα, we transfected COS-7 cells with a mutant full length TNFα construct resistant to cleavage by TACE. Co-culture of COS-7 cells expressing the mutant TNFα with microglial cells led to microglial cell activation indicated by increased OX-42 immunoreactivity and release of macrophage chemoattractant peptide 1 (CCL2) by direct cell-cell contact. These results suggest a novel pathway through which release of SP by primary afferents activates microglial expression of mTNFα, establishing a feed-forward loop that may contribute to the establishment of chronic pain. PMID:20609516

CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus.

PubMed

Dudzinska, Dominika; Luzak, Boguslawa; Boncler, Magdalena; Rywaniak, Joanna; Sosnowska, Dorota; Podsedek, Anna; Watala, Cezary

2014-09-01

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1-15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

PubMed Central

Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

PubMed

Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

2007-07-15

(-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

Leukotriene B4 induces EMT and vimentin expression in PANC-1 pancreatic cancer cells: Involvement of BLT2 via ERK2 activation.

PubMed

Kim, You Ri; Park, Mi Kyung; Kang, Gyeong Jin; Kim, Hyun Ji; Kim, Eun Ji; Byun, Hyun Jung; Lee, Moo-Yeol; Lee, Chang Hoon

2016-12-01

Leukotriene B 4 (LTB 4 ) is a leukocyte chemoattractant and plays a major role controlling inflammatory responses including pancreatitis. LTB 4 is known to be correlated with cancer progression. LTB 4 induces keratin phosphorylation and reorganization by activating extracellular regulated kinase (ERK) in PANC-1 pancreatic cancer cell lines. However, the role of LTB 4 in epithelial mesenchymal transition (EMT) and vimentin expression in pancreatic cancer cells is unknown. We examined whether LTB 4 induces EMT and vimentin expression by Western blot, si-RNA, and RT-PCR. LTB 4 induced morphological change, decreased E-cadherin expression and increased N-cadherin and vimentin expression. LTB4 increased migration and invasion of PANC-1 cancer cells. LTB 4 dose-dependently upregulated expression of vimentin in PANC-1 cancer cells. LTB 4 -induced vimentin expression was suppressed by LY255283 (BLT2 antagonist). Comp A, a BLT2 agonist, further increased vimentin expression. Gene silencing of BLT2 suppressed LTB 4 -or Comp A-induced vimentin expression in PANC-1 cells. The MEK inhibitor, PD98059 suppressed Comp A-induced vimentin expression. Comp A or transfection of plasmid containing BLT2 cDNA (pC BLT2 ) activated ERK, and BLT2 gene silencing suppressed Comp A-induced ERK activation. ERK2 siRNA abrogated Comp A-induced vimentin expression and ERK2 overexpression enhanced vimentin expression. One of well-known cause of ras mutation, cigarette smoke extracts increased BLT2 expression in PANC-1 cancer cells. Taken together, these results suggest that BLT2 is involved in LTB 4 -induced vimentin expression through ERK2 in PANC-1 cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reconstitution of a secondary cell wall in a secondary cell wall-deficient Arabidopsis mutant.

PubMed

Sakamoto, Shingo; Mitsuda, Nobutaka

2015-02-01

The secondary cell wall constitutes a rigid frame of cells in plant tissues where rigidity is required. Deposition of the secondary cell wall in fiber cells contributes to the production of wood in woody plants. The secondary cell wall is assembled through co-operative activities of many enzymes, and their gene expression is precisely regulated by a pyramidal cascade of transcription factors. Deposition of a transmuted secondary cell wall in empty fiber cells by expressing selected gene(s) in this cascade has not been attempted previously. In this proof-of-concept study, we expressed chimeric activators of 24 transcription factors that are preferentially expressed in the stem, in empty fiber cells of the Arabidopsis nst1-1 nst3-1 double mutant, which lacks a secondary cell wall in fiber cells, under the control of the NST3 promoter. The chimeric activators of MYB46, SND2 and ANAC075, as well as NST3, reconstituted a secondary cell wall with different characteristics from those of the wild type in terms of its composition. The transgenic lines expressing the SND2 or ANAC075 chimeric activator showed increased glucose and xylose, and lower lignin content, whereas the transgenic line expressing the MYB46 chimeric activator showed increased mannose content. The expression profile of downstream genes in each transgenic line was also different from that of the wild type. This study proposed a new screening strategy to identify factors of secondary wall formation and also suggested the potential of the artificially reconstituted secondary cell walls as a novel raw material for production of bioethanol and other chemicals. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

An AKT activity threshold regulates androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines.

PubMed

Paliouras, Miltiadis; Diamandis, Eleftherios P

2008-06-01

The androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancer in vitro model. Using the cell line PC3(AR)(2), we stimulated cells with 5-alpha-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)(2) cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose an in vitro androgen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

PubMed

Kang, Lianguo; Zhang, Xintian; Xie, Yan; Tu, Yaping; Wang, Dong; Liu, Zhenming; Wang, Zhao-Yi

2010-04-01

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Activation status of mucosal-associated invariant T cells reflects disease activity and pathology of systemic lupus erythematosus.

PubMed

Chiba, Asako; Tamura, Naoto; Yoshikiyo, Kazunori; Murayama, Goh; Kitagaichi, Mie; Yamaji, Ken; Takasaki, Yoshinari; Miyake, Sachiko

2017-03-14

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes constituting a large proportion of peripheral blood T cells expressing αβ T-cell receptor in humans. In this study, we aimed to investigate their involvement in systemic lupus erythematosus (SLE). Peripheral blood MAIT cells from patients with SLE were assessed for their frequency, activation markers, and cell death by flow cytometry. The correlation between plasma cytokine levels and CD69 expression on MAIT cells was analyzed. The major histocompatibility complex class I-related protein MR1-restricted antigen-presenting capacity of antigen-presenting cells was investigated. Cytokine-mediated activation of MAIT cells in the absence of exogenous antigens was also examined. The frequency of MAIT cells was markedly reduced in SLE. The reduced number of MAIT cells was not attributable to the downregulation of surface markers, but it was partially due to the enhanced cell death of MAIT cells, possibly by activation-induced cell death. The CD69 expression levels on MAIT cells in SLE correlated with disease activity. Moreover, monocytes from patients with SLE exhibited increased ability to induce MAIT cell activation. The plasma concentration of interleukin (IL)-6, IL-18, and interferon (IFN)-α positively correlated with the expression levels of CD69 on MAIT cells in SLE. MAIT cells were activated by cytokines, including IFN-α, IL-15, and IL-12 plus IL-18, in the absence of exogenous antigens. These results suggest that MAIT cells reflect the pathological condition of SLE and that their activated status correlates with presence of disease.

Stimulatory role of interleukin 10 in CD8+ T cells through STATs in gastric cancer.

PubMed

Xi, Jianjun; Xu, Mingzheng; Song, Zongchang; Li, Hongqiang; Xu, Shumin; Wang, Chunmei; Song, Haihan; Bai, Jianwen

2017-05-01

CD8 + T cells are considered to be critical in tumor surveillance and elimination. Increased CD8 + T cell frequency and function is associated with better prognosis in cancer patients. Interleukin 10 is a cytokine with controversial roles in CD8 + T cell-mediated anti-tumor immunity. We therefore examined the interleukin 10 expression and consumption in CD8 + T cells harvested from the peripheral blood and resected tumors of gastric cancer patients of stages II-IV. We found that the gastric cancer patients presented significantly elevated frequencies of interleukin 10-expressing cells in both CD4 + and CD8 + T cells compared to healthy controls. But distinctive from the interleukin 10-expressing CD4 + T cells, which increased in frequency in advanced cancer, the interleukin 10-expressing CD8 + T cells did not increase with cancer stage in the peripheral blood and actually decreased with cancer stage in resected tumor. Interleukin 10 and interleukin 10 receptor expression was also enriched in interferon gamma-expressing activated CD8 + T cells. Compared to interleukin 10-nonexpressing CD8 + T cells, interleukin 10 receptor-expressing CD8 + T cells secreted significantly elevated interferon gamma levels. Treatment of anti-CD3/CD28-stimulated, purified CD8 + T cells with interleukin 10 alone could significantly enhance CD8 + T cell survival, an effect dependent on interleukin 10 receptor expression. Interleukin 10 also increased CD8 + T cell proliferation synergistically with interferon gamma but not alone. Analysis of downstream signal transducer and activator of transcription molecules showed that interleukin 10 treatment significantly increased the phosphorylation of signal transducer and activator of transcription 3 and signal transducer and activator of transcription 1 to lesser extent. Together, these results demonstrate that interleukin 10 possessed stimulatory roles in activated CD8 + T cells from gastric cancer patients.

Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK/AMPK Activation in Bupivacaine-Treated Neuro2a Cells

PubMed Central

Huang, Lei; Kondo, Fumio; Gosho, Masahiko; Feng, Guo-Gang; Harato, Misako; Xia, Zhong-yuan; Ishikawa, Naohisa; Fujiwara, Yoshihiro; Okada, Shoshiro

2014-01-01

We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. PMID:24859235

Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity.

PubMed

Montero, Juan Carlos; López-Pérez, Ricardo; San Miguel, Jesús F; Pandiella, Atanasio

2008-06-01

c-Kit is expressed in the plasma cells from 30% of patients with multiple myeloma. Two different isoforms of c-Kit, characterized by the presence or absence of the tetrapeptide sequence GNNK in the extracellular domain, have been described. However, their expression and function in myeloma cells are unknown. We explored the function and expression of these c-Kit isoforms in myeloma cells. Expression of c-Kit isoforms was investigated by reverse transcriptase polymerase chain reaction in fresh plasma cells from patients and cell lines. The function of these c-Kit isoforms was analyzed upon expression in myeloma cells. Signaling was investigated by western blotting using antibodies specific for activated forms of several signaling proteins. The impact of c-Kit on the action of drugs commonly used in the treatment of multiple myeloma was investigated by MTT proliferation assays. Fresh plasma cells from patients as well as myeloma cell lines expressed the two isoforms of c-Kit. Retroviral infection of myeloma cells with vectors that code for c-Kit-GNNK+ or c-Kit-GNNK- forms demonstrated differences in the kinetics of phosphorylation between these isoforms. Stem cell factor-induced activation of the GNNK- form was faster and more pronounced than that of the GNNK+ form, whose activation, however, lasted for longer. The c-Kit receptors weakly activated the Erk1/2 and Erk5 pathways. Both receptors, however, efficiently coupled to the PI3K/Akt pathway, and stimulated p70S6K activation. The latter was sensitive to the mTOR inhibitor, rapamycin. Studies of drug sensitivity indicated that cells expressing the GNNK- form were more resistant to the anti-myeloma action of bortezomib and melphalan. Our data indicate that c-Kit expression in multiple myeloma cells is functional, and coupled to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.

Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells.

PubMed

Gordon, Jonathan A R; Sodek, Jaro; Hunter, Graeme K; Goldberg, Harvey A

2009-08-15

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF. (c) 2009 Wiley-Liss, Inc.

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent anti-tumor activity

PubMed Central

Caruso, Hillary G.; Hurton, Lenka V.; Najjar, Amer; Rushworth, David; Ang, Sonny; Olivares, Simon; Mi, Tiejuan; Switzer, Kirsten; Singh, Harjeet; Huls, Helen; Lee, Dean A.; Heimberger, Amy B.; Champlin, Richard E.; Cooper, Laurence J. N.

2015-01-01

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells. PMID:26330164

Activated but not resting T cells or thymocytes express colony-stimulating factor 1 mRNA without co-expressing c-fms mRNA.

PubMed

Cerdan, C; Courcoul, M; Razanajaona, D; Pierrès, A; Maroc, N; Lopez, M; Mannoni, P; Mawas, C; Olive, D; Birg, F

1990-02-01

Following the observation that, besides acute myeloid leukemia cells, acute lymphoid leukemia cells of either B or T phenotype could express the transcript for the colony-stimulating factor 1 (CSF-1), a growth factor known to be restricted to the monocytic-macrophage lineage, various sources of resting and/or activated T cells and thymocytes were screened for expression of this hemopoietic growth factor. We report here that the CSF-1 transcript was rapidly (7 h) induced in T cells by a variety of stimuli, but was not detectable in either resting T cells or thymocytes. In addition, secretion of CSF-1 was detectable in the supernatants of activated T cells by 72 h, with a peak around 92-120 h. In contrast to activated monocytes, the transcript of the c-fms proto-oncogene, the product of which is the receptor for CSF-1, was not detectable in either resting or activated T cells. This observation could be relevant to the intimate relationships between T cells and antigen-presenting cells during immune responses.

Activation of Toll-like receptor-9 promotes cellular migration via up-regulating MMP-2 expression in oral squamous cell carcinoma.

PubMed

Ruan, Min; Zhang, Zun; Li, Siyi; Yan, Min; Liu, Shengwen; Yang, Wenjun; Wang, Lizheng; Zhang, Chenping

2014-01-01

Activation of Toll like receptors (TLRs) signaling has been implicated in promoting malignant cell invasion and metastatic potential. Previously we demonstrated that increased TLR-9 expression predicted poor survival in oral cancer patients. The objective of this study is to further investigate the roles and potential molecular mechanisms of TLR-9 signaling in human oral cancer cell invasion. Cell migration, invasion and protein expression were detected by wound healing assay, Transwell chambers model and western blot. The secretion and activity levels of metalloproteinases-2/9 were quantified by ELISA and Gelatin zymography. EMSA and ChIP assays were employed to detect the activity of AP-1signal pathway. TLR-9 siRNA transfection was used to regulate the expression and activity of TLR-9 in oral cancer cell line HB cells. The results of both wound healing assay and in vitro Transwell assay revealed that activation of TLR-9 induced dose- and time- dependent migration and invasion of HB cells. An increased expression, secretion and activity of MMP-2 were observed upon the treatment of CpG-ODN. The TLR-9 signaling-mediated MMP-2 expression appeared to be a consequence of AP-1 activation, because that their DNA binding activity was enhanced by CpG-ODN treatment. All these influences were efficiently repressed by the knockdown of TLR-9 through siRNA or pretreatment of an AP-1 inhibitor. Activation of TLR-9 signaling could promote human oral cancer HB cells invasion with the induction of MMP-2 presentation by attenuating AP-1 binding activity, suggesting a novel anti-metastatic application for TLR-9 targeted therapy in oral cancer in the future.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yilin; Yang, Yang; Cai, Yanyan

Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. Inmore » this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.« less

Nickel-induced down-regulation of {Delta}Np63 and its role in the proliferation of keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Zhuo, E-mail: zhuo.zhang@uky.edu; Li Wenqi; Cheng Senping

2011-06-15

Epidemiological, animal, and cell studies have demonstrated that nickel compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. p63, a close homologue of the p53 tumor suppressor protein, has been linked to cell fate determination and/or maintenance of self-renewing populations in several epithelial tissues, including skin, mammary gland, and prostate. {Delta}Np63, a dominant negative isoform of p63, is amplified in a variety of epithelial tumors including squamous cell carcinomas and carcinomas of the prostate and mammary glands. The present study shows that nickel suppressed {Delta}Np63 expression in a short-time treatment (up to 48 h). Nickelmore » treatment caused activation of NF-{kappa}B. Blockage of NF-{kappa}B partially reversed nickel-induced {Delta}Np63 suppression. Nickel decreased interferon regulatory factor (IRF) 3 and IRF7, IKK{epsilon}, and Sp100. Over-expression of IRF3 increased {Delta}Np63 expression suppressed by nickel. Nickel was able to activate p21, and its activation was offset by the over-expression of {Delta}Np63. In turn, elevated p63 expression counteracted the ability of nickel to restrict cell growth. The present study demonstrated that nickel decreased interferon regulatory proteins IRF3 and IRF7, and activated NF-{kappa}B, resulting in {Delta}Np63 suppression and then p21 up-regulation. {Delta}Np63 plays an important role in nickel-induced cell proliferation. - Highlights: > Ni suppressed {Delta}Np63 expression in HaCat cells. > Ni activated NF-{kappa}B, decreased expressions of IRF3 and IRF7, IKK{epsilon}, and Sp100. > Over-expression of IRF3 increased {Delta}Np63 expression suppressed by Ni. > Ni activated p21, and its activation was offset by over-expression of {Delta}Np63. > Elevated p63 expression counteracted the ability of nickel to restrict cell growth.« less

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

PubMed Central

2013-01-01

Background The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. Methods The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. Results ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. Conclusions The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior. PMID:23648148

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

PubMed Central

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

PubMed

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Friend spleen focus-forming virus transforms rodent fibroblasts in cooperation with a short form of the receptor tyrosine kinase Stk

PubMed Central

Nishigaki, Kazuo; Hanson, Charlotte; Jelacic, Tanya; Thompson, Delores; Ruscetti, Sandra

2005-01-01

Friend spleen focus-forming virus (SFFV) causes rapid erythroleukemia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator erythropoietin (Epo) because of constitutive activation of Epo signal transduction pathways. Although SFFV infects many cell types, deregulation of cell growth occurs only when SFFV infects erythroid cells, suggesting that these cells express unique proteins that the virus requires to mediate its biological effects. Not only do erythroid cells express the Epo receptor (EpoR), but those from mice susceptible to SFFV-induced erythroleukemia also express a short form of the receptor tyrosine kinase Stk (sf-Stk). In erythroid cells, SFFV gp55 interacts with the EpoR complex and sf-Stk, leading to activation of the kinase and constitutive activation of signal transducing molecules. In this study, we demonstrate that SFFV gp55 can also deregulate the growth of nonerythroid cells when it is coexpressed with sf-Stk. Expression of SFFV gp55 in rodent fibroblasts engineered to express sf-Stk induced their transformation, as demonstrated by focus formation and anchorage-independent growth in vitro. This transformation by SFFV gp55 requires the kinase activity of sf-Stk and the presence of its extracellular domain but not expression of the EpoR or the tyrosine kinase Jak2, which is required for activation of signal transduction pathways through the EpoR. Thus, expression of SFFV gp55 in nonerythroid cells coexpressing sf-Stk results in their uncontrolled growth, demonstrating a previously unrecognized mechanism for retrovirus transformation of rodent fibroblasts and providing insight into SFFV-induced disease. PMID:16223879

Polymethoxylated flavones potentiate the cytolytic activity of NK leukemia cell line KHYG-1 via enhanced expression of granzyme B.

PubMed

Saito, Takeshi; Abe, Daigo; Nogata, Yoichi

2015-01-16

Polymethoxylated flavones (PMFs) are found in the peel tissues of some citrus species. Here, we report that PMFs, such as nobiletin, potentiate the cytolytic activity of KHYG-1 natural killer (NK) leukemia cells. Nobiletin markedly enhanced the expression of granzyme B, a serine protease that plays critical roles in the cytolytic activity of NK cells. The potentiated cytolytic activity induced by nobiletin was canceled by the granzyme B inhibitor Z-AAD-CMK. Nobiletin also increased the levels of phosphorylated CREB, ERK1/2, and p38 MAPK in KHYG-1 cells, which are known to participate in NK cell function. Inhibition of an upstream kinase of ERK1/2 failed to reduce the granzyme B expression and KHYG-1 cytolytic activity. Meanwhile, inhibition of p38 MAPK attenuated both granzyme B expression and KHYG-1 cytolytic activity. These results suggest that the primary role of nobiletin in KHYG-1 cytolytic activity lies in upregulation of granzyme B expression, at least in part, mediated through p38 MAPK function. Copyright © 2014 Elsevier Inc. All rights reserved.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

PubMed Central

Lai, Jian-Ping; Lai, Saien; Tuluc, Florin; Tansky, Morris F.; Kilpatrick, Laurie E.; Leeman, Susan E.; Douglas, Steven D.

2008-01-01

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-κB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-κB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCδ but inhibited phosphorylation of PKCδ in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1–2 min), whereas truncated NK1R-mediated activation was slower (peak at 20–30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists. PMID:18713853

Viral antigen mediated NKp46 activation of NK cells results in tumor rejection via NK-DC crosstalk

PubMed Central

Chinnery, Fay; King, Catherine A.; Elliott, Tim; Bateman, Andrew R.; James, Edward

2012-01-01

Natural killer (NK) cells play a critical role in antitumor immunity, their activation being regulated through NK cell receptors. Although the endogenous ligands for these receptors are largely unknown, viral ligands have been identified. We investigated the ability of an activating NK receptor ligand derived from the mumps virus, haemagglutinin-neuraminidase (HN) to enhance NK activation against tumor cells. HN-expressing B16.OVA tumor cells induced stronger activation of NK cells compared with B16.OVA cells and also promoted dendritic cell (DC) activation toward a DC1 phenotype, in vitro. Moreover, incubation of DCs, NK cells and HN-expressing B16-OVA cells further enhanced NK cell activation through the NK-DC crosstalk, in a cell-to-cell contact- and IL-12-dependent fashion. Immunization of mice with HN-expressing B16-OVA cells resulted in > 85% survival rate after subsequent challenge with parental B16 or B16.OVA tumor cells. Tumor rejection was dependent on both NK and CD8+ T cells but not on CD4+ T cells, demonstrating induction of an effective adaptive immune response through innate immune cell activation. Our data indicate the potential of using robust NK cell activation, which through the NK-DC crosstalk stimulates effective antitumor responses, providing an alternate vaccine strategy. PMID:23162755

Expression of Tlx in both stem cells and transit amplifying progenitors regulates stem cell activation and differentiation in the neonatal lateral subependymal zone.

PubMed

Obernier, Kirsten; Simeonova, Ina; Fila, Tatiana; Mandl, Claudia; Hölzl-Wenig, Gabriele; Monaghan-Nichols, Paula; Ciccolini, Francesca

2011-09-01

Niche homeostasis in the postnatal subependymal zone of the lateral ventricle (lSEZ) requires coordinated proliferation and differentiation of neural progenitor cells. The mechanisms regulating this balance are scarcely known. Recent observations indicate that the orphan nuclear receptor Tlx is an intrinsic factor essential in maintaining this balance. However, the effect of Tlx on gene expression depends on age and cell-type cues. Therefore, it is essential to establish its expression pattern at different developmental ages. Here, we show for the first time that in the neonatal lSEZ activated neural stem cells (NSCs) and especially transit-amplifying progenitors (TAPs) express Tlx and that its expression may be regulated at the posttranscriptional level. We also provide evidence that in both cell types Tlx affects gene expression in a positive and negative manner. In activated NSCs, but not in TAPs, absence of Tlx leads to overexpression of negative cell cycle regulators and impairment of proliferation. Moreover, in both cell types, the homeobox transcription factor Dlx2 is downregulated in the absence of Tlx. This is paralleled by increased expression of Olig2 in activated NSCs and glial fibrillary acidic protein in TAPs, indicating that in both populations Tlx decreases gliogenesis. Consistent with this, we found a higher proportion of cells expressing glial makers in the neonatal lSEZ of mutant mice than in the wild type counterpart. Thus, Tlx playing a dual role affects the expression of distinct genes in these two lSEZ cell types. Copyright © 2011 AlphaMed Press.

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

PubMed Central

Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

2014-01-01

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

Human T-lymphotropic virus type I tax regulates the expression of the human lymphotoxin gene.

PubMed

Tschachler, E; Böhnlein, E; Felzmann, S; Reitz, M S

1993-01-01

Human T-lymphotropic virus type-I (HTLV-I)-infected T-cell lines constitutively produce high levels of lymphotoxin (LT). To analyze the mechanisms that lead to the expression of LT in HTLV-I-infected cell lines, we studied regulatory regions of the human LT promoter involved in the activation of the human LT gene. As determined by deletional analysis, sequences between +137 and -116 (relative to the transcription initiation site) are sufficient to direct expression of a reporter gene in the HTLV-I-infected cell line MT-2. Site-directed mutation of a of the single kappa B-like motif present in the LT promoter region (positions -99 to -89) completely abrogated LT promoter activity in MT-2 cells, suggesting that this site plays a critical role in the activation of the human LT gene. Transfection of LT constructs into HTLV-I-uninfected and -unstimulated Jurkat and U937 cell lines showed little to no activity of the LT promoter. Cotransfection of the same constructs with a tax expression plasmid into Jurkat cells led to detectable promoter activity, which could be significantly increased by stimulation of the cells with phorbol myristate acetate (PMA). Similarly, cotransfection of the LT promoter constructs and the tax expression plasmid into U937 cells led to significant promoter activity upon stimulation with PMA. These data suggest that HTLV-I tax is involved in the upregulation of LT gene expression in HTLV-I-infected cells.

Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Mi Hee; Min, Do Sik, E-mail: minds@pusan.ac.kr

Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety ofmore » cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.« less

Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

PubMed Central

Cole, Sara L.; Dagg, Rebecca A.; Lau, Loretta M. S.; Duncan, Emma L.; Moy, Elsa L.; Reddel, Roger R.

2012-01-01

Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT. PMID:23185534

Tax-Independent Constitutive IκB Kinase Activation in Adult T-Cell Leukemia Cells1

PubMed Central

Hironaka, Noriko; Mochida, Kanako; Mori, Naoki; Maeda, Michiyuki; Yamamoto, Naoki; Yamaoka, Shoji

2004-01-01

Abstract Adult T-cell leukemia (ATL) is a fatal T-cell malignancy that arises long after infection with human T-cell leukemia virus type I (HTLV-I). We reported previously that nuclear factor-κB (NF-κB) was constitutively activated in ATL cells, although expression of the viral proteins was barely detectable, including Tax, which was known to persistently activate NF-κB. Here we demonstrate that ATL cells that do not express detectable Tax protein exhibit constitutive IκB kinase (IKK) activity. Transfection studies revealed that a dominant-negative form of IKK1, and not of IKK2 or NF-κB essential modulator (NEMO), suppressed constitutive NFκB activity in ATL cells. This IKK activity was accompanied by elevated expression of p52, suggesting that the recently described noncanonical pathway of NF-κB activation operates in ATL cells. We finally show that specific inhibition of NF-κB by a super-repressor form of IκBα (SR-IκBα) in HTLV-I-infected T cells results in cell death regardless of Tax expression, providing definitive evidence of an essential role for NF-κB in the survival of ATL cells. In conclusion, the IKK complex is constitutively activated in ATL cells through a cellular mechanism distinct from that of Tax-mediated IKK activation. Further elucidation of this cellular mechanism should contribute to establishing a rationale for treatment of ATL. PMID:15153339

Comparison of NSAIDs activity in COX-2 expressing and non-expressing 2D and 3D pancreatic cancer cell cultures

PubMed Central

Čeponytė, Ugnė; Paškevičiūtė, Miglė; Petrikaitė, Vilma

2018-01-01

Purpose In this study, we evaluated the anticancer activity of non-steroidal anti-inflammatory drugs (NSAIDs) in BxPC-3 and MIA PaCa-2 pancreatic cancer cell cultures. Methods To test the effect of compounds on the viability of cells, the MTT assay was used. The activity of NSAIDs in 3D cell cultures was evaluated by measuring the size change of spheroids. The type of cell death was identified by cell staining with Hoechst 33342 and propidium iodide. To evaluate the effect on the colony-forming ability of cancer cells, the clonogenic assay was used. Results Five out of seven tested NSAIDs reduced the viability of BxPC-3 and MIA PaCa-2 cancer cells. Fenamates were more active against cyclooxygenase-2 expressing BxPC-3 than cyclooxygenase-2 non-expressing MIA PaCa-2 cell line. Fenamates and coxibs exerted higher activity in monolayer cultured cells, whereas salicylates were more active in 3D cultures. Fenamates and coxibs induced dose-dependent apoptosis and necrosis. NSAIDs also inhibited the colony-forming ability of cancer cells. Meclofenamic acid, niflumic acid, and parecoxib possessed higher activity on BxPC-3, and celecoxib possessed higher activity on MIA PaCa-2 cell colony formation. Conclusion Our results show that fenamates, coxibs, and salicylates possess anticancer activity on human pancreatic cancer BxPC-3 and MIA PaCa-2 cell cultures. PMID:29942156

CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

PubMed

Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

2006-05-01

We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

Activated CD4+ and CD8+ cells in the colonic mucosa of ulcerative colitis patients: their relationship to HLA-DR antigen expression on the colonic epithelium and serum soluble CD25 levels.

PubMed

Sasakawa, T; Takizawa, H; Bannai, H; Narisawa, R; Asakura, H

1995-01-01

This study was performed to clarify the relationship between activated (HLA-DR-expressing) CD4+ and CD8+ cells in the colonic lamina propria of ulcerative colitis and other immunological factors, i.e., epithelial DR expression, serum soluble CD25 levels, and colonic mucosal CD25+ cells. The frequency of epithelial DR expression was positively correlated with the numbers of CD4+ and CD8+ cells. The percentages activated CD4+/CD4+ cells were higher in mucosae with DR- epithelium than in mucosae with DR+ epithelium. The serum soluble CD25 levels were increased in ulcerative colitis, and there was an inverse correlation between these levels and the relative number of activated CD4+ cells in untreated active disease. These results suggest that interactions among mucosal CD4+ cells, colonic epithelium, and serum soluble CD25 might play an important role in the pathogenesis of ulcerative colitis.

Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wenjie; Zhang, Xiaomei, E-mail: zhangxm667@163.com; Lu, Hong

2013-05-10

Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cellmore » HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.« less

Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye.

PubMed

Johnston, Robert J; Otake, Yoshiaki; Sood, Pranidhi; Vogt, Nina; Behnia, Rudy; Vasiliauskas, Daniel; McDonald, Elizabeth; Xie, Baotong; Koenig, Sebastian; Wolf, Reinhard; Cook, Tiffany; Gebelein, Brian; Kussell, Edo; Nakagoshi, Hideki; Desplan, Claude

2011-06-10

How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification. Copyright © 2011 Elsevier Inc. All rights reserved.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

PubMed

Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

2015-05-01

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

A cell-ELISA for the quantification of adherent murine macrophages and the surface expression of antigens.

PubMed

Nibbering, P H; Van de Gevel, J S; Van Furth, R

1990-07-20

The present study was performed in order to establish whether a cell-ELISA could be used to determine the expression of antigens by adherent murine peritoneal macrophages and also quantify the numbers of such macrophages. Accurate determination of the number of adherent macrophages proved to be possible with a cell-ELISA designed to assess complement receptor type III (CRIII) expression. Expression of CRIII was considerably more sensitive than determination of the cell-protein or DNA content as a measure of the number of adherent macrophages. For the calculation of the expression of CRIII, Ia antigen, and antigen F4/80 by resident and activated macrophages, use was made of the linear part of the curve obtained when the numbers of macrophages were plotted against the absorbance values for each of the antigens. The values for CRIII expression did not differ significantly between resident macrophages, macrophages activated with recombinant interferon-gamma (rIFN-gamma) and macrophages activated with BCG/PPD. IFN-gamma-activated and BCG/PPD-activated macrophages expressed Ia antigen significantly more intensely than did resident peritoneal macrophages. In contrast the activated macrophages expressed F4/80 significantly less intensely than resident peritoneal macrophages.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines.

PubMed

Nagakawa, O; Murakami, K; Yamaura, T; Fujiuchi, Y; Murata, J; Fuse, H; Saiki, I

2000-07-31

Membrane-type metalloproteinase-1 (MT1-MMP) is a transmembrane metalloproteinase, which activates proMMP-2 and expressed on the cell surface in many invasive cancer cells. We investigated the expression of MT1-MMP in prostate cancer cell lines. MT1-MMP protein and mRNA were expressed in PC-3, DU-145 and TSU-pr1 cells (androgen-independent prostate cancer cell lines), but in LNCaP cells (androgen-dependent prostate cancer cell line). MT1-MMP protein was negative and mRNA was low to detect by RT-PCR. Cell lysate of PC-3 cleaved proMMP-2 to the active form. In addition, both hepatocyte growth factor (HGF) and gastrin-releasing peptide (GRP) increased Matrigel invasion and induced the expression of MT1-MMP protein in DU-145 prostate cancer cells. These results suggest that MT1-MMP is indeed the tumor-specific activator of proMMP-2 in androgen-independent prostate cancer cells and plays an important role in the invasive properties of prostate cancer cells.

Inhibition of Survivin Influences the Biological Activities of Canine Histiocytic Sarcoma Cell Lines

PubMed Central

Hoshino, Yuki; Hosoya, Kenji; Okumura, Masahiro

2013-01-01

Canine histiocytic sarcoma (CHS) is an aggressive malignant neoplasm that originates from histiocytic lineage cells, including dendritic cells and macrophages, and is characterized by progressive local infiltration and a very high metastatic potential. Survivin is as an apoptotic inhibitory factor that has major functions in cell proliferation, including inhibition of apoptosis and regulation of cell division, and is expressed in most types of human and canine malignant neoplasms, including melanoma and osteosarcoma. To investigate whether survivin was expressed at high levels in CHS and whether its expression was correlated with the aggressive biological behavior of CHS, we assessed relation between survivin expression and CHS progression, as well as the effects of survivin inhibition on the biological activities of CHS cells. We comparatively analyzed the expression of 6 selected anti-apoptotic genes, including survivin, in specimens from 30 dogs with histiocytic sarcoma and performed annexin V staining to evaluate apoptosis, methylthiazole tetrazolium assays to assess cell viability and chemosensitivity, and latex bead assays to measure changes in phagocytic activities in 4 CHS cell lines and normal canine fibroblasts transfected with survivin siRNA. Survivin gene expression levels in 30 specimens were significantly higher than those of the other 6 genes. After transfection with survivin siRNA, apoptosis, cell growth inhibition, enhanced chemosensitivity, and weakened phagocytic activities were observed in all CHS cell lines. In contrast, normal canine fibroblasts were not significantly affected by survivin knockdown. These results suggested that survivin expression may mediate the aggressive biological activities of CHS and that survivin may be an effective therapeutic target for the treatment of CHS. PMID:24260303

RIPK3 expression in cervical cancer cells is required for PolyIC-induced necroptosis, IL-1α release, and efficient paracrine dendritic cell activation.

PubMed

Schmidt, Susanne V; Seibert, Stefanie; Walch-Rückheim, Barbara; Vicinus, Benjamin; Kamionka, Eva-Maria; Pahne-Zeppenfeld, Jennifer; Solomayer, Erich-Franz; Kim, Yoo-Jin; Bohle, Rainer M; Smola, Sigrun

2015-04-20

Previous studies have shown that cervical cancer cells only release low levels of pro-inflammatory cytokines owing to infection with human papillomaviruses. This results in low immunogenicity of the cancer cells. The viral dsRNA analog PolyIC has been suggested as a promising adjuvant for cervical cancer immunotherapy. However, little is known about the molecular requirements resulting in successful immune activation. Here, we demonstrate that stimulation of cervical cancer cells with PolyIC induced necroptotic cell death, which was strictly dependent on the expression of the receptor-interacting protein kinase RIPK3. Necroptotic cancer cells released interleukin-1α (IL-1α), which was required for powerful activation of dendritic cells (DC) to produce IL-12, a cytokine critical for anti-tumor responses. Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells. Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas. In summary, our study identified a novel RIPK3-dependent mechanism that explains how PolyIC-treatment of cervical cancer cells leads to potent DC activation. Our findings suggest that the RIPK3 expression status in cervical cancer cells might critically influence the outcome of PolyIC-based immunotherapeutic approaches and should therefore be assessed prior to immunotherapy.

Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

PubMed Central

Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

2016-01-01

The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

P16/p53 expression and telomerase activity in immortalized human dental pulp cells

PubMed Central

Egbuniwe, Obi; Idowu, Bernadine D; Funes, Juan M; Grant, Andrew D; Renton, Tara

2011-01-01

Introduction Residing within human dental pulp are cells of an ectomesenchymal origin that have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). Results With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN and OPN), as did nDPSCs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed, as well as low telomerase activity readings. Methods Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured, and their telomerase activities were determined using a telomerase quantification assay. Also examined, were the expression of genes involved in proliferation and mineralization, such as human alkaline phosphatase (ALP), β-actin, collagen I (col-1), core binding factor (cbfa)-1, dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphate activity was also assayed in both cell groups. Conclusion These results indicate maintenance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression. PMID:22067611

Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*

PubMed Central

G. Lavoie, Elise; Dranoff, Jonathan A.

2017-01-01

Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression. PMID:28898276

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells

PubMed Central

Freund, Ariane; Jolivel, Valérie; Durand, Sébastien; Kersual, Nathalie; Chalbos, Dany; Chavey, Carine; Vignon, Françoise; Lazennec, Gwendal

2004-01-01

We have recently reported that Interleukin-8 (IL-8) expression was inversely correlated to estrogen-receptor (ER)-status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-κB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knock-down of NF-κB and AP-1 activities by adenovirus-mediated expression of a NF-κB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPα and C/EBPβ expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-κB, AP-1 and C/EBP transcription factors. PMID:15208657

A role for RNA post-transcriptional regulation in satellite cell activation

PubMed Central

2012-01-01

Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation. PMID:23046558

Induced Expression of Rnd3 Is Associated with Transformation of Polarized Epithelial Cells by the Raf–MEK–Extracellular Signal-Regulated Kinase Pathway†

PubMed Central

Hansen, Steen H.; Zegers, Mirjam M. P.; Woodrow, Melissa; Rodriguez-Viciana, Pablo; Chardin, Pierre; Mostov, Keith E.; McMahon, Martin

2000-01-01

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf–MEK–extracellular signal-regulated kinase pathway in epithelial oncogenesis. PMID:11094087

Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reimers, Kerstin; Buchholz, Katja; Werchau, Hermann

2005-01-20

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cellsmore » revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.« less

Enhanced expression by the brain matrix of P-glycoprotein in brain capillary endothelial cells.

PubMed

Tatsuta, T; Naito, M; Mikami, K; Tsuruo, T

1994-10-01

P-glycoprotein (PGP), an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in brain capillary endothelium and could be functionally involved in the blood-brain barrier. To study the regulatory mechanism of PGP expression in brain capillary endothelium, various mouse tissue matrices were tested for their abilities to enhance the expression of PGP in mouse brain capillary endothelial cells (MBEC), which express relatively small amounts of PGP. Of the four tissue matrices we examined, PGP expression in MBEC cultured on the brain matrix increased 2.0-fold. The PGP-inducing activity was similarly detected in bovine brain matrix, and the activity was enriched in the fraction of pl 9.0 by isoelectric focusing. The fraction, named PIC-fraction (PGP-inducing component), increased the PGP expression in MBEC 3.5-fold. By Northern blot analysis, a 3.3-fold enhancement of mdr gene expression was observed in MBEC cultured on the PIC-fraction. The PGP-inducing activity of the PIC-fraction was reduced by the treatment with trypsin but not with collagenase, suggesting that a proteinaceous factor distinct from type I collagen might be responsible for the PGP-inducing activity of PIC-fraction. Although the PIC-fraction increased the PGP expression in other mouse brain capillary endothelial cells, the PIC-fraction did not increase PGP expression in mouse aortic endothelial cells and KB carcinoma cell lines expressing various amounts of PGP. These observations suggest that PGP expression in brain capillary endothelium is specifically regulated by a tissue-specific factor in the brain matrix.

CD26 expression and adenosine deaminase activity in regulatory T cells (Treg) and CD4+ T effector cells in patients with head and neck squamous cell carcinoma

PubMed Central

Mandapathil, Magis; Szczepanski, Miroslaw; Harasymczuk, Malgorzata; Ren, Jin; Cheng, Dongmei; Jackson, Edwin K.; Gorelik, Elieser; Johnson, Jonas; Lang, Stephan; Whiteside, Theresa L

2012-01-01

Adenosine deaminase (ADA) is responsible for the deamination of immunosuppressive adenosine to inosine. In human T lymphocytes, ADA is associated with dipeptidyl peptidase IV (CD26). ADA expression and activity were evaluated in regulatory T cells (Treg) and CD4+ T effector cells (Teff) of patients with head and neck squamous cell cancer (HNSCC). CD4+CD39+ and CD4+CD39neg T cells were isolated by single-cell sorting from the peripheral blood of 15 HNSCC patients and 15 healthy donors (NC). CD26/ADA expression in these cells was studied by multicolor flow cytometry, confocal microscopy, RT-PCR and immunohistochemistry in tumor tissues. ADA activity was evaluated by mass spectrometry, suppression of Teff proliferation in CFSE assays and cytokine production by Luminex. CD4+CD39+ Treg had low and CD4+CD39neg Teff high CD26/ADA expression and ADA activity in NC or HNSCC. The frequency and suppressor activity of CD39+CD26neg Treg were elevated in patients relative to NC (p < 0.01). However, ADA activity in patients’ CD4+CD39neg Teff was decreased (p < 0.05), resulting in extracellular adenosine accumulation. Also, patients’ Teff were more sensitive to inhibitory signals delivered via adenosine receptors. IL-2, IL12 and INFγ upregulated ADA expression and activity in CD4+CD39neg Teff, whereas IL-10, PGE2 and CADO downregulated it. The differentially expressed CD26/ADA can serve as surface markers for functionally-active CD39+CD26neg Treg. PMID:22934258

COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells

PubMed Central

Litchfield, Lacey M.; Appana, Savitri N.; Datta, Susmita; Klinge, Carolyn M.

2016-01-01

Reduced COUP-TFII expression contributes to endocrine resistance in breast cancer cells. Endocrine-resistant breast cancer cells have higher NFkappa B (NFκB) activity and target gene expression. The goal of this study was to determine if COUP-TFII modulates NFκB activity. Endocrine-resistant LCC9 cells with low endogenous COUP-TFII displayed ~5-fold higher basal NFκB activity than parental endocrine-sensitive MCF-7 breast cancer cells. Transient transfection of LCC9 cells with COUP-TFII inhibited NFκB activation and reduced NFκB target gene expression. COUP-TFII and NFκB were inversely correlated in breast cancer patient samples. Endogenous COUP-TFII coimmunoprecipitated with NFκB subunits RelB and NFκB1 in MCF-7 cells. COUP-TFII inhibited NFκB-DNA binding in vitro and impaired coactivator induced NFκB transactivation. LCC9 cells were growth-inhibited by an NFκB inhibitor and 4-hydroxytamoxifen compared to MCF-7 cells. Together these data indicate a novel role for COUP-TFII in suppression of NFκB activity and explain, in part, why decreased COUP-TFII expression results in an endocrine-resistant phenotype. PMID:24141032

Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients1

PubMed Central

Jury, Elizabeth C.; Eldridge, Jillian; Isenberg, David A.; Kabouridis, Panagiotis S.

2008-01-01

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity. PMID:18025246

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells.

PubMed

Oike, Hideaki; Wakamori, Minoru; Mori, Yasuo; Nakanishi, Hiroki; Taguchi, Ryo; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

2006-09-01

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.

Tangeretin Inhibits IL-12 Expression and NF-κB Activation in Dendritic Cells and Attenuates Colitis in Mice.

PubMed

Eun, Su-Hyeon; Woo, Je-Te; Kim, Dong-Hyun

2017-04-01

In the preliminary study, tangeretin (5,6,7,8,4'-pentamethoxy flavone), a major constituent of the pericarp of Citrus sp., inhibited TNF- α , IL-12, and IL-23 expression and nuclear factor kappa-B activation in lipopolysaccharide-stimulated dendritic cells; however, it did not affect IL-10 expression. Furthermore, tangeretin (5, 10, and 20 µM) suppressed the activation and translocation of nuclear factor kappa-B (p65) into the nuclei in vitro by inhibiting the binding of lipopolysaccharide on dendritic cells. Oral administration of tangeretin (10 and 20 mg/kg) suppressed the inflammatory responses, such as nuclear factor kappa-B and mitogen-activated protein kinase activation and myeloperoxidase activity, in the colon of mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed expression of tight junction proteins occludin, claudin-1, and ZO-1. Tangeretin also inhibited 2,4,6-trinitrobenzene sulfonic acid-induced differentiation of Th1 and Th17 cells as well as the expression of T-bet, ROR γ t, interferon- γ , IL-12, IL-17, and TNF- α . However, tangeretin increased 2,4,6-trinitrobenzene sulfonic acid-suppressed differentiation of regulatory T cells as well as the expression of Foxp3 and IL-10. These results suggest that oral administration of tangeretin may attenuate colitis by suppressing IL-12 and TNF- α expression and nuclear factor kappa-B activation through the inhibition of lipopolysaccharide binding on immune cells such as dendritic cells. Georg Thieme Verlag KG Stuttgart · New York.

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

PubMed

Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation. These events are manifest in inflammatory diseases, such as atherosclerosis. Therefore, our results suggest that DBMSCs could be usefully employed as a therapeutic strategy for atherosclerosis.

African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

PubMed

Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2002-04-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

PubMed Central

Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

2002-01-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells.

PubMed

Kim, Hyung Gyun; Jin, Sun Woo; Kim, Yong An; Khanal, Tilak; Lee, Gi Ho; Kim, Se Jong; Rhee, Sang Dal; Chung, Young Chul; Hwang, Young Jung; Jeong, Tae Cheon; Jeong, Hye Gwang

2017-08-01

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E 2 . Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

PubMed

Knittel, T; Aurisch, S; Neubauer, K; Eichhorst, S; Ramadori, G

1996-08-01

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

PubMed

Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

2014-05-01

In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

PubMed

Pan, Yinbing; Sun, Xiaodi; Jiang, Lai; Hu, Liang; Kong, Hong; Han, Yuan; Qian, Cheng; Song, Chao; Qian, Yanning; Liu, Wentao

2016-11-17

Tolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine. The microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests. We found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice. Metformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.

Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong

2014-05-15

Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1more » expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.« less

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promotermore » activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.« less

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

Oncogenic activation of c-Abl in non-small cell lung cancer cells lacking FUS1 expression: inhibition of c-Abl by the tumor suppressor gene product Fus1.

PubMed

Lin, J; Sun, T; Ji, L; Deng, W; Roth, J; Minna, J; Arlinghaus, R

2007-10-25

In lung cancer, frequent loss of one allele of chromosome 3p is seen in both small cell lung cancer and non-small cell lung cancer (NSCLC), providing evidence of tumor suppressor genes (TSGs) in this chromosomal region. The mechanism of Fus1 tumor suppressor activity is unknown. We have found that a Fus1 peptide inhibits the Abl tyrosine kinase in vitro (IC(50) 35 microM). The inhibitory Fus1 sequence was derived from a region that was deleted in a mutant FUS1 gene (FUS1 (1-80)) detected in some lung cancer cell lines. Importantly, a stearic acid-modified form of this peptide was required for the inhibition, but stearic acid alone was not inhibitory. Two NSCLC cell lines, which lack expression of wild-type Fus1, contain activated c-Abl. Forced expression of an inducible FUS1 cDNA in H1299 NSCLC cells decreased levels of activated c-Abl and inhibited its tyrosine kinase activity. Similarly, treatment of c-Abl immune complexes with the inhibitory Fus1 peptide also reduced the level of c-Abl in these immune complexes. The size and number of colonies of the NSCLC cell line, H1,299, in soft agar was strongly inhibited by the Abl kinase inhibitor imatinib mesylate. Co-expression of FUS1 and c-ABL in COS1 cells blocked activation of c-Abl tyrosine kinase. In contrast, co-expression of mutant FUS1 (1-80) with c-ABL had little inhibitory activity against c-Abl. These findings provide strong evidence that c-Abl is a possible target in NSCLC patients that have reduced expression of Fus1 in their tumor cells.

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

PubMed

Arosa, F A; de Jesus, O; Porto, G; Carmo, A M; de Sousa, M

1999-06-11

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.

Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

PubMed

Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

2015-07-01

Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

PubMed

Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

2012-08-01

Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis

PubMed Central

Wormley, Floyd L.; Chaiban, Joseph; Fidel, Paul L.

2001-01-01

Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection. PMID:11447188

Aberrantly Expressed OTX Homeobox Genes Deregulate B-Cell Differentiation in Hodgkin Lymphoma.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2015-01-01

In Hodgkin lymphoma (HL) we recently reported that deregulated homeobox gene MSX1 mediates repression of the B-cell specific transcription factor ZHX2. In this study we investigated regulation of MSX1 in this B-cell malignancy. Accordingly, we analyzed expression and function of OTX homeobox genes which activate MSX1 transcription during embryonal development in the neural plate border region. Our data demonstrate that OTX1 and OTX2 are aberrantly expressed in both HL patients and cell lines. Moreover, both OTX loci are targeted by genomic gains in overexpressing cell lines. Comparative expression profiling and subsequent pathway modulations in HL cell lines indicated that aberrantly enhanced FGF2-signalling activates the expression of OTX2. Downstream analyses of OTX2 demonstrated transcriptional activation of genes encoding transcription factors MSX1, FOXC1 and ZHX1. Interestingly, examination of the physiological expression profile of ZHX1 in normal hematopoietic cells revealed elevated levels in T-cells and reduced expression in B-cells, indicating a discriminatory role in lymphopoiesis. Furthermore, two OTX-negative HL cell lines overexpressed ZHX1 in correlation with genomic amplification of its locus at chromosomal band 8q24, supporting the oncogenic potential of this gene in HL. Taken together, our data demonstrate that deregulated homeobox genes MSX1 and OTX2 respectively impact transcriptional inhibition of (B-cell specific) ZHX2 and activation of (T-cell specific) ZHX1. Thus, we show how reactivation of a specific embryonal gene regulatory network promotes disturbed B-cell differentiation in HL.

Upregulation of cathepsin C expression contributes to endothelial chymase activation in preeclampsia.

PubMed

Gu, Yang; Lewis, David F; Alexander, J Steven; Wang, Yuping

2017-12-01

Chymase is an ACE (angiotensin-converting enzyme)-independent angiotensin II-forming enzyme whose expression is increased in the maternal vascular endothelium in preeclampsia. However, mechanisms underlying chymase activation in preeclampsia remain unclear. Cathepsin C is a key enzyme in the activation of several serine proteases including chymase. In this study, we determined whether increased cathepsin C expression/activity might be responsible for the upregulation of chymase expression in preeclampsia. Maternal vascular cathepsin C, chymase and ACE expression were examined through immunohistochemical staining of subcutaneous fat tissue sections of normal and preeclamptic pregnant women. The role of cathepsin C in endothelial chymase and ACE expression was determined in cells treated with cathepsin C. Consequences of chymase activation were then determined by measurement of angiotensin II production in cells treated with the ACE inhibitor captopril and the chymase inhibitor chymostatin, separately and in combination. Expression of both cathepsin C and chymase, but not ACE expression, was markedly increased in the maternal vascular endothelium in subjects with preeclampsia compared with normal pregnant controls. Exogenous cathepsin C induced a dose-dependent increase in expression of mature cathepsin C and chymase, but not ACE, in endothelial cells. Moreover, angiotensin II production was significantly inhibited in cells treated with captopril or chymostatin alone and was further inhibited in cells treated with both inhibitors. These results suggest that cathepsin C upregulation induces chymase activation and subsequently promotes angiotensin II generation in endothelial cells. These data also provide evidence of upregulation of the cathepsin C-chymase-angiotensin signaling axis in maternal vasculature in preeclampsia.

1,25-dihydroxyvitamin D3 induces CCR10 expression in terminally differentiating human B cells.

PubMed

Shirakawa, Aiko-Konno; Nagakubo, Daisuke; Hieshima, Kunio; Nakayama, Takashi; Jin, Zhe; Yoshie, Osamu

2008-03-01

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

PubMed

Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

2017-07-03

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

PubMed

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

The role of human chorionic gonadotropin in regulation of naïve and memory T cells activity in vitro.

PubMed

Zamorina, S A; Litvinova, L S; Yurova, K A; Khaziakhmatova, O G; Timganova, V P; Bochkova, M S; Khramtsov, P V; Rayev, M B

2018-01-01

The role of human chorionic gonadotropin (hCG) in the regulation of molecular genetics factors determining the functional activity of human naïve and memory T cells in vitro was studied. It was found that hCG (10 and 100IU/ml) inhibited CD28 and CD25 expression on the naïve T cells (CD45RA+) and CD25 expression on the memory T cells (CD45R0+). hCG didn't affect the CD71 proliferation marker expression in total. Nevertheless, hCG reduced the percentage of proliferating memory T cells with simultaneous suppression of CD71 expression on proliferating CD45R0+cells. In parallel, expression of U2af1l4, Gfi1, and hnRNPLL genes, which are Ptprc gene alternative splicing regulators was evaluated. It was established that hCG stimulated the expression of U2af1l4 and hnRNPLL genes, responsible for the assembly of CD45R0 in memory T cells, but reduced the expression of Gfi1 in these cells. In general, hCG promotes the differentiation of memory T cells by increasing of CD45R0 expression, but inhibits proliferation and CD25 expression which reflects their functional activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Induction of Tissue Factor Pathway Inhibitor 2 by hCG Regulates Periovulatory Gene Expression and Plasmin Activity.

PubMed

Puttabyatappa, Muraly; Al-Alem, Linah F; Zakerkish, Farnosh; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2017-01-01

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation. Copyright © 2017 by the Endocrine Society.

Induction of Tissue Factor Pathway Inhibitor 2 by hCG Regulates Periovulatory Gene Expression and Plasmin Activity

PubMed Central

Puttabyatappa, Muraly; Al-Alem, Linah F.; Zakerkish, Farnosh; Rosewell, Katherine L.; Brännström, Mats

2017-01-01

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation. PMID:27813674

Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

PubMed

Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

2000-10-31

We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

CD94/NKG2C is a killer effector molecule in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis.

PubMed

Morel, Esther; Escamochero, Salvador; Cabañas, Rosario; Díaz, Rosa; Fiandor, Ana; Bellón, Teresa

2010-03-01

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, bullous cutaneous diseases with uncertain pathogenesis, although cytotoxic T cells seem to be involved. Natural killer (NK)-like activity has been found in blister infiltrates. Cytotoxic T lymphocytes (CTLs) with NK-like activity (NK-CTLs) have been shown to express T-cell receptors restricted by the HLA-Ib molecule HLA-E. Alternatively, the HLA-E-specific activating receptor CD94/NKG2C can trigger T-cell receptor-independent cytotoxicity in CTLs. Our aim was to test whether HLA-E expression sensitizes keratinocytes to killing by CTLs with NK-like activity and to explore the expression of activating receptors specific for HLA-E in blister cytotoxic lymphocytes. We used flow cytometry and immunohistochemistry to analyze HLA-E expression in keratinocytes from affected skin in patients with SJS, TEN, and other less severe drug-induced exanthemas. The expression of CD94/NKG2C was analyzed by means of flow cytometry in PBMCs and blister cells from patients. PBMCs and blister cells were analyzed for their ability to kill HLA-E-expressing cells. Involvement of CD94/NKG2C in triggering degranulation of cytolytic cells was explored by means of CD107a mobilization assays and standard cytotoxicity chromium release assays. We found that keratinocytes from affected skin expressed HLA-E and that cell-surface HLA-E sensitizes keratinocytes to killing by CD94/NKG2C(+) CTLs. Frequencies of CD94/NKG2C(+) peripheral blood T and NK cells were increased in patients with SJS and TEN during the acute phase. Moreover, activated blister T and NK lymphocytes expressed CD94/NKG2C and were able to degranulate in response to HLA-E(+) cells in an NKG2C-dependent manner. CD94/NKG2C might be involved in triggering cytotoxic lymphocytes in patients with SJS and TEN.

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression.

PubMed

Gee, Katrina; Lim, Wilfred; Ma, Wei; Nandan, Devki; Diaz-Mitoma, Francisco; Kozlowski, Maya; Kumar, Ashok

2002-11-15

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

2001-01-01

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

The orphan nuclear receptor NR4A1 attenuates oxidative stress-induced β cells apoptosis via up-regulation of glutathione peroxidase 1.

PubMed

Yang, Yingfeng; Xie, Fangyu; Qin, Dandan; Zong, Chen; Han, Feng; Pu, Zeqing; Liu, Dong; Li, Xia; Zhang, Yuchao; Liu, Yuantao; Wang, Xiangdong

2018-06-15

Our previous study showed that NR4A1 protects against oxidative stress-induced cell apoptosis. However, the targets downstream of NR4A1 are incompletely known. Glutathione peroxidase 1 (GPX1) is the most common antioxidant enzyme in the glutathione peroxidase class. In this study, we aimed to investigate whether GPX1 is a mediator of the protective effects of NR4A1 in pancreatic β cells. A pancreatic β cell line, MIN6, was used to generate NR4A1 over-expression cell line. GPX1 expression and GPX1 promoter trans-activation in these cells was determined. These cells were then treated with H 2 O 2 , and the active caspase3 level was determined. NR4A1 over-expression in MIN6 cells resulted in increased GPX1 expression at both mRNA and protein levels. Dual luciferase assay showed that NR4A1 over-expression was able to enhance the trans-activation of GPX1 promoter, and the critical regulatory elements were narrowed down between 0 to -2000 bp in GPX1 promoter with a putative NR4A1 binding site (-273 to -268). ChIP assays demonstrated that NR4A1 physically associates with the GPX1 promoter. Over-expression of GPX1 reduced the active level of Caspase3 after H 2 O 2 treatment. NR4A1 increases the expression of GPX1 by enhancing the trans-activation of GPX1 promoter through binding to the putative binding site on GPX1 promoter. NR4A1 potentially protects pancreatic β cells against oxidative stress-induced apoptosis by increasing GPX1 expression. Copyright © 2018 Elsevier Inc. All rights reserved.

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

PubMed

Zhang, Danfeng; Wang, Chunhui; Li, Zhenxing; Li, Yiming; Dai, Dawei; Han, Kaiwei; Lv, Liquan; Lu, Yicheng; Hou, Lijun; Wang, Junyu

2018-01-01

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue ( N  = 31), whereas the lowest expression was in high-grade glioma tissue ( N  = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2006-03-01

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

PubMed

Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

2017-09-01

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

A gene expression signature that correlates with CD8+T cell expansion in acute Epstein Barr virus infection1

PubMed Central

Greenough, Thomas C.; Straubhaar, Juerg R.; Kamga, Larisa; Weiss, Eric R.; Brody, Robin M.; McManus, Margaret M.; Lambrecht, Linda K.; Somasundaran, Mohan; Luzuriaga, Katherine F.

2015-01-01

Virus specific CD8+ T cells expand dramatically during acute Epstein Barr virus (EBV) infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of ten individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and Eomesodermin (Eomes), were upregulated and maintained at similar levels in both AIM and CONV; by contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Altogether, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus-specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and Eomes may help to maintain effector mechanisms in activated cells, and to enable proliferation and transition to earlier differentiation states in CONV. PMID:26416268

Successful TB treatment induces B-cells expressing FASL and IL5RA mRNA.

PubMed

van Rensburg, Ilana C; Wagman, Chandre; Stanley, Kim; Beltran, Caroline; Ronacher, Katharina; Walzl, Gerhard; Loxton, Andre G

2017-01-10

Activated B-cells increase T-cell behaviour during autoimmune disease and other infections by means of cytokine production and antigen-presentation. Functional studies in experimental autoimmune encephalomyelitis (EAE) indicate that B-cell deficiencies, and a lack of IL10 and IL35 leads to a poor prognosis. We hypothesised that B-cells play a role during tuberculosis. We evaluated B-cell mRNA expression using real-time PCR from healthy community controls, individuals with other lung diseases and newly diagnosed untreated pulmonary TB patients at three different time points (diagnosis, month 2 and 6 of treatment).We show that FASLG, IL5RA, CD38 and IL4 expression was lower in B-cells from TB cases compared to healthy controls. The changes in expression levels of CD38 may be due to a reduced activation of B-cells from TB cases at diagnosis. By month 2 of treatment, there was a significant increase in the expression of APRIL and IL5RA in TB cases. Furthermore, after 6 months of treatment, APRIL, FASLG, IL5RA and CD19 were upregulated in B-cells from TB cases. The increase in the expression of APRIL and CD19 suggests that there may be restored activation of B-cells following anti-TB treatment. The upregulation of FASLG and IL5RA indicates that B-cells expressing regulatory genes may play an important role in the protective immunity against M.tb infection. Our results show that increased activation of B-cells is present following successful TB treatment, and that the expression of FASLG and IL5RA could potentially be utilised as a signature to monitor treatment response.

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

PubMed

Resnick, M S; Kang, B S; Luu, D; Wickham, J T; Sando, J J; Hahn, C S

1998-10-16

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

PubMed

Sha, Wei Hong; Zeng, Xiao Hui; Min, Lu

2014-05-01

This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D(+) NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

Deciphering the role of the signal- and Sty1 kinase-dependent phosphorylation of the stress-responsive transcription factor Atf1 on gene activation.

PubMed

Salat-Canela, Clàudia; Paulo, Esther; Sánchez-Mir, Laura; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2017-08-18

Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast ( Schizosaccharomyces pombe ), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daviau, Alex; Couture, Jean-Philippe; Blouin, Richard, E-mail: Richard.Blouin@USherbrooke.ca

Highlights: {yields} Role of DLK in cell proliferation. {yields} Modulation of DLK expression during cell cycle progression. {yields} DLK knockdown induces proliferation arrest and senescence. {yields} DLK-depleted cells display loss of cyclin D1 and up-regulation of p21. {yields} DLK participates in cell proliferation by modulating cell cycle regulator expression. -- Abstract: DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, althoughmore » little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.« less

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

PubMed

Sun, Jingping; Law, Gabriela P; McKallip, Robert J

2012-03-01

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

PubMed

Shibahara, Daisuke; Tanaka, Kentaro; Iwama, Eiji; Kubo, Naoki; Ota, Keiichi; Azuma, Koichi; Harada, Taishi; Fujita, Jiro; Nakanishi, Yoichi; Okamoto, Isamu

2018-03-27

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

An aberrant NOTCH2-BCR signaling axis in B cells from patients with chronic GVHD.

PubMed

Poe, Jonathan C; Jia, Wei; Su, Hsuan; Anand, Sarah; Rose, Jeremy J; Tata, Prasanthi V; Suthers, Amy N; Jones, Corbin D; Kuan, Pei Fen; Vincent, Benjamin G; Serody, Jonathan S; Horwitz, Mitchell E; Ho, Vincent T; Pavletic, Steven Z; Hakim, Frances T; Owzar, Kouros; Zhang, Dadong; Blazar, Bruce R; Siebel, Christian W; Chao, Nelson J; Maillard, Ivan; Sarantopoulos, Stefanie

2017-11-09

B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8 , each critical to B-cell differentiation and fate. All- trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4 -to- IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5 , but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

PubMed Central

Wang, Lihong; Cao, Hailong; Lu, Ning; Liu, Liping; Wang, Bangmao; Hu, Tianhui; Israel, Dawn A.; Peek, Richard M.; Polk, D. Brent; Yan, Fang

2013-01-01

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells. PMID:23457600

Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

PubMed Central

Holmes, Tim D.; Wilson, Erica B.; Black, Emma V. I.; Benest, Andrew V.; Vaz, Candida; Tan, Betty; Tanavde, Vivek M.; Cook, Graham P.

2014-01-01

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues. PMID:25512551

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

PubMed Central

Lieberman, Richard; Kranzler, Henry R.; Levine, Eric S.; Covault, Jonathan

2016-01-01

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual. PMID:27915167

The Hippo pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells

PubMed Central

Judson, Robert N.; Tremblay, Annie M.; Knopp, Paul; White, Robert B.; Urcia, Roby; De Bari, Cosimo; Zammit, Peter S.; Camargo, Fernando D.; Wackerhage, Henning

2012-01-01

Summary Satellite cells are the resident stem cells of skeletal muscle. Mitotically quiescent in mature muscle, they can be activated to proliferate and generate myoblasts to supply further myonuclei to hypertrophying or regenerating muscle fibres, or self-renew to maintain the resident stem cell pool. Here, we identify the transcriptional co-factor Yap as a novel regulator of satellite cell fate decisions. Yap expression increases during satellite cell activation and Yap remains highly expressed until after the differentiation versus self-renewal decision is made. Constitutive expression of Yap maintains Pax7+ and MyoD+ satellite cells and satellite cell-derived myoblasts, promotes proliferation but prevents differentiation. In contrast, Yap knockdown reduces the proliferation of satellite cell-derived myoblasts by ≈40%. Consistent with the cellular phenotype, microarrays show that Yap increases expression of genes associated with Yap inhibition, the cell cycle, ribosome biogenesis and that it represses several genes associated with angiotensin signalling. We also identify known regulators of satellite cell function such as BMP4, CD34 and Myf6 (Mrf4) as genes whose expression is dependent on Yap activity. Finally, we confirm in myoblasts that Yap binds to Tead transcription factors and co-activates MCAT elements which are enriched in the proximal promoters of Yap-responsive genes. PMID:23038772

TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jing; Liu, Su-zhi; Lin, Yan

Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significantmore » when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.« less

Effects of Carbocysteine and Beclomethasone on Histone Acetylation/Deacetylation Processes in Cigarette Smoke Exposed Bronchial Epithelial Cells.

PubMed

Pace, Elisabetta; Di Vincenzo, Serena; Ferraro, Maria; Siena, Liboria; Chiappara, Giuseppina; Dino, Paola; Vitulo, Patrizio; Bertani, Alessandro; Saibene, Federico; Lanata, Luigi; Gjomarkaj, Mark

2017-10-01

Histone deacetylase expression/activity may control inflammation, cell senescence, and responses to corticosteroids. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity. The effects of cigarette smoke extracts (CSE), carbocysteine, and beclomethasone dipropionate on chromatin remodeling processes in human bronchial epithelial cells are largely unknown. The present study was aimed to assess the effects of cigarette smoke, carbocysteine, and beclomethasone dipropionate on histone deacetylase 3 (HDAC3) expression/activity, N-CoR (nuclear receptor corepressor) expression, histone acetyltransferases (HAT) (p300/CBP) expression, p-CREB and IL-1 m-RNA expression, neutrophil chemotaxis. Increased p-CREB expression was observed in the bronchial epithelium of smokers. CSE increased p-CREB expression and decreased HDAC3 expression and activity and N-CoR m-RNA and protein expression. At the same time, CSE increased the expression of the HAT, p300/CBP. All these events increased acetylation processes within the cells and were associated to increased IL-1 m-RNA expression and neutrophil chemotaxis. The incubation of CSE exposed cells with carbocysteine and beclomethasone counteracted the effects of cigarette smoke on HDAC3 and N-CoR but not on p300/CBP. The increased deacetylation processes due to carbocysteine and beclomethasone dipropionate incubation is associated to reduced p-CREB, IL-1 m-RNA expression, neutrophil chemotaxis. These findings suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure. J. Cell. Physiol. 232: 2851-2859, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hemoglobin enhances tissue factor expression on human malignant cells.

PubMed

Siddiqui, F A; Amirkhosravi, A; Amaya, M; Meyer, T; Biggerstaff, J; Desai, H; Francis, J L

2001-04-01

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells

PubMed Central

Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F.; Schett, Georg

2014-01-01

The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression. PMID:25288397

Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells.

PubMed

Ye, Xueting; Xie, Jing; Huang, Hang; Deng, Zhexian

2018-01-01

Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling. © 2018 The Author(s). Published by S. Karger AG, Basel.

Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

PubMed Central

Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

2015-01-01

SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

Strong fascin expression promotes metastasis independent of its F-actin bundling activity.

PubMed

Heinz, Lisa S; Muhs, Stefanie; Schiewek, Johanna; Grüb, Saskia; Nalaskowski, Marcus; Lin, Yuan-Na; Wikman, Harriet; Oliveira-Ferrer, Leticia; Lange, Tobias; Wellbrock, Jasmin; Konietzny, Anja; Mikhaylova, Marina; Windhorst, Sabine

2017-12-15

High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo , showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules ( MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells.

PubMed

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group and blank group (P<0.05), demonstrating that after the down-regulation of Tiam1 gene expression, the speed of cell proliferation was inhibited. MTT assay results showed that the total growth speed in experimental group was significantly lower than that in control group and blank group (P<0.05), indicating that the proliferation activity of cholangiocarcinoma cells was inhibited after targeted inhibition of Tiam1 gene expression. Transwell detection results showed that the metastasis rate in experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that targeted inhibition of Tiam1 gene expression could significantly inhibit migration ability of RBE cells. Tiam1 expression significantly increased in cholangiocarcinoma tissues, and increased along with the degree of malignancy of cholangiocarcinoma. Targeted silencing Tiam1 expression could inhibit proliferation and migration activity of cholangiocarcinoma cells.

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells

PubMed Central

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

Objective: To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Methods: Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. Results: The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group and blank group (P<0.05), demonstrating that after the down-regulation of Tiam1 gene expression, the speed of cell proliferation was inhibited. MTT assay results showed that the total growth speed in experimental group was significantly lower than that in control group and blank group (P<0.05), indicating that the proliferation activity of cholangiocarcinoma cells was inhibited after targeted inhibition of Tiam1 gene expression. Transwell detection results showed that the metastasis rate in experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that targeted inhibition of Tiam1 gene expression could significantly inhibit migration ability of RBE cells. Conclusion: Tiam1 expression significantly increased in cholangiocarcinoma tissues, and increased along with the degree of malignancy of cholangiocarcinoma. Targeted silencing Tiam1 expression could inhibit proliferation and migration activity of cholangiocarcinoma cells. PMID:26884821

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays

NASA Astrophysics Data System (ADS)

Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

2014-06-01

Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation. Electronic supplementary information (ESI) available: (1) ESEM cross-sectional view images of the flat silicon and SiNW substrates. (2) Bright field morphology images of fibroblasts cultured in Petri dishes. (3) FIB/SEM 52° tilt images of fibroblasts cultured on SiNW 2 and SiNW 3. (4) Immunofluorescence images of FAP expression in fibroblasts re-cultured in Petri dishes after detachment from flat silicon and a series of SiNW substrates. (5) ESEM images of cells re-cultured in Petri dishes after detachment from each group. See DOI: 10.1039/c4nr01415d

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

PubMed

Sumitomo, M; Shen, R; Goldberg, J S; Dai, J; Navarro, D; Nanus, D M

2000-12-01

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit.

PubMed

Tanaka, Junji; Sugita, Junichi; Kato, Naoko; Toubai, Tomomi; Ibata, Makoto; Shono, Yusuke; Ota, Shuichi; Kondo, Takeshi; Kobayashi, Takahiko; Kobayashi, Masanobu; Asaka, Masahiro; Imamura, Masahiro

2007-10-01

Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas α1-adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na+,K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. PMID:20380860

Stat6 activity-related Th2 cytokine profile and tumor growth advantage of human colorectal cancer cells in vitro and in vivo.

PubMed

Li, Ben Hui; Xu, Shuang Bing; Li, Feng; Zou, Xiao Guang; Saimaiti, Abudukeyoumu; Simayi, Dilixia; Wang, Ying Hong; Zhang, Yan; Yuan, Jia; Zhang, Wen Jie

2012-03-01

Signal transducer and activator of transcription 6 (Stat6) is critical in Th2 polarization of immune cells and active Stat6 activity has been suggested in anti-tumor immunity in animal models. The present study aims at investigating the impact of natural Stat6 activity on tumor microenvironment in human colorectal cancer cells in vitro and in vivo. Using colorectal cancer cell lines HT-29 and Caco-2 whose IL-4/Stat6 activities were known and nude mice as a model, we examined correlative relationships between Stat6 activities and gene expression profiles together with cellular behaviors in vitro and in vivo. HT-29 cells carrying active Stat6 signaling displayed spontaneous expression profiles favoring Th2 cytokines, cell cycle promotion, anti-apoptosis and pro-metastasis with increased mRNA levels of IL-4, IL-13, GATA-3, CDK4, CD44v6 and S100A4 using RT-PCR. In contrast, Caco-2 cells carrying defective Stat6 signaling exhibited spontaneous expression profiles favoring Th1 and Th17 cytokines, cell cycle inhibition, pro-apoptosis and anti-metastasis with elevated mRNA expression of IFNγ, TNFα, IL-12A, IL-17, IL-23, T-bet, CDKN1A, CDKNIB, CDKN2A and NM23-H1. Xenograft tumors of Stat6-active HT-29 cells showed a growth advantage over those of Stat6-defective Caco-2 cells. Furthermore, mice bearing HT-29 tumors expressed increased levels of Th2 cytokines IL-4 and IL-5 in the blood and pro-growth and/or pro-metastasis proteins CDK4 and CD44v6 in the tumor. To the contrary, mice bearing Caco-2 tumors expressed heightened levels of Th1 cytokines IFNγ and TNF in the blood and pro-apoptosis and anti-metastatic proteins p53 and p27(kip1) in the tumor. Colorectal cancer cells carrying active Stat6 signaling may create a microenvironment favoring Th2 cytokines and promoting expression of genes related to pro-growth, pro-metastasis and anti-apoptosis, which leads to a tumor growth advantage in vivo. These findings may imply why Stat6 pathway is constitutively activated in a number of human malignancies. Copyright © 2011 Elsevier Inc. All rights reserved.

T cell activation responses are differentially regulated during clinorotation and in spaceflight

NASA Technical Reports Server (NTRS)

Hashemi, B. B.; Penkala, J. E.; Vens, C.; Huls, H.; Cubbage, M.; Sams, C. F.

1999-01-01

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.

Constitutive androstane receptor activation evokes the expression of glycolytic genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarushkin, Andrei A.; Kazantseva, Yuliya A.; Prokopyeva, Elena A.

It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in amore » mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation. - Highlights: • CAR-mediated liver growth is correlated with increased expression of cMyc. • CAR activation increased the expression of glycolytic genes in mouse livers. • CAR activation increased the level of Pkm2 in mouse livers.« less

Ginkgolide B Reduces LOX-1 Expression by Inhibiting Akt Phosphorylation and Increasing Sirt1 Expression in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

PubMed Central

Chen, Beidong; Li, Xingguang; Qi, Ruomei

2013-01-01

Oxidized low-density lipoprotein (ox-LDL) is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1) expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells. PMID:24069345

Human Naive T Cells Express Functional CXCL8 and Promote Tumorigenesis.

PubMed

Crespo, Joel; Wu, Ke; Li, Wei; Kryczek, Ilona; Maj, Tomasz; Vatan, Linda; Wei, Shuang; Opipari, Anthony W; Zou, Weiping

2018-05-25

Naive T cells are thought to be functionally quiescent. In this study, we studied and compared the phenotype, cytokine profile, and potential function of human naive CD4 + T cells in umbilical cord and peripheral blood. We found that naive CD4 + T cells, but not memory T cells, expressed high levels of chemokine CXCL8. CXCL8 + naive T cells were preferentially enriched CD31 + T cells and did not express T cell activation markers or typical Th effector cytokines, including IFN-γ, IL-4, IL-17, and IL-22. In addition, upon activation, naive T cells retained high levels of CXCL8 expression. Furthermore, we showed that naive T cell-derived CXCL8 mediated neutrophil migration in the in vitro migration assay, supported tumor sphere formation, and promoted tumor growth in an in vivo human xenograft model. Thus, human naive T cells are phenotypically and functionally heterogeneous and can carry out active functions in immune responses. Copyright © 2018 by The American Association of Immunologists, Inc.

L1CAM stimulates glioma cell motility and proliferation through the fibroblast growth factor receptor.

PubMed

Mohanan, Vishnu; Temburni, Murali K; Kappes, John C; Galileo, Deni S

2013-04-01

The L1CAM cell adhesion/recognition molecule (L1, CD171) and fibroblast growth factor receptor (FGFR) both are expressed by human high-grade glioma cells, but their potential actions in controlling cell behavior have not been linked. L1 actions in cancer cells have been attributed mainly to integrin receptors, and we demonstrated previously that L1-stimulated glioma cell migration correlates with integrin expression, increased focal adhesion kinase activation and focal complex turnover. Our analyses of datasets revealed FGFR is overexpressed in glioma regardless of grade, while ADAM10 metalloprotease expression increases with glioma grade. Here, we used dominant-negative and short hairpin RNA approaches to inhibit the activation of FGFR1 and expression of L1, respectively. An L1 peptide that inhibits L1-FGFR interaction and PD173074, a chemical inhibitor of FGFR1 activity, also were used to elucidate the involvement of L1-FGFR interactions on glioma cell behavior. Time-lapse cell motility studies and flow cytometry cell cycle analyses showed that L1 operates to increase glioma cell motility and proliferation through FGFR activation. Shutdown of both L1 expression and FGFR activity in glioma cells resulted in a complete termination of cell migration in vitro. These studies show for the first time that soluble L1 ectodomain (L1LE) acts on glioma cells through FGFRs, and that FGFRs are used by glioma cells for increasing motility as well as proliferation in response to activation by L1LE ligand. Thus, effective treatment of high-grade glioma may require simultaneous targeting of L1, FGFRs, and integrin receptors, which would reduce glioma cell motility as well as proliferation.

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica

Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition,more » EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.« less

CARMA3 is overexpressed in colon cancer and regulates NF-{kappa}B activity and cyclin D1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Zhifeng; Zhao, Tingting; Wang, Zhenning

2012-09-07

Highlights: Black-Right-Pointing-Pointer CARMA3 expression is elevated in colon cancers. Black-Right-Pointing-Pointer CARMA3 promotes proliferation and cell cycle progression in colon cancer cells. Black-Right-Pointing-Pointer CARMA3 upregulates cyclinD1 through NF-{kappa}B activation. -- Abstract: CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression andmore » TNM stage (p = 0.0383), lymph node metastasis (p = 0.0091) and Ki67 proliferation index (p = 0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-I{kappa}B levels and NF-{kappa}B activity and its overexpression increased p-I{kappa}B expression and NF-{kappa}B activity. NF-{kappa}B inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-{kappa}B mediated upregulation of cyclin D1.« less

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

PubMed Central

Gomes, J A P; Dua, H S; Rizzo, L V; Nishi, M; Joseph, A; Donoso, L A

2004-01-01

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes. PMID:14736792

Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis.

PubMed

Lim, Su-Min; Jeong, Jin-Ju; Choi, Hyun Sik; Chang, Hwan Bong; Kim, Dong-Hyun

2016-05-01

In the previous study, 80% ethanol extract of the rhizome mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC) and its main constituent mangiferin improved TNBS-induced colitis in mice by inhibiting macrophage activation related to the innate immunity. In the preliminary study, we found that AC could inhibit Th17 cell differentiation in mice with TNBS-induced colitis. Therefore, we investigated whether AC and it main constituent mangiferin are capable of inhibiting inflammation by regulating T cell differentiation related to the adaptive immunity in vitro and in vivo. AC and mangiferin potently suppressed colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis. They also suppressed TNBS-induced Th17 cell differentiation and IL-17 expression, but increased TNBS-suppressed Treg cell differentiation and IL-10 expression. Moreover, AC and mangiferin strongly inhibited the expression of TNF-α and IL-17, as well as the activation of NF-κB. Furthermore, mangiferin potently inhibited the differentiation of splenocytes into Th7 cells and increased the differentiation into Treg cells in vitro. Mangiferin also inhibited RORγt and IL-17 expression and STAT3 activation in splenocytes and induced Foxp3 and IL-10 expression and STAT5 activation. Based on these findings, mangiferin may ameliorate colitis by the restoration of disturbed Th17/Treg cells and inhibition of macrophage activation. Copyright © 2016 Elsevier B.V. All rights reserved.

Pleiotropic effect of sigE over-expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803.

PubMed

Osanai, Takashi; Kuwahara, Ayuko; Iijima, Hiroko; Toyooka, Kiminori; Sato, Mayuko; Tanaka, Kan; Ikeuchi, Masahiko; Saito, Kazuki; Hirai, Masami Yokota

2013-11-01

Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Expression of K2P5.1 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients.

PubMed

Bittner, Stefan; Bobak, Nicole; Feuchtenberger, Martin; Herrmann, Alexander M; Göbel, Kerstin; Kinne, Raimund W; Hansen, Anker J; Budde, Thomas; Kleinschnitz, Christoph; Frey, Oliver; Tony, Hans-Peter; Wiendl, Heinz; Meuth, Sven G

2011-02-11

CD4+ T cells express K(2P)5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K(2P)5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K(2P)5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients. Expression levels of K(2P)5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K(2P)5.1. K(2P)5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K(2P)5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K(2P)5.1 expression to disease activity parameters during a longitudinal follow-up for six months. Disease activity in RA patients correlates strongly with K(2P)5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K(2P)5.1 as a potential biomarker for disease activity and differential diagnosis.

B29 Gene Silencing in Pituitary Cells is Regulated by Its 3′ Enhancer

PubMed Central

Malone, Cindy S.; Kuraishy, Ali I.; Fike, Francesca M.; Loya, Ruchika G.; Mikkili, Minil R.; Teitell, Michael A.; Wall, Randolph

2007-01-01

Summary B cell-specific B29 (Igβ, CD79b) genes in rat, mouse, and human are situated between the 5′ growth hormone (GH) locus control region (LCR) and the 3′ GH gene cluster. The entire GH genomic region is DNase1 hypersensitive in GH-expressing pituitary cells, which predicts an “open” chromatin configuration, and yet B29 is not expressed. The B29 promoter and enhancers exhibit histone deacetylation in pituitary cells, but histone deacetylase inhibition failed to activate B29 expression. The B29 promoter and a 3′ enhancer showed local dense DNA methylation in both pituitary and non-lymphoid cells consistent with gene silencing. However, DNA methyltransferase inhibition did not activate B29 expression either. B29 promoter constructs were minimally activated in transfected pituitary cells. Co-transfection of the B cell-specific octamer transcriptional co-activator Bob1 with the B29 promoter construct resulted in high level promoter activity in pituitary cells comparable to B29 promoter activity in transfected B cells. Unexpectedly, inclusion of the B29 3′ enhancer in B29 promoter constructs strongly inhibited B29 transcriptional activity even when pituitary cells were co-transfected with Bob1. Both Oct-1 and Pit-1 bind the B29 3′ enhancer in in vitro EMSA and in in vivo chromatin immunoprecipitation analyses. These data indicate that the GH locus-embedded, tissue-specific B29 gene is silenced in GH-expressing pituitary cells by epigenetic mechanisms, the lack of a B cell-specific transcription factor, and likely by the B29 3′ enhancer acting as a powerful silencer in a context and tissue-specific manner. PMID:16920149

Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

PubMed

Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M; Litvinov, Ivan V; Fredholm, Simon; Petersen, David L; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P; Sasseville, Denis; Wasik, Mariusz A; Bonefeld, Charlotte M; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B; Odum, Niels

2016-03-10

Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. © 2016 by The American Society of Hematology.

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

PubMed

Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

2013-01-01

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

Dasatinib is preclinically active against Src-overexpressing human transitional cell carcinoma of the urothelium with activated Src signaling.

PubMed

Levitt, Jonathan M; Yamashita, Hideyuki; Jian, Weiguo; Lerner, Seth P; Sonpavde, Guru

2010-05-01

Dasatinib is an orally administered multitargeted kinase inhibitor that targets Src family tyrosine kinases, Abl, c-Kit, and PDGFR. A preclinical study was conducted to evaluate dasatinib alone or combined with cisplatin for human transitional cell carcinoma (TCC). Expression of Src in a human TCC tissue microarray was evaluated by immunohistochemistry. The activity of dasatinib and/or cisplatin was evaluated in six human TCC cell lines. Western blot was done to assess Src and phosphorylated-Src (p-Src) expression. The activity of dasatinib alone and in combination with cisplatin was determined in murine subcutaneous xenografts. Sixty-two percent to 75% of human TCC expressed Src. Dasatinib displayed significant antiproliferative activity at nanomolar concentrations against two human TCC cell lines (RT4 and Hu456) that exhibited high Src and p-Src expression and were cisplatin-resistant. RT4 cells were the most sensitive and displayed the highest level of Src pathway activation (p-Src/Src ratio). Dasatinib downregulated p-Src in either sensitive or resistant cells. TCC cells that were sensitive to cisplatin (5637 and TCC-SUP) were highly resistant to dasatinib and exhibited low Src expression. Dasatinib showed antitumor activity in RT4 murine xenografts, and the combination of dasatinib and cisplatin was significantly more active than placebo. Combination dasatinib plus cisplatin significantly inhibited proliferation and promoted apoptosis in vivo. In conclusion, dasatinib displayed significant preclinical antitumor activity against Src-overexpressing human TCC with active Src signaling and was highly active in combination with cisplatin in vivo. Further clinical development might be warranted in selected human subjects.

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediatedmore » toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.« less

Expression of CXCR6 on CD8(+) T cells was up-regulated in allograft rejection.

PubMed

Jiang, Xiaofeng; Sun, Wenyu; Zhu, Lei; Guo, Dawei; Jiang, Honglei; Ma, Dongyan; Jin, Junzhe; Zhao, Yu; Liang, Jian

2010-02-01

CXCL16/SR-PSOX is a novel transmembrane-type chemokine, which was also identified as a novel scavenger receptor for oxidized low density lipoprotein. Its receptor CXCR6 expresses on activated CD8(+) T cells, type 1-polarized CD4(+), and constitutively expresses on NKT cells. Moreover, it has been shown that CXCL16 accumulated activated CD8(+) T cells to sites of inflammation. To date, the effect of CXCL16 (SR-PSOX)/CXCR6 on CD8(+) T cells and its role in allograft rejection/acceptance are not well understood. In the current study, we show that rejected allografts showed higher expressions of CXCR6 and CXCL16. More importantly, expression of CXCR6 on CD8(+) T cells was also up-regulated by rejection. However, the blockade of CXCL16(SR-PSOX)/CXCR6 interaction could not inhibit cytotoxic activity of CD8(+) T cells, and therefore, could not prolong the cardiac graft survival time. Copyright 2009 Elsevier B.V. All rights reserved.

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells.

PubMed

Müller, Tina; Uherek, Christoph; Maki, Guitta; Chow, Kai Uwe; Schimpf, Annemarie; Klingemann, Hans-Georg; Tonn, Torsten; Wels, Winfried S

2008-03-01

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcgammaRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3zeta chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

Augmented Rac1 Expression and Activity are Associated with Oxidative Stress and Decline of β Cell Function in Obesity.

PubMed

Zhou, Shutong; Yu, Dongni; Ning, Shangyong; Zhang, Heli; Jiang, Lei; He, Lei; Li, Miao; Sun, Mingxiao

2015-01-01

The aim of this study was to clarify the relationship among Rac1 expression and activation, oxidative stress and β cell dysfunction in obesity. In vivo, serum levels of glucose, insulin, oxidative stress markers and Rac1 expression were compared between ob/ob mice and C57BL/6J controls. Then, these variables were rechecked after the administration of the specific Rac1 inhibitor-NSC23766 in ob/ob mice. In vitro, NIT-1 β cells were cultured in a hyperglycemic and/or hyperlipidemic state with or without NSC23766, and the differences of Rac1 expression and translocation, NADPH oxidase(Nox) enzyme activity, reactive oxygen species (ROS) and insulin mRNA were observed. ob/ob mice displayed abnormal glycometabolism, oxidative stress and excessive expression of Rac1 in the pancreas. NSC23766 injection inhibited the expression of Rac1 in the pancreas, along with amelioration of oxidative stress and glycometabolism in obese mice. Under hyperglycemic and/or hyperlipidemic conditions, Rac1 translocated to the cellular membrane, induced activation of the NADPH oxidase enzyme and oxidative stress, and simultaneously reduced the insulin mRNA expression in NIT-1 β cells. Inhibiting Rac1 activity could alleviate oxidative stress and meliorate the decline of insulin mRNA in β cells. Rac1 might contribute to oxidative stress systemically and locally in the pancreas in obesity. The excessive activation and expression of Rac1 in obesity were associated with β cell dysfunction through ROS production. © 2015 S. Karger AG, Basel.

Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation

PubMed Central

Hrdličková, Radmila; Nehyba, Jiří; Liss, Andrew S.; Bose, Henry R.

2006-01-01

Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS. PMID:16352553

Fibroblast Growth Factor signaling regulates the expansion of A6-expressing hepatocytes in association with AKT-dependent β-catenin activation

PubMed Central

Utley, Sarah; James, David; Mavila, Nirmala; Nguyen, Marie V.; Vendryes, Christopher; Salisbury, S. Michael; Phan, Jennifer; Wang, Kasper S.

2014-01-01

Background & Aims Fibroblast Growth Factors (FGFs) promote the proliferation and survival of hepatic progenitor cells (HPCs) via AKT-dependent β-catenin activation. Moreover, the emergence of hepatocytes expressing the HPC marker A6 during 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury is mediated partly by FGF and β-catenin signaling. Herein, we investigate the role of FGF signaling and AKT-mediated β-catenin activation in acute DDC liver injury. Methods Transgenic mice were fed DDC chow for 14 days concurrent with either Fgf10 over-expression or inhibition of FGF signaling via expression of soluble dominant-negative FGF Receptor (R)-2IIIb. Results After 14 days of DDC treatment, there was an increase in periportal cells expressing FGFR1, FGFR2, and AKT-activated phospho-Serine 552 (pSer552) β-CATENIN in association with up-regulation of genes encoding FGFR2IIIb ligands, Fgf7, Fgf10, and Fgf22. In response to Fgf10 over-expression, there was an increase in the number of pSer552-β-CATENIN(positive)+ive periportal cells as well as cells co-positive for A6 and hepatocyte marker, Hepatocyte Nuclear Factor-4α (HNF4α). A similar expansion of A6+ive cells was observed after Fgf10 over-expression with regular chow and after partial hepatectomy during ethanol toxicity. Inhibition of FGF signaling increased the periportal A6+iveHNF4α+ive cell population while reducing centrolobular A6+ive HNF4α+ive cells. AKT inhibition with Wortmannin attenuated FGF10-mediated A6+iveHNF4α+ive cell expansion. In vitro analyses using FGF10 treated HepG2 cells demonstrated AKT-mediated β-CATENIN activation but not enhanced cell migration. Conclusion During acute DDC treatment, FGF signaling promotes the expansion of A6-expressing liver cells partly via AKT-dependent activation of β-CATENIN expansion of A6+ive periportal cells and possibly by reprogramming of centrolobular hepatocytes. PMID:24365171

Systems biology of lupus: mapping the impact of genomic and environmental factors on gene expression signatures, cellular signaling, metabolic pathways, hormonal and cytokine imbalance, and selecting targets for treatment.

PubMed

Perl, Andras

2010-02-01

Systemic lupus erythematosus (SLE) is characterized by the dysfunction of T cells, B cells, and dendritic cells, the release of pro-inflammatory nuclear materials from necrotic cells, and the formation of antinuclear antibodies (ANA) and immune complexes of ANA with DNA, RNA, and nuclear proteins. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key factor in abnormal activation of T and B cells in SLE. In T cells, increased production of nitric oxide and mitochondrial hyperpolarization (MHP) were identified as metabolic checkpoints upstream of mTOR activation. mTOR controls the expression T-cell receptor-associated signaling proteins CD4 and CD3zeta through increased expression of the endosome recycling regulator Rab5 and HRES-1/Rab4 genes, enhances Ca2+ fluxing and skews the expression of tyrosine kinases both in T and B cells, and blocks the expression of Foxp3 and the generation of regulatory T cells. MHP, increased activity of mTOR, Rab GTPases, and Syk kinases, and enhanced Ca2+ flux have emerged as common T and B cell biomarkers and targets for treatment in SLE.

Suppression of hepatic stellate cell activation by microRNA-29b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekiya, Yumiko; Ogawa, Tomohiro; Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka

Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has beenmore » previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.« less

PSCs and GLP-1R: occurrence in normal pancreas, acute/chronic pancreatitis and effect of their activation by a GLP-1R agonist

PubMed Central

Nakamura, Taichi; Ito, Tetsuhide; Uchida, Masahiko; Hijioka, Masayuki; Igarashi, Hisato; Oono, Takamasa; Kato, Masaki; Nakamura, Kazuhiko; Suzuki, Koichi; Jensen, Robert T.; Takayanagi, Ryoichi

2013-01-01

Background and Aims There is increasing concern about the development of pancreatitis in patients with diabetes mellitus who received long-term GLP-1 analog treatment. Its pathogenesis is unknown. The effects of GLP-1 agonists on pancreatic endocrine cells is well studied, however there is little information on effects on other pancreatic tissues that might be involved in inflammatory processes. Pancreatic stellate cells (PSCs) can play an important role in pancreatitis, secreting various inflammatory cytokines/chemokines, as well as collagen. In this study, we investigated GLP-1R occurrence in normal pancreas, acute/chronic pancreatitis, and the effects of GLP-1 analog on normal PSCs, their ability to stimulate inflammatory mediator secretion or proliferation. Methods GLP-1R expression/localization in normal pancreas and pancreatitis (acute/chronic) tissues were evaluated with histological/immunohistochemical analysis. PSCs were isolated from male Wistar rats. GLP1R expression and effects of GLP-1 analog on activated PSCs was examined with realtime PCR, MTS assays and Western Blotting. Results In normal pancreas, pancreatic β cells expressed GLP-1R, with only low expression in acinar cells, whereas in acute or chronic pancreatitis, acinar cells, ductal cells and activated PSCs expressed GLP-1R. With activation of normal PSCs, GLP-1R is markedly increased, as is multiple other incretin-related receptors. The GLP-1 analog, liraglutide, did not induce inflammatory genes expression in activated PSCs, but induced proliferation. Liraglutide activated multiple signaling cascades in PSCs, and the ERK pathway mediated the PSCs proliferation. Conclusions GLP-1Rs are expressed in normal pancreas and there is marked enhanced expression in acute/chronic pancreatitis. GLP-1-agonist induced cell proliferation of activated PSCs without increasing release of inflammatory mediators. These results suggest chronic treatment with GLP-1R agonists could lead to proliferation/chronic activation of PSCs, which may lead to important effects in the pancreas. PMID:24217090

Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and B regulatory cells in patients with Inflammatory Bowel Disease.

PubMed

Fonseca-Camarillo, Gabriela; Furuzawa-Carballeda, Janette; Yamamoto-Furusho, Jesús K

2015-10-01

The aim of the study was to characterize and to quantify peripheral and tissue. IL-35- and IL-37-producing cells in Inflammatory Bowel Disease (IBD) patients. We studied a total of 38 active UC, 31 inactive UC, 17 active CD, and 13 inactive CD and 50 non-inflamed tissues as control group. Gene expression was measured by real time polymerase chain reaction (RT-PCR) and protein expression was evaluated in tissue by immunohistochemistry and in peripheral blood mononuclear cells by flow cytometry. Higher levels of IL-35 was produced by intestinal regulatory B cells and circulating regulatory CD4(+) and CD8(+) T cells in active vs. inactive disease or healthy donors (P<0.05). The IL-37 was conspicuously synthesized by circulating B cells, active natural killer cells and monocytes. These results suggest that down-regulation of inflammation in active IBD patients might be based on the increased expression of IL-35 and IL-37. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE PAGES

Wang, Wei; Li, Eryang; Porth, Ilga; ...

2016-02-02

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Li, Eryang; Porth, Ilga

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

Ly49 Receptors: Innate and Adaptive Immune Paradigms

PubMed Central

Rahim, Mir Munir A.; Tu, Megan M.; Mahmoud, Ahmad Bakur; Wight, Andrew; Abou-Samra, Elias; Lima, Patricia D. A.; Makrigiannis, Andrew P.

2014-01-01

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity. PMID:24765094

Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kappaB activation and calcium elevation.

PubMed

Hsuan, S L; Kannan, M S; Jeyaseelan, S; Prakash, Y S; Malazdrewich, C; Abrahamsen, M S; Sieck, G C; Maheswaran, S K

1999-05-01

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity. Copyright 1999 Academic Press.

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

PubMed Central

Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

2015-01-01

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1.

PubMed

Cheng, Mingrong; Zhi, Kangkang; Gao, Xiaoyan; He, Bing; Li, Yingchun; Han, Jiang; Zhang, Zhiping; Wu, Yan

2013-12-18

Cancer is both a systemic and a genetic disease. The pathogenesis of cancer might be related to dampened immunity. Host immunity recognizes nascent malignant cells - a process referred to as immune surveillance. Augmenting immune surveillance and suppressing immune escape are crucial in tumor immunotherapy. A recombinant plasmid capable of co-expressing granulocyte-macrophage colony- stimulating factor (GM-SCF), interleukin-21 (IL-21), and retinoic acid early transcription factor-1 (Rae-1) was constructed, and its effects determined in a mouse model of subcutaneous liver cancer. Serum specimens were assayed for IL-2 and INF-γ by ELISA. Liver cancer specimens were isolated for Rae-1 expression by RT-PCR and Western blot, and splenocytes were analyzed by flow cytometry. The recombinant plasmid inhibited the growth of liver cancer and prolonged survival of tumor-loaded mice. Activation of host immunity might have contributed to this effect by promoting increased numbers and cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) following expression of GM-SCF, IL-21, and Rae-1. By contrast, the frequency of regulatory T cells was decreased, Consequently, activated CTL and NK cells enhanced their secretion of INF-γ, which promoted cytotoxicity of NK cells and CTL. Moreover, active CTL showed dramatic secretion of IL-2, which stimulates CTL. The recombinant expression plasmid also augmented Rae-1 expression by liver cancer cells. Rae-1 receptor expressing CTL and NK cells removed liver cancer. The recombinant expression plasmid inhibited liver cancer by a mechanism that involved activation of cell-mediated immunity and Rae-1 in liver cancer.

Artesunate protects pancreatic beta cells against cytokine-induced damage via SIRT1 inhibiting NF-κB activation.

PubMed

Yu, L; Chen, J F; Shuai, X; Xu, Y; Ding, Y; Zhang, J; Yang, W; Liang, X; Su, D; Yan, C

2016-01-01

Artesunate (ART) has been known as the most effective and safe reagents to treat malaria for many years. In this study, we explored whether ART could protect pancreatic beta-cell against cytokine-induced damage. The production of nitrite (NO) was detected with the Griess Assay Kit. SIRT1 and inducible nitric oxide synthase (iNOS) expression were determined with Western blot. The transcriptional activity of NF-κB was evaluated by luciferase reporter assay. The expression of Sirt1 was silenced by RNA interference. Glucose-stimulated insulin secretion (GSIS) and potassium-stimulated insulin secretion (KSIS) assays were performed to measure the effect of ART on pancreatic beta-cells' function. The effect of ART on beta-cells apoptosis was evaluated by using Hochest/PI staining and TUNEL assay. ART enhanced GSIS (KSIS) and reduced apoptosis of pancreatic beta-cells induced by IL-1β. Further study showed that ART inhibited IL-1β-induced increase of NF-κB activity, iNOS expression, and NO production. Moreover, ART up-regulated SIRT1 expression in INS-1 cells and islets exposed to IL-1β. Inhibition of SIRT1 expression could partially abolished the inhibitory effect of ART on NF-κB activity in IL-1β-treated beta-cells. More importantly, the protective effect of ART on cytokine-induced damage was reversed by silencing SIRT1 expression. ART can elicit a protective effect on beta-cells exposed to IL-1β by stimulating SIRT1 expression, which resulted in the decrease of NF-κB activity, iNOS expression, and NO production. Hence, ART might be an effective drug for diabetes.

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

DTIC Science & Technology

2007-12-01

CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with... surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the DCs...cell surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H.; Morton, Derrick J.

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, wemore » examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. - Highlights: • ID4 expression induces AR expression in PC3 cells, which generally lack AR. • ID4 expression increased apoptosis and decreased cell proliferation and invasion. • Overexpression of ID4 reduces tumor growth of subcutaneous xenografts in vivo. • ID4 induces p21 and FKBP51 expression- co-factors of AR tumor suppressor activity.« less

Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1, 25(OH)(2)D(3), which is functionally linked to activation of a nonselective cation channel.

Wnt/beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression

DTIC Science & Technology

2014-07-01

NT1 cells that over-expressing Foxa2. The reason we used NT1 cells for the Foxa2 over-expressing experiments is that NT1 is an AR-expressing... cells . We have also established NT1 cells over-expressing a dominant active beta-catenin. We have characterized these cells . Our research found: 1...expression profiles of control NT1 , NT1 /Foxa2, and NT1 /beta-catenin cells Figure 1. We did RT-PCR to examine the expression of key

Regulation of the expression of GARP/latent-TGF-β1 complexes on mouse T cells and their role in Regulatory T Cell and Th17 differentiation1

PubMed Central

Edwards, Justin P.; Fujii, Hodaka; Zhou, Angela X.; Creemers, John; Unutmaz, Derya; Shevach, Ethan M.

2013-01-01

GARP/LRRC32 has previously been defined as a marker of activated human regulatory T-cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation, but in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent-TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of T conventional cells in vitro. Activated Tregs expressing GARP/latent-TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17 producing cells and Treg is preferentially induced by Tregs expressing the latent-TGF-β1/GARP complex on their cell surface rather than by secreted latent-TGF-β1. PMID:23645881

Regulation of the expression of GARP/latent TGF-β1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation.

PubMed

Edwards, Justin P; Fujii, Hodaka; Zhou, Angela X; Creemers, John; Unutmaz, Derya; Shevach, Ethan M

2013-06-01

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-β1/GARP complex on their cell surface rather than by secreted latent TGF-β1.

Suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression and TNFα-mediated NFκB activation in piceatannol-treated human leukemia U937 cells.

PubMed

Liu, Wen-Hsin; Chang, Long-Sen

2012-09-01

To address the mechanism of piceatannol in inhibiting TNFα-mediated pathway, studies on piceatannol-treated human leukemia U937 cells were conducted. Piceatannol treatment reduced TNFα shedding and NFκB activation and decreased the release of soluble TNFα into the culture medium of U937 cells. Moreover, ADAM17 expression was down-regulated in piceatannol-treated cells. Over-expression of ADAM17 abrogated the ability of piceatannol to suppress TNFα-mediated NFκB activation. Piceatannol-evoked β-TrCP up-regulation promoted Sp1 degradation, thus reducing transcriptional level of ADAM17 gene in U937 cells. Piceatannol treatment induced p38 MAPK phosphorylation but inactivation of Akt and ERK. In contrast to p38 MAPK inhibitor or restoration of ERK activation, transfection of constitutive active Akt abolished the effect of piceatannol on β-TrCP, Sp1 and ADAM17 expression. Piceatannol-elicited down-regulation of miR-183 expression was found to cause β-TrCP up-regulation. Inactivation of Akt resulted in Foxp3 down-regulation and reduced miR-183 expression in piceatannol-treated cells. Knock-down of Foxp3 and chromatin immunoprecipitating revealed that Foxp3 genetically regulated transcription of miR-183 gene. Taken together, our data indicate that suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression in piceatannol-treated U937 cells. Consequently, piceatannol suppresses TNFα shedding, leading to inhibition of TNFα/NFκB pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.

PubMed

Shin, Youngmi; Cho, Nam Jeong

2014-04-01

Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.

Cross talk between the TM4SF5/focal adhesion kinase and the interleukin-6/STAT3 pathways promotes immune escape of human liver cancer cells.

PubMed

Ryu, Jihye; Kang, Minkyung; Lee, Mi-Sook; Kim, Hye-Jin; Nam, Seo Hee; Song, Haeng Eun; Lee, Doohyung; Lee, Jung Weon

2014-08-01

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph

2007-01-01

Osteoclast differentiation is tightly regulated by receptor activator of NF-{kappa}B ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity inmore » RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to - 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to - 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from - 446 bp to - 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (- 1123 bp to - 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.« less

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation

PubMed Central

Sivori, Simona; Falco, Michela; Marcenaro, Emanuela; Parolini, Silvia; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro

2002-01-01

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor. PMID:11917118

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

PubMed Central

Zhou, Yuning; Wang, Qingding; Weiss, Heidi L.; Evers, B. Mark

2014-01-01

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway. PMID:25057011

Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK.

PubMed

Kim, Ha-Na; Kim, Do-Hee; Kim, Eun-Hee; Lee, Mee-Hyun; Kundu, Joydeb Kumar; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

2014-08-28

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK-IKKβ. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effect of LED irradiation on the expression of MMP-3 and MMP-13 in SW1353 cells in vitro

NASA Astrophysics Data System (ADS)

Zeng, Chang-chun; Guo, Zhou-yi; Zhang, Feng-xue; Deng, Wen-di; Liu, Song-hao

2007-05-01

Matrix Metalloproteinase (MMP) plays an active role in remodeling cartilage in osteoarthritic cartilage. To find an effective method of prevention of osteoclasia, this in vitro study focuses on the expression of MMP-3 and MMP-13 in the SW1353 cells by LED irradiation. The human chondrosarcoma cell line SW1353 were stimulated with the proinflammatory cytokine IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and were received the irradiation of LED (632nm, 4mW/cm2). The cell count was assessed over a 96-hour period by using Trypan blue dye exclusion assay, and the cell activity was evaluated with a Cell Counting Kit-8 Assays. The subsequent expression of MMP-3 and MMP-13 was quantified. Results of this experiment showed that the cultural cell activity was decreased, and the expression of MMP-3 and MMP-13 was increased by being stimulated with IL-1beta or TNF-alpha. After received LED irradiation, the death rate of cultural cell was increased and the expression of MMP-3 and MMP-13 was decreased significantly. The present study concluded that particular LED irradiation stimulates SW1353 cell proliferation activity and inhibit the MMP-3 and MMP-13 enzymatic activity. These findings might be clinically relevant, indicating that the low power laser irradiation treatment is likely to achieve the repair of articular cartilage in clinic.

Id1 expression promotes peripheral CD4{sup +} T cell proliferation and survival upon TCR activation without co-stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chen; Jin, Rong; Wang, Hong-Cheng

2013-06-21

Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïvemore » CD4{sup +} cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.« less

Effects of tissue factor, PAR-2 and MMP-9 expression on human breast cancer cell line MCF-7 invasion.

PubMed

Lin, Zeng-Mao; Zhao, Jian-Xin; Duan, Xue-Ning; Zhang, Lan-Bo; Ye, Jing-Ming; Xu, Ling; Liu, Yin-Hua

2014-01-01

This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2 (PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence on invasiveness. Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established. TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasiveness was evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. TF protein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expression was significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR- 2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TF ShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TF and MMP-9 expression was positively correlated with the invasiveness of tumor cells. TF, PAR-2, and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Il-Rae; Koh, Sang Seok; Department of Functional Genomics, University of Science and Technology, Daejeon 305-333

2012-06-29

Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, knownmore » to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1-mediated degradation of {beta}-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of {beta}-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of {beta}-catenin.« less

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

PubMed

Cao, Liyan; Cheng, Shan; Du, Juan; Guo, Yanhai; Huang, Xiaofeng

2017-04-01

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H 2 O 2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H 2 O 2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H 2 O 2 . Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

PubMed Central

Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

2013-01-01

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value. PMID:23613998

Distinct regulation of Snail in two muscle lineages of the ascidian embryo achieves temporal coordination of muscle development.

PubMed

Tokuoka, Miki; Kobayashi, Kenji; Satou, Yutaka

2018-06-06

The transcriptional repressor Snail is required for proper differentiation of the tail muscle of ascidian tadpole larvae. Two muscle lineages (B5.1 and B6.4) contribute to the anterior tail muscle cells, and are consecutively separated from a transcriptionally quiescent germ cell lineage at the 16- and 32-cell stages. Concomitantly, cells of these lineages begin to express Tbx6.b ( Tbx6-r.b ) at the 16- and 32-cell stages, respectively. Meanwhile, Snail expression begins in these two lineages simultaneously at the 32-cell stage. Here, we show that Snail expression is regulated differently between these two lineages. In the B5.1 lineage, Snail was activated through Tbx6.b , which is activated by maternal factors, including Zic-r.a. In the B6.4 lineage, the MAPK pathway was cell-autonomously activated by a constitutively active form of Raf, enabling Zic-r.a to activate Snail independently of Tbx6.b As a result, Snail begins to be expressed at the 32-cell stage simultaneously in these two lineages. Such shortcuts might be required for coordinating developmental programs in embryos in which cells become separated progressively from stem cells, including germline cells. © 2018. Published by The Company of Biologists Ltd.

Regulatory effects of resveratrol on antioxidant enzymes: a mechanism of growth inhibition and apoptosis induction in cancer cells.

PubMed

Khan, Md Asaduzzaman; Chen, Han-Chun; Wan, Xin-Xing; Tania, Mousumi; Xu, Ai-Hua; Chen, Fang-Zhi; Zhang, Dian-Zheng

2013-03-01

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-3 cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK293T kidney epithelial cells were treated with a wide range of RSV concentrations (10-100 μM) for 24-72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 μM) significantly increased superoxide dismutase (SOD) activity in PC-3, HepG2 and MCF-7 cells, but not in HEK293T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced SOD2 expression was observed in cancer cells, although the expression of SOD1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-3 and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of SOD, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis.

Regulatory Effects of Resveratrol on Antioxidant Enzymes: a Mechanism of Growth Inhibition and Apoptosis Induction in Cancer Cells

PubMed Central

Khan, Md. Asaduzzaman; Chen, Han-chun; Wan, Xin-xing; Tania, Mousumi; Xu, Ai-hua; Chen, Fang-zhi; Zhang, Dian-zheng

2013-01-01

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-3 cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK293T kidney epithelial cells were treated with a wide range of RSV concentrations (10–100 μM) for 24–72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 μM) significantly increased superoxide dismutase (SOD) activity in PC-3, HepG2 and MCF-7 cells, but not in HEK293T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced SOD2 expression was observed in cancer cells, although the expression of SOD1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-3 and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of SOD, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis. PMID:23456297

Induction of CD69 expression by cagPAI-positive Helicobacter pylori infection

PubMed Central

Mori, Naoki; Ishikawa, Chie; Senba, Masachika

2011-01-01

AIM: To investigate and elucidate the molecular mechanism that regulates inducible expression of CD69 by Helicobacter pylori (H. pylori) infection. METHODS: The expression levels of CD69 in a T-cell line, Jurkat, primary human peripheral blood mononuclear cells (PBMCs), and CD4+ T cells, were assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry. Activation of CD69 promoter was detected by reporter gene. Nuclear factor (NF)-κB activation in Jurkat cells infected with H. pylori was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling in H. pylori-induced CD69 expression was analyzed using inhibitors of NF-κB and dominant-negative mutants. The isogenic mutants with disrupted cag pathogenicity island (cagPAI) and virD4 were used to elucidate the role of cagPAI-encoding type IV secretion system and CagA in CD69 expression. RESULTS: CD69 staining was detected in mucosal lymphocytes and macrophages in specimens of patients with H. pylori-positive gastritis. Although cagPAI-positive H. pylori and an isogenic mutant of virD4 induced CD69 expression, an isogenic mutant of cagPAI failed to induce this in Jurkat cells. H. pylori also induced CD69 expression in PBMCs and CD4+ T cells. The activation of the CD69 promoter by H. pylori was mediated through NF-κB. Transfection of dominant-negative mutants of IκBs, IκB kinases, and NF-κB-inducing kinase inhibited H. pylori-induced CD69 activation. Inhibitors of NF-κB suppressed H. pylori-induced CD69 mRNA expression. CONCLUSION: The results suggest that H. pylori induces CD69 expression through the activation of NF-κB. cagPAI might be relevant in the induction of CD69 expression in T cells. CD69 in T cells may play a role in H. pylori-induced gastritis. PMID:21990950

Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells.

PubMed

Shibata, Ayano; Tanabe, Eriko; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2013-04-12

Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide. Copyright © 2013 Elsevier Inc. All rights reserved.

Methods of expressing and detecting activity of expansin in plant cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hood, Elizabeth E.; Yoon, Sangwoong

A method of expressing heterologous expansin in a plant cell is provided where a nucleic acid molecule encoding expansin is introduced into the plant cell and in an embodiment is operably linked to a promoter preferentially expressing in the seed tissue of the plant, and in another embodiment is linked to a promoter preferentially expressing in the embryo tissue of the seed. An embodiment provides the nucleic acid molecule is operably linked to a second nucleic acid molecule that directs expression to the endoplasmic reticulum, vacuole or cell wall. Plants and plant parts expressing expansin are provided. An assay formore » detection of expansin activity is also provided.« less

Increased Soluble CD226 in Sera of Patients with Cutaneous T-Cell Lymphoma Mediates Cytotoxic Activity against Tumor Cells via CD155.

PubMed

Takahashi, Naomi; Sugaya, Makoto; Suga, Hiraku; Oka, Tomonori; Kawaguchi, Makiko; Miyagaki, Tomomitsu; Fujita, Hideki; Inozume, Takashi; Sato, Shinichi

2017-08-01

Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances. CD155 is expressed in various types of cancer, and this surface molecule on tumor cells functions either as a co-stimulatory molecule or a co-inhibitory molecule, depending on its receptor. CD226, a CD155 ligand, is mainly expressed on natural killer cells and CD8 + T cells, playing important roles in natural killer cell-mediated cytotoxicity. In this study, we investigated the expression and function of CD155 and CD226 in cutaneous T-cell lymphoma (CTCL). CD155 was strongly expressed on tumor cells and CD155 mRNA expression levels were increased in CTCL lesional skin. CD226 expression on natural killer cells and CD8 + cells in peripheral blood of CTCL patients was decreased. On the other hand, serum CD226 levels were significantly elevated in CTCL patients, strongly reflecting disease activity, suggesting that soluble CD226 in sera was generated by shedding of its membrane form. Recombinant CD226 itself showed cytotoxic activity against CD155-expressing CTCL cells in vitro. These data suggest that soluble CD226 elevated in sera of CTCL patients would be important for tumor immunity by interacting with CD155 on tumor cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Expression and function of the atypical cadherin FAT1 in chronic liver disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valletta, Daniela; Czech, Barbara; Thasler, Wolfgang E.

Highlights: Black-Right-Pointing-Pointer The expression of the atypical cadherin FAT1 is increased in chronic liver disease. Black-Right-Pointing-Pointer FAT1 expression goes up during the activation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Activated HSCs are the cellular source of enhanced FAT1 expression in diseased livers. Black-Right-Pointing-Pointer FAT1 enhanced NFkB activity and resistance to apoptosis in activated HSCs. Black-Right-Pointing-Pointer FAT1 is a new therapeutic target for prevention and treatment of hepatic fibrosis. -- Abstract: Hepatic fibrosis can be considered as wound healing process in response to hepatocellular injury. Activation of hepatic stellate cells (HSCs) is a key event of hepatic fibrosis since activated HSCsmore » are the cellular source of enhanced extracellular matrix deposition, and reversion of liver fibrosis is accompanied by clearance of activated HSCs by apoptosis. The atypical cadherin FAT1 has been shown to regulate diverse biological functions as cell proliferation and planar cell polarity, and also to affect wound healing. Here, we found increased FAT1 expression in different murine models of chronic liver injury and in cirrhotic livers of patients with different liver disease. Also in hepatic tissue of patients with non-alcoholic steatohepatitis FAT1 expression was significantly enhanced and correlated with collagen alpha I(1) expression. Immunohistochemistry revealed no significant differences in staining intensity between hepatocytes in normal and cirrhotic liver tissue but myofibroblast like cells in fibrotic septa of cirrhotic livers showed a prominent immunosignal. Furthermore, FAT1 mRNA and protein expression markedly increased during in vitro activation of primary human and murine HSCs. Together, these data indicated activated HSCs as cellular source of enhanced FAT1 expression in diseased livers. To gain insight into the functional role of FAT1 in activated HSCs we suppressed FAT1 in these cells by siRNA. We newly found that FAT1 suppression in activated HSCs caused a downregulation of NF{kappa}B activity. This transcription factor is critical for apoptosis resistance of HSCs, and consequently, we detected a higher apoptosis rate in FAT1 suppressed HSCs compared to control cells. Our findings suggest FAT1 as new therapeutic target for the prevention and treatment of hepatic fibrosis in chronic liver disease.« less

Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

PubMed

Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

2013-01-01

Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.

PubMed

Rosa, Paolo; Sforna, Luigi; Carlomagno, Silvia; Mangino, Giorgio; Miscusi, Massimo; Pessia, Mauro; Franciolini, Fabio; Calogero, Antonella; Catacuzzeno, Luigi

2017-09-01

Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478-2488, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Intracellular staining for analysis of the expression and phosphorylation of signal transducers and activators of transcription (STATs) in NK cells.

PubMed

Miyagi, Takuya; Lee, Seung-Hwan; Biron, Christine A

2010-01-01

Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFNgamma, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predisposes for pSTAT4 activation by type 1 IFNs. The work has also shown, however, that total STAT1 levels are induced during viral infections as a result of IFN exposure, and that this change acts to promote the activation of STAT1 but limit both the activation of STAT4 and IFNgamma expression. The intracellular staining approaches used for the studies described here have utility in characterizing other mechanisms regulating cytokine-mediated signaling, and defining additional pathways shaping cellular responses to cytokines.

Tumor-infiltrating Tim-3+ T cells proliferate avidly except when PD-1 is co-expressed: Evidence for intracellular cross talk

PubMed Central

Li, Jing; Shayan, Gulidanna; Avery, Lyndsay; Jie, Hyun-Bae; Gildener-Leapman, Neil; Schmitt, Nicole; Lu, Bin Feng; Kane, Lawrence P.; Ferris, Robert L.

2016-01-01

ABSTRACT Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors highly expressed on tumor infiltrating T lymphocytes (TIL). PD-1 inhibits T cell activation and type-1 T cell responses, while Tim-3 is proposed to mark more extensively exhausted cells, although the mechanisms underlying Tim-3 function are not clear. Trials of anti-PD-1 therapy have identified a large subset of non-responder patients, likely due to expression of alternative checkpoint molecules like Tim-3. We investigated the phenotypic and functional characteristics of T cells with differential expression of PD-1 (high/low) and Tim-3 (positive/negative), using TIL directly isolated from head and neck squamous cell carcinomas (HNSCC). Unexpectedly, we found that expression of Tim-3 alone does not necessarily mark TIL as dysfunctional/exhausted. In Tim-3-TIL, PD-1 levels correlate with T cell dysfunction, with a PD-1low/intermed phenotype identifying recently activated and still functional cells, whereas PD-1hiTim-3− T cells are actually exhausted. Nonetheless, PD-1intermed cells are still potently suppressed by PD-L1. PD-1 expression was associated with reduced phosphorylation of ribosomal protein S6 (pS6), whereas Tim-3 expression was associated with increased pS6. Using a novel mouse model for inducible Tim-3 expression, we confirmed that expression of Tim-3 does not necessarily render T cells refractory to further activation. These results suggest the existence of PD-1 and Tim-3 crosstalk in regulating antitumor T cell responses, with important implications for anti-PD-1 immunotherapy. PMID:27853635

Effect of polysaccharide of dendrobium candidum on proliferation and apoptosis of human corneal epithelial cells in high glucose

PubMed Central

Li, Qiangxiang; Chen, Jing; Li, Yajia; Chen, Ting; Zou, Jing; Wang, Hua

2017-01-01

Abstract Background: The aim of the study was to observe the effect of polysaccharide of dendrobium candidum (PDC) and high glucose on proliferation, apoptosis of human corneal epithelial cells (HCEC). Methods: The MTT method was used to screen and take the optimal high-glucose concentration, treatment time, and PDC concentration using HCEC and divide it into 4 groups: control group (C), high glucose group (HG), PDC group, and HG + PDC group. We observed and compared the effect of the 4 groups on HCEC proliferation by MTT, apoptosis by Annexin V-FITC/PI double fluorescent staining and flow cytometry (FCM), and expression of bax mRNA and bcl-2 mRNA by RT-qPCR. Results: Compared with the control group, proliferative activity of HCEC cells was reduced; the cells apoptosis ratio was increased; the expression of bax mRNA was increased, and the expression of bcl-2 mRNA was reduced in the HG group. Proliferative activity of HCEC cells in the PDC group was increased, and the expression of bcl-2 mRNA was increased but that of bax mRNA was decreased. Proliferative activity of HCEC cells in the HG + PDC group was increased, but it could not restore to the normal level; the expression of bax mRNA was significantly decreased but the expression of bcl-2 mRNA was significantly increased. Conclusions: Our results demonstrate that high glucose can inhibit proliferative activity and induce apoptosis of HCEC. PDC can improve the proliferative activity of HCEC cells under the high glucose environment and reduce the apoptosis of cells by regulating the expression of bax and bcl-2. PDC play a very important role on protecting and repairing of corneal epithelial cells damage in high glucose. PMID:28796073

Staphylococcal enterotoxin A-activated regulatory T cells promote allergen-specific TH2 response to intratracheal allergen inoculation.

PubMed

Zeng, Wei-Ping; McFarland, Margaret M; Zhou, Baohua; Holtfreter, Silva; Flesher, Susan; Cheung, Ambrose; Mallick, Avishek

2017-02-01

T H 2 responses are implicated in asthma pathobiology. Epidemiologic studies have found a positive association between asthma and exposure to staphylococcal enterotoxins. We used a mouse model of asthma to determine whether staphylococcal enterotoxins promote T H 2 differentiation of allergen-specific CD4 conventional T (Tcon) cells and asthma by activating allergen-nonspecific regulatory T (Treg) cells to create a T H 2-polarizing cytokine milieu. Ovalbumin (OVA)-specific, staphylococcal enterotoxin A (SEA)-nonreactive naive CD4 Tcon cells were cocultured with SEA-reactive allergen-nonspecific Treg or CD4 Tcon cells in the presence of OVA and SEA. The OVA-specific CD4 T cells were then analyzed for IL-13 and IFN-γ expression. SEA-activated Treg cells were analyzed for the expression of the T H 2-polarizing cytokine IL-4 and the T-cell activation markers CD69 and CD62L. For asthma induction, mice were intratracheally sensitized with OVA or cat dander extract (CDE) alone or together with SEA and then challenged with OVA or CDE. Mice were also subject to transient Treg cell depletion before sensitization with OVA plus SEA. Asthma features and T H 2 differentiation in these mice were analyzed. SEA-activated Treg cells induced IL-13 but suppressed IFN-γ expression in OVA-specific CD4 Tcon cells. SEA-activated Treg cells expressed IL-4, upregulated CD69, and downregulated CD62L. Sensitization with OVA plus SEA but not OVA alone induced asthma, and SEA exacerbated asthma induced by CDE. Depletion of Treg cells abolished these effects of SEA and IL-13 expression in OVA-specific T cells. SEA promoted T H 2 responses of allergen-specific T cells and asthma pathogenesis by activating Treg cells. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

PubMed Central

2013-01-01

Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

PubMed

Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

2017-01-01

In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulated expression of telomerase activity in human T lymphocyte development and activation

PubMed Central

1996-01-01

Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion. PMID:8676067

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells.more » MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: Black-Right-Pointing-Pointer Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. Black-Right-Pointing-Pointer MLK3 is required for MMP expression and activity in ovarian cancer cells. Black-Right-Pointing-Pointer MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. Black-Right-Pointing-Pointer MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.« less

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

PubMed

Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

2009-01-01

The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells

PubMed Central

Watanabe, Akira; Ogiwara, Hideki; Ehata, Shogo; Mukasa, Akitake; Ishikawa, Shumpei; Maeda, Daichi; Ueki, Keisuke; Ino, Yasushi; Todo, Tomoki; Yamada, Yasuhiro; Fukayama, Masashi; Saito, Nobuhito; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells. PMID:21750150

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells.

PubMed

Jakob, F; Homann, D; Adamski, J

1995-12-01

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

PubMed

Pan, Yao; Wei, Xuetao; Hao, Weidong

2015-08-28

Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression inJurkat Cells

PubMed Central

Pan, Yao; Wei, Xuetao; Hao, Weidong

2015-01-01

Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells. PMID:26343699

Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-κB pathway in sertoli TM4 cells.

PubMed

Liu, Xiaozhen; Nie, Shaoping; Huang, Danfei; Xie, Mingyong

2015-09-01

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)-2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX-2 protein expression and interleukin-6 (IL)-6 and prostaglandin E2 (PGE2) secretion in a dose-dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), attenuated NP-induced ROS production, COX-2 expression, and IL-6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF-κB, whereas the NF-κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP-enhanced COX-2 expression and IL-6 and PGE2 release in TM4 cells in a dose-dependent manner. Furthermore, NAC blocked NP-induced activation of NF-κB. In addition, inhibition of COX-2 mitigated NP-induced IL-6 release. In conclusion, NP induced ROS generation, activation of NF-κB pathway, COX-2 upregulation, and IL-6 and PGE2 secretion in TM4 cells. NP may regulate COX-2 expression via ROS-activated NF-κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc.

Leptin modulates dose-dependently the metabolic and cytolytic activities of NK-92 cells.

PubMed

Lamas, Bruno; Goncalves-Mendes, Nicolas; Nachat-Kappes, Rachida; Rossary, Adrien; Caldefie-Chezet, Florence; Vasson, Marie-Paule; Farges, Marie-Chantal

2013-06-01

Leptin, a hormone-cytokine produced primarily in the adipose tissue, has pleiotropic effects on many biological systems and in several cell types, including immune cells. Hyperleptinemia is associated with immune dysfunction and carcinogenesis. Natural killer (NK) cells are critical mediators of anti-tumor immunity, and leptin receptor deficiency in mice leads to impaired NK function. It was thus decided to explore the in vitro effects of leptin on human NK cell function. NK-92 cells were cultured during 48 h with different leptin concentrations [absence, 10 (physiological), 100 (obesity), or 200 ng/ml (pharmacology)]. Their metabolic activity was assessed using the resazurin test. NK-92 cell cytotoxicity and intracellular IFN-γ production were analyzed by flow cytometry. NK-92 cell mRNA and protein expression levels of cytotoxic effectors were determined by RT-qPCR and Western blot. In our conditions, leptin exerted a dose-dependent stimulatory effect on NK-92 cell metabolic activity. In addition, high leptin concentrations enhanced NK-92 cell cytotoxicity against K562-EGFP and MDA-MB-231-EGFP target cells and inversely reduced cytotoxicity against the MCF-7-EGFP target. At 100 ng/ml, leptin up-regulated both NK cell granzyme B and TRAIL protein expressions and concomitantly down-regulated perforin expression without affecting Fas-L expression. In response to PMA/ionomycin stimulation, the proportion of IFN-γ expressing NK-92 cells increased with 100 and 200 ng/ml of leptin. In conclusion, leptin concentration, at obesity level, variably increased NK-92 cell metabolic activity and modulated NK cell cytotoxicity according to the target cells. The underlying mechanisms are partly due to an up-regulation of TRAIL and IFN-γ expression and a down-regulation of perforin. Copyright © 2012 Wiley Periodicals, Inc.

PPARbeta/delta agonist stimulates human lung carcinoma cell growth through inhibition of PTEN expression: the involvement of PI3K and NF-kappaB signals.

PubMed

Han, ShouWei; Ritzenthaler, Jeffrey D; Zheng, Ying; Roman, Jesse

2008-06-01

Recent studies suggest that activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) promotes cancer cell survival. We previously demonstrated that a selective PPARbeta/delta agonist, GW501516, stimulated human non-small cell lung carcinoma (NSCLC) cell growth. Here, we explore the mechanisms responsible for this effect. We show that GW501516 decreased phosphate and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor known to decrease cell growth and induce apoptosis. Activation of PPARbeta/delta and phosphatidylinositol 3-kinase (PI3K)/Akt signaling was associated with inhibition of PTEN. GW501516 increased NF-kappaB DNA binding activity and p65 protein expression through activation of PPARbeta/delta and PI3K/Akt signals and enhanced the physical interactions between PPARbeta/delta and p65 protein. Conversely, inhibition of PI3K and silencing of p65 by small RNA interference (siRNA) blocked the effect of GW501516 on PTEN expression and on NSCLC cell proliferation. GW501516 also inhibited IKBalpha protein expression. Silencing of IKBalpha enhanced the effect of GW501516 on PTEN protein expression and on cell proliferation. It also augmented the GW501516-induced complex formation of PPARbeta/delta and p65 proteins. Overexpression of PTEN suppressed NSCLC cell growth and eliminated the effect of GW501516 on phosphorylation of Akt. Together, our observations suggest that GW501516 induces the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PPARbeta/delta, which stimulates PI3K/Akt and NF-kappaB signaling. Overexpression of PTEN overcomes this effect and unveils PPARbeta/delta and PTEN as potential therapeutic targets in NSCLC.

Invariant NKT cells from HIV-1 or Mycobacterium tuberculosis-infected patients express an activated phenotype.

PubMed

Montoya, Carlos J; Cataño, Juan C; Ramirez, Zoraida; Rugeles, Maria T; Wilson, S Brian; Landay, Alan L

2008-04-01

The frequency, subsets and activation status of peripheral blood invariant NKT (iNKT) cells were evaluated in pulmonary tuberculosis (TB) patients and in chronically HIV-1-infected subjects. The absolute numbers of iNKT cells were significantly decreased in TB patients and in HIV-1+ individuals who were antiretroviral therapy naive or had detectable viremia despite receiving HAART. iNKT cell subset analysis demonstrated a decreased percentage of CD4(+) iNKT cells in HIV-1+ subjects, and a decreased percentage of double negative iNKT cells in TB patients. Peripheral blood iNKT cells from HIV-1+ and TB patients had significantly increased expression of CD69, CD38, HLA-DR, CD16, CD56, and CD62L. The expression of CD25 was significantly increased only on iNKT cells from TB patients. These findings indicate that peripheral blood iNKT cells in these two chronic infections show an up-regulated expression of activation markers, suggesting their role in the immune response to infection.

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

PubMed

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Profibrotic Effect of Interleukin-18 in HK-2 Cells Is Dependent on Stimulation of the Toll-like Receptor 4 (TLR4) Promoter and Increased TLR4 Expression*

PubMed Central

Meldrum, Kirstan K.; Zhang, Hongji; Hile, Karen L.; Moldower, Lyle L.; Dong, Zizheng; Meldrum, Daniel R.

2012-01-01

IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-β1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling. PMID:23027874

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

PubMed Central

Gilbert, Guillaume; Carvalho, Gabrielle; Trian, Thomas; Ozier, Annaig; Gillibert-Duplantier, Jennifer; Ousova, Olga; Maurat, Elise; Thumerel, Matthieu; Quignard, Jean-François; Girodet, Pierre-Olivier; Marthan, Roger; Berger, Patrick

2014-01-01

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation. BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days. Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells. In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation. PMID:24551046

Expression of HES and HEY genes in infantile hemangiomas.

PubMed

Adepoju, Omotinuwe; Wong, Alvin; Kitajewski, Alex; Tong, Karen; Boscolo, Elisa; Bischoff, Joyce; Kitajewski, Jan; Wu, June K

2011-08-11

Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.

Antiosteoporotic compounds from seeds of Cuscuta chinensis.

PubMed

Yang, Lijuan; Chen, Qianfeng; Wang, Fei; Zhang, Guolin

2011-05-17

The seeds of Cuscuta chinensis (Tu-Si-Zi, TSZ) have long been used for the treatment of osteoporosis in China and some Asian countries. The compounds in TSZ responsible for the antiosteoporotic activity are still poorly understood. The present study was designed to investigate the osteogenic compounds in TSZ, and to evaluate their antiosteoporotic effects in osteoblastic cells. Osteoblast-like UMR-106 cells were used for bioactivity-guided isolation of the active compounds. The activity of alkaline phosphatase (ALP) in UMR-106 cells was measured by p-nitrophenyl sodium phosphate assay. The proliferation of UMR-106 cells was assayed by Alamar-Blue method. Estrogenic activity of the extracts and isolated compounds was evaluated by activation of estrogen response element (ERE) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 5×ERE luciferase reporter plasmid. Antiestrogenic activity of the extracts and isolated compounds were evaluated by activation of activator protein-1 (AP-1) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 6×AP-1 luciferase reporter plasmid. ALP-guided fractionation led to the isolation of five known flavonoids, quercetin, kaempferol, isorhamnetin, hyperoside and astragalin from the crude ethanolic extract of TSZ. Further study showed that kaempferol and hyperoside significantly increased the ALP activity in UMR-106 cells. Astragalin promoted the proliferation of UMR-106 cells whereas other compounds had no such effect. The isolated compounds showed estrogenic activity but quercetin, kaempferol and isorhamnetin showed more potent ERβ agonist activity. However, compared with their ER agonist activity, only quercetin and kaempferol showed potent ER antagonist activity by activating ERα/β-mediated AP-1 reporter expression. Our findings validated the clinical use of TSZ in the treatment of osteoporosis, and demonstrated that kaempferol and hyperoside are the active compounds in TSZ for the osteogenic effect. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma

PubMed Central

Beldi-Ferchiou, Asma; Lambert, Marion; Dogniaux, Stéphanie; Vély, Frédéric; Vivier, Eric; Olive, Daniel; Dupuy, Stéphanie; Levasseur, Frank; Zucman, David; Lebbé, Céleste; Sène, Damien; Hivroz, Claire; Caillat-Zucman, Sophie

2016-01-01

Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance. PMID:27662664

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

PubMed Central

Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Evers, Maurits; Kohler, Christian; Shirneshan, Katayoon; Vockerodt, Martina; Klapper, Wolfram; Szczepanowski, Monika; Murray, Paul G.; Bastians, Holger; Trümper, Lorenz; Spang, Rainer; Kube, Dieter

2016-01-01

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery. PMID:27166259

Analysis of Ly49 gene transcripts in mature NK cells supports a role for the Pro1 element in gene activation, not gene expression.

PubMed

McCullen, M V; Li, H; Cam, M; Sen, S K; McVicar, D W; Anderson, S K

2016-09-01

The variegated expression of murine Ly49 loci has been associated with the probabilistic behavior of an upstream promoter active in immature cells, the Pro1 element. However, recent data suggest that Pro1 may be active in mature natural killer (NK) cells and function as an enhancer element. To assess directly if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA-sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49(+) NK cells that contained high levels of Pro2 transcripts. Low levels of Ly49g Pro1 transcripts were found in both Ly49G(+) and Ly49G(-) populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in interleukin-2. Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on Pro2 in a mature Ly49-expressing cell line. Taken together, the results are consistent with Pro1 transcription having a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Expression Levels of ALA Dehydratase as a Marker of ALA-PDT Efficacy

NASA Astrophysics Data System (ADS)

Avital, Schauder; Tamar, Feuerstein; Zvi, Malik

2010-05-01

Accelerated synthesis of protoporphyrinIX (PpIX) following ALA pre-treatment followed by light irradiation is the principle of ALA-PDT. Several limiting enzymes were suggested to control PpIX accumulation and PDT efficacy, among them porphobilinogen deaminase (PBGD) and ferrochelatase. Here we reveal the centrality of ALA dehydratase (ALAD) activity in predicting ALA-PDT efficacy. Silencing of ALAD expression and activity was carried out in leukemic cells using shRNA plasmid transfection or Pb2+ intoxication. ALAD activity, porphyrin synthesis and mitochondrial activity were determined versus PDT efficacy. In K562 ALAD-silenced cells, ALAD activity and expression were reduced and as a result, PpIX synthesis was almost abolished. Following ALA treatment and irradiation, ALAD-silenced cells depicted normal mitochondrial activity, in contrast to control and non-silencing transfected cells where accumulated PpIX and irradiation caused ROS formation and mitochondrial damage. Morphological analysis by scanning electron microscopy (SEM) of ALA-PDT treated cells showed no morphological changes in ALAD-silenced cells, while controls exhibited cell deformations and lysis. Annexin V-FITC/PI staining as well as LDH-L leakage testing showed that membrane integrity was undamaged following ALA-PDT in ALAD silenced cells. Pb2+ treatment in MEL cells impaired ALAD activity and reduced PpIX synthesis but to a lesser extent. In conclusion, we show that a dramatic reduction in PpIX accumulation following down regulation of ALAD expression prevents an efficient PDT. Thus, ALAD has a major role in regulating PpIX synthesis and ALA-PDT therapeutic outcome. Monitoring ALAD expression or activity in various tumors may be useful as prognostic tool to predict PDT efficacy.

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays.

PubMed

Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

2014-07-21

Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.

Repression of GSK3 restores NK cell cytotoxicity in AML patients

PubMed Central

Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A.; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A.; Gupta, Kalpana; deLima, Marcos; Beck, Rose C.; Wald, David N.

2016-01-01

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy. PMID:27040177

Development of MGD007, a gpA33 x CD3 bispecific DART® protein for T-cell immunotherapy of metastatic colorectal cancer.

PubMed

Moore, Paul A; Shah, Kalpana; Yang, Yinhua; Alderson, Ralph; Roberts, Penny; Long, Vatana; Liu, Daorong; Li, Jonathan C; Burke, Steve; Ciccarone, Valentina; Li, Hua; Fieger, Claudia B; Hooley, Jeff; Easton, Ann; Licea, Monica; Gorlatov, Sergey; King, Kathleen L; Young, Peter; Adami, Arash; Loo, Deryk; Chichili, Gurunadh R; Liu, Liqin; Smith, Douglas H; Brown, Jennifer G; Chen, Francine Z; Koenig, Scott; Mather, Jennie; Bonvini, Ezio; Johnson, Syd

2018-06-04

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART® protein designed to redirect T-cells to target gpA33 expressing colon cancer. The gpA33 target was selected based on an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic CRC specimens, including putative cancer stem cell populations. MGD007 displays the anticipated bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T-cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 µg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33 expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD 1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 µg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Copyright ©2018, American Association for Cancer Research.

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

PubMed

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

O-GlcNAcylation of NF-κB Promotes Lung Metastasis of Cervical Cancer Cells via Upregulation of CXCR4 Expression.

PubMed

Ali, Akhtar; Kim, Sung Hwan; Kim, Min Jun; Choi, Mee Young; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Jun-Young; Choi, Wan Sung

2017-07-31

C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-κB regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-κB promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-κB in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-κB p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.

Curcumin Inhibits CD4+ T Cell Activation, but Augments CD69 Expression and TGF-β1-Mediated Generation of Regulatory T Cells at Late Phase

PubMed Central

Kim, Girak; Jang, Mi Seon; Son, Young Min; Seo, Min Ji; Ji, Sang Yun; Han, Seung Hyun; Jung, In Duk; Park, Yeong-Min; Jung, Hyun Jung; Yun, Cheol-Heui

2013-01-01

Background Curcumin is a promising candidate for a natural medicinal agent to treat chronic inflammatory diseases. Although CD4+ T cells have been implicated in the pathogenesis of chronic inflammation, whether curcumin directly regulates CD4+ T cells has not been definitively established. Here, we showed curcumin-mediated regulation of CD2/CD3/CD28-initiated CD4+ T cell activation in vitro. Methodology/Principal Findings Primary human CD4+ T cells were stimulated with anti-CD2/CD3/CD28 antibody-coated beads as an in vitro surrogate system for antigen presenting cell-T cell interaction and treated with curcumin. We found that curcumin suppresses CD2/CD3/CD28-initiated CD4+ T cell activation by inhibiting cell proliferation, differentiation and cytokine production. On the other hand, curcumin attenuated the spontaneous decline of CD69 expression and indirectly increased expression of CCR7, L-selectin and Transforming growth factor-β1 (TGF-β1) at the late phase of CD2/CD3/CD28-initiated T cell activation. Curcumin-mediated up-regulation of CD69 at late phase was associated with ERK1/2 signaling. Furthermore, TGF-β1 was involved in curcumin-mediated regulation of T cell activation and late-phase generation of regulatory T cells. Conclusions/Significance Curcumin not merely blocks, but regulates CD2/CD3/CD28-initiated CD4+ T cell activation by augmenting CD69, CCR7, L-selectin and TGF-β1 expression followed by regulatory T cell generation. These results suggest that curcumin could directly reduce T cell-dependent inflammatory stress by modulating CD4+ T cell activation at multiple levels. PMID:23658623

Interleukin-8 (IL-8) over-production and autocrine cell activation are key factors in monomethylarsonous acid [MMA(III)]-induced malignant transformation of urothelial cells.

PubMed

Escudero-Lourdes, C; Wu, T; Camarillo, J M; Gandolfi, A J

2012-01-01

The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation. Copyright © 2011 Elsevier Inc. All rights reserved.

Doxycycline down-regulates matrix metalloproteinase expression and inhibits NF-κB signaling in LPS-induced PC3 cells.

PubMed

Ogut, Deniz; Reel, Buket; Gonen Korkmaz, Ceren; Arun, Mehmet Zuhuri; Cilaker Micili, Serap; Ergur, Bekir Ugur

2016-01-01

Matrix metalloproteinase enzymes (MMPs) play important role in inflammation, malignant cell proliferation, invasion and angiogenesis by mediating extracellular matrix degradation. Doxycycline, a synthetic tetracycline, behaves as a MMP inhibitor at a subantimicrobial dose and inhibits tumor cell proliferation, invasion and angiogenesis. The aberrant activity of nuclear factor kappa B (NF-κB) causes activation of MMPs and thereby proliferation and invasion of cancer cells. The aim of this study was to investigate the effects of doxycycline on the expression of MMPs in lipopolysaccharide (LPS)-induced PC3 human prostate cancer cells and the possible role of NF-κB signaling. PC3 cells were incubated with LPS (0.5 μg/mL) for 24 h in the presence or absence of doxycycline (5 μg/mL). The effects of LPS and doxycycline on the expressions of MMP-2, MMP-8, MMP-9, MMP-10, NF-κB/p65, IκB-α, p-IκB-α, IKK-β were examined by Western blotting and immunohistochemistry in PC3 cells. Furthermore, relative proteinase activities of MMP-2 and MMP-9 were determined by gelatin zymography. LPS increased expression and activity of MMP-9 and expression of MMP-8, MMP-10, NF-κB /p65, p-IκB-α, IKK-β and doxycycline down-regulated its effects with the exception of MMP-10 expression. The expression of MMP-2 and IκB-α was affected by neither LPS nor doxycycline. Our findings indicate that doxycycline inhibits the expression of various MMPs and NF-κB signaling may play a role in the regulation of MMPs expression in LPS-induced PC3 human prostate cancer cells.

Hedgehog restricts its expression domain in the Drosophila wing

PubMed Central

Bejarano, Fernando; Pérez, Lidia; Apidianakis, Yiorgos; Delidakis, Christos; Milán, Marco

2007-01-01

The stable subdivision of Drosophila limbs into anterior and posterior compartments is a consequence of asymmetrical signalling by Hedgehog (Hh), from the posterior to anterior cells. The activity of the homeodomain protein Engrailed in posterior cells helps to generate this asymmetry by inducing the expression of Hh in the posterior compartment and, at the same time, repressing the expression of the essential downstream component Cubitus interruptus (Ci). Therefore, only anterior cells that receive the Hh signal across the compartment boundary will respond by stabilizing Ci. Here, we describe a new molecular mechanism that helps to maintain the Hh-expressing and Hh-responding cells in different non-overlapping cell populations. Master of thickveins (mtv)—a target of Hh activity encoding a nuclear zinc-finger protein—is required to repress hh expression in anterior cells. Mtv exerts this action in a protein complex with Groucho (Gro)—the founding member of a superfamily of transcriptional corepressors that are conserved throughout eukaryotes. Therefore, Hh restricts its own expression domain in the Drosophila wing through the activity of Mtv and Gro. PMID:17571073

v-Src-driven transformation is due to chromosome abnormalities but not Src-mediated growth signaling.

PubMed

Honda, Takuya; Morii, Mariko; Nakayama, Yuji; Suzuki, Ko; Yamaguchi, Noritaka; Yamaguchi, Naoto

2018-01-18

v-Src is the first identified oncogene product and has a strong tyrosine kinase activity. Much of the literature indicates that v-Src expression induces anchorage-independent and infinite cell proliferation through continuous stimulation of growth signaling by v-Src activity. Although all of v-Src-expressing cells are supposed to form transformed colonies, low frequencies of v-Src-induced colony formation have been observed so far. Using cells that exhibit high expression efficiencies of inducible v-Src, we show that v-Src expression causes cell-cycle arrest through p21 up-regulation despite ERK activation. v-Src expression also induces chromosome abnormalities and unexpected suppression of v-Src expression, leading to p21 down-regulation and ERK inactivation. Importantly, among v-Src-suppressed cells, only a limited number of cells gain the ability to re-proliferate and form transformed colonies. Our findings provide the first evidence that v-Src-driven transformation is attributed to chromosome abnormalities, but not continuous stimulation of growth signaling, possibly through stochastic genetic alterations.

Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma.

PubMed

Rounseville, M P; Davis, T P

2000-08-01

A hallmark of small cell lung carcinoma (SCLC) is the expression of autocrine growth factors such as neurotensin and gastrin-releasing peptide, which bind to cellular receptors and stimulate cell division. The biological activity of autocrine growth factors requires the concurrent expression of prohormone convertases that cleave the growth factors to their active form, suggesting the expression of these genes is linked in SCLCs. RNase protection assays were used to detect the expression of autocrine growth factor and prohormone convertase mRNAs in a panel of lung cancer cell lines. These mRNAs are coexpressed in SCLC and lung carcinoid cell lines, but not in normal lung epithelium or in non-small cell lung cancers. These findings, together with earlier results from our laboratory, suggest the expression of prohormone convertases has an important role in the development and maintenance of the SCLC phenotype and that autocrine growth factor and prohormone convertase genes respond to a common transcriptional activator in SCLC.

Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

PubMed Central

Ju, Xiao-Ning; Mu, Wei-Na; Liu, Yuan-Tao; Wang, Mei-Hong; Kong, Feng; Sun, Chao; Zhou, Qing-Bo

2015-01-01

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. PMID:26823714

The Plasma Concentration of the B Cell Activating Factor Is Increased in Children With Acute Malaria

PubMed Central

Nduati, Eunice; Gwela, Agnes; Karanja, Henry; Mugyenyi, Cleopatra; Langhorne, Jean; Marsh, Kevin

2011-01-01

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells. PMID:21849293

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

PubMed Central

Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

2016-01-01

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway.

PubMed

Hou, Gang; Zhao, Huiqing; Teng, Haijun; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Guo, Zhiliang; Wei, Li; Yao, Hui; Xu, Yichun

2018-01-01

Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured by SA-β-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-β-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration. © 2018 The Author(s). Published by S. Karger AG, Basel.

The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.

PubMed

Paul, N L; Millet, I; Ruddle, N H

1993-07-01

The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.

Small molecule activation of PKM2 in cancer cells induces serine auxotrophy.

PubMed

Kung, Charles; Hixon, Jeff; Choe, Sung; Marks, Kevin; Gross, Stefan; Murphy, Erin; DeLaBarre, Byron; Cianchetta, Giovanni; Sethumadhavan, Shalini; Wang, Xiling; Yan, Shunqi; Gao, Yi; Fang, Cheng; Wei, Wentao; Jiang, Fan; Wang, Shaohui; Qian, Kevin; Saunders, Jeff; Driggers, Ed; Woo, Hin Koon; Kunii, Kaiko; Murray, Stuart; Yang, Hua; Yen, Katharine; Liu, Wei; Cantley, Lewis C; Vander Heiden, Matthew G; Su, Shinsan M; Jin, Shengfang; Salituro, Francesco G; Dang, Lenny

2012-09-21

Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Small Molecule Activation of PKM2 in Cancer Cells Induces Serine Auxotrophy

PubMed Central

Kung, Charles; Hixon, Jeff; Choe, Sung; Marks, Kevin; Gross, Stefan; Murphy, Erin; DeLaBarre, Byron; Cianchetta, Giovanni; Sethumadhavan, Shalini; Wang, Xiling; Yan, Shunqi; Gao, Yi; Fang, Cheng; Wei, Wentao; Jiang, Fan; Wang, Shaohui; Qian, Kevin; Saunders, Jeff; Driggers, Ed; Woo, Hin Koon; Kunii, Kaiko; Murray, Stuart; Yang, Hua; Yen, Katharine; Liu, Wei; Cantley, Lewis C.; Vander Heiden, Matthew G.; Su, Shinsan M.; Jin, Shengfang; Salituro, Francesco G.; Dang, Lenny

2013-01-01

SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. PMID:22999886

B-cell activating factor detected on both naïve and memory B cells in bullous pemphigoid.

PubMed

Qian, Hua; Kusuhara, Masahiro; Li, Xiaoguang; Tsuruta, Daisuke; Tsuchisaka, Atsunari; Ishii, Norito; Koga, Hiroshi; Hayakawa, Taihei; Ohara, Koji; Karashima, Tadashi; Ohyama, Bungo; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

2014-08-01

B-cell activating factor (BAFF), an important immune regulatory cytokine, is involved in development of autoimmune diseases. Although BAFF is expressed in various cells, including dendritic cells (DCs) and monocytes, BAFF expression on B cells has not been well documented. In the present study, BAFF molecules on DCs and naïve and memory B cells in autoimmune bullous diseases, including pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid (BP), were analysed by flow cytometry. Compared with healthy controls (HC), BAFF expression on naïve and memory B cells increased significantly in BP. No difference in BAFF receptor expression in naïve and memory B cells was shown among all study groups. Furthermore, BAFF expression in both naïve and memory B cells of BP, but not HC, was detected by confocal microscopic analysis. These results implied that BAFF expressed by B cells may play a pathogenic role in autoimmune bullous diseases, particularly BP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

PubMed

Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells.

PubMed

Chen, Ying-Jung; Liu, Wen-Hsin; Chang, Long-Sen

2017-02-01

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5 μM HQ for 24 h, and the cells were re-suspended in serum-containing medium without HQ for 2 days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5 μM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced β-TrCP mRNA stability and suppressed β-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

PubMed

Alexandrova, A; Ivanov, A; Chumakov, P; Kopnin, B; Vasiliev, J

2000-11-23

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovalova, Natalia, E-mail: kovalova@msu.edu

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized thatmore » TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes. - Highlights: • Kovalova TAAP Highlights Nov. 2016 • RNA-Seq identified TCDD-induced gene expression in PWM-activated primary B cells. • TCDD elicited differential expression of 515 human, 2371 mouse and 712 rat orthologs. • 28 orthologs were differentially expressed in response to TCDD in all three species. • TCDD elicits mostly species-specific gene expression changes in activated B cells.« less

Quercetin Has Antimetastatic Effects on Gastric Cancer Cells via the Interruption of uPA/uPAR Function by Modulating NF-κb, PKC-δ, ERK1/2, and AMPKα.

PubMed

Li, Hai; Chen, Chen

2018-06-01

Gastric cancer (GC) is a malignancy with few effective treatment options after metastasis occurs. Quercetin (Qu) intake has been associated with reduced incidence and slow development of GC, probably due to its anti-proliferative and apoptotic effects, but it is unclear whether Qu can inhibit the metastatic activity. The urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system plays an important role in cancer metastasis. In this study, we measured both uPA activity and uPAR expression in GC and pericarcinous tissues, and we investigated the correlation between uPAR expression and the migratory and invasive activities of various GC cell lines. GC BGC823 and AGS cells were subjected to treatment with 10 μM Qu for 72 hours and uPAR knockdown, alone or in combination, before evaluating cell metastasis. The results showed that uPA activity and uPAR expression were higher in GC tissues than in pericarcinous tissues. Migratory and invasive activities of GC cell lines positively correlated with uPAR expression. Qu treatment decreased BGC823 and AGS cell migration and invasion, accompanied by reduced uPA and uPAR protein expression. Both Qu treatment and uPAR knockdown decreased matrix metalloproteinase-2 and -9 activity and blocked Pak1-Limk1-cofilin signaling. Qu treatment was associated with inhibition of NF-κb, PKC-δ, and ERK1/2, and with AMPKα activation. Specific inhibitors of NF-κb, PKC, and ERK1/2, and an AMPKα activator suppressed uPA and uPAR expression in GC cells. Collectively, Qu showed an antimetastatic effect on GC cells via the interruption of uPA/uPAR function and modulation of NF-κb, PKC-δ, ERK1/2, and AMPKα. This suggests that Qu is a promising agent against GC metastasis.

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

NF-κB dynamics show digital activation and analog information processing in cells

NASA Astrophysics Data System (ADS)

Tay, Savas; Hughey, Jake; Lee, Timothy; Lipniacki, Tomasz; Covert, Markus; Quake, Stephen

2010-03-01

Cells operate in ever changing environments using extraordinary communication capabilities. Cell-to-cell communication is mediated by signaling molecules that form spatiotemporal concentration gradients, which requires cells to respond to a wide range of signal intensities. We used high-throughput microfluidic cell culture, quantitative gene expression analysis and mathematical modeling to investigate how single mammalian cells respond to different concentrations of the signaling molecule TNF-α via the transcription factor NF-κB. We measured NF-κB activity in thousands of live cells under TNF-α doses covering four orders of magnitude. In contrast to population studies, the activation is a stochastic, switch-like process at the single cell level with fewer cells responding at lower doses. The activated cells respond fully and express early genes independent of the TNF-α concentration, while only high dose stimulation results in the expression of late genes. Cells also encode a set of analog parameters such as the NF-κB peak intensity, response time and number of oscillations to modulate the outcome. We developed a stochastic model that reproduces both the digital and analog dynamics as well as the gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-α induced NF-κB signaling in various types of cells.

Activation of the Endoplasmic Reticulum Stress-Associated Transcription Factor X Box-Binding Protein-1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive B-Cell Lymphomas with Prognostic Implications

PubMed Central

Balague, Olga; Mozos, Ana; Martinez, Daniel; Hernandez, Luis; Colomo, Lluis; Mate, Jose Luis; Teruya-Feldstein, Julie; Lin, Oscar; Campo, Elias; Lopez-Guillermo, Armando; Martinez, Antonio

2009-01-01

X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4+/Bcl-6−/Pax-5− B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas. PMID:19389935

NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15Rα Expression

PubMed Central

Braun, Monika; Björkström, Niklas K.; Gupta, Shawon; Sundström, Karin; Ahlm, Clas; Klingström, Jonas; Ljunggren, Hans-Gustaf

2014-01-01

Clinical infection with hantaviruses cause two severe acute diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases are characterized by strong immune activation, increased vascular permeability, and up to 50% case-fatality rates. One prominent feature observed in clinical hantavirus infection is rapid expansion of natural killer (NK) cells in peripheral blood of affected individuals. We here describe an unusually high state of activation of such expanding NK cells in the acute phase of clinical Puumala hantavirus infection. Expanding NK cells expressed markedly increased levels of activating NK cell receptors and cytotoxic effector molecules. In search for possible mechanisms behind this NK cell activation, we observed virus-induced IL-15 and IL-15Rα on infected endothelial and epithelial cells. Hantavirus-infected cells were shown to strongly activate NK cells in a cell-cell contact-dependent way, and this response was blocked with anti-IL-15 antibodies. Surprisingly, the strength of the IL-15-dependent NK cell response was such that it led to killing of uninfected endothelial cells despite expression of normal levels of HLA class I. In contrast, hantavirus-infected cells were resistant to NK cell lysis, due to a combination of virus-induced increase in HLA class I expression levels and hantavirus-mediated inhibition of apoptosis induction. In summary, we here describe a possible mechanism explaining the massive NK cell activation and proliferation observed in HFRS patients caused by Puumala hantavirus infection. The results add further insights into mechanisms behind the immunopathogenesis of hantavirus infections in humans and identify new possible targets for intervention. PMID:25412359

Dexamethasone reduces mitomycin C-related inflammatory cytokine expression without inducing further cell death in corneal fibroblasts.

PubMed

Chang, Shu-Wen; Chou, San-Fang; Yu, Shuen-Yuen

2010-01-01

The purpose of this study was to investigate the effect of dexamethasone (DEX) on mitomycin C (MMC)-induced inflammatory cytokine expression in corneal fibroblasts. Primary human corneal fibroblasts were treated with MMC, dexamethasone, or in combination. Morphological changes and cell growth were documented using phase-contrast microscopy and PicoGreen assay, respectively. Cell apoptosis was evaluated by annexin V/propidium iodide staining, whereas viability was tested by the live/dead assay and analyzed by flow cytometry. The relative expression of interleukin-8 and monocyte chemoattractant protein-1 was investigated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Mitogen-activated protein kinase activation and mitogen-activated protein kinase phosphatase-1 expression were documented by Western blot analysis. We found that MMC induced corneal fibroblast elongation, apoptosis, and retarded cell growth, whereas DEX did not significantly alter cell morphology or viability. The combination of DEX and MMC did not induce additional apoptosis and cell death. DEX dose dependently down-regulated basal and MMC-induced interleukin-8 and monocyte chemoattractant protein-1 mRNA expression and protein secretion. DEX attenuated MMC-induced p38 and Jun N-terminal kinases activation and up-regulated expression. These suggested that DEX may inhibit MMC-induced interleukin-8 and monocyte chemoattractant protein-1 by up-regulating MKP-1 expression, which subsequently deactivated p38 and Jun N-terminal kinases activation. Combined MMC and DEX treatment may facilitate corneal wound healing.

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

PubMed

Kirby, R W; Martelli, A; Calderone, V; McKay, N G; Lawson, K

2013-07-15

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Yadav, Sanjiv Kumar; Foo, Chuan Han Jonathan; Sethi, Gautam; Qiang, Yu; Bellot, Gregory L; Pervaiz, Shazib

2016-05-10

We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Epidermal patterning genes are active during embryogenesis in Arabidopsis.

PubMed

Costa, Silvia; Dolan, Liam

2003-07-01

Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

Expression of interleukin-8 gene in inflammatory bowel disease is related to the histological grade of active inflammation.

PubMed Central

Mazzucchelli, L.; Hauser, C.; Zgraggen, K.; Wagner, H.; Hess, M.; Laissue, J. A.; Mueller, C.

1994-01-01

Interleukin-8 (IL-8) is a potent cytokine for recruitment and activation of neutrophils. To visualize its distribution in the intestinal mucosa and to understand better its possible role in the induction and promotion of inflammatory bowel disease, expression of the IL-8 gene was analyzed in resected bowel segments of 14 patients with active Crohn's disease or ulcerative colitis. In situ hybridization with IL-8 anti-sense RNA probes revealed strong and specific signals in the histologically affected mucosa. The number of cells expressing IL-8 gene correlated with the histological grade of active inflammation. In accordance with the characteristic histological signs of active disease, IL-8-expressing cells were diffusely distributed over the entire affected mucosa in patients with ulcerative colitis, whereas in patients with Crohn's disease, IL-8-expressing cells showed a focal distribution pattern. Cells expressing IL-8 were mainly located at the base of ulcers, in inflammatory exudates on mucosal surfaces, in crypt abscesses, and at the border of fistulae. Analysis of semi-serial sections pointed to macrophages, neutrophils, and epithelial cells as possible sources of this cytokine in active inflammatory bowel disease. We consistently failed to detect IL-8 messenger RNA in the mucosa of uninvolved bowel segments and in normal-appearing control mucosa of patients with colon cancer. In contrast, tissue specimens from two patients with acute appendicitis displayed IL-8-expressing cells in the mucosa. These results support the notion that IL-8 plays and important but nonspecific role in the pathogenesis of inflammatory bowel disease and that the production of IL-8 messenger RNA is restricted to areas with histological signs of inflammatory activity and mucosal destruction. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8178948

[The expression of interferon-lambda1 in CHO cell].

PubMed

Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

2013-06-01

To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

Ovarian steroid hormones modulate the expression of progesterone receptors and histone acetylation patterns in uterine leiomyoma cells.

PubMed

Sant'Anna, Gabriela Dos Santos; Brum, Ilma Simoni; Branchini, Gisele; Pizzolato, Lolita Schneider; Capp, Edison; Corleta, Helena von Eye

2017-08-01

Uterine leiomyomas are the most common benign smooth muscle cell tumors in women. Estrogen (E2), progesterone (P4) and environmental factors play important roles in the development of these tumors. New treatments, such as mifepristone, have been proposed. We evaluated the gene expression of total (PRT) and B (PRB) progesterone receptors, and the histone acetyltransferase (HAT) and deacetylase (HDAC) activity after treatment with E2, P4 and mifepristone (RU486) in primary cell cultures from uterine leiomyoma and normal myometrium. Compared to myometrium, uterine leiomyoma cells showed an increase in PRT mRNA expression when treated with E2, and increase in PRB mRNA expression when treated with E2 and P4. Treatment with mifepristone had no significant impact on mRNA expression in these cells. The HDAC activity was higher in uterine leiomyoma compared to myometrial cells after treatment with E2 and E2 + P4 + mifepristone. HAT activity was barely detectable. Our results suggest that ovarian steroid hormones modulate PR, and mifepristone was unable to decrease PRT and PRB mRNA. The higher activity of HDAC leiomyoma cells could be involved in transcriptional repression of genes implicated in normal myometrium cell function, contributing to the maintenance and growth of uterine leiomyoma.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

Activation of D4 dopamine receptor decreases AT1 angiotensin II receptor expression in rat renal proximal tubule cells

PubMed Central

Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

2014-01-01

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031

Malat1 activates autophagy and promotes cell proliferation by sponging miR-101 and upregulating STMN1, RAB5A and ATG4D expression in glioma.

PubMed

Fu, Zhenqiang; Luo, Wenzheng; Wang, Jingtao; Peng, Tao; Sun, Guifang; Shi, Jingyu; Li, Zhihong; Zhang, Boai

2017-10-21

The long noncoding RNA Malat1 has been reported to be an oncogene that promotes tumor progress and correlates with prognosis in glioma. Growing evidence shows that autophagy plays a very important role in tumorigenesis and tumor cell survival, but whether Malat1 regulates autophagy in glioma is still unclear. In this study, we found that Malat1 expression and autophagy activity were significantly increased in glioma tissues compared with adjacent normal tissues. Additionally, Malat1 level was positively correlated with the expression of LC3-II (autophagy marker) mRNA in vivo. In vitro assays revealed that Malat1 significantly promoted autophagy activation and cell proliferation in glioma cells. More importantly, inhibition of autophagy by 3-MA relieved Malat1-induced cell proliferation. These data demonstrated that Malat1 activates autophagy and increases cell proliferation in glioma. We further investigated the molecular mechanisms whereby Malat1 functioned on glioma cell autophagy and proliferation. We found that Malat1 served as an endogenous sponge to reduce miR-101 expression by directly binding to miR-101. Moreover, Malat1 abolished the suppression effects of miR-101 on glioma cell autophagy and proliferation, which involved in upregulating the expression of miR-101 targets STMN1, RAB5A and ATG4D. Overall, our study elucidated a novel Malat1-miR-101-STMN1/RAB5A/ATG4D regulatory network that Malat1 activates autophagy and promotes cell proliferation by sponging miR-101 and upregulating STMN1, RAB5A and ATG4D expression in glioma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression ofmore » Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.« less

Comparative characterization of stem cell marker expression, metabolic activity and resistance to doxorubicin in adherent and spheroid cells derived from the canine prostate adenocarcinoma cell line CT1258.

PubMed

Liu, Wen; Moulay, Mohammed; Willenbrock, Saskia; Roolf, Catrin; Junghanss, Christian; Ngenazahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

2015-04-01

Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Feline Glycoprotein A Repetitions Predominant Anchors Transforming Growth Factor Beta on the Surface of Activated CD4+CD25+ Regulatory T Cells and Mediates AIDS Lentivirus-Induced T Cell Immunodeficiency

PubMed Central

Miller, Michelle M.; Fogle, Jonathan E.; Ross, Peter

2013-01-01

Abstract Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP+TGFb+ Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP+ Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb+ Treg-mediated T cell immune suppression during lentivirus infection. PMID:23373523

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

PubMed

Miller, Michelle M; Fogle, Jonathan E; Ross, Peter; Tompkins, Mary B

2013-04-01

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Mechanosensory hair cells express two molecularly distinct mechanotransduction channels

PubMed Central

Zhao, Bo; Cunningham, Christopher; Harkins-Perry, Sarah; Coste, Bertrand; Ranade, Sanjeev; Zebarjadi, Navid; Beurg, Maryline; Fettiplace, Robert; Patapoutian, Ardem; Mueller, Ulrich

2016-01-01

Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. Surprisingly, we report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair-cell stereocilia and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS/LHFPL5, TMIE and TMC1/2. We show here that the second ion channel is expressed at the apical surface of hair cells and contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca2+ concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distribution. PMID:27893727

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor.

PubMed

Sordat, B; Reiter, L; Cajot, J F

1990-12-02

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.

T Cell Activation Thresholds are Affected by Gravitational

NASA Technical Reports Server (NTRS)

Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

1999-01-01

T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

The Chinese Herbal Medicine Sophora flavescens Activates Pregnane X Receptor

PubMed Central

Wang, Laiyou; Li, Feng; Lu, Jie; Li, Guodong; Li, Dan; Zhong, Xiao-bo; Guo, Grace L.

2010-01-01

Sophora flavescens (SF) is an herbal medicine widely used for the treatment of viral hepatitis, cancer, viral myocarditis, gastrointestinal hemorrhage, and skin diseases. It was recently reported that SF up-regulates CYP3A expression. The mechanism of SF-induced CYP3A expression is unknown. In the current study, we tested the hypothesis that SF-induced CYP3A expression is mediated by the activation of pregnane X receptor (PXR). We used two cell lines, DPX2 and HepaRG, to investigate the role of PXR in SF-induced CYP3A expression. The DPX2 cell line is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter. In DPX2 cells, SF activated PXR in a concentration-dependent manner. We used a metabolomic approach to identify the chemical constituents in SF, which were further analyzed for their effect on PXR activation and CYP3A regulation. One chemical in SF, N-methylcytisine, was identified as an individual chemical that activated PXR. HepaRG is a highly differentiated hepatoma cell line that mimics human hepatocytes. In HepaRG cells, N-methylcytisine significantly induced CYP3A4 expression, and this induction was suppressed by the PXR antagonist sulforaphane. These results suggest that SF induces CYP3A expression via the activation of PXR. PMID:20736322

Early induction of c-Myc is associated with neuronal cell death.

PubMed

Lee, Hyun-Pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2011-11-14

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

PubMed

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Inhibition of Protein Farnesylation Arrests Adipogenesis and Affects PPARγ Expression and Activation in Differentiating Mesenchymal Stem Cells

PubMed Central

Rivas, Daniel; Akter, Rahima; Duque, Gustavo

2007-01-01

Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARγ2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARγ, and SREBP-1 were determined by western blot. Finally, DNA binding PPARγ activity was determined using an ELISA-based PPARγ activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARγ expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARγ activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARγ expression and activity. PMID:18274630

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

PubMed

Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

2013-03-01

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of human blood levels of two PAH mixtures on the AHR signalling activation pathway and CYP1A1 and COMT target genes in granulosa non-tumor and granulosa tumor cell lines.

PubMed

Zajda, Karolina; Ptak, Anna; Rak, Agnieszka; Fiedor, Elżbieta; Grochowalski, Adam; Milewicz, Tomasz; Gregoraszczuk, Ewa L

2017-08-15

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB. Copyright © 2017. Published by Elsevier B.V.

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1.

PubMed

Ambler, Carrie A; Watt, Fiona M

2010-11-01

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.

PubMed

Abruzzese, Maria Pia; Bilotta, Maria Teresa; Fionda, Cinzia; Zingoni, Alessandra; Soriani, Alessandra; Vulpis, Elisabetta; Borrelli, Cristiana; Zitti, Beatrice; Petrucci, Maria Teresa; Ricciardi, Maria Rosaria; Molfetta, Rosa; Paolini, Rossella; Santoni, Angela; Cippitelli, Marco

2016-12-01

Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells. Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138 + MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays. Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA. These findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed Central

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity

PubMed Central

Simonetti, Giorgia; Carette, Amanda; Silva, Kathryn; Wang, Haowei; De Silva, Nilushi S.; Heise, Nicole; Siebel, Christian W.; Shlomchik, Mark J.

2013-01-01

The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity. PMID:24323359

Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

NASA Technical Reports Server (NTRS)

Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

PubMed Central

2009-01-01

Background Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Methods Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. Results We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Conclusion Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis. PMID:19527490

[Effect of GPER on the activation of PI3K/Akt induced by 17β-estradiol in endometrial carcinoma cells].

PubMed

Zhang, Yan-Cai; Guo, Rui-Xia; Ge, Xin; Qiao, Yu-Huan

2012-04-01

To investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17β-estradiol (17β-E(2))in endometrial carcinoma cells, Ishikawa and HEC-1A. Expressions of GPER, ERα and ERβ protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method. Levels of GPER, ERα and ERβ were examined by western blot in Ishikawa and HEC-1A cells after treated with 1×10(-6) mol/L 17β-E(2) at different time (0, 15, 30, 60, 120 minutes). GPER was positive expressed in Ishikawa and HEC-1A cells. ERα and ERβ were both positive expressed in Ishikawa cells. While, ERα was weakly expressed and ERβ was almost negatively expressed in HEC-1A cells. Western blot analysis showed that 1×10(-6) mol/L 17β-E(2) treatment, the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P < 0.05), which Ishikawa 30 minutes, when cells reached the highest level (0.192 ± 0.004), HEC-1A cells for 15 minutes and reached the highest level (0.184 ± 0.006); Ishikawa and HEC-1A cells, Akt, activation of 15 minutes from the treatment start was significantly increased (P < 0.05), which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021), HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1A cells, ERα and ERβ protein expression did not change significantly (P > 0.05). GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells, Ishikawa and HEC-1A.

CD4+ T cells defined by their Vβ T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis

PubMed Central

Keesen, T S L; Antonelli, L R V; Faria, D R; Guimarães, L H; Bacellar, O; Carvalho, E M; Dutra, W O; Gollob, K J

2011-01-01

Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vβ region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vβ region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4+ T cells expressing Vβ 5·2 and Vβ 24 in CL compared to controls; (2) a Leishmania antigen-induced increase in CD4+ T cells expressing Vβ 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4+ Vβ 5·2-, 11- and 24-positive T cells, with higher expression of CD45RO, HLA-DR, interferon-γ, tumour necrosis factor-α and interleukin-10 compared to other Vβ-expressing subpopulations; (4) a positive correlation between higher frequencies of CD4+Vβ5·2+ T cells and larger lesions; and (5) biased homing of CD4+ T cells expressing Vβ 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long-lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology. PMID:21726211

Regulation of androgen receptor transactivity and mTOR-S6 kinase pathway by Rheb in prostate cancer cell proliferation.

PubMed

Kobayashi, Takashi; Shimizu, Yosuke; Terada, Naoki; Yamasaki, Toshinari; Nakamura, Eijiro; Toda, Yoshinobu; Nishiyama, Hiroyuki; Kamoto, Toshiyuki; Ogawa, Osamu; Inoue, Takahiro

2010-06-01

Ras homolog-enriched in brain (Rheb), a small GTP-binding protein, is associated with prostate carcinogenesis through activating mammalian target of rapamycin (mTOR) signaling pathway. This study aimed to elucidate whether Rheb promotes proliferation of prostate cancer cells and can act as a potent therapeutic target in prostate cancer. Prostate cancer cell lines and human prostatic tissues were examined for the expression of Rheb. The effects of forced expression or knockdown of Rheb on cell proliferation were also examined. Semi-quantitative and quantitative RT-PCR were performed to evaluate mRNA expression. Western blotting was used to examine protein expression. Cell count and WST-1 assay were used to measure cell proliferation. Fluorescence-activated cell sorting was used to assess the cell cycle. Rheb mRNA and protein expression was higher in more aggressive, androgen-independent prostate cancer cell lines PC3, DU145, and C4-2, compared with the less aggressive LNCaP. Rheb expression was higher in cancer tissues than in benign prostatic epithelia. Forced expression of Rheb in LNCaP cells accelerated proliferation without enhancing androgen receptor transactivity. Attenuation of Rheb expression or treatment with the mTOR inhibitor rapamycin decreased proliferation of PC3 and DU145 cells, with a decrease in the activated form of p70S6 kinase, one of the main targets of mTOR. Rheb potentiates proliferation of prostate cancer cells and inhibition of Rheb or mTOR can lead to suppressed proliferation of aggressive prostate cancer cell lines in vitro. Rheb and the mTOR pathway are therefore probable targets for suppressing prostate cancer.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

PubMed

Matveeva, Natalia M; Fishman, Veniamin S; Zakharova, Irina S; Shevchenko, Alexander I; Pristyazhnyuk, Inna E; Menzorov, Aleksei G; Serov, Oleg L

2017-12-22

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells.

PubMed

Nakajima, Nakako Izumi; Niimi, Atsuko; Isono, Mayu; Oike, Takahiro; Sato, Hiro; Nakano, Takashi; Shibata, Atsushi

2017-08-01

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low‑dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage‑dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage‑dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

PubMed

Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

2015-08-01

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

The roles of ERAS during cell lineage specification of mouse early embryonic development.

PubMed

Zhao, Zhen-Ao; Yu, Yang; Ma, Huai-Xiao; Wang, Xiao-Xiao; Lu, Xukun; Zhai, Yanhua; Zhang, Xiaoxin; Wang, Haibin; Li, Lei

2015-08-01

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development. © 2015 The Authors.

CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brand, Stephan; Dambacher, Julia; Beigel, Florian

2005-10-15

Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting inmore » increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.« less

Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

PubMed Central

Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna; Hoffmann, Else Kay

2016-01-01

The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21Waf1/Cip1, Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21Waf1/Cip1 as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21Waf1/Cip1 and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A. PMID:26984736

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells.

PubMed

Mihaylova, Ivana; DeRuyter, Marcel; Rummens, Jean-Luc; Bosmans, Eugene; Maes, Michael

2007-08-01

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

PubMed

Shozu, M; Zhao, Y; Simpson, E R

2000-02-25

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

PubMed Central

Kojima, Hiroyuki; Takeda, Yukimasa; Muromoto, Ryuta; Takahashi, Miki; Hirao, Toru; Takeuchi, Shinji; Jetten, Anton M.; Matsuda, Tadashi

2018-01-01

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10−6 M to 1 × 10−5 M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells. PMID:25583575

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines.

PubMed

Watanabe, Masatada; Ohno, Shuji; Nakajin, Shizuo

2012-04-05

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells

PubMed Central

Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B.

2014-01-01

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors. PMID:24810746

Blocking signaling at the level of GLI regulates downstream gene expression and inhibits proliferation of canine osteosarcoma cells.

PubMed

Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B

2014-01-01

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.

Erythroblast Transformation by the Friend Spleen Focus-Forming Virus Is Associated with a Block in Erythropoietin-Induced STAT1 Phosphorylation and DNA Binding and Correlates with High Expression of the Hematopoietic Phosphatase SHP-1

PubMed Central

Nishigaki, Kazuo; Hanson, Charlotte; Ohashi, Takashi; Spadaccini, Angelo; Ruscetti, Sandra

2006-01-01

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein. PMID:16731906

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

PubMed Central

Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

2012-01-01

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

PubMed

Lanuza, Pilar M; Vigueras, Alan; Olivan, Sara; Prats, Anne C; Costas, Santiago; Llamazares, Guillermo; Sanchez-Martinez, Diego; Ayuso, José María; Fernandez, Luis; Ochoa, Ignacio; Pardo, Julián

2018-01-01

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

PubMed

Drozd, Ewa; Krzysztoń-Russjan, Jolanta; Marczewska, Jadwiga; Drozd, Janina; Bubko, Irena; Bielak, Magda; Lubelska, Katarzyna; Wiktorska, Katarzyna; Chilmonczyk, Zdzisław; Anuszewska, Elżbieta; Gruber-Bzura, Beata

2016-10-01

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Expression of activation-induced cytidine deaminase is associated with a poor prognosis of diffuse large B cell lymphoma patients treated with CHOP-based chemotherapy.

PubMed

Kawamura, Kiyoko; Wada, Akihiko; Wang, Ji-Yang; Li, Quanhai; Ishii, Akihiro; Tsujimura, Hideki; Takagi, Toshiyuki; Itami, Makiko; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2016-01-01

Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation and class switch recombination processes in the antibody formation. The AID activity induces gene mutations and could be associated with transformation processes of B cells. Nevertheless, the relation between AID expression and the prognosis of B cell lymphoma patients remains uncharacterized. We examined expression levels of the AID gene in 89 lymph node specimens from lymphoma and non-lymphoma patients with Northern blot analysis and investigated an association with their survival. The AID gene was preferentially expressed in B cell lymphoma in particular in diffuse large B cell lymphoma and follicular lymphoma. We confirmed AID protein expression in the mRNA-positive but not in the negative specimens with Western blot analysis and immunohistochemical staining. Survival of the patients treated with cyclophosphamide-/doxorubicin-/vincristine-/prednisone-based chemotherapy demonstrated that the prognosis of diffuse large B cell patients was unfavorable in the mRNA-positive group compared with the negative group, and that AID expression levels were correlated with the poor prognosis. In contrast, AID expression was not linked with the prognosis of follicular lymphoma patients. AID expression is a predictive marker for an unfavorable outcome in DLBCL patients treated with the chemotherapy.

Tissue factor expression by endothelial cells in sickle cell anemia.

PubMed

Solovey, A; Gui, L; Key, N S; Hebbel, R P

1998-05-01

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

PubMed

Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

2015-01-01

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanism of Hormonal Regulation of CAD Gene Expression in Human Breast Cancer Cells

DTIC Science & Technology

2006-05-01

showed that PGJ2, troglitazone, and ciglitazone inhibit E2-stimulated cell proliferation of a leiomyoma -derived cell line and human primary leiomyoma ...cultures and also inhibit ER-mediated gene expression and protein expression. These results suggested that in uterine leiomyomas PPARγ activation is...peroxisome proliferator-activated receptor gamma ligands in uterine leiomyoma . Cancer Res. 63, 1221-1227. Hulka, BS., Liu, ET., and Lininger, RA., 1994

LIGHT: A Novel Immunotherapy for Primary and Metastatic Prostate Cancer

DTIC Science & Technology

2015-11-01

R), is predominantly expressed on activated immune cells , signaling via LTR is required for the formation of organized lymphoid tissues while...expressed on activated immune cells . Signaling via LTβR is required for the formation of organized lymphoid tissues while signaling via HVEM...required for the formation of organized lymphoid tissues. Forced expression of LIGHT recruits naive T cells into tumors and is capable of establishing

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway, but not AP-1, in MCF-7 breast cancer cells.

PubMed

Lee, Young-Rae; Noh, Eun-Mi; Han, Ji-Hey; Kim, Jeong-Mi; Hwang, Bo-Mi; Kim, Byeong-Soo; Lee, Sung-Ho; Jung, Sung Hoo; Youn, Hyun Jo; Chung, Eun Yong; Kim, Jong-Suk

2013-04-01

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells.

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway, but not AP-1, in MCF-7 breast cancer cells

PubMed Central

Lee, Young-Rae; Noh, Eun-Mi; Han, Ji-Hey; Kim, Jeong-Mi; Hwang, Bo-Mi; Kim, Byeong-Soo; Lee, Sung-Ho; Jung, Sung Hoo; Youn, Hyun Jo; Chung, Eun Yong; Kim, Jong-Suk

2013-01-01

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206] PMID:23615261

Real-time monitoring of artemin in vivo chaperone activity using luciferase as an intracellular reporter.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Asghari, S Mohsen

2016-11-15

Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones. Copyright © 2016 Elsevier Inc. All rights reserved.

Antitumor action of lymphokin-activated cells of patients with soft tissue sarcomas and melanomas in dependence on expression of MHC classes I and II antigenes.

PubMed

Berezhnaya, N M; Vinnichuk, U D; Belova, O B; Baranovich, V V

2006-09-01

To study expression of major histocompatibility complex (MHC) classes and antigens and CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA on lymphocytes and tumor cells and antitumor action of lymphocytes activated with IL-2. Tumor explants (soft tissue sarcoma, n = 20, melanoma, n = 25) were co-cultivated in diffusion chambers with autologous lymphocytes; antitumor action was evaluated by morphologic patterns of explant's growth. Expression of CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA was evaluated by the method of indirect fluorescence using respective monoclonal antibodies. The highest antitumor action of lymphocytes toward soft tissue sarcoma and melanoma cells is observed if tumor cells are expressing MHC class I antigens. In the cases of soft tissue sarcoma no correlation between the level of antitumor activity of lymphocytes and expression of CD25, CD71, Ki-67, CD54, CD56, CD11b, PCNA has been found, whilst in the case of melanoma it is associated with the high level of CD11b expression. There is a direct correlation between sensitivity of soft tissue sarcoma and melanoma cells to action of lymphokin-activated killer cells and the level of MHC class I antigens.

Quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet‐induced obese mice

PubMed Central

Takahashi, Yumiko; Sakurai, Mutsumi; Akimoto, Yukari; Tsushida, Tojiro; Oike, Hideaki; Ippoushi, Katsunari

2015-01-01

Scope To examine the effect of dietary quercetin on the function of epididymal adipose tissue (EAT) in Western diet‐induced obese mice. Methods and results C57BL/6J mice were fed a control diet; a Western diet high in fat, cholesterol, and sucrose; or the same Western diet containing 0.05% quercetin for 18 weeks. Supplementation with quercetin suppressed the increase in the number of macrophages, the decrease in the ratio of CD4+ to CD8+ T cells in EAT, and the elevation of plasma leptin and tumor necrosis factor α levels in mice fed the Western diet. Comprehensive gene expression analysis revealed that quercetin suppressed gene expression associated with the accumulation and activation of immune cells, including macrophages and lymphocytes in EAT. It also improved the expression of the oxidative stress‐sensitive transcription factor NFκB, NADPH oxidases, and antioxidant enzymes. Quercetin markedly increased gene expression associated with mitochondrial oxidative phosphorylation and mitochondrial DNA content. Conclusion Quercetin most likely universally suppresses the accumulation and activation of immune cells, including antiinflammatory cells, whereas it specifically increased gene expression associated with mitochondrial oxidative phosphorylation. Suppression of oxidative stress and NFκB activity likely contributed to the prevention of the accumulation and activation of immune cells and resulting chronic inflammation. PMID:26499876

Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen.

PubMed Central

Fox, D A; Millard, J A; Kan, L; Zeldes, W S; Davis, W; Higgs, J; Emmrich, F; Kinne, R W

1990-01-01

Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis. Images PMID:2212003

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR

DOE PAGES

Wang, Yunshan; Zhang, Pengju; Liu, Ziming; ...

2014-11-21

CUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear. Expression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively. We found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpressionmore » of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities. In conclusion, our results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.« less

Human NK cells: From surface receptors to clinical applications.

PubMed

Moretta, Lorenzo; Pietra, Gabriella; Vacca, Paola; Pende, Daniela; Moretta, Francesca; Bertaina, Alice; Mingari, Maria Cristina; Locatelli, Franco; Moretta, Alessandro

2016-10-01

Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK cells derived from donor HSC. Another promising approach is based on the use of anti-KIR blocking monoclonal antibodies (mAbs), rendering alloreactive any KIR(+) NK cells. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Molecular expression in transfected corneal endothelial cells

NASA Astrophysics Data System (ADS)

Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

2017-10-01

To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Kyung Sook; Jo, Ji Yoon; Kim, Su Jin

2011-04-29

Highlights: {yields} Elafin expression is epigenetically silenced in human melanoma cells. {yields} Foxa2 expression in melanoma cells is silenced by promoter hypermethylation. {yields} Foxa2 directs activation of the elafin promoter in vivo. {yields} Foxa2 expression induces apoptosis of melanoma cells via elafin re-expression. -- Abstract: Elafin, a serine protease inhibitor, induces the intrinsic apoptotic pathway in human melanoma cells, where its expression is transcriptionally silenced. However, it remains unknown how the elafin gene is repressed in melanoma cells. We here demonstrate that elafin expression is modulated via epigenetically regulated expression of the transcription factor Foxa2. Treatment of melanoma cells withmore » a DNA methyltransferase inhibitor induced elafin expression, which was specifically responsible for reduced proliferation and increased apoptosis. Suppression of Foxa2 transcription, mediated by DNA hypermethylation in its promoter region, was released in melanoma cells upon treatment with the demethylating agent. Luciferase reporter assays indicated that the Foxa2 binding site in the elafin promoter was critical for the activation of the promoter. Chromatin immunoprecipitation assays further showed that Foxa2 bound to the elafin promoter in vivo. Analyses of melanoma cells with varied levels of Foxa2 revealed a correlated expression between Foxa2 and elafin and the ability of Foxa2 to induce apoptosis. Our results collectively suggest that, in melanoma cells, Foxa2 expression is silenced and therefore elafin is maintained unexpressed to facilitate cell proliferation in the disease melanoma.« less

Hippo signaling promotes JNK-dependent cell migration.

PubMed

Ma, Xianjue; Wang, Hongxiang; Ji, Jiansong; Xu, Wenyan; Sun, Yihao; Li, Wenzhe; Zhang, Xiaoping; Chen, Juxiang; Xue, Lei

2017-02-21

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam -Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicidemore » gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.« less

Different induction of LPA receptors by chemical liver carcinogens regulates cellular functions of liver epithelial WB-F344 cells.

PubMed

Hirane, Miku; Ishii, Shuhei; Tomimatsu, Ayaka; Fukushima, Kaori; Takahashi, Kaede; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

2016-11-01

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA 1 to LPA 6 ) mediates a variety of cellular functions, including cell motility. In the present study, we investigated the effects of LPA receptors on cell motile activity during multi-stage hepatocarcinogenesis in rat liver epithelial WB-F344 cells treated with chemical liver carcinogens. Cells were treated with a initiator (N-nitrosodiethylamine (DEN)) and three promoters (phenobarbital (PB), okadaic acid (OA) and clofibrate) every 24 h for 2 days. Cell motile activity was elevated by DEN, correlating with Lpar3 expression. PB, OA, and clofibrate elevated Lpar1 expression and inhibited cell motile activity. To evaluate the effects of long-term treatment on cell motility, cells were treated with DEN and/or PB for at least 6 months. Lpar3 expression and cell motile activity were significantly elevated by the long-term DEN treatment with or without further PB treatment. In contrast, long-term PB treatment with or without further DEN elevated Lpar1 expression and inhibited cell motility. When the synthesis of extracellular LPA was blocked by a potent ATX inhibitor S32826 before cell motility assay, the cell motility induced by DEN and PB was markedly suppressed. These results suggest that activation of the different LPA receptors may regulate the biological functions of cells treated with chemical carcinogens. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors

PubMed Central

Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

2015-01-01

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

The transcriptome of the human mast cell leukemia cells HMC-1.2: an approach to identify specific changes in the gene expression profile in KitD816V systemic mastocytosis.

PubMed

Haenisch, B; Herms, S; Molderings, G J

2013-05-01

To circumvent the costly isolation procedure associated with tissue mast cells, human mast cell lines such as HMC-1 are employed in mastocytosis research, but their relation to mutated mast cells in systemic mastocytosis has not been investigated systematically. In the present study, we determined the transcriptome of HMC-1.2 cells and compared the expression data with those reported in the literature for normal human resting lung and tonsillar mast cells as well as leukocytes from peripheral blood and mononuclear cells from bone marrow aspirates of patients with D816 V-positive systemic mastocytosis. Our results suggest that HMC-1.2 cells are an appropriate model for the investigation of this variant of systemic mast cell activation disease. The data confirm previous suggestions that the pathologically increased activity of mast cells in patients with D816 V-positive systemic mastocytosis can be deduced from the detection of mutation-related changes in the gene expression profile in leukocytes from peripheral blood and in mononuclear cells from bone marrow aspirates. Thus, mutation-related changes of the expression profile can serve as surrogates (besides clustering of mast cells, expression of CD25, and increased release of tryptase) for the presence of the mutation D816 V in tyrosine kinase Kit in patients with systemic mastocytosis according to the WHO criteria. Whether this also holds true for systemic mast cell activation disease caused by other mutations in Kit or other mast cell activity-related genes is a subject for future studies.

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

PubMed

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Chacon, Jessica A; Figini, Mariangela; Powell, Daniel J

2016-07-20

The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.

CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

PubMed

Jia, Xiaoyi; Wei, Fang; Sun, Xiaojing; Chang, Yan; Xu, Shu; Yang, Xuezhi; Wang, Chun; Wei, Wei

2016-08-02

Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability to inhibit the expression of BAFF-R in CD4(+) T cells in a co-culture. These observations might provide a scientific basis for the development of new drugs for the treatment of autoimmune diseases by CP-25. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[Long-term expansion of multipotent mesenchymal stromal cells under reduced oxygen tension].

PubMed

Rylova, Iu V; Buravkova, L B

2013-01-01

We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.

Novel function of STAT1beta in B cells: induction of cell death by a mechanism different from that of STAT1alpha.

PubMed

Najjar, Imen; Schischmanoff, Pierre Olivier; Baran-Marszak, Fanny; Deglesne, Pierre-Antoine; Youlyouz-Marfak, Ibtissam; Pampin, Mathieu; Feuillard, Jean; Bornkamm, Georg W; Chelbi-Alix, Mounira K; Fagard, Remi

2008-12-01

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp; Haniu, Hisao

2012-08-03

Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO,more » DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.« less

β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

PubMed

Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

2015-06-01

β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells. © 2014 Wiley Periodicals, Inc.

17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.

PubMed

Girgert, Rainer; Emons, Günter; Gründker, Carsten

2017-02-01

Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptor α expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclin D1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑ and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200 nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7 days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200 nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70, 147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets

PubMed Central

2011-01-01

Background It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells. Results We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells. Conclusion Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response. PMID:21914188

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

PubMed

Lindholm, Paul F; Lu, Yi; Adley, Brian P; Vladislav, Tudor; Jovanovic, Borko; Sivapurapu, Neela; Yang, Ximing J; Kajdacsy-Balla, André

2010-11-01

Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

Temporal Characterization of Microglia/Macrophage Phenotypes in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

PubMed Central

Hellström Erkenstam, Nina; Smith, Peter L. P.; Fleiss, Bobbi; Nair, Syam; Svedin, Pernilla; Wang, Wei; Boström, Martina; Gressens, Pierre; Hagberg, Henrik; Brown, Kelly L.; Sävman, Karin; Mallard, Carina

2016-01-01

Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury. PMID:28018179

mNotch1 signaling and erythropoietin cooperate in erythroid differentiation of multipotent progenitor cells and upregulate beta-globin.

PubMed

Henning, Konstanze; Schroeder, Timm; Schwanbeck, Ralf; Rieber, Nikolaus; Bresnick, Emery H; Just, Ursula

2007-09-01

In many developing tissues, signaling mediated by activation of the transmembrane receptor Notch influences cell-fate decisions, differentiation, proliferation, and cell survival. Notch receptors are expressed on hematopoietic cells and cognate ligands on bone marrow stromal cells. Here, we investigate the role of mNotch1 signaling in the control of erythroid differentiation of multipotent progenitor cells. Multipotent FDCP-mix cell lines engineered to permit the conditional induction of the constitutively active intracellular domain of mNotch1 (mN1(IC)) by the 4-hydroxytamoxifen (OHT)-inducible system were used to analyze the effects of activated mNotch1 on erythroid differentiation and on expression of Gata1, Fog1, Eklf, NF-E2, and beta-globin. Expression was analyzed by Northern blotting and real-time polymerase chain reaction. Enhancer activity of reporter constructs was determined with the dual luciferase system in transient transfection assays. Induction of mN1(IC) by OHT resulted in increased and accelerated differentiation of FDCP-mix cells along the erythroid lineage. Erythroid maturation was induced by activated Notch1 also under conditions that normally promote self-renewal, but required the presence of erythropoietin for differentiation to proceed. While induction of Notch signaling rapidly upregulated Hes1 and Hey1 expression, the expression of Gata1, Fog1, Eklf, and NF-E2 remained unchanged. Concomitantly with erythroid differentiation, activated mNotch1 upregulated beta-globin RNA. Notch signaling transactivated a reporter construct harboring a conserved RBP-J (CBF1) binding site in the hypersensitive site 2 (HS2) of human beta-globin. Transactivation by activated Notch was completely abolished when this RBP-J site was mutated to prevent RBP-J binding. Our results show that activation of mNotch1 induces erythroid differentiation in cooperation with erythropoietin and upregulates beta-globin expression.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Decidual activin: its role in the apoptotic process and its regulation by prolactin.

PubMed

Tessier, Christian; Prigent-Tessier, Anne; Bao, Lei; Telleria, Carlos M; Ferguson-Gottschall, Susan; Gibori, Gil B; Gu, Yan; Bowen-Shauver, Jennifer M; Horseman, Nelson D; Gibori, Geula

2003-05-01

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

EFFECT OF THAI SARAPHI FLOWER EXTRACTS ON WT1 AND BCR/ABL PROTEIN EXPRESSION IN LEUKEMIC CELL LINES.

PubMed

Sangkaruk, Rungkarn; Rungrojsakul, Methee; Tima, Singkome; Anuchapreeda, Songyot

2017-01-01

Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC 50 values of 2.6 and 77.6 μg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WTlprotein expressions by 75 and 49.5%, respectively when compared to vehicle control. The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

PubMed Central

Knight, B L; Patel, D D; Soutar, A K

1983-01-01

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow. PMID:6305342

Statins meditate anti-atherosclerotic action in smooth muscle cells by peroxisome proliferator-activated receptor-γ activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuda, Kazuki; Matsumura, Takeshi, E-mail: takeshim@gpo.kumamoto-u.ac.jp; Senokuchi, Takafumi

Highlights: • Statins induce PPARγ activation in vascular smooth muscle cells. • Statin-induced PPARγ activation is mediated by COX-2 expression. • Statins suppress cell migration and proliferation in vascular smooth muscle cells. • Statins inhibit LPS-induced inflammatory responses by PPARγ activation. • Fluvastatin suppress the progression of atherosclerosis and induces PPARγ activation in the aorta of apoE-deficient mice. - Abstract: The peroxisome proliferator-activated receptor-γ (PPARγ) is an important regulator of lipid and glucose metabolism, and its activation is reported to suppress the progression of atherosclerosis. We have reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) activate PPARγ in macrophages. However,more » it is not yet known whether statins activate PPARγ in other vascular cells. In the present study, we investigated whether statins activate PPARγ in smooth muscle cells (SMCs) and endothelial cells (ECs) and thus mediate anti-atherosclerotic effects. Human aortic SMCs (HASMCs) and human umbilical vein ECs (HUVECs) were used in this study. Fluvastatin and pitavastatin activated PPARγ in HASMCs, but not in HUVECs. Statins induced cyclooxygenase-2 (COX-2) expression in HASMCs, but not in HUVECs. Moreover, treatment with COX-2-siRNA abrogated statin-mediated PPARγ activation in HASMCs. Statins suppressed migration and proliferation of HASMCs, and inhibited lipopolysaccharide-induced expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in HASMCs. These effects of statins were abrogated by treatment with PPARγ-siRNA. Treatment with statins suppressed atherosclerotic lesion formation in Apoe{sup −/−} mice. In addition, transcriptional activity of PPARγ and CD36 expression were increased, and the expression of MCP-1 and TNF-α was decreased, in the aorta of statin-treated Apoe{sup −/−} mice. In conclusion, statins mediate anti-atherogenic effects through PPARγ activation in SMCs. These effects of statins on SMCs may be beneficial for the prevention of atherosclerosis.« less

FoxP3 Expression in Macrophages, Cancer, and B Cells-Is It Real?

PubMed

Vadasz, Zahava; Toubi, Elias

2017-06-01

During the last decade, B regulatory cells are appreciated to have a central role in preventing autoimmunity and maintaining self-tolerance. They are characterized by expressing different phenotypic markers and the production of either IL-10 or TGF-β or both. The recent recognition of Fas ligand expressing B regulatory cells as "killer" cells established their role in maintaining viral persistence by preventing effective antiviral immune responses. The forkhead lineage-transcription factor (FoxP3) was considered for many years to be a highly specific intracellular regulatory marker of CD4+CD25+ T regulatory cells. The possibility of FoxP3 being expressed in B regulatory cells was suggested in many studies. Though controversial, FoxP3 expression was also reported in macrophages and cancer cells. Aiming to avoid artifact staining, many researchers required the usage of FoxP3 messenger RNA (mRNA) and PCR in order to prove a true expression of FoxP3 in these different cells. In addition, most studies' report on that FoxP3 expression in all abovementioned cells is related to their status of activation since naïve (non-activated cells) were found poorly FoxP3 expressing. In this review, we present the existing data on FoxP3 expression in non-T-regulatory cells, but we suggest that further studies are needed to better establish this concept.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

PubMed Central

Domae, Eisuke; Hirai, Yuya; Ikeo, Takashi; Goda, Seiji; Shimizu, Yoji

2017-01-01

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells. PMID:28521284

Inhibition of cell proliferation and induction of apoptosis by oleanane triterpenoid (CDDO-Me) in pancreatic cancer cells is associated with the suppression of hTERT gene expression and its telomerase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo

2012-06-15

Highlights: Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT gene expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT protein expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT telomerase activity. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT regulatory proteins. -- Abstract: Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a multifunctional oleanane synthetic triterpenoid with potent anti-inflammatory and antitumorigenic properties. The mechanisms of the antisurvival and apoptosis-inducing activities of CDDO-Me and related derivatives of oleanolic acid have been defined; however, to date, no study has been carried out on the effect of CDDOs on human telomerase reverse transcriptase (hTERT) gene or telomerase activity. Here we report for the first time that inhibition of cell proliferation and induction of apoptosismore » by CDDO-Me in pancreatic cancer cell lines is associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT expression and activity. Furthermore, abrogation or overexpression of hTERT protein altered the susceptibility of tumor cells to CDDO-Me. These findings suggest that telomerase (hTERT) is a relevant target of CDDO-Me in pancreatic cancer cells.« less

IRE1α links Nck1 deficiency to attenuated PTP1B expression in HepG2 cells.

PubMed

Li, Hui; Li, Bing; Larose, Louise

2017-08-01

PTP1B, a prototype of the non-receptor subfamily of the protein tyrosine phosphatase superfamily, plays a key role in regulating intracellular signaling from various receptor and non-receptor protein tyrosine kinases. Previously, we reported that silencing Nck1 in human hepatocellular carcinoma HepG2 cells enhances basal and growth factor-induced activation of the PI3K-Akt pathway through attenuating PTP1B expression. However, the underlying mechanism by which Nck1 depletion represses PTP1B expression remains unclear. In this study, we found that silencing Nck1 attenuates PTP1B expression in HepG2 cells through down-regulation of IRE1α. Indeed, we show that silencing Nck1 in HepG2 cells leads to decreased IRE1α expression and signaling. Accordingly, IRE1α depletion using siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation, reproducing the effects of silencing Nck1 on activation of this pathway. In addition, depletion of IRE1α also leads to reduced PTP1B expression, which was rescued by ectopic expression of IRE1α in Nck1-depleted cells. Mechanistically, we found that silencing either Nck1 or IRE1α in HepG2 cells decreases PTP1B mRNA levels and stability. However, despite miR-122 levels, a miRNA targeting PTP1B 3' UTR and inducing PTP1B mRNA degradation in HepG2 cells, are increased in both Nck1- and IRE1α-depleted HepG2 cells, a miR-122 antagomir did not rescue PTP1B expression in these cells. Overall, this study highlights an important role for Nck1 in fine-tuning IRE1α expression and signaling that regulate PTP1B expression and subsequent activation of the PI3K-Akt pathway in HepG2 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model.

PubMed

Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

2016-09-01

Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo , and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

PubMed

Liu, Jun; Ben, Qi-Wen; Yao, Wei-Yan; Zhang, Jian-Jun; Chen, Da-Fan; He, Xiang-Yi; Li, Lei; Yuan, Yao-Zong

2012-06-01

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

Role of P-glycoprotein on CD69+CD4+ cells in the pathogenesis of proliferative lupus nephritis and non-responsiveness to immunosuppressive therapy

PubMed Central

Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

Introduction P-glycoprotein (P-gp) expression on activated lymphocytes in systemic lupus erythematosus (SLE) plays a role in active efflux of intracellular drugs, resulting in drug resistance. The role of P-gp-expressing lymphocytes in the pathogenesis of SLE remains unclear. The aim of this study was to determine the importance of P-gp+CD4+ cells in organ manifestations in refractory SLE. Methods The proportion of P-gp+CD4+ cells was determined by flow cytometry in peripheral blood of patients with SLE (n=116) and healthy adults (n=10). Renal biopsy specimens were examined by immunohistochemistry for P-gp expression. Results CD69 is a marker of CD4 cell activation. The proportion of both P-gp-expressing CD4+ cells and CD69-expressing CD4+ cells in peripheral blood was higher in SLE than control. The proportion of P-gp+CD69+CD4+ cells correlated with Systemic Lupus Erythematosus Disease Activity Index and was higher in poor responders to corticosteroids. Furthermore, the proportion of P-gp+CD69+CD4+ cells was significantly higher in proliferative lupus nephritis (LN) with poor response to corticosteroids. The efficacy of immunosuppressive therapy depended on the regulation of the proportion of P-gp+CD69+CD4+ cells. Marked accumulation of P-gp+CD4+ cells in renal interstitial tissue and high proportion of peripheral P-gp+CD69+CD4+ cells were noted in patients with proliferative LN. Conclusions The results showed high proportion of P-gp+CD69+CD4+ cells in peripheral blood and their accumulation in renal tissue in patients with proliferative LN refractory to CS therapy, suggesting that P-gp expression on activated CD4+ T cells is a potentially useful marker for refractoriness to treatment and a novel target for treatment. PMID:29225917

Role of P-glycoprotein on CD69+CD4+ cells in the pathogenesis of proliferative lupus nephritis and non-responsiveness to immunosuppressive therapy.

PubMed

Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Tanaka, Yoshiya

2017-01-01

P-glycoprotein (P-gp) expression on activated lymphocytes in systemic lupus erythematosus (SLE) plays a role in active efflux of intracellular drugs, resulting in drug resistance. The role of P-gp-expressing lymphocytes in the pathogenesis of SLE remains unclear. The aim of this study was to determine the importance of P-gp + CD4 + cells in organ manifestations in refractory SLE. The proportion of P-gp + CD4 + cells was determined by flow cytometry in peripheral blood of patients with SLE (n=116) and healthy adults (n=10). Renal biopsy specimens were examined by immunohistochemistry for P-gp expression. CD69 is a marker of CD4 cell activation. The proportion of both P-gp-expressing CD4 + cells and CD69-expressing CD4 + cells in peripheral blood was higher in SLE than control. The proportion of P-gp + CD69 + CD4 + cells correlated with Systemic Lupus Erythematosus Disease Activity Index and was higher in poor responders to corticosteroids. Furthermore, the proportion of P-gp + CD69 + CD4 + cells was significantly higher in proliferative lupus nephritis (LN) with poor response to corticosteroids. The efficacy of immunosuppressive therapy depended on the regulation of the proportion of P-gp + CD69 + CD4 + cells. Marked accumulation of P-gp + CD4 + cells in renal interstitial tissue and high proportion of peripheral P-gp + CD69 + CD4 + cells were noted in patients with proliferative LN. The results showed high proportion of P-gp + CD69 + CD4 + cells in peripheral blood and their accumulation in renal tissue in patients with proliferative LN refractory to CS therapy, suggesting that P-gp expression on activated CD4 + T cells is a potentially useful marker for refractoriness to treatment and a novel target for treatment.

[Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage].

PubMed

Chen, Juan-Juan; Huang, Si-Yong; Ma, Peng-Fei; Wu, Bi-Jia; Zhou, Si-Lang; Zhao, Yong-Xing; Gong, Jun-Mei; Liang, Ying-Min

2018-04-01

To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni 2+ -beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin - Scal-1 + c-Kit + cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.

Monocrotophos Induces the Expression and Activity of Xenobiotic Metabolizing Enzymes in Pre-Sensitized Cultured Human Brain Cells

PubMed Central

Tripathi, Vinay K.; Kumar, Vivek; Singh, Abhishek K.; Kashyap, Mahendra P.; Jahan, Sadaf; Pandey, Ankita; Alam, Sarfaraz; Khan, Feroz; Khanna, Vinay K.; Yadav, Sanjay; Lohani, Mohtshim; Pant, Aditya B.

2014-01-01

The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics. PMID:24663500

The Role of Cholecystokinin in Peripheral Taste Signaling in Mice

PubMed Central

Yoshida, Ryusuke; Shin, Misa; Yasumatsu, Keiko; Takai, Shingo; Inoue, Mayuko; Shigemura, Noriatsu; Takiguchi, Soichi; Nakamura, Seiji; Ninomiya, Yuzo

2017-01-01

Cholecystokinin (CCK) is a gut hormone released from enteroendocrine cells. CCK functions as an anorexigenic factor by acting on CCK receptors expressed on the vagal afferent nerve and hypothalamus with a synergistic interaction between leptin. In the gut, tastants such as amino acids and bitter compounds stimulate CCK release from enteroendocrine cells via activation of taste transduction pathways. CCK is also expressed in taste buds, suggesting potential roles of CCK in taste signaling in the peripheral taste organ. In the present study, we focused on the function of CCK in the initial responses to taste stimulation. CCK was coexpressed with type II taste cell markers such as Gα-gustducin, phospholipase Cβ2, and transient receptor potential channel M5. Furthermore, a small subset (~30%) of CCK-expressing taste cells expressed a sweet/umami taste receptor component, taste receptor type 1 member 3, in taste buds. Because type II taste cells are sweet, umami or bitter taste cells, the majority of CCK-expressing taste cells may be bitter taste cells. CCK-A and -B receptors were expressed in both taste cells and gustatory neurons. CCK receptor knockout mice showed reduced neural responses to bitter compounds compared with wild-type mice. Consistently, intravenous injection of CCK-Ar antagonist lorglumide selectively suppressed gustatory nerve responses to bitter compounds. Intravenous injection of CCK-8 transiently increased gustatory nerve activities in a dose-dependent manner whereas administration of CCK-8 did not affect activities of bitter-sensitive taste cells. Collectively, CCK may be a functionally important neurotransmitter or neuromodulator to activate bitter nerve fibers in peripheral taste tissues. PMID:29163209

Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression.

PubMed

Hancock, Meaghan H; Corcoran, Jennifer A; Smiley, James R

2006-08-15

HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.

Vitamin D Receptor Expression in Plasmablastic Lymphoma and Myeloma Cells Confers Susceptibility to Vitamin D

PubMed Central

Lyne, Linden; Spearman, Hayley; Buffa, Francesca M.; Soilleux, Elizabeth J.; Banham, Alison H.

2017-01-01

Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphomaDLBCL. These diseases can be difficult to diagnose and treat, and they lack well-characterized cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORA, IGF1, and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumor cells and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyzes active vitamin D3 production. Although Vdr and Cyp27b1 transcription in normal B cells were activated by interleukin 4 (IL-4) and CD40 signaling, respectively, unstimulated malignant plasmablastic cells lacking IL-4 expressed both VDR and CYP27B1. Positive autoregulation evidenced intact VDR function in all plasmablastic lines, and inhibition of growth by active vitamin D3 was both dependent on MYC protein inhibition and could be enhanced by cotreatment with a synthetic ROR ligand SR-1078. Furthermore, a VDR polymorphism, FOK1, was associated with greater vitamin D3–dependent growth inhibition. In summary, HLY-1 provides an important model of strongly plasmablastic lymphoma, and disruption of VDR pathway activity may be of therapeutic benefit in both plasmablastic lymphoma and myeloma. PMID:28001444

[Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

PubMed

Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

2017-04-01

This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
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Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jiahua; Jedinak, Andrej; Sliva, Daniel, E-mail: dsliva@iuhealth.org

2011-11-18

Highlights: Black-Right-Pointing-Pointer Ganodermanontriol (GDNT), a Ganoderma mushroom alcohol, inhibits growth of breast cancer cells. Black-Right-Pointing-Pointer CDC20 is over-expressed in tumors but not in the tumor surrounding tissue in breast cancer patients. Black-Right-Pointing-Pointer GDNT inhibits expression of CDC20 in breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits cell adhesion, cell migration and cell invasion of breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits secretion of uPA and down-regulates expression of uPAR in breast cancer cells. -- Abstract: Ganoderma lucidum is a medicinal mushroom that has been recognized by Traditional Chinese Medicine (TCM). Although some of the direct anticancer activities are attributed to the presence ofmore » triterpenes-ganoderic and lucidenic acids-the activity of other compounds remains elusive. Here we show that ganodermanontriol (GDNT), a Ganoderma alcohol, specifically suppressed proliferation (anchorage-dependent growth) and colony formation (anchorage-independent growth) of highly invasive human breast cancer cells MDA-MB-231. GDNT suppressed expression of the cell cycle regulatory protein CDC20, which is over-expressed in precancerous and breast cancer cells compared to normal mammary epithelial cells. Moreover, we found that CDC20 is over-expressed in tumors when compared to the tissue surrounding the tumor in specimens from breast cancer patients. GDNT also inhibited invasive behavior (cell adhesion, cell migration, and cell invasion) through the suppression of secretion of urokinase-plasminogen activator (uPA) and inhibited expression of uPA receptor. In conclusion, mushroom GDNT is a natural agent that has potential as a therapy for invasive breast cancers.« less

Differential Expression of FAK and Pyk2 in Metastatic and Non-metastatic EL4 Lymphoma Cell Lines

PubMed Central

Zhang, Zhihong; Knoepp, Stewart M.; Ku, Hsun; Sansbury, Heather M.; Xie, Yuhuan; Chahal, Manpreet S.; Tomlinson, Stephen; Meier, Kathryn E.

2011-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells. PMID:21533871

Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines.

PubMed

Zhang, Zhihong; Knoepp, Stewart M; Ku, Hsun; Sansbury, Heather M; Xie, Yuhuan; Chahal, Manpreet S; Tomlinson, Stephen; Meier, Kathryn E

2011-08-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Comparative study of cathepsins D and S in rat IPE and RPE cells.

PubMed

Sugano, Eriko; Tomita, Hiroshi; Abe, Toshiaki; Yamashita, Asahi; Tamai, Makoto

2003-08-01

To investigate differences between activities related to phagocytosis in iris pigment epithelial (IPE) and retinal pigment epithelial (RPE) cells, an aspartic protease, cathepsin D (cat D), and a cysteine protease, cathepsin S (cat S), of IPE and RPE were studied. IPE and RPE cells were isolated from Long Evans rat eyes. The origin of the isolated cells was determined by pigmentation and cytokeratin labelling. The mRNA expressions of cat D and cat S in cultured IPE or RPE cells were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activities of cat D and cat S in IPE or RPE cells were measured by using specific fluorogenic substrates, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(Dnp)D-Arg-NH2 and Z-Val-Val-Arg-MCA, respectively. Western blot analysis of both proteins was also performed. The cultured cells, both of IPE and RPE cells were pigmented and showed positive labelling with an anti-cytokeratin monoclonal antibody. The cat D activity in RPE cells was 37 times that in IPE cells. The cat S activity in RPE cells was four times that in IPE cells. On the other hand, mRNA expression levels of cat D in RPE cells were at the same level with IPE cells, cat S mRNA expression in RPE cells were 10 times that in IPE cells. These results were also correlated with the Western blot analysis. In this study, we measured the characteristic expressions of cat D and S in IPE and RPE cells for the first time to compare their lysosomal activities. IPE cells have the lysosomal activities like RPE cells, however, the function of lysosomal activity in IPE cells is beneath RPE's. These results indicated that the ability of ROS digestion in IPE cells was not same as RPE cells.

Classical and alternative activation of rat hepatic sinusoidal endothelial cells by inflammatory stimuli.

PubMed

Liu, Yinglin; Gardner, Carol R; Laskin, Jeffrey D; Laskin, Debra L

2013-02-01

The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2 h with 5 mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui

2015-04-01

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressedmore » c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.« less

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

PubMed

Qian, Jiaying; Niu, Jiangong; Li, Ming; Chiao, Paul J; Tsao, Ming-Sound

2005-06-15

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Expression and function of the atypical cadherin FAT1 in chronic liver disease.

PubMed

Valletta, Daniela; Czech, Barbara; Thasler, Wolfgang E; Müller, Martina; Bosserhoff, Anja-Katrin; Hellerbrand, Claus

2012-09-28

Hepatic fibrosis can be considered as wound healing process in response to hepatocellular injury. Activation of hepatic stellate cells (HSCs) is a key event of hepatic fibrosis since activated HSCs are the cellular source of enhanced extracellular matrix deposition, and reversion of liver fibrosis is accompanied by clearance of activated HSCs by apoptosis. The atypical cadherin FAT1 has been shown to regulate diverse biological functions as cell proliferation and planar cell polarity, and also to affect wound healing. Here, we found increased FAT1 expression in different murine models of chronic liver injury and in cirrhotic livers of patients with different liver disease. Also in hepatic tissue of patients with non-alcoholic steatohepatitis FAT1 expression was significantly enhanced and correlated with collagen alpha I(1) expression. Immunohistochemistry revealed no significant differences in staining intensity between hepatocytes in normal and cirrhotic liver tissue but myofibroblast like cells in fibrotic septa of cirrhotic livers showed a prominent immunosignal. Furthermore, FAT1 mRNA and protein expression markedly increased during in vitro activation of primary human and murine HSCs. Together, these data indicated activated HSCs as cellular source of enhanced FAT1 expression in diseased livers. To gain insight into the functional role of FAT1 in activated HSCs we suppressed FAT1 in these cells by siRNA. We newly found that FAT1 suppression in activated HSCs caused a downregulation of NFκB activity. This transcription factor is critical for apoptosis resistance of HSCs, and consequently, we detected a higher apoptosis rate in FAT1 suppressed HSCs compared to control cells. Our findings suggest FAT1 as new therapeutic target for the prevention and treatment of hepatic fibrosis in chronic liver disease. Copyright © 2012 Elsevier Inc. All rights reserved.

UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-β1-FAP-α.

PubMed

Wäster, Petra; Orfanidis, Kyriakos; Eriksson, Ida; Rosdahl, Inger; Seifert, Oliver; Öllinger, Karin

2017-08-08

Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-α is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-α is not yet completely revealed. Expression of FAP-α was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-β1. Fibroblast activation protein-α expression was induced by UVR in melanocytes of human skin. The FAP-α expression was regulated by UVR-induced release of TGF-β1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-α mediated ECM degradation and facilitated tumour cell dissemination. Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-β1 and FAP-α expression, promoting cancer cell dissemination and melanoma metastatic spread.

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

NASA Astrophysics Data System (ADS)

Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

2002-08-01

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation

PubMed Central

Yang, Li-yun; He, Chang-yu; Chen, Xue-hua; Su, Li-ping; Liu, Bing-ya; Zhang, Hao

2016-01-01

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment. PMID:27356739

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Youn-hee; Kim, Donghern; Dai, Jin

Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 uponmore » acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit reduced activities of p53 and p21. • Cr(VI)-transformed cells exhibit increased activities of p53 and p21.« less

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

PubMed Central

Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

2014-01-01

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10–50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24 hours showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 hours, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2Kb 5′. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 minutes in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions. PMID:24518819

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

PubMed

Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

2014-03-01

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of Neonicotinoid Pesticides on Promoter-Specific Aromatase (CYP19) Expression in Hs578t Breast Cancer Cells and the Role of the VEGF Pathway.

PubMed

Caron-Beaudoin, Élyse; Viau, Rachel; Sanderson, J Thomas

2018-04-26

Aromatase (CYP19) is a key enzyme in estrogens biosynthesis. In the mammary gland, CYP19 gene is expressed at low levels under the regulation of its I.4 promoter. In hormone-dependent breast cancer, fibroblast cells surrounding the tumor express increased levels of CYP19 mRNA due to a decrease of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter activity. Little is known about the effects of environmental chemicals on the promoter-specific CYP19 expression. We aimed to determine the effects of two neonicotinoids (thiacloprid and imidacloprid) on promoter-specific CYP19 expression in Hs578t breast cancer cells and understand the signaling pathways involved. Hs578t cells were exposed to various signaling pathway stimulants or neonicotinoids for 24 h. Promoter-specific expression of CYP19 was determined by real-time quantitative polymerase chain reaction and catalytic activity of aromatase by tritiated water release assay. To our knowledge, we are the first to demonstrate that the normal I.4 promoter and the breast cancer-relevant PII, I.3, and I.7 promoters of CYP19 are active in these cells. We found that the expression of CYP19 via promoters PII, I.3, and I.7 in Hs578t cells was, in part, dependent on the activation of two VEGF signaling pathways: mitogen-activated protein kinase (MAPK) 1/3 and phospholipase C (PLC). Exposure of Hs578t cells to environmental concentrations of imidacloprid and thiacloprid resulted in a switch in CYP19 promoter usage, involving inhibition of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter-mediated CYP19 expression and aromatase catalytic activity. Greater effects were seen at lower concentrations. Our results suggest that thiacloprid and imidacloprid exert their effects at least partially by inducing the MAPK 1/3 and/or PLC pathways. We demonstrated in vitro that neonicotinoids may stimulate a change in CYP19 promoter usage similar to that observed in patients with hormone-dependent breast cancer. https://doi.org/10.1289/EHP2698.

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

PubMed Central

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-01-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with ≤ 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3− CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0·001) but increased to a normal level during the 24 months’ follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0·001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0·05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells. PMID:16045743

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

PubMed

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-09-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

PubMed

Ueno, Koji; Hirata, Hiroshi; Majid, Shahana; Tabatabai, Z Laura; Hinoda, Yuji; Dahiya, Rajvir

2011-11-15

The Wnt/β-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ β-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and β-catenin expression was increased. Also the β-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis. Copyright © 2011 UICC.

Reactivation of Embryonic Nodal Signaling is Associated with Tumor Progression and Promotes the Growth of Prostate Cancer Cells

PubMed Central

Lawrence, Mitchell G.; Margaryan, Naira V.; Loessner, Daniela; Collins, Angus; Kerr, Kris M.; Turner, Megan; Seftor, Elisabeth A.; Stephens, Carson R.; Lai, John; BioResource, APC; Postovit, Lynne-Marie; Clements, Judith A.; Hendrix, Mary J.C.

2011-01-01

Background Nodal is a member of the Transforming Growth Factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity. Methods Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. Results Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal’s co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. Conclusions An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells. PMID:21656830

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense.

PubMed

Hansdottir, Sif; Monick, Martha M; Hinde, Sara L; Lovan, Nina; Look, Dwight C; Hunninghake, Gary W

2008-11-15

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.

2007-06-29

Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells,more » not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.« less

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

PubMed

Huang, Zi-Ming; Kang, Jhi-Kai; Chen, Chih-Yu; Tseng, Tz-Hau; Chang, Chien-Wen; Chang, Yung-Chi; Tai, Shyh-Kuan; Hsieh, Shie-Liang; Leu, Chuen-Miin

2012-06-15

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonen, Nitzan; Bram, Eran E.; Assaraf, Yehuda G.

2008-11-28

The proton-coupled folate transporter (PCFT/SLC46A1) displays optimal and prominent folate and antifolate transport activity at acidic pH in human carcinoma cells but poor activity in leukemia cells. Consistently herein, human leukemia cell lines expressed poor PCFT transcript levels, whereas various carcinoma cell lines showed substantial PCFT gene expression. We identified a CpG island with high density at nucleotides -200 through +100 and explored its role in PCFT promoter silencing. Leukemia cells with barely detectable PCFT transcripts consistently harbored 85-100% methylation of this CpG island, whereas no methylation was found in carcinoma cells. Treatment with 5-Aza-2'-deoxycytidine which induced demethylation but notmore » with the histone deacetylase inhibitor trichostatin A, restored 50-fold PCFT expression only in leukemia cells. These findings constitute the first demonstration of the dominant epigenetic silencing of the PCFT gene in leukemia cells. The potential translational implications of the restoration of PCFT expression in chemotherapy of leukemia are discussed.« less

[Tryptase inhibits cell apoptosis through upregulating PAR-2 and Rho kinase in rheumatoid arthritis synovial fibroblasts].

PubMed

Zheng, Qianqian; Li, Shigang; Jia, Yunli; Liu, Wei; Yu, Lingling; Chen, Xianyong; Wang, Jinling

2016-12-01

Objective To investigate the regulatory effects of tryptase on protease-activated receptors 2 (PAR-2), Rho signal pathway and apoptosis of MH7A rheumatoid arthritis synovial fibroblasts. Methods In MH7A cells, the expression of PAR-2 was measured by flow cytometry; cell apoptosis was examined by annexin V- FITC/PI staining combined with flow cytometry; the expression of Rho kinase was detected by Pull-down assay and Western botting. Results Tryptase upregulated the expression of PAR-2 in MH7A cells, and suppressed Fas-mediated apoptosis of MH7A cells in a dose-dependent manner. Meanwhile, PAR-2 inhibitor, FSLLRY-NH2 significantly reduced anti-apoptotic effects of tryptase in MH7A cells, which was related with the increase of activated Rho kinase expression. Conclusion Tryptase plays a role in resistance to the apoptosis of MH7A cells through raising PAR-2 and activating Rho kinase.

The Effects of TLR Activation on T-Cell Development and Differentiation

PubMed Central

Jin, Bo; Sun, Tao; Yu, Xiao-Hong; Yang, Ying-Xiang; Yeo, Anthony E. T.

2012-01-01

Invading pathogens have unique molecular signatures that are recognized by Toll-like receptors (TLRs) resulting in either activation of antigen-presenting cells (APCs) and/or costimulation of T cells inducing both innate and adaptive immunity. TLRs are also involved in T-cell development and can reprogram Treg cells to become helper cells. T cells consist of various subsets, that is, Th1, Th2, Th17, T follicular helper (Tfh), cytotoxic T lymphocytes (CTLs), regulatory T cells (Treg) and these originate from thymic progenitor thymocytes. T-cell receptor (TCR) activation in distinct T-cell subsets with different TLRs results in differing outcomes, for example, activation of TLR4 expressed in T cells promotes suppressive function of regulatory T cells (Treg), while activation of TLR6 expressed in T cells abrogates Treg function. The current state of knowledge of regarding TLR-mediated T-cell development and differentiation is reviewed. PMID:22737174

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhishan; Humphries, Brock; Xiao, Hua

2013-08-15

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchialmore » epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro-angiogenic effect. • EMT in arsenic-transformed cells activates β-catenin. • β-Catenin activation increases VEGF expression in arsenic-transformed cells. • β-Catenin activation is likely independent of canonical Wnt signaling. • EMT in arsenic-transformed cells promotes angiogenesis via β-catenin–VEGF pathway.« less

FGF-1-induced matrix metalloproteinase-9 expression in breast cancer cells is mediated by increased activities of NF-kappaB and activating protein-1.

PubMed

Lungu, Gina; Covaleda, Lina; Mendes, Odete; Martini-Stoica, Heidi; Stoica, George

2008-06-01

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK. (c) 2007 Wiley-Liss, Inc.

Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

PubMed Central

Zweidler-McKay, Patrick A.; He, Yiping; Xu, Lanwei; Rodriguez, Carlos G.; Karnell, Fredrick G.; Carpenter, Andrea C.; Aster, Jon C.; Allman, David; Pear, Warren S.

2005-01-01

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies. PMID:16118316

Interleukin-5 regulates genes involved in B-cell terminal maturation.

PubMed

Horikawa, Keisuke; Takatsu, Kiyoshi

2006-08-01

Interleukin (IL)-5 induces CD38-activated splenic B cells to differentiate into immunoglobulin M-secreting cells and undergo micro to gamma 1 class switch recombination (CSR) at the DNA level, resulting in immunoglobulin G1 (IgG1) production. Interestingly, IL-4, a well-known IgG1-inducing factor does not induce immunoglobulin production or micro to gamma 1 CSR in CD38-activated B cells. In the present study, we implemented complementary DNA microarrays to investigate the contribution of IL-5-induced gene expression in CD38-stimulated B cells to immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR. IL-5 and IL-4 stimulation of CD38-activated B cells induced the expression of 418 and 289 genes, respectively, that consisted of several clusters. Surprisingly, IL-5-inducible 78 genes were redundantly regulated by IL-4. IL-5 and IL-4 also suppressed the gene expression of 319 and 325 genes, respectively, 97 of which were overlapped. Genes critically regulated by IL-5 include immunoglobulin-related genes such as J chain and immunoglobulinkappa, and genes involved in B-cell maturation such as BCL6, activation-induced cytidine deaminase (Aid) and B lymphocyte-induced maturation protein-1 (Blimp-1) and tend to be induced slowly after IL-5 stimulation. Intriguingly, among genes, the retroviral induction of Blimp-1 and Aid in CD38-activated B cells could induce IL-4-dependent maturation to Syndecan-1+ antibody-secreting cells and micro to gamma 1 CSR, respectively, in CD38-activated B cells. Taken together, preferential Aid and Blimp-1 expression plays a critical role in IL-5-induced immunoglobulin-secreting cell differentiation and micro to gamma 1 CSR in CD38-activated B cells.

The effect of inhibitory signals on the priming of drug-hapten-specific T-cells that express distinct Vβ receptors

PubMed Central

Gibson, Andrew; Faulkner, Lee; Lichtenfels, Maike; Ogese, Monday; Al-Attar, Zaid; Alfirevic, Ana; Esser, Philipp R.; Martin, Stefan F.; Pirmohamed, Munir; Park, B. Kevin; Naisbitt, Dean J.

2017-01-01

Drug hypersensitivity involves the activation of T-cells in an HLA allele-restricted manner. Since the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T-cell response. Thus, we have utilized a T-cell priming assay and nitroso sulfamethoxazole (SMX-NO) as a model antigen to investigate (1) the activation of specific T-cell receptor (TCR)Vβ subtypes, (2) the impact of PD-1, CTLA4 and TIM-3 co-inhibitory signalling on activation of naïve and memory T-cells and (3) the ability of Tregs to prevent responses. An expansion of the TCR repertoire was observed for nine different Vβ subtypes, while spectratyping revealed that SMX-NO-specific T-cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vβ-restricted antigen-specific T-cell responses is governed by regulatory signals. Blockade of PDL-1/CTLA4 signalling dampened activation of SMX-NO-specific naïve and memory T-cells, while blockade of TIM-3 produced no effect. PD-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T-cell activation and expression of each receptor was enhanced on dividing T-cells. As these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T-cell activation was investigated. Tregs significantly dampened the priming of T-cells. In conclusion, our findings demonstrate that distinct TCR Vβ subtypes, dysregulation of co-inhibitory signalling pathways and dysfunctional Tregs may influence predisposition to hypersensitivity. PMID:28687658

Epigenetic hierarchy governing Nestin expression.

PubMed

Han, Dong Wook; Do, Jeong Tae; Araúzo-Bravo, Marcos J; Lee, Sung Ho; Meissner, Alexander; Lee, Hoon Taek; Jaenisch, Rudolf; Schöler, Hans R

2009-05-01

Nestin is an intermediate filament protein expressed specifically in neural stem cells and progenitor cells of the central nervous system. DNA demethylation and histone modifications are two types of epigenetic modifications working in a coordinate or synergistic manner to regulate the expression of various genes. This study investigated and elucidated the epigenetic regulation of Nestin gene expression during embryonic differentiation along the neural cell lineage. Nestin exhibits differential DNA methylation and histone acetylation patterns in Nestin-expressing and nonexpressing cells. In P19 embryonic carcinoma cells, activation of Nestin expression is mediated by both trichostatin A and 5-aza-2'-deoxycytidine treatment, concomitant with histone acetylation, but not with DNA demethylation. Nestin transcription is also mediated by treatment with retinoic acid, again in the absence of DNA demethylation. Thus, histone acetylation is sufficient to mediate the activation of Nestin transcription. This study proposed that the regulation of Nestin gene expression can be used as a model to study the epigenetic regulation of gene expression mediated by histone acetylation, but not by DNA demethylation.

Gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells.

PubMed

Chen, Ying-Jung; Lin, Ku-Nan; Jhang, Li-Mei; Huang, Chia-Hui; Lee, Yuan-Chin; Chang, Long-Sen

2016-05-25

Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

AMPK regulates energy metabolism through the SIRT1 signaling pathway to improve myocardial hypertrophy.

PubMed

Dong, H-W; Zhang, L-F; Bao, S-L

2018-05-01

We investigated the correlations of adenosine monophosphate-activated protein kinase (AMPK), Silence information regulator 1 (SIRT1) and energy metabolism with myocardial hypertrophy. Myocardial hypertrophy experimental model was established via transverse aortic constriction (TAC)-induced myocardial hypertrophy and phenylephrine (PE)-induced hypertrophic myocardial cell culture. After activation of AMPK, the messenger ribonucleic acid (mRNA) expressions in myocardial tissue- and myocardial cell hypertrophy-related genes, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC), were detected. The production rate of 14C-labeled 14CO2 from palmitic acid was quantitatively determined to detect the fatty acid and glucose oxidation of hypertrophic myocardial tissues or cells, and the glucose uptake of myocardial cells was studied using [14C] glucose. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to detect the changes in SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues. Moreover, SIRT1 small interfering ribonucleic acid (siRNA) was used to interfere in SIRT1 expression to further investigate the role of SIRT1 in the effect of AMPK activation on myocardial hypertrophy. AMPK activation could significantly reduce the mRNA expressions of ANP and β-MHC in vitro and in vivo. AMPK could increase the ejection fraction (EF) and decrease the protein synthesis rate in myocardial cells in mice with myocardial hypertrophy. Besides, AMPK activation could increase the fatty acid oxidation, improve the glucose uptake and reduce the glucose oxidation. After AMPK activation, both SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues and myocardial cells were increased. After SIRT1 siRNA was further used to interfere in SIRT1 expression in myocardial cells, it was found that mRNA expressions and protein synthesis rates of ANP and β-MHC were increased. The activation of AMPK can inhibit the myocardial hypertrophy, which may be realized through regulating the myocardial energy metabolism via SIRT1 signaling pathway.

The B-cell receptor controls fitness of MYC-driven lymphoma cells via GSK3β inhibition.

PubMed

Varano, Gabriele; Raffel, Simon; Sormani, Martina; Zanardi, Federica; Lonardi, Silvia; Zasada, Christin; Perucho, Laura; Petrocelli, Valentina; Haake, Andrea; Lee, Albert K; Bugatti, Mattia; Paul, Ulrike; Van Anken, Eelco; Pasqualucci, Laura; Rabadan, Raul; Siebert, Reiner; Kempa, Stefan; Ponzoni, Maurilio; Facchetti, Fabio; Rajewsky, Klaus; Casola, Stefano

2017-06-08

Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR - ) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR + ) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR + tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3β) activity to support MYC-controlled gene expression. BCR - tumour cells exhibit increased GSK3β activity and are rescued from their competitive growth disadvantage by GSK3β inhibition. BCR - lymphoma variants that restore competitive fitness normalize GSK3β activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR - tumour cells.

An essential role for IL-2 receptor in regulatory T cell function

PubMed Central

Levine, Andrew G; Fan, Xiying; Klein, Ulf; Zheng, Ye; Gasteiger, Georg; Feng, Yongqiang; Fontenot, Jason D.; Rudensky, Alexander Y.

2016-01-01

Regulatory T (Treg) cells, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature implied a key role for a simple network based on IL-2 consumption by Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg cell lineage specification factor Foxp3, confounding experimental efforts to understand the role of IL-2R expression and signaling in Treg suppressor function. Using genetic gain and loss of function approaches, we demonstrate that IL-2 capture is dispensable for control of CD4+ T cells, but is important for limiting CD8+ T cell activation, and that IL-2R dependent STAT5 transcription factor activation plays an essential role in Treg cell suppressor function separable from T cell receptor signaling. PMID:27595233

Cholesterol depletion in cell membranes of human airway epithelial cells suppresses MUC5AC gene expression.

PubMed

Song, Kee Jae; Kim, Na Hyun; Lee, Gi Bong; Kim, Ji Hoon; Kwon, Jin Ho; Kim, Kyung-Su

2013-05-01

If cholesterol in the cell membrane is depleted by treating cells with methyl-β-cyclodextrin (MβCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1β is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1β showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. After NCI-H292 cells were pretreated with 1% MβCD before adding IL-1β for 24 hours, MUC5AC mRNA expression was determined by reverse transcription- polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MβCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. Cholesterol in the cell membrane was significantly depleted by treatment with MβCD on cells. IL-1β-induced MUC5AC mRNA expression was decreased by MβCD and this decrease occurred IL-1-receptor- specifically. Moreover, we have shown that MβCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1β. This result suggests that MβCD-mediated suppression of IL-1β-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.

A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts

PubMed Central

Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna; Schrøder, Henrik Daa

2013-01-01

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts. PMID:23717533

Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome.

PubMed

Parvaiz, Fahed; Manzoor, Sobia; Iqbal, Jawed; McRae, Steven; Javed, Farrakh; Ahmed, Qazi Laeeque; Waris, Gulam

2014-05-01

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

PubMed Central

Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

2016-01-01

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

Repression of the expression of PPP3CC by ZEB1 confers activation of NF-κB and contributes to invasion and growth in glioma cells.

PubMed

Wang, Hongquan; Zhao, Shuli; Chen, Bo; Fu, Chuhua; Dang, Yanwei; Fang, Peihai; Wang, Jun; Wang, Ning; Liu, Lijun

2018-02-01

Gliomas are highly malignant brain tumors. Aberrant activation of NF-κB plays a crucial role in tumor progression. ELISA assay was used to detect NF-κB activity in glimoas cells with different treatments. PPP3CC expression was evaluated by qRT-PCR and western blot assay. Kaplan-Meier analysis estimated the overall survival rates according to the protein level of PPP3CC. Transwell and MTS assay were performed to determine cell invasion and growth. Chromatin immunoprecipitation combined with luciferase reporter assays illustrated the transcriptional regulation of PPP3CC. We showed that PPP3CC decrease was responsible for constitutive activation of NF-κB in gliomas. Restored PPP3CC expression inhibited activation of NF-κB. PPP3CC was frequently decreased in gliomas and that repression of the expression of PPP3CC correlated glioma progression. The ectopic expression of PPP3CC inhibited the invasive potential of glioma cells, and inhibited glioma cells proliferation in vitro and growth in vivo. Additionally, the expression of Zinc finger E-box-binding homeobox 1(ZEB1) was increased in gliomas and was negatively correlated with clinical outcomes of glioma patients. ZEB1 inversely correlated with the expression of PPP3CC. ZEB1 was also confirmed to physically bind to the PPP3CC promoter. ZEB1 knockdown resulted in an increase in the expression of PPP3CC and elevation of PPP3CC promoter activity in glioma cells. These findings indicated that the down-regulation of PPP3CC by ZEB1 resulted in activation of NF-κB is a critical oncogenic event in gliomas. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.

PubMed

Lam, Adeline R; Bert, Nina Le; Ho, Samantha Sw; Shen, Yu J; Tang, Li Fm; Xiong, Gordon M; Croxford, John L; Koo, Christine X; Ishii, Ken J; Akira, Shizuo; Raulet, David H; Gasser, Stephan

2014-04-15

The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3(+/-);Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3(+/+);Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance. ©2014 AACR.

Effect of β-glucan on MUC4 and MUC5B expression in human airway epithelial cells.

PubMed

Kim, Yong-Dae; Bae, Chang Hoon; Song, Si-Youn; Choi, Yoon Seok

2015-08-01

β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan-induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of p38 MAPK and NF-κB. These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4-p38 MAPK-NF-κB signaling pathway. © 2015 ARS-AAOA, LLC.

Casein Kinase II Regulation of the Hot1 Transcription Factor Promotes Stochastic Gene Expression*

PubMed Central

Burns, Laura T.; Wente, Susan R.

2014-01-01

In Saccharomyces cerevisiae, Hog1 MAPK is activated and induces a transcriptional program in response to hyperosmotic stress. Several Hog1-responsive genes exhibit stochastic transcription, resulting in cell-to-cell variability in mRNA and protein levels. However, the mechanisms governing stochastic gene activity are not fully defined. Here we uncover a novel role for casein kinase II (CK2) in the cellular response to hyperosmotic stress. CK2 interacts with and phosphorylates the Hot1 transcription factor; however, Hot1 phosphorylation is not sufficient for controlling the stochastic response. The CK2 protein itself is required to negatively regulate mRNA expression of Hot1-responsive genes and Hot1 enrichment at target promoters. Single-cell gene expression analysis reveals altered activation of Hot1-targeted STL1 in ck2 mutants, resulting in a bimodal to unimodal shift in expression. Together, this work reveals a novel CK2 function during the hyperosmotic stress response that promotes cell-to-cell variability in gene expression. PMID:24817120

HSP70 in human polymorphonuclear and mononuclear leukocytes: comparison of the protein content and transcriptional activity of HSPA genes.

PubMed

Boyko, Anna A; Azhikina, Tatyana L; Streltsova, Maria A; Sapozhnikov, Alexander M; Kovalenko, Elena I

2017-01-01

Cell-type specific variations are typical for the expression of different members of the HSP70 family. In circulating immune cells, HSP70 proteins interact with units of signaling pathways involved in the immune responses and may promote cell survival in sites of inflammation. In this work, we compared basal HSP70 expression and stress-induced HSP70 response in polymorphonuclear and mononuclear human leukocytes. The intracellular content of inducible and constitutive forms of HSP70 was analyzed in relation to the transcriptional activity of HSPA genes. Hyperthermia was used as the stress model for induction of HSP70 synthesis in the cells. Our results demonstrated that granulocytes (mainly neutrophils) and mononuclear cells differ significantly by both basal HSP70 expression and levels of HSP70 induction under hyperthermia. The differences were observed at the levels of HSPA gene transcription and intracellular HSP70 content. The expression of constitutive Hsс70 protein was much higher in mononuclear cells consisting of monocytes and lymphocytes than in granulocytes. At the same time, intact neutrophils showed increased expression of inducible Hsp70 protein compared to mononuclear cells. Heat treatment induced additional expression of HSPA genes in leukocytes. The most pronounced increase in the expression was observed in polymorphonuclear and mononuclear leukocytes for HSPA1A/B. However, in granulocytes, the induction of the transcription of the HSPA8 gene encoding the Hsc70 protein was significantly higher than in mononuclear cells. These variations in transcriptional activity of HSPA genes and intracellular HSP70 content in different populations of leukocytes may reflect specified requirements for the chaperone activity in the cells with a distinct functional role in the immune system.

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

PubMed

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. © 2013 Elsevier Inc. All rights reserved.

Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons

RXRα and LXR activate two promoters in placenta- and tumor-specific expression of PLAC1

PubMed Central

Chen, Yaohui; Moradin, Adi; Schlessinger, David; Nagaraja, Ramaiah

2011-01-01

PLAC1 expression, first characterized as restricted to developing placenta among normal tissues, is also found in a wide range of tumors and transformed cell lines. To understand the basis for its unusual expression profile, we have analyzed the gene structure and its mode of transcription. We find that the gene has a hitherto unique feature, with two promoters, P1 and P2, separated by 105 kb. P2 has been described before. Here we define P1 and show that it and P2 are activated by RXRα in conjunction with LXRα or LXRβ. In placenta, P2 is the preferred promoter, whereas various tumor cell lines tend to express predominantly either one or the other promoter. Furthermore, when each promoter is fused to a luciferase reporter gene and transfected into cancer cell lines, the promoter corresponding to the more active endogenous promoter is preferentially transcribed. Joint expression of activating nuclear receptors can partially account for the restricted expression of PLAC1 in placenta, and may be co-opted for preferential P1 or P2 PLAC1 expression in various tumor cells. PMID:21937108

Regorafenib inhibited gastric cancer cells growth and invasion via CXCR4 activated Wnt pathway.

PubMed

Lin, Xiao-Lin; Xu, Qi; Tang, Lei; Sun, Li; Han, Ting; Wang, Li-Wei; Xiao, Xiu-Ying

2017-01-01

Regorafenib is an oral small-molecule multi kinase inhibitor. Recently, several clinical trials have revealed that regorafenib has an anti-tumor activity in gastric cancer. However, only part of patients benefit from regorafenib, and the mechanisms of regorafenib's anti-tumor effect need further demonstrating. In this study, we would assess the potential anti-tumor effects and the underlying mechanisms of regorafenib in gastric cancer cells, and explore novel biomarkers for patients selecting of regorafenib. The anti-tumor effects of regorafenib on gastric cancer cells were analyzed via cell proliferation and invasion. The underlying mechanisms were demonstrated using molecular biology techniques. We found that regorafenib inhibited cell proliferation and invasion at the concentration of 20μmol/L and in a dose dependent manner. The anti-tumor effects of regorafenib related to the decreased expression of CXCR4, and elevated expression and activation of CXCR4 could reverse the inhibition effect of regorafenib on gastric cancer cells. Further studies revealed that regorafenib reduced the transcriptional activity of Wnt/β-Catenin pathway and led to decreased expression of Wnt pathway target genes, while overexpression and activation of CXCR4 could attenuate the inhibition effect of regorafenib on Wnt/β-Catenin pathway. Our findings demonstrated that regorafenib effectively inhibited cell proliferation and invasion of gastric cancer cells via decreasing the expression of CXCR4 and further reducing the transcriptional activity of Wnt/β-Catenin pathway.

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuyama, Yoshiko; Tokuhara, Daisuke; Division of Mucosal Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639

Highlights: Black-Right-Pointing-Pointer Nasal Ad-FL effectively up-regulates APC function by CD11c{sup +} DCs in mucosal tissues. Black-Right-Pointing-Pointer Nasal Ad-FL induces Notch ligand (L)-expressing CD11c{sup +} DCs. Black-Right-Pointing-Pointer Notch L-expressing DCs support the induction of Th1- and Th2-type cytokine responses. -- Abstract: Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c{sup +} dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c{sup +} DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FLmore » activated CD11c{sup +} DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c{sup +} DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4{sup +} T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-{gamma}, IL-2 and IL-4 producing CD4{sup +} T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c{sup +} DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.« less

Xanthine Oxidase Induces Foam Cell Formation through LOX-1 and NLRP3 Activation.

PubMed

Dai, Yao; Cao, Yongxiang; Zhang, Zhigao; Vallurupalli, Srikanth; Mehta, Jawahar L

2017-02-01

Xanthine oxidase catalyzes the oxidation of xanthine to uric acid. This process generates excessive reactive oxygen species (ROS) that play an important role in atherogenesis. Recent studies show that LRR and PYD domains-containing protein 3 (NLRP3), a component of the inflammasome, may be involved in the formation of foam cells, a hallmark of atherosclerosis. This study was designed to study the role of various scavenger receptors and NLRP3 inflammasome in xanthine oxidase and uric acid-induced foam cell formation. Human vascular smooth muscle cells (VSMCs) and THP-1 macrophages were treated with xanthine oxidase or uric acid. Xanthine oxidase treatment (of both VSMCs and THP-1 cells) resulted in foam cell formation in concert with generation of ROS and expression of cluster of differentiation 36 (CD36) and oxidized low density lipoprotein (lectin-like) receptor 1 (LOX-1), but not of scavenger receptor A (SRA). Uric acid treatment resulted in foam cell formation, ROS generation and expression of CD36, but not of LOX-1 or SRA. Further, treatment of cells with xanthine oxidase, but not uric acid, activated NLRP3 and its downstream pro-inflammatory signals- caspase-1, interleukin (IL)-1β and IL-18. Blockade of LOX-1 or NLRP3 inflammasome with specific siRNAs reduced xanthine oxidase-induced foam cell formation, ROS generation and activation of NLRP3 and downstream signals. Xanthine oxidase induces foam cell formation in large part through activation of LOX-1 - NLRP3 pathway in both VSMCs and THP-1 cells, but uric acid-induced foam cell formation is exclusively through CD36 pathway. Further, LOX-1 activation is upstream of NLRP3 activation. Graphical Abstract Steps in the formation of foam cells in response to xanthine oxidase and uric acid. Xanthine oxidase stimulates LOX-1 expression on the cell membrane of macrophages and vascular smooth muscle cells (VSMCs) and increases generation of ROS, which activate NLRP3 inflammasome and downstream pro-inflammatory mediators such as Caspase-1, IL-1β and IL-18. Xanthine oxidase also induces CD36 expression. Activation of both LOX-1 and CD36 (LOX-1> > CD36) participates in the transformation of macrophages and VSMCs into foam cells. Uric acid formed from xanthine-xanthine oxidase interaction stimulates CD36 expression and triggers foam cell formation independent of NLRP3 activation.

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: Elucidation of a novel glycosphingolipid-signaling pathway

PubMed Central

Dasgupta, Somsankar; Wang, Guanghu; Yu, Robert K.

2011-01-01

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid (GSL) of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the HNK-1 sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell death (apoptosis) and the maintenance of blood-brain or blood-nerve barrier (BBB or BNB) integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase (ERK) activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. While SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IKB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and BBB/BNB barrier function. PMID:21916893

A non-neuronal cholinergic system regulates cellular ATP levels to maintain cell viability.

PubMed

Oikawa, Shino; Iketani, Mitsue; Kakinuma, Yoshihiko

2014-01-01

We previously suggested that a non-neuronal cholinergic system modulates energy metabolism through the mitochondria. However, the mechanisms responsible for making this system crucial remained undetermined. In this study, we developed a fusion protein expression vector containing a luciferase gene fused to the folic acid receptor-α gene. This protein of the vector was confirmed to target the plasma membrane of transfected HEK293 cells, and vector-derived luciferase activities and ATP levels in viable cells were positively correlated (r = 0.599). Using this luciferase vector, choline acetyltransferase (ChAT)-expressing cells (i.e., cells with an activated non-neuronal cholinergic system) had increased cellular ATP levels. ChAT-expressing cells also had upregulated IGF-1R and Glut-1 protein expressions as well as increased glucose uptake. This activated non-neuronal cholinergic system with efficient glucose metabolism rendered cells resistant to serum depletion-induced cell death. Our results indicate that a non-neuronal cholinergic system is involved in sustaining ATP levels to render cells resistant to a nutrient-deficient environment. © 2014 S. Karger AG, Basel.

Endocytosis as a mechanism of regulating natural killer cell function: unique endocytic and trafficking pathway for CD94/NKG2A.

PubMed

Peruzzi, Giovanna; Masilamani, Madhan; Borrego, Francisco; Coligan, John E

2009-01-01

Natural killer (NK) cells are lymphocytes generally recognized as sentinels of the innate immune system due to their inherent capacity to deal with diseased (stressed) cells, including malignant and infected. This ability to recognize many potentially pathogenic situations is due to the expression of a diverse panel of activation receptors. Because NK cell activation triggers an aggressive inflammatory response, it is important to have a means of throttling this response. Hence, NK cells also express a panel of inhibitory receptors that recognize ligands expressed by "normal" cells. Little or nothing is known about the endocytosis and trafficking of NK cell receptors, which are of great relevance to understanding how NK cells maintain the appropriate balance of activating and inhibitory receptors on their cell surface. In this review, we focus on the ITIM-containing inhibitory receptor CD94/NKG2A showing that it is endocytosed by a previously undescribed macropinocytic-like process that may be related to the maintenance of its surface expression.

MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Teng, E-mail: tengyu33@yahoo.com; Ji, Jiang; Guo, Yong-li

2013-11-08

Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen speciesmore » (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.« less

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

PubMed Central

Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

PubMed

Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

2017-03-01

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaver, Laura M., E-mail: beaverl@onid.orst.edu; School of Biological and Population Health Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331; Yu, Tian-Wei, E-mail: david.yu@oregonstate.edu

2012-09-15

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast,more » DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis. -- Highlights: ► DIM inhibits HDAC activity and decreases HDAC2 expression in prostate cancer cells. ► DIM is significantly more effective than I3C at inhibiting HDAC activity. ► I3C has no effect on HDAC protein expression. ► Inhibition of HDAC activity by DIM is associated with increased p21 expression. ► HDAC inhibition may be a novel epigenetic mechanism for cancer prevention with DIM.« less

Artemin protects cells and proteins against oxidative and salt stress.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

2017-02-01

Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H 2 O 2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells.

PubMed

Han, Min Ae; Min, Kyoung-Jin; Woo, Seon Min; Seo, Bo Ram; Kwon, Taeg Kyu

2016-10-04

Eupafolin, a flavone found in Artemisia princeps, has been reported for its anti-tumor activity in several cancer cells. In this study, we examined whether eupafolin could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that eupafolin alone and TRAIL alone had no effect on apoptosis. However, combined treatment with eupafolin and TRAIL markedly induced apoptosis in human renal carcinoma (Caki) cells, glioma cells (U251MG), and prostate cancer cells (DU145), but not normal cells [mesangial cells (MC) and normal mouse kidney cells (TCMK-1)]. Eupafolin induced down-regulation of Mcl-1 expression at the post-translational levels in cathepsin S-dependent manner, and over-expression of Mcl-1 markedly blocked apoptosis induced by combined treatment with eupafolin and TRAIL. In addition, eupafolin increased Bim expression at the post-translational levels via AMP-activated protein kinase (AMPK)-mediated inhibition of proteasome activity. Knock-down of Bim expression by siRNA inhibited eupafolin plus TRAIL-induced apoptosis. Furthermore, combined treatment with eupafolin and TRAIL reduced tumor growth in xenograft models. Taken together, these results suggest that eupafolin enhanced TRAIL-mediated apoptosis via down-regulation of Mcl-1 and up-regulation of Bim in renal carcinoma Caki cells.