Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PI3K beta Plays a Critical Role in Neutrophil Activation by Immune Complexes

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (Fc gamma Rs). Here, we used genetic and pharmacological approaches to define a selective role for the beta isoform of phosphoinositide 3-kinase (PI3K beta) in Fc gamma R-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3K beta alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3K beta and PI3K delta, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3K beta by immune complexes involved cooperation between Fc gamma Rs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B-4. Coincident activation by a tyrosine kinase-coupled receptor (Fc gamma R) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the beta isoform of PI3K. PI3K beta-deficient mice were highly protected in an Fc gamma R-dependent model of autoantibody-induced skin blistering and were partially protected in an Fc gamma R-dependent model of inflammatory arthritis, whereas combined deficiency of PI3K beta and PI3K delta resulted in near-complete protection in the latter case. These results define PI3K beta as a potential therapeutic target in inflammatory disease.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Differential roles of integrins alpha(2)beta(1), and alpha(IIb)beta(3) in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha(2)beta(1) and possibly to other receptors. In this study, we have compared the role of integrins alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A(2) (TxA(2)) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alpha(IIb)beta(3) activation. This adhesion was substantially mediated by alpha(2)beta(1). The alpha(IIb)beta(3) antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alpha(IIb)beta(3) outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA(2) greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha(2)beta(1).

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Connective tissue growth factor antagonizes transforming growth factor-beta 1/Smad signalling in renal mesangial cells

The critical involvement of TGF-beta 1 (transforming growth factor-beta 1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-beta 1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-beta 1 and its physiological significance. CTGF was determined to bind directly to the T beta RIII (TGF-beta type III receptor) and antagonize TGF-beta 1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-beta 1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-beta 1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-beta 1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF Knockdown of T beta RIII restored TGF-beta 1-mediated Smad signalling and cell contractility, suggesting that T beta RIII is key for CTGF-mediated regulation of TGF-beta 1. Comparison of gene expression profiles from CTGF/TGF-beta 1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-beta 1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Cell-to-Cell Interactions and Signals Involved in the Reconstitution of Peripheral CD8(+) T-CM and T-EM Cell Pools

We here describe novel aspects of CD8(+) and CD4(+) T cell subset interactions that may be clinically relevant and provide new tools for regulating the reconstitution of the peripheral CD8(+) T cell pools in immune-deficient states. We show that the reconstitution capacity of transferred isolated naive CD8(+) T cells and their differentiation of effector functions is limited, but both dramatically increase upon the co-transfer of CD4(+) T cells. This helper effect is complex and determined by multiple factors. It was directly correlated to the number of helper cells, required the continuous presence of the CD4(+) T cells, dependent on host antigen-presenting cells (APCs) expressing CD40 and on the formation of CD4/CD8/APC cell clusters. By comparing the recovery of (CD44(+)CD62L(high)) T-CM and (CD44(+)CD62L(low)) T-EM CD8(+) T cells, we found that the accumulation of TCM and TEM subsets is differentially regulated. T-CM-cell accumulation depended mainly on type I interferons, interleukin (IL)-6, and IL-15, but was independent of CD4(+) T-cell help. In contrast, TEM-cell expansion was mainly determined by CD4(+) T-cell help and dependent on the expression of IL-2R beta by CD8 cells, on IL-2 produced by CD4(+) T-cells, on IL-15 and to a minor extent on IL-6.

Gingival fibroblast response to cyclosporin A and transforming growth factor beta(1)

This study investigates a potential role for TGF beta(1), in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGF beta(1) was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGF beta(1) was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGF beta(1), (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGF beta(1). In monolayer culture TGF beta(1) significantly increased protein and collagen production in all cell strains (p

Apoptosis and chemotherapy in bladder cancer in bladder cancer

Purpose: To discuss the role of apoptosis, gene directed self-destruction of a cell, in the response of transitional cell carcinoma of the bladder cells to chemotherapy. Methods: A directed MEDLINE literature search of apoptosis, bladder cancer and chemotherapy was performed to extract the relevant information, which was reviewed. The characteristics of apoptotic cells were defined and the methods in common use to detect these traits were described. The role of the key mediators of the apoptotic process in bladder cancer is discussed in the context of chemosensitivity and stage of disease. The importance of induction of apoptosis post chemotherapy is highlighted. Results: On stimulus by appropriate external or internal signals, a cell may alter the expression of genes coding for proteins associated with the apoptotic process. The development of apoptosis depends on the balance between pro- and anti- apoptotic proteins. Key alterations in genes and proteins related to apoptosis within bladder cancer result in a shift away from an ability to undergo apoptosis towards a cell with increased survival properties that is chemoresistant. Conclusions: Much current research in bladder cancer is aimed at restoring chemosensitivity by shifting the balance in a cell towards a pro-apoptotic phenotype. Successful translation of this work into clinical practice may improve survival in patients in whom prognosis is currently poor.

PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling.

BACKGROUND:
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear.

METHODS:
To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies.

RESULTS:
The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis.

CONCLUSION:
Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.

The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.