Ionising radiation is a potent human carcinogen. Epidemiological studies have shown that adolescent and young women are at increased risk of developing breast cancer following exposure to ionising radiation compared with older women, and that risk is dose-dependent. Although it is well understood which individuals are at risk of radiation-induced breast carcinogenesis, the molecular genetic mechanisms that underlie cell transformation are less clear. To identify genetic alterations potentially responsible for driving radiogenic breast transformation, we exposed the human breast epithelial cell line MCF-10A to fractionated doses of X-rays and examined the copy number and cytogenetic alterations. We identified numerous alterations of c-MYC that included high-level focal amplification associated with increased protein expression. c-MYC amplification was also observed in primary human mammary epithelial cells following exposure to radiation. We also demonstrate that the frequency and magnitude of c-MYC amplification and c-MYC protein expression is significantly higher in breast cancer with antecedent radiation exposure compared with breast cancer without a radiation aetiology. Our data also demonstrate extensive intratumor heterogeneity with respect to c-MYC copy number in radiogenic breast cancer, suggesting continuous evolution at this locus during disease development and progression. Taken together, these data identify c-MYC as a radiosensitive locus, implicating this oncogenic transcription factor in the aetiology of radiogenic breast cancer.

Introduction Oesophageal adenocarcinoma commonly arises from a premalignant lesion known as Barrett's oesophagus. Many patients are asymptomatic and present to the clinic with very advanced disease and poor prognosis. Deoxycholic acid (DCA) is a component of gastric refluxate, implicated as a tumour promoter for oesophageal adenocarcinoma. We had previously demonstrated that DCA disrupts Golgi structure and consequently impairs protein secretion and glycosylation processes. Impairment of these fundamental cell processes are implicated in metaplasia, dysplasia and carcinogenesis. To exploit this phenomenon in order to identify a novel biomarker we used an informatic approach. We identified a Golgi-associated protein, GOLPH2 whose expression is elevated in tissue from patients with Barrett's oesophagus and oesophageal adenocarcinoma. Aims/Background GOLPH2 is localised to the Golgi and is not normally secreted. It has been found to be secreted and detected in serum from patients with hepatocellular carcinoma. We hypothesised that DCA disruption of the Golgi structure would result in cleavage and secretion of GOLPH2 thus acting as a potential serum biomarker. The localisation of GOLPH2 to the Golgi membrane suggests it functions in protein processing. We sought to determine the expression and localisation of GOLPH2 in patient tissue and to elucidate the mechanisms of secretion in oesophageal cell line models of squamous, metaplasia dysplasia and adenocarcinoma. Method Golgi structure and GOLPH2 expression were examined in tissue from patients with Barrett' metaplasia, high grade dysplasia (HGD) and adenocarcinoma by immuno fluorescence. GOLPH2 expression, localisation and secretion was assessed in normal squamous, Barrett's oesophageal and adenocarcinoma cell lines in response to DCA To determine the mechanism of GOLPH2 secretion, GOLPH2 mutant constructs were used. Results The Golgi structure was intact in normal oesophageal and metaplastic tissue but fragmented in dysplastic and adenocarcinoma tissue. GOLPH2 was localised to the Golgi in normal and metaplastic tissue whereas localisation of GOLPH2 with the Golgi was lost in dysplastic and adenocarcinoma tissue. GOLPH2 expression was up-regulated in areas of differentiation and invasion in tissue from Barrett's patients. GOLPH2 was localised to the Golgi in all oesophageal cell lines but endogenously secreted by Barrett's oesophagus and adenocarcinoma cell lines only. DCA altered cellular localisation of GOLPH2 and caused secretion from normal oesophageal cells. To determine the mechanism of GOLPH2 secretion, GOLPH2 mutant constructs were used and the cleavage site was identified as the Pro-protein convertase (PC) site. Conclusion In conclusion, altered expression, localisation and secretion of GOLPH2 in Barrett's oesophagus and oesophageal adenocarcinoma suggests its potential use as a serum biomarker to identify asymptomatic patients with oesophageal disease. Up-regulation of GOLPH2 expression at sites of differentiation and invasion suggests a role in these processes in progression of this disease.

INTRODUCTION: Substituted indoles and related structures have been shown to exhibit potent anticancer activity against breast cancer cell lines. Here, the effects of structurally similar substituted indoles against the human glial cancer cell lines, 1321N1 and U87MG, have been investigated by comparing the effects of these compounds to conventional anti cancer drugs. METHODS: Cell viability in the presence of the test compounds was measured using an MTS assay and corroborated by an ATP cell proliferation assay as well as a Trypan blue exclusion test. The significance of reactive oxygen species (ROS) in the process was determined using an Image-iT® LIVE ROS kit from Invitrogen. RESULTS: Both cell lines were treated with four commercial anticancer drugs and IC50 values were only reached at concentrations of 20 µM for cisplatin and 50 µM for gemcitabine over 48hrs on the 1321N1 cell line. However, the more malignant U87MG cell line was resistant to all the drugs, except for cisplatin where the IC50 value was reached at 300 µM after treatment for 48hrs. Similar studies were carried out with various substituted indoles and the cytotoxicity results on both cell lines showed that the IC50 value was reached within 90 minutes for the most potent compound at a concentration of 600 µM (1321N1) and 800 µM (U87MG). The idea that the mechanism of action of these compounds may work through the generation of ROS was investigated and this was confirmed over a similar time course using a suitable fluorogenic marker. Moreover, it was shown that the addition of an antioxidant (ascorbic acid) abolished the potency of the most active compound. CONCLUSION: Here, it has been demonstrated that certain substituted indoles are able to have a rapid, deleterious effect on the viability of two glioma cell lines and indicated that ROS generation may induce cell death.

Introduction Hydrophobic bile acids have been identified as aetiological agents in inflammatory oesophageal diseases. Ursodeoxycholic acid (UDCA) has been shown to attenuate the inflammatory effects of hydrophobic bile acids in numerous cell lines. This has been shown to be partly mediated by UDCA activation of the glucocorticoid receptor (GR). Here, we investigate the potential role of UDCA in modulating the GR in oesophageal cell lines. Methods Western blot analysis and RT-PCR were used to characterise GR expression in oesophageal cell lines (HET1A, SKGT-4). High Content Analysis (GE In-Cell analyser 1000) was used to investigate nuclear translocation of the GR in response to UDCA stimulation. Trans-activation of glucocorticoid response elements (GRE) was determined in a transient transfection assay using a construct of the human GRE promoter ligated to a firefly luciferase reporter gene Similarly trans-repression of NFKB was determined using a construct of the human NFKB promoter ligated to a luciferase reporter gene. Results GR was found to be present in the oesophageal cell lines used at the mRNA and protein levels. No GRβ was expressed at the mRNA level suggesting that the GR present is GRα. UDCA induced translocation of the GR in a time- and concentration-dependent manner (EC50 of 298.68 μM in SKGT-4 and 320 μM in HET1A cell lines, 54% and 36% efficacy of dexamethasone 100 nM, respectively, both p<0.05 relative to untreated control). UDCA activated the GRE at 300 μM (22% efficacy of dexamethasone 100 nM, p<0.05). UDCA was found to inhibit TNF-α stimulated NFKB activation in HEK-293 cells at 300 μM (100% efficacy of dexamethasone 100 nM, p<0.05). Conclusion Glucocorticoids are used as anti-inflammatory agents for treating a spectrum of diseases. Their chronic use is limited due to serious side effects, primarily thought to be due to GR-mediated transactivation. Here we show that UDCA can act as a GR modulator, with the ability to differentiate between transrepression and transactivation pathways. As a result UDCA may be of benefit in treating inflammatory conditions of the oesophagus.

BACKGROUND & AIMS:The ability of viruses to escape the host immune response represents a globally important problem related to a wide variety of pathogens. Hepatitis C is one of the major causes of liver disease worldwide. Clearance rates of this virus are low, and this condition normally involves a chronic inflammatory process. This raises a possibility that the virus may have developed mechanisms enabling it to evade T-cell-mediated immune surveillance. The aim of this study was to investigate the effect of the hepatitis C envelope protein E2 on LFA-1-stimulated T-cell migration and macrophage inflammatory protein (MIP-1alpha, MIP-1beta) secretion.METHODS:T cells were stimulated through the leukocyte function-associated molecule-1 (LFA-1) receptor by incubating with either intracellular adhesion molecule 1 (ICAM-1)-Fc fusion protein or anti-LFA-1 immobilized on 8-well chamber slides. Subcellular localization of protein kinase C (PKC)-beta, CD81, and LFA-1 was determined by immunofluorescence analysis. Lipid raft formation was assessed using the Cellomics Kineticscan reader. MIP-1alpha and MIP-1beta levels were detected by enzyme-linked immunosorbent assay.RESULTS:We report that the hepatitis C envelope protein E2 can dramatically inhibit T-lymphocyte motility and chemokine release induced via LFA-1 integrin ligation. We have demonstrated a novel T-lymphocyte-directed viral inhibitory mechanism involving the PKC-beta enzyme as a definitive intracellular target. E2-CD81 interaction stimulates translocation of PKC-beta to lipid rafts, thereby preventing its association with the centrosome and microtubule cytoskeleton, which is crucial to the process of T-cell migration.CONCLUSIONS:These studies identify a mechanism whereby the hepatitis C virus can evade the host immune response by inhibition of T-cell migration.

T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C beta and delta isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the beta(1) integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.

To assess the effects of dietary supplementation using two isomeric blends of conjugated linoleic acid (CLA) on immune function in healthy human volunteers. Double-blind, randomised, placebo-controlled intervention trial. A total of 55 healthy volunteers (n=20 males, n=35 females) were randomised into one of three study groups who received 3 g/day of a fatty acid blend containing a 50:50 cis-9, trans-11: trans-10, cis-12 CLA isomer blend (2 g CLA), and 80:20 cis-9, trans-11: trans-10, cis-12 (80:20) CLA isomer blend (1.76 g CLA) or linoleic acid (control, 2 g linoleic acid) for 8 weeks. Supplementation with the 80:20 CLA isomer blend significantly (P≤0.05) enhanced PHA-induced lymphocyte proliferation. CLA decreased basal interleukin (IL)-2 secretion (P≤0.01) and increased PHA-induced IL-2 and tumor necrosis factor α (TNFα) production (P≤0.01). However, these effects were not solely attributable to CLA as similar results were observed with linoleic acid. CLA supplementation had no significant effect on peripheral blood mononuclear cells IL-4 production, or on serum-soluble intercellular adhesion molecule-1 (sICAM-1) or plasma prostaglandin E2 (PGE2) or leukotreine B4 (LTB4) concentrations. This study shows that CLA supplementation had a minimal effect on the markers of human immune function. Furthermore, supplementation with CLA had no immunological benefit compared with linoleic acid. CLA supplements were provided by Loders Croklaan.

Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC‐β 1 , ‐ϵ and ‐δ and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC‐ϵ and ‐δ but not PKC‐β. DCA treatment results in endogenous PKC‐ϵ translocation but not PKC‐δ after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC‐β 1 , PKC‐ϵ and PKC‐δ functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 μM DCA induces rapid translocation of PKC‐β 1 ‐GFP and PKC‐ϵ‐GFP but not PKC‐δ‐GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC‐β 1 ‐GFP and PKC‐ϵ‐GFP translocates to the plasma membrane in 15 min whereas PKC‐δ‐GFP localisation remains unaltered. Immunofluorescence shows that PKC‐β 1 ‐GFP and PKC‐ϵ‐GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html © 2004 Wiley‐Liss, Inc.

CD44 is a receptor for hyaluronic acid and is found on the surface of hematopoetic cells and in mesenchymal tissue. It is also expressed on endothelial cells (EC). Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins in EC. Here we show that engagement of CD44 with signaling monoclonal antibodies (mAbs) or its natural ligand hyaluronic acid induces COX-2 and prostacyclin (PGI2) formation in human EC. This induction was blocked by mAbs that have been shown to inhibit CD44-mediated intracellular signaling. COX-1 induction was not observed after CD44 ligation. CD44-stimulated COX-2 activation/PGI2 production was accompanied by the production of the potent endothelial mitogen, vascular endothelial growth factor (VEGF) and was inhibited by a neutralizing VEGF antibody. Moreover, this COX-2 induction was also associated with an increase in EC proliferation that was inhibited by the blocking anti-CD44 mAbs and a COX-2-specific inhibitor. This is the first study to show that engagement of CD44 with mAbs or its natural ligand induces COX-2, generates VEGF, and thus leads to an increase in EC proliferation. Results from this study may have important and widespread implications for the development of novel therapeutic agents for modulating blood vessel growth during ischemic heart disease, during inflammation, or around solid tumors.

Deoxycholic acid (DCA) has been implicated in colonic carcinogenesis through effects mediated by protein kinase C (PKC) activation. By contrast, ursodeoxycholic acid (UDCA) is reported to reduce colon cancer incidence in ulcerative colitis. The aim of this study was to investigate whether UDCA modulated DCA-induced PKC isoenzyme translocation to its site of activity. HCT116 cells were treated with DCA, UDCA alone or pre-treated with UDCA followed by DCA. Analysis of translocation of endogenous and enhanced green fluorescent protein (EGFP) constructs of PKC isoenzymes was performed. Both DCA and phorbol myristate acetate (PMA) but not UDCA caused translocation of endogenous PKC α, ε and δ and transfected PKC β1-, ε- and δ-EGFP from cytosol to plasma membrane, reflecting isoenzyme activation. Furthermore, UDCA inhibited DCA-induced translocation of PKC isoenzymes. Inhibition of DCA-induced PKC translocation may be a mechanism for UDCA-mediated chemoprevention of colon carcinogenesis.

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC βI and δ are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC θ is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC θ demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC θ signalling.

Factors contributing to the high rate of HCV chronicity are not fully understood. Interleukin 2(IL-2) is critical for the generation of broad-range antigen-specific host T-cell responses, essential for HCV clearance. Many viruses target IL-2 as part of their immune-evasion strategy, however, specific inhibition of IL-2 by HCV has not been demonstrated. CD81, a widely expressed tetraspanin, binds HCV envelope and inhibits NK cells, but, also acts as a co-stimulatory signal for IL-2 production by T cells. Thus, CD81:HCV interactions appear to favour the host and their role in viral persistence is paradoxical. We aimed to determine the role of IL-2 in HCV chronicity. We show that pre-engagement of CD81, using recombinant HCV-envelope or anti-CD81 antibodies, in a manner that mimics in vivo infection, inhibits IL-2 production by T cells. In addition HCV infectious serum produces the same inhibitory effect. We also demonstrate that hepatic IL-2 is significantly reduced in HCV compared to cirrhotic controls. These data support our model of HCV persistence whereby, CD81:HCV engagement prior to T-cell activation compromises host T cell responses. We describe a novel immune evasion strategy employed by HCV. Low-dose IL-2 in combination with current treatment regimes may lead to successful elimination of HCV.