Báo cáo khoa học: The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury pdf

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Báo cáo khoa học: The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury pdf

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The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury Joanne Darragh1,*, Mairi Hunter1, Elizabeth Pohler2, Lenny Nelson2, John F Dillon1, Rudolf Nenutil3, Borek Vojtesek3, Peter E Ross1, Neil Kernohan1 and Ted R Hupp2 Division of Pathology and Neurosciences, University of Dundee, UK University of Edinburgh Cancer Centre, CRUK Cell Signalling Unit, UK Masaryk Memorial Cancer Institute, BRNO Czech Republic Keywords Barrett’s apoptosis; calcium; deoxycholic acid; SEP53; stress response Correspondence T R Hupp, University of Edinburgh Cancer Centre, CRUK Cell Signalling Unit, South Crewe Road, Edinburgh EH4 2XR, UK E-mail: ted.hupp@ed.ac.uk *Present address MRC Protein Phosphorylation Unit, University of Dundee, UK (Received 12 December 2005, revised February 2006, accepted 28 February 2006) doi:10.1111/j.1742-4658.2006.05206.x Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53) Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53mediated responses to damage To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development Human cancers develop through a multistage process involving morphological changes in tissue, mutations in oncogenes and tumour suppressor genes, and epigenetic programmes that give rise to enhanced survival in a stressed microenvironment [1] The development of human cancer is proving to be a tissue-specific process involving an interaction between mutated cells and the unique conditions within a particular local matrix and microenvironment Such local cellular stresses include hypoxia, acidification, pro-oxidants from the diet, genome instability and altered autocrine responses This evolutionary path relies on the developing tumour cell to repair, survive and overcome intrinsic tumour-suppressing signals that normally are used to kill abnormal cells and maintain tissue integrity The mechanisms underlying tissue-specific responses to local environment in cancer development are largely undefined Abbreviations Bis-I, bisindolylmaleimide I; Bis-V, bisindolylmaleimide V; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HRP, horse radish peroxidase; LCA, lithocholic; PKC, protein kinase C; PPI, proton pump inhibitor; SEP53, squamous epithelial-induced stress protein of 53 kDa; UDCA, ursodeoxycholic acid; YFP, yellow fluorescent protein 1930 FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al In developing physiologically relevant models of stress protein dysregulation in developing human cancers, a key clinical model that is giving novel molecular mechanistic insight is adenocarcinoma of the oesophagus [2] This cancer is one of the fastest rising cancers in the west, is taking the place of squamous cell carcinoma as a more common type of oesophageal cancer, and is associated in part with stresses induced by environmental damaging agents including acid and bile reflux [3–5] Furthermore, the transition from squamous epithelium to adenocarcinoma appears to proceed through the well-characterized epithelial intermediate (named Barrett’s) and is associated with increases in proliferation due to an acidified microenvironment [5] In addition to acid as a key microenvironmental stress implicated in disease progression, bile is present within the lumen of the gut and is a naturally occurring agent that may act in different ways to facilitate carcinogenesis [6,7] In particular bile acids such as deoxycholic acid (DCA) can stimulate cell proliferation, migration, DNA damage and apoptosis in gut epithelial cells [8–15] Cells of the normal human oesophageal squamous epithelium are under relatively unique environmental pressures being exposed to thermal stresses, pro-oxidants, and refluxed acid and bile adducts These cells have therefore presumably evolved specific mechanisms to tolerate and repair injury induced by exposure to these and other damaging agents that are relatively unique to this tissue We have defined previously the stress-responsive pathways in normal squamous oesophageal epithelial cells using a ‘functional proteomics’ approach The first studies indicated that ex vivo stressed squamous cells in organ culture did not synthesize the classic stressed-induced protein HSP70 after stress, suggesting a novel type of stress response in this cell type [16] Further ex vivo organ culture in conjunction with specific stresses, including ethanol and heat shock, identified using mass-spectrometric methods a novel class of stress protein in normal squamous epithelium; these include SEP70, squamous epithelialinduced stress protein of 53 kDa (SEP53) and glutamine–glutamyl transferase [17] SEP70 is induced by acidified extracellular conditions and is a glucose-regulated protein [17] SEP53 was originally cloned as a gene expressed in normal oesophagus but downregulated in oesophageal cancers and was named Clone open reading frame 10 [18] The SEP53 gene is located on chromosome 1q21 within a group of proteins named the epidermal differentiation complex fusedgene family that it silenced as part of a general mechanism that apparently suppresses genes from this locus in cancer cells [19,20] The function and regulation of SEP53 attenuates deoxycholate-mediated injury SEP53 are not yet clear In this study, we present data indicating that SEP53 can function as a survival factor and that it does so in part by attenuating DCA-mediated calcium release and cell death SEP53 is a rapidly evolving gene with < 50% identity to its murine orthologue suggesting that the antiapoptotic activity of SEP53 is evolving in relation to selection pressures resulting from environmental stress in squamous epithelium Results SEP53 protein is expressed in human squamous epithelium Having previously shown, using a functional proteomics approach, that SEP53 is one of the major proteins induced by ex vivo stress to normal squamous epithelium [17], we needed to confirm that the SEP53 protein is in fact expressed in normal human squamous epithelium of the oesophagus We first needed to develop antibodies to SEP53 and the human SEP53 gene was cloned into a bacterial and insect cell-expression system for the purification and acquisition of full-length protein for immunization, and to develop a panel of monoclonal antibodies (MAb) A tryptic digest of pure full-length SEP53 protein (Fig 1A, lane 1) gave rise to a ladder of bands (as in Fig 1A, lane 2) that was used to define the number of unique MAb clones Three distinct classes of MAbs were grouped according to binding activity to different tryptic fragments (Fig 1A, lanes 2, 4, 6, 8, and 10) Class A MAb produced a unique pattern of immunoreactive bands (Fig 1A, lane 2) that was distinct from Class B MAb (Fig 1A, lane 4), whilst the Class C MAb epitope was destroyed by the trypsinization as effectively no ladder of bands was produced (Fig 1A, lane 6, and 10) We next investigated whether SEP53 protein was expressed in squamous epithelium using these immunochemical reagents The SEP53 protein is highly expressed in normal squamous epithelium under conditions in which Anterior Gradient-2 is relatively low (Fig 1B, Normal) As a control for the integrity of the Barrett’s cell population, the Anterior Gradient-2 protein is confirmed to be highly overexpressed in Barrett’s samples [21] compared with normal squamous epithelium from the same patient (Fig 1D, Barrett’s versus Normal) SEP53 immunostaining can also be observed in the suprabasal layer of squamous epithelium (Fig 1F), where immunoreactivity is generally cytoplasmic granular staining with minor epimembranous staining in maturing and mature squamous cells Furthermore, SEP53 is variably expressed in Barrett’s FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1931 SEP53 attenuates deoxycholate-mediated injury A J Darragh et al Classes of SEP53 MAB B C D E F Fig Development of a panel of monoclonal antibodies (MAbs) to the major squamous-cell specific stress protein SEP53 (A) Characterization of SEP53 MAb Purified SEP53 protein (1 lg) was incubated without (lanes 1, 3, 5, and 9) or with (10 ng, lanes 2, 4, 6, and 10) trypsin in a buffer containing 25 mM Hepes (pH 7.5) and at 30 °C for Reactions were quenched with SDS sample buffer and protein was separated on a 12% SDS polyacrylamide gel Protein was immunoblotted and probed with different antibodies (1 lgỈmL)1) giving rise to the three classes, as indicated The arrows highlight the unique proteolytic fragments produced that are recognized by the respective antibodies (B–E) Expression of SEP53 in normal squamous epithelium Lysates were obtained from normal and Barrett’s tissue (defined endoscopically and histochemically) from the same patient (as indicated by the numbering between the panels) and protein was immunoblotted for (B) SEP53 protein, (C) SEP70 protein, (D) AG-2 protein and (E) loading controls for squamous and Barrett’s biopsies (ink stain immunoblot to normalize for protein loading), as indicated (F) Immunostaining in normal squamous oesophageal epithelium shows SEP53 expression predominantly in the suprabasal layer of the epithelium in cytoplasmic or perinuclear regions where Anterior Gradient-2 protein is relatively high (Fig 1B, Barrett’s) However, this expression of SEP53 enriched in biopsies endoscopically defined as Barrett’s epithelium might be due to a contamination of normal squamous epithelium in the biopsy The variable expression of the acid- and glucose-regulated SEP70 protein [17] (Fig 1C, Barrett’s), under conditions where SEP53 protein is variable (Fig 1B, Barrett’s), highlights heterogeneity in the Barrett’s samples with respect to all three stress proteins Nevertheless, the SEP53 protein is in fact expressed in normal human squamous epithelium and this prompted us to continue studying the gene to define a possible molecular function for the protein in stress-responsive pathways Developing cell models to examine effects of DCA on cell death SEP53 was originally identified as a protein synthesized ex vivo after heat or ethanol stress [17] The 1932 physiological stress the SEP53 responds to in cells is, however, undefined, as heat exposure to the oesophagus and ethanol are unlikely to be evolutionary adaptations The oesophagus is an organ that is commonly exposed to bile acids and the structure of normal oesophageal epithelium is altered by bile exposure [22] Developing knowledge of the effects that these chemicals may have on oesophageal epithelial cells and apoptotic pathways might be relevant to understanding the molecular function of SEP53 We were therefore interested in determining whether the SEP53 gene had any effects on modifying DCA-induced cell stresses However, prior to examining the effects of DCA on SEP53-mediated apoptotic responses, we wanted to confirm that DCA was in fact a significant constituent of gastric fluid To analyse gastric fluid samples for bile acid content, bile acids were extracted, derivatized and then analysed by gas chromatography The relative retention times of peaks present in the gastric fluid sample FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al SEP53 attenuates deoxycholate-mediated injury A Bile Acid Std ratio Cholesterol Lithocholic Deoxycholic Chenodeoxycholic Ursodeoxycholic Cholic 7-Ketolithocholate Fig Concentration of naturally occurring bile acids (A) Data from a representative chromatogram indicating the retention times of each bile acid Peaks: 1, cholesterol; 2, LCA; 3, DCA; 4, CDCA; 5, UDCA; 6, CA; 7, 7-ketolithocholic acid (internal standard) The retention (Rt) times and relative retention times (RRt) of the bile acid standards are shown and were used as a standard to quantify the bile acids from patients Standard ratios represent the peak area of each mgỈmL)1 standard compared with the peak area of the internal standard (B) Summary of the range of the total bile acid concentrations found in gastric fluid samples (C) Percentage of patients with bile acid concentrations as indicated (D) Percentage of patients with unconjugated bile acids concentrations as indicated (E) Ratio of bile acids to the levels of cholesterol present in gastric fluid samples The range, mean and median concentrations of cholesterol are as indicated Cholesterol made up 40% of all the components measured in gastric fluid, while the various bile acids contributed 60%, giving a bile acid to cholesterol ratio of : Rt (Min) RRt 4.31 5.55 6.35 7.19 7.84 8.89 10.74 1.69 1.65 1.11 1.15 1.63 1.16 1.00 0.40 0.52 0.59 0.67 0.73 0.83 1.00 B Bile Acid Range uM (cong) Mean uM (cong) Range uM (uncong) Mean uM (uncong) CA CDCA DCA LCA UDCA Total 1–2447 1–3655 1–1592 1–515 1–860 1–6386 118 112 63 17 13 323 1–211 1–121 1–115 1–82 1–720 1–978 C 3 18 D 32% 8% 23% 9% 200 uM 37% 53% Cholic 22% E were calculated (Fig 2A) and the relevant bile acid peaks identified by comparison with values from the standard mix of pure lipids (data not shown) Bile acids were detected in 92% (158 ⁄ 172) of patient samples and the concentration and ⁄ or composition of the bile acid pool varied considerably between patient samples (Fig 2B) In samples with detectable levels, the concentration of total bile acids ranged from lm to 6.4 mm, with a mean of 323 lm (Fig 2B) In total, 31% of samples contained no or low concentrations of bile acids, with 32% having high concentrations in excess of 200 lm, and the remaining 37% of cases having concentrations ranging between 20 and 200 lm (Fig 2C,D) The majority of patient samples contained a mixture of bile acids (as well as cholesterol, Fig 2E), including DCA, chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA) and cholic acid (CA), with both conjugated and unconjugated (Fig 2B,E) forms being identified The Cholesterol 40% Ursodeoxy cholic 2% Chenodeoxy cholic 21% Deoxy cholic 12% Lithocholic 3% primary bile acids, CA and CDCA, with mean concentrations of 118 and 112 lm, respectively, were present in a higher concentrations than the secondary bile acids, with the mean concentration of DCA being 63 lm and LA levels averaging 17 lm (Fig 2B) The proportion of DCA to CA in gastric juice was higher than anticipated (Fig 2E), as in normal duodenal fluid the DCA levels have been found to be one fifth of cholate [23] DCA was present in gastric samples and the range of DCA was from lm to over 1.5 mm (Fig 2B) The physiological levels of DCA that are associated with injury are not known, as patients fast before entering the clinic for sample collection Furthermore, it is not known whether chronic exposure to low levels that are not acutely toxic induces a worse or better indicator than single supratoxic acute doses over time Despite this heterogeneity in bile levels in gastric fluid, it is difficult to extrapolate to in vivo concentrations, however, FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1933 SEP53 attenuates deoxycholate-mediated injury J Darragh et al using rabbit oesophageal mucosa as a model, the epithelium concentrates bile acids up to 7· lumenal concentrations [24] Thus, given the range of DCA in patients (1 lm to > mm) and given that bile can be concentrated from the lumen up to 7· [24], the possible concentration of DCA in cells might be from lm to 10 mm Furthermore, Zhang et al [25] evaluated the range of bile acids (as in Fig 2) and found that $ 500 lm of selected bile acids were required to give rise to significant apoptosis These latter levels were in the range we used (Figs and 3) and given this, we titrated DCA from low lm to > mm to determine A I B C whether it was toxic in our cell assays and whether it was modified by SEP53 We next evaluated the effects of these key bile acids present in gastric fluid on the cell-cycle parameters a set of relatively well-characterized oesophageal cancer cell lines (OE21, KYSE 30, OE 19 and OE33), particularly to determine whether DCA was able to significantly induce injury In the presence of DCA up to a concentration of 500 lm, no significant apoptotic response was obtained in the OE21 or KYSE 30 squamous cell lines [Fig 3A and C versus Fig 4G (OE21 cells)], in contrast to the oesophageal cancer Eca109 J D K L M N O P Q R E F 1934 G H FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al cell line in which this dose gives rise to 22% apoptotic cells [25] The adenocarcinoma cell lines (OE 19 and OE33 cells) did, however, demonstrate a dosedependent death response following exposure to DCA (Fig 3B,D versus control Fig 4A,G) The production of these sub-G1 fragments detected by FACS after DCA exposure was confirmed to be apoptotic by characteristic nuclear morphology changes (Fig 4M,N and Q,R) Titration of DCA up to 500 lm demonstrated a dose-dependent increase in sub-G1 fragments which can be observed selectively in OE33 and OE19 cells (Fig 3E) and is consistent with data published recently in a different oeopshageal cancer cell line [25] DCA-mediated apoptosis is mediated by a PKC-dependent pathway and is p53 independent One of the principal biological functions of the tumour-suppressor protein p53 is as a mediator of apoptosis in response to cellular stress and DNA damage [26] Because DCA can induce DNA damage [14], the role of p53 in mediating DCA-dependent apoptosis was investigated using a pair of isogenic p53+ and p53– cell lines [27], in order to determine whether we needed to consider the p53 status in dissecting DCAmediated signalling The HCT116 (p53+) isogenic cell line was incubated with increasing concentrations of DCA (0–500 lm) for h and the resultant stressed cells were then fixed, stained with PI and the mean SEP53 attenuates deoxycholate-mediated injury (± SEM) (n ¼ 3) percentage of apoptotic cells measured by flow cytometry (Fig 3F–H) Under these conditions, apoptosis was elevated, in a dose-dependent manner, from to 58% of the cell population as defined by sub-G1 fragments Both of the HCT116 (p53+ and p53–) cell lines were equally sensitive to DCA-induced apoptosis (data not shown) indicating that DCA-induced apoptosis does not require signalling via p53 in these colonic cell lines Furthermore, because the OE33 and OE19 cell lines have mutant p53 (data not shown), p53-independent apoptosis operates under these conditions In order to define a positive mechanism for DCAmediated apoptosis in OE33 versus OE21 cells, we evaluated a set of common protein kinase inhibitors for an attenuation of the response in OE33 cells (data not shown) One striking observation was made using the protein kinase C (PKC) inhibitor bisindolylmaleimide I (Bis-I), which inhibited DCA-dependent apoptosis (Fig 3I) The control inactive version of the inhibitor bisindolylmaleimide V (Bis-V) was unable to block the apoptosis (Fig 3J), demonstrating the selectivity in the response Because the PKC pathway was being activated to induce apoptosis in the OE33 cell line, but not in the OE21 cell line, we reasoned that differential activation of key components of the PKC pathway, the pro-apoptotic GSK3 or pro-survival PKB kinases might account for the altered DCA-mediated apoptotic response [28,29] Consistent with this, Fig Cell-cycle parameters in deoxycholic acid-treated cells (A–D) Effects of bile on cell cycle parameters Representative FACS profiles of each cell line stressed with 500 lM deoxycholic acid for h are shown with untreated controls from the same experiment for OE33 in Fig 4A and for O21 in Fig 4G Histograms show the number of cells on the y-axis against the level of fluorescence (FL3-H) on the x-axis, with the different stages of the cell cycle highlighted [sub-G1 (apoptotic), G1, S and G2–M] The percentage figures indicate the number of cells in the sub-G1 peak (apoptotic), which are similar for the two adenocarcinoma cell lines (OE33 and OE19) The squamous cell carcinoma lines (OE21 and KYSE30) retained a normal DNA profile following the deoxycholic acid stress (E) Titration of DCA Cells were treated and processed as in (A–D) and the sub-G1 cell number was quantified (% apoptosis) and plotted as a function of cell line and level of DCA added (from to 500 lM) (F–H) p53 independence in apoptosis induced by DCA In addition to analysing the effects of DCA stress on the OE oesophageal cell (A–E), HCT116 (p53 wild-type and p53-null) colon cancer cells were used to examine p53 dependence in apoptosis HCT116 cells were incubated with 250 and 500 lM DCA for h Cells were then fixed, stained with PI and sub-G1 peaks quantitated by flow cytometry, as indicated (I, J) Attenuation of DCA-induced apoptosis by a PKC inhibitor OE33 cells were treated as indicated without chemical, with DMSO control, with DCA (I), Bis-I (1 lM) or (J) Bis-V (1 lM), and DCA with Bis-I (1 lM) or Bis-V(1 lM) The apoptotic cell number was quantified by FASC (as indicated in Fig 3A–D) (K–R) Analysis of GSK3-PKB modification in DCA-treated OE33 and OE21 cells (K–N) DCA stimulates GSK3 activation and mediates PKB attenuation in OE33 cells Following serum starvation, OE33 cells were stressed without or with 500 lM DCA for h followed by a treatment of 100 ngỈmL)1 EGF for 10 The cells were lysed, and the lysate was then subjected to electrophoresis on a 4–12% NuPAGE gel, transferred to nitrocellulose and 20 lg immunoblotted with either a (K) phospho-PKB antibody or (L) the antibody specific for the native form of PKB Blots were reprobed for actin (lower bands) to show equal loading of protein Cell lysates were also used to determine the levels of (M) phosphorylated GSK3a and b and (N) total cellular levels of GSK3b (O–R) Deoxycholic acid increases GSK3 inactivation and maintains PKB phosphorylation in OE21 cells Following serum starvation, OE21 cells were stressed without or with 500 lM DCA for h followed by a treatment of 100 ngỈmL)1 EGF for 10 The cells were lysed, and the lysate was then subjected to electrophoresis on a 4–12% NuPAGE gel, transferred to nitrocellulose and 20 lg immunoblotted with either a (O) phospho-PKB antibody or (P) the antibody specific for the native form of PKB Blots were reprobed for actin (lower bands) to show equal loading of protein Cell lysates were also used to determine the levels of (Q) phosphorylated GSK3a and b and (R) total cellular levels of GSK3b FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1935 SEP53 attenuates deoxycholate-mediated injury A B Control Cholic Acid D Cheno-Deoxycholic Acid 25% M OE33 cells O OE21 cells G H Control 1% Urso-Deoxycholic Acid I Tauro-Deoxycholic Acid F Lithocholic Acid 1% K Cheno-Deoxycholic Acid 2% 23% N OE33 + DCA P OE21 + DCA 1% J Urso-Deoxycholic Acid Cholic Acid 6% 3% E Tauro-Deoxycholic Acid 1% 3% C J Darragh et al 1% L Lithocholic Acid 1% Q OE33 R OE33 + DCA Fig Cell-cycle parameters in bile acid-treated cells (A–F) Apoptosis after bile acid exposure in OE33 cells Under normal growth conditions (A), apoptotic debris is rarely identified among OE33 cells following staining with the nuclear dye Cytox Addition of 500 lM of the indicated bile acid (B–F) for h leads to changes in cell-cycle parameters as indicated (G–L) Reduced apoptosis after bile acid exposure in OE21 cells Addition of 500 lM of the indicated bile acid (H–L) for h leads to little changes in cell-cycle parameters as indicated The (%) of cells in apoptosis is indicated in the top left corner of each panel Characterization of nuclear morphology following DCA stress (M–P) Morphology of OE cells OE33 (M, N) and OE21 (O, P) cells were treated with DCA (500 lM) for h Cells were then fixed, the nuclei stained with Cytox and the fluorescence measured using confocal microscopy Control OE33 cells were dividing, but following the DCA stress, small early apoptotic nuclei (red arrow), and late apoptotic nuclear fragments (white arrows) were visualized In OE21 cells treated with DCA, no nuclear fragmentation was visualized, and only a few sparse small apoptotic nuclei were present (white arrow) (Q, R) Electron microscopic analysis of OE33 oesophageal cells treated with DCA OE33 cells (Q) were treated with 500 lM DCA for h and analysed by electron microscopy OE33 cells showed characteristic signs of apoptosis following the DCA stress, as shown by their small, isolated, spherical shape (R) The multiple regions of darkly stained nuclei also indicate that nuclear condensation and fragmentation has occurred in these cells OE21 cells retained normal histology following DCA stress, indicating they were nonapoptotic (data not shown) 1936 FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al DCA attenuated phosphorylation of the normally prosurvival PKB at the activating site of PKB in OE33 cells (Fig 3K, lane versus 2) By contrast, basal inactivating phosphorylation of GSK3 was reduced in OE33 cells (Fig 3M, lane versus 2) The opposite SEP53 attenuates deoxycholate-mediated injury occurs in the OE21 cells: DCA did not block phosphorylation of PKB in the resistant OE21 cells (Fig 3O, lane versus 2), although GSK phosphorylation actually increased in OE21 cells (Fig 3Q, lane versus 2) The data suggest that the GSK3–PKB–PKC Fig SEP53 enhances colony survival in tumour cell lines (A–D) Survival activity in tumour cell lines H1299 cells (p53-null) (A, B) and A375 cells (wt p53) (C, D) were transfected with the indicated DNA vector (1 lg) and one day after transfection, cells were split and plated in media containing Geneticin to select for cell containing vector DNA After three weeks, the number of cells was determined by fixing cells and staining with dye: vector only, p53 and SEP53 (E) Homology of SEP53 to other genes imbedded in the epidermal differentiation complex on chromosome 1q21 including THH, REP, PFG, HORN and BBBAS Amino acid and DNA homology (%) are as indicated (F) Homology of the EF-hand domain between members of the Homo sapiens epidermal differentiation complex loci (G) Deletion of the calcium-binding EF-hand domain of SEP53 inhibits its activity in a clonogenic assay H1299 cells (p53-null) were transfected with the indicated YFP-DNA vector (1 lg) and one day after transfection, cells were split and plated in media containing Geneticin to select for cell containing vector DNA After three weeks, the number of cells were determined by fixing cells and staining with dye and quantified in (H) The lower molecular mass of DCa–YFP–SEP53 compared with full-length SEP53 is depicted in (I) Individual colonies from a different plate (vector only, SEP53 transfected or YFP–SEP53 transfected) were cloned and propagated for use in the assays described in other experiments FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1937 SEP53 attenuates deoxycholate-mediated injury J Darragh et al pathway axis, rather than p53, is a primary mediator of the differential apoptotic response of the two cell lines Gastric fluid contains a mixture of different bile acids in addition to DCA (as in Fig 2B) These have different biochemical properties and in terms of biological effect they have been shown to vary in their ability to induce apoptosis in colorectal cancer cell lines, although DCA is the prime bile used in generalized research [11,13,25,30] Therefore, the effect of several conjugated and unconjugated bile acids on the induction of apoptosis in both the sensitive OE33 and resistant OE21 oesophageal cell lines was investigated (Fig 4) The sensitivity of the adenocarcinoma cell line, OE33 to deoxycholic acid-induced apoptosis was abrogated when this bile acid was conjugated to taurine (taurodeoxycholic acid; Fig 4B versus Fig 4D) Similarly the addition of CA, a trihydroxy bile acid or ursodeoxycholic (UDCA) a 3a:7b dihydroxy bile acid had no damaging effect on OE33 cells (Fig 4C,D) However, CDCA and LA did induce apoptosis in the OE33 cell line, with the percentage of sub-G1 cells increasing to 25 and 23%, respectively (Fig 4E,F) Furthermore, the levels of apoptosis induced by these two bile acids were similar to levels obtained following a DCA stress in this same cell line (25%, Fig 3D,E versus Fig 4A) OE21 cells remained resistant to all bile acids studied, irrespective of their hydrophobicity (Fig 4G–L) Thus, CDCA, DCA and LA were the three most potent cell death-inducers and the mean concentration of these in gastric fluid was 112, 63 and 17 lm, respectively The data indicate that DCA is in fact the second-most abundant toxic effector, exerts a similar toxicity to the other two bile acids, and affirms its use as a model damaging agent SEP53 functions as a survival factor in a clonogenic assay The key stresses thought to predominate in oesophageal squamous epithelium and cause tissue injury include heat shock [31], low pH [5] and DCA [14] We examined specifically whether SEP53 protein modifies the DCA death response, as this is proving to be a physiologically relevant DNA damaging agent [14,25] We had first analysed a range of tumour cell lines for SEP53 protein levels and have not found one cell that expressed the protein including the OE panel described here (data not shown) This may relate to the fact that the SEP53 gene is located on chromosome 1q21 within a group of proteins named the epidermal differentiation complex fused-gene family and that this locus might be silenced by chromatin remodelling as part of 1938 a general mechanism that suppresses genes from this locus in cancer cells [19,20] Furthermore, the OE oesophageal cancer cell lines were not easily transfected with the SEP53 gene to make protein, so alternate model cells had to be used to study SEP53 gene function For example, although the transfected SEP53 gene can be transcribed into a stable RNA species in OE19 or OE33 cells (Fig 6A, left, lanes and 5), we could not detect SEP53 protein in these OE cell panels (data not shown) This contrasts with, for example, HCT116 cells, in which untagged or HIS-tagged SEP53 protein could be easily detected in wild-type p53 or p53-null cells (Fig 6A, middle, lanes 2, 3, and versus and 5) We first chose the H1299 cell as a model because it is well characterized with regards to its apoptotic pathway, is p53-null (which is not required for DCA-induced death) (Fig 5), does not express endogenous SEP53 protein (data not shown), has been used previously to characterize the Barrett’s oesophageal antigen Anterior Gradient-2 [21], and can express transfected SEP53 protein (see below) Using this cell model, the transfection of the tumour suppressor p53 gene into cells can suppress the number of colonies formed, relative to vector DNA only control (Fig 5A,B), whereas SEP53 enhances colony formation in this assay (Fig 5A,B), indicating that SEP53 can function like a survival factor rather than a growth suppressor like p53 The survival activity is apparently not modified by p53 because A375 cells containing a wild-type p53 pathway also exhibit similar enhanced survival in response to DCA -mediated cell death (Fig 5C,D) The survival-promoting activity of SEP53 is consistent with its role as a stress-induced protein where cells might recruit the protein to maintain cell integrity The mechanism whereby SEP53 functions as a survival factor is not defined, but is consistent with the function of other unrelated stress proteins In order to begin to develop a mechanism to explain how SEP53 functions as a survival factor, we thought that analysing the functional domains of SEP53 might gives clues to the signalling pathways linked to its function The SEP53 gene is located on chromosome 1q21 within a group of proteins – the ‘fused gene’ family These proteins are of similar structure to SEP53 containing an N-terminal EF-hand calcium-binding domain and multiple C-terminal amino acid repeat sequences Using a protein BLAST search, several proteins on the 1q21 locus demonstrated limited homology to SEP53 (Fig 5E) The greatest similarity between these proteins was within the first 90 amino acids, which contain the two helix–turn–helix sequences of the EF-hand calcium-binding motifs The calcium-binding FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al SEP53 attenuates deoxycholate-mediated injury SEP53 expression in cancer cells H1299 SEP53 H1299 HIS-SEP53 GST-SEP53 control native SEP53 HCT116 p21 -/GST-SEP53 control HIS-SEP53 GST-SEP53 HCT116 p53 -/native SEP53 control control HIS-SEP53 HCT116 p53 +/+ SEP53 control Middle panel (Protein expression in transfected HCT116 cells) Right panel (Protein expression in transfected H1299 cells) OE21 SEP53 OE19 control Left panel (RT-PCR of transfected SEP53 in OE cells) native SEP53 A SEP53 SEP53 amplimer actin B C Deoxycholic acid only 100 80 60 Sep-53 40 80 60 Sep-53 40 Cell viability (%) 100 Cell Viability (%) 120 20 20 0 0.5 Sep-53 E SEP53 and ∆Ca-SEP53 protein F Cell Viability (%) Actin Deletion of the Calcium binding domain attenuates SEP53 function after DCA exposure expression in stable cells SEP53 ∆ca-SEP53 0.5 Fixed DCA + increasing Ethanol (% ) Ethanol (% ) 90 80 70 60 50 40 30 20 10 0 0 Hours of incubation with DCA DCA with Ethanol D Ethanol only 120 Cell Viability (%) 1 34 56 78 80 70 60 50 40 30 20 10 SEP53- SEP53+ Dca- Dca+ stable cell genotype Fig Cell viability in response to DCA damage is enhanced by SEP53 (A) SEP53 gene expression in transfected tumour cell lines Vector or SEP53 gene (1 lg) was transfected into: (a) left panel, OE190 and OE21 cells; (b) middle panel, HCT116 cells; and (c) right panel, H1299 cells In the left panel, SEP53 protein production could not be observed (data not shown), but RNA was isolated for RT-PCR analysis where the expression of the gene can be detected (lanes and versus and 4) In the middle panel, SEP53 expression vectors were used in HCT116 cells without a tag (lanes and 6), with a HIS-tag (lanes and 7), or GST tag (lanes and 8) and immunoblotted with the SEP53 antibody In the right panel, SEP53 protein was detected in H1299 cells, relative to the control (B–D) Viability of H1299 cells after exposure to selected stresses The H1299 panel without or with SEP53 protein (see immunoblot in the Fig 6A, right panel) was treated with the indicated combination of (B) fixed concentrations of DCA over the indicated time (500 lM), (C) increasing concentrations of ethanol (for h), or (D) combination of fixed DCA (500 lM) and increasing concentrations of ethanol for h Viability was determined as indicated in Experimental procedures using Trypan Blue (E) Development of stable cell lines expressing wt SEP53 and DCa–SEP53 H1299 cells (p53-null) were transfected with the indicated DNA vector (1 lg) and one day after transfection, cells were split and plated in media containing Geneticin to select for cell containing vector DNA (as in Fig 5A,G) After three weeks, the number of cells was determined by fixing cells and staining with dye Individual colonies from a different plate: (a) YFP-vector only (SEP53-negative clones); (b) YFP–SEP53 (lane 1); and (c) DCa–YFP– SEP53 (lane 2) were cloned, propagated, and amount of SEP53 quantified by immunoblotting as indicated (F) Deletion of the EF-hand domain inhibits the survival activity of SEP53 Cell panels were exposed to DCA and processed to analyse for toxicity by Trypan Blue staining The data reflect cell survival (%) as a function of genotype: from left SEP53– ⁄ –, SEP53+, DCa-SEP53– ⁄ –, and DCa-SEP53+ sites of the proteins all share 45–50% identity with SEP53’s calcium-binding site (Fig 5B) The EF-hand in SEP53 homologues is also well conserved (data not shown), although the remaining 80% of the protein has < 30% identity with its murine counterpart This bioinformatics analysis suggests that calcium binding might be central to the function of SEP53 and as such we analysed whether deletion of the calcium-binding domain of SEP53 alters its specific activity in the clonogenic assay Yellow fluorescent protein (YFP)- FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1939 SEP53 attenuates deoxycholate-mediated injury J Darragh et al fusion constructs of wild-type SEP53 was transfected into cells and this fusion protein induced a similar survival activity to untagged SEP53 in a clonogenic survival assay relative to YFP-control (Fig 5G,H) Deletion of the calcium-binding domain in SEP53 strikingly reduced the survival activity, possibly into a dominant negative form of the protein that actually functioned as a growth suppressor, relative to the control (Fig 5G,H) SEP53 functions as a survival factor after DCA exposure in a viability assay Another standard assay was used to evaluate SEP53 function as a stress protein, involving alterations in cell viability as measured by Trypan Blue exclusion When SEP53neg ⁄ H1299 cells were exposed to increasing concentrations of DCA, there was a time- and dosedependent increase in nonviable cells within 4–6 h (Fig 6B) However the SEP53+ ⁄ H1299 cells had a higher degree of resistance to DCA-induced toxicity (Fig 6B), especially at and h post treatment, again consistent with SEP53 functioning like a classic stress protein and promoting cell survival under a stressful stimuli We examined a range of treatments and ⁄ or chemicals relevant to acid-reflux disease control for SEP53-modified effects alone and in combination with DCA (data not shown) These include pro-oxidizing agents, chemicals that alter chromatin deacetylation and methylation, thermal stresses, ethanol, heavy metals and acidified versions of such treatments In particular, ethanol may be an associated risk factor for tissue injury, however, ethanol alone at up to 4% (v ⁄ v) does not effect cell viability in SEP53+ ⁄ neg cells (Fig 6C) However, the combined action of ethanol with fixed DCA is more toxic than DCA alone (Fig 6D) and SEP53 overproduction reduces the toxic effect of this treatment (Fig 6D) Together, these date demonstrate that SEP53 can function as a survival factor, in particular modifying the DCA-viability response Because deletion of the calcium-binding domain of SEP53 attenuated is activity as a survival factor in a clonogenic assay, we also evaluated whether the response to DCA required the calcium-binding domain Stable cells overexpressing full-length YFP– SEP53 fusion protein and the YFP–DCa:SEP53 variant were also constructed in order to analyse differences in DCA response in a nontransient cell system An example of the set of cell clones acquired is given in Fig 6E, and one representative pair was used expressing equivalent levels of both full-length YFP–SEP53 fusion protein and the YFP–DCa:SEP53 variant In 1940 response to DCA challenge, the full-length YFP– SEP53 stable cell enhanced cell survival as defined by Trypan Blue staining (Fig 6F), similar to that observed in transient systems or in stable cells overproducing the untagged version of the protein By contrast, the YFP–DCa:SEP53 variant was unable to protect cells from death induced by exposure to DCA (Fig 6F) Finally, SEP53 was also able to attenuate DCA-induced apoptotic response in HCT116 cells (Fig 3F–H), from 33 to 16% apoptotic cells (data not shown) This attenuation was lost using the transfected DCa:SEP53 (data not shown), which is again consistent with a survival activity of the protein The mechanism whereby SEP53 protects cells from DCA-mediated injury is not clear and we sought to define such a mechanism DCA is known to induce DNA damage [14], but whether DNA damage-independent pathways are linked to SEP53 function is further undefined The mechanism underlying the effects of the calcium-binding domain in SEP53 on its protective function was examined by determining whether SEP53 alters calcium-signalling pathways in cells For example, SEP53 might function as a sensor of calcium perturbation and this might recruit the protein to function in a protective pathway Fura-2 can be used to quantify the concentration of free calcium in cells (Fig 7A,B) Stresses that activate cell death often induce the release of calcium that acts like a signalling molecule and triggers apoptotic cascades [32,33] Although not reported previously, DCA induces release of calcium in cells in a dose-dependent manner up to $ 40 nm (Fig 7C) Furthermore, in cells overexpressing SEP53, there was an attenuation in the release of calcium in response to DCA (Fig 7C) By contrast, cells expressing the YFP–DCa:SEP53 variant were unable to prevent the release of calcium in response to DCA (Fig 7D) These data together provide a correlation between the protective function of SEP53 in response to DCA, calcium release suppressed by SEP53, and its highly conserved calcium-binding domain Discussion Tissue-specific stress responses control cell injury, disease development, and related cancer progression rates The environmental agents that play a role in cancer development are defined in only a few types of cancers, including those of the skin, breast and gut, mainly because patients present with complications representing intermediates that can be analysed by endoscopy The oesophageal squamous epithelium is one such tissue amenable to study and is subject to damage from FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al Emission Spectra for FURA-2 calibration C Em=510 nm 1.35 0.602 39 nM free 0.351 Calcium 0.225 0.150 0.100 2000 1000 0 Increases in Calcium concentration (nM) Fluorescence Excitation A SEP53 attenuates deoxycholate-mediated injury 0.065 0.038 0.017 300 350 400 450 nm SEP53 reduces calcium release after DCA treatment 50 45 40 35 30 25 20 15 10 Calibration plot for FURA-2 Log (bound/free) 1.00 0.75 r2=1 y=0.958x+0.95 x intercept = -0.9921 Kd=0.102 25 0.50 0.25 0.00 -1.5 -0.25 -1.0 The calcium binding domain of SEP53 is required to suppress calcium release D Increases in calcium concentration (nM) 1.25 Sep-53 0.25 0.5 DCA concentration (mM) Wavelength (nm) B -0.5 0.0 20 15 10 2+ SEP53- log([Ca ]free SEP53+ Dca- Dca+ stable cell genotype -0.50 -0.75 -1.00 Fig The EF-hand domain of SEP53 is required to suppress calcium release after DCA exposure (A, B) Calibration for Fura-2 was developed as described in the Experimental procedures (C) DCA-induced calcium release is attenuated by SEP53 The SEP53– ⁄ – and SEP53+ cell pair was exposed to increasing concentrations of DCA for h and cells were analysed for free calcium changes based on the calibration in (A) and (B) (D) Deletion of the EF-hand domain permits DCA-induced calcium release Cell panels as indicated were exposed to DCA and processed to analyse for free calcium changes based on the calibration in (C) and (D) The data reflect changes in calcium release (in nanomolar concentrations) as a function of genotype: from left SEP53– ⁄ –, SEP53+, DCa–SEP53– ⁄ – and DCa–SEP53+ refluxate containing deoxycholate and related bile acids that might play a role in promoting normal tissue injury [22] In this report, we cloned the uncharacterized squamous cell-specific stress gene SEP53, developed antibodies to the gene product, examined its expression, and developed cell lines to determine whether the gene product had protective effects from DCA damage in vitro Cellular stress protein responses play a key role in minimizing cell injury and maintaining tissue integrity in response to damaging levels of an environmental agent As such, the integrity of this system plays a role in modifying progression of diseases associated with ageing, DNA or protein damage and chronic injury Although the HSP genes are evolutionarily conserved and presumably have a ubiquitous function in all cellular repair processes [38], a surprising observation in metazoans is that there is a relatively high degree of cell and tissue specificity in HSP and related stress-activated transcription factor induction [39–44] Consistent with this, our initial analysis of the basic stress response in squamous epithelium indicated that the FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1941 SEP53 attenuates deoxycholate-mediated injury J Darragh et al classic HSP70 was strikingly downregulated after stresses like heat shock or ethanol exposure [16] Many tissues also exhibit uncoupled HSP gene expression and HSP protein induction many hours after stresses including hyperthermia as well as endotoxin exposure [45–47] Some animals not show any evidence of HSP gene expression after stress in some cell types [48–52], similar to the squamous epithelium as summarized above In Drosophila melanogaster, Malpighian tubules an atypical stress response and can remarkably induce a novel HSP60 family member after heat shock, but only in this tissue type [44], indicating that some cells have evolved unique stress responses presumably due to unique microenvironmental pressures The mechanisms underlying this tissue and cell-specific control on rates of stress protein induction in metazoans is not clear We therefore used a functional proteomics approach with normal oesophageal squamous epithelium, a relatively unique tissue with respect to the types of environmental agents to which it is exposed Three major proteins were identified as stress proteins and of these, the SEP53 has the most unresolved function [17] As such, we focused on the study of this gene product because identifying its function might give more insight into squamous cellular stress responses The SEP53 gene was originally cloned as a gene named C1orf10 (clone open reading frame 10) expressed in normal but not oesophageal cancers [18], whereas we identified it independently as a protein induced by heat shock or ethanol treatment ex vivo in normal squamous epithelium [17] A more recent study has indicated that the protein is expressed by immunohistochemical methods in skin keratinocytes and it was speculated that the protein may play a role in epidermal differentiation [53] In this report we provide the first functional information to explain why SEP53 protein might be induced by stress in the oesophagus: it can function as a survival factor that might allow cells to tolerate normally lethal levels of DCA SEP53 protein expression will presumably help maintain the barrier function in squamous epithelium in response to injury The mechanism whereby DCA can induce cell injury was recently shown to involve in part DNA damage [14] Our study also shows that free calcium concentrations are elevated after exposure to DCA (Fig 7) and that the apoptotic response requires a PKC-dependent pathway (Fig 3) The fact that SEP53 can attenuate DCA-mediated elevations in calcium (Fig 7) suggest that it can at the least function in between the signal cascade initiated by exposure and the trigger that releases calcium leading to PKC-dependent apoptosis What will the biological relevance of SEP53 entail? Because the gene is not well-conserved during evolu1942 tion, being, as far as we can tell, confined to mammals with the murine homologue being only $ 50% identical, it is not clear whether SEP53 will function in DCA responses in other species Because microarray analyses from genomic consortium indicate that murine SEP53 is expressed in cervical squamous epithelium and skin (data not shown), it might have evolved initially a ‘barrier’ function in tissues other than the oesophagus The N-terminal calcium-binding domain of SEP53 is highly conserved suggesting this is central to its function Accordingly, viral infection or oxidant stresses in cervix, bladder or skin that effect calcium release might reflect the conditions under which SEP53 evolved originally The response to DCA in human cells might have been acquired later in evolution, and because the toxic effects of bile in humans might also lead to calcium release (Fig 7), the highly conserved calcium-binding domain of SEP53 might play an important role in this sensing In relation to this, our previous proteomics approach comparing normal squamous to Barrett’s epithelium noted a relatively high level of calcium-binding proteins differentially expressed [21], which might relate to the importance of calcium signalling in oesophageal epithelial homeostasis Future research in this area will involve identifying novel SEP53-binding proteins that can in turn be evaluated biologically to ascertain how SEP53 protein might function as a protective stress-responsive protein Experimental procedures Chemicals and reagents Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK) Bisindolylmaleimide I (Bis-I) and the negative control bisindolylmaleimide V (Bis-V) were from Calbiochem All solvents and acids were obtained from BDH (Merck Ltd, Dorset, UK) Tissue culture medium, sterile NaCl ⁄ Pi, fetal bovine serum (FBS), Trypsin EDTA and Lipofectamine were all purchased from Gibco BRL (Paisley, UK) Analysis of bile acids in gastric juice by gas chromatography Aspirates of gastric fluid were obtained at endoscopy from 172 patients being investigated for GORD following informed consent and with the approval of the local Tayside Medical Ethics Committee Gastric juice aspirates were collected from patients during a routine upper gastro-intestinal (GI) endoscopy at Ninewells Hospital and Medical School, Dundee, UK All patients were participating in the FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al Barrett’s Oesophagus Risk Evaluation Database (BORED) study, which was approved by the Tayside Medical Ethics Committee As part of this study, written consent was given to obtain pinch biopsies, gastric juice aspirates and blood from all patients In addition, questionnaires giving detailed information on patient age, sex, medical and drug prescribing history, alcohol consumption and diet were provided An endoscopy examination was carried out to obtain tissue for diagnosis, during which gastric juice was aspirated from the gastric fundic region using a suction trap, and then stored at )20 °C until analysed Gastric juice samples were taken during a routine diagnostic endoscopic procedure Although all patients had symptoms of gastro-oesophageal reflux disease, 52 patients had no indicators of disease by endoscopy Forty-eight patients had oesophagitis with varying degrees of severity and 60 patients were diagnosed with Barrett’s oesophagus Clinical information was unavailable for 12 of the patients No significant difference was found when total and unconjugated bile acid concentrations of gastric juice were compared for the patient groups (data not shown and published in the PhD thesis of J Darragh, University of Dundee) Similar levels of bile acids were detected in each group, however, a small number of patients displayed unusually high concentrations The concentrations of cholesterol, CA, CDCA, DCA, LCA and UDCA were analysed individually with regard to the different patient groups The presence of a particular bile acid was not associated with disease state as determined by one-way anova (data not shown and published in the PhD thesis of J Darragh, University of Dundee) Although no difference in specific bile acid concentrations was observed between the various diagnostic groups, the ratio of the secondary bile acid DCA and its hydroxylated primary precursor CA was determined for each group to determine any possible changes in proportion In addition possible changes in the proportion of conjugated to unconjugated bile acids were analysed No statistically significant change in bile acid ratios was found (data not shown and published in the PhD thesis of J Darragh, University of Dundee) The effect of proton pump inhibitor (PPI) medication on bile acid levels was also investigated The total bile acid concentrations of patients who had, and had not taken PPIs within a month of endoscopy were compared Furthermore, the change in the conjugated to unconjugated bile acid ratio was determined for these two groups No significant difference in either concentration or composition of bile acids was demonstrated following analysis with a Student’s paired t-test It is, however, important to note that the numbers of patients in each group differed considerably, as only 42 patients were not on any PPI treatment compared with 113 patients on medication Gastric juice samples were taken during a routine diagnostic endoscopic procedure The bile acid composition of the samples was determined by gas chromatography using a method described previously [34] Known concentrations (0.5, 1, mgỈmL)1) of SEP53 attenuates deoxycholate-mediated injury standard CA, CDCA DCA, UDCA and LCA bile acids were used for calibration (Fig 1A,B) For detection by gas chromatography, bile acids had to be initially hydrolysed to remove glycine and taurine conjugates, and subsequently extracted and derivatized [34] Cell culture Cell lines were cultured in a 95% O2, 5% CO2 incubator at 37 °C in the indicated medium: H1299 cells (lung carcinoma cells were a gift from D Lane, University of Dundee, UK) were cultured in RPMI 1640, 25 mm Hepes, mm l-glutamine, and 10% FBS; A375 cells (melanoma cells were a gift from J Blaydes Vogelstein, Southampton University, UK) were maintained in Dulbecco’s modified Eagle’s medium and 10% FBS; and HCT116 cells (p53+ and p53– derivative colon carcinoma cells were a gift from B Vogelstein, Johns Hopkins University, USA) were maintained in McCoys’s media containing 10% FBS All human oesophageal cancer cell lines were obtained from the European Collection of Cell Cultures (ECACC), Salisbury, UK, and grown in RPMI OE19 (ECACC no 96071721) and OE33 (ECACC no 96070808) cells were derived from adenocarcinomatous tumours of the oesophagus, whereas the OE21 (ECACC no 96062201) and KYSE30 (ECACC no 94072011) cell lines were derived from oesophageal squamous carcinoma Cells were transfected at 70% confluency with DNA as indicated and the plasmid DNA solution was diluted to a final concentration of lgỈmL)1 in prewarmed serum-free medium Tfx reagent (Promega Corp., Madison, WI) was thawed at room temperature and added to the DNA ⁄ medium mixture, with 4.5 lL of Tfx added for every lg of DNA used (a charge ratio of : 1, Tfx ⁄ DNA) The DNA ⁄ Tfx reaction mixture was incubated for 15 at room temperature, made up to mL with serum-free medium, and then added to each flask of medium-free cells Cells were then placed in the 37 °C incubator for h to enable the transfection to occur Following the h incubation, mL of medium containing 20% fetal calf serum (FCS) was added to the cells, giving a final concentration of 10% FCS To develop colony formation assays, cells were transfected with lg of DNA per well and 24 h post transfection, equal numbers of cells were seeded into 10 cm plates with Geneticin (antibiotic G418) selection at mgỈmL)1 Colonies were fixed with methanol (10 at room temperature) and stained with a ⁄ 20 (v ⁄ v) dilution of Geisma stain for 20 at room temperature To create stable cell lines expressing SEP53 protein, H1299 cells were transfected with lg DNA (untagged SEP53 or tagged: pEYFP–C ⁄ N, pEYFP–C ⁄ N–DCa:SEP53 and pEYFP– C ⁄ N:SEP53) When single colonies reached 1–2 mm in size, colonies were trypsinized using cloning cylinders (Sigma, UK) Trypan Blue staining was carried out as described [35] FACS analysis was performed as described previously [36] A FACScan flow cytometer system (Becton Dickinson, Europe) was used to count the individual cells FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1943 SEP53 attenuates deoxycholate-mediated injury J Darragh et al (30 000 events) The flow cytometer measured the following parameters: forward light scatter (FCS), side light scatter (NaCl ⁄ Cit) and fluorescence of the DNA–PI complex at 620 nm using the FL3 lens Immunochemical methods Pinch biopsies were obtained from patients during a routine upper GI endoscopy at Ninewells Hospital and Medical School, Dundee, UK All patients were taking part in the BORED study, which was approved by Tayside Medical Ethics Committee All patients gave informed consent before samples were taken Samples were snap frozen in liquid nitrogen and stored at )70 °C until analysis Frozen cell pellets were developed as described previously [21] Protein concentrations were determined by the method of Bradford [37] Antibodies used for immunoblotting and fluorescent microscopy include: a-b-actin (Abcam, Cambridge, UK), a-SEP53 polyclonal (Moravian Biotechnologies, Czech Republic.), a-SEP53 monoclonals [developed in this study and unless indicated the MAbs immunoblotted for SEP53 were 4.1 (Class B), since it was of highest titre when grown as ascites and could detect cleaved SEP53 (as could class A)], DO1 (a-p53), PKB ⁄ AKT IgG, phospho-specific PKB ⁄ AKT IgG, and phospho-specific GSK3 IgG were from Cell Signalling Technology (UK), anti-GSK3 IgG was from BD Transduction Laboratories, Europe Horseradish peroxidase (HRP)anti-mouse (Dako Ltd, Ely, UK), HRP-anti-rabbit (Dako), and Alexa-FluorÒ 594 anti-mouse sera (Molecular Probes, Invitrogen, Paisley, UK) Stained slides were viewed with an Eclipse E600 microscope (Nikon, Kingston upon Thames, UK) Fluorescent micrographs were produced using spot advanced software (Diagnostic Instruments, Sterling Heights, MI) Recombinant SEP53 gene construction Total RNA was isolated from normal oesophageal tissue (RNeasy Mini Kit, Qiagen, Crawley UK) and lg used for reverse transcription using Omniscript and oligo(dT)15 primer (Qiagen) The sequences of the oligonucleotides used in the amplification of SEP53 are: forward 5¢-CATA GCTCGAGCTATGCCTCAGTTACTGCAAAACATT-3¢; reverse 5¢-CAGTCAAGCTTCATGGCTTGGTGCTTCT CAAGT-3¢ Oligonucleotides used in PCR amplification of this SEP53 fragment to introduce attB sequences for cloning into the Gateway cloning system (Invitrogen) were: forward 5¢-GGGGACAAGTTTGTACAAAAAAGCAG GCTCCATGCCTCAGTTACTGCAAAACATTAATGGG ATCATCGAGGCC-3¢; reverse 5¢-GGGGACCACTTTGT ACAAGAAAGCTGGGTCGGCCAGCGGCTTAAGGTT TTATTGATGCATTAGGGTAGATGGGGC-3¢ Human SEP53 gene was subcloned into the Gateway entry vector pDONR201 (Invitrogen) and the sequence confirmed by DNA sequence analysis To subclone SEP53 with the 1944 calcium binding site deleted (DCa–SEP53) PCR primers were designed with restriction sites BglII at the N-terminus, XbaI at the C-terminus and start and stop codons These primers were used to amplify a DCa–SEP53 PCR product The XbaI site in the pEYFP-C1 vector is methylated and for cloning into this vector, plasmid DNA was transformed, into the dam– Escherichia coli strain GM2163 (New England Biolabs, UK) DNA was isolated using a QIAPREP Spin mini prep kit (Qiagen) The unmethylated pEYFP-C1 DNA and DCa–SEP53 PCR product were then digested with BglII and XbaI restriction enzymes The vector and PCR product were then ligated and transformed into competent cells SEP53 expression was analysed using the primers: (a) full-length SEP53, forward 5¢)3¢ CAGTC AAGCTTATGCCTCAGTTACTGCAAAAC and reverse 5¢)3¢ CATAGCTCGAGTCATGGCTTGGTGCTTCTC; (b) DCa–SEP53, forward 5¢)3¢ TGCTAGAATTCAGATC TATGAGCGAGAGTGCTGAGGGA and reverse 5¢)3¢ TGCTATCTAGATCATGGCTTGGTGCTTCT HIS-tagged SEP53 in the vector pDEST17 (Invitrogen) was transformed into E.coli BL21 AI cells (Invitrogen, Paisley, UK) and purified by nickel affinity chromatography A panel of MAb obtained by immunization of mice with full length HIS-tagged SEP53 protein were developed by Moravian Biotechnologies Calcium determinations The fluorescent indicator Fura-2 (Molecular Probes) was used to measure cytosolic-free calcium levels in cells When Fura-2 is bound to calcium it is excited at 340 nm and when it is free it is exited at 380 nm and this difference can be used to ascertain the intracellular calcium by measuring the fluorescence emission at 510 nm for both these excitation wavelengths (A, excitation and emission at 340 nm when Fura-2 is bound to calcium; B, excitation and emission at 380 nm when Fura-2 is free) Intracellular calcium levels can then be calculated from Eqn (1) where Kd is the dissociation constant of the indicator, R is the ratio of fluorescence (F) at 340 nm and 380 nm (F340 nm ⁄ F380 nm), Q is the ratio of Fmin ⁄ Fmax at 380 nm ẵCa2ỵ ẳ Kd QR Rmin Þ=ðRmax À RÞÞ ð1Þ The Kd value was obtained by calibrating the potassium salt form of Fura-2 in cell-free solutions using a calcium calibration buffer kit #1 (Molecular Probes) The kit contains two buffers: 10 mm K2EGTA-buffered solution (‘zero’ free Ca2+) and 10 mm CaEGTA-buffered solution (40 lm free Ca2+) Equal amounts of dye (10 lm) were added to each buffer solution Emission spectra at 510 nm were measured over the excitation spectra 300–450 nm to create a series of curves using a fluorescent spectrophotometer (Hitachi) Data were analysed using fl solutions software (Hitachi) The emission fluorescence at 510 nm for excitation at 340 and 380 nm was then used to calculate FEBS Journal 273 (2006) 1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS J Darragh et al the Kd value The log of the [Ca2+] free (x-axis) is plotted against the log of bound ⁄ free dye {(R ) Rmin) ⁄ (Rmax ) R)} (F2 ⁄ F2 max) (y-axis) This can then be used to calculate Kd of the indicator, which is the inverse log of the x-intercept To label cells with Fura-2, Fura-2AM was used, as the addition of the AM group results in an uncharged molecule that can permeate cell membranes Once the molecule has entered the cell the AM group is cleaved by nonspecific esterases in the cells, resulting in a charged molecule that leaks out far slower than its parent compound A mm stock solution of Fura-2AM prepared in dimethyl sulfoxide (DMSO) was diluted in an equal volume of the nonionic detergent Pluronic F-127 (20% solution in DMSO; Molecular Probes) This solution was then diluted in serum-free, phenol red-free RPMI to a final concentration of lm and added to cells that were incubated at room temperature for 20 with gentle shaking Cells were then washed twice in NaCl ⁄ Pi and resuspended in phenol red-free RPMI with 10% FBS and incubated for 30 at room temperature with gentle shaking Cell suspensions were then used for intracellular calcium measurements Calcium levels were measured on a fluorescent spectrophotometer F4500 (Hitachi) using the intracellular cation scan mode The effects of bile acids were measured by adding deoxycholic acid dissolved in methanol to the cell suspension to the desired concentration Methanol alone was used as a control Data were analysed using fl solutions software (Hitachi) The fluorescent spectrophotometer settings for intracellular cation scan were as follows: excitation wavelength k1 (340 nm), excitation wavelength k2 (380 nm), emission wavelength (510 nm), excitation slit (5 nm), emission slit (5 nm), PMT voltage (700 V); cycle time (0.7 s) and 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1930–1947 ª 2006 The Authors Journal compilation ª 2006 FEBS 1947 ... Cellular stress protein responses play a key role in minimizing cell injury and maintaining tissue integrity in response to damaging levels of an environmental agent As such, the integrity of this... protein to maintain cell integrity The mechanism whereby SEP53 functions as a survival factor is not defined, but is consistent with the function of other unrelated stress proteins In order to begin... using the protein kinase C (PKC) inhibitor bisindolylmaleimide I (Bis-I), which inhibited DCA-dependent apoptosis (Fig 3I) The control inactive version of the inhibitor bisindolylmaleimide V (Bis-V)

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