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 st
Trang 1is 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 Kernohan1and Ted R Hupp2
1 Division of Pathology and Neurosciences, University of Dundee, UK
2 University of Edinburgh Cancer Centre, CRUK Cell Signalling Unit, UK
3 Masaryk Memorial Cancer Institute, BRNO Czech Republic
Human cancers develop through a multistage process
involving morphological changes in tissue, mutations
in oncogenes and tumour suppressor genes, and
epi-genetic programmes that give rise to enhanced survival
in a stressed microenvironment [1] The development
of human cancer is proving to be a tissue-specific
pro-cess 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 devel-oping tumour cell to repair, survive and overcome intrinsic tumour-suppressing signals that normally are used to kill abnormal cells and maintain tissue integ-rity The mechanisms underlying tissue-specific responses to local environment in cancer development are largely undefined
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 2
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 SEP53-mediated 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 intracel-lular free calcium Deletion of the highly conserved EF-hand calcium-bind-ing domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and per-mits 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
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.
Trang 2In developing physiologically relevant models of
stress protein dysregulation in developing human
can-cers, a key clinical model that is giving novel molecular
mechanistic insight is adenocarcinoma of the
oesopha-gus [2] This cancer is one of the fastest rising cancers
in the west, is taking the place of squamous cell
carci-noma 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
inter-mediate (named Barrett’s) and is associated with
increases in proliferation due to an acidified
microenvi-ronment [5] In addition to acid as a key
microenviron-mental 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
facili-tate carcinogenesis [6,7] In particular bile acids such
as deoxycholic acid (DCA) can stimulate cell
prolifer-ation, migrprolifer-ation, 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-oxi-dants, 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
oeso-phageal epithelial cells using a ‘functional proteomics’
approach The first studies indicated that ex vivo
stressed squamous cells in organ culture did not
syn-thesize 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
conjunc-tion 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
epithelial-induced stress protein of 53 kDa (SEP53) and
gluta-mine–glutamyl transferase [17] SEP70 is induced by
acidified extracellular conditions and is a
glucose-regu-lated protein [17] SEP53 was originally cloned as
a gene expressed in normal oesophagus but
downregu-lated in oesophageal cancers and was named Clone 1
open reading frame 10 [18] The SEP53 gene is located
on chromosome 1q21 within a group of proteins
named the epidermal differentiation complex
fused-gene family that it silenced as part of a fused-general
mech-anism that apparently suppresses genes from this locus
in cancer cells [19,20] The function and regulation of
SEP53are 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-medi-ated calcium release and cell death SEP53 is a rapidly evolving gene with < 50% identity to its murine ortho-logue suggesting that the antiapoptotic activity of SEP53 is evolving in relation to selection pressures resulting from environmental stress in squamous epi-thelium
Results
SEP53 protein is expressed in human squamous epithelium
Having previously shown, using a functional proteo-mics approach, that SEP53 is one of the major pro-teins 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 squa-mous 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-expres-sion 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 frag-ments (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, 8 and 10)
We next investigated whether SEP53 protein was expressed in squamous epithelium using these immuno-chemical reagents The SEP53 protein is highly expressed in normal squamous epithelium under condi-tions 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 pro-tein is confirmed to be highly overexpressed in Barr-ett’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 epithe-lium (Fig 1F), where immunoreactivity is generally cytoplasmic granular staining with minor epimembra-nous staining in maturing and mature squamous cells Furthermore, SEP53 is variably expressed in Barrett’s
Trang 3where 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
func-tion 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
synthes-ized ex vivo after heat or ethanol stress [17] The
physiological stress the SEP53 responds to in cells is, however, undefined, as heat exposure to the oesopha-gus and ethanol are unlikely to be evolutionary adap-tations The oesophagus is an organ that is commonly exposed to bile acids and the structure of normal oeso-phageal epithelium is altered by bile exposure [22] Developing knowledge of the effects that these chemi-cals 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 con-tent, bile acids were extracted, derivatized and then analysed by gas chromatography The relative retent-ion times of peaks present in the gastric fluid sample
B
C
D
E F
Fig 1 Development of a panel of monoclonal antibodies (MAbs) to the major squamous-cell specific stress protein SEP53 (A) Characteriz-ation of SEP53 MAb Purified SEP53 protein (1 lg) was incubated without (lanes 1, 3, 5, 7 and 9) or with (10 ng, lanes 2, 4, 6, 8 and 10) trypsin in a buffer containing 25 mM Hepes (pH 7.5) and at 30 C for 5 min 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 lgÆmL)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 immunoblot-ted for (B) SEP53 protein, (C) SEP70 protein, (D) AG-2 protein and (E) loading controls for squamous and Barrett’s biopsies (ink stain immu-noblot 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.
Trang 4were 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
sam-ples 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 1 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
primary bile acids, CA and CDCA, with mean concen-trations 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 1 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 dif-ficult to extrapolate to in vivo concentrations, however,
8%
23%
37%
1-20 u M 20-200 u M
> 200 u M
36%
53%
9% 2%
2%
Cholesterol 40%
Lithocholic 3%
Deoxy cholic 12%
Chenodeoxy cholic 21%
Ursodeoxy cholic
Cholic 22%
A
Bile Acid Rt (Min) RRt Std ratio
Chenodeoxycholic 7.19 0.67 1.15 Ursodeoxycholic 7.84 0.73 1.63
7-Ketolithocholate 10.74 1.00 1.00
B
E
Bile Acid Range u M (cong) Mean u M (cong) Range u M (uncong) Mean u M (uncong)
Fig 2 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 (R t ) 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
Stand-ard ratios represent the peak area of each
1 mgÆmL)1standard compared with the
peak area of the internal standard (B)
Sum-mary of the range of the total bile acid
con-centrations found in gastric fluid samples.
(C) Percentage of patients with bile acid
concentrations as indicated (D) Percentage
of patients with unconjugated bile acids
con-centrations 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 3 : 2.
Trang 5using rabbit oesophageal mucosa as a model, the
epi-thelium concentrates bile acids up to 7· lumenal
con-centrations [24] Thus, given the range of DCA in
patients (1 lm to > 1 mm) and given that bile can be
concentrated from the lumen up to 7· [24], the
poss-ible concentration of DCA in cells might be from 7 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 2 and 3) and given this, we
titrated DCA from low lm to > 1 mm to determine
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), partic-ularly to determine whether DCA was able to signifi-cantly 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 squa-mous cell lines [Fig 3A and C versus Fig 4G (OE21 cells)], in contrast to the oesophageal cancer Eca109
N M
F
E
Trang 6cell line in which this dose gives rise to 22%
apop-totic cells [25] The adenocarcinoma cell lines (OE 19
and OE33 cells) did, however, demonstrate a
dose-dependent 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
char-acteristic 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
dam-age [26] Because DCA can induce DNA damdam-age [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
DCA-mediated signalling The HCT116 (p53+) isogenic cell
line was incubated with increasing concentrations of
DCA (0–500 lm) for 6 h and the resultant stressed
cells were then fixed, stained with PI and the mean
(± SEM) (n¼ 3) percentage of apoptotic cells meas-ured by flow cytometry (Fig 3F–H) Under these con-ditions, apoptosis was elevated, in a dose-dependent manner, from 2 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 signal-ling via p53 in these colonic cell lines Furthermore, because the OE33 and OE19 cell lines have mutant p53 (data not shown), p53-independent apoptosis oper-ates under these conditions
In order to define a positive mechanism for DCA-mediated 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 bisindolylmalei-mide I (Bis-I), which inhibited DCA-dependent apop-tosis (Fig 3I) The control inactive version of the inhibitor bisindolylmaleimide V (Bis-V) was unable to block the apoptosis (Fig 3J), demonstrating the selec-tivity 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-medi-ated apoptotic response [28,29] Consistent with this,
Fig 3 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 6 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 0 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 6 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 6 h followed by a treatment of 100 ngÆmL)1EGF for 10 min 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 6 h followed by a treatment of 100 ngÆmL)1EGF for 10 min 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.
Trang 7M OE33
R
Q
OE33 + DCA
N
B A
F E
Cheno-Deoxycholic Acid
Urso-Deoxycholic Acid Cholic Acid
Lithocholic Acid
1%
1%
1%
2%
Tauro-Deoxycholic Acid
Urso-Deoxycholic Acid
K I
J
L
Control
Cheno-Deoxycholic Acid Cholic Acid
Lithocholic Acid
Fig 4 Cell-cycle parameters in bile acid-treated cells (A–F) Apoptosis after bile acid exposure in OE33 cells Under normal growth condi-tions (A), apoptotic debris is rarely identified among OE33 cells following staining with the nuclear dye Cytox Addition of 500 lM of the indi-cated bile acid (B–F) for 6 h leads to changes in cell-cycle parameters as indiindi-cated (G–L) Reduced apoptosis after bile acid exposure in OE21 cells Addition of 500 lM of the indicated bile acid (H–L) for 6 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) Mor-phology of OE cells OE33 (M, N) and OE21 (O, P) cells were treated with DCA (500 lM) for 6 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 6 h and analysed by electron micro-scopy 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).
Trang 8DCA attenuated phosphorylation of the normally
pro-survival PKB at the activating site of PKB in OE33
cells (Fig 3K, lane 4 versus 2) By contrast, basal
inac-tivating phosphorylation of GSK3 was reduced in
OE33 cells (Fig 3M, lane 4 versus 2) The opposite
occurs in the OE21 cells: DCA did not block phos-phorylation of PKB in the resistant OE21 cells (Fig 3O, lane 4 versus 2), although GSK phosphorylat-ion actually increased in OE21 cells (Fig 3Q, lane 4 versus 2) The data suggest that the GSK3–PKB–PKC
Fig 5 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 differ-entiation complex on chromosome 1q21 including THH, REP, PFG, HORN and BBBAS Amino acid and DNA homology (%) are as indica-ted (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.
Trang 9pathway 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
biolo-gical effect they have been shown to vary in their
abil-ity 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
induc-tion of apoptosis in both the sensitive OE33 and
resist-ant 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
tau-rine (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
oesopha-geal 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
differenti-ation complex fused-gene family and that this locus
might be silenced by chromatin remodelling as part of
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 func-tion For example, although the transfected SEP53 gene can be transcribed into a stable RNA species in OE19 or OE33 cells (Fig 6A, left, lanes 3 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, 6 and 7 versus 1 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 suppres-sor p53 gene into cells can suppress the number of colonies formed, relative to vector DNA only control (Fig 5A,B), whereas SEP53 enhances colony forma-tion 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 survi-val 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 analy-sing 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 pro-teins 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
Trang 10sites 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
SEP53
actin
SEP53 expression in cancer cells
Left panel (RT-PCR of transfected SEP53 in OE cells) Middle panel (Protein expression in transfected HCT116 cells) Right panel (Protein expression in transfected H1299 cells)
SEP53 amplimer
OE19 OE21
HCT116 p53 + /+ HCT116p53- /
-HCT116 p21 - /
HCT116 p53 + /+ HCT116p53- /
-HCT116 p21 - /
-1 2 3 4 5 6 7 8
A
D
F E
DCA with Ethanol
0 10 20 30 40 50 60 70 80 90
Fixed DCA + increasing Ethanol (% )
con Sep-53
0 20 40 60 80 100 120
Ethanol (% )
con Sep-53
0
20
40
60
80
100
120
Hours of incubation with DCA
con Sep-53
Deletion of the Calcium binding domain attenuates SEP53 function after DCA exposure
0 10 20 30 40 50 60 70 80
SEP53- SEP53+ Dca- Dca+
stable cell genotype
SEP53
∆ca-SEP53 Actin
SEP53 and ∆Ca-SEP53 protein expression in stable cells
1 2
Fig 6 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 5 and 3 versus 2 and 4) In the middle panel, SEP53 expression vectors were used in HCT116 cells without a tag (lanes 2 and 6), with a HIS-tag (lanes 3 and 7), or GST tag (lanes 4 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 indi-cated combination of (B) fixed concentrations of DCA over the indiindi-cated time (500 lM), (C) increasing concentrations of ethanol (for 6 h), or (D) combination of fixed DCA (500 lM) and increasing concentrations of ethanol for 2 h Viability was determined as indicated in Experimen-tal 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 stain-ing The data reflect cell survival (%) as a function of genotype: from left SEP53 – ⁄ – , SEP53 + , DCa-SEP53 – ⁄ – , and DCa-SEP53 +