2004 Summer Meeting - Amsterdam - The Pathological Society of ...

205
Wnt Expression In Colonic Subepithelial Myofibroblasts
S Bamba 1 , WR Otto 1 , M Brittan 1 , SL Preston 1 , S Ahmed 2 ,
PJ Pollard 1 , NA Wright 1
1 Cancer Research UK, London, United Kingdom, 2 Barts and The London
Hospital, London, United Kingdom
BACKGROUND: Colonic subepithelial myofibroblasts (SEMFs) are present
immediately beneath the epithelial cells, and speculation is rife that these
maintain the stem cell niche. In this study we show that SEMFs are the source
of Wnt signalling in the colon.
METHOD: Colonic SEMFs and colonic crypts were isolated from C57/BL and
IL-10 knockout mice (which develop colitis spontaneously) using EDTA, and
mRNA expression of Wnt and its receptor Frizzled (Fzd) was studied using RT-
PCR. To evaluate the regulation of Wnt mRNA, EGF was used to stimulate
SEMFs.
RESULTS: RT-PCR studies revealed (i) 10 out of 19 Wnt mRNAs were
expressed in SEMFs from C57/BL mice and IL-10 knockout mice; (ii) Wnt
mRNA expression was observed mainly in colonic SEMFs, on the other hand
the expression of Fzd mRNA was observed in both colonic SEMFs and crypt
epithelium. (iii) IL-10 knockout mice-derived SEMFs tended to express more
Wnt mRNA than C57/BL-derived SEMFs. (iv) EGF specifically upregulated
Wnt2 mRNA expression.
CONCLUSION: The source of Wnt signalling in the colon is predominantly
from the colonic SEMFs, consistent with a role for these cells in the
maintenance of the stem cell niche. Fzd mRNA expression in SEMFs suggests
that Wnt proteins secreted from SEMFs can act not only in a paracrine manner
but also in an autocrine manner. IL-10 knockout mice-derived SEMFs
displayed different Wnt mRNA expression patterns from C57//BL-derived
mice, implying immature characteristics of SEMFs in inflammation.
206
Expression of osteoprotegerin (OPG) and TRAIL in
oesophageal carcinoma and non-neoplastic gastric and
oesophageal mucosa
A Spencer , SS Cross , I Holen , R Ackroyd , CJ Stoddard , JP
Bury
University of Sheffield, Sheffield, United Kingdom
Osteoprotegerin (OPG) is a member of the Tumour Necrosis Factor (TNF)
superfamily, and acts a soluble decoy receptor for the Receptor Activator of
Nuclear Factor-B Ligand (RANKL) and TNF-related apoptosis-inducing
ligand (TRAIL). OPG inhibits the differentiation of osteoclast precursors, and
has been widely studied within the context of bone metastasis, particularly in
breast and prostate cancers. OPG expression has also been demonstrated in
gastric carcinomas, and has been shown to correlate with poor survival. We
studied the expression of OPG and TRAIL in oesophageal carcinomas and
surrounding normal gastric and oesophageal mucosa. We mounted 1mm cores
from carcinomas and from background oesophageal and gastric mucosa in a
tissue microarray and performed immunohistochemistry for OPG and TRAIL.
OPG was expressed in 11 of the 20 tumours studied (55%). TRAIL was
expressed in 12 of 19 tumours studied (63%; cores from one tumour were not
retained on the array). No consistent pattern of co-expression or inverse
expression of OPG and TRAIL was discernible within tumour tissue. Neither
OPG nor TRAIL was detected in normal oesophageal mucosa. We observed
OPG staining in normal background gastric mucosa, which was markedly more
prominent in oxyntic cells. TRAIL staining was also positive in background
gastric mucosa, and interestingly was markedly less prominent in oxyntic cells.
At least one previous study has demonstrated OPG in gastric carcinomas whilst
failing to do so in normal gastric epithelium, using an antibody to the N-
terminal region of the protein. This study used an antibody to the C-terminal
region of the protein, raising the possibility that OPG in gastric mucosa exists in
an altered form to that found in gastric carcinomas.
207
Molecular interactions between E-cadherin and a2b1 integrins
in epithelial cells.
N Ockendon , A Buda , M Pignatelli
Department of Pathology and Microbiology, University of Bristol, Bristol,
United Kingdom
E-cadherin is a transmembrane glycoprotein, which mediates epithelial cell-cell
adhesion. Its adhesive function requires association with -catenin and -
catenin linking to the actin cytoskeleton. Integrins are transmembrane
glycoproteins that bind extracellular matrix (ECM). Both integrins and E-
cadherin are localised at the cell-cell junctions.
The aim of this study was to investigate the sub-cellular distribution of E-
cadherin-catenin complex and 1 integrin in oesophageal and colonic cancer
derived cell lines and their physical and/or functional interactions.
Two colonic carcinoma cell lines (HT29 and LS174T) and two oesophageal
carcinoma cell lines (KYSE30 and OE33) were employed. Protein expression
was assessed by Western blotting (WB). Sub-cellular distribution was
illustrated by immuno-fluorescence followed by confocal microscopy (IF).
Molecular interaction was investigated by sequential co-immunoprecipitation
(IP) and WB. Cytoskeletal protein association was detected by extraction of
Triton X-100 soluble vs. insoluble cellular fractions.
Indirect IF showed E-cadherin and 1 integrin co-localising with F-actin at cell
peripheries in HT29, KYSE30, and OE33 cells. KYSE30 and OE33 also
showed cytoplasmic localisation. Co-immunoprecipitation experiments
identified a truncated form of 1 integrin associated with E-cadherin. Extraction
of Triton X-100 soluble vs. insoluble sub-cellular fractions.
In conclusion, E-cadherin and integrins are both localised at the cell-cell
junctions but only E-cadherin appears to be associated with the actin
cytoskeleton. Further functional studies will need to elucidate the biological
significance of this novel association.
208
A PROTECTIVE ROLE FOR THE P50-SUBUNIT OF NFB
IN THE AGING LIVER
NS Mungalsingh , F Oakley , DA Mann , J Mann , H Millward-
Sadler
1 Liver Group, University of Southampton, Level D, South Academic
Block, Southampton General Hospital, Tremona Road SO16 6YD,
Southampton, United Kingdom, 2 Department of Pathology, Level D, South
Block, Southampton General Hospital, Tremona Road SO16 6YD,
Southampton, United Kingdom
NFB (p50:p65 dimer) regulates the expression of multiple inflammatory
genes. Targeted disruption of NFB components has revealed critical roles. For
example, knockout of p50 results in susceptibility to infection. There are reports
of tissue-specific changes in NFB activity as humans and animals age,
however to date there is no clear evidence that NFB plays a definitive role in
age-related diseases.
Hypothesis – the p50 subunit of NFB protective against inflammation in
aged mouse livers.
Aims – To examine aged livers of wild type (WT) and p50-knockout mice
for histological evidence of inflammation.
Methods – W.T. and p50-knockout mice were aged for 1 year in SPF
conditions. Livers were removed and processed for haemotoxylin and eosin and
neutrophil staining. Slides were randomised and analysed double-blinded with a
consultant histopathologist. The livers of the p50-knockouts were also
compared with those of 12 week old W.T. mice.
Results – 12 week old mouse livers showed no inflammatory change from
normal. Livers of 5 of the 6 aged W.T. mice displayed no significant
inflammatory change. 4 out of the 5 p50-knockouts displayed significant
inflammatory changes (2 severe, 2 mild).
Conclusion – Loss of the p50 subunit of NFB is likely to be associated with
age-related inflammation of the liver.
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