Download An N-Terminally Truncated Cytoplasmic Form of Oxygen

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Conclusion: Lifestyle risk factors for colonic neoplsaia (obesity,
physical inactivity, low fibre, high fat diets and a past history of
smoking) are all associated with elevations in faecal calprotectin.
Bowel inflammation may be the common mechanism whereby diet
and lifestyle influences risk of colonic neoplasia.
M118
An N-terminally truncated cytoplasmic form of oxygenregulated protein 150 (orplSO), the major intracellular ligand for
the anti-proliferative mushroom lectin, is essential for nuclear
localisation sequence (nls)-dependent nuclear protein import in
human intestinal cancer cells
Yu LG and Rhodes JM,
Department of Medicine, University of Liverpool, Liverpool L69
3GA
The classical NU-dependent nuclear import system, which mediates
import of large nuclear proteins, is fundamentally important for
maintaining nuclear function. Our previous studies have shown that
the inhibition of cell proliferation of mushroom Agaricus bisporus
lectin (ABL) (Cancer Res. 1993,53:4627) is linked to its
internalisation and selective blockade of NU-dependent nuclear
protein import (J.Biol.Chern.1999, 274:4890). One of the major
intracellular ABL-binding ligands is an N-terminally truncated
cytoplasmic
form
of
Orp150
[Gusrroenrerology
2001,12O(suppll):3579]. Orp150 is a stress-related protein and is up
regulated in tumours and highly expressed in cancer cell lines. In this
study we investigated the role of Orp150 in nuclear protein import.
Nuclear protein import was performed in digitonin semipermeabilized human colon cancer HT29 and gastric cancer AGS
cells using a fluorescein-conjugated synthetic NLS peptidehovine
albumin complex (NLS-BSA-FITC) as a transport marker. It was
found that introduction of an anti-Orp150 antibody, but not other
irrelevant antibodies, into the transport system resulted in 57% and
48% reduction of nuclear accumulation of NLS in HT29 and AGS
cells respectively. Removal of cytosolic Orp150 from the transport
system caused over 40% reduction of NLS nuclear accumulation.
The ras-related nuclear transport factor Ran was identified in the
Orp150 immunoprecipitate. Orp150 was also identified by
immunoblotting in the immunoprecipitates of Ran but not in the
immunoprecipitates of other Ran-associated proteins (Ran-BPI ,
RCCI, Ran-GAP1 and NTF2).
This result suggests that the truncated cytoplasmic Orp150 has a
crucial role in NU-dependent nuclear protein import probably by
direct interaction with Ran.
M119
ALTERED COLONIC GLYCOPROTEIN EXPRESSION IN
UNAFFECTED
MONOZYGOTIC
TWINS
OF
INFLAMMATORY BOWEL DISEASE PATIENTS
Bodger K*, Dodson AR**, Tysk C***, Campbell F**, Lindherg
E***, JLnerot G***, Rhodes JM*.
Depts of Medicine* & Pathology**, University of Liverpool,
UK; ***OrebroMedical Centre Hospital, Orebro, Sweden
INTRODUCTION Alterations in epithelial glycoprotein expression
in inflammatory bowel disease (IBD) include increased expression of
the oncofetal carbohydrate antigens, TF and sialyl-Tn (sTn).
Previous chromatographic colonic mucin analysis in monozygotic
twins with IBD suggested a possible genetic basis for these changes
(Tysk ef a1 Gusrroenferology 1991;100:419). l l e present study
aimed to explore this further by assessing mucosal expression of
specific glycoprotein epitopes amongst these IBD twins.
METHODS Formalin-fixed rectal biopsies from 22 twin pairs with
IBD were studied (6 pairs concordant for UC or C D 16 healthy
twins). Affected twins were in clinical and endoscopic remission.
Expression of TF and sTn was assessed by PNA-histochemistry and
immunohistochemistry (anti-sTn mAb) respectively, and compared
with expression in rectal biopsies from normal mucosa controls
@=I4 sTn; n=17 TF). T F positivity was scored from 0 to 3.
RESULTS Compared to normal controls, healthy twins showed
greater TF positivity and a trend towards greater sTn positivity.
IBD affected
Healthy
Normal controls
twin
twin
TF-Dositive
TF&w (meanlsdl)
Sialyl-Tn positive
22/22
1.91 [0.56]
12/22
15116
1.50 10.631
5/16
5/20
0.30 l0.571
1/14
(Healthy twin versus Nonnal controls: T F p=<O.OOOI; sTn ~ 4 . 1 8 )
Of the 5 healthy twins who were sTn+, 4 had twins with UC and 1
with CD. One of the sTn+lTF+ healthy twins has since developed
uc.
CONCLUSIONS The positive findings in healthy twins support
previous evidence of a biochemical mucin defect. This could be the
result either of a direct genetically determined alteration in
glycosylation or of a secondary (eg cytokine-mediated) alteration in
glycosylation. The altered glycosylation could be. relevant in
determining changes in the mucosa-associated bacterial flora.
M120
Stress protein expression in human hepatocyte cell lines cultured
as in three-dimensional(3-D)spheroids in alginate.
S Choudhury, C Selden, M Hubank and HJF Hodgson.
Expression and maintenance of differentiated hepatocytic function
by the HepG2 cell line is enhanced by 3-D spheroid culture in
alginate cf. monolayer culture. We have previously shown a 3-10fold increase in secreted protein synthetic, eg albumin, prothrombin,
alpha-I -antitrypsin, and a 5-fold increase in detoxificatory function
eg cytochrome P450 and steroid metabolism, in 3-D cultures cf.
monolayer. Aim: We have investigated the expression of stress
proteins comparing monlayer cultures and spheroidal cultures at
times of optimal function (day 8). and later (day 15) where spheroids
exhibit decreased function despite maintained viability and
proliferation. Methods: RNA prepared from HepG2 cells cultured
as monolayers, 8-day 3-D spheroids, and 15-day 3-D spheroids was
subjected to microarray analysis using the U95Av2 Affymetrix gene
chips (n=3 for each condition). Chips were interrogated for changes
in expression of genes associated with cellular stress. A >2-fold
change in 2 44% of chips was considered baseline for a true change.
Results: At times of maximal expression of differentiated function,
cornparing 8-day 3-D cultures with monolayer, the most prominent
changes in stress proteins were an increuse in Heat-shock protein 70
(HSWO in 77% 3.46*2.5), catalase (in 100% 2.44k0.3) and MHC
class 1 (in 100% 3.2H.68) and a decrease in HSP 47 (in 55% 3.15k0.94) and RNA polymerase I1 (in 55% -2.3850.24). At times of
loss of differentiated function, despite continued viability (Day15 vs.
Day8) the most prominent changes were an increuse in vascular
endothelial cell growth factor (VEGF in 100%. 1.97kO.19) and
decreases in Gluathione-s-transferase(in 44%. -3.83&1.98), MAPK
protein kinase 3 (in 44%. 4.45k1.39) and the transcription factor
HNF3alpha (in 44%. -2.17M.22). Conclusion: Gene arrays