received eradication therapy CAPZA overexpressing cells br a
746
received eradication therapy. CAPZA1-overexpressing cells
805
against
acetylated
histone
H3.
We
used
quantitative
749
Figure 3. (See previous page). Enhanced expression of ESRP1 and b-catenin in CAPZA1-overexpressing PCI 32765 con-
808
tributes to the development of CD44v9-positive cells. (A) AGS cells were transfected with pCMV-ctrl or pCMV-CAPZA1,
751
infected with H pylori G27 or H pylori G27 DcagPAI for 5 hours (multiplicity of infection, 50), and incubated in antibiotic-
810
752
containing medium for 24 hours. The intracellular CagA level and expression of CD44v9, CD44s, ESRP1, and b-catenin
811
753
were examined by Western blot. (B) AGS cells were transfected with pCMV-ctrl or pCMV-CAPZA1, infected with H pylori G27
812
754
or H pylori G27 DcagPAI for 5 hours (multiplicity of infection, 50), and incubated in antibiotic-containing medium for 24 hours.
813
b-catenin localization in the cytoplasmic (Cyt) and nuclear (Nuc) fractions was examined. Data are presented as the means ±
757
incubated in antibiotic-containing medium for 24 hours, and stained for b-catenin. Scale bar: 20 mm. (D) AGS cells were
816
758
transfected with pCMV-CAPZA1, infected with H pylori ATCC700392 for 5 hours (multiplicity of infection, 50), and incubated in
817
AGS cells were transfected with pCMV-ctrl or pCMV-CAPZA1, and incubated in CCT031374-containing medium for 24 hours.
Expression of CAPZA1, b-catenin, and ESRP1 was examined. (F) AGS cells were transfected with pCMV-CAPZA1, subse-
quently transfected with control siRNA (-) or 2 different ESRP1 siRNAs 1 and 2, infected with H pylori ATCC700392 for 5 hours,
and incubated in antibiotic-containing medium for 24 hours. Expression of ESRP1, CD44v9, CD44s, and b-catenin was
8
Tsugawa et al
Cellular and Molecular Gastroenterology and Hepatology Vol. -, No. -
825
855 Figure 4. Biological oxidants induce CAPZA1 expression by enhancing histone H3 acetylation of the CAPZA1 promoter. 914
856
(A) AGS cells were incubated with hydrogen peroxide or di-tert-butyl peroxide for 2 hours, and then the CAPZA1 expression
915
level was analyzed. (B) AGS cells were incubated with NAC for 24 hours before treatment with hydrogen peroxide or di-tert-
butyl peroxide, and then CAPZA1 expression was analyzed. (C) AGS cells were incubated with NAC for 24 hours before
treatment with hydrogen peroxide or di-tert-butyl peroxide. Thereafter, these cells were analyzed by a ChIP assay with an
859
anti–histone H3 (acetyl K9) antibody or IgG obtained from rabbit serum. Real-time PCR showed the relative enrichment of the
918
860
CAPZA1 promoter in the DNA fragments pulled down by an anti–histone H3 (acetyl K9) antibody. Data are presented as the
919
862
aldehyde-3-phosphate dehydrogenase.
864
were detected in tissues not only of H pylori–infected pa-
H pylori eradication therapy and that the expression pat-
923
865
tients, but also of H pylori–eradicated patients (Figure 7A).
terns of CD44v9 and CAPZA1 co-localized (Figure 7A and C,
924
867
group), we assessed the staining intensity of CAPZA1 per
overexpression in gastric epithelium contributes to the
926
868
cell. The data points in Figure 7B represent the average
development of CD44v9-positive cells in H pylori–infected
927
869
CAPZA1 staining intensity per cell in each individual image.
human gastric mucosa. Moreover, expression of ESRP1 was
928
870
The level of CAPZA1 expression per cell tended to be
detected in gastric tissues containing CD44v9-positive cells
929
871
reduced in gastric biopsy specimens from patients who had
and the
expression patterns of CAPZA1
and ESRP1
930
872
received H pylori eradication therapy; however, this differ-
co-localized (Figure 8A). In addition, nuclear localization of
931
873
ence was not statistically significant (Figure 7B). These bi-
b-catenin
was detected in CAPZA1-overexpressing
cells
932
874
opsy specimens were taken from patients with a mild-
(Figure 8B). These biopsy specimens were
taken
from
933
875
to-moderate level of neutrophil infiltration. Therefore,
inflamed tissue that did not show dysplasia, and these pa-
934
876
continuation of the inflammatory response after eradication
tients had not progressed to gastric cancer. Nevertheless,
935
877
therapy is thought to contribute to induction of CAPZA1
CD44v9 expression was detected in CAPZA1-overexpressing
936
878
overexpression. CAPZA1 overexpression was detected