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