1. Volume 127, Issue 2, Pages 428-443 (August 2004)
Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid 
Atsushi Nobuoka, Tetsuji Takayama, Koji Miyanishi, Tsutomu Sato, Kunihiro Takanashi, Tsuyoshi Hayashi, Takehiro Kukitsu, Yasushi Sato, Minoru Takahashi, Tetsuro Okamoto, Takuya Matsunaga, Junji Kato, Masayuki Oda, Takachika Azuma, Yoshiro Niitsu 
Gastroenterology 
Volume 127, Issue 2, Pages (August 2004)
DOI: /j.gastro

2. Figure 1
Immunohistochemical stains for GSTP1-1 and COX-2 in ACF and adenoma. Serial sections are shown of a representative ACF specimen (A-D) and an adenoma specimen (E-H). The dotted lines show the borders between normal epithelium and ACF or adenoma. Original magnification, A-H, 100×. Representative sections with different staining intensities for GSTP1-1 (I-K) are also shown. Original magnification, I-K, 200×. In these sections, nuclei were not counterstained so that the staining intensities could be quantitatively assessed by the KS-400 image-analysis system.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
GSTP1-1 mRNA expression in ACF quantified by TaqMan real-time RT-PCR. The GSTP1-1 and GAPDH relative messages to the calibrator ([GSTP1-1] c and [GAPDH] c, respectively) were quantified by measuring the threshold cycle and by using a standard curve. The final relative mRNA expression level was expressed as follows: [GSTP1-1] c/[GAPDH] c.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
TUNEL staining of normal colorectal epithelium, ACF, and adenoma with or without DCA (1 mmol/L) treatment. Specimens of ACF treated (I-L) or not treated (A-D) with DCA and of adenoma treated (M-P) or not treated (E-H) with DCA were stained by either the H&E method (A, E, I, M) or the TUNEL method (B-D, F-H, J-L, N-P). In each panel of H&E staining, normal epithelium and ACF, or adenoma are separated by dotted lines. NE, normal epithelium; Ad, adenoma. (Original magnification: A, B, E, F, I, J, M, N, 100×; C, D, G, H, K, L, O, P, 200×.)
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Percentage of apoptotic cells (TUNEL-positive cells) in normal colorectal epithelium, ACF, and adenoma specimens treated with or without DCA. Specimens treated with DCA (1 mmol/L) for 3 hours or those without DCA treatment were fixed, sectioned, and stained for TUNEL positivity as described in Materials and Methods. N.S., not significant.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Effect of a GSTP1-1-specific inhibitor (TER199) on DCA-induced apoptosis in ACF specimens. (A) Specimens of ACF were treated with DCA (1 mmol/L) and TER199 (100 μmol/L) or DCA (1 mmol/L) alone for 3 hours. Apoptotic cells were detected by the TUNEL method. Representative specimens of TUNEL staining of ACF treated with DCA and TER199 and that with DCA alone are shown. (B) The percentage of apoptotic cells in each of 14 ACF specimens treated with both DCA and TER199 and that in each of 14 ACF specimens treated with DCA alone is shown.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
GSTP1-1 expression of M7609 cells and HEL cells transfected with GSTP1-1 complementary DNA. GSTP1-1 expression was detected by Western blotting for M7609 and its transfectants (A) and HEL and its transfectants (B). KD, kilodaltons.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Cytotoxic effects of DCA on M7609 and HEL cells and their neomycin transfectants and GSTP1-1 transfectants. Cells were treated with DCA at 125–2000 μmol/L, and the percentage survival was measured as detected by the dye-uptake method. (A) M7609 and its transfectants. (B) HEL and its transfectants. Error bars show ±SD from 6 independent experiments. ∗P < 0.01; ∗∗P < 0.05 compared with M7609 and M7609-neo or HEL and HEL-neo.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Binding of DCA to GSTP1-1 verified by circular dichroism and immunoprecipitation. In circular dichroic spectra (A), the observed ellipticity (θ obs millidegrees) per 10−6 mol/L GSTP1-1, which was saturated by bilirubin (refer to Materials and Methods), decreased as the concentrations of DCA added increased. The inset shows the relative decrease in the ellipticity extrema at 465 nm. Binding of [3H]DCA to GSTP1-1 in M7609-neo, M7609-GST-1, and M7609-GST-2 cells was assessed by a coimmunoprecipitation assay with the anti-GSTP1-1 antibody (B). When the cells were incubated with [3H]DCA at a final concentration of 3.8 μmol/L, as described in Materials and Methods, approximately 1/270 of the total radioactivity was taken up into the cells. The radioactivity co-precipitated with GSTP1-1 by anti-GSTP1-1 antibody treatment was measured, and the percentage of the radioactivity in total cytosol was calculated. Error bars show ±SD in 5 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Annexin V/PI staining positivity of neomycin- transfected M7609, neomycin-transfected HEL, GSTP1-1-transfected M7609, and GSTP1-1-transfected HEL cells. Cells treated with DCA 250 μmol/L for 12 hours in the presence or absence of TER199 were analyzed for annexin V and PI double staining by flow cytometry. (A) Neomycin-transfected M7609 (M7609-neo). (B and C) GSTP1-1-transfected M7609 (M7609-GST-1 and M7609-GST-2). (D) Neomycin-transfected HEL (HEL-neo). (E and F) GSTP1-1-transfected HEL (HEL-GST-1 and HEL-GST-2).
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
Activities of caspase-8, -9, and -3 in M7609-neo, M7609-GST-1, and M7609-GST-2 cells treated with DCA. The activity of caspase-8 (A), -9 (B) and -3 (C) in cells in the presence or absence of TER199 was determined by the colorimetric protease method by using each corresponding substrate.
Gastroenterology  , DOI: ( /j.gastro )

12. Figure 11
Binding of sulindac sulfone and sulindac sulfide to GSTP1-1 (A and B) and the effect of sulindac on DCA-induced apoptosis of ACF tissues (C). Binding of sulindac sulfone (A) and that of sulindac sulfide (B) to GSTP1-1 was assessed by an enzyme inhibition assay with a substrate of 1-chloro-2,4-dinitrobenzene (CDNB) or glutathione (GSH). ◊, 1 μmol/L; □, 10 μmol/L; ▵, 100 μmol/L. (C) Percentages of apoptotic cells in 13 ACF specimens from adenoma patients treated with sulindac sulfone (10 μmol/L) in the presence of DCA (1 mmol/L) for 3 hours were measured by TUNEL staining.
Gastroenterology  , DOI: ( /j.gastro )

13. Figure 12
Effect of oral sulindac administration on epithelial cell apoptosis in normal epithelium, ACF, and adenoma from patients with adenoma. Serial sections of representative ACF (A-D) and those of adenoma (E-H) from adenoma patients who received oral sulindac administration for 4 days are shown. Sections of A and E were stained with H&E, and those of B, C, D, F, G, and H were stained with TUNEL. The dotted lines show the borders between normal epithelium and ACF or adenoma. C and D (magnification, 400×) are magnified images of corresponding squared areas of B (magnification, 100×), and G and H are magnified images of corresponding squared areas of F. (I) Percentages of apoptotic cells in normal epithelium, ACF, and adenoma biopsied from 15 adenoma patients after 4 days of oral administration of sulindac and those from nontreated patients are shown. (J) The number of ACF in the patients who underwent continuous treatment of sulindac for 2 or 3 months was examined, whereas the number of ACF in 14 patients who had not received sulindac was also examined.
Gastroenterology  , DOI: ( /j.gastro )