Volume 121, Issue 4, Pages 865-874 (October 2001) Glutathione S-transferase-π overexpression is closely associated with K-ras mutation during human colon carcinogenesis  Koji Miyanishi, Tetsuji Takayama, Motoh Ohi, Tsuyoshi Hayashi, Atsushi Nobuoka, Takaharu Nakajima, Rishu Takimoto, Katsuhisa Kogawa, Junji Kato, Sumio Sakamaki, Yoshiro Niitsu  Gastroenterology  Volume 121, Issue 4, Pages 865-874 (October 2001) DOI: 10.1053/gast.2001.27982 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Immunohistochemical analysis for GSTP1-1 and p21K-ras expression in colorectal carcinoma, adenoma, and ACF. (A) Serial sections of the colon carcinoma tissue. Diffuse staining for GSTP1-1 was found throughout the lesion. (B) The staining for p21K-ras was also found to be diffuse. (C) No staining was detected in the negative control, which had not been treated with primary antibodies. (D–G) Serial sections of the colon adenoma tissue. (D and E) Both stainings for GSTP1-1 and (F and G) p21K-ras in the adenoma portion were more intense than those in normal epithelial cells. The staining pattern for GSTP1-1 and p21K-ras in the lesion was similar and rather multifocal. (H and I) Serial sections of ACF. (H) The ACF showed apparent GSTP1-1 staining, particularly in basal parts of the cells compared with normal adjacent crypts. (I) p21K-ras staining was similar to that of GSTP1-1. Original magnification 100× in A–D and F, 200× in H and I, and 400× in E and G. Gastroenterology 2001 121, 865-874DOI: (10.1053/gast.2001.27982) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Two-step PCR-RFLP analysis of K-ras mutation in colorectal carcinoma, adenoma, and ACF. A pancreatic cancer cell line, APSC (ATCC CRL1682; American Tissue Culture Collection, Rockville, MD), which is known to have a K-ras point mutation, was used as a positive control. A normal colonic mucosa was used as a negative control. K-ras mutation was detected in (A) 70.6% of colorectal carcinomas, (B) 64.3% of colorectal adenomas, and (C) 89.0% of ACF. Gastroenterology 2001 121, 865-874DOI: (10.1053/gast.2001.27982) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 GSTP1-1 mRNA expression in colorectal carcinoma, adenoma, and ACF quantified by real-time quantitative RT-PCR. The GSTP1-1 and GAPDH relative messages to the calibrator in unknown samples ([GSTP1-1] c and [GAPDH] c, respectively) were quantified by measuring threshold cycle (Ct) and using a standard curve. The final relative mRNA expression level was expressed as follows: [GSTP1-1] c / [GAPDH] c. The relative mRNA level of normal colonic mucosa was 0.62 ± 0.14. The cut-off level for normal GSTP1-1 mRNA level was defined as mean + 2SD (i.e., 0.90). Nine of 10 ACF showed high levels of GSTP1-1. All of the colorectal adenomas and carcinomas that contained mutated K-ras showed high levels of GSTP1-1 mRNA. On the other hand, only 1 of 4 colorectal adenomas and 1 of 5 colorectal carcinomas with wild-type K-ras showed high levels of GSTP1-1 mRNA. Gastroenterology 2001 121, 865-874DOI: (10.1053/gast.2001.27982) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Increased activity of AP-1 responsive element in RPMI-ras-1 and RPMI-ras-2 cells. (A) Schematic representation of the GSTP1-1 promoter deletion fragments and the mutated GSTP1-1 promoter fragments, which were ligated upstream of the SEAP reporter gene. (B) A summary of SEAP assay results. Each value represents the mean of 6 determinations. pSEAP.Basic (contains neither SV40 enhancer nor promoter) and pSEAP.Control (contains SV40 enhancer and promoter) were used as negative and positive controls, respectively. All results were expressed relative to the activity of p73SEAP in RPMI-neo cells (given a value of 1.0 for each experiment). Results were compared between 2 v-K-ras transfectants and mock-transfectant by correcting for pSEAP.Control activity levels. Gastroenterology 2001 121, 865-874DOI: (10.1053/gast.2001.27982) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Identification of an AP-1 complex bound to the GSTP1-1 promoter. EMSA demonstrating the nuclear complexes from RPMI-neo, RPMI-ras-1, and RPMI-ras-2 cells that bind to the GSTP1-1 promoter region (−73 to −54). Addition of either anti-c-Fos or anti-c-Jun antibody to nuclear extracts clearly retarded the migration of these complexes. Gastroenterology 2001 121, 865-874DOI: (10.1053/gast.2001.27982) Copyright © 2001 American Gastroenterological Association Terms and Conditions