1. Volume 133, Issue 2, Pages 599-607 (August 2007)
Guanylyl Cyclase C Suppresses Intestinal Tumorigenesis by Restricting Proliferation and Maintaining Genomic Integrity 
Peng Li, Stephanie Schulz, Alessandro Bombonati, Juan P. Palazzo, Terry M. Hyslop, Yihuan Xu, Amy A. Baran, Linda D. Siracusa, Giovanni M. Pitari, Scott A. Waldman 
Gastroenterology 
Volume 133, Issue 2, Pages (August 2007)
DOI: /j.gastro
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2. Figure 1
GCC opposes tumorigenesis in ApcMin/+ mice. (A) Tumors (arrows) were enumerated in colons of ApcMin/+Gcc+/+ and ApcMin/+Gcc−/− mice and their (B) incidence (percentage of animals developing tumors) and (C) multiplicity (number of tumors per animal), and (D) size (diameter2) quantified. (E) Colon tumor burden in each animal was calculated as the sum of the sizes of all tumors. Similarly, tumor (F) incidence, (G) multiplicity, (H) size, and (I) burden were quantified in small intestine in ApcMin/+Gcc+/+ and ApcMin/+Gcc−/− mice. **, P < .01; ***, P < ApcMin/+Gcc+/+ mice, n = 32; ApcMin/+Gcc−/− mice, n = 27.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
GCC opposes tumorigenesis in mice treated with AOM. (A) Tumors (arrows) were enumerated in colons of mice treated with AOM, and their (B) incidence, (C) multiplicity, and (D) size were quantified to calculate (E) tumor burden, as described in Figure 1. *, P < .05; **, P < .01. Apc+/+Gcc+/+ mice, n = 25; Apc+/+Gcc−/− mice, n = 25.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
GCC opposes colonic tumorigenesis in ApcMin/+ mice by regulating genomic integrity in the context of suppressed apoptosis. (A) β-catenin accumulated in tumors but not in (B) normal colonocytes in ApcMin/+Gcc+/+ and ApcMin/+Gcc−/− mice. Accordingly, there were no differences in normal colonocyte proliferation quantified by (C–E) Ki67 immunohistochemistry or (F) immunoblot analysis of mediators of the cell cycle, including cyclin D1, cdk4, phosphorylated Rb, β-catenin or cyclin E in ApcMin/+ mice. (E) By comparison, the genotoxic agent AOM induced hyperproliferation in colonocytes in Apc+/+Gcc−/− compared to Apc+/+Gcc+/+ mice. (G–I) In contrast, double-strand DNA breaks, quantified by immunohistochemistry analysis of phosphorylated histone H2AX (γ-H2AX), were more abundant in colonocytes from ApcMin/+Gcc−/− compared to ApcMin/+Gcc+/+ mice. (I) By comparison, AOM induced comparable elevated levels of DNA damage in colonocytes from Apc+/+Gcc+/+ and Apc+/+Gcc−/− mice. (J–K) Further, analysis of cells from normal epithelium, normal epithelium adjacent to tumors (NAT), and tumors by PCR revealed that APC LOH occurred more frequently in tumors from ApcMin/+Gcc−/− compared to ApcMin/+Gcc+/+ mice. Here, a ratio of the WT allele (Apcwt) to the ApcMin allele <1 indicated LOH (Supplementary Figure 4A–C). (L) Conversely, compensatory apoptosis in colonocytes, quantified by TUNEL analysis, observed upon elimination of Gcc was abrogated in ApcMin/+ mice and following AOM treatment, in a GCC-independent fashion. Bars in A–D, G, H represent 50 μm, *P < .05; **, P < .01; ***, P < Data are represented as mean ± SEM (n ≥ 5 mice/genotype), except in K where each bar represents 1 tumor, mean ± SD (n = 3 for each tumor).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
GCC suppresses tumor growth in the small intestine of ApcMin/+ mice by restricting proliferation in the absence of excess DNA damage. (A–C) In contrast to the colon, elimination of Gcc induced hyperproliferation of epithelial cells quantified by Ki67 IHC in the small intestine in Apc+/+Gcc−/− and ApcMin/+Gcc−/− compared to Apc+/+Gcc+/+ and ApcMin/+Gcc+/+ mice, respectively, (D) associated with increases in protein mediators of the cell cycle. (E, F) Conversely, double-strand DNA breaks were equivalent in normal epithelia in small intestines of ApcMin/+Gcc+/+ and ApcMin/+Gcc−/− mice, comparable to Apc+/+Gcc+/+ and Apc+/+Gcc−/− mice, quantified by γ-H2AX IHC staining. (G) Moreover, LOH of Apc in the small intestine was equally abundant in tumors from ApcMin/+Gcc+/+ and ApcMin/+Gcc−/− mice. (H) As in the colon, compensatory apoptosis induced by eliminating Gcc in epithelial cells in small intestine2 was abrogated in ApcMin/+ mice in a Gcc-independent fashion. Bars in A, B, and E represent 50 μm. *, P < .05; ***, P < Data are represented as mean ± SEM (n ≥ 5 mice/genotype).
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
GCC opposes colonic tumorigenesis in mice treated with AOM by regulating proliferation and genomic integrity. (A–C) AOM induced hyperproliferation of colonocytes in Apc+/+Gcc−/− compared to Apc+/+Gcc+/+ mice, quantified by (A, B) Ki67 IHC and (C) immunoblot analysis of mediators of the cell cycle, including cyclin D1, cdk4, and phosphorylated Rb. (D, E) AOM induced accumulation of β-catenin in tumors in a Gcc-independent fashion. (F) Further, sequence analysis revealed greater β-catenin single bp mutation rates in this AOM target gene in Apc+/+Gcc−/− compared to Apc+/+Gcc+/+ mice. Bars in A, B, D, and E represent 50 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions