Beta-Catenin mutations in a mouse model of inflammation-related colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sodium sulfate

Abstract

In a previous study, we developed a novel mouse model for colitis-related carcinogenesis, utilizing a single dose of azoxymethane (AOM) followed by dextran sodium sulfate (DSS) in drinking water. In the present study, we investigated whether colonic neoplasms can be developed in mice initiated with a single injection of another genotoxic colonic carcinogen 1,2-dimethylhydrazine (DMH), instead of AOM and followed by exposure of DSS in drinking water. Male crj: CD-1 (ICR) mice were given a single intraperitoneal administration (10, 20 or 40 mg/kg body weight) of DMH and 1-week oral exposure (2% in drinking water) of a non-genotoxic carcinogen, DSS. All animals were killed at week 20, histological alterations and immunohistochemical expression of beta-catenin, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) were examined in induced colonic epithelial lesions (colonic dysplasias and neoplasms). Also, the beta-catenin gene mutations in paraffin-embedded colonic adenocarcinomas were analyzed by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. The incidences of colonic neoplasms with dysplastic lesions developed were 100% with 2.29+/-0.95 multiplicity, and 100% with 10.38+/-4.00 multiplicity in mice given DMH at doses of 10 mg/kg or 20 mg/kg and 2%DSS, respectively. Although approximately half of the mice given DMH at a dose of 40 mg/kg bodyweight were dead after 2-3 days after the injection, mice who received DMH 40 mg/kg and 2%DSS had 100% incidence of colonic neoplasms with 9.75+/-6.29 multiplicity. Immunohistochemical investigation revealed that adnocarcinomas, induced by DMH at all doses and 2%DSS, showed positive reactivities against beta-catenin, COX-2 and iNOS. In DMH/DSS-induced adenocarcinomas, 10 of 11 (90.9%) adenocacrcinomas had beta-catenin gene mutations. Half of the mutations were detected at codon 37 or 41, encoding serine and threonine that are direct targets for phosphorylation by glycogen synthase kinase-3beta. The present results suggests that, as in the previously reported model (AOM/DSS) our experimental protocol, DMH initiation followed by DSS, may provide a novel and useful mouse model for investigating inflammation-related colon carcinogenesis and for identifying xenobiotics with modifying effects.

Figure 1

Representative macroscopic view of the colonc from group 2 (1,2‐dimethylhydrazine [DMH] 20 mg/kg body weight →2% dextran sodium sulfate [2%DSS]). Note the numerous polypoid tumors in the colon.

Figure 2

Histopathology of colonic neoplasms developed in mice. (a) adenocarcinoma (b) adenoma, and (c) dysplasia. Hematoxylin–eosion stain. Original magnification (a) ×4, (b) ×10, and (c) ×20.

Figure 3

Immunohistochemistry of (a) β‐catenin, (b) cyclooxygenase (COX‐2), and (c) nitric oxide synthase (iNOS), and immunofluorescent staining of β‐catenin in colonic adenocarcinoma in mice. Original magnification (a–c) ×10.

Figure 4

Polymerase chain reaction–single strand conformation polymorphism (PCR‐SSCP) analysis of the β‐catenin gene in mouse colon adenocarcinomas. 1,2‐Dimethylhydrazine/dextran sodium sulfate (DMH/DSS)‐induced mouse colon adenocarcinomas (lanes 1–11). Lanes 1–3: DMH (10 mg/kg body weight)/DSS‐induced mouse colon adenocarcinoma samples. Lanes 4–11: DMH (20 mg/kg body weight)/DSS‐induced mouse colon adenocarcinoma samples. Lane N_D: DMH (20 mg/kg body weight)‐induced mouse colon mucosa sample. Lane N_C: negative control mouse colon mucosa sample. Arrowheads indicate tumor‐specific bands.

Figure 5

Restriction fragment length polymorphism (RFLP) analysis of the β‐catenin gene in mouse colon adenocarcinomas. Lanes 1–11: 1,2‐dimethylhydrazine/dextran sodium sulfate (DMH/DSS)‐induced mouse colon adenocarcinomas samples. Lanes 1–3: DMH (10 mg/kg bodyweight)/DSS‐induced mouse colon adenocarcinoma samples. Lanes 4–11: DMH (20 mg/kg body weight)/DSS‐induced mouse colon adenocarcinoma samples. Lane M: DNA size markers. Lane N: Negative control mouse colon mucosa sample. Arrowheads indicate tumor‐specific bands.