Hege Benedikte Ølstørn

Connexin43 is overexpressed in ApcMin/+-mice adenomas and colocalises with COX-2 in myofibroblasts

The expression of gap junction proteins, connexins, in the intestine and their role in tumorigenesis are poorly characterised. Truncating mutations in the tumour suppressor gene adenomatous polyposis coli (APC) are early and important events, both in inheritable (familial adenomatous polyposis, FAP) and spontaneous forms of intestinal cancer. Multiple intestinal neoplasia (Min) mice, a FAP model with inherited heterozygous mutation in Apc, spontaneously develop numerous intestinal adenomas. We recently reported reduced expression of connexin32 in Paneth cells of Min‐mice. We further examine the expression of connexin43 (Cx43) and other connexins as a function of heterozygous and homozygous Apc mutation in normal intestinal tissues and adenomas of Min‐mice. Qualitative analysis of connexin mRNA in intestine revealed a similar expression pattern in Min‐ and wild‐type (wt) mice. Connexin26 and connexin40 proteins were found in equal amounts in Min and wt epithelia of large and small intestine, respectively. Interestingly, the connexin43 level was increased in the stroma of Min‐mice adenomas, in close proximity to epithelial cells with nuclear β‐catenin staining. Cx43 and COX‐2 were located to the same areas of the adenomas, and immunostaining exhibited coexpression in the myofibroblasts. Prostaglandin E2 induces Cx43 expression and COX‐2 is the rate‐limiting enzyme in the prostaglandin synthesis. However, the COX‐2‐specific inhibitor, celecoxib, did not reduce Cx43 expression. Although both Cx43 and COX‐2 are target genes for β‐catenin, they were overexpressed in stromal cells but not in epithelial tumour cells. We hypothesise that gap junctions may be of importance in the transfer of signals between epithelium and stroma.

Identification of flat dysplastic aberrant crypt foci in the colon of azoxymethane-treated A/J mice

The role of aberrant crypt foci (ACF) as preneoplastic lesions in colon carcinogenesis is not clear. In Min/+ mice and their wild‐type littermates treated with azoxymethane (AOM), we previously identified a subgroup of flat ACF that seem more immediate precursors of tumors than the classical elevated ACF. In the present study, we identified a similar subgroup of flat ACF in AOM‐treated A/J mice and compared them with nascent tumors and classical elevated ACF. At week 1 and 2 after birth, A/J mice were injected subcutaneously with AOM (10 mg/kg bw/injection). At weeks 7–14, we examined the luminal surface of unsectioned colon preparations stained with methylene blue in the inverse light microscope. The lesions were also examined by histopathology and immunohistochemistry. Surface examination revealed flat ACF, classical elevated ACF and nascent tumors. Since flat ACF were not observed as elevated structures, their bright blue appearance and compressed pit pattern of crypt openings seen with transillumination were used as criteria for their identification. Flat ACF and nascent tumors displayed a uniform picture of severe dysplasia, compressed pit pattern, overexpression of cytoplasmic/nuclear β‐catenin and nuclear overexpression of cyclin D1. Apparently, flat ACF and tumors represented the same type of dysplastic lesions at different stages of crypt multiplication. In contrast, classical elevated ACF did not seem to be as clearly related to tumorigenesis. They infrequently (1/20) possessed severe dysplasia, overexpression of cytoplasmic/nuclear β‐catenin, or nuclear overexpression of cyclin D1, and they did not have compressed crypt openings. Furthermore, flat ACF grew significantly faster than classical elevated ACF. In conclusion, our data indicate a development from flat ACF to adenoma characterized by aberrant activation of the Wnt signaling pathway and fast crypt multiplication. Classical elevated ACF do not seem to be as closely related to tumorigenesis.