Frequent Mutation of Apc Gene in Rat Colon Tumors and Mucin-Depleted Foci, Preneoplastic Lesions in Experimental Colon Carcinogenesis

Abstract

Mucin-depleted foci (MDF) are microscopic dysplastic lesions induced in the colon of rodents by specific colon carcinogens. Most MDF show Wnt pathway activation, whereas only a subset shows mutations in the Ctnnb1 gene, coding for B-catenin. Because Apc is a member of the Wnt pathway and the most frequent mutated gene in human colon cancer, we tested whether MDF harbor Apc mutations. F344 rats were treated twice with 150 mg/kg of 1,2-dimethylhydrazine. After 15 or 28 weeks, MDF, aberrant crypt foci (ACF), and tumors were collected. We screened a segment of the Apc gene comprising the region homologous to the mutation cluster region (MCR) of human APC, which frequently shows mutations in experimental colon tumors. Mutations were identified by PCR amplification and sequencing in 6:24 MDF (25%), 7:23 tumors (30%), 0:24 ACF (0%). Most of the mutations (92%) in MDF and tumors were localized in a region upstream from the MCR. All mutations were singlebase substitutions and mainly formed by G:C ! A:T and C:G ! T:A transitions. The pattern of nucleotide changes was similar in MDF and tumors, and, interestingly, the same mutation in codon 1047 was found in two MDF and in three tumors. Four out of the six mutations found in MDF were nonsense mutations, and two were missense. All mutations in tumors determined a protein truncation. These results show that Apc mutations are present in MDF with a frequency similar to that of tumors, strengthening the evidence that they are precancerous lesions in colon carcinogenesis. [Cancer Res 2007;67(2):445-9]

Mucin‐depleted foci show strong activation of inflammatory markers in 1,2‐dimethylhydrazine‐induced carcinogenesis and are promoted by the inflammatory agent sodium dextran sulfate

“…Constitutive activation of Wnt-pathway has been reported by us in MDF, 3 and is caused, at least for a part of these lesions, by genetic mutations in Apc and b-catenin genes. 3,4 Also, it is relevant to note that Wnt activation may downregulate MUC2 expression in vitro and that both COX-2 and i-NOS have been reported as target genes upregulated by Wnt. [40][41][42] It has also been reported that prostaglandin E2 (PGE2), produced by COX-2, may activate Wnt-signalling acting by its cognate G-coupled receptor.…”

“…4 Due to the low amount of RNA available we studied only the expression of i-NOS and we found (Fig. 3) that it was expressed in all the MDF analysed, with a strong expression in the large MDF formed by 200 crypts (MDF4 in the fifth lane of the gel).…”

“…The colons intended for IH experiments were excised, washed with saline, opened longitudinally, fixed with 70% ethanol and then stained with Alcian Blue-Neutral Red for MDF and ACF visualization as previously described. 4 The colon intended for the RT-PCR experiment was processed as described in the next section. Twenty-eight weeks after carcinogen induction, the remaining group of rats (n 5 10) was killed to harvest tumours.…”

“…Fragments were deposited luminal side up on cold histological slides and the mucosa was constantly moistened with RNAlater (Qiagen, S.p.A, Milan, Italy) during microscopic observation. MDF, identified as previously described, 4 were plucked out using sterile insulin needles under a dissecting microscope and immediately transferred in a lysis RLT Buffer Plus with b-mercaptoethanol (Qiagen, S.p.A, Italy). Total RNA extraction from MDF and apparently normal mucosa in the same colon was carried out using the RNeasy Plus Micro Kit (Protocol for microdissected cryosections, Qiagen).…”

“…1,2 MDF harbour mutations in genes affecting colon carcinogenesis and, like colonic tumours, show Wnt signalling activation, features suggesting that these lesions are precancerous. 3,4 Recently, we also reported that the expression of MUC2, the most abundant mucin in the colon, is dramatically reduced in MDF. 5 This last result, led us to speculate that the focal loss of the protective mucous layer might activate local inflammation, since MDF would be more exposed to noxious agents present in the colonic lumen.…”