Frequent Mutation of Apc Gene in Rat Colon Tumors and Mucin-Depleted Foci, Preneoplastic Lesions in Experimental Colon Carcinogenesis Angelo Pietro Femia, 1 Piero Dolara, 1 Augusto Giannini, 3 Maddalena Salvadori, 1 Annibale Biggeri, 2 and Giovanna Caderni 1 1 Department of Pharmacology, University of Florence, 2 Department of Statistics, University of Florence and Biostatistics Unit, Centro per lo Studio e la Prevenzione Oncologica, Florence, Italy; and 3 Department of Pathology, General Hospital of Prato-Azienda Sanitaria Locale 4, Prato, Italy 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 single- base 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] Introduction Mucin-depleted foci (MDF) are dysplastic lesions originally identified in the colon of azoxymethane-treated rats (1). MDF are dose-dependently induced by colon carcinogens (2), and their occurrence is correlated to the number of tumors after the administration of promoters or chemopreventive agents (3–6), thus suggesting that these lesions are preneoplastic. We previously reported that most MDF, like tumors, show constitutive activation of the Wnt-signaling pathway with cytoplasmic and nuclear accumulation of h-catenin (2). However, because only a fraction of MDF (25%) harbors mutations in the rat gene coding for h-catenin ( Ctnnb1 gene), other components of the Wnt pathway might be altered in these lesions. The Apc gene is a component of the Wnt-signaling pathway as a member of the macromolecular complex which degrades h-catenin (7). Mutations in either the Apc or Ctnnb1 gene make h-catenin resistant to degradation, thus causing its accumulation in the nuclei and activation of the Wnt-signaling pathway. Germ line APC mutations are responsible for the inherited predisposition for familial adenomatous polyposis (FAP) and for intestinal tumors in the Min mouse carcinogenesis model (8, 9). Somatic APC mutations are also common in sporadic colorectal cancers (8) and are present with variable frequencies in chemically induced colon cancer of rodents (10, 11). On the basis of these considerations, we studied Apc mutations in rat MDF as a possible cause of Wnt pathway activation observed in these lesions. Moreover, the presence of a mutation so relevant for colon carcinogenesis such as the Apc would strengthen the hypothesis that MDF are preneoplastic. To compare MDF with more advanced stages of transformation or with other purported preneoplastic lesions, we also studied Apc mutations in colon tumors and aberrant crypt foci (ACF), well characterized in rodents and humans (12, 13). Materials and Methods Carcinogenesis induction. Male F344 rats, 4 to 5 weeks old, were obtained from Nossan (Correzzana, Milan, Italy). They were housed according to the European Union Regulations on the Care and Use of Laboratory Animals (14) and fed a high-fat diet (23% corn oil w/w) based on the American Institute of Nutrition-76 diet (2). The experimental protocol was approved by the Commission for Animal Experimentation of the Italian Ministry of Health. At 6 to 7 weeks of age, rats were treated twice, 1 week apart, with s.c. injections of 1,2-dimethylhydrazine (DMH; 150 mg/kg  2). Rats were divided into two groups. One group was sacrificed by CO 2 asphyxiation 15 weeks after the first injection, and the other was sacrificed 28 weeks afterward. The colons were excised and opened longitudinally. Those lesions which were visible by the naked eye at sacrifice were defined as tumors following subsequent histopathologic examination as described (2). To collect MDF, ACF, or tumors, the colons were then fixed flat, as reported (1, 2), using 70% ethanol for no more than 3 h. Identification of ACF and MDF. MDF are foci of crypts depleted of mucins in a background of normal crypts using the high-iron diamine Alcian blue (HID-AB) technique, which allows differentiation between sialomucin and sulfomucin production (1). MDF have also been identified by other authors using Alcian blue (AB), which stains in blue all acid mucins (6). However, we found that both staining methods (HID-AB or AB) degrade DNA, making it unsuitable for relatively long amplification by PCR (data not shown). Therefore, we used a milder staining procedure (Alcian blue-neutral red [AB-NR]) consisting of a shorter AB incubation (10 min versus 30 min Requests for reprints: Giovanna Caderni, Dipartimento di Farmacologia Preclinica e Clinica della Universita` di Firenze, Viale G. Pieraccini, 6, 50139 Florence, Italy. Phone: 39-55-4271319; Fax: 39-55-4271280; E-mail: giovanna.caderni@unifi.it. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3861 www.aacrjournals.org 445 Cancer Res 2007; 67: (2). January 15, 2007 Priority Report Research. on November 29, 2015. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from