RESUMEN
A differential susceptibility phenotype to the organotropic colon carcinogen azoxymethane (AOM) has been described in mice. The following studies were undertaken to test the hypothesis that intraspecific susceptibility can be accounted for by the specific complement of genetic alterations acquired by precancerous colon lesions referred to as aberrant crypt foci (ACF). As an initial approach to this question, mutations in codons 12 and 13 of the Ki-ras proto-oncogene were assessed in ACF, normal-appearing AOM-treated colonic epithelium, and tumors from A/J and SWR/J (susceptible) as well as AKR/J (resistant) mice. Four-week-old male mice were injected intraperitonealy, with AOM once a week for a total of 6 wk and killed 4 and 24 wk after the last injection. DNA was isolated from microdissected tissue, and polymerase chain reaction (PCR)-amplified products of Ki-ras exon 1 (codons 12 and 13) were directly sequenced from microdissected tissues. At 4 wk after AOM exposure, there was no significant difference in the frequency of Ki-ras activation (20-33%) between the three strains. Ki-ras mRNA expression was also evaluated by reverse transcription (RT)-PCR analysis and was comparably reduced (40-50%) in all three strains at the 4 wk time point. However, Ki-ras expression returned to normal by 24 wk after treatment. Finally, to gain further insight into the molecular pathogenesis underlying this experimental tumor model, analysis of the adenomatous polyposis coli (APC) protein within the colonic epithelium was undertaken by using an immunohistochemical approach. Although the APC protein was lost to a varying extent in tumors from A/J and SWR/J mice, the full-length form of the protein was still present in precancerous ACF isolated from each of the three strains, regardless of the degree of dysplasia of the lesion. A further molecular genetic analyses of ACF will be required to gain a more complete understanding of the molecular basis of tumor susceptibility phenotype in this murine model.
Asunto(s)
Azoximetano/farmacología , Colon/efectos de los fármacos , Regulación de la Expresión Génica/genética , Genes ras , Animales , Secuencia de Bases , Colon/metabolismo , Cartilla de ADN , Exones , Inmunohistoquímica , Masculino , Ratones , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Inbred mice exhibit differential susceptibility to colon carcinogens. The following study addresses the possibility that differences are intrinsic to colonic mucosa (cell autonomous) or are mediated by extracolonic systemic factors (e.g. liver activation of carcinogens). Our approach was to construct mouse aggregation chimeras, mice whose tissues are a mosaic of cells derived from two parental genotypes, from a susceptible (SWR) and a resistant (DBA/2) strain. Forty-five embryo aggregations yielded 11 viable pups, four of which were chimeric by coat color. Six-week-old SWR<-->BA/2 chimeras were injected i.p. with azoxymethane (AOM) once a week for 8 weeks (5 and 7.5 mg/kg body wt for 2 weeks followed by 10 mg/kg for 6 weeks) and tumor incidence in distal colon was evaluated 15 weeks after the last injection. Additional groups of parental mice received the same treatment. In the parental SWR treatment group, 1.7 +/- 0.82 tumors/colon were found. No tumors were observed in AOM-treated DBA/2 mice. In SWR<-->DBA/2 chimeras exposed to AOM, 2.8 +/- 2.1 tumors/colon were found. Tumor lineage was examined in paraffin sections stained with Dolichos biflorus agglutinin-peroxidase, a cell surface specific marker that stains intestinal endothelial cells of SWR and epithelial cells of DBA/2. Cellular lineage of tumors was further evaluated by microsatellite analysis of DNA isolated by microdissection. There was no significant difference in tumor incidence between SWR parental and chimera treatment groups. Histochemical analysis of tumor tissue in chimeras suggested that most tumors were derived from SWR. However, subsequent genetic analysis of tumors indicated mixed parental composition. These preliminary studies suggest that DBA/2 resistance mechanisms are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of AOM.