Strain-specific homeostatic responses during early stages of Azoxymethane-induced colon tumorigenesis in mice.

Identifying molecular changes that predict the risk for developing colon cancer is critical for designing effective prevention strategies. In the present study, we determined early-stage molecular alterations within the colonic epithelium of A/J and AKR/J mice that are sensitive and resistant to Azoxymethane (AOM)-initiated tumor development, respectively. Six week-old male mice were injected intraperitoneally with AOM (10 mg/kg body weight) once a week for six weeks. One week after the last injection, distal colons from both strains were analyzed for cell proliferation using a proliferating cell nuclear antigen (PCNA) assay. Unlike AKR/J, a significant increase (2.5-fold, p<0.05) in the number of PCNA-positive cells within the upper third of the crypt compartment was observed in the A/J colons. This proliferative response was associated with a sizeable increase in the levels of c-myc mRNA, quantified by RNase protection assay. cDNA sequencing, protein expression and localization of beta-catenin, an upstream activator of c-myc, however, showed no aberrant changes within AOM-exposed A/J colons. Interestingly, TdT-mediated dUTP nick-end labeling assay revealed a significant increase (4-fold) in the number of apoptotic colonocytes in A/J mice following AOM treatment. Consistent with this finding, a modest increase in the expression of pro-apoptotic Bak was limited to the sensitive A/J colons. In summary, the current study suggests that a significant alteration in the rate of cell turnover in the normal appearing colonic mucosa, as observed in susceptible A/J mice, may be one of the earliest events predisposing the colon to neoplastic growth.

Carcinogen-induced colon tumors in mice are chromosomally stable and are characterized by low-level microsatellite instability.

The azoxymethane (AOM)-induced mouse colon tumor model recapitulates many of the histopathological features associated with the multistage progression of human sporadic colorectal cancers (CRCs). To better define the genetic events associated with tumorigenesis in this murine model, we analysed tumors from A/J mice for chromosomal (CIN) and microsatellite (MSI) instabilities, two fundamental pathways of genomic instability that play a critical role in the pathogenesis of human CRCs. Male A/J mice, 6-week old, were injected with either AOM (n=5) (10 mg/kg b.w., i.p.) or vehicle (n=5) (0.9% NaCl solution) once a week for 6 weeks. At 32 weeks after the last dose, comparative genomic hybridization (CGH) was performed on 16 tumors harvested from five animals. Although 25% of the tumors displayed either a gain of chromosome 2 or loss of Y, the majority (75%) showed no genomic imbalances. Further analysis of chromosomal aberrations, using CGH and spectral karyotyping (SKY) was performed in our recently established A/J colon tumor-derived cell line, AJ02-NM0. Results showed a pseudotetraploid karyotype with loss of only the Y chromosome in these cultured cells, thereby providing additional evidence for the minimal role of CIN in the primary AOM-induced tumors. Interestingly, the majority (81%) of A/J tumors displayed low-level microsatellite instability (MSI-L) when analysed using mono- and dinucleotide repeat markers, and showed a significant expansion to high-level instability (MSI-H) in the AJ02-NM0 cells. This finding in cultured cells additionally provides evidence that a mild mutator pathway may contribute to the development of behaviorally benign carcinomas in situ in A/J mice. To better understand the tumorigenic process in the A/J colons, we screened for mutational alterations in key regions of the K-ras and Apc genes. Results showed a very low frequency (6%) of K-ras activating mutations, together with the absence of Apc truncation mutations in primary tumors and AJ02-NM0 cells. However, these tumors displayed intense nuclear accumulation of beta-catenin protein, indicating activation of the Wnt signaling pathway. Based on our molecular and cytogenetic findings, we propose that carcinogen-induced tumors may develop via mechanisms independent of the 'classical' CIN or MSI pathways.