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Azoxymethane induces KI-ras activation in the tumor resistant AKR/J mouse colon
A B Bolt1, A Papanikolaou, D A Delker
1Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, The University of Connecticut, Storrs, CT 06269-2092, USA.
Abstract:
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.
Insights
Genetic alterations in aberrant crypt foci (ACF) may explain differing susceptibility to colon cancer in mice. Ki-ras mutations and APC protein levels in ACF did not fully account for this differential susceptibility, suggesting further molecular analysis is needed.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- A differential susceptibility to the colon carcinogen azoxymethane (AOM) exists in mice.
- Aberrant crypt foci (ACF) are precancerous lesions in the colon.
- The genetic basis for differential susceptibility to AOM-induced colon cancer is not fully understood.
Purpose of the Study:
- To test the hypothesis that intraspecific susceptibility to AOM is linked to genetic alterations in ACF.
- To investigate mutations in the Ki-ras proto-oncogene and adenomatous polyposis coli (APC) protein expression in ACF and tumors from mice with varying susceptibility.
Main Methods:
- Mice (A/J, SWR/J, AKR/J) were injected with AOM weekly for 6 weeks.
- DNA from microdissected ACF, normal epithelium, and tumors was analyzed for Ki-ras mutations (codons 12 and 13) using PCR and sequencing.
- Ki-ras mRNA expression was assessed by reverse transcription (RT-PCR).
- APC protein expression in colonic epithelium was evaluated using immunohistochemistry.
Main Results:
- No significant difference in Ki-ras activation frequency (20-33%) was observed in ACF across strains at 4 weeks post-AOM.
- Ki-ras mRNA expression was transiently reduced in all strains at 4 weeks, returning to normal by 24 weeks.
- APC protein was present in ACF from all strains, but lost in tumors from susceptible strains (A/J, SWR/J).
Conclusions:
- Ki-ras mutations and APC protein loss in tumors do not fully explain the differential susceptibility to AOM-induced colon cancer in this murine model.
- Further molecular genetic analyses of ACF are necessary to elucidate the basis of differential tumor susceptibility.