Application of inter–simple sequence repeat PCR to mouse models: Assessment of genetic alterations in carcinogenesis

Abstract

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.

Evaluation of Microsatellite Instability and Apoptosis in Gall Bladder Malignancy from Patients of a Cohort Exposed to Methylisocyanate

“…Biological evolution ensures that several backup mechanisms always exist to prevent the failure of cellular processes. In response to genotoxic exposure or other adverse environmental conditions cell cycle checkpoints are initiated in an attempt to prevent the fixation of mutations from one generation to the next [27]. Microsatellite instability or genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity.…”

Mechanistic data and cancer risk assessment: The need for quantitative molecular endpoints

“…Again, this method would appear to have a broader application for use with a range of specific point mutations in genes associated with tumor formation. Benavides et al [2002] utilized an intersimple sequence repeat PCR to assess a range of genetic alterations in spontaneous and induced mouse tumors. The general aim of the method is to use PCR amplification of DNA regions between microsatellite repeats using microsatellite-anchored primers.…”

Loss of heterozygosity analysis of mouse pulmonary adenomas induced by coal tar

“…This PCR-based multilocus fingerprinting technique was used to assess the number of mutational events during tumor development in the coal tar-induced mouse pulmonary tumors as previously described [Benavides et al, 2002]. In brief, genomic DNA was obtained from frozen (nonmicrodissected) lung adenomas and analyzed by inter-SSR PCR by use of (CA) 8 RY, (CA) 8 RG, (AAC) 6 Y, and (AGC) 4 Y primers (R ϭ purine; Y ϭ pyrimidine).…”

“…Since frozen normal tissues were not available we used DNA from standard B6C3F1 mice (genetically identical to the mice used for the tumor bioassay) as normal DNA in the comparisons. Because changes in intensity of some bands among different individuals within inbred strains were previously described [Benavides et al, 2002], we primarily looked for band shifts, new bands, and band losses. These band alterations need not be limited to insertions or deletions between primer binding sites, nor to the deletion of a binding site itself.…”

“…Figure 2 shows fingerprints originated from 20 lung adenomas and the control B6C3F1 DNA amplified with primer (CA) 8 RY and stained with silver staining. In previous work, inter-SSR PCR proved to be useful in identifying nonrandom changes (altered bands that reappear in different tumors) in N-methyl-N-nitrosourea-induced thymic lymphomas generated in p53 knockout mice, where both copies of the p53 gene are inactivated [Benavides et al, 2002].…”