A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer. Proc Natl Acad Sci USA

Department of Biostatistics and Medical Informatics, Section of Gastroenterology and Hepatology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53726, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2007; 104(10):4036-41. DOI: 10.1073/pnas.0611690104
Source: PubMed


Progress toward the understanding and management of human colon cancer can be significantly advanced if appropriate experimental platforms become available. We have investigated whether a rat model carrying a knockout allele in the gatekeeper gene Adenomatous polyposis coli (Apc) recapitulates familial colon cancer of the human more closely than existing murine models. We have established a mutagen-induced nonsense allele of the rat Apc gene on an inbred F344/NTac (F344) genetic background. Carriers of this mutant allele develop multiple neoplasms with a distribution between the colon and small intestine that closely simulates that found in human familial adenomatous polyposis patients. To distinguish this phenotype from the predominantly small intestinal phenotype found in most Apc-mutant mouse strains, this strain has been designated the polyposis in the rat colon (Pirc) kindred. The Pirc rat kindred provides several unique and favorable features for the study of colon cancer. Tumor-bearing Pirc rats can live at least 17 months, carrying a significant colonic tumor burden. These tumors can be imaged both by micro computed tomography scanning and by classical endoscopy, enabling longitudinal studies of tumor genotype and phenotype as a function of response to chemopreventive and therapeutic regimes. The metacentric character of the rat karyotype, like that of the human and unlike the acrocentric mouse, has enabled us to demonstrate that the loss of the wild-type Apc allele in tumors does not involve chromosome loss. We believe that the Pirc rat kindred can address many of the current gaps in the modeling of human colon cancer.

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Available from: Mark Reichelderfer, Oct 05, 2015
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    • "mplification, purification and sequencing were carried out as described previously (Kanter-Smoler et al., 2008). Primers used for direct sequencing were identical to those used in the amplification reactions. The primer sequences for the exons and promoter regions are given in Supplementary Table 3, and the cDNA primers are described in De Rosa et al. (2007). CN detection by MLPA was performed according to the protocol provided by the supplier using the SALSA MLPA kit P043 APC (version B1, MRC-Holland, Amsterdam, the Netherlands ). The MLPA data were analyzed using GeneMapper 4.0 genotyping software (Applied Biosystem) and SeqPilot version 3.3.2 (JSI medical systems, GmBH, Kippenheim, Germa"
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    ABSTRACT: Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance of promoter 1B in normal colorectal mucosa (from controls), expression levels of specific transcripts from each of the promoters, 1A and 1B, were examined, and the expression from 1B was significantly higher compared with 1A. Significant amounts of transcripts generated from promoter 1B were also determined in a panel of 20 various normal tissues examined. In FAP-related tumors, the APC germline mutation is proposed to dictate the second hit. Mutations leaving two or three out of seven 20-amino-acid repeats in the central domain of APC intact seem to be required for tumorigenesis. We examined adenomas from mutation carriers in Family 1 for second hits in the entire gene without any findings, however, loss of the residual expression of the deleterious allele was observed. Three major conclusions of significant importance in relation to the function of APC can be drawn from this study; (i) germline inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require homozygous inactivation of APC allowing for alternative genetic models as basis for adenoma formation.
    Oncogene 06/2011; 30(50):4977-89. DOI:10.1038/onc.2011.201 · 8.46 Impact Factor
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    • "Based on these observations, both groups concluded that homozygous loss of APC alone is insufficient for nuclear accumulation of β-catenin. In agreement with these findings, recent analysis of microadenomas and late adenomas taken from a novel rat model (PIRC) carrying a targeted Apc truncation revealed detectable levels of nuclear β-catenin only in the late adenomas (Amos-Landgraf et al., 2007). Taken together, these findings suggest that Wnt activation, as assessed by nuclear accumulation of β-catenin, may require events in addition to loss of APC. "
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    ABSTRACT: Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, we show that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS. These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human FAP adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas.
    Cell 06/2009; 137(4):623-34. DOI:10.1016/j.cell.2009.02.037 · 32.24 Impact Factor
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    • "However, in Arabidopsis and C. elegans chemically induced (EMS) point mutation frequencies are as high as 1 in 100 kb [13,14] and in zebrafish ENU-induced frequencies of 1 in 150–250 kb can be obtained routinely [15] (E.C., unpublished results), suggesting that the maximum mutation load in a vertebrate genome that is compatible with viability, is much higher than what is currently reached in both rat and mouse. Although about 10 rat knockouts were successfully generated by ENU-driven target-selected mutagenesis [5,6,16] the relatively low mutation frequency makes the target-selected mutagenesis procedure laborious and costly. "
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    ABSTRACT: Background The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.
    BMC Genomics 11/2008; 9(1):460. DOI:10.1186/1471-2164-9-460 · 3.99 Impact Factor
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