Fas, a member of the tumor necrosis family, is responsible for initiating the apoptotic pathway when bound to its ligand, Fas-L. Defects in the Fas-mediated apoptotic pathway have been reported in colorectal cancer.
In the present study, a variant of the ApcMin/+ mouse, a model for the human condition, Familial Adenomatous Polyposis (FAP), was generated with an additional deficiency of Fas (ApcMin/+/Faslpr) by cross-breeding ApcMin/+ mice with Fas deficient (Faslpr) mice. One of the main limitations of the ApcMin/+ mouse model is that it only develops benign polyps. However, ApcMin/+/Faslpr mice presented with a dramatic increase in tumor burden relative to ApcMin/+ mice and invasive lesions at advanced ages. Proliferation and apoptosis markers revealed an increase in cellular proliferation, but negligible changes in apoptosis, while p53 increased at early ages. Fas-L was lower in ApcMin/+/Faslpr mice relative to ApcMin/+ cohorts, which resulted in enhanced inflammation.
This study demonstrated that imposition of a Fas deletion in an ApcMin/+ background results in a more aggressive phenotype of the ApcMin/+ mouse model, with more rapid development of invasive intestinal tumors and a decrease in Fas-L levels.
Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) ApcMin/+ mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. ApcMin/+ mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The ApcMin model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter ApcMin-mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 ApcMin/+ mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than ApcMin/+ controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: (1) Mom12, a dominant modifier linked to the congenic region on chromosome 6 and (2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.
adenomatous polyposis coli; modifier of min; congenic mice; caveolin-1; cancer susceptibility
Ets transcription factors have been widely implicated in the control of tumorigenesis, with most studies suggesting tumor-promoting roles. However, few studies have examined Ets tumorigenesis-modifying functions in vivo using model genetic systems.
Using mice expressing a previously characterized Ets dominant repressor transgene in the intestinal epithelium (Villin-En/Erm), we examined the consequences of blocking endogenous Ets-mediated transcriptional activation on tumorigenesis in the ApcMin model of intestinal carcinoma.
En/Erm expression in the intestine, at levels not associated with overt crypt-villus dysmorphogenesis, results in a marked increase in tumor number in ApcMin animals. Moreover, when examined histologically, tumors from En/Erm-expressing animals show a trend toward greater stromal invasiveness. Detailed analysis of crypt-villus homeostasis in these En/Erm transgenic animals suggests increased epithelial turnover as one possible mechanism for the enhanced tumorigenesis.
Our findings provide in vivo evidence for a tumor-restricting function of endogenous Ets factors in the intestinal epithelium.
M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apcmin/+ mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apcmin/+ mice and compared intestinal morphology and tumor number in 12-week-old Apcmin/+Chrm3−/− and Apcmin/+Chrm3+/+ control mice. Compared with Apcmin/+Chrm3+/+ mice, Apcmin/+Chrm3−/− mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, β-catenin nuclear staining was reduced in Apcmin/+Chrm3−/− compared with Apcmin/+Chrm3+/+ mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apcmin/+Chrm3−/− mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apcmin/+ mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and β-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.
With the advent of high-density DNA marker data sets for the mouse and other model systems, 100 or more genotypes are routinely generated from large groups of mice. Issues of the accuracy and reliability of the genotyping are extremely important but often not addressed until genetic analysis is conducted. Simple tests that rely on the robust predictions arising from Mendelian genetics can be made quickly in the molecular laboratory as the data are generated, and require only a spreadsheet program. In this report, genotype data from 392 mice tested at 96 marker sites were analyzed for errors that are typical when handling large volumes of data generated in a repetitive process. The testing consisted of: (1) repeating the genotyping of approximately 1% of the samples; (2) examining the deviation from the expected segregation ratio (1:2:1) on a marker-by-marker basis; and (3) testing the correlation of the genotype at one marker with that at neighboring genetic markers on a chromosome. These three steps allowed analysis at the level of the microtiter plate, where errors are most likely to occur. A set of 96 dinucleotide repeat markers that are polymorphic between the C57BL/6J and DBA/2J mouse strains and can be multiplexed is reported for use in other genotyping projects.
Hardy–Weinberg equilibrium; inbred lines; C57; DBA; multiplex marker set
TGF-β signaling is frequently altered in colorectal cancer. Using a novel model of mice heterozygous for a targeted null mutation of Tgfbr1 crossed with ApcMin/+ mice, we show that ApcMin/+;Tgfbr1+/− mice develop twice as many intestinal tumors as ApcMin/+ ;Tgfbr1+/+ mice as well as adenocarcinoma of the colon, without loss of heterozygosity at the Tgfbr1 locus. Decreased Smad2 and Smad3 phosphorylation and increased cellular proliferation are observed in the colonic epithelium crypts of ApcMin/+ ;Tgfbr1+/− mice. Smad-mediated TGF-β signaling is preserved in both ApcMin/+ ;Tgfbr1+/+ and ApcMin/+ ;Tgfbr1+/− intestinal tumors but cyclin D1 expression and cellular proliferation are significantly higher in ApcMin/+ ;Tgfbr1+/− tumors. These results show that reduced Tgfbr1-mediated TGF-β signaling significantly enhances colorectal cancer development and results in increased tumor cell proliferation. These findings provide a plausible molecular mechanism for colorectal cancer development in individuals with constitutively altered TGFBR1 expression, a recently identified common form of human colorectal cancer.
Tgfbr1; Transforming growth factor beta; haploinsufficiency; colon cancer; SMAD
We have previously reported that sulindac, a non-steroidal anti-inflammatory drug, inhibited tumor formation in the small intestine but increased tumors in the colon of ApcMin/+ mice, a model of human familial adenomatous polyposis. To further explore intestinal regional responses, we studied effects of sulindac on additional gene-targeted mouse models of human intestinal tumorigenesis; these were (i) Apc1638N/+ mouse (chain termination mutation in exon 15 of the Apc gene); (ii) Mlh1+/− mouse (DNA mismatch repair deficiency, a mouse model of human hereditary non-polyposis colorectal cancer) and (iii) double-heterozygous Mlh1+/−Apc1638N/+ mutant mouse. Mice were fed AIN-76A control diet with or without 0.02% sulindac for 6 months. Intestinal regional tumor incidence, multiplicity, volume and degree of inflammation were used as end points. The results showed the following: (i) sulindac inhibited tumor development in the small intestine of Apc1638N/+ mice; (ii) in contrast, sulindac increased tumors in the small intestine of Mlh1 mutant mice, a neoplastic effect which persisted in heterozygous compound Mlh1+/−Apc1638N/+ mutant mice; (iii) sulindac increased tumors in the cecum of all mice regardless of genetic background; (iv) sulindac decreased inflammation in the small intestine of Apc1638N/+ mice, but it increased inflammation in the small intestine of Mlh1+/− mice and Mlh1+/−Apc1638N/+ mice and (v) sulindac enhanced inflammation in the cecum of all mutant mice. Findings indicate that the effects of sulindac in the intestine of these mutant mouse models are probably related to genetic background and appear to be associated with its inflammatory-inducing response.
AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for enterobacterial repetitive intergenic consensus (ERIC)-PCR detection.
METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR.
RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.
CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost-effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.
DNA extraction; Intestinal microflora
Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis, but the mechanism remains unclear. The APC/β-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose is this study is to explore the interactions of vitamin D and APC/β-catenin pathways in intestinal tumor development. APCmin/+ mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APCmin/+ and APCmin/+VDR−/− mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APCmin/+ and APCmin/+VDR−/− mice at 3, 4 and 6–7 months of age. The size of the tumors, however, was significantly increased in APCmin/+VDR−/− mice in all age groups. Immunostaining showed a significant increases in β-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APCmin/+VDR−/− tumors compared to APCmin/+ tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APCmin/+VDR−/− mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and β-catenin signaling pathways in intestinal tumorigenesis.
vitamin D receptor; beta-catenin; intestinal tumor; colon cancer; Apc(min/+)
Brown rice is a staple dietary constituent in Asia, whereas rice consumed in the Western world is generally white, obtained from brown rice by removal of the bran. We tested the hypothesis that rice bran interferes with development of tumours in TAg, TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) or ApcMin mice, genetic models of mammary, prostate and intestinal carcinogenesis, respectively. Mice received rice bran (30%) in AIN-93G diet throughout their post-weaning lifespan. In TAg and TRAMP mice, rice bran did not affect carcinoma development. In TRAMP or wild-type C57Bl6/J mice, dietary rice bran increased kidney weight by 18 and 20%, respectively. Consumption of rice bran reduced numbers of intestinal adenomas in ApcMin mice by 51% (P<0.01), compared to mice on control diet. In parallel, dietary rice bran decreased intestinal haemorrhage in these mice, as reflected by increased haematocrit. At 10% in the diet, rice bran did not significantly retard ApcMin adenoma development. Likewise, low-fibre rice bran (30% in the diet) did not affect intestinal carcinogenesis, suggesting that the fibrous constituents of the bran mediate chemopreventive efficacy. The results suggest that rice bran might be beneficially evaluated as a putative chemopreventive intervention in humans with intestinal polyps.
chemoprevention; diet; rice bran; genetic models of carcinogenesis
Aberrant activation of the Wnt/β-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear β-catenin and the induction of its target genes. Apcmin/+ mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear β-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear β-catenin levels in colon cancer in vivo. By appropriate breeding of Apcmin/+ mice and Vdr+/− mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apcmin/+Vdr-/- mice had increased nuclear β-catenin and the tumors reached a larger size than those of Apcmin/+Vdr+/+. Both ACF and carcinomas in Apcmin/+Vdr-/- mice showed higher expression of β-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced β-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)2D3 to promote the relocation of β-catenin from the nucleus to the plasma membrane and to inhibit β-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear β-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/β-catenin pathway.
Both mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMin mutation or intestine-specific expression of the KRASV12 gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV12 double transgenic mice to develop intestinal tumors.
At 12 weeks of age, ApcMin/KRASV12 mice had three times as many intestinal tumors as ApcMin mice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV12/Klf5+/- mice was also statistically significant compared to ApcMin mice alone, with a 75% decrease. Compared with ApcMin/KRASV12, tumors from both ApcMin/KRASV12/Klf5+/- and ApcMin mice were smaller. In addition, tumors from ApcMin mice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMin and ApcMin/KRASV12 mice but were attenuated in ApcMin/KRASV12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV12/Klf5+/- mice when compared to ApcMin/KRASV12 mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV12 mice and modestly reduced in ApcMin/KRASV12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.
Results of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV12 mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.
AIM: To investigate whether Recql5, a DNA helicase that plays an important role in the maintenance of genome integrity, is a tumor suppressor in the gastrointestinal tract in mice.
METHODS: We generated cohorts of both Recql5-proficient and Recql5-deficient Apcmin/+ mice and compared the tumor susceptibility in their gastrointestinal tracts.
RESULTS: Recql5 deficiency in Apcmin/+ mice resulted in a significant increase in the tumor incidence in both the colon (P = 0.0162) and the small intestine (P < 0.01). These findings have provided the first genetic evidence for a tumor suppression role of Recql5 in the gastrointestinal tract of mice. Importantly, since mouse Recql5 and human RECQL5 are highly conserved, these findings also suggest that RECQL5 may be a tumor suppressor for human colon cancer.
CONCLUSION: Recql5 has a tumor suppression role in the mouse gastrointestinal tract.
Recql5; Apc; Tumor suppressor; Genome instability; Colon cancer; Apcmin/+ mice
Developing and evaluating new technology that enables researchers to recover gene-expression levels of colonic cells from fecal samples could be key to a non-invasive screening tool for early detection of colon cancer. The current study, to the best of our knowledge, is the first to investigate and report the reproducibility of fecal microarray data. Using the intraclass correlation coefficient (ICC) as a measure of reproducibility and the preliminary analysis of fecal and mucosal data, we assessed the reliability of mixture density estimation and the reproducibility of fecal microarray data. Using Monte Carlo-based methods, we explored whether ICC values should be modeled as a beta-mixture or transformed first and fitted with a normal-mixture. We used outcomes from bootstrapped goodness-of-fit tests to determine which approach is less sensitive toward potential violation of distributional assumptions.
The graphical examination of both the distributions of ICC and probit-transformed ICC (PT-ICC) clearly shows that there are two components in the distributions. For ICC measurements, which are between 0 and 1, the practice in literature has been to assume that the data points are from a beta-mixture distribution. Nevertheless, in our study we show that the use of a normal-mixture modeling approach on PT-ICC could provide superior performance.
When modeling ICC values of gene expression levels, using mixture of normals in the probit-transformed (PT) scale is less sensitive toward model mis-specification than using mixture of betas. We show that a biased conclusion could be made if we follow the traditional approach and model the two sets of ICC values using the mixture of betas directly. The problematic estimation arises from the sensitivity of beta-mixtures toward model mis-specification, particularly when there are observations in the neighborhood of the the boundary points, 0 or 1. Since beta-mixture modeling is commonly used in approximating the distribution of measurements between 0 and 1, our findings have important implications beyond the findings of the current study. By using the normal-mixture approach on PT-ICC, we observed the quality of reproducible genes in fecal array data to be comparable to those in mucosal arrays.
Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the ApcMin/+ mouse, a model for spontaneous intestinal polyposis. PRKO-ApcMin/+mice exhibited no change in polyp number, size or localization compared to ApcMin/+. To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.
Here we assessed whether silibinin, a nontoxic chemopreventive agent, inhibits spontaneous intestinal tumorigenesis in APCmin/+ mouse model, a genetically predisposed animal model of human familial adenomatous polyposis (FAP).
Materials and Methods
Six-week old APCmin/+ mice were divided into four groups and orally gavaged with 0.2 ml vehicle, or 250, 500 and 750 mg silibinin/kg body weight in 0.2 ml vehicle for five days/week. After 6 weeks, polyp burden was analyzed and tissues examined for molecular alterations.
Silibinin treatments decreased total number of intestinal polyps by 34% (P<0.01), 42% (P<0.01) and 55% (P<0.001), respectively. Immunohistochemical analysis showed that silibinin dose-dependently decreases (P<0.001) proliferation and induces (P<0.001) apoptosis only in intestinal polyps without any considerable effects on normal crypt-villi in APCmin/+ or wild-type mice. Further analysis of polyps showed that silibinin decreases β-catenin, cyclin D1, c-Myc and phospho-glycogen synthase kinase-3β expression. Silibinin treatment also decreased phospho-Akt, cyclooxygenase-2, inducible nitric oxide synthase, nitrotyrosine and nitrite levels in polyps, the well-known mediators of intestinal/colon carcinogenesis.
Together, these results establish silibinin efficacy in a well-established genetic model of FAP, APCmin/+ mouse, and suggest that this natural agent modulates various molecular pathways including β-catenin in its overall chemopreventive efficacy against intestinal carcinogenesis.
colon cancer; chemoprevention; silibinin; COX-2; beta-catenin
In human pancreatic cancers, promoter CpG island hypermethylation is observed in both benign and malignant tumors. It is thought that silencing of key growth-controlling genes by promoter hypermethylation may play a role in pancreatic oncogenesis. We have shown previously that sufficient levels of DNA methyltransferase (Dnmt) 1 expression are required for the development of murine intestinal tumors. Here, we report the results of a large-scale triple cross (progeny n = 761) between ApcMin/+, Trp53−/− and Dnmt1 hypomorphic mice to investigate the role of Dnmt levels in the ApcMin/+, Trp53−/− mouse models of acinar cell pancreatic cancer. Mutations of both APC and TP53 are observed in human pancreatic cancer. We found that tumor burden, but not tumor size, is significantly reduced with decreasing Dnmt1 levels, suggesting that DNA methylation is involved in pancreatic tumorigenesis in this mouse model. Detailed analyses showed that the reduction in tumor burden is the result of a decrease in both early- and late-stage lesions. We observed decreased levels of DNA methylation at candidate genes in the normal pancreas of Dnmt1 hypomorphic mice. Some of these genes showed increased methylation associated with tumorigenesis, suggesting that the tumor-suppressive effects of Dnmt1 hypomorphic alleles may be mediated in part through reduced promoter hypermethylation. Our work is the first in vivo study to show the effects of reduced Dnmt levels on pancreatic tumor development.
The domestic dog presents an attractive model system for the study of the genetic basis of disease. The development of resources such as the canine genome sequence and SNP genotyping platforms has allowed for the implementation of canine genetic studies. Successful implementation of such studies depends not only on the quality of individual DNA samples, but also on the number of samples obtained. The latter can be maximized using a non-invasive DNA collection method that can increase study participation. We compared the DNA yield and quality obtained from blood and buccal swabs to those obtained using a non-invasive saliva collection kit (Oragene ®•ANIMAL kit). We also assessed the success rate of PCR amplification and genotyping accuracy of DNA isolated using these collection methods.
Comparison of DNA yields from matched saliva, blood and buccal swab samples showed that yields from saliva were significantly higher than those from blood (p = 0.0198) or buccal swabs (p = 0.0008). Electrophoretic analysis revealed that blood and saliva produced higher quality DNA than buccal swabs. In addition, a 1.1-kb PCR fragment was successfully amplified using the paired DNA samples and genotyping by PCR-RFLP yielded identical results.
We demonstrate that DNA yields from canine saliva are higher than those from blood or buccal swabs. The quality of DNA extracted from saliva is sufficient for successful amplification of a 1.1-kb fragment and for accurate SNP genotyping by PCR-RFLP. We conclude that saliva presents a non-invasive alternative source of high quantities of canine genomic DNA suitable for genotyping studies.
Ionising radiation is a carcinogen capable of inducing tumours, including colorectal cancer, in both humans and animals. By backcrossing a recombinant line of ApcMin/+ mice to the inbred BALB/c mouse strain, which is unusually sensitive to radiation–induced tumour development, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites at ∼15 cM density and phenotyping by counting adenomas in the small intestine. Interval and composite interval mapping along with permutation testing identified five significant susceptibility quantitative trait loci (QTLs) responsible for radiation induced tumour multiplicity in the small intestine. These were defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (log of odds, LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p<0.00001, 6.2, p<0.00001) and two regions within chromosome 16 respectively (LOD 4.1, p = 4×10−5, 4.8, p<0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p<0.005). Genes containing BALB/c specific non-synonymous polymorphisms were identified within Mrip regions and prediction programming used to locate potentially functional polymorphisms. Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Casc5, and Bub1), DNA repair (Recc1 and Prkdc) or inflammation (Duox2, Itgb2l and Cxcl5). Our study demonstrates use of in silico analysis in candidate gene identification as a way of reducing large-scale backcross breeding programmes.
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, but currently available non-invasive screening programs have achieved only a modest decrease in mortality. MicroRNAs (miRNAs) play important role in a wide array of biological processes and are commonly dysregulated in neoplasia. We aimed to evaluate the feasibility of fecal miRNAs as biomarkers for colorectal neoplasia screening.
Material and methods
Total RNA was extracted from freshly collected stool samples from 8 healthy volunteers and 29 FOBT collected feces from subjects with normal colonoscopies, colon adenomas and CRCs. miRNA expression analysis were performed with TaqMan qRT-PCR for a subset of miRNAs. Illumina miRNA microarray profiling was performed to evaluate the differences in expression patterns between normal colonic mucosa tissues and stool samples from healthy subjects.
We efficiently extracted miRNAs from stool specimens using our developed protocol. Data from independent experiments showed high reproducibility for miRNA extraction and expression. miRNA expression patterns were similar in stool specimens among healthy volunteers and reproducible in stool samples that were collected serially in time from the same individuals. miRNA expression profiles from 29 patients demonstrated higher expression of miR-21 and -106a in patients with adenomas and CRCs, compared with individuals free of colorectal neoplasia.
Our data indicate that miRNAs can be extracted from stool easily and reproducibly. The stools of patients with colorectal neoplasms have unique and identifiable patterns of miRNA expression.
Fecal miRNAs may be an excellent candidate for the development of a non-invasive screening test for colorectal neoplasms.
microRNA; stool biomarker; colon cancer; cancer screening
Genomic-level analyses of DNA from non-invasive sources would facilitate powerful conservation and evolutionary studies in natural populations of endangered and otherwise elusive species. However, the typical low quantity and poor quality of DNA that is extracted from non-invasive samples have generally precluded such work. Here we apply a modified DNA capture protocol that, when used in combination with massively-parallel sequencing technology, facilitates efficient and highly-accurate resequencing of megabases of specified nuclear genomic regions from fecal DNA samples. We validated our approach by comparing genetic variants identified from corresponding fecal and blood DNA samples of six western chimpanzees (Pan troglodytes verus) across more than 1.5 megabases of chromosome 21, chromosome X, and the complete mitochondrial genome. Our results suggest that it is now feasible to conduct genomic studies in natural populations for which constraints on invasive sampling have otherwise long been a barrier. The data we collected also provided an opportunity to examine western chimpanzee genetic diversity at unprecedented scale. Despite high mitochondrial genome diversity (π = 0.585%), western chimpanzees have a low ratio (0.42) of X chromosomal (π = 0.034%) to autosomal (chromosome 21 π = 0.081%) sequence diversity, a pattern that may reflect an unusual demographic history of this subspecies.
molecular ecology; population genetics; non-invasive sampling; conservation genomics
The chemokine receptor CXCR2 is a key mediator of neutrophil migration that also plays a role in tumor development. However, CXCR2 influences tumors through multiple mechanisms and might promote or inhibit tumor development depending on context. Here, we used several mouse models of spontaneous and inflammation-driven neoplasia to define indispensable roles for CXCR2 in benign and malignant tumors. CXCR2-activating chemokines were part of the secretome of cultured primary benign intestinal adenomas (ApcMin/+) and highly expressed by all tumors in all models. CXCR2 deficiency profoundly suppressed inflammation-driven tumorigenesis in skin and intestine as well as spontaneous adenocarcinoma formation in a model of invasive intestinal adenocarcinoma (AhCreER;Apcfl/+;Ptenfl/fl mice). Pepducin-mediated CXCR2 inhibition reduced tumorigenesis in ApcMin/+ mice. Ly6G+ neutrophils were the dominant source of CXCR2 in blood, and CXCR2 deficiency attenuated neutrophil recruitment. Moreover, systemic Ly6G+ cell depletion purged CXCR2-dependent tumor-associated leukocytes, suppressed established skin tumor growth and colitis-associated tumorigenesis, and reduced ApcMin/+ adenoma formation. CXCR2 is thus a potent protumorigenic chemokine receptor that directs recruitment of tumor-promoting leukocytes into tissues during tumor-inducing and tumor-driven inflammation. Similar leukocyte populations were also found in human intestinal adenomas, which suggests that CXCR2 antagonists may have therapeutic and prophylactic potential in the treatment of cancer.
c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, ApcMin mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes.
Cancer occurs when cells change their behavior and start to divide in an uncontrolled manner. To achieve this altered behavior, cells need to change their metabolism to be able to grow even when nutrient and oxygen supplies are limiting. Therefore, targeting metabolic pathways could be used to treat patients suffering from cancer. Here we studied a gene called MYC, which can regulate many metabolic pathways. By using genetically modified mice we can show that tumors have a remarkable ability to change their metabolism, even if key enzymes are removed. Taken together, our data suggest that metabolic disturbance by drugs in the clinic may present a future challenge.
Myc is a transcription factor frequently found deregulated in human cancer. The Myc-mediated cellular transformation process is associated with fast proliferative cells and inherent genomic instability, giving rise to malignant, invasive neoplasms with poor prognosis for survival. Transcription-independent functions of Myc include stimulation of replication. Excessive Myc expression stimulates a replication-associated DNA damage response that signals via the phosphoinositide-3-kinase (PI3K)-related protein kinases (PIKKs) ATM and ATR. These, in turn, activate the DNA damage transducers Chk1 and Chk2. Here, we show that Myc can stimulate Chek2 transcript indirectly in vitro as well as in B cells of λ-Myc transgenic mice or in the intestine of ApcMin mice. However, Chk2 is dispensable for Myc's ability to transform cells in vitro and for the survival of established lymphoma cells from λ-Myc transgenic mice. Chk2 deficiency induces polyploidy and slow growth, but the cells are viable and protected against DNA damage. Furthermore, inhibition of both Chk1/Chk2 with AZD7762 induces cell death and significantly delays disease progression of transplanted lymphoma cells in vivo. DNA damage recruits PARP family members to sites of DNA breaks that, in turn, facilitate the induction of DNA repair. Strikingly, combining Chk2 and PARP inhibition elicits a synergistic lethal response in the context of Myc overexpression. Our data indicates that only certain types of chemotherapy would give rise to a synergistic lethal response in combination with specific Chk2 inhibitors, which will be important if Chk2 inhibitors enter the clinic.
lymphoma; Myc; Chk1; Chk2; PARP; DNA damage; AZD-7762; ABT-888
The numbers of obese people and diabetic patients are ever increasing. Obesity and diabetes are high-risk conditions for chronic diseases, including certain types of cancer, such as colorectal cancer (CRC). The aim of this study was to develop a novel animal model in order to clarify the pathobiology of CRC development in obese and diabetic patients. We developed an animal model of obesity and colorectal cancer by breeding the C57BL/KsJ-db/db (db/db) mouse, an animal model of obesity and type II diabetes, and the C57BL/6J-ApcMin/+ (Min/+) mouse, a model of familial adenomatous polyposis. At 15 weeks of age, the N9 backcross generation of C57BL/KsJ-db/db-ApcMin/+ (db/db-Min/+) mice developed an increased incidence and multiplicity of adenomas in the intestinal tract when compared to the db/m-Min/+ and m/m-Min/+ mice. Blood biochemical profile showed significant increases in insulin (8.3-fold to 11.7-fold), cholesterol (1.2-fold to 1.7-fold), and triglyceride (1.2-fold to 1.3-fold) in the db/db-Min/+ mice, when compared to those of the db/m-Min/+ and m/m-Min/+ mice. Increases (1.4-fold to 2.6-fold) in RNA levels of insulin-like growth factor (IGF)-1, IRF-1R, and IGF-2 were also observed in the db/db- Min/+ mice. These results suggested that the IGFs, as well as hyperlipidemia and hyperinsulinemia, promoted adenoma formation in the db/db-Min/+ mice. Our results thus suggested that the db/db-Min/+ mice should be invaluable for studies on the pathogenesis of CRC in obese and diabetes patients and the therapy and prevention of CRC in these patients.
C57BL/KsJ-db/db; C57BL/6J-ApcMin/+; Type 2 diabetes mellitus; colon carcinogenesis; animal model