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PKCa -/- vs wt small intestine

ABSTRACT: Members of the PKC family of serine / threonine kinases play key regulatory roles in numerous cellular processes including differentiation and proliferation. Of the eleven mammalian PKC isoforms known several have been implicated in tumor development and progression. However, in most cases isotype specificity is poorly defined and even contrary functions for a single PKC have been reported, mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report we therefore used PKC genetic targeting to study the role of PKCa and PKCz in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of ApcMin/+ mice. A deletion of PKCz did not affect tumorigenesis in this animal model. In contrast, PKCa deficient ApcMin/+ mice developed more aggressive tumors and died significantly earlier than their PKCa proficient littermates. Even without an additional Apc mutation PKCa knock out mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating EGFR signaling and proposed a synergistic mechanism for EGFR / AP-1 and WNT / APC pathways in mediating intestinal tumor development. Keywords: genetic modification

ORGANISM(S): Mus musculus

PROVIDER: GSE3915 | GEO | 2006/07/01

SECONDARY ACCESSION(S): PRJNA94171

REPOSITORIES: GEO

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Project description:Members of the PKC family of serine / threonine kinases play key regulatory roles in numerous cellular processes including differentiation and proliferation. Of the eleven mammalian PKC isoforms known several have been implicated in tumor development and progression. However, in most cases isotype specificity is poorly defined and even contrary functions for a single PKC have been reported, mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report we therefore used PKC genetic targeting to study the role of PKCa and PKCz in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of ApcMin/+ mice. A deletion of PKCz did not affect tumorigenesis in this animal model. In contrast, PKCa deficient ApcMin/+ mice developed more aggressive tumors and died significantly earlier than their PKCa proficient littermates. Even without an additional Apc mutation PKCa knock out mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating EGFR signaling and proposed a synergistic mechanism for EGFR / AP-1 and WNT / APC pathways in mediating intestinal tumor development. Experiment Overall Design: Adult male animals (PKCa -/- and congenic wt controls; 50-60 days old) were sacrificed by cervical dislocation. Small intestines were dissected and total RNA (~5Âµg per sample) was extracted using the QIAGEN RNeasy kit. Experiment Overall Design: Sample processing (performed at the RZPD, Deutsches Ressourcenzentrum fÃ¼r Genomforschung GmbH, Heubnerweg 6, 14059 Berlin) included cRNA generation and labeling with biotin. cRNA samples were hybridized to the GeneChips and chips stained with a streptavidine-phycoerythrine conjugate, Experiment Overall Design: washed and scanned. Hybridization images were analyzed using Experiment Overall Design: GCOSâ¢ 1.1.

Project description:Mutation of the adenomatous polyposis coli (APC gene), an early event in the adenoma-carcinoma sequence, is present in 70-80% of sporadic human colorectal adenomas and carcinomas. To test the hypothesis that mutation of the APC gene alters microbial interactions with host intestinal mucosa prior to the development of polyposis, culture-independent methods (targeted qPCR assays and Illumina sequencing of the 16S rRNA gene V1V2 hypervariable region) were used to compare the intestinal microbial composition of 30 six-week old C57BL/6 APCMin/+ and 30 congenic wild type (WT) mice. The results demonstrate that similar to 12-14 week old APCMin/+ mice with intestinal neoplasia, 6 week old APCMin/+ mice with no detectable neoplasia, exhibit an increased relative abundance of Bacteroidetes spp in the colon. Parallel mouse RNA sequence analysis, conducted on a subset of proximal colonic RNA samples (6 APCMin/+, 6 WT) revealed 130 differentially expressed genes (DEGs, fold change ? 2, FDR <0.05). Hierarchical clustering of the DEGs was carried out by using 1-r dissimilarity measurement, where r stands for the Pearson correlation, and Ward minimum variance linkage, in order to reduce the number of input variables. When the cluster centroids (medians) were included along with APC genotype as input variables, analysis of variance and covariance (ANCOVA) with stepwise variable selection, selected two of seven mouse gene clusters, in addition to APC genotype, as significantly associated (p = 0.011 and p = 0.004) with the increased relative abundance of Bacteroidetes spp. One of the two clusters includes several downregulated genes encoding immunoglobulin variable regions and non-protein coding RNAs. These results support the concept that mutation of the APC gene alters colonic-microbial interactions prior to polyposis. It remains to be determined whether interventions directed at ameliorating dysbiosis in APCMin/+mice, such as through probiotics, prebiotics or antibiotics, could reduce tumor formation. Comparing the differential gene expressions in proximal colonic mucosal samples from 6 six-week-old female Apc Min/+ and 6 six-week-old female WT mice

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PKCa -/- vs wt small intestine

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