GSK-3B mutations in canine colon carcinomas

Abstract

Neoplasias of the gastrointestinal tract are uncommon in small animals. In dogs, gastrointestinal tumors most commonly develop in the rectum and colon. The cells of the mucosa are closely packed in the crypts of Lieberkühn. At the base of the crypts stem cells are present. These stem cells continually divide to replace the epithelium. A loss of proliferation control in the crypts is thought to lead to colorectal cancer. The Wnt signaling pathway is known to regulate the proliferation of the stem cells. The Wnt signaling pathway inhibits the formation of the multi-protein complex of APC, GSK-3β and Axin. Without the multiprotein complex, β-catenin accumulates in the cytoplasm and it translocates to the nucleus where β-catenin forms nuclear complexes with Tcf/Lef family. This leads to transcription of several target genes which play an important part in the regulation of cell proliferation, differentiation and apoptosis. The hypothesis of this research is that a mutation in the gene that codes for GSK-3β makes GSK-3β inactive. Therefore, there are higher levels of free β-catenin present and as a result the continuing of cell proliferation in the epithelia of the colon in dogs with colon carcinomas. After development of the primers and reversing the RNA of the 10 samples (6 tumor samples and 4 control samples) to cDNA, PCR was performed. The PCR products were purified and after that the concentration of DNA molecules were measured. An exact amount of DNA was used to perform the tercycle, after which the sequence was determined of all the samples. There were eight variants know of GSK-3β. It was at first determined that the variant which were present in all the samples were variant 2. After determination of the sequence of all the samples, they were all compared to variant 2 and the tumor samples were compared to the control samples. All the samples showed a difference with the sequence a variant 2. At the level of nucleotide 1779 and nucleotide 2235 there were wobbles or mismatches found, for glycine and alanine respectively. At the level of nucleotide 1009 of the samples C2 and C4 there was a Single Nucleotide Polymorphism found at first. The codon at that point could be GGG, coding for glycine, or AGG, coding for arginine. Glycine is a non polar amino acid and arginine is a positively charged amino acid. This could mean that the protein could have a different configuration. But it was only seen in two control samples. After sequencing with a reverse primer there were no SNP found. On the basis of these findings it was concluded that GSK-3β is not mutated in colon carcinomas in the dog. In previous research on colon carcinomas in the dog there were no mutations found in APC and β-catenin. Both these proteins count for the most of the mutations in colon carcinomas in humans. After the research into mutations in APC, β-catenin and GSK-3β the logical continuation is to investigate the other proteins involving the ubiquitin-degradation of β-catenin, namely Axin. After possibly ruling out Axin, the proteins that inhibit the degradation of β-catenin, for example like K-ras, Frat1 and Dvl, could be investigated next.