J.E. Crabtree & P.A. Robinson, Email: j.crabtree@leeds.ac.uk
H. pylori is a major risk factor for gastric carcinogenesis. The mechanisms by which H. pylori promotes gastric cancer are multiple. The long-term chronic inflammatory response and epithelial hyperproliferative changes are considered to increase the risk of mutational changes. Whilst chronic inflammation is generally considered the driving force initiating gastric mutations, H. pylori also induces the aberrant expression of the DNA editing enzyme activation-induced cytidine deaminase (AID) in gastric epithelial cells in a cag PAI dependent manner. As epithelial expression of AID is upregulated in patients with chronic gastritis, aberrant expression of this DNA editing enzyme may be a key factor linking H. pylori cag positive strains with increased risk of developing gastric cancer. The specific role of H. pylori virulence factors and associated upregulation of activation induced cytidine deaminase in inducing mutations in p53 will be assessed in in vivo and in vitro cell culture systems.
The frequency of mutations in Helicobacter infected gastric tissue and the specific epithelial cell populations in which mutations arise will be determined. Gastric tissue from model systems exposed to parental H. pylori strains or isogenic mutant strains lacking key virulence genes such as cagA and the cag pathogenicity island will be examined. The use of a model system containing the bacterial transgene lacI/lacZ will allow in vivo analysis of mutational changes in DNA following Helicobacter infection. Immunohistological labelling will be used to identify potential stem cell populations for laser capture microscopy and analysis of gastric mutations. Mutations in the p53 gene in selected epithelial cell populations harvested using laser capture microscopy will be examined as well as proliferative and apoptotic responses of gastric epithelial cells and mucosal gene expression. In addition, sequencing of lacI will allow determination of the mutational frequency in different mouse epithelial cell populations.
The PhD student will gain experience in a wide range of molecular and cellular biology experimental techniques including cell and bacterial culture, mutational analysis, laser capture microscopy, immunohistochemistry and RT-PCR.