Study of the APC gene function in the mouse APC+/APC1638N model

Familial adenomatous polyposis (FAP) is an autosomal dominant disease characterized by the presence of hundreds to thousands of benign polyps in the colon. If not removed prophylactically they represent a risk of developing malignant cancer with an almost 100% penentrance. FAP is induced by germline mutation in the APC gene. Tumorigenesis launched a second somatic mutation of APC gene allele, leading to synthesis of non-functional APC protein. One of the possibilities of cancer prevention could be an alternative gene therapy using bacteria as vectors for delivery of therapeutic protein molecules.

For this purpose mice model APC+/APC1638N with mutation in one allele murine homolog of the APC gene were used. Mice were fed orally commercial nutrition enriched with 0.5 ml PBS buffer with 5% milk containing 5×108 recombinant bacterial cells DE3plys6 bearing plasmid with cloned APC gene twice a week during 42 weeks. Afterwords mice were killed by thiopental, gastrointestinal tracts were removed, microscopically, macroscopically inspected for polyps/neoplastic lesions and immunohistochemically investigated with polyclonal rabbit antibody against APC protein.

We have cloned full-lenght APC gene into vector for expression in bacterial cells Escherichia coli BL21(DE3) and BL21(DE3) pLysS. Expression of the APC protein, induced by IPTG, was detected in protein extracts of three bacterial clones: DE3104-11, DE3pLys5, DE3pLys6. APC protein was identified by Western blot analysis using monoclonal and polyclonal antibodies against the APC protein. Bacteria of clone DE3pLys6 were orally administered to APC+/APC1638N mice with mutations in the APC gene. All transgenic mice without therapy developed adenomatous polyps in the gastrointestinal tract. Transgenic mice treated by oral administration of bacteria expressing functional APC protein developed polyps in 33.3%. The remaining four mice 66.7% were without polyps development.

Administration of APC gene expressing by bacteria to transgenic mice with mutation in APC gene leads to reduction in the number of mice developing polyps in the gastrointestinal tract. The effect of bacterially expressed APC protein in elimination of intestinal polyps or tumors has been monitored. These are our preliminary results and for possible confirmation of our hypotheses still more research is needed.