4808

The Clostridium perfringens bacterial strain type A produces a single polypeptide enterotoxin (CPE) that is originally associated with food poisoning causing gastroenteritis. It was shown, that the target receptor of the CPE-protein is the cellular transmembrane protein claudin 3 and claudin 4. However, detailed analyses determined the strong preference of CPE-protein for binding to claudin 4. The claudins 3 and 4 are transmembrane proteins found in tight junctions, which are involved in control of paracellular permeability of cells. They were shown to be highly upregulated in their expression in several human cancers, including breast, ovarian, pancreatic and colon carcinoma. The bacterial CPE-toxin binds specifically to the surface receptor claudin 3 and 4 and initiates the formation of large multiprotein membrane pore complexes, which results in the rapid cell lysis. Previous studies have demonstrated the antitumoral activity of treatment with local application of the recombinant bacterial CPE-protein. In our recent study we went one step further and used the CPE cDNA for the specific gene therapy of claudin 3 and 4 overexpressing colon carcinoma cells. To approach this, we first isolated the CPE cDNA from the total Clostridium perfringens bacterial DNA (kindly provided by M.-R. Popoff, Inst. Pasteur, Paris, France) by using specific primers for PCR amplification. For the eucaryotic expression of the toxin gene, the respective Kozak-sequence was added to the CPE cDNA. The 990 bp CPE gene was cloned into an eucaryotic expression vector. Prior to in vitro transfection experiments, different human colon carcinoma cell lines were screened by specific PCR for claudin 3 and 4 expression. This screen revealed, that the colon carcinoma line HCT116 expresses both, claudin 3 and 4. Therefore, this model was used for the in vitro transfection experiments. The transfection of the CPE-expressing vector into HCT116 cells resulted in the expression of CPE-specific mRNA and the 35 kDa CPE-protein, as shown by RT-PCR and by Western blotting. More importantly, transfection of the CPE-expressing vector caused the rapid cytotoxic effect in HCT116 cells. The CPE-expression resulted in cell death of about 50% of the cells as soon as 24 hours after transfection. By contrast, this cytotoxic effect was not observed in control cells that lack claudin 3 and 4 expression. These first in vitro results provide indication, that the CPE-mediated cell killing effect can be exploited for targeted specific gene therapy of tumors that overexpress claudin 3 and/or claudin 4.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA