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BifidobactErial Selective Targeting - Cytosine Deaminase (BEST-CD) therapy is an ingenious strategy for cancer therapy. In our previous studies, a strain of Bifidobacterium longum (B.longum), which is nonpathogenic, anaerobic and domestic, selectively localized and proliferated in solid tumors after the systemic administration, and B.longum transferred with cytosine deaminase (CD) gene efficiently expressed active CD, which converted pro-drug 5-fluorocytosine (5FC) to fully active drug 5-fluorouracil (5FU). We also reported the antitumor efficacy with the plasmid in several animal experiments. This strategy, called BEST-CD has been noted as a tumor targeting therapy. In this study, we developed a novel shuttle-plasmid pAV001-HU-eCD to enhance the CD activities, based on the sequence of Bifidobacterium wild type plasmid pTB6, which was recently analyzed. The shuttle-plasmid pAV001-HU-eCD was designed to replicate in both Bifidobacterium and Escherichia coli efficiently, and the average copy number of the plasmids in B. longum was 2 to 5 folds higher than previously reported shuttle-plasmid pBLES100-S-eCD. When compared with pBLES100-S-eCD, the amount of CD expressed by B. longum with pAV001-HU-eCD was 2 to 8 folds higher than that by B. longum with pBLES100-S-eCD, thus the activity of CD produced by B. longum with pAV001-HU-eCD being significantly higher than that of pBLES100-S-eCD. Furthermore, in human breast carcinoma MCF7 apoptosis was strongly induced by the converted 5FU, and B. longum with pAV001-HU-eCD was apparently more forceful in inducing apoptotic effects than that of pBLES100-S-eCD. All these results indicated that a novel shuttle plasmid pAV001-HU-eCD was able to highly express exogenous CD in B. longum and could be more useful for our BEST-CD strategy.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]