Enzyme/prodrug approach is one of the actively developing areas for cancer therapy. In an effort to develop more effective enzyme/prodrug systems, cell-permeable cytosine deaminase was produced by fusing yeast cytosine deaminase (yCD) in frame with RKKRRQRRR domain of HIV-1 Tat which is an efficient delivery peptide of the foreign proteins into cells. The purified Tat-yCD fusion protein expressed in Escherichia coli was readily transduced into mammalian cells in a time- and dose-dependent manner. A significant level of the transduced Tat-yCD protein was recovered in the cell and was stable for 24 h as indicated by both results of the enzymatic assay of 5-fluorocytosine (5-FC) conversion to 5-fluorouracil (5-FU) and Western blot analysis. The cells transduced with Tat-yCD become highly sensitive to the cytotoxicity of 5-FC, while cells treated with yCD are unaffected by 5-FC. In addition, a strong bystander effect was observed with conditioned media from cells transduced with Tat-yCD added to non-transduced cells. Tat-yCD fusion protein demonstrated here for its ability to transduce into cells and convert nontoxic prodrug 5-FC to the toxic antimetabolite 5-FU, may be a useful approach for cancer therapy.
Animals ; Antimetabolites/*metabolism/pharmacology ; Bystander Effect ; Cytosine Deaminase/genetics/*metabolism ; Flucytosine/*metabolism/pharmacology ; Gene Products, tat/chemistry/genetics/*metabolism ; Genetic Vectors/genetics/metabolism ; HIV-1/metabolism ; Hela Cells/drug effects/physiology ; Humans ; Prodrugs/metabolism/therapeutic use ; Recombinant Fusion Proteins/genetics/*metabolism ; Research Support, Non-U.S. Gov't ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; *Transduction, Genetic