Abstract
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.
Highlights
Enzyme/prodrug therapy is being developed as treatment for cancer and other pathological conditions
We constructed pET-yeast cytosine deaminase (yCD) expression vector which expresses the yCD fusion protein without the basic domain of HIV-1 Tat by inserting the same polymerase chain reaction (PCR) product digested with XhoI into XhoI-digested pET15-b (Invitrogen, Carlsbad, CA)
Purified fusion proteins denatured with 6 M of urea did not show any detectable levels of yCD activity. These results indicate that the expressed and refolded yCD and Tat-yCD fusion proteins in E. coli had been renatured into native structural conformation and were possessed of enzyme activity as native Cytosine deaminase (CD), respectively
Summary
Enzyme/prodrug therapy is being developed as treatment for cancer and other pathological conditions. CD has the ability to deaminate the nontoxic prodrug 5-fluorocytosine (5-FC) into the highly toxic compound 5fluorouracil (5-FU) which eventually inhibits DNA synthesis leading to cell death (Mullen et al, 1992) Both bacterial and yeast CDs have been exploited in CD/5-FC enzyme prodrug gene therapy. The enhanced conversion efficiency for 5-FC by yeast CD increased the bystander effect as well as the efficacy of radiotherapy (Kievit et al, 2000) These data suggested that the use of yCD increase the therapeutic potential of the CD/5-FC treatment strategy. For potential use of cell-permeable cytosine deaminase in the chemotherapy of cancer by a combination of CD enzyme and 5-FC, recombinant cytosine deaminase fusion protein, Tat-yCD, was obtained by expressing yeast CD in frame with HIV-1 Tat basic domain. The bystander effect was observed when nontransduced cells were cultured with supernatant from cells transduced with Tat-yCD in the presence of 5-FC
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