Transduction of yeast cytosine deaminase mediated by HIV-1 Tat basic domain into tumor cells induces chemosensitivity to 5-fluorocytosine.
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.
Lee
Hakjoo
H
Division of Life Sciences, College of Natural Sciences and College of Medicine, Hallym University Chuncheon 200-702, Korea.
Ryu
Jiyoon
J
Kim
Kyung-Ae
KA
Lee
Kil Soo
KS
Lee
Jae Young
JY
Park
Jae-Bong
JB
Park
Jinseu
J
Choi
Soo Young
SY
D000818:Animals; D000963:Antimetabolites / Q000378:metabolism* / Q000494:pharmacology; D024201:Bystander Effect; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism*; D005437:Flucytosine / Q000378:metabolism* / Q000494:pharmacology; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D011355:Prodrugs / Q000378:metabolism / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D000963:Antimetabolites; D015696:Gene Products, tat; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D005437:Flucytosine; D043525:Cytosine Deaminase