HIV-1 TAT protein transduction domain mediates enhancement of enzyme prodrug cancer gene therapy in vitro: a study with TAT-TK-GFP triple fusion construct.
Protein transduction domain (PTD) from HIV-1 TAT protein has been reported to translocate across the mammalian cell membrane, also as a part of fusion proteins. However, the true nature of TAT-mediated intercellular spreading is still under debate because it has been claimed to be a fixation artifact. To study the spreading of TAT fusion proteins and their potency to enhance thymidine kinase/ ganciclovir (HSV-TK/GCV) cancer gene therapy, we constructed a novel triple fusion protein containing TAT PTD, HSV-TK and green fluorescent protein (TAT-TK-GFP). This fusion protein has three functional domains in the same polypeptide, allowing reliable determination of the relationship between transduction rate and cell killing efficiency. TAT-TK-GFP was cloned into a lentivirus vector and used for analyses of TAT-mediated protein translocation and enhancement of HSV-TK/GCV cytotoxicity. The triple fusion protein was expressed correctly in vitro, but cell-to-cell translocation was not observed in rat glioma cells (BT4C). However, TAT-TK-GFP made BT4C and SKOV3.ip1 (human ovarian carcinoma) cells significantly more sensitive to ganciclovir than TK-GFP, whereas the effect in PC-3 human prostate carcinoma cells was more subtle. It was also observed that growth in lower serum concentration (2.5-5%) abolished the enhancement in BT4C cells, suggesting that high proliferation rate is one of the factors that contribute to TAT PTD-mediated enhancement of cytotoxicity. In summary, our results indicate that TAT PTD fusion proteins do not translocate intercellularly at detectable levels, but enhancement of the HSV-TK/GCV cytotoxicity can be detected in rat and human tumor cell lines in vitro.
Meriläinen
Outi
O
A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine, University of Kuopio, Finland.
Hakkarainen
Tanja
T
Wahlfors
Tiina
T
Pellinen
Riikka
R
Wahlfors
Jarmo
J
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015774:Ganciclovir / Q000494:pharmacology; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005910:Glioma / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016086:Lentivirus / Q000235:genetics; D008856:Microscopy, Fluorescence; D011355:Prodrugs / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics
D000998:Antiviral Agents; D015696:Gene Products, tat; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir