title: Ku80 attentuates cytotoxicity induced by green fluorescent protein transduction independently of non-homologous end joining.
abstract: The green fluorescent protein (GFP) is the most commonly used reporter protein for monitoring gene expression and protein localization in a variety of living and fixed cells, including not only prokaryotes, but also eukaryotes, e.g., yeasts, mammals, plants and fish. In general, it is thought that GFP is nontoxic to cells, although there are some reports on the side effect of GFP. Further, details of the molecular mechanism concerning the side effect of GFP remain unclear. Here we show that Ku80, but not XRCC4, plays an important role in the mechanism of the resistance to cytotoxicity induced by enhanced GFP (EGFP). EGFP inhibited both cell proliferation and colony formation, and induced cell death in Ku80-deficient hamster cells, i.e., xrs-6 cells. In addition, Ku80 attenuated EGFP-induced cytotoxicity in the xrs-6 cells. No EGFP-induced cytotoxicity was observed in the NHEJ core protein XRCC4-deficient hamster cells, i.e., XR-1 cells. Furthermore, EGFP markedly enhanced X-ray-induced cytotoxicity in the xrs-6 cells. These results suggest that Ku80 plays a key role in the novel NHEJ-independent defense mechanism against EGFP-induced cytotoxicity. Caution should be taken in considering of the potential influence by the stress response mechanism, namely, the Ku80-dependent elimination mechanism of EGFP-induced cytotoxicity, being activated, even when using EGFP-expressing cells in which Ku80 functions normally.
authors.affiliation: DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Koike
authors.forename: Yasutomo
authors.initials: Y
authors.lastname: Yutoku
authors.forename: Aki
authors.initials: A
authors.lastname: Koike
keywords: Cell death; Cytotoxicity; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DSBs, DNA double-strand breaks; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; GFP; GFP, green fluorescent protein; Ku; NHEJ, nonhomologous end-joining; XRCC4