A Cytoplasmic Form of Gaussia luciferase Provides a Highly Sensitive Test for Cytotoxicity

Abstract

The elimination of unfavorable chemicals from our environment and commercial products requires a sensitive and high-throughput in vitro assay system for drug-induced hepatotoxicity. Some previous methods for evaluating hepatotoxicity measure the amounts of cytoplasmic enzymes secreted from damaged cells into the peripheral blood or culture medium. However, most of these enzymes are proteolytically digested in the extracellular milieu, dramatically reducing the sensitivity and reliability of such assays. Other methods measure the decrease in cell viability following exposure to a compound, but such endpoint assays are often confounded by proliferation of surviving cells that replace dead or damaged cells. In this study, with the goal of preventing false-negative diagnoses, we developed a sensitive luminometric cytotoxicity test using a stable form of luciferase. Specifically, we converted Gaussia luciferase (G-Luc) from an actively secreted form to a cytoplasmic form by adding an ER-retention signal composed of the four amino acids KDEL. The bioluminescent signal was >30-fold higher in transgenic HepG2 human hepatoblastoma cells expressing G-Luc+KDEL than in cells expressing wild-type G-Luc. Moreover, G-Luc+KDEL secreted from damaged cells was stable in culture medium after 24 hr at 37°C. We evaluated the accuracy of our cytotoxicity test by subjecting identical samples obtained from chemically treated transgenic HepG2 cells to the G-Luc+KDEL assay and luminometric analyses based on secretion of endogenous adenylate kinase or cellular ATP level. Time-dependent accumulation of G-Luc+KDEL in the medium increased the sensitivity of our assay above those of existing tests. Our findings demonstrate that strong and stable luminescence of G-Luc+KDEL in human hepatocyte-like cells, which have high levels of metabolic activity, make it suitable for use in a high-throughput screening system for monitoring time-dependent cytotoxicity in a limited number of cells.

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

“…In this study, we used the following two types of luciferase genes to generate stable cell lines: (i) beetle luciferase from P. termitilluminans (ELuc), which emits light by oxidizing d ‐luciferin as its substrate and localizes in peroxisomes due to the naturally existing peroxisome localization signal (Ser‐Lys‐Leu) at its C‐terminus; (ii) secretion‐type copepod luciferase from G. princeps , which emits light by oxidizing coelenterazine as its substrate and localizes in ER due to a fused in‐frame ER retention signal (Lys‐Asp‐Glu‐Leu) at its C‐terminus (GLuc‐KDEL) . As reported previously, GLuc‐KDEL is released from damaged cells accompanied by cytotoxicity, and stably existing in the culture medium owing to the high stability of the protein …”

“…The site‐specific insertion of expression plasmids into the MI‐MAC vector and the expression of the luciferases in HepG2 cells were confirmed by genomic PCR and bioluminescence measurement ( λ max = 538 and 485 nm for ELuc and GLuc‐KDEL, respectively) (data not shown), respectively. The bioluminescence of GLuc‐KDEL in the cells, whose light intensity was superior to that in culture medium, was also confirmed (Figure b) as observed in a previous report …”

“…[18] As reported previously, GLuc-KDEL is released from damaged cells accompanied by cytotoxicity, and stably existing in the culture medium owing to the high stability of the protein. [19] To generate stable cell lines in which ELuc and GLuc-KDEL are expressed under the control of CAG promoter, we used human hepatoblastoma HepG2 cells harboring the MI-MAC vector. [13] First, an expression plasmid carrying ELuc was inserted by homologous recombination into the R4 attP site of the MI-MAC vector, in which the expression cassette was flanked by a tandem repeat of HS4 (chicken hypersensitive site 4) insulators (Figure 1a), and the transfected cells were selected with blasticidin S. Next, an expression plasmid carrying GLuc-KDEL was further inserted into the φC31 attP site of the MI-MAC vector and the cells were selected with G418.…”

“…Furthermore, it has been reported that beetle luciferases, including firefly luciferase from Photinus pyralis and click beetle luciferase from Pyrearinus termitilluminans (Emerald luciferase, ELuc), have been utilized for continuous long‐term cytotoxicity monitoring in three‐dimensional spheroids by repetitive non‐destructive bioluminescence measurements, by utilizing the high stability and permeability properties of their substrate, d ‐luciferin . On the other hand, endoplasmic reticulum (ER)‐targeted secretion‐type copepod luciferase from Gaussia princeps , in which the ER retention signal (Lys‐Asp‐Glu‐Leu; KDEL) is fused in‐frame to the extreme C‐terminus of GLuc (hereinafter referred to as GLuc‐KDEL), has been used for high‐sensitivity cytotoxicity monitoring by measuring the extracellular release of GLuc‐KDEL by damaged cells …”

“…[16,17] On the other hand, endoplasmic reticulum (ER)-targeted secretion-type copepod luciferase from Gaussia princeps, [18] in which the ER retention signal (Lys-Asp-Glu-Leu; KDEL) is fused in-frame to the extreme C-terminus of GLuc (hereinafter referred to as GLuc-KDEL), has been used for high-sensitivity cytotoxicity monitoring by measuring the extracellular release of GLuc-KDEL by damaged cells. [19] Extracellular release of high mobility group box 1 (HMGB1), a representative of damage-associated molecular pattern (DAMP) molecules, from damaged cells is considered the key molecule in the evaluation of cytotoxicity potential of toxicant. HMGB1 is a non-histone nuclear protein that acts as a transcriptional regulator in intact cells.…”