Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

Abstract

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

The BlueScreen-384 Assay as an Indicator of Genotoxic Hazard Potential in Early-Stage Drug Discovery

“…This apparatus is in routine use for apoaequorin-based measurement of intracellular calcium concentration, typically following activation of heterologously expressed G-protein-coupled receptors or ion channels. 17 For the BlueScreen-384 assay, conditions were identical to those described for the BlueScreen HC 96-well protocol, 12 except volumes were scaled to 50 µL/well. Concentrationdependent induction of GADD45a-luciferase of three-to fourfold over basal was detected in TK6 cells treated with known genotoxins NQO, acridine orange (ACO), and MTX, after integrated addition of the flash luminescence substrate (Fig.…”

“…6,9 Optimal cell density in the BlueScreen-384 protocol was 10 6 cells/mL (data not shown), the same as described for the BlueScreen HC 96-well protocol. 12 BlueScreen HC involves cell lysis after the flash luminescence read and addition of thiazole orange to stain DNA and permit a fluorescence measurement to estimate cell number. 12 For the 384-well protocol, we also included cell lysis and thiazole orange treatment steps and compared data gathered on fluorescence plate-reading devices.…”

“…12 BlueScreen HC involves cell lysis after the flash luminescence read and addition of thiazole orange to stain DNA and permit a fluorescence measurement to estimate cell number. 12 For the 384-well protocol, we also included cell lysis and thiazole orange treatment steps and compared data gathered on fluorescence plate-reading devices. The Envision (Perkin Elmer) apparatus was considered optimal, based on minimizing the coefficient of variation across plates containing 196 wells of vehicletreated cells and 196 wells of 20 µM NQO-treated cells (data not shown).…”

“…We tested whether induction of the GADD45a-Gaussia luciferase (GADD45a-GLuc) reporter in TK6 cells 12 was detected using a 384-well compatible luminometer. Lumilux (Perkin Elmer, Cambridge, UK) permits concurrent detection of flash luminescence from all wells of a 384-well microplate with integrated liquid handling.…”

“…12 The bright flash luminescence of the GLuc reaction, and use of a nucleic acid stain (thiazole orange) rather than turbidity to determine cell number, make BlueScreen HC amenable to miniaturization. Here, we describe a 384-well version of the BlueScreen HC assay (BlueScreen-384), which is adopted to detect the genotoxic hazard potential during the hit-tolead and lead-to-candidate phases of drug discovery.…”