Firefly Luciferase and Rluc8 Exhibit Differential Sensitivity to Oxidative Stress in Apoptotic Cells

Abstract

Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H2O2), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H2O2 to HeLa cells also led to a reduction in fLuc bioluminescence, while H2O2 scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings.

Noninvasive Bioluminescence Imaging of α-Synuclein Oligomerization in Mouse Brain Using Split Firefly Luciferase Reporters

“…2C,D), underscoring that the Fluctags do not significantly affect the aggregation behavior of ␣SYN under oxidative stress conditions. Of note, ␣SYN oligomerization could not be monitored with the bioluminescent PCA under oxidative stress conditions because Fluc activity is sensitive to reactive oxygen species (Czupryna and Tsourkas, 2011; and data not shown).…”

Xanthine oxidase‐generated hydrogen peroxide is a consequence, not a mediator of cell death

“…S1). It has previously been shown that changes in the ratio RLuc8 : fLuc correlate qualitatively with the level of H 2 O 2 in cells [21].…”

“…ROS levels were recorded using the ROS sensor CM‐H 2 DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase (fLuc) to H 2 O 2 , compared with a stable variant of Renilla luciferase (RLuc), RLuc8 [21]. Specifically, the bioluminescent ratio, RLuc8 : fLuc, was used to provide insight into intracellular H 2 O 2 levels.…”

In Vivo Cell Tracking with Bioluminescence Imaging

“…In addition, differential sensitivity of FLuc to oxidative stress in apoptotic cells has also been reported. Czupryna et al [29] suggested that reactive oxygen species (ROS) were responsible for the reduction of FLuc activity and could be substantially altered in studies where pH levels and ROS levels become elevated. Also, Kitamaya et al [30] reported on the thermal instability and pH-sensitive spectral property of firefly luciferase which can affect its use as a sensitive multicolor luminescence label or bioluminescence resonance energy transfer donor.…”