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Firefly luciferase and RLuc8 exhibit differential sensitivity to oxidative stress in apoptotic cells.

Czupryna J, Tsourkas A - PLoS ONE (2011)

Bottom Line: 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.Consistent with these findings, the direct application of H(2)O(2) to HeLa cells also led to a reduction in fLuc bioluminescence, while H(2)O(2) scavengers stabilized fLuc activity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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 (H(2)O(2)), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H(2)O(2) to HeLa cells also led to a reduction in fLuc bioluminescence, while H(2)O(2) 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.

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Dose response of HeLa-fR cells to H2O2-related scavengers/inhibitors.HeLa-fR cells were pretreated with a dosage range of (a) catalase, (b) allopurinol, or (c) aspirin for 1 hour prior to incubating with 10 µM STS (solid lines, +STS) or PBS (dotted lines, -STS) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were pretreated for 1 hour with PBS, 50 U/mL catalase, 100 µM allopurinol or 1 mM aspirin (columns 2–5) prior to the administration 10 µM STS for 24 hours. A western blot was subsequently performed with anti-fLuc and anti-RLuc antibodies. β-actin is shown as a loading control.
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pone-0020073-g004: Dose response of HeLa-fR cells to H2O2-related scavengers/inhibitors.HeLa-fR cells were pretreated with a dosage range of (a) catalase, (b) allopurinol, or (c) aspirin for 1 hour prior to incubating with 10 µM STS (solid lines, +STS) or PBS (dotted lines, -STS) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were pretreated for 1 hour with PBS, 50 U/mL catalase, 100 µM allopurinol or 1 mM aspirin (columns 2–5) prior to the administration 10 µM STS for 24 hours. A western blot was subsequently performed with anti-fLuc and anti-RLuc antibodies. β-actin is shown as a loading control.

Mentions: To validate the protective effect of H2O2 inhibitors/scavengers on fLuc activity, HeLa-fR cells were pretreated with increasing doses of catalase (Figure 4a), allopurinol (Figure 4b) or aspirin (Figure 4c) for 1 hour prior to the addition of 10 µM STS. After a 24 hr incubation period with the various H2O2 inhibitors/scavengers and STS, the bioluminescent ratio was measured. It was found that each inhibitor/scavenger effectively rescued fLuc activity, thus reducing the Rluc8:fLuc ratio, in a dose-dependent manner. A Western blot was also performed to directly determine the effect of the various H2O2 inhbitors/scavengers on bioluminescent protein levels. Consistent with the observed recovery in fLuc activity, STS-treated HeLa-fR cells that were pretreated with catalase, allopurinol, or aspirin exhibited higher levels of fLuc protein (Figure 4d, column 3–5) compared to cells treated with STS in the absence of inhibitor (i.e. PBS, column 2).


Firefly luciferase and RLuc8 exhibit differential sensitivity to oxidative stress in apoptotic cells.

Czupryna J, Tsourkas A - PLoS ONE (2011)

Dose response of HeLa-fR cells to H2O2-related scavengers/inhibitors.HeLa-fR cells were pretreated with a dosage range of (a) catalase, (b) allopurinol, or (c) aspirin for 1 hour prior to incubating with 10 µM STS (solid lines, +STS) or PBS (dotted lines, -STS) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were pretreated for 1 hour with PBS, 50 U/mL catalase, 100 µM allopurinol or 1 mM aspirin (columns 2–5) prior to the administration 10 µM STS for 24 hours. A western blot was subsequently performed with anti-fLuc and anti-RLuc antibodies. β-actin is shown as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094452&req=5

pone-0020073-g004: Dose response of HeLa-fR cells to H2O2-related scavengers/inhibitors.HeLa-fR cells were pretreated with a dosage range of (a) catalase, (b) allopurinol, or (c) aspirin for 1 hour prior to incubating with 10 µM STS (solid lines, +STS) or PBS (dotted lines, -STS) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were pretreated for 1 hour with PBS, 50 U/mL catalase, 100 µM allopurinol or 1 mM aspirin (columns 2–5) prior to the administration 10 µM STS for 24 hours. A western blot was subsequently performed with anti-fLuc and anti-RLuc antibodies. β-actin is shown as a loading control.
Mentions: To validate the protective effect of H2O2 inhibitors/scavengers on fLuc activity, HeLa-fR cells were pretreated with increasing doses of catalase (Figure 4a), allopurinol (Figure 4b) or aspirin (Figure 4c) for 1 hour prior to the addition of 10 µM STS. After a 24 hr incubation period with the various H2O2 inhibitors/scavengers and STS, the bioluminescent ratio was measured. It was found that each inhibitor/scavenger effectively rescued fLuc activity, thus reducing the Rluc8:fLuc ratio, in a dose-dependent manner. A Western blot was also performed to directly determine the effect of the various H2O2 inhbitors/scavengers on bioluminescent protein levels. Consistent with the observed recovery in fLuc activity, STS-treated HeLa-fR cells that were pretreated with catalase, allopurinol, or aspirin exhibited higher levels of fLuc protein (Figure 4d, column 3–5) compared to cells treated with STS in the absence of inhibitor (i.e. PBS, column 2).

Bottom Line: 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.Consistent with these findings, the direct application of H(2)O(2) to HeLa cells also led to a reduction in fLuc bioluminescence, while H(2)O(2) scavengers stabilized fLuc activity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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 (H(2)O(2)), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H(2)O(2) to HeLa cells also led to a reduction in fLuc bioluminescence, while H(2)O(2) 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.

Show MeSH
Related in: MedlinePlus