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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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Related in: MedlinePlus

Assessment of fLuc and RLuc8 protein levels in HeLa-fR cells pre-treated with proteasome inhibitors prior to the administration of STS.HeLa-fR cells were pretreated for 1 hour with PBS, 20 µM MG-132, 10 µM Epoxomicin or 10 µM Lactacystin prior to the administration of PBS (-) or 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-g007: Assessment of fLuc and RLuc8 protein levels in HeLa-fR cells pre-treated with proteasome inhibitors prior to the administration of STS.HeLa-fR cells were pretreated for 1 hour with PBS, 20 µM MG-132, 10 µM Epoxomicin or 10 µM Lactacystin prior to the administration of PBS (-) or 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: It is widely acknowledged that oxidatively-modified proteins can be targeted to the proteasome for degradation [25]. To investigate whether the proteasome was ultimately responsible for the degradation of fLuc following ROS-mediated inactivation in STS-treated cells, a western blot was performed on cells that were pretreated with the proteasome inhibitors MG-132, epoxomicin or lactacystin prior to the administration of STS. As shown in Figure 7, none of the proteasome inhibitors examined were capable of preventing fLuc degradation. These findings provide strong evidence that fLuc was not degraded by the proteasome secondary to inactivation. This leaves open the possibility that oxidatively-modified proteins are specifically degraded via other proteases; however, this seems unlikely considering the degradation of oxidatively-modified proteins is generally a well accepted physiological function of the proteasomal system, with proteolysis by the 20S proteasome being the major pathway.[17], [25], [26] Taking this into consideration, we suspect that ROS may be directly responsible for the degradation of fLuc. It has been well documented that peptide bond cleavage can occur as a result of ROS attack.[27], [28] The formation of protein aggregates and/or cross-linking of fLuc may also contribute to the apparent loss in the wild-type fLuc band on western blots of STS-treated cells,[25] but it is hypothesized that protein cleavage is the predominant mechanism considering that cross-linking was only observed on western blots after long exposures to STS (Figure 2).


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

Czupryna J, Tsourkas A - PLoS ONE (2011)

Assessment of fLuc and RLuc8 protein levels in HeLa-fR cells pre-treated with proteasome inhibitors prior to the administration of STS.HeLa-fR cells were pretreated for 1 hour with PBS, 20 µM MG-132, 10 µM Epoxomicin or 10 µM Lactacystin prior to the administration of PBS (-) or 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094452&req=5

pone-0020073-g007: Assessment of fLuc and RLuc8 protein levels in HeLa-fR cells pre-treated with proteasome inhibitors prior to the administration of STS.HeLa-fR cells were pretreated for 1 hour with PBS, 20 µM MG-132, 10 µM Epoxomicin or 10 µM Lactacystin prior to the administration of PBS (-) or 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: It is widely acknowledged that oxidatively-modified proteins can be targeted to the proteasome for degradation [25]. To investigate whether the proteasome was ultimately responsible for the degradation of fLuc following ROS-mediated inactivation in STS-treated cells, a western blot was performed on cells that were pretreated with the proteasome inhibitors MG-132, epoxomicin or lactacystin prior to the administration of STS. As shown in Figure 7, none of the proteasome inhibitors examined were capable of preventing fLuc degradation. These findings provide strong evidence that fLuc was not degraded by the proteasome secondary to inactivation. This leaves open the possibility that oxidatively-modified proteins are specifically degraded via other proteases; however, this seems unlikely considering the degradation of oxidatively-modified proteins is generally a well accepted physiological function of the proteasomal system, with proteolysis by the 20S proteasome being the major pathway.[17], [25], [26] Taking this into consideration, we suspect that ROS may be directly responsible for the degradation of fLuc. It has been well documented that peptide bond cleavage can occur as a result of ROS attack.[27], [28] The formation of protein aggregates and/or cross-linking of fLuc may also contribute to the apparent loss in the wild-type fLuc band on western blots of STS-treated cells,[25] but it is hypothesized that protein cleavage is the predominant mechanism considering that cross-linking was only observed on western blots after long exposures to STS (Figure 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