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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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Response of HeLa-fR cells to a hypoxanthine (HX)-xanthine oxidase (XO) reaction and the affect of adenovirus-catalase on STS-treated cells.(a) HeLa-fR cells were subjected to the HX-XO reaction (50 µM HX, 25 mU/mL XO) or PBS (Control) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (b) A TUNEL assay was used to determine the percent DNA fragmentation, following exposure to HX-XO. (c) HeLa-fR cells were treated with adenovirus-catalase (Adv-Cat) or empty adenovirus (Adv-E) for 24 hours, washed, and incubated in serum-containing media for another 24 hours. Then, 10 µM STS or PBS was added to the cells. After 24 hours the RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were subjected to the same conditions as in (c) but assayed for H2O2 levels using CM-H2DCFDA.
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pone-0020073-g006: Response of HeLa-fR cells to a hypoxanthine (HX)-xanthine oxidase (XO) reaction and the affect of adenovirus-catalase on STS-treated cells.(a) HeLa-fR cells were subjected to the HX-XO reaction (50 µM HX, 25 mU/mL XO) or PBS (Control) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (b) A TUNEL assay was used to determine the percent DNA fragmentation, following exposure to HX-XO. (c) HeLa-fR cells were treated with adenovirus-catalase (Adv-Cat) or empty adenovirus (Adv-E) for 24 hours, washed, and incubated in serum-containing media for another 24 hours. Then, 10 µM STS or PBS was added to the cells. After 24 hours the RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were subjected to the same conditions as in (c) but assayed for H2O2 levels using CM-H2DCFDA.

Mentions: Having shown that various inhibitors/scavengers of H2O2 exhibited a stabilizing affect on fLuc activity in apoptotic cells, additional studies were performed that dealt with H2O2 more directly. First, we investigated the response of HeLa-fR cells to the hypoxanthine (HX)-XO reaction, which allowed for the continual extracellular production of H2O2. After 24 hours, it was found that the bioluminescent ratio increased dramatically, reaching levels that were significantly higher than what was observed previously with STS treatment (Figure 6a). Additionally, it was found that the HX-XO reaction did not cause cell death, as indicated by a TUNEL assay (Figure 6b). These results provide additional evidence that the observed loss in fLuc activity is specifically associated with elevated levels of ROS as opposed to other mechanisms that are a consequence of cell death.


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

Czupryna J, Tsourkas A - PLoS ONE (2011)

Response of HeLa-fR cells to a hypoxanthine (HX)-xanthine oxidase (XO) reaction and the affect of adenovirus-catalase on STS-treated cells.(a) HeLa-fR cells were subjected to the HX-XO reaction (50 µM HX, 25 mU/mL XO) or PBS (Control) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (b) A TUNEL assay was used to determine the percent DNA fragmentation, following exposure to HX-XO. (c) HeLa-fR cells were treated with adenovirus-catalase (Adv-Cat) or empty adenovirus (Adv-E) for 24 hours, washed, and incubated in serum-containing media for another 24 hours. Then, 10 µM STS or PBS was added to the cells. After 24 hours the RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were subjected to the same conditions as in (c) but assayed for H2O2 levels using CM-H2DCFDA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094452&req=5

pone-0020073-g006: Response of HeLa-fR cells to a hypoxanthine (HX)-xanthine oxidase (XO) reaction and the affect of adenovirus-catalase on STS-treated cells.(a) HeLa-fR cells were subjected to the HX-XO reaction (50 µM HX, 25 mU/mL XO) or PBS (Control) for 24 hours. The RLuc8:fLuc ratio was calculated and reported. (b) A TUNEL assay was used to determine the percent DNA fragmentation, following exposure to HX-XO. (c) HeLa-fR cells were treated with adenovirus-catalase (Adv-Cat) or empty adenovirus (Adv-E) for 24 hours, washed, and incubated in serum-containing media for another 24 hours. Then, 10 µM STS or PBS was added to the cells. After 24 hours the RLuc8:fLuc ratio was calculated and reported. (d) HeLa-fR cells were subjected to the same conditions as in (c) but assayed for H2O2 levels using CM-H2DCFDA.
Mentions: Having shown that various inhibitors/scavengers of H2O2 exhibited a stabilizing affect on fLuc activity in apoptotic cells, additional studies were performed that dealt with H2O2 more directly. First, we investigated the response of HeLa-fR cells to the hypoxanthine (HX)-XO reaction, which allowed for the continual extracellular production of H2O2. After 24 hours, it was found that the bioluminescent ratio increased dramatically, reaching levels that were significantly higher than what was observed previously with STS treatment (Figure 6a). Additionally, it was found that the HX-XO reaction did not cause cell death, as indicated by a TUNEL assay (Figure 6b). These results provide additional evidence that the observed loss in fLuc activity is specifically associated with elevated levels of ROS as opposed to other mechanisms that are a consequence of cell death.

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