Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of allopurinol pretreament on STS-treated HeLa cells.HeLa-fR cells were pretreated with either 100 µM allopurinol or PBS for 1 hour prior to the administration of 10 µM STS or PBS. After 24 hours, (a) the RLuc8:fLuc ratio, (b) intracellular H2O2 levels as measured by CM-H2DCFDA, and (c) DNA fragmentation levels, as measured using a TUNEL assay, were recorded for each sample. Statistical significance: *(p<0.05), **(p<0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3094452&req=5

pone-0020073-g005: Effect of allopurinol pretreament on STS-treated HeLa cells.HeLa-fR cells were pretreated with either 100 µM allopurinol or PBS for 1 hour prior to the administration of 10 µM STS or PBS. After 24 hours, (a) the RLuc8:fLuc ratio, (b) intracellular H2O2 levels as measured by CM-H2DCFDA, and (c) DNA fragmentation levels, as measured using a TUNEL assay, were recorded for each sample. Statistical significance: *(p<0.05), **(p<0.01).

Mentions: Although pretreating HeLa-fR cells with allopurinol, prior to the addition of STS, helped decrease the Rluc8:fLuc ratio by stabilizing fLuc activity (Figure 5a) and reducing intracellular H2O2 levels as determined by CM-H2DCFDA (Figure 5b), TUNEL assays revealed that allopurinol did not provide protection against cell death (Figure 5c). These findings demonstrate that the processes associated with fLuc inactivation are separate and distinct from cell death, further supporting the notion that the various proteases that are activated/upregulated in apoptotic cells are not responsible for fLuc inactivation. Moreover, considering that allopurinol specifically inhibits xanthine oxidase, a significant source of hydrogen peroxide (and superoxide and hydroxyl radicals to a lesser extent), these findings provide strong evidence that ROS are primarily responsible for fLuc inactivation in apoptotic cells. It is also interesting to note that since significant reductions in the intracellular level of H2O2 did not prevent cell death, these results also indicate that H2O2 is not essential for the progression of programmed cell death in STS-treated HeLa cells, but rather is a downstream byproduct.


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

Czupryna J, Tsourkas A - PLoS ONE (2011)

Effect of allopurinol pretreament on STS-treated HeLa cells.HeLa-fR cells were pretreated with either 100 µM allopurinol or PBS for 1 hour prior to the administration of 10 µM STS or PBS. After 24 hours, (a) the RLuc8:fLuc ratio, (b) intracellular H2O2 levels as measured by CM-H2DCFDA, and (c) DNA fragmentation levels, as measured using a TUNEL assay, were recorded for each sample. Statistical significance: *(p<0.05), **(p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020073-g005: Effect of allopurinol pretreament on STS-treated HeLa cells.HeLa-fR cells were pretreated with either 100 µM allopurinol or PBS for 1 hour prior to the administration of 10 µM STS or PBS. After 24 hours, (a) the RLuc8:fLuc ratio, (b) intracellular H2O2 levels as measured by CM-H2DCFDA, and (c) DNA fragmentation levels, as measured using a TUNEL assay, were recorded for each sample. Statistical significance: *(p<0.05), **(p<0.01).
Mentions: Although pretreating HeLa-fR cells with allopurinol, prior to the addition of STS, helped decrease the Rluc8:fLuc ratio by stabilizing fLuc activity (Figure 5a) and reducing intracellular H2O2 levels as determined by CM-H2DCFDA (Figure 5b), TUNEL assays revealed that allopurinol did not provide protection against cell death (Figure 5c). These findings demonstrate that the processes associated with fLuc inactivation are separate and distinct from cell death, further supporting the notion that the various proteases that are activated/upregulated in apoptotic cells are not responsible for fLuc inactivation. Moreover, considering that allopurinol specifically inhibits xanthine oxidase, a significant source of hydrogen peroxide (and superoxide and hydroxyl radicals to a lesser extent), these findings provide strong evidence that ROS are primarily responsible for fLuc inactivation in apoptotic cells. It is also interesting to note that since significant reductions in the intracellular level of H2O2 did not prevent cell death, these results also indicate that H2O2 is not essential for the progression of programmed cell death in STS-treated HeLa cells, but rather is a downstream byproduct.

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