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S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism.

Kumar A, Oskouian B, Fyrst H, Zhang M, Paris F, Saba JD - Cell Death Dis (2011)

Bottom Line: S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism.SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death.Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609-1673, USA.

ABSTRACT
The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

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SPL delays the kinetics of DNA repair. (a) Control and SPLhi cells were left untreated or irradiated and harvested 1 or 30 min after radiation exposure. Extent of DNA damage was analyzed by comet assay using alkaline electrophoresis. In each sample, 50 cells were scored. (b) Olive tail moment for control (open bar) and SPLhi (solid bar) cells was analyzed using Komet 6.0 software. (c) Control and SPLlo cells were left untreated or irradiated and harvested 30 min after radiation exposure and comet assay was done using neutral electrophoresis. (d) Olive tail moment for control (open bar) and SPLlo (solid bar) was analyzed. Photomicrographs and histograms are representative of three independent experiments. (b and d) data represents mean ±S.D. of three independent experiments (*P<0.01)
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fig6: SPL delays the kinetics of DNA repair. (a) Control and SPLhi cells were left untreated or irradiated and harvested 1 or 30 min after radiation exposure. Extent of DNA damage was analyzed by comet assay using alkaline electrophoresis. In each sample, 50 cells were scored. (b) Olive tail moment for control (open bar) and SPLhi (solid bar) cells was analyzed using Komet 6.0 software. (c) Control and SPLlo cells were left untreated or irradiated and harvested 30 min after radiation exposure and comet assay was done using neutral electrophoresis. (d) Olive tail moment for control (open bar) and SPLlo (solid bar) was analyzed. Photomicrographs and histograms are representative of three independent experiments. (b and d) data represents mean ±S.D. of three independent experiments (*P<0.01)

Mentions: The normal cellular response to DNA damage involves both cell cycle arrest and activation of DNA repair mechanisms. To explore the effects of SPL on DNA repair, we investigated the extent of DNA damage and kinetics of DNA repair after IR in control cells and cells containing high and low levels of SPL using Comet assay. In Figure 6a, Comet assays show that control and SPLhi cells contain mostly intact DNA under baseline conditions. One minute after IR, control and SPLhi cells demonstrate evidence of damaged DNA, illustrated by the comet tail. By 30 min, most of the DNA damage has been repaired in control cells, as shown by the lack of comet tails. In contrast, SPLhi cells continue to exhibit evidence of DNA damage 30 min after IR. These results are depicted quantitatively using the olive tail moment measurement, as shown in Figure 6b. Conversely, SPLlo cells exhibited a greater degree of DNA repair compared with control cells, as shown in Figure 6c and quantitatively in Figure 6d. These findings indicate that endogenous SPL has a negative influence on the kinetics and/or efficiency of DNA repair, whereas reducing SPL expression hastens DNA repair after IR.


S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism.

Kumar A, Oskouian B, Fyrst H, Zhang M, Paris F, Saba JD - Cell Death Dis (2011)

SPL delays the kinetics of DNA repair. (a) Control and SPLhi cells were left untreated or irradiated and harvested 1 or 30 min after radiation exposure. Extent of DNA damage was analyzed by comet assay using alkaline electrophoresis. In each sample, 50 cells were scored. (b) Olive tail moment for control (open bar) and SPLhi (solid bar) cells was analyzed using Komet 6.0 software. (c) Control and SPLlo cells were left untreated or irradiated and harvested 30 min after radiation exposure and comet assay was done using neutral electrophoresis. (d) Olive tail moment for control (open bar) and SPLlo (solid bar) was analyzed. Photomicrographs and histograms are representative of three independent experiments. (b and d) data represents mean ±S.D. of three independent experiments (*P<0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: SPL delays the kinetics of DNA repair. (a) Control and SPLhi cells were left untreated or irradiated and harvested 1 or 30 min after radiation exposure. Extent of DNA damage was analyzed by comet assay using alkaline electrophoresis. In each sample, 50 cells were scored. (b) Olive tail moment for control (open bar) and SPLhi (solid bar) cells was analyzed using Komet 6.0 software. (c) Control and SPLlo cells were left untreated or irradiated and harvested 30 min after radiation exposure and comet assay was done using neutral electrophoresis. (d) Olive tail moment for control (open bar) and SPLlo (solid bar) was analyzed. Photomicrographs and histograms are representative of three independent experiments. (b and d) data represents mean ±S.D. of three independent experiments (*P<0.01)
Mentions: The normal cellular response to DNA damage involves both cell cycle arrest and activation of DNA repair mechanisms. To explore the effects of SPL on DNA repair, we investigated the extent of DNA damage and kinetics of DNA repair after IR in control cells and cells containing high and low levels of SPL using Comet assay. In Figure 6a, Comet assays show that control and SPLhi cells contain mostly intact DNA under baseline conditions. One minute after IR, control and SPLhi cells demonstrate evidence of damaged DNA, illustrated by the comet tail. By 30 min, most of the DNA damage has been repaired in control cells, as shown by the lack of comet tails. In contrast, SPLhi cells continue to exhibit evidence of DNA damage 30 min after IR. These results are depicted quantitatively using the olive tail moment measurement, as shown in Figure 6b. Conversely, SPLlo cells exhibited a greater degree of DNA repair compared with control cells, as shown in Figure 6c and quantitatively in Figure 6d. These findings indicate that endogenous SPL has a negative influence on the kinetics and/or efficiency of DNA repair, whereas reducing SPL expression hastens DNA repair after IR.

Bottom Line: S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism.SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death.Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609-1673, USA.

ABSTRACT
The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

Show MeSH
Related in: MedlinePlus