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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 sensitizes cells to apoptosis after DNA damage by depleting S1P and increasing ceramide levels. (a) Total cellular S1P content in control (open bar) and SPLhi (solid bar) cells was quantitated by LC–MS and normalized to total phospholipids of whole cell extracts. Data are shown as mean±S.D. of three different experiments. *P<0.01. (b) Sphk1 (SK1) was overexpressed in SPLhi cells using a pBabe mammalian expression system. SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were left untreated or subjected to IR, and 36 h later apoptosis was assessed by measuring caspase-3 activity. Data are shown as mean±S.D. (n=3). (c) Whole cell lysates from SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were immunoblotted with SK1 or actin antibodies. (d) Control and SPLhi cells were grown on poly--lysine coated culture slides for 24 h. Immunolabeling was performed for ceramide in fixed and permeabilized cells. Images were acquired using a laser-scanning microscope (LSM 710). (e) Fluorescence intensity for ceramide was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)
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fig3: SPL sensitizes cells to apoptosis after DNA damage by depleting S1P and increasing ceramide levels. (a) Total cellular S1P content in control (open bar) and SPLhi (solid bar) cells was quantitated by LC–MS and normalized to total phospholipids of whole cell extracts. Data are shown as mean±S.D. of three different experiments. *P<0.01. (b) Sphk1 (SK1) was overexpressed in SPLhi cells using a pBabe mammalian expression system. SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were left untreated or subjected to IR, and 36 h later apoptosis was assessed by measuring caspase-3 activity. Data are shown as mean±S.D. (n=3). (c) Whole cell lysates from SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were immunoblotted with SK1 or actin antibodies. (d) Control and SPLhi cells were grown on poly--lysine coated culture slides for 24 h. Immunolabeling was performed for ceramide in fixed and permeabilized cells. Images were acquired using a laser-scanning microscope (LSM 710). (e) Fluorescence intensity for ceramide was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)

Mentions: SPL catalyzes the conversion of S1P into hexadecanal and ethanolamine phosphate in the final step of the sphingolipid degradation biochemical pathway. SPL could mediate its radiosensitizing effects by lowering intracellular S1P, by increasing the products of S1P breakdown, or through some indirect effect. As shown in Figure 3a, SPLhi cells contain low S1P levels. However, addition of S1P did not rescue the IR-induced cell death in SPLhi cells (data not shown). To avoid issues related to ineffective delivery of exogenous S1P to cells, we employed a genetic strategy to increase intracellular S1P through overexpression of sphingosine kinase 1 (SK1). As shown in Figures 3b and c, the IR-sensitivity of SPLhi cells was partially reversed by SK1 overexpression. These findings indicate that S1P depletion is partially but not completely responsible for the effect of SPL on radiation responses. Radiation-induced ceramide generation contributes to cellular apoptosis and acute radiation syndromes.7 Therefore, we also measured intracellular ceramide content at baseline and after IR of control and SPLhi cells via mass spectrometry (Table 1). As expected, both cell lines showed an increase in ceramide levels after IR. Surprisingly, however, SPLhi cells contained 50% greater ceramide content compared with control cells under baseline conditions as well as after IR. Immunofluorescent staining of permeabilized cells using a ceramide-specific antibody further confirmed the high ceramide levels in SPLhi cells compared with control cells (Figures 3d and e). These findings confirm that SPL expression leads to an increase in ceramide levels. Ceramide can be generated by de novo biosynthesis or hydrolysis of membrane sphingomyelin. To explore whether SPL radiosensitizes cells in a ceramide-dependent fashion, apoptosis was quantitated in irradiated control and SPLhi cells that had been treated before IR with either desipramine, which causes proteolysis of the ASMase protein and thereby blocks ceramide formation from SM, or inhibitors of de novo ceramide biosynthesis including myriocin (which inhibits serine palmitoyltransferase) or fumonisin B1 (which inhibits ceramide synthase). As shown in Figure 4a, only desipramine pretreatment blocked apoptotic responses to IR in SPLhi cells, indicating that SPL promotes IR-induced apoptosis through a process involving ASMase-dependent generation of ceramide from SM.


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 sensitizes cells to apoptosis after DNA damage by depleting S1P and increasing ceramide levels. (a) Total cellular S1P content in control (open bar) and SPLhi (solid bar) cells was quantitated by LC–MS and normalized to total phospholipids of whole cell extracts. Data are shown as mean±S.D. of three different experiments. *P<0.01. (b) Sphk1 (SK1) was overexpressed in SPLhi cells using a pBabe mammalian expression system. SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were left untreated or subjected to IR, and 36 h later apoptosis was assessed by measuring caspase-3 activity. Data are shown as mean±S.D. (n=3). (c) Whole cell lysates from SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were immunoblotted with SK1 or actin antibodies. (d) Control and SPLhi cells were grown on poly--lysine coated culture slides for 24 h. Immunolabeling was performed for ceramide in fixed and permeabilized cells. Images were acquired using a laser-scanning microscope (LSM 710). (e) Fluorescence intensity for ceramide was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)
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Related In: Results  -  Collection

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fig3: SPL sensitizes cells to apoptosis after DNA damage by depleting S1P and increasing ceramide levels. (a) Total cellular S1P content in control (open bar) and SPLhi (solid bar) cells was quantitated by LC–MS and normalized to total phospholipids of whole cell extracts. Data are shown as mean±S.D. of three different experiments. *P<0.01. (b) Sphk1 (SK1) was overexpressed in SPLhi cells using a pBabe mammalian expression system. SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were left untreated or subjected to IR, and 36 h later apoptosis was assessed by measuring caspase-3 activity. Data are shown as mean±S.D. (n=3). (c) Whole cell lysates from SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were immunoblotted with SK1 or actin antibodies. (d) Control and SPLhi cells were grown on poly--lysine coated culture slides for 24 h. Immunolabeling was performed for ceramide in fixed and permeabilized cells. Images were acquired using a laser-scanning microscope (LSM 710). (e) Fluorescence intensity for ceramide was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)
Mentions: SPL catalyzes the conversion of S1P into hexadecanal and ethanolamine phosphate in the final step of the sphingolipid degradation biochemical pathway. SPL could mediate its radiosensitizing effects by lowering intracellular S1P, by increasing the products of S1P breakdown, or through some indirect effect. As shown in Figure 3a, SPLhi cells contain low S1P levels. However, addition of S1P did not rescue the IR-induced cell death in SPLhi cells (data not shown). To avoid issues related to ineffective delivery of exogenous S1P to cells, we employed a genetic strategy to increase intracellular S1P through overexpression of sphingosine kinase 1 (SK1). As shown in Figures 3b and c, the IR-sensitivity of SPLhi cells was partially reversed by SK1 overexpression. These findings indicate that S1P depletion is partially but not completely responsible for the effect of SPL on radiation responses. Radiation-induced ceramide generation contributes to cellular apoptosis and acute radiation syndromes.7 Therefore, we also measured intracellular ceramide content at baseline and after IR of control and SPLhi cells via mass spectrometry (Table 1). As expected, both cell lines showed an increase in ceramide levels after IR. Surprisingly, however, SPLhi cells contained 50% greater ceramide content compared with control cells under baseline conditions as well as after IR. Immunofluorescent staining of permeabilized cells using a ceramide-specific antibody further confirmed the high ceramide levels in SPLhi cells compared with control cells (Figures 3d and e). These findings confirm that SPL expression leads to an increase in ceramide levels. Ceramide can be generated by de novo biosynthesis or hydrolysis of membrane sphingomyelin. To explore whether SPL radiosensitizes cells in a ceramide-dependent fashion, apoptosis was quantitated in irradiated control and SPLhi cells that had been treated before IR with either desipramine, which causes proteolysis of the ASMase protein and thereby blocks ceramide formation from SM, or inhibitors of de novo ceramide biosynthesis including myriocin (which inhibits serine palmitoyltransferase) or fumonisin B1 (which inhibits ceramide synthase). As shown in Figure 4a, only desipramine pretreatment blocked apoptotic responses to IR in SPLhi cells, indicating that SPL promotes IR-induced apoptosis through a process involving ASMase-dependent generation of ceramide from SM.

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