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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 is a radioresponsive protein that increases radiation-induced cell death. (a) HEK293T and NIH3T3 cells were left untreated or irradiated with 10 Gy of X-rays. Cells were harvested 9 h (HEK293T) or 24 h (NIH3T3) after radiation exposure and whole cell lysate was immunoblotted with actin and anti-human SPL, or anti-murine SPL antibodies, respectively. (b and c) Control and SPLhi cells were exposed with indicated dose of IR. After 24 h of incubation, cells were harvested, and apoptosis was assessed by measuring caspase-3 activity (b) and cell lysate was immunoblotted for PARP (Poly (ADP-ribose) polymerase). (c) Caspase-3 activity experiment represents mean±S.D. of three independent experiments. The immunoblot is representative of at least three independent experiments. (d) Control and SPLhi cells were left untreated or subjected to 5 Gy dose of X-rays and cells were harvested 12, 24 and 36 h after radiation exposure. Apoptosis was determined by flow cytometry using propidium iodide staining. Values are shown as mean±S.D. of three independent experiments. (e) Control, SPLhi, and SPLhi cells expressing a shRNA specific for Sgpl1 (SPLhi/shRNA) were exposed to 10 Gy IR. After 24 h of incubation, caspase-3 activity was measured in whole cell lysate. Sgpl1 knockdown was confirmed by western blotting (inset). Data represents mean±S.D. of three different experiments done in duplicates. (f) Endogenous knockdown of Sgpl1 induces radio-resistance. HEK293T control cells with lentiviral vector (control, open bar) or with shRNA specific for sgpl1 (SPLlo; solid bar) were exposed to 10 Gy X-rays and caspase-3 activity was measured 36 h after radiation exposure. Sgpl1 knockdown was confirmed by RT-PCR (inset). Data are shown as mean±S.D. (n=3, *P<0.01). Each experiment was repeated at least three times. Data points indicate average±S.D. of representative experiment
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fig1: SPL is a radioresponsive protein that increases radiation-induced cell death. (a) HEK293T and NIH3T3 cells were left untreated or irradiated with 10 Gy of X-rays. Cells were harvested 9 h (HEK293T) or 24 h (NIH3T3) after radiation exposure and whole cell lysate was immunoblotted with actin and anti-human SPL, or anti-murine SPL antibodies, respectively. (b and c) Control and SPLhi cells were exposed with indicated dose of IR. After 24 h of incubation, cells were harvested, and apoptosis was assessed by measuring caspase-3 activity (b) and cell lysate was immunoblotted for PARP (Poly (ADP-ribose) polymerase). (c) Caspase-3 activity experiment represents mean±S.D. of three independent experiments. The immunoblot is representative of at least three independent experiments. (d) Control and SPLhi cells were left untreated or subjected to 5 Gy dose of X-rays and cells were harvested 12, 24 and 36 h after radiation exposure. Apoptosis was determined by flow cytometry using propidium iodide staining. Values are shown as mean±S.D. of three independent experiments. (e) Control, SPLhi, and SPLhi cells expressing a shRNA specific for Sgpl1 (SPLhi/shRNA) were exposed to 10 Gy IR. After 24 h of incubation, caspase-3 activity was measured in whole cell lysate. Sgpl1 knockdown was confirmed by western blotting (inset). Data represents mean±S.D. of three different experiments done in duplicates. (f) Endogenous knockdown of Sgpl1 induces radio-resistance. HEK293T control cells with lentiviral vector (control, open bar) or with shRNA specific for sgpl1 (SPLlo; solid bar) were exposed to 10 Gy X-rays and caspase-3 activity was measured 36 h after radiation exposure. Sgpl1 knockdown was confirmed by RT-PCR (inset). Data are shown as mean±S.D. (n=3, *P<0.01). Each experiment was repeated at least three times. Data points indicate average±S.D. of representative experiment

Mentions: To determine whether IR influences SPL expression, we subjected human embryonic kidney (HEK293T) cells and NIH3T3 fibroblasts to 10 Gy IR and SPL protein levels were examined by immunoblotting. As shown in Figure 1a, SPL protein levels were elevated in response to IR in both cell lines. SPL induction in response to IR was even more pronounced at higher dose (Supplementary Figure 1). In contrast to SPL protein levels, SPL message levels were not consistently altered under these conditions (data not shown), which suggests that SPL protein accumulates in response to IR through a translational or posttranslational mechanism. To investigate the ramifications of SPL upregulation on irradiated cells, HEK293T cells exhibiting constitutively high SPL activity by virtue of stable overexpression of human SPL (SPLhi cells) and HEK293T vector control cells containing low endogenous SPL levels17 were irradiated, and apoptosis was evaluated. As shown in Figure 1b, SPLhi cells exhibited enhanced sensitivity to IR as shown by an increase in apoptosis measured by caspase-3 activity and by the presence of PARP cleavage products (Figure 1c) and sub-G1 peak (Figure 1d). To determine the role of SPL in apoptotic signaling pathways, cytochrome c release into the cytosol was examined by immunoblotting. In response to IR, SPLhi cells and control cells both released cytochrome c into the cytoplasm (Supplementary Figure 2). However, SPLhi cells showed greater cytochrome c release into the cytosol compared with control cells. Further, treatment with inhibitors of caspases, including those involved in extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways, attenuated radiation-induced apoptosis in SPLhi cells, as shown in Supplementary Figure 3. To confirm that the IR-sensitive phenotype of SPLhi cells is specifically attributed to SPL overexpression and not an acquired genetic modification associated with stable transformation, SPL overexpression was reversed by lentiviral expression of a shRNA molecule that specifically targeted human SPL. As shown in Figure 1e, SPL knockdown reversed the radiosensitive phenotype of SPLhi cells. Further, knockdown of endogenous SPL in HEK293T cells (SPLlo cells) conferred an IR-resistant phenotype, demonstrating a role for endogenous SPL in radiation-induced apoptosis (Figure 1f). Together, these findings reveal SPL to be a radiation-responsive protein that sensitizes cells to IR through an apoptotic pathway or pathways involving mitochondrial permeability transition and activation of caspases.


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 is a radioresponsive protein that increases radiation-induced cell death. (a) HEK293T and NIH3T3 cells were left untreated or irradiated with 10 Gy of X-rays. Cells were harvested 9 h (HEK293T) or 24 h (NIH3T3) after radiation exposure and whole cell lysate was immunoblotted with actin and anti-human SPL, or anti-murine SPL antibodies, respectively. (b and c) Control and SPLhi cells were exposed with indicated dose of IR. After 24 h of incubation, cells were harvested, and apoptosis was assessed by measuring caspase-3 activity (b) and cell lysate was immunoblotted for PARP (Poly (ADP-ribose) polymerase). (c) Caspase-3 activity experiment represents mean±S.D. of three independent experiments. The immunoblot is representative of at least three independent experiments. (d) Control and SPLhi cells were left untreated or subjected to 5 Gy dose of X-rays and cells were harvested 12, 24 and 36 h after radiation exposure. Apoptosis was determined by flow cytometry using propidium iodide staining. Values are shown as mean±S.D. of three independent experiments. (e) Control, SPLhi, and SPLhi cells expressing a shRNA specific for Sgpl1 (SPLhi/shRNA) were exposed to 10 Gy IR. After 24 h of incubation, caspase-3 activity was measured in whole cell lysate. Sgpl1 knockdown was confirmed by western blotting (inset). Data represents mean±S.D. of three different experiments done in duplicates. (f) Endogenous knockdown of Sgpl1 induces radio-resistance. HEK293T control cells with lentiviral vector (control, open bar) or with shRNA specific for sgpl1 (SPLlo; solid bar) were exposed to 10 Gy X-rays and caspase-3 activity was measured 36 h after radiation exposure. Sgpl1 knockdown was confirmed by RT-PCR (inset). Data are shown as mean±S.D. (n=3, *P<0.01). Each experiment was repeated at least three times. Data points indicate average±S.D. of representative experiment
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fig1: SPL is a radioresponsive protein that increases radiation-induced cell death. (a) HEK293T and NIH3T3 cells were left untreated or irradiated with 10 Gy of X-rays. Cells were harvested 9 h (HEK293T) or 24 h (NIH3T3) after radiation exposure and whole cell lysate was immunoblotted with actin and anti-human SPL, or anti-murine SPL antibodies, respectively. (b and c) Control and SPLhi cells were exposed with indicated dose of IR. After 24 h of incubation, cells were harvested, and apoptosis was assessed by measuring caspase-3 activity (b) and cell lysate was immunoblotted for PARP (Poly (ADP-ribose) polymerase). (c) Caspase-3 activity experiment represents mean±S.D. of three independent experiments. The immunoblot is representative of at least three independent experiments. (d) Control and SPLhi cells were left untreated or subjected to 5 Gy dose of X-rays and cells were harvested 12, 24 and 36 h after radiation exposure. Apoptosis was determined by flow cytometry using propidium iodide staining. Values are shown as mean±S.D. of three independent experiments. (e) Control, SPLhi, and SPLhi cells expressing a shRNA specific for Sgpl1 (SPLhi/shRNA) were exposed to 10 Gy IR. After 24 h of incubation, caspase-3 activity was measured in whole cell lysate. Sgpl1 knockdown was confirmed by western blotting (inset). Data represents mean±S.D. of three different experiments done in duplicates. (f) Endogenous knockdown of Sgpl1 induces radio-resistance. HEK293T control cells with lentiviral vector (control, open bar) or with shRNA specific for sgpl1 (SPLlo; solid bar) were exposed to 10 Gy X-rays and caspase-3 activity was measured 36 h after radiation exposure. Sgpl1 knockdown was confirmed by RT-PCR (inset). Data are shown as mean±S.D. (n=3, *P<0.01). Each experiment was repeated at least three times. Data points indicate average±S.D. of representative experiment
Mentions: To determine whether IR influences SPL expression, we subjected human embryonic kidney (HEK293T) cells and NIH3T3 fibroblasts to 10 Gy IR and SPL protein levels were examined by immunoblotting. As shown in Figure 1a, SPL protein levels were elevated in response to IR in both cell lines. SPL induction in response to IR was even more pronounced at higher dose (Supplementary Figure 1). In contrast to SPL protein levels, SPL message levels were not consistently altered under these conditions (data not shown), which suggests that SPL protein accumulates in response to IR through a translational or posttranslational mechanism. To investigate the ramifications of SPL upregulation on irradiated cells, HEK293T cells exhibiting constitutively high SPL activity by virtue of stable overexpression of human SPL (SPLhi cells) and HEK293T vector control cells containing low endogenous SPL levels17 were irradiated, and apoptosis was evaluated. As shown in Figure 1b, SPLhi cells exhibited enhanced sensitivity to IR as shown by an increase in apoptosis measured by caspase-3 activity and by the presence of PARP cleavage products (Figure 1c) and sub-G1 peak (Figure 1d). To determine the role of SPL in apoptotic signaling pathways, cytochrome c release into the cytosol was examined by immunoblotting. In response to IR, SPLhi cells and control cells both released cytochrome c into the cytoplasm (Supplementary Figure 2). However, SPLhi cells showed greater cytochrome c release into the cytosol compared with control cells. Further, treatment with inhibitors of caspases, including those involved in extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways, attenuated radiation-induced apoptosis in SPLhi cells, as shown in Supplementary Figure 3. To confirm that the IR-sensitive phenotype of SPLhi cells is specifically attributed to SPL overexpression and not an acquired genetic modification associated with stable transformation, SPL overexpression was reversed by lentiviral expression of a shRNA molecule that specifically targeted human SPL. As shown in Figure 1e, SPL knockdown reversed the radiosensitive phenotype of SPLhi cells. Further, knockdown of endogenous SPL in HEK293T cells (SPLlo cells) conferred an IR-resistant phenotype, demonstrating a role for endogenous SPL in radiation-induced apoptosis (Figure 1f). Together, these findings reveal SPL to be a radiation-responsive protein that sensitizes cells to IR through an apoptotic pathway or pathways involving mitochondrial permeability transition and activation of caspases.

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