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The role of protein kinase C alpha translocation in radiation-induced bystander effect.

Fang Z, Xu A, Wu L, Hei TK, Hong M - Sci Rep (2016)

Bottom Line: However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear.Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells.Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China.

ABSTRACT
Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

No MeSH data available.


Related in: MedlinePlus

PKC translocation is involved in radiation-induced bystander effect.(a,b) PKCα expression in different fractions of bystander AL cells. Cells were irradiated with a 50-cGy dose and cells on the aluminum-wrapped half of the dish were collected at time points indicated. Membrane fraction proteins were extracted and separated with SDS-PAGE, transferred to PVDF membrane and probed with PKCα antibody (a). Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). In (b), cells were fixed with 4% paraformadehyde, probed with PKCα antibody followed by detection with Alexa Fluor 488 secondary antibody. (c) Effect of PKC inhibitor chelerythrine chloride on the mutagencity of bystander cells. Exponentially growing AL cells were irradiated as described above. Chelerythrine chloride (10 μM) was added into the cultures 1 hr before irradiation. Data are pooled from four independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between the treated and untreated control (p = 0.03). (d.e) Effect of chelerythrine chloride on ERK activity and COX-2 expression. Proteins from bystander cells were extracted with RIPA buffer at the indicated time points after irradiation and probed with phosphor-ERK1/2 or COX-2 antibody. Left panels, untreated controls; right panels, cells were treated with chelerythrine chloride before irradiation. Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05).
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f1: PKC translocation is involved in radiation-induced bystander effect.(a,b) PKCα expression in different fractions of bystander AL cells. Cells were irradiated with a 50-cGy dose and cells on the aluminum-wrapped half of the dish were collected at time points indicated. Membrane fraction proteins were extracted and separated with SDS-PAGE, transferred to PVDF membrane and probed with PKCα antibody (a). Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). In (b), cells were fixed with 4% paraformadehyde, probed with PKCα antibody followed by detection with Alexa Fluor 488 secondary antibody. (c) Effect of PKC inhibitor chelerythrine chloride on the mutagencity of bystander cells. Exponentially growing AL cells were irradiated as described above. Chelerythrine chloride (10 μM) was added into the cultures 1 hr before irradiation. Data are pooled from four independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between the treated and untreated control (p = 0.03). (d.e) Effect of chelerythrine chloride on ERK activity and COX-2 expression. Proteins from bystander cells were extracted with RIPA buffer at the indicated time points after irradiation and probed with phosphor-ERK1/2 or COX-2 antibody. Left panels, untreated controls; right panels, cells were treated with chelerythrine chloride before irradiation. Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05).

Mentions: PKC translocation has been implicated in the responses of multiple stimuli including ionizing radiation. It was shown that gamma-radiation could induce apoptosis, growth arrest through activation and translocation of PKCα, PKCε and PKCδ1623. To see if PKCα translocates in bystander responses, we investigated PKCα level in different cellular fraction of bystander AL cells. As shown in Fig. 1a, PKCα level in cytosol decreased while its level in the membrane fraction increased significantly in bystander cells, suggesting the responsiveness of PKCα in bystander effect. PKCα level changed as early as 15 min after irradiation and sustained as long as 2 hrs. Immunocytochemistry analysis also showed that PKCα translocates from cytosol to cell membrane in bystander cells. PKCα distributed homogenously before irradiation within AL cells, while its signal intensified along the cell membrane in bystander cells after irradiation (Fig. 1b).


The role of protein kinase C alpha translocation in radiation-induced bystander effect.

Fang Z, Xu A, Wu L, Hei TK, Hong M - Sci Rep (2016)

PKC translocation is involved in radiation-induced bystander effect.(a,b) PKCα expression in different fractions of bystander AL cells. Cells were irradiated with a 50-cGy dose and cells on the aluminum-wrapped half of the dish were collected at time points indicated. Membrane fraction proteins were extracted and separated with SDS-PAGE, transferred to PVDF membrane and probed with PKCα antibody (a). Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). In (b), cells were fixed with 4% paraformadehyde, probed with PKCα antibody followed by detection with Alexa Fluor 488 secondary antibody. (c) Effect of PKC inhibitor chelerythrine chloride on the mutagencity of bystander cells. Exponentially growing AL cells were irradiated as described above. Chelerythrine chloride (10 μM) was added into the cultures 1 hr before irradiation. Data are pooled from four independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between the treated and untreated control (p = 0.03). (d.e) Effect of chelerythrine chloride on ERK activity and COX-2 expression. Proteins from bystander cells were extracted with RIPA buffer at the indicated time points after irradiation and probed with phosphor-ERK1/2 or COX-2 antibody. Left panels, untreated controls; right panels, cells were treated with chelerythrine chloride before irradiation. Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05).
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Related In: Results  -  Collection

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f1: PKC translocation is involved in radiation-induced bystander effect.(a,b) PKCα expression in different fractions of bystander AL cells. Cells were irradiated with a 50-cGy dose and cells on the aluminum-wrapped half of the dish were collected at time points indicated. Membrane fraction proteins were extracted and separated with SDS-PAGE, transferred to PVDF membrane and probed with PKCα antibody (a). Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05). In (b), cells were fixed with 4% paraformadehyde, probed with PKCα antibody followed by detection with Alexa Fluor 488 secondary antibody. (c) Effect of PKC inhibitor chelerythrine chloride on the mutagencity of bystander cells. Exponentially growing AL cells were irradiated as described above. Chelerythrine chloride (10 μM) was added into the cultures 1 hr before irradiation. Data are pooled from four independent experiments. Bars indicate ± S.D. of means. Asterisk indicates significant difference between the treated and untreated control (p = 0.03). (d.e) Effect of chelerythrine chloride on ERK activity and COX-2 expression. Proteins from bystander cells were extracted with RIPA buffer at the indicated time points after irradiation and probed with phosphor-ERK1/2 or COX-2 antibody. Left panels, untreated controls; right panels, cells were treated with chelerythrine chloride before irradiation. Three independent experiments were performed and a representative blot was shown. Ratios of the corresponding band intensity compared with that of untreated control were measured and calculated with Image J and indicated under each band. Asterisk indicates significant difference between untreated control and the treated groups (p < 0.05).
Mentions: PKC translocation has been implicated in the responses of multiple stimuli including ionizing radiation. It was shown that gamma-radiation could induce apoptosis, growth arrest through activation and translocation of PKCα, PKCε and PKCδ1623. To see if PKCα translocates in bystander responses, we investigated PKCα level in different cellular fraction of bystander AL cells. As shown in Fig. 1a, PKCα level in cytosol decreased while its level in the membrane fraction increased significantly in bystander cells, suggesting the responsiveness of PKCα in bystander effect. PKCα level changed as early as 15 min after irradiation and sustained as long as 2 hrs. Immunocytochemistry analysis also showed that PKCα translocates from cytosol to cell membrane in bystander cells. PKCα distributed homogenously before irradiation within AL cells, while its signal intensified along the cell membrane in bystander cells after irradiation (Fig. 1b).

Bottom Line: However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear.Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells.Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China.

ABSTRACT
Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2.

No MeSH data available.


Related in: MedlinePlus