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Regulation of p53 expression, phosphorylation and subcellular localization by a G-protein-coupled receptor.

Solyakov L, Sayan E, Riley J, Pointon A, Tobin AB - Oncogene (2009)

Bottom Line: In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells.This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53.This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
G-protein-coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This superfamily of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M(3)-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

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Related in: MedlinePlus

M3-muscarinic receptors protect CHO-m3 cells from etoposide-induced apoptosis and attenuated the up-regulation of p53 expressionCHO cells stably expressing the human M3-muscarinic (CHO-m3) receptor were exposed to etoposide (250μM; Eto) for 16hrs in the absence or presence of methycholine (1mM; Met) and atropine (Atr; 10μM). Atropine was added at the same time as methylcholine. The cells were then lysed and either (A) caspase 3 activity determined as described in materials and methods or (B) the lysate (20μg protein) was Western blotted for p53 expression with α-tubulin probed as a loading control. Shown in the right hand panel is quantification of p53 expression levels. (C) CHO-m3 cells were transfected with either control or p53 specific siRNA duplexes (50pmols). 48 hours following transfection cells were treated with or without etoposide (250μM; Eto) in the presence or absence of methacholine (1mM; Met) for 16 hours after which cell lysates were prepared and either processed for caspase 3 activity or probed in Western blots for p53 expression. (D) Gene expression changes measured by RT-PCR, of Bax, Puma, Mdm2 and Noxa genes following etoposide (250μM; Eto) treatment in the absence and presence of methacholine (1mM; Met). (E) CHO-m3 cells were treated with etoposide (250μM; Met) for 4 or 16 hours. Methylcholine (1mM; Met) was applied simultaneously with etoposide. The action of methylcholine was stopped after four hours by the addition of atropine (10μM). Etoposide treatment was either stopped at the point of atropine addition (i.e. 4 hours) or allowed to continue for a total of 16 hours. Cell lysates were then prepared and p53 levels determined by Western blot. (F) Following etoposide (250μM; Eto) treatment in the absence or presence of methacholine (1mM; Met) for 16 hours protein synthesis was inhibited by the addition of cyclohexamide (2μg/ml) and the incubation continued for the times indicated. The incubations were stopped by preparation of a cell lysate that was then probed for p53 expression in Western blots.. (G and H) CHO-m3 cells were stimulated in the presence and absence of etoposide (250mM; Eto) with or without methacholine (1mM; Met). At the times indicated stimulation with methylcholine was stopped by the addition of atropine (10μM) and the incubation with etoposide continued for a total of 16 hours. Cells were then lysed and lysates probed in Western blots for p53 and serine-15 phosphorylation (G) or p53 and serine-392 phosphorylation (H).Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of 6 independent experiments. *** p<0.001,**p<0.01; *p<0.05, paired Students t-test; represents significant difference from etoposide only treatment except for figure 1C where * represents significant difference from etoposide treatment in the presence of control siRNA..
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Figure 1: M3-muscarinic receptors protect CHO-m3 cells from etoposide-induced apoptosis and attenuated the up-regulation of p53 expressionCHO cells stably expressing the human M3-muscarinic (CHO-m3) receptor were exposed to etoposide (250μM; Eto) for 16hrs in the absence or presence of methycholine (1mM; Met) and atropine (Atr; 10μM). Atropine was added at the same time as methylcholine. The cells were then lysed and either (A) caspase 3 activity determined as described in materials and methods or (B) the lysate (20μg protein) was Western blotted for p53 expression with α-tubulin probed as a loading control. Shown in the right hand panel is quantification of p53 expression levels. (C) CHO-m3 cells were transfected with either control or p53 specific siRNA duplexes (50pmols). 48 hours following transfection cells were treated with or without etoposide (250μM; Eto) in the presence or absence of methacholine (1mM; Met) for 16 hours after which cell lysates were prepared and either processed for caspase 3 activity or probed in Western blots for p53 expression. (D) Gene expression changes measured by RT-PCR, of Bax, Puma, Mdm2 and Noxa genes following etoposide (250μM; Eto) treatment in the absence and presence of methacholine (1mM; Met). (E) CHO-m3 cells were treated with etoposide (250μM; Met) for 4 or 16 hours. Methylcholine (1mM; Met) was applied simultaneously with etoposide. The action of methylcholine was stopped after four hours by the addition of atropine (10μM). Etoposide treatment was either stopped at the point of atropine addition (i.e. 4 hours) or allowed to continue for a total of 16 hours. Cell lysates were then prepared and p53 levels determined by Western blot. (F) Following etoposide (250μM; Eto) treatment in the absence or presence of methacholine (1mM; Met) for 16 hours protein synthesis was inhibited by the addition of cyclohexamide (2μg/ml) and the incubation continued for the times indicated. The incubations were stopped by preparation of a cell lysate that was then probed for p53 expression in Western blots.. (G and H) CHO-m3 cells were stimulated in the presence and absence of etoposide (250mM; Eto) with or without methacholine (1mM; Met). At the times indicated stimulation with methylcholine was stopped by the addition of atropine (10μM) and the incubation with etoposide continued for a total of 16 hours. Cells were then lysed and lysates probed in Western blots for p53 and serine-15 phosphorylation (G) or p53 and serine-392 phosphorylation (H).Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of 6 independent experiments. *** p<0.001,**p<0.01; *p<0.05, paired Students t-test; represents significant difference from etoposide only treatment except for figure 1C where * represents significant difference from etoposide treatment in the presence of control siRNA..

Mentions: Treatment of CHO-m3 cells with etoposide for 16 hours resulted in a ~2.5-fold increase in caspase activity over control levels. Stimulation of the M3-muscarinic receptor using the full agonist methylcholine significantly reduced caspase activation by 69.4 ±8.9% (p<0.01, n=6) (Fig.1A); an observation consistent with previous studies (Budd et al., 2004; Tobin and Budd, 2003). The effect of methylcholine was blocked by the muscarinic specific antagonist, atropine (Fig 1A). The ability of M3-muscarinic receptor stimulation to reduce etoposide-mediated caspase activation correlated with changes in the expression levels of p53. Levels of p53 increased in CHO-m3 cells challenged with etoposide and this was attenuated by the stimulation of the M3-muscarinic receptor (Fig 1B).


Regulation of p53 expression, phosphorylation and subcellular localization by a G-protein-coupled receptor.

Solyakov L, Sayan E, Riley J, Pointon A, Tobin AB - Oncogene (2009)

M3-muscarinic receptors protect CHO-m3 cells from etoposide-induced apoptosis and attenuated the up-regulation of p53 expressionCHO cells stably expressing the human M3-muscarinic (CHO-m3) receptor were exposed to etoposide (250μM; Eto) for 16hrs in the absence or presence of methycholine (1mM; Met) and atropine (Atr; 10μM). Atropine was added at the same time as methylcholine. The cells were then lysed and either (A) caspase 3 activity determined as described in materials and methods or (B) the lysate (20μg protein) was Western blotted for p53 expression with α-tubulin probed as a loading control. Shown in the right hand panel is quantification of p53 expression levels. (C) CHO-m3 cells were transfected with either control or p53 specific siRNA duplexes (50pmols). 48 hours following transfection cells were treated with or without etoposide (250μM; Eto) in the presence or absence of methacholine (1mM; Met) for 16 hours after which cell lysates were prepared and either processed for caspase 3 activity or probed in Western blots for p53 expression. (D) Gene expression changes measured by RT-PCR, of Bax, Puma, Mdm2 and Noxa genes following etoposide (250μM; Eto) treatment in the absence and presence of methacholine (1mM; Met). (E) CHO-m3 cells were treated with etoposide (250μM; Met) for 4 or 16 hours. Methylcholine (1mM; Met) was applied simultaneously with etoposide. The action of methylcholine was stopped after four hours by the addition of atropine (10μM). Etoposide treatment was either stopped at the point of atropine addition (i.e. 4 hours) or allowed to continue for a total of 16 hours. Cell lysates were then prepared and p53 levels determined by Western blot. (F) Following etoposide (250μM; Eto) treatment in the absence or presence of methacholine (1mM; Met) for 16 hours protein synthesis was inhibited by the addition of cyclohexamide (2μg/ml) and the incubation continued for the times indicated. The incubations were stopped by preparation of a cell lysate that was then probed for p53 expression in Western blots.. (G and H) CHO-m3 cells were stimulated in the presence and absence of etoposide (250mM; Eto) with or without methacholine (1mM; Met). At the times indicated stimulation with methylcholine was stopped by the addition of atropine (10μM) and the incubation with etoposide continued for a total of 16 hours. Cells were then lysed and lysates probed in Western blots for p53 and serine-15 phosphorylation (G) or p53 and serine-392 phosphorylation (H).Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of 6 independent experiments. *** p<0.001,**p<0.01; *p<0.05, paired Students t-test; represents significant difference from etoposide only treatment except for figure 1C where * represents significant difference from etoposide treatment in the presence of control siRNA..
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Figure 1: M3-muscarinic receptors protect CHO-m3 cells from etoposide-induced apoptosis and attenuated the up-regulation of p53 expressionCHO cells stably expressing the human M3-muscarinic (CHO-m3) receptor were exposed to etoposide (250μM; Eto) for 16hrs in the absence or presence of methycholine (1mM; Met) and atropine (Atr; 10μM). Atropine was added at the same time as methylcholine. The cells were then lysed and either (A) caspase 3 activity determined as described in materials and methods or (B) the lysate (20μg protein) was Western blotted for p53 expression with α-tubulin probed as a loading control. Shown in the right hand panel is quantification of p53 expression levels. (C) CHO-m3 cells were transfected with either control or p53 specific siRNA duplexes (50pmols). 48 hours following transfection cells were treated with or without etoposide (250μM; Eto) in the presence or absence of methacholine (1mM; Met) for 16 hours after which cell lysates were prepared and either processed for caspase 3 activity or probed in Western blots for p53 expression. (D) Gene expression changes measured by RT-PCR, of Bax, Puma, Mdm2 and Noxa genes following etoposide (250μM; Eto) treatment in the absence and presence of methacholine (1mM; Met). (E) CHO-m3 cells were treated with etoposide (250μM; Met) for 4 or 16 hours. Methylcholine (1mM; Met) was applied simultaneously with etoposide. The action of methylcholine was stopped after four hours by the addition of atropine (10μM). Etoposide treatment was either stopped at the point of atropine addition (i.e. 4 hours) or allowed to continue for a total of 16 hours. Cell lysates were then prepared and p53 levels determined by Western blot. (F) Following etoposide (250μM; Eto) treatment in the absence or presence of methacholine (1mM; Met) for 16 hours protein synthesis was inhibited by the addition of cyclohexamide (2μg/ml) and the incubation continued for the times indicated. The incubations were stopped by preparation of a cell lysate that was then probed for p53 expression in Western blots.. (G and H) CHO-m3 cells were stimulated in the presence and absence of etoposide (250mM; Eto) with or without methacholine (1mM; Met). At the times indicated stimulation with methylcholine was stopped by the addition of atropine (10μM) and the incubation with etoposide continued for a total of 16 hours. Cells were then lysed and lysates probed in Western blots for p53 and serine-15 phosphorylation (G) or p53 and serine-392 phosphorylation (H).Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of 6 independent experiments. *** p<0.001,**p<0.01; *p<0.05, paired Students t-test; represents significant difference from etoposide only treatment except for figure 1C where * represents significant difference from etoposide treatment in the presence of control siRNA..
Mentions: Treatment of CHO-m3 cells with etoposide for 16 hours resulted in a ~2.5-fold increase in caspase activity over control levels. Stimulation of the M3-muscarinic receptor using the full agonist methylcholine significantly reduced caspase activation by 69.4 ±8.9% (p<0.01, n=6) (Fig.1A); an observation consistent with previous studies (Budd et al., 2004; Tobin and Budd, 2003). The effect of methylcholine was blocked by the muscarinic specific antagonist, atropine (Fig 1A). The ability of M3-muscarinic receptor stimulation to reduce etoposide-mediated caspase activation correlated with changes in the expression levels of p53. Levels of p53 increased in CHO-m3 cells challenged with etoposide and this was attenuated by the stimulation of the M3-muscarinic receptor (Fig 1B).

Bottom Line: In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells.This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53.This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
G-protein-coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This superfamily of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M(3)-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

Show MeSH
Related in: MedlinePlus