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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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The M3-muscarinic receptor protects SH-SY5Y cells from DNA-damage induced apoptosisSH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 2-16 hours. After treatment, cells were lysed and lysates processed either for (A) caspase-3 activity, (B) Western blotting for PARP cleavage or (C) Western blotted for cytsolic cytochrome C, cleaved caspase 9 and cleaved caspase 3 together with Western blots for Bid and caspase 8. (D) SH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 4 hours. During this period methylcholine stimulation was stopped by addition of the antagonist atropine (10μM) at the indicated times. Lysates were prepared at the end of the experiment and caspase activity determined.The Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of at least 3 independent experiments. *** p<0.001, * p<0.05; paired Students t-test; represents significant difference from etoposide only treatment.
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Figure 2: The M3-muscarinic receptor protects SH-SY5Y cells from DNA-damage induced apoptosisSH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 2-16 hours. After treatment, cells were lysed and lysates processed either for (A) caspase-3 activity, (B) Western blotting for PARP cleavage or (C) Western blotted for cytsolic cytochrome C, cleaved caspase 9 and cleaved caspase 3 together with Western blots for Bid and caspase 8. (D) SH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 4 hours. During this period methylcholine stimulation was stopped by addition of the antagonist atropine (10μM) at the indicated times. Lysates were prepared at the end of the experiment and caspase activity determined.The Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of at least 3 independent experiments. *** p<0.001, * p<0.05; paired Students t-test; represents significant difference from etoposide only treatment.

Mentions: Consistent with previous reports (De Sarno et al., 2003; De Sarno et al., 2005) we show here that the M3-muscarinic receptor endogenously expressed in the human neuroblastoma cell line SH-SY5Y was able to protect cells from etoposide induced cell death. Treatment with etoposide (25μM) for 4 and 8 hours resulted in 6-8 fold increase in caspase activity which was significantly reduced by methylcholine (90.2 ± 0.1% (P<0.001, n=5) and 59.3 ± 0.7% (P<0.05, n=3), respectively (Fig 2A)). Furthermore, muscarinic receptor stimulation was able to attenuate caspase-mediated PARP cleavage in response to etoposide treatment (Fig 2B), caspase 3 and caspase 9 processing together and cytochrome C release into the cytoplasm (Fig 2C). We did not, however, observe any changes in caspase 8 processing nor cleavage of the caspase 8 substrate Bid (Fig 2C).


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)

The M3-muscarinic receptor protects SH-SY5Y cells from DNA-damage induced apoptosisSH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 2-16 hours. After treatment, cells were lysed and lysates processed either for (A) caspase-3 activity, (B) Western blotting for PARP cleavage or (C) Western blotted for cytsolic cytochrome C, cleaved caspase 9 and cleaved caspase 3 together with Western blots for Bid and caspase 8. (D) SH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 4 hours. During this period methylcholine stimulation was stopped by addition of the antagonist atropine (10μM) at the indicated times. Lysates were prepared at the end of the experiment and caspase activity determined.The Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of at least 3 independent experiments. *** p<0.001, * p<0.05; paired Students t-test; represents significant difference from etoposide only treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875175&req=5

Figure 2: The M3-muscarinic receptor protects SH-SY5Y cells from DNA-damage induced apoptosisSH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 2-16 hours. After treatment, cells were lysed and lysates processed either for (A) caspase-3 activity, (B) Western blotting for PARP cleavage or (C) Western blotted for cytsolic cytochrome C, cleaved caspase 9 and cleaved caspase 3 together with Western blots for Bid and caspase 8. (D) SH-SY5Y cells were treated with etoposide (Eto, 25μM) in the presence or absence of methylcholine (Met, 100μM) for 4 hours. During this period methylcholine stimulation was stopped by addition of the antagonist atropine (10μM) at the indicated times. Lysates were prepared at the end of the experiment and caspase activity determined.The Western blots shown are typical of at least three experiments. The graphical results represent the mean (± SE) of at least 3 independent experiments. *** p<0.001, * p<0.05; paired Students t-test; represents significant difference from etoposide only treatment.
Mentions: Consistent with previous reports (De Sarno et al., 2003; De Sarno et al., 2005) we show here that the M3-muscarinic receptor endogenously expressed in the human neuroblastoma cell line SH-SY5Y was able to protect cells from etoposide induced cell death. Treatment with etoposide (25μM) for 4 and 8 hours resulted in 6-8 fold increase in caspase activity which was significantly reduced by methylcholine (90.2 ± 0.1% (P<0.001, n=5) and 59.3 ± 0.7% (P<0.05, n=3), respectively (Fig 2A)). Furthermore, muscarinic receptor stimulation was able to attenuate caspase-mediated PARP cleavage in response to etoposide treatment (Fig 2B), caspase 3 and caspase 9 processing together and cytochrome C release into the cytoplasm (Fig 2C). We did not, however, observe any changes in caspase 8 processing nor cleavage of the caspase 8 substrate Bid (Fig 2C).

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