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p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase.

Li S, Banck M, Mujtaba S, Zhou MM, Sugrue MM, Walsh MJ - PLoS ONE (2010)

Bottom Line: Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis.Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence.These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

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Analysis of p53 in subcellular fractions from senescent EJ-p53 cells.(A) Immunoblots using 50 µg protein of total cell lysate and subcellular fractions (mitochondria and nuclear fraction obtained from a sucrose gradient) were subject to SDS-PAGE and subsequently transferred to PVDF filters (Immobilon, Millipore Corp.). Blots were then blocked with BSA and initially probed using a mouse monoclonal antibody specific for p53 (clone 1801, Oncogene Research) followed by HRP-conjugated anti-mouse IgG (Roche) and then developed using ECL kit (GE Healthcare/Amersham). Blots were reprobed using mouse monoclonal specific for the mitochondrial marker, Cox II (Molecular Probes, Invitrogen) and nuclear marker PCNA (Calbiochem) followed by HRP-conjugated anti-mouse IgG and then developed using the ECL kit (GE Healthcare/Amersham). (B) Laser confocal image of p53 immunohistochemistry and CMTMR, YOYO fluorescence in EJ-p53 cells. EJ-p53 cells were maintained in (+) tet (no p53 expression) and (-)tet 2 hours through 4days (overexpression of wt p53). p53-specific mAb(Oncogene research) and Cy5-labled anti-mouse were used to immunohistochemistry localize p53 in EJ-p53 cells. YOYO was used to stain nuclei and CMTMR was to stain mitochondria. For each image, p53 was re-colored to red, Nuclei was re-colored to blue, and mitochondria was re-colored to green. p53 localization in p53-induced senescence in EJ-p53 cells was determined by overlay.
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pone-0010486-g001: Analysis of p53 in subcellular fractions from senescent EJ-p53 cells.(A) Immunoblots using 50 µg protein of total cell lysate and subcellular fractions (mitochondria and nuclear fraction obtained from a sucrose gradient) were subject to SDS-PAGE and subsequently transferred to PVDF filters (Immobilon, Millipore Corp.). Blots were then blocked with BSA and initially probed using a mouse monoclonal antibody specific for p53 (clone 1801, Oncogene Research) followed by HRP-conjugated anti-mouse IgG (Roche) and then developed using ECL kit (GE Healthcare/Amersham). Blots were reprobed using mouse monoclonal specific for the mitochondrial marker, Cox II (Molecular Probes, Invitrogen) and nuclear marker PCNA (Calbiochem) followed by HRP-conjugated anti-mouse IgG and then developed using the ECL kit (GE Healthcare/Amersham). (B) Laser confocal image of p53 immunohistochemistry and CMTMR, YOYO fluorescence in EJ-p53 cells. EJ-p53 cells were maintained in (+) tet (no p53 expression) and (-)tet 2 hours through 4days (overexpression of wt p53). p53-specific mAb(Oncogene research) and Cy5-labled anti-mouse were used to immunohistochemistry localize p53 in EJ-p53 cells. YOYO was used to stain nuclei and CMTMR was to stain mitochondria. For each image, p53 was re-colored to red, Nuclei was re-colored to blue, and mitochondria was re-colored to green. p53 localization in p53-induced senescence in EJ-p53 cells was determined by overlay.

Mentions: EJ-p53 cells were harvested at different time points as indicated (Fig. 1A) (+tet, -tet 2 hours, 4 hours, 6 hours,1 day, 2 days,3 days and 4 days). Using a subcellular fractionation and sucrose step gradient, total cell lysates, nuclear and mitochondrial fractions were obtained. Specific nuclear and mitochondrial subcellular organelle markers were used to trace p53 alongside the fractionation procedure. Upon tetracycline withdrawal p53 was detected in mitochondrial fractions as early as 4 h after and persisted in all subsequent time points analyzed, e.g. -tet 6 h, 1 d, 2 d, 3 d, 4 d. From –tet 1 day to 4 days, p53 levels dimished (Fig. 1A). However, p53 levels were higher in nuclear and total cell lysates prepared from EJ-p53 cells over the same time course. Immunohistochemical analysis using confocal imaging showed p53 exclusively in mitochondria at –tet 2 h (Fig. 1B). At –tet 4 h, p53 was detected at higher levels in the mitochondria with concomitant appearance of p53 in the nucleus. p53 persisted in mitochondria and the nucleus for the duration of the time course studied (from −4 h through −4 d).


p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase.

Li S, Banck M, Mujtaba S, Zhou MM, Sugrue MM, Walsh MJ - PLoS ONE (2010)

Analysis of p53 in subcellular fractions from senescent EJ-p53 cells.(A) Immunoblots using 50 µg protein of total cell lysate and subcellular fractions (mitochondria and nuclear fraction obtained from a sucrose gradient) were subject to SDS-PAGE and subsequently transferred to PVDF filters (Immobilon, Millipore Corp.). Blots were then blocked with BSA and initially probed using a mouse monoclonal antibody specific for p53 (clone 1801, Oncogene Research) followed by HRP-conjugated anti-mouse IgG (Roche) and then developed using ECL kit (GE Healthcare/Amersham). Blots were reprobed using mouse monoclonal specific for the mitochondrial marker, Cox II (Molecular Probes, Invitrogen) and nuclear marker PCNA (Calbiochem) followed by HRP-conjugated anti-mouse IgG and then developed using the ECL kit (GE Healthcare/Amersham). (B) Laser confocal image of p53 immunohistochemistry and CMTMR, YOYO fluorescence in EJ-p53 cells. EJ-p53 cells were maintained in (+) tet (no p53 expression) and (-)tet 2 hours through 4days (overexpression of wt p53). p53-specific mAb(Oncogene research) and Cy5-labled anti-mouse were used to immunohistochemistry localize p53 in EJ-p53 cells. YOYO was used to stain nuclei and CMTMR was to stain mitochondria. For each image, p53 was re-colored to red, Nuclei was re-colored to blue, and mitochondria was re-colored to green. p53 localization in p53-induced senescence in EJ-p53 cells was determined by overlay.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864751&req=5

pone-0010486-g001: Analysis of p53 in subcellular fractions from senescent EJ-p53 cells.(A) Immunoblots using 50 µg protein of total cell lysate and subcellular fractions (mitochondria and nuclear fraction obtained from a sucrose gradient) were subject to SDS-PAGE and subsequently transferred to PVDF filters (Immobilon, Millipore Corp.). Blots were then blocked with BSA and initially probed using a mouse monoclonal antibody specific for p53 (clone 1801, Oncogene Research) followed by HRP-conjugated anti-mouse IgG (Roche) and then developed using ECL kit (GE Healthcare/Amersham). Blots were reprobed using mouse monoclonal specific for the mitochondrial marker, Cox II (Molecular Probes, Invitrogen) and nuclear marker PCNA (Calbiochem) followed by HRP-conjugated anti-mouse IgG and then developed using the ECL kit (GE Healthcare/Amersham). (B) Laser confocal image of p53 immunohistochemistry and CMTMR, YOYO fluorescence in EJ-p53 cells. EJ-p53 cells were maintained in (+) tet (no p53 expression) and (-)tet 2 hours through 4days (overexpression of wt p53). p53-specific mAb(Oncogene research) and Cy5-labled anti-mouse were used to immunohistochemistry localize p53 in EJ-p53 cells. YOYO was used to stain nuclei and CMTMR was to stain mitochondria. For each image, p53 was re-colored to red, Nuclei was re-colored to blue, and mitochondria was re-colored to green. p53 localization in p53-induced senescence in EJ-p53 cells was determined by overlay.
Mentions: EJ-p53 cells were harvested at different time points as indicated (Fig. 1A) (+tet, -tet 2 hours, 4 hours, 6 hours,1 day, 2 days,3 days and 4 days). Using a subcellular fractionation and sucrose step gradient, total cell lysates, nuclear and mitochondrial fractions were obtained. Specific nuclear and mitochondrial subcellular organelle markers were used to trace p53 alongside the fractionation procedure. Upon tetracycline withdrawal p53 was detected in mitochondrial fractions as early as 4 h after and persisted in all subsequent time points analyzed, e.g. -tet 6 h, 1 d, 2 d, 3 d, 4 d. From –tet 1 day to 4 days, p53 levels dimished (Fig. 1A). However, p53 levels were higher in nuclear and total cell lysates prepared from EJ-p53 cells over the same time course. Immunohistochemical analysis using confocal imaging showed p53 exclusively in mitochondria at –tet 2 h (Fig. 1B). At –tet 4 h, p53 was detected at higher levels in the mitochondria with concomitant appearance of p53 in the nucleus. p53 persisted in mitochondria and the nucleus for the duration of the time course studied (from −4 h through −4 d).

Bottom Line: Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis.Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence.These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

Show MeSH
Related in: MedlinePlus