Labeling methods for the study of poly- and mono(ADP-ribose) metabolism in cultured cells

Aboul-Ela, Nasreen; Jacobson, Elaine L.; Jacobson, Myron K.

doi:10.1016/0003-2697(88)90541-6

Cited by 53 publications

(30 citation statements)

References 27 publications

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“…To induce ADP-ribose polymer synthesis, cells were incubated in 136 M MNNG at 37°C for 20 min, followed by addition of 20% (w/v) trichloroacetic acid at 4°C. The trichloroacetic acid-insoluble material was collected by centrifugation and applied to a dihydroxyboronyl-Bio-Rex column as described previously (11). The eluate containing ADP-ribose polymers was lyophilized to dryness and resuspended in 2 ml of 50 mM MOPS buffer, 5 mM MgCl 2 , pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

“…3B) and as a function of MNNG dose 240 min following treatment (Fig. 3A) ]adenine and examined for the presence of ADP-ribose polymers using a method previously described (11). In this method, ADP-ribose polymers are isolated using a boronyl resin and digested with snake venom phosphodiesterase and alkaline phosphatase, which results in the generation of ribosyladenosine (rAdo), a nucleoside unique to the ribosyl-ribosyl linkages of ADP-ribose polymers (14).…”

Section: Mnng-induced Nadmentioning

confidence: 99%

“…Quantitative comparisons of ADP-ribose polymer accumulation following MNNG treatment in 3T3 and primary embryo cells of various PARP genotypes are shown in Table I. The quantification of ADP-ribose polymer residues was determined by first measuring the specific radioactivity of the NAD ϩ pools in each cell type (11), because the different growth rates of the cells result in slightly different specific radioactivities. The amount of polymer accumulated was similar in both primary embryo cells and 3T3 cells from the PARP ϩ/ϩ genotype following MNNG treatment.…”

Section: Adp-ribose Polymer Synthesis In Parp Null Cells 30070mentioning

confidence: 99%

“…Cells were treated with 136 M MNNG, and ADP-ribose polymers were isolated and quantified as described (11). Benzamide concentration was 1 mM.…”

Section: Table I Quantification Of Adp-ribose Polymers In Mnng-treatementioning

confidence: 99%

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Poly(ADP-ribose) Polymerase Null Mouse Cells Synthesize ADP-ribose Polymers

Shieh¹,

Amé²,

Wilson³

et al. 1998

Journal of Biological Chemistry

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271

190

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Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD؉ , is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to ␥-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP ؊/؊ mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP ؊/؊ cells were indistinguishable from that of PARP ؉/؉ cells by several criteria. First, they bound to a boronate resin selective for ADPribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP ؊/؊ cells in a DNA damagedependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADPribose polymers in PARP ؊/؊ cells.Poly(ADP-ribose) polymerase (PARP) 1 (EC 2.4.2.30) is a nuclear enzyme that catalyzes the formation of complex homopolymers of ADP-ribose from NAD ϩ on nuclear proteins in response to DNA damage. Recently, mice homozygous for a disrupted PARP gene (PARP Ϫ/Ϫ ) have been generated (1-3). These animals and cells derived from them are being widely studied to better understand the potential roles of ADP-ribose polymer metabolism in cellular functions including DNA repair (4) and apoptosis (5, 6). The PARP Ϫ/Ϫ mice show normal fetal and postnatal development (1, 2) but have inherent genomic instability and are highly sensitive to DNA damage induced by ␥-radiation and alkylating agents (2, 4, 5). These results indicate that PARP is involved in maintaining genomic integrity and cell survival, especially following genotoxic insults. However, viability of the null genotype mice suggests that PARP is not essential for growth, development, or differentiation. Because there has been only a single gene product reported with PARP activity, we have used the PARP null genotype to address whether any other activities might be present that are capable of generating ADP-ribose polymers. Here, we show that cells derived from PARP Ϫ/Ϫ animals form ADP-ribose polymers, indicating the presence of enzymatic activity capable of catalyzing ADP-ribose polymer synthesis, which may represent heretofore unreported family member(s) of PARP. EXPERIMENTAL PROCEDURESCell Culture-PARP ϩ/ϩ and PARP Ϫ/Ϫ mice (strain C57BL/6) developed by Wang et al. (1) were used for the studies described here. Primary mouse embryo or immort...

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Section: Methodsmentioning

confidence: 99%

Section: Mnng-induced Nadmentioning

confidence: 99%

Section: Adp-ribose Polymer Synthesis In Parp Null Cells 30070mentioning

confidence: 99%

“…Cells were treated with 136 M MNNG, and ADP-ribose polymers were isolated and quantified as described (11). Benzamide concentration was 1 mM.…”

Section: Table I Quantification Of Adp-ribose Polymers In Mnng-treatementioning

confidence: 99%

See 2 more Smart Citations

Poly(ADP-ribose) Polymerase Null Mouse Cells Synthesize ADP-ribose Polymers

Shieh¹,

Amé²,

Wilson³

et al. 1998

Journal of Biological Chemistry

Self Cite

271

190

View full text Add to dashboard Cite

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“…PAR was purified essentially as described (Aboul-Ela et al 1988). Briefly, 5 3 10 6 human embryonic kidney (HEK) 293T cells were grown to confluency in 150-mm plates.…”

Section: Par Purificationmentioning

confidence: 99%

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

et al. 2012

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Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormoneresponsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

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Nonisotopic Methods for Determination of Poly(ADP‐Ribose) Levels and Detection of Poly(ADP‐Ribose) Polymerase

2003

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Poly(ADP‐ribosyl)ation is a post‐translational modification catalyzed mostly by the 116‐kDa enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1), a nuclear enzyme that transfers an ADP‐ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500‐fold increase in net poly(ADP‐ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP‐ribose) synthesis is PARP‐1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD+ depletion. It has also been proposed that the presence of negatively charged poly(ADP‐ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP‐ribose) in cells using nonisotopic reagents and for identifying the poly(ADP‐ribose) polymerase enzymes present in cells.

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Labeling methods for the study of poly- and mono(ADP-ribose) metabolism in cultured cells

Cited by 53 publications

References 27 publications

Poly(ADP-ribose) Polymerase Null Mouse Cells Synthesize ADP-ribose Polymers

Poly(ADP-ribose) Polymerase Null Mouse Cells Synthesize ADP-ribose Polymers

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

Nonisotopic Methods for Determination of Poly(ADP‐Ribose) Levels and Detection of Poly(ADP‐Ribose) Polymerase

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