Immunodot Blot Method for the Detection of Poly(ADP-ribose) Synthesizedin Vitroandin Vivo

Affar, El Bachir; Duriez, Patrick J.; Shah, Rashmi G.; Sallmann, Frédéric R.; Bourassa, Sylvie; Küpper, Jan-Heiner; Bürkle, Alexander; Poirier, Guy G.

doi:10.1006/abio.1998.2664

Cited by 44 publications

(34 citation statements)

References 8 publications

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“…Poly(ADP-ribose) was measured, using a dot blot technique with the LP96 -10 antibody as described previously (20). To determine the sizes of the polymers in the apoptotic cells, 5 ϫ 10 7 HL60 cells were treated for 3 h with 68 M VP-16.…”

Section: Materials-[mentioning

confidence: 99%

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Cleavage of Automodified Poly(ADP-ribose) Polymerase during Apoptosis

Germain¹,

Affar²,

D’Amours³

et al. 1999

Journal of Biological Chemistry

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412

269

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The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADPribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.

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Section: Materials-[mentioning

confidence: 99%

“…An immunological method recently developed in our laboratory (20) was then used to directly measure the endogenous polymer levels. This technique relies on affinity chromatography purification of the polymers followed by their specific immunodetection.…”

Section: Parp Cleavage By Caspases At the Time Of Its Activation-mentioning

confidence: 99%

Cleavage of Automodified Poly(ADP-ribose) Polymerase during Apoptosis

Germain¹,

Affar²,

D’Amours³

et al. 1999

Journal of Biological Chemistry

Self Cite

412

269

View full text Add to dashboard Cite

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“…Analysis of pADPr Levels-pADPr determination was done using the immunodot-blot technique as described previously (45). Briefly, dihydroxyboronyl Bio-Rex purified pADPr was diluted in 0.4 M NaOH containing 10 mM EDTA and loaded on Hybond Nϩ membrane using a dot-blot manifold system (Life Technologies, Inc.).…”

Section: Materials-[mentioning

confidence: 99%

Caspase-3-mediated Processing of Poly(ADP-ribose) Glycohydrolase during Apoptosis

Affar¹,

Germain²,

Winstall³

et al. 2001

Journal of Biological Chemistry

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115

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Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fasinduced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID 256 2 V and the unconventional site MDVD 307 2 N. Kinetic studies have shown similar maximal velocity (V max ) and affinity (K m ) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.Poly(ADP-ribose) polymerase (PARP-1) 1 synthesizes poly-(ADP-ribose) (pADPr) in response to DNA strand breaks. This nuclear enzyme, present in most eukaryotic cells, is involved in the maintenance of the DNA integrity (1, 2). Recently, other pADPr synthesizing enzymes were identified, suggesting the presence within mammalian cells of a PARP-1-like enzyme family. A protein named tankyrase with homology to ankyrins and to the catalytic domain of PARP-1 was isolated from human tissue and shown to be associated with telomeres (3). Other proteins homologous to the catalytic domain of PARP-1 have also been reported (4 -8).Cells display a low basal level of pADPr, which can increase dramatically in response to DNA damaging agents (9 -11). This increase in pADPr synthesis is transient and is followed by a rapid degradation by poly(ADP-ribose) glycohydrolase (PARG) (10, 12, 13). Two forms of PARG (74 and 59 kDa) have previously been purified from various tissues (14 -19). However, the PARG cDNA recently isolated encodes an active protein of 111 kDa (20). Furthermore, we have recently reported the presence of only the 111-kDa form of PARG, which is localized mostly in the cytoplasm of the cells (21,22). These findings raise questions about the cellular mechanism of pADPr catabolism and the physiological significance of the 59-and 74-kDa forms of PARG.Programmed cell death, or apoptosis, is an essential mechanism for appropriate embryogenesis, normal cell turnover, and the selection of lymphocytes (23,24). Apoptosis is characterized b...

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“…The amount of synthesized pADPr was estimated by immunodot blot (40), while the analysis and measurements of NAD ϩ were performed using an enzyme cycling assay (39).…”

Section: Methodsmentioning

confidence: 99%

“…Wild type and mutant cells were treated with 100 M MNNG or UVC irradiated with a dose of 30 J/m 2 for different periods of time up to 60 min. After purification by affinity chromatography, the amount of pADPr at each time point was measured by an immunodot blot assay as described earlier (39,40). Table I (Fig.…”

Section: Padpr Synthesis In Parp-1(ϫ/ϫ) Cells After Genotoxicmentioning

confidence: 99%