Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Mar;13(3):978-88.
doi: 10.1091/mbc.01-05-0272.

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Marek Los  1 , Malgorzata Mozoluk, Davide Ferrari, Anna Stepczynska, Christopher Stroh, Andrea Renz, Zdenko Herceg, Zhao-Qi Wang, Klaus Schulze-Osthoff
Affiliations

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Marek Los et al. Mol Biol Cell. 2002 Mar.

Abstract

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Distinct forms of cell death induced by either anti-CD95 or TNF in L929 fibroblasts. L929 cells were treated with either anti-CD95 (1 μg/ml) or TNF (40 ng/ml) in the presence of actinomycin D. (A) Different morphological alterations after 6-h treatment of cells with anti-CD95 or TNF (magnification, ×200). (B) Detection of apoptotic DNA laddering after TNF or anti-CD95 treatment. Cells were incubated with either anti-CD95 or TNF; after the indicated time genomic DNA was separated on a 1.5% agarose gel. (C) Differences between flow cytometric measurement of cell death by PI uptake as a marker for increased cell membrane permeability and apoptotic hypodiploid nuclei 6 h after death induction by TNF and anti-CD95. Data represents the mean of four independent experiments.
Figure 2
Figure 2
Time course of caspase activation and the effect of caspase inhibition on TNF- and CD95-mediated cell death. (A) L929 cells were incubated with TNF or anti-CD95 as described in Figure 1. After the indicated time cells were harvested and analyzed by immunoblotting for the processing of procaspase-3 into the active p17 active subunit (top panel) and PARP-1 cleavage (bottom panel). (B) Effect of the caspase inhibitor zVAD: Cells were preincubated for 30 min with the indicated concentrations of zVAD or the medium control and then stimulated with either TNF or anti-CD95. Cell death was determined in triplicates by PI staining and flow cytometry 5 h after cell treatment. SDs were <9%.
Figure 3
Figure 3
Inhibition of PARP-1 cleavage potentiates TNF-induced death. Immortalized murine fibroblasts were triggered with TNF for 8 h. (A) Cells expressing the caspase-resistant PARP-1 D214N mutant were more sensitive to TNF-induced death than wild-type or PARP-1–deficient cells. (B) Effect of the caspase inhibitor zVAD on TNF-induced cell death in the different immortalized cells. Potentiation of TNF-induced death by zVAD was only observed in PARP-1(+/+) fibroblasts but not in PARP-1–deficient cells. SDs were <11%. (C) Expression of PARP-1 and -2 in immortalized wild-type fibroblasts and PARP knockout cells retransfected the caspase-resistant PARP D214N mutant, the vector control or wild-type (wt) PARP-1 cDNA. Cell lysates were separated by SDS polyacrylamide electrophoresis and immunoblotted with antibodies specific for PARP-1 and PARP-2, respectively. The immunoblots show the full-length form of PARP–1 (116 kDa) and -2 (62 kDa). No compensatory upregulation of PARP-2 expression was observed in the different cell clones.
Figure 4
Figure 4
Inhibition of PARP activity counteracts potentiation of TNF-induced death and ATP depletion after caspase inhibition. (A and B) Effect of zVAD and the PARP inhibitor 3-aminobenzamide (3AB) on TNF- and anti-CD95–induced cell death. L929 cells were preincubated with 40 μM zVAD, 3 mM 3AB, or a combination thereof and then stimulated for 5 h with the indicated concentrations of TNF (A) or anti-CD95 (B). (C) Potentiation of TNF-induced ATP depletion by zVAD. Cells were stimulated with or without zVAD (40 μM) in the presence or absence of TNF. After 4 h intracellular ATP levels were determined by the luciferase method. (D) Potentiation of TNF-triggered ATP depletion by the caspase inhibitor zVAD is prevented by 3AB. Cells were treated as described in C in the presence and absence of 3AB and analyzed for the intracellular ATP content 4 h after TNF stimulation. SDs in the experiments were <10%.
Figure 5
Figure 5
Involvement of ROS formation in death receptor-induced PARP activation. (A and B) Effect of TNF and anti-CD95 on ROS formation. L929 cells were triggered with TNF (A, 40 ng/ml) or anti-CD95 (B, 1 μg/ml) for the indicated time. The antioxidant BHA (150 μM) and zVAD (50 μM) were added 15 min before treatment with the death stimuli. ROS production was measured with the dye DFCH and flow cytometry. Similar data were obtained using rhodamine-123. SDs in the above experiments were <12%. (C) Different effect of BHA on TNF- and anti-CD95–induced cell death. Cells were stimulated as described in (A) with TNF or anti-CD95 in the presence or absence of 150 μM BHA. Cell death was determined after 5 h by measurement of PI uptake. (D) Visualization of PARP activity by immunodetection of poly(ADP-ribose) chains. Cells were either stimulated with TNF (40 ng/ml) in the presence and absence of BHA or zVAD or incubated with 1 μg/ml anti-CD95 or 2 mM H2O2. PARP activation was detected with a antibody specific for poly(ADP-ribose) polymers. Note the similarity of TNF-induced and zVAD-potentiated PARP activity with the immunocytochemical signal obtained after H2O2 treatment.
Figure 6
Figure 6
Schematic representation of the role of ROS, PARP, caspases, and intracellular ATP on the mode of cell death. Cells will die by necrosis when ROS-induced DNA damage, for instance in response to TNF, causes elevated PARP activity, resulting in subsequent energy depletion. Apoptosis will occur when caspases are activated early upon death induction. Caspases proteolytically inactivate a number of proteins including PARP-1 and thereby preserve ATP levels that are required for the apoptotic process. This scenario may also explain why caspase inhibitors prevent apoptosis but increase necrotic forms of cell death after death receptor stimulation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ame JC, Rolli V, Schreiber V, Niedergang C, Apiou F, Decker P, Muller S, Hoger T, Menissier-de Murcia J, de Murcia G. PARP-2, a novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J Biol Chem. 1999;274:17860–17868. - PubMed
    1. Avila MA, Velasco JA, Smulson ME, Dritschilo A, Castro R, Notario V. Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation. Yeast. 1994;10:1003–1017. - PubMed
    1. Beneke R, Geisen C, Zevnik B, Bauch T, Muller WU, Kupper JH, Moroy T. DNA excision repair and DNA damage-induced apoptosis are linked to poly(ADP-ribosyl)ation but have different requirements for p53. Mol Cell Biol. 2000;20:6695–6703. - PMC - PubMed
    1. Bürkle A. Physiology and pathophysiology of poly(ADP-ribosyl)ation. Bioessays. 2001;9:795–806. - PubMed
    1. Cohen GM. Caspases: the executioners of apoptosis. Biochem J. 1997;326:1–16. - PMC - PubMed

Publication types

MeSH terms