The roles of poly(ADP-ribose)-metabolizing enzymes in alkylation-induced cell death

Abstract.: Poly(ADP-ribose) (PAR) has been identified as a DNA damage-inducible cell death signal upstream of apoptosis-inducing factor (AIF). PAR causes the translocation of AIF from mitochondria to the nucleus and triggers cell death. In living cells, PAR molecules are subject to dynamic changes pending on internal and external stress factors. Using RNA interference (RNAi), we determined the roles of poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2) and poly(ADP-ribose) glycohydrolase (PARG), the key enzymes configuring PAR molecules, in cell death induced by an alkylating agent. We found that PARP-1, but not PARP-2 and PARG, contributed to alkylation-induced cell death. Likewise, AIF translocation was only affected by PARP-1. PARP-1 seems to play a major role configuring PAR as a death signal involving AIF translocation regardless of the death pathway involve

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca2+ fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca2+ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca2+ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca2+ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and protein

The roles of poly(ADP-ribose)-metabolizing enzymes in alkylation-induced cell death

Poly(ADP-ribose) (PAR) has been identified as a DNA damage-inducible cell death signal upstream of apoptosis-inducing factor (AIF). PAR causes the translocation of AIF from mitochondria to the nucleus and triggers cell death. In living cells, PAR molecules are subject to dynamic changes pending on internal and external stress factors. Using RNA interference (RNAi), we determined the roles of poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2) and poly(ADP-ribose) glycohydrolase (PARG), the key enzymes configuring PAR molecules, in cell death induced by an alkylating agent. We found that PARP-1, but not PARP-2 and PARG, contributed to alkylation-induced cell death. Likewise, AIF translocation was only affected by PARP-1. PARP-1 seems to play a major role configuring PAR as a death signal involving AIF translocation regardless of the death pathway involved

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca2+ fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca2+ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca2+ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca2+ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins

Selective Down-Regulation of Nuclear Poly(ADP-Ribose) Glycohydrolase

The formation of ADP-ribose polymers on target proteins by poly(ADP-ribose) polymerases serves a variety of cell signaling functions. In addition, extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) is a dominant cause of cell death in ischemia-reperfusion, trauma, and other conditions. Poly(ADP-ribose) glycohydrolase (PARG) degrades the ADP-ribose polymers formed on acceptor proteins by PARP-1 and other PARP family members. PARG exists as multiple isoforms with differing subcellular localizations, but the functional significance of these isoforms is uncertain.Primary mouse astrocytes were treated with an antisense phosphorodiamidate morpholino oligonucleotide (PMO) targeted to exon 1 of full-length PARG to suppress expression of this nuclear-specific PARG isoform. The antisense-treated cells showed down-regulation of both nuclear PARG immunoreactivity and nuclear PARG enzymatic activity, without significant alteration in cytoplasmic PARG activity. When treated with the genotoxic agent MNNG to induced PARP-1 activation, the antisense-treated cells showed a delayed rate of nuclear PAR degradation, reduced nuclear condensation, and reduced cell death.These results support a preferentially nuclear localization for full-length PARG, and suggest a key role for this isoform in the PARP-1 cell death pathway

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health

The role of poly(ADP-ribose) glycohydrolase (PARG) in cell death

The post-translational modification of proteins with poly(ADP-ribose) (PAR) occurs as an early response to genotoxic insults. The nuclear poly(ADP-ribose) polymerase 1 (PARP-1) reacts to DNA nicks, activating its catalytical function and thereby producing large amounts of PAR. The substrate for this reaction is NAD+. Negatively charged linear PAR chains of up to 200 ADP-ribose monomers with maximal 3% branching are formed within minutes. Most of PAR is covalently bound on PARP-1 itself. Only 5-10% of PAR molecules are attached to other acceptor molecules. The turnover of PAR is tightly regulated. Its catabolic half-life ranges from seconds to hours depending on polymer size, complexity, protein association and the particular stress conditions to which cells are exposed. PAR glycohydrolase (PARG) degrades PAR via endo- and exoglycosidic activity. PARG produces primarily monomeric ADP-ribose albeit a few PAR polymers may arise. The coordinate action of PARP and PARG is required for proper cellular response to DNA damage and maintenance of genomic stability. Although the role of PARP-1 has been extensively studied, the biological function of PARG is not well established so far. Several studies with inhibitors and partial knockout models of PARG pointed to a possible role for PARG enzyme in the regulation of cell death or survival after alkylation- and oxidant-induced DNA damage. However, the lack of potent and cell permeable inhibitory molecules for PARG and the lethal phenotype of parg-/- mutants prevented the discovery of distinct functions in the cell death and survival network. Therefore we developed a RNA interference (RNAi) approach against PARG to reduce the amount of PARG protein in human and murine cells. Silencing of the parg gene resulted in a time-dependent reduction of parg mRNA, PARG protein and enzymatic activity which was maximal after 72 h of siRNA transfection. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions as cell proliferation and DNA damage repair after sublethal doses of oxidative stress (H2O2). Cell survival following higher concentrations of H2O2 was increased in cells lacking PARG. In fact, the resident time of PAR molecules after oxidative stress was prolonged in cells silenced for PARG resulting in a protective phenotype. Dawson and collaborators demonstrated that PAR may act as a cell death signal upstream of apoptosis-inducing factor (AIF). In particular, PAR was found to mediate between DNA damage and translocation of AIF from mitochondria to the nucleus. On the basis of this information we used siRNA in human cells (HeLa) to selectively down-regulate the PAR metabolizing enzymes PARP-1, PARP-2 and PARG, either separately or in combination, to determine their individual contribution to modulate PAR as a cell death factor. Cell death mediated by the alkylating agent MNNG is characterized by the activation of PAR synthesis, moderate changes in ATP/ADP ratio of cells, activation of caspases 7 and 9 and finally AIF translocation from mitochondria to nucleus. We observed that only PARP-1, and not PARP-2 and PARG, are involved in this type of alkylation-induced cell death. PAR synthesized by PARP-2 as well as modified PAR molecules due to PARG reduction were unable to promote AIF translocation. We further investigated molecular signal cascades in oxidant-induced cell death in the presence or absence of PARG. As previously shown, PARG silenced cells are less sensitive to H2O2-induced cytotoxicity. The hypothesis was that specific PAR metabolites can act as a signalling factor in cell death, in particular monomeric ADP-ribose, that has been shown to bind to specific Ca2+ channels in vitro. We established a fluorescence spectroscopic determination of the influx of Ca2+ ions into the cytosol as an immediate response to an oxidative insult in mouse embryonic fibroblasts (MEFs) with a manipulated PAR metabolism. Using a set of PARP inhibitors, RNAi against PARP-1 and parp-1-/- cells, we investigated a PARP-dependent elevation of cytosolic Ca2+ after lethal doses of H2O2. Moreover, the translocation of AIF from mitochondria to nuclei was abolished when PARP function was eliminated leading to reduced cell death rates. Melastatin-like transient receptor potential channels 2 (TRPM2) were identified earlier required for Ca2+ gating from the extracellular space into the cytosol after oxidative insults. TRPM2 channels become activated by monomeric ADP-ribose, but not PAR and have been reported to act downstream of PARP-1. As free ADP-ribose is the final product of PARP/PARG cycle we tested the hypothesis, that PARG inhibition by RNAi could abolish the Ca2+ gating function of TRPM2. Indeed, we could demonstrate that cytososlic Ca2+ elevations after H2O2 were diminished when cells are silenced for the parg gene leading to a reduced cytotoxicity mediated by an impaired AIF translocation. Moreover, RNAi against TRPM2 as well as chemical inhibition of this channel showed a similar pattern in cytosolic Ca2+ fluxes after an oxidative insult. ADP-ribose loading of cells induces TRPM2 mediated Ca2+ fluxes in the absence of oxidative stress, suggesting that ADP-ribose is the key metabolite of the PARP/PARG cycle regulating TRPM2. We conclude that the interplay of PARP-1 and PARG controls a cell death pathway that operates between nucleus, cytoplasm, cell membrane and mitochondria and communicates via three types of chemical messengers: a nucleotide, a cation and proteins. Overall the results of this study contribute to the understanding of the specific involvements of PARP-1, PARP-2 and especially PARG in the response to DNA damage and the subsequent signal cascade that translates the duration of genotoxic insults into the cell fate of programmed cell death

Messung von zytosolischem Ca2+ als Indikator toxischer Zellschädigung

Oxidativer Stress ist toxisch und reduziert dosisabhängig den Anteil vitaler Zellen. Der erhöhte zytosolische Spiegel freier Ca2+-Ionen ist ein unmittelbarer Parameter nach der Applikation des ROS-Agens H2O2. Der Anstieg des zytosolischen Ca2+-Spiegels aus internen sowie auch aus externen Ca2+-Quellen kann mittels fluoreszenzspektrometrischer Analyse des Kalzium-bindenden Farbstoffs Fluo-4, AM bestimmt werden