Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death

Abstract

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.

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

“…PARP-1 silencing increased HeLa cell survival from 13AE4.3 % to 27AE5.6 %, while PARP-2 and PARG silencing had no significant effect. Similar findings were obtained in mouse embryonic fibroblasts show- ing that PARP-1 silencing protected against MNNGinduced cell death, whereas PARG silencing did not [30]. Simultaneous down-regulation of PARP-1 and PARP-2 offered no additional cytoprotection in HeLa cells as compared to PARP-1-silenced cells.…”

“…However, at this level, we see a dramatic accumulation of PAR molecules (Fig. 2, [30]), suggesting that silencing is sufficient to visibly reduce PAR catabolism, and this is sufficient to provide a substantial protection of both human and murine cells against oxidant-induced apoptosis ( [30], unpublished data). Thus, it seems that the type of PAR molecules relevant for MNNG-induced cell death is primarily determined by PARP-1.…”

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

“…There have been studies that have followed the kinetics of PAR chain accumulation after treatment with the alkylating agent MNNG or the oxidant H 2 O 2 20 but none on the increase of PAR following temozolomide treatment. However, studies using RNA interference have been able to show a delay in hydrolysis of nuclear PAR after treatment with H 2 O 2 and knockdown of PARG 21 .…”

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

“…In some experiments, cells were transfected at 80% confluence with Metafectene-Pro (Biointex) coated plasmid DNA, and incubation lasted o/n to allow expression of the recombinant protein. Cytosolic and nuclear extracts were prepared as described [19]. Cytosolic and mitochondrial fractions were prepared with the Qproteome Mitochondria Isolation Kit (Qiagen).…”