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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

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2+/+ and p53+/+. HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT + PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT + PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72 h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors. © 2010 Elsevier Inc. All rights reserved.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a molecular DNA damage sensor that catalyzes the synthesis of the complex biopolymer poly(ADP-ribose) (PAR) under consumption of NAD(+). PAR engages in fundamental cellular processes such as DNA metabolism and transcription and interacts noncovalently with specific binding proteins involved in DNA repair and regulation of chromatin structure. A factor implicated in DNA repair and chromatin organization is the DEK oncoprotein, an abundant and conserved constituent of metazoan chromatin, and the only member of its protein class. We have recently demonstrated that DEK, under stress conditions, is covalently modified with PAR by PARP-1, leading to a partial release of DEK into the cytoplasm. Additionally, we have also observed a noncovalent interaction between DEK and PAR, which we detail here. Using sequence alignment, we identify three functional PAR-binding sites in the DEK primary sequence and confirm their functionality in PAR binding studies. Furthermore, we show that the noncovalent binding to DEK is dependent on PAR chain length as revealed by an overlay blot technique and a PAR electrophoretic mobility shift assay. Intriguingly, DEK promotes the formation of a defined complex with a 54mer PAR (K(D) = 6 x 10(-8) M), whereas no specific interaction is detected with a short PAR chain (18mer). In stark contrast to covalent poly(ADP-ribosyl)ation of DEK, the noncovalent interaction does not affect the overall ability of DEK to bind to DNA. Instead the noncovalent interaction interferes with subsequent DNA-dependent multimerization activities of DEK, as seen in South-Western, electrophoretic mobility shift, topology, and aggregation assays. In particular, noncovalent attachment of PAR to DEK promotes the formation of DEK-DEK complexes by competing with DNA binding. This was seen by the reduced affinity of PAR-bound DEK for DNA templates in solution. Taken together, our findings deepen the molecular understanding of the DEK-PAR interplay and support the existence of a cellular "PAR code" represented by PAR chain length.

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.

Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

This chapter emphasizes that along the chain of events that induce DNA methylation-dependent chromatin condensation, a post-synthetic modification other than histone acetylation, poly(ADP-ribosyl)ation, participates in the establishment and maintenance of methylation-free regions of chromatin. In fact, several lines of in vitro and in vivo evidence have shown that poly(ADP-ribosyl)ation is involved in the control of DNA methylation pattern, protecting genomic DNA from full methylation. More recent studies have provided some clues to the understanding of the molecular mechanism(s) connecting poly(ADP-ribosyl)ation with DNA methylation. We aim here to demonstrate the direct correlation existing between inhibition of poly(ADP-ribose) polymerases and DNA hypermethylation, and to describe some possible mechanisms underlying this molecular link. We will then present our hypothesis that the inhibition of the poly(ADP-ribosyl)ation process in the cell may be responsible for the anomalous hypermethylation of oncosuppressor gene promoters during tumorigenesis and to suggest the possibility that an active poly(ADP-ribosyl)ation process is also involved in maintaining the unmethylated state of CpG islands in normal cells.

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.

Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.

After Zn2+ finger-mediated binding to a DNA break, poly(ADP-ribose) polymerase becomes automodified with long polymers of ADP-ribose. These nucleic acid-like polymers may facilitate DNA repair by noncovalently interacting with neighboring proteins. Using a novel screening technique, we have identified histones as the predominant poly(ADP-ribose)-binding species in human keratinocytes, rat hepatocytes, frog eggs, and yeast. Polymer binding is confined specifically to the histone domains responsible for DNA condensation, i.e. histone tails. Our results indicate that polymers of ADP-ribose are targeted to sites of DNA strand breaks by poly(ADP-ribose) polymerase and subsequently function to alter chromatin conformation through noncovalent interactions with histone tails.