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Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have entered the clinics for their promising anticancer effect as adjuvant in chemo- and radiotherapy and as single agent on BRCA-mutated tumours. Poly(ADP-ribose) glycohydrolase (PARG) deficiency was also shown to potentiate the cytotoxicity of genotoxic agents and irradiation. The aim of this study is to investigate the effect of PARG deficiency on BRCA1- and/or PTEN-deficient tumour cells. METHODS: Since no PARG inhibitors are available for in vivo studies, PARG was depleted by siRNA in several cancer cell lines, proficient or deficient for BRCA1 and/or PTEN. The impact on cell survival was evaluated by colony formation assay and short-term viability assays. The effect of simultaneous PARG and BRCA1 depletion on homologous recombination (HR) efficacy was evaluated by immunodetection of RAD51 foci and using an in vivo HR assay. RESULTS: The BRCA1-deficient cell lines MDA-MB-436, HCC1937 and UWB1.289 showed mild sensitivity to PARG depletion, whereas no sensitivity was observed for the BRCA1-proficient MDA-MB-231, MDA-MB-468, MCF10A and U2OS cell lines. However, the BRCA1-reconstituted UWB1.289 cell lines was similarly sensitive to PARG depletion than the BRCA1-deficient UWB1.289, and the simultaneous depletion of PARG and BRCA1 and/or PTEN in MDA-MB-231 or U2OS cells was not more cytotoxic than depletion of BRCA1 or PTEN only. CONCLUSIONS: Some tumour cells displayed slight sensitivity to PARG deficiency, but this sensitivity could not be correlated to BRCA1- or PTEN-deficiency. Therefore, PARG depletion cannot be considered as a strategy to kill tumours cells mutated in BRCA1 or PTEN.

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Whereas the role of PARP1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP2, has not been studied so far. Aim. To find out specific DNA targets of PARP2. Methods. The EMSA and the PARP activity tests were used. Results. We evaluated Kd values of PARP2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes and tested these DNA as «activators» of PARP1 and PARP2 in poly(ADP-ribose) synthesis. Conclusions. Like PARP1, PARP2 does not show correlation between the activation efficiency and Kd values for DNA. PARP2 was activated most effectively in the presence of over5DNA. Keywords: PARP1, PARP2, poly(ADP-ribosyl)ation, DNA binding. Полі(ADP-рибозил)ювання – це тип посттрансляційної модифікації білків, який є важливим для забезпечення стабільності геному та виживання клітин у відповідь на пошкодження ДНК. Полі (ADP-рибозил)ювання каталізується полі(ADP-рибоза)полімеразами (PARP). У той час як роль PARP1 у клітинній відповіді на пошкодження ДНК детально досліджено, внесок іншої ДНК-залежної полі(ADP-рибоза)полімерази – PARP2 – вивчено поки що слабко. Мета. Виявити специфічні ДНК-мішені PARP2. Методи. Метод «затримки в гелі» (EMSA) і тест активності PARP. Результати. Проведено оціночні виміри значень Kd комплексів PARP2–ДНК для деяких структур ДНК, імітуючих інтермедіати різних процесів метаболізму ДНК, а також ці ДНК проаналізовано як «активатори» PARP1 і PARP2 у синтезі полі(ADP-рибози). Висновки. Як і для PARP1, для PARP2 не спостерігається кореляції між ефективністю активації та значенням Kd для різних ДНК. Найефективніше PARP2 активується за присутності over5DNA. Ключові слова: PARP1, PARP2, полі(ADP-рибозил)ювання, зв’язування ДНК.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in the regulation of chromatin structure, DNA metabolism, cell division and cell death. Through the hydrolysis of poly(ADP-ribose) (PAR), Poly(ADP-ribose) glycohydrolase (PARG) has a crucial role in the control of life-and-death balance following DNA insult. Comprehension of PARG function has been hindered by the existence of many PARG isoforms encoded by a single gene and displaying various subcellular localizations. To gain insight into the function of PARG in response to irradiation, we constitutively and stably knocked down expression of PARG isoforms in HeLa cells. PARG depletion leading to PAR accumulation was not deleterious to undamaged cells and was in fact rather beneficial, because it protected cells from spontaneous single-strand breaks and telomeric abnormalities. By contrast, PARG-deficient cells showed increased radiosensitivity, caused by defects in the repair of single- and double-strand breaks and in mitotic spindle checkpoint, leading to alteration of progression of mitosis. Irradiated PARG-deficient cells displayed centrosome amplification leading to mitotic supernumerary spindle poles, and accumulated aberrant mitotic figures, which induced either polyploidy or cell death by mitotic catastrophe. Our results suggest that PARG could be a novel potential therapeutic target for radiotherapy.

1471-0072 (Print) Journal Article Review; The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD(+), is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.

Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.

Poly(ADP-ribosyl)ation (PARylation), a protein post-translational modification that was originally connected to the DNA damage response, is now known to engage in a continuously increasing number of biological processes. Despite extensive research and ceaseless, important findings about its role and mode of action, poly(ADP-ribose) remains an enigma regarding its structural complexity and diversity. The recent identification and structural characterization of four different poly(ADP-ribose) binding motifs represents a quantum leap in the comprehension of how this molecule can be decoded. Moreover, the recent discovery of a direct connection between PARylation and poly-ubiquitylation in targeting proteins for degradation by the proteasome has paved the way for a new interpretation of this protein modification. These two novel aspects, poly(ADP-ribose) recognition and readout by the ubiquitylation/proteasome system are developed here.

Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.

H1 RNA, the RNA component of the human nuclear RNase P, is encoded by a unique gene transcribed by RNA polymerase III (Pol III). In this work, cis-acting elements and trans-acting factors involved in human H1 gene transcription were characterized by transcription assays of mutant templates and DNA binding assays of recombinant proteins. Four elements, lying within 100 bp of 5′-flanking sequences, were defined to be essential for maximal in vitro and in vivo expression, consisting of the octamer, Staf, proximal sequence element (PSE) and TATA motifs. These are also encountered in the promoter elements of vertebrate snRNA genes, where the first two constitute the distal sequence element (DSE). In all the genes examined so far, the DSE is distant from the PSE and TATA box that compose the basal promoter. However, we observed a fundamental difference in the organization of the H1 RNA and snRNA gene promoters with respect to the relative spacing of the DSE and PSE. Indeed, the H1 promoter is unusually compact, with the octamer motif and Staf binding site adjacent to the PSE and TATA motifs. It thus appears that the human RNase P RNA gene has adopted a unique promoter strategy placing the DSE immediately adjacent to the basal promoter.

Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms ofPARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.

Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the β-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183–193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californicacADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki = 148 µM determined for the recombinant extracellular protein domain and Ki = 180 µM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis = 47 µM and Kii = 198 µM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californicacADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells.

The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage. We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover. PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa. Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA. The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein. A single transcript of approximately 4. 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa. Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa. Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene. Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein. The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.