Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.     

Involvement of poly (ADP-ribose) in the radiation response of mammalian cells

The evidence implicating poly (ADP-ribose) in the radiation response of mammalian cells is reviewed. It is concluded that the apparently conflicting results using inhibitors of ADP-ribosyl transferase (ADPRT) can be explained by a working hypothesis. This hypothesis maintains that poly (ADP-ribose) is required for repair of radiation damage (presumably to facilitate ligation). In most cells the synthesis of poly (ADP-ribose) is not rate limiting for repair and therefore, an almost complete inhibition of ADPRT activity is required to potentiate the radiation response. In radiation-sensitive cells (e.g. resting lymphocytes, L5178Y-S cells) with a deficient poly (ADP-ribose) metabolism, its synthesis can become rate limiting for repair. In such cells even a partial inhibition of ADPRT activity may enhance radiation-induced cell killing. It is suggested that if such differences exist between normal and cancer cells, they can be utilized to improve the therapeutic ratio of radiotherapy.     

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.     

DNA-ligase IV and DNA-protein kinase play a critical role in deficient caspases activation in apoptosis-resistant cancer cells by using doxorubicin

Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.     

Poly(ADP-ribose) and the response of cells to ionizing radiation

The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.     

Poly(ADP-ribose)polymerase: a perplexing participant in cellular responses to DNA breakage

Poly(ADP-ribose) polymerase is a major nuclear protein of 116 kd, coded by a gene on chromosome 1, that plays a role in cellular responses to DNA breakage. The polymerase binds to DNA at single- and double-strand breaks and synthesizes long branched chains of poly(ADP-ribose), which covalently, but transiently, modifies itself and numerous other cellular proteins and depletes cells of NAD+. This much is known, but the physiological role of the polymerization-degradation cycle is still unclear. Poly(ADP-ribosyl)ation of proteins generally inhibits their function and can dissociated chromatin proteins from DNA. Inhibition of poly(ADP-ribose) polymerase increases to toxicity of alkylating agents and some other DNA-damaging agents and increases sister-chromatid exchange frequencies. During repair of alkylation damage, inhibition of poly(ADP-ribose) polymerase makes no change in excision of damaged products. increases the total number of repair patches, accelerates the rejoining of DNA breaks, and makes variable increases or decreases in net break frequencies. The polymerization cycle consequently is a major player in the response of cells to DNA breakage, but the game it plays is yet to be explained.     

Poly ADP-ribosylation--a cellular emergency reaction?

We propose that the activation of poly(ADP-ribose) synthetase by DNA damage serves to decrease rapidly and transiently the cellular level of NAD (by production therefrom of poly ADP-ribose). The result is a slow-down of energy-requiring reactions, in particular of replicative DNA synthesis giving cells more time to repair the damage. We do not attribute any specific role to poly ADP-ribosylated proteins in this reaction beyond their action as acceptors for poly ADP-ribose.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.     

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using ³²P-labeled NAD⁺ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.     

The rise and fall of poly(ADP-ribose): An enzymatic perspective

Human cells respond to DNA damage with an acute and transient burst in production of poly(ADP-ribose), a posttranslational modification that expedites damage repair and plays a pivotal role in cell fate decisions. Poly(ADP-ribose) polymerases (PARPs) and glycohydrolase (PARG) are the key set of enzymes that orchestrate the rise and fall in cellular levels of poly(ADP-ribose). In this perspective, we focus on recent structural and mechanistic insights into the enzymes involved in poly(ADP-ribose) production and turnover, and we highlight important questions that remain to be answered.     

DNA repair deficiency as a therapeutic target in cancer

Inhibitors of DNA repair proteins have been used in cancer therapy, mostly to potentiate the effects of cytotoxic agents. However, tumor cells frequently exhibit deficiencies in the signalling or repair of DNA damage. These deficiencies probably contribute to pathogenesis of the disease, but they also present an opportunity to target the tumor. Recently, inhibitors of poly(ADP-ribose) polymerase (PARP) have been shown to be highly selective for tumor cells with defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, particularly in the context of BRCA1 or BRCA2 mutation. It seems likely that other DNA repair processes can be targeted in a similar manner. These synthetic lethal approaches highlight how an understanding of DNA repair processes can be used in the development of novel cancer treatments.     

Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used 2 isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX, and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G₂ checkpoint abrogation, inhibition of HRR, and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 ‘deficient-like’ phenotype in p53 mutant tumor cells, while sparing normal tissue.     

A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage

The molecular role of poly (ADP-ribose) polymerase-1 in DNA repair is unclear. Here, we show that the single-strand break repair protein XRCC1 is rapidly assembled into discrete nuclear foci after oxidative DNA damage at sites of poly (ADP-ribose) synthesis. Poly (ADP-ribose) synthesis peaks during a 10 min treatment with H₂O₂ and the appearance of XRCC1 foci peaks shortly afterwards. Both sites of poly (ADP-ribose) and XRCC1 foci decrease to background levels during subsequent incubation in drug-free medium, consistent with the rapidity of the single-strand break repair process. The formation of XRCC1 foci at sites of poly (ADP-ribose) was greatly reduced by mutation of the XRCC1 BRCT I domain that physically interacts with PARP-1. Moreover, we failed to detect XRCC1 foci in Adprt1^–/– MEFs after treatment with H₂O₂. These data demonstrate that PARP-1 is required for the assembly or stability of XRCC1 nuclear foci after oxidative DNA damage and suggest that the formation of these foci is mediated via interaction with poly (ADP-ribose). These results support a model in which the rapid activation of PARP-1 at sites of DNA strand breakage facilitates DNA repair by recruiting the molecular scaffold protein, XRCC1.     

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]     

The role of poly(ADP-ribose) in the DNA damage signaling network.

DNA damage signaling is crucial for the maintenance of genome integrity. In higher eukaryotes a NAD+-dependent signal transduction mechanism has evolved to protect cells against the genome destabilizing effects of DNA strand breaks. The mechanism involves 2 nuclear enzymes that sense DNA strand breaks, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2). When activated by DNA breaks, these PARPs use NAD+ to catalyze their automodification with negatively charged, long and branched ADP-ribose polymers. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell survival-cell death pathways. A number of proteins, including histones, DNA topoisomerases, DNA methyltransferase-1 as well as DNA damage repair and checkpoint proteins (p23, p21, DNA-PK, NF-kB, XRCC1, and others) can be targeted in this manner; the interaction involves a specific poly(ADP-ribose)-binding sequence motif of 20-26 amino acids in the target domains.