Increased poly(ADP-ribose) polymerase activity during repair of ({+/-})-anti-benzo[a]pyrene diolepoxide-induced DNA damage in human peripheral blood lymphocytes in vitro

Poly(ADP-ribose) polymerase, which catalyzes the formation ofpoly(ADP-ribose) polymers, is an enzyme involved in cell proliferation,differentiation and transformation as well as in recovery fromDNA damage. Poly(ADP-ribose) polymers are rapidly synthesizedfrom the ADP-ribose moieties from intracellular NAD⁺ which,as a consequence, is depleted. It has been shown that DNA strandbreaks are required for enzyme activation and it is suggestedthat one of the functions of poly(ADP-ribosylation) is to improveaccessibility of damaged sites to other DNA repair enzymes.The aim of this study was to investigate whether poly(ADP-ribosylation)is involved in repair of (±)-7ß,8     

Targeting poly(ADP-ribose) polymerase: a two-armed strategy for cancer therapy.

The DNA repair pathways are protective of the host genome in normal cells; however, in cancer cells, these pathways may be disrupted and predispose to tumorigenesis or their activity may overcome the potentially cytotoxic damage caused by anticancer agents and be a mechanism of resistance. Poly(ADP-ribose) polymerase inhibitors, which block base excision repair of single-strand breaks, have entered the clinic in the last few years. This article discusses the interactions between the pathways of single- and double-strand break repair, which explain the two clinical development strategies for this class of drugs.     

Poly(ADP-ribose)polymerase (PARP) Inhibitors: From Bench to Bedside

Poly(ADP-ribose)polymerase (PARP) inhibitors are a novel class of anticancer agents that target the DNA damage response pathways. The enzyme target, PARP, plays a key role in signalling DNA single-strand breaks. Clinical development to date has focused on their potential role in combination with DNA-damaging chemotherapy, where efficacy has been limited by enhanced normal tissue toxicity, and as single agents in the context of synthetic lethality. This article reviews these data in the context of future development as radio-potentiating agents.     

Reduced poly(ADP-ribosyl)ation in lymphocytes of laryngeal cancer patients: results of a case-control study

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme that is catalytically activated by DNA strand breaks, plays a complex role in DNA repair. Using NAD(+) as a precursor, it catalyzes the formation of ADP-ribose polymers, which are attached to various proteins. Defects in DNA repair pathways have been associated with increased risks for cancer in humans. We investigated whether differences in the activity of PARP are associated with the risk for laryngeal cancer. In a case-control study on genetic, lifestyle and occupational risk factors for laryngeal cancer, PARP activity was assessed as DNA damage-induced poly(ADP-ribose) formation in human peripheral blood lymphocytes by quantitative immunofluorescence analysis. Polymer formation was determined as the cellular response to bleomycin, a well-known inducer of DNA strand breaks, in lymphocytes from 69 laryngeal cancer patients and 125 healthy controls. The frequency of bleomycin-induced polymer formation, measured as mean pixel intensity, was significantly lower in cases (74.6, SE = 3.7) than in controls (94.5, SE = 3.5) and not influenced by smoking, age or sex. There was no significant difference between cases (59.1, SE = 5.2) and controls (50.5, SE = 3.7) in basal polymer formation (in cells not treated with bleomycin). When the highest tertile of polymer formation was used as the reference, the odds ratio for the lowest tertile of bleomycin-induced polymer formation was 3.79 (95% confidence interval 1.37-10.47, p = 0.01). Peripheral blood lymphocytes from laryngeal cancer patients thus showed significantly less bleomycin-induced poly(ADP-ribose) formation. Our results suggest that a reduced capacity of somatic cells to synthesize poly(ADP-ribose) might be associated with an increased risk for laryngeal cancer. The underlying mechanism remains to be investigated.     

Functional overexpression of human poly(ADP-ribose) polymerase in transfected rat tumor cells

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a nuclear enzyme possibly involved in DNA base excision repair. The presence of single- or double-strand breaks in DNA stimulates this enzyme to covalently modify acceptor proteins with poly(ADP-ribose) in a reaction that uses NAD+ as substrate. To test the hypothesis that increased PARP activity could promote resistance towards DNA-damaging agents and gamma- radiation, we established stable rat cell transfectants that constitutively express human PARP. A number of subclones that showed different levels of PARP activity were isolated from two primary transfectants of different clonal origin. PARP activity was determined in permeabilized cells after maximal stimulation with a short, double- stranded oligonucleotide. Activity in different human PARP-expressing subclones was increased 1.6- to 3.1-fold compared with non-expressing subclones. In vivo labeling of poly(ADP-ribose) was performed in one of these subclones, revealing that the level of poly(ADP-ribose) accumulation after the same treatment with N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) was four times higher in the human PARP- expressing subclone compared with both non-expressing transfected control cells and parental cells. Clonal survival assays revealed a sensitization upon treatment with gamma-radiation (up to 1.4-fold) or MNNG (up to 2.7-fold) of several subclones expressing human PARP; in some others survival was not changed. Survival after cisplatin (DDP) treatment remained essentially unchanged. A protective effect against DNA-damage was never observed. We conclude that human PARP overexpression in rodent cells leads to increased poly(ADP- ribosyl)ation capacity and does not promote survival after gamma- radiation or treatment with the DNA-damaging agents MNNG or DDP.      

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers

Background

DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects.

Methods

Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures.

Results

Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity.

Conclusions

It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.     

Poly(ADP-ribose) polymerase in DNA damage-response pathway: implications for radiation oncology

Poly(ADP-ribose) polymerase (PARP) catalyzes the transfer of successive units of ADP-ribose moiety from NAD(+) covalently to itself and other nuclear acceptor proteins. PARP is a zinc finger-containing protein, allowing the enzyme to bind to either double- or single-strand DNA breaks without any apparent sequence preference. The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification. Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with the breaking and rejoining of DNA strands, and plays a pivotal role in DNA damage repair. Recent advances in apoptosis research identified PARP as one of the intracellular "death substrates" and demonstrated the involvement of polymerase in the execution of programmed cell death. This review summarizes the biological effects of PARP function that may have a potential for targeted sensitization of tumor cells to genotoxic agents and radiotherapy. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 59-67 (2000).     

The potential for poly (ADP-ribose) polymerase inhibitors in cancer therapy

The modulation of DNA repair pathways for therapeutic benefit in cancer has now become a reality with the development of poly (ADP-ribose) polymerase inhibitors (PARPi). PARP is involved in single-strand DNA breaks, which in the presence of defective homologous recombination repair lead to double-strand DNA breaks, the most lethal form of DNA damage. These agents therefore may be the drugs of choice for BRCA mutant breast and ovarian cancers. PARPi result in synergistic antitumor effects when combined with cisplatin, temozolomide, topoisomerase inhibitors and ionizing radiation. The indications for PARPi lie beyond BRCA mutations and may include genomic and functional defects in DNA repair and damage response pathways. Several PARPi are in the clinical development phase at this time and, given the recent failure of a phase III clinical trial of iniparib in triple-negative breast cancer, the identification of structural and functional differences between these inhibitors becomes critical. Acquired resistance to PARPi is being noted and represents an important limitation in this field. A concise review of the literature in this field is presented.     

Current development of clinical inhibitors of poly(ADP-ribose) polymerase in oncology.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that signals the presence of DNA damage by catalyzing the addition of ADP-ribose units to DNA, histones, and various DNA repair enzymes and by facilitating DNA repair. PARP has been gaining increasing interest as a therapeutic target for many diseases and especially for cancer. Inhibition of PARP potentiates the activity of DNA-damaging agents, such as alkylators, platinums, topoisomerase inhibitors, and radiation in in vitro and in vivo models. In addition, tumors with DNA repair defects, such as those arising from patients with BRCA mutations, may be more sensitive to PARP inhibition. At least five different companies have now initiated oncology clinical trials with PARP inhibitors, ranging in stage from phase 0 to phase 2. This review summarizes the preclinical and clinical data currently available for these agents and some of the challenges facing the clinical development of these agents.     

DNA ligation and changes in chromatin structure associated with repair patches under conditions of inhibition of poly (ADP-ribose) synthesis

The rate of intracellular ligation of excision repair patcheshas been measured under conditions of inhibition of poly(ADP-ribose)synthesis by 3-aminobenzamide. Excision repair patches in DNAof cells damaged by methyl methane-sulfonate were labeled with(³H]thymidine and blocked at an intermediate stage by aphidicolin,an inhibitor of DNA polymerase alpha. Nearly half of the [³H]thymidinelabel in the repair patches was sensitive to rapid digestionby exo-nudease III, indicating that the label was at unligated3' termini of repair sites. Removal of [³H]thymidine and aphidicolinpermitted the intracellular ligation rate to be determined.From analysis of chromatin, ligation appeared to occur rapidly,independent of the effect of 3-aminobenzamide. Analysis of purifiedDNA, however, indicated that high doses of methyl methanesulfonateresulted in slow ligation rates but that 3-aminobenzamide acceleratedthe rates of ligation. The analysis of chromatin, therefore,indicates that unligated repair sites are sites of protein accretionwhich block exo-nuclease III action. The results from analysisof DNA indicate that poly(ADP-ribose) synthesis and associatedpool depletion inhibits ligation rates; 3-aminobenzamide preventspoly(ADP-ribose) synthesis, maintains pool levels high and facilitatesrapid ligation.     

Absence of stimulation of poly(ADP-ribose) polymerase activity in patients predisposed to colon cancer.

Poly(ADP-ribose)polymerase (PARP) has been implicated in DNA repair mechanisms and the associated activity shown to markedly increase after DNA damage in carcinogen-treated cells. A defective DNA repair has been associated to the aetiology of human cancers. In order to assess the potential role of this enzyme in cellular response to DNA damage by gamma-radiation, we studied the activity of PARP in patients with familial adenomatous polyposis (FAP). We compared poly(ADP-ribose)polymerase activity by the rate of incorporation of radioactivity from [3H]adenine-NAD+ into acid-insoluble material in permeabilized leucocytes from FAP patients and healthy volunteers. Concomitantly, the intracellular levels of NAD+--the substrate for the PARP--and the reduced counterpart NADH were determined using an enzymatic cycling assay 30 min after [60Co] gamma-ray cells irradiation. Our results demonstrate that a marked stimulation of PARP activity is produced upon radiation of the cells from healthy subjects but not in the FAP leucocytes, which concomitantly show a marked decrease in total NAD-/NADH content. Our observations point to a role of PARP in the repair of the gamma-radiation-induced DNA lesions through a mechanism that is impaired in the cells from FAP patients genetically predisposed to colon cancer. The differences observed in PARP activation by gamma-radiation in patients and healthy individuals could reflect the importance of PARP activity dependent on treatment with gamma-rays. The absence of this response in FAP patients would seem to suggest a possible defect in the role of PARP in radiation-induced DNA repair in this cancer-prone disease.     

Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice

Poly(ADP-ribose) polymerase (PARP) is a constitutive factorof the DNA damage surveillance network in dividing cells. Basedon its capacity to bind to DNA strand breaks, PARP plays a regulatoryrole in their resolution in vivo. ATM belongs to a large familyof proteins involved in cell cycle progression and checkpointsin response to DNA damage. Both proteins may act as sensorsof DNA damage to induce multiple signalling pathways leadingto activation of cell cycle checkpoints and DNA repair. To determinea possible relationship between PARP and ATM, we examined thePARP response in an ATM-null background. We demonstrated thatATM deficiency does not affect PARP activity in human cell linesor Atm-deficient mouse tissues, nor does it alter PARP activityinduced by oxidative damage or -irradiation. Our results supporta model in which PARP and ATM could be involved in distinctpathways, both effectors transducing the damage signal to cellcycle regulators.     

PARP抑制剂的临床研究进展

肿瘤细胞对化疗药物耐药是导致化疗失败的重要原因。肿瘤细胞DNA修复基因的过表达引起DNA损伤后修复并最终导致了耐药性的产生。聚腺苷酸二磷酸核糖基聚合酶（PARP）是具有碱基切除修复功能的DNA单链损伤修复酶,由于其在肿瘤细胞内过表达并在肿瘤细胞DNA损伤修复过程中发挥着重要作用,被认为是肿瘤靶向治疗领域的重要靶点。近年来关于PARP抑制剂的研究屡见报道,发现PARP抑制剂不仅对放化疗具有一定的增敏效果,且在特定基因型瘤种中还具有显著的单独抗肿瘤效应。本文旨在对近年来PARP抑制剂的最新临床研究进展进行综述。     

Poly(ADP-ribose) polymerase-1 and ionizing radiation: sensor, signaller and therapeutic target

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that binds rapidly to single and double-strand breaks in DNA and consequently modifies a range of nuclear proteins involved in the cellular response to ionising radiation. PARP-1 knockout mice are highly sensitive to ionising radiation, and inhibition or depletion of PARP-1 brings about modest sensitisation of cells in culture to radiation doses of 2 Gy and above. In certain cell lines, chemical inhibition of PARP activity is also associated with marked sensitisation to very low doses of radiation (<0.5 Gy). The mechanisms underlying these effects are discussed, and possible therapeutic applications of PARP-1 manipulation in combination with ionising radiation are considered.     

Poly(ADP-ribose)polymerase-1 (PARP-1) in carcinogenesis: potential role of PARP inhibitors in cancer treatment

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear, zinc-finger, deoxyribonucleic acid (DNA)-binding protein that detects specifically DNA strand breaks generated by different genotoxic agents. Whereas activation of PARP-1 by mild genotoxic stimuli facilitates DNA repair and cell survival, severe DNA damage triggers different pathways of cell death, including PARP-mediated cell death through the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. Pharmacological inhibition or genetic ablation of PARP-1 results in a clear benefit in cancer treatment by different mechanisms, including selective killing of homologous recombinationdeficient tumor cells, downregulation of tumor-related gene expression, and decrease in the apoptotic threshold in the cotreatment with chemo- and radiotherapy. We summarize in this review the findings and concepts for the role of PARP-1 and poly(ADP-ribosylation) in the regulation of carcinogenesis and some of the preclinical and clinical data available for these agents, together with the challenges facing the clinical development of these agents.     

TSL-1502, a glucuronide prodrug of a poly (ADP-ribose) polymerase (PARP) inhibitor,exhibits potent anti-tumor activity in preclinical models

Poly (ADP-ribose) polymerase (PARP) enzymes play an important role in the cellular response to DNA damage and the inhibition of PARP causes synthetic lethality in homologous recombination (HR)-deficient cancer. Multiple PARP inhibitors have been developed and have shown remarkable clinical benefits. However, treatment-related toxicities, especially the hematologic toxicities, are common and restrict the clinical applications of PARP inhibitors. In this study, we designed the first glucuronide prodrug of PARP inhibitor, TSL-1502, based on a novel and highly potent PARP inhibitor TSL-1502M. TSL-1502M exhibited promising inhibitory activity on PARP1/2, significantly induced DNA double strand breaks, G2/M arrest and apoptosis in HR-deficient cells, selectively inhibited the proliferation of HR-deficient cancer cells and sensitized both HR-deficient and HR-proficient cancer cells to conventional chemotherapy. Notably, TSL-1502M was superior to olaparib, the first-in-class PARP inhibitor, in all these processes. TSL-1502 had no inhibitory effects on PARP1/2 itself, but could selectively liberate the active drug TSL-1502M in tumor after administration in nude mice. Moreover, TSL-1502 elicited significant more potent inhibitory effects than olaparib in HR-deficient tumors, and sensitized chemotherapy in both HR-deficient and HR-proficient tumors. No severe toxicities were caused by TSL-1502 in this study. Based on the encouraging preclinical antitumor activity and the selective decomposition characteristic of TSL-1502, a clinical phase I study was initiated in China, and an Investigational New Drug (IND) was granted by the US FDA. TSL-1502 could represent a new potential therapeutic choice of PARP inhibitors.     

Poly(ADP-ribose) polymerase (PARP) inhibitors: Exploiting a synthetic lethal strategy in the clinic

Poly(ADP-ribose) polymerase (PARP) is an attractive antitumor target because of its vital role in DNA repair. The homologous recombination (HR) DNA repair pathway is critical for the repair of DNA double-strand breaks and HR deficiency leads to a dependency on error-prone DNA repair mechanisms, with consequent genomic instability and oncogenesis. Tumor-specific HR defects may be exploited through a synthetic lethal approach for the application of anticancer therapeutics, including PARP inhibitors. This theory proposes that targeting genetically defective tumor cells with a specific molecular therapy that inhibits its synthetic lethal gene partner should result in selective tumor cell killing. The demonstration of single-agent antitumor activity and the wide therapeutic index of PARP inhibitors in BRCA1 and BRCA2 mutation carriers with advanced cancers provide strong evidence for the clinical application of this approach. Emerging data also indicate that PARP inhibitors may be effective in sporadic cancers bearing HR defects, supporting a substantially wider role for PARP inhibitors. Drugs targeting this enzyme are now in pivotal clinical trials in patients with sporadic cancers. In this article, the evidence supporting this antitumor synthetic lethal strategy with PARP inhibitors is reviewed, evolving resistance mechanisms and potential molecular predictive biomarker assays are discussed, and the future development of these agents is envisioned.