Inhibition Of PARP Activity Research Articles

Abstract C295: Update on first-in-man trial of novel oral PARP inhibitor BMN 673 in patients with solid tumors.

Abstract Background: BMN 673 is the most potent and specific inhibitor of PARP1/2 in clinical development (IC50<1nM). In tumors genetically dependent on DNA repair by homologous recombination PARP inhibition induces synthetic lethality. PARP inhibition has also been shown to be effective in preclinical models of small cell lung cancer and Ewing sarcoma, possibly related to high PARP expression. Methods: Pharmacokinetics (PK), pharmacodynamics (PD), safety and anti-tumor activity of BMN 673 were evaluated in a 2-stage escalation/expansion study. Expansion included cohorts of pts with BRCA-related cancers, small cell lung cancer (SCLC) and Ewing sarcoma (ES). Results: (As of July 29, 2013) In escalation, 39 pts were enrolled in 9 cohorts from 25 to 1100 µg/d with 1000 µg/d being the MTD related to dose-limiting hematologic toxicity. 38 pts have been enrolled in expansion including 25 patients with BRCA related cancers. For all 77 (63F/14M) pts, median age (range) is 52 (18-81), PS, 0 (0-1) and # of prior therapies 4 (1-13). Eight ES, 7 SCLC and 48 patients with deleterious BRCA mutations and breast (n=18), ovarian (n=28) or pancreatic (n=2) cancer were enrolled. BMN 673 demonstrated good oral bioavailability and a long half-life supporting daily dosing (ASCO 2013 Abstract 2580). One related grade 4 toxicity (thrombocytopenia) occurred. Related grade 3 adverse events have included fatigue, anemia, neutropenia and thrombocytopenia, all in fewer than 15% of patients. Dose-related inhibition of PARP activity in PBMC's was observed. Response, reduction in neutrophils and platelets and the need for blood transfusions and dose reductions appear to correlate with BMN 673 exposure.Overall, the objective response (including CA-125) and clinical benefit response rates are for all BRCA patients are 60.4% and 81%, respectively. Treatment is ongoing in 5 of 7 SCLC patients and 2 of 8 ES patients with no objective responses observed yet. Conclusions: BMN 673 is well tolerated with high objective and clinical benefit response rates in heavily pre-treated ovarian and breast cancer pts with deleterious germline BRCA mutations. A phase 3 trial in metastatic breast cancer is underway. Clinicaltrial.gov ID: NCT01286987 BRCA Breast and Ovarian CancerNBRCA Tumor TypeResponse18BreastCRPRSD > 12 weeksCBR*18Breast1 (6%)9 (50%)∧14 (78%)Response - 25 RECIST evaluable; 27 CA-125 evaluable28Ovarian28OvarianCRPRCA-1251 (4%)10 (40%)19 (70%)2PancreasCRPRSD ≥ 12 weeks2Pancreas0 (0%)0 (0%)2 (100%)**∧3 breast cancer responses are not yet confirmed; *CBR: clinical benefit response; **CA19-9 normalized in 1 patient Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C295. Citation Format: Zev A. Wainberg, Johann S. de Bono, Lida Mina, Jasgit Sachdev, Lauren Averett Byers, Rashmi Chugh, Charlie Zhang, Joshua W. Henshaw, Andrew Dorr, John Glaspy, Ramesh Ramanathan. Update on first-in-man trial of novel oral PARP inhibitor BMN 673 in patients with solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C295.

First-in-human trial of novel oral PARP inhibitor BMN 673 in patients with solid tumors.

2580 Background: BMN 673 is the most potent and specific inhibitor of PARP1/2 in clinical development (IC50<1nM). In tumors genetically dependent on DNA repair by homologous recombination PARP inhibition induces synthetic lethality. Methods: Pharmacokinetics (PK), pharmacodynamics (PD), safety and anti-tumor activity of BMN 673 were evaluated in a 2-stage dose-escalation study with 3-6 patients (pts)/dose level. In dose escalation (Stage 1) cycle 1 was 6 wks, with drug taken on days 1 and 8-35, for PK and PD assays, followed by daily continuous dosing in 4-wk cycles. Stage 2 (expansion at MTD) recruits pts with tumors defective in DNA repair: Ewing sarcoma, small cell lung cancer or tumors associated with BRCA mutation (mut). Results: 39 pts (33F/6M) were enrolled in 9 cohorts from 25 to 1100 µg/d that defined a MTD of 1000 µg/d. Median (range) age was 58 (19-81), PS 0 (0-1) and # of prior therapies 4 (1-13). Tumors (# with deleterious BRCA 1/2 mut) included 23 ovarian/primary peritoneal (17); 8 breast (6); 3 pancreas; 2 colon; 1 prostate (1), and 1 mullerian carcinosarcoma. 17 and 8 pts had BRCA 1 and 2 mut, respectively. Dose-limiting thrombocytopenia occurred in 1/6 and 2/5 pts at 900 and 1100 µg/d, respectively. Potentially-related adverse events in >10% of pts (# grade 1 and 2/grade 3 and 4) included fatigue (10/0); nausea (10/0); flatulence (4/0); anemia (5/2); neutropenia (4/3); thrombocytopenia (1/3); and grade 1 alopecia (10). Inhibition of PARP activity in PBMCs was observed at doses ≥ 100 µg/d. BMN 673 plasma concentrations peaked 1-2 hrs post-dose; exposure increased dose proportionally. Steady state plasma concentrations were reached by the end of the 2nd week of daily dosing; mean Cmax: 0.30 - 25.4 ng/mL and AUC0-24: 3.96 - 203 ng-hr/mL across the 25 to 1100 µg/d dose range after 28d of daily dosing. RECIST and/or CA-125 responses occurred at doses ≥ 100 µg/d in 11/17 BRCA carrier ovarian/peritoneal cancer pts. Objective responses occurred in 2/6 BRCA-carrier breast cancer pts. Conclusions: BMN 673 is well tolerated with impressive anti-tumor activity in pts with BRCA mut with a single agent recommended Phase II trial dose of 1000 µg/d due to dose-limiting thrombocytopenia. Clinical trial information: NCT01286987.

Effect of PARP inhibitors on sensitivity of lung and breast cancer cells to trans-platinum as compared to cis-platinum.

e22115 Background: Poly(ADP-ribose) polymerase inhibitors (PARP-i) represent promising strategy in treatment of cisplatin resistant lung cancer and triple-negative breast cancer. We compared PARP inhibition by 3-aminobenzamide (3-AB) or NU1025, on activity of cis- and trans-platinum on NSCLC cells (A549) and breast cancer cells (MDA-MB-361), and correlated with p53 and ERCC1 status. Methods: Cell cycle distribution was determined by FACS-flow and PI-staining, while morphological changes were followed by light microscopy. ERCC1 siRNA transfection was performed by HiperFect reagent, and mRNA was analyzed by qRT-PCR. Protein expression levels were determined by Western blot. PARP-inhibition was quantified as NAD(P)H depletion using colorimetric XTT assay. Results: Trans-platinum compound with planar ammine ligands, trans-[PtCl2(4-acetylpyridine)2] showed cytotoxic activity comparable to cis-platin and exhibited enhanced action in combination with PARP-i. 3-AB enhanced the activity of trans more efficient comparing to cis-platinum in MDA-MB-361 cells. At equitoxic concentrations corresponding to IC50 values in A549 cells, cis-platin and trans-platinum compound differently affected cell survival in combination with PARP-i. XTT assay confirmed efficient inhibition of PARP activity at 200 μM NU1025 and 2 mM 3-AB in both cell lines. ERCC1 silencing in p53 proficient A549 cells, alone or in combination with NU1025, almost completely diminished resistance to trans-[PtCl2(4-acetylpyridine)2], decreasing cell survival to 50 % following 24 h action and more than 85 % following 48 h treatment, comparing to siRNA/NU1025 treated control. By contrast, sensitivity to cisplatin was much less altered. Simultaneous alteration of ERCC1 and PARP itself did not affect cell cycle progression, but prolonged sub-G1 fraction by trans-platinum as well as G2-M fraction by cis-platin. Conclusions: PARP-inhibition enhanced tumor cell sensitivity to trans-platinum drug, stronger than cis-platinum. Knockdown of ERCC1 further increases sensitivity to platinum drugs, irrelevant to p53 status. Trans-platinum complexes should be further explored as good candidates for combining with PARP-i.

205 POLY(ADP-RIBOSE) POLYMERASE-14 INTERACTS WITH TRISTETRAPROLIN TO SELECTIVELY REGULATE TISSUE FACTOR MRNA STABILITY: A NOVEL ROLE FOR ADP-RIBOSYLATION IN REGULATING MRNA TURNOVER AND THROMBOSIS

BackgroundTargeting post-transcriptional pathways is now emerging as a promising therapeutic strategy in a wide spectrum of diseases. Monocyte-derived tissue factor (TF) plays critical roles in atherothrombosis, but little is known...

Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1

Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity.

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.

Abstract 1774: Dihydroartemisinin (DHA): A potent enhancer of PARP inhibitors and DNA-damaging agents activity in human tumor models

Abstract Synthetic lethality (SL) is a cellular condition in which two or more non-allelic and non-essential mutations, which are not lethal on their own, become deadly when present within the same cell. PARP inhibitors (PARPi) represent a paradigmatic example of therapeutic application of the SL approaches. In cancer cells with BRCA1 or BRCA2 loss of function, inhibition of PARP1 activity leads to an accumulation of single strand breaks converted to double strand breaks but cannot be repaired by homologous recombination. However, preclinical and preliminary clinical evidences suggest a potentially broader scope for PARPi. Currently, several PARPi are under different phases of clinical investigation as monotherapy or in combination with DNA damaging agents. Although PARPi in monotherapy are quite safe, their combination with cytotoxic agents leads to exacerbation of the typical cytotoxic-related side effects, thus reducing the potentiality to improve the therapeutic index (T.I.) of these combinations. The study aim was to assess if the combination of PARPi and DNA-damaging agents with the widely used antimalarial agent DHA could improve both their antitumor activity and T.I. We have preliminary observed, in different cancer cells, that DHA was an inducer of endoplasmic reticulum (ER) stress (increased expression of typical ER stress-related genes), activation of checkpoint kinases and transcriptional up-regulation of different p53-target genes and of mitochondrial membrane depolarization. A consequence is PARP activation that, in absence of other factors, leads the cells to recover from damage and to their rescue. On the contrary, we observed that the simultaneous use of DHA with a PARPi or cytotoxic agents causes a massive cell death. More specifically a strong synergism was observed on tumor cells, when DHA was combined with different drugs: doxorubicin and platinum compounds on NCI-H460, temozolomide (TMZ) on MDA-MB436 and CAPAN-1. In addition, a synergistic interaction was identified between DHA and the PARPi (ABT-888 or AZD-2281) on various tumor cells (e.g., A431, HCT116, SW620, MDA-MB436, NCI-H460, A2780/DDP, CAPAN-1). In vivo, the combination of DHA (delivered at 200 mg/kg p.o. according to the schedule qdx5/w) with ABT-888 or AZD-2281, resulted in a significantly higher tumor growth inhibition than with monotherapy in NCI-H460 NSCLC, HCT116 colon ca. and MDA-MB436 triple-negative breast cancer BRCA1 negative xenografted in nude mice. DHA was also able to synergize in vivo with the alkylating agent TMZ against MDA-MB436 tumor model. No toxic effects of administrations of DHA in combination with chemotherapeutics in terms of body weight recordings were found throughout the experiments.Taken together, these results clearly pinpoint the clinical potential offered by DHA in the SL approaches in developing less toxic and more efficacious therapies to treat cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1774. doi:1538-7445.AM2012-1774

Iniparib Nonselectively Modifies Cysteine-Containing Proteins in Tumor Cells and Is Not a Bona Fide PARP Inhibitor

PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are β-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite. Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins. All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.

Increased PARP-1 levels in nuclear matrix isolated from heat shock treated rat liver

Poly(ADP-ribose) polymerase-1 (PARP-1), a chromatin-associated enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers onto a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. PARP-1 levels in the nuclear matrix vary depending on the matrix isolation method used. The nuclear matrix appears to be the most thermosensitive nuclear structure during heat shock. Here we provide evidence for the extensive translocation of PARP-1 from chromatin to the nuclear matrix during heat shock. This translocation is accompanied by inhibition of PARP activity in the nucleus and elevation of PARP activity in the nuclear matrix. Our data suggest that thermal destabilization of the nuclear matrix is less likely to contribute to the translocation of PARP-1 to the nuclear matrix.

All PARP inhibitors are not equal: An in vitro mechanistic comparison of PF-01367338 to iniparib.

e13576 Background: PARP is a key enzyme involved in base-excision repair. Pre-clinically it has been shown that BRCA1/2 deficient cells are hyper-sensitive to PARP inhibition. These cells can’t repair replication associated double-strand breaks that result from PARP inhibition due to defects in homologous recombination repair. Iniparib (BSI-201) is an allosteric PARP inhibitor that, in its activated form, reacts with PARP in the DNA binding domain. This interaction inhibits DNA damage induced activation of isolated PARP protein resulting in inhibition in vitro. PF-01367338 is an enzymatic active site directed inhibitor. This study compared iniparib with a potent competitive inhibitor, PF-01367338 (formerly AG014699), in a cellular environment. Methods: PARP inhibition was evaluated using purified enzyme and cell extracts in standard biochemical assays. Inhibition of poly(ADP-ribose)(PAR) formation in live cells (MDA-MB-231/436) was determined using an anti-PAR antibody ELISA assay. PARP activity in tumor cell homogenates was measured by radioactive polymer formation assay. Results: PF-01367338 inhibited PARP enzymatic activity and PAR formation in cells with a potency of 5 nM. PF-01367338 inhibited proliferation of MDA-MB-436 cells with an IC50 of 1.2 uM. In addition, PAR formation in tumor-bearing mice was fully inhibited within 30 min at 10 mg/kg. In contrast, iniparib did not inhibit enzymatic activity or PAR formation in vitro. Incubation of iniparib with cell lysates resulted in inhibition of PARP activity with an IC50 of 200 nM, demonstrating that the active nitroso compound can form in these cells. However, iniparib did not induce antiproliferative activity in treated cells. Conclusions: The lack of PARP inhibition by iniparib in two BRCA deficient cell lines suggests that while this inhibitor can inhibit PARP in vitro, it may not be able to inhibit PARP in a complex cellular environment. Additionally, iniparib inhibits other enzymes including GAPDH and caspase-3, suggesting that results seen for iniparib in the clinic may not be due solely to, or reflect PARP inhibition. In contrast, the primary mechanism-of-action of catalytic PARP inhibitors such as PF-01367338 is likely to be related to PARP inhibition.

Cordycepin Blocks Lung Injury-Associated Inflammation and Promotes BRCA1-Deficient Breast Cancer Cell Killing by Effectively Inhibiting PARP

Cordycepin has been shown to interfere with a myriad of molecular processes from RNA elongation to kinase activity, and prevents numerous inflammatory processes in animal models. Here we show in a mouse model of LPS-induced acute lung injury that cordycepin prevents airway neutrophilia via a robust blockade of expression of several inflammatory genes, including the adhesion molecule ICAM-1 and VCAM-1, the cytokine/chemokine MCP-1, MIP-1α, MIP-2 and KC, and the chemokine receptor CXCR2. Such a blockade appears to be related to a severe reduction in TNF-α expression. Interestingly, in an in vitro system of A549 epithelial cell inflammation, cordycepin effectively blocked LPS-induced, but not TNF-α-induced, VCAM-1 expression. Such effects correlated with a marked reduction in p65-NF-κB activation as assessed by its phosphorylation at serine-536 but without an apparent effect on its nuclear translocation. The effects of cordycepin on the expression of VCAM-1 and ICAM-1, and of NF-κB activation and nuclear translocation upon TNF-α stimulation resembled the effects achieved upon poly(ADP-ribose) polymerase (PARP) inhibition, suggesting that cordycepin may function as a PARP inhibitor. Indeed, cordycepin blocked H(2)O(2)-induced PARP activation in A549 cells. In a cell-free system, cordycepin inhibited PARP-1 activity at nanomolar concentrations. Similar to PARP inhibitors, cordycepin significantly induced killing of breast cancer susceptibility gene (BRCA1)-deficient MCF-7 cells, supporting its therapeutic use for the treatment of BRCA-deficient breast cancers. With added antiinflammatory characteristics, therapies that include cordycepin may prevent potential inflammation triggered by traditional chemotherapeutic drugs. Cordycepin, to the best of our knowledge, represents the first natural product possessing PARP inhibitory traits.

PARP-2 Regulates SIRT1 Expression and Whole-Body Energy Expenditure

SIRT1 is a NAD(+)-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here, we tested whether deletion of PARP-2, an alternative NAD(+)-consuming enzyme, impacts on NAD(+) bioavailability and SIRT1 activity. Our results indicate that PARP-2 deficiency increases SIRT1 activity in cultured myotubes. However, this increase was not due to changes in NAD(+) levels, but to an increase in SIRT1 expression, as PARP-2 acts as a direct negative regulator of the SIRT1 promoter. PARP-2 deletion in mice increases SIRT1 levels, promotes energy expenditure, and increases mitochondrial content. Furthermore, PARP-2(-/-) mice were protected against diet-induced obesity. Despite being insulin sensitized, PARP-2(-/-) mice were glucose intolerant due to a defective pancreatic function. Hence, while inhibition of PARP activity promotes oxidative metabolism through SIRT1 activation, the use of PARP inhibitors for metabolic purposes will require further understanding of the specific functions of different PARP family members.

Poly(ADP-ribosyl)ation affects stabilization of Che-1 protein in response to DNA damage

Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes a post-translational modification that plays a crucial role in coordinating the signalling cascade in response to stress stimuli. During the DNA damage response, phosphorylation by ataxia telangiectasia mutated (ATM) kinase and checkpoint kinase Chk2 induces the stabilization of Che-1 protein, which is critical for the maintenance of G2/M arrest. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after the inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo.

Lipoxygenases and Poly(ADP-Ribose) Polymerase in Amyloid Beta Cytotoxicity

The 12/15-lipoxygenase(s) (LOX), poly(ADP-ribose) polymerase (PARP-1) activity and mitochondrial apoptosis inducing factor (AIF) protein in the amyloid β (Aβ) toxicity were investigated in PC12 cells that express either wild-type (APPwt) or double Swedish mutation (APPsw) forms of human Aβ precursor protein. Different levels of Aβ secretion and free radicals formation characterize these cells. The results demonstrated a relationship between the Aβ levels and LOX protein expression and activity. High Aβ concentration in APPsw cells correlated with a significant increase in free radicals and LOX activation, which leads to translocation of p65/NF-κB into the nucleus. An increase in AIF expression in mitochondria was observed concurrently with inhibition of PARP-1 activity in the nuclear fraction of APPsw cells. We suggested that AIF accumulation in mitochondria may be involved in adaptive/protective processes. However, inhibition of PARP-1 may be responsible for the disturbances in transcription and DNA repair as well as the degeneration of APP cells. Under conditions of increased nitrosative stress, evoked by the nitric oxide donor, sodium nitroprusside (SNP, 0.5 mM), 70–80% of all cells types died after 24 h, significantly more in APPsw cells. There was no further significant change in mitochondrial AIF level and PARP-1 activity compared to corresponding non-treated cells. Only one exception was observed in PC12 control, where SNP significantly inhibits PARP-1 activity. Moreover, SNP significantly activated gene expression for 12/15-LOX in all types of investigated cells. Inhibitors of all LOX isoforms and specific inhibitor of 12-LOX enhanced the survival of cells that were subjected to SNP. We conclude that the LOX pathways may play a role in Aβ toxicity and in nitrosative-stress-induced cell death and that inhibition of these pathways offers novel protective strategies.

HPuf-A/KIAA0020 Modulates PARP-1 Cleavage upon Genotoxic Stress

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3.

Abstract 4985: Identification of PARP-1 as a novel KLF8 interacting and regulating protein

Abstract KLF8 transcription factor regulates important genes including cyclin D1 and E-cadherin and plays a critical role in cell cycle progression, oncogenic transformation and EMT. Transcription factors need to interact with both DNA and other proteins in order to regulate transcription. However, little is known about KLF8-protein interaction. Using mass spectrometry and co-immunoprecipitation (co-IP), we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as one of novel proteins that interact with KLF8. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interation with PARP1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interation was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of the wild-type mouse embryonic fibroblasts (MEF), but was mislocalized in the perinuclear cytoplasm of the PARP-1−/− MEFs, which could be rescued by re-expression of PARP1. Interestingly, while the PARP1-interation deficient KLF8 mutant was also mislocalized in the cytoplasm of the wild-type MEFs, inhibition of PARP1 activity in the MEFs did not affect the nuclear localization of wild-type KLF8. Cychloheximade (CHX)-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted. Ubiquitination assay showed heavier ubiquitin chains in PARP-1−/− MEFs or when KLF8-PARP-1 interaction was disrupted. Furthermore, DNA damage assay under Doxorubin treatment indicated KLF8 involved in PARP-1-dependent DNA damage repair pathway. Those results suggest that PARP-1 work as a co-activator in KLF8's transcriptional activity on Cyclin D1 promoter, keep KLF8's nuclear localization, inhibit KLF8's ubiquitination and contribute to KLF8's stability. This work has identified PARP1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, function and stability in the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4985.

Abstract 1974: Nuclear BCL2 inhibits PARP1 enzymatic activity, DNA repair, and necrotic cell death

Abstract BCL-2 is overexpressed in many types of cancer through a variety of genetic, epigenetic and posttranscriptional mechanisms. The physiologic role of BCL2 is to suppress apoptosis by sequestering BH3 domain-containing pro-apoptotic factors. We demonstrate that some tumor cells with BCL2 overexpression have constitutive localization of BCL2 within the nucleoplasm and that DNA damage induces mobilization of BCL2 to the chromatin. To identify factors that mediate this response, we irradiated lymphoid tumor cells and then performed immunoprecipitation of the chromatin protein fraction using an anti-BCL2 antibody. Mass spectrometry of a unique protein isolated only after DNA damage identified PARP1 (poly(ADP) ribose polymerase 1), an enzyme involved in various cellular processes, including base excision repair and alternative nonhomologous end-joining. In response to DNA damage, PARP1 utilizes NAD to add poly(ADP) ribose (PAR) onto acceptor proteins including itself, which provides a scaffold for additional damage response factors. We confirmed the BCL2-PARP1 interaction in additional tumor cell lines and transgenic mouse embryonic fibroblasts that overexpress BCL2 but lack the apoptosis effectors BAX and BAK. Purified BCL2 and PARP1 interacted directly in vitro independently of both DNA and NAD. Small molecule PARP inhibitors did not interfere with the BCL2-PARP1 complex, suggesting that the interaction is not dependent on PAR. In contrast, the BH3 mimetic ABT-737 blocked the BCL2-PARP1 interaction in a dose-dependent manner, indicating that PARP1 binding by BCL2 involves the BCL2 BH3-binding pocket. In fact, PARP1 contains 9 consensus BH3 motifs (LXXXXD). Interaction with PARP1 was specific to BCL2 and was not observed with the anti-apoptotic factors BCL-xL or MCL1. Using an anti-PAR enzyme immunoassay, the addition of BCL2 to PARP1 inhibited PARP1 activity in vitro. This effect was reversed by adding ABT-737. In addition, we used the alkaline comet assay to determine the effect of BCL2-PARP1 interaction on base excision repair. PARP1-mediated repair of single-strand DNA breaks induced by hydrogen peroxide was attenuated in BCL2 overexpressing cells. ABT-737 increased PARP1-mediated repair, both in tumor cells rendered resistant to apoptosis through the addition of a caspase inhibitor and in embryonic fibroblasts that overexpress BCL2 but lack BAX and BAK. In the latter cells, restoration of PARP1-dependent repair by ABT-737 promoted necrotic cell death through the exhaustion of cellular NAD. This suggests that nuclear BCL2 blocks clastogen-mediated killing by inhibiting PARP1 activity, even in cells that are resistant to BCL2 inhibitors through loss of apoptosis effectors or overexpression of antiapoptotic factors like MCL1. As such, inhibition of BCL2 interactions could synergize with DNA damaging agents independently of effects on apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1974.

Inhibition of PARP activity by PJ‐34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment.

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM

By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited the duration of the S phase delay. The combination of MMS and 4-AN resulted in ATM and Chk2 phosphorylation and the time course of phosphorylation for both kinases correlated with the S phase delay. Chk2 phosphorylation was reduced in the absence of ATM activity. The Chk2 phosphorylation that remained in the absence of ATM appeared to be dependent on ATR and DNA-PK. The results demonstrate that, following initiation of base excision repair and inhibition of PARP activity, ATM activation is critical for preventing the cell from progressing through S phase, and for protection against MMS-induced cytotoxicity.

Aprataxin, poly-ADP ribose polymerase 1 (PARP-1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1 (AOA1), is a DNA repair protein that processes the product of abortive ligations, 5' adenylated DNA. In addition to its interaction with the single-strand break repair protein XRCC1, aprataxin also interacts with poly-ADP ribose polymerase 1 (PARP-1), a key player in the detection of DNA single-strand breaks. Here, we reveal reduced expression of PARP-1, apurinic endonuclease 1 (APE1) and OGG1 in AOA1 cells and demonstrate a requirement for PARP-1 in the recruitment of aprataxin to sites of DNA breaks. While inhibition of PARP activity did not affect aprataxin activity in vitro, it retarded its recruitment to sites of DNA damage in vivo. We also demonstrate the presence of elevated levels of oxidative DNA damage in AOA1 cells coupled with reduced base excision and gap filling repair efficiencies indicative of a synergy between aprataxin, PARP-1, APE-1 and OGG1 in the DNA damage response. These data support both direct and indirect modulating functions for aprataxin on base excision repair.