F. Javier Oliver

PARP-1 is involved in autophagy induced by DNA damage

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. PARP-1 has been implicated in different pathways leading to cell death and its inhibition potentiates chemotherapy-induced cell death. Whether PARP-1 participates in the cell’s decision to commit to autophagy following DNA damage is still not known. To address this issue PARP-1 wild type and deficient cells have been treated with a dose of doxorubicin that induces autophagy. Electron microscopy examination and GFP-LC3 transfection revealed autophagic vesicles and increased expression of genes involved in autophagy (bnip-3, cathepsin b and l and beclin-1) in wild type cells treated with doxo but not in parp-1-/- cells or cells treated with a PARP inhibitor. Mechanistically the lack of autophagic features in PARP-1 deficient/PARP inhibited cells is attributed to prevention of ATP and NAD+ depletion and to the activation of the key autophagy regulator mTOR. Pharmacological or genetical inhibition of autophagy results in increased cell death, suggesting a protective role of autophagy induced by doxorubicin. These results suggest that autophagy might be cytoprotective during the response to DNA damage and suggest that PARP-1 activation is involved in the cell’s decision to undergo autophagy.

PARP inhibitors: New partners in the therapy of cancer and inflammatory diseases

Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD(+) to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, which currently comprises 18 members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress. PARP is involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Inhibitors of PARP-1 have been shown to enhance the cytotoxic effects of ionizing radiation and DNA-damaging chemotherapy agents, such as the methylating agents and topoisomerase I inhibitors. There are currently at least five PARP inhibitors in clinical trial development. Recent in vitro and in vivo evidence suggests that PARP inhibitors could be used not only as chemo/radiotherapy sensitizers, but also as single agents to selectively kill cancers defective in DNA repair, specifically cancers with mutations in the breast cancer-associated genes (BRCA1 and BRCA2). PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes and promotes an inflammatory response associated with multiple organ failure. Inhibition of PARP activity is protective in a wide range of inflammatory and ischemia-reperfusion-associated diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. The aim of this review is to overview the emerging data in the literature showing the role of PARP in the pathogenesis of cancer and inflammatory diseases and unravel the solid body of literature that supports the view that PARP is an important target for therapeutic intervention in critical illness.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cells response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.

PARP-1 modifies the effectiveness of p53-mediated DNA damage response.

The tumour supressor protein p53 plays a key role in the cells decision to arrest the cell cycle or undergo apoptosis following a genotoxic insult. p53 is stabilized and activated after DNA damage, however the cascade of events signalling from DNA lesions to p53 stabilization and activation is still controversial. Poly (ADP-ribosylation) of different nuclear acceptors by PARP-1 is an early event when a single strand DNA lesion is produced. We present here evidences that interplay between PARP-1 and p53 is dependent on the type of damage induced to DNA. Primary mouse embryonic fibroblasts derived from parp-1−/− mice exhibited decreased p53 accumulation and activation following γ-irradiation compared to parp-1 proficient cells. On the other hand, treatment with the single alkylating agent 2′-methyl-2′-nitrose-urea (MNU), resulted in the rapid and sustained accumulation and activation of p53 in parp-1-deficient cells, while very little accumulation was observed in parp-1+/+ cells. After IR, the turnover of the p53 inhibitory protein MDM-2 is perturbed and the level of phosphorylation of p53 at serine-15 is blunted in parp-1−/− cells. PARP-1 is determinant in the cytotoxic response to alkylating agents but only partially contributes to radiation-induced cell killing, as determined by colony forming assay. Altogether, these results suggest that PARP-1 participates in the p53 response following irradiation, resides upstream of p53 and indirectly modulates the level of phosphorylation of key substrates in this pathway while treatment with MNU results in an enhanced p53-mediated response in parp-1-null cells.

Inhibition of Poly(ADP-Ribose) Polymerase Modulates Tumor-Related Gene Expression, Including Hypoxia-Inducible Factor-1 Activation, during Skin Carcinogenesis

Poly(ADP-ribose) polymerase (PARP)-1, an enzyme that catalyzes the attachment of ADP ribose to target proteins, acts as a component of enhancer/promoter regulatory complexes. In the present study, we show that pharmacologic inhibition of PARP-1 with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) results in a strong delay in tumor formation and in a dramatic reduction in tumor size and multiplicity during 7,12-dimethylbenz(a)anthracene plus 12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis. This observation was parallel with a reduction in the skin inflammatory infiltrate in DPQ-treated mice and tumor vasculogenesis. Inhibition of PARP also affected activator protein-1 (AP-1) activation but not nuclear factor-kappaB (NF-kappaB). Using cDNA expression array analysis, a substantial difference in key tumor-related gene expression was found between chemically induced mice treated or not with PARP inhibitor and also between wild-type and parp-1 knockout mice. Most important differences were found in gene expression for Nfkbiz, S100a9, Hif-1alpha, and other genes involved in carcinogenesis and inflammation. These results were corroborated by real-time PCR. Moreover, the transcriptional activity of hypoxia-inducible factor-1alpha (HIF-1alpha) was compromised by PARP inhibition or in PARP-1-deficient cells, as measured by gene reporter assays and the expression of key target genes for HIF-1alpha. Tumor vasculature was also strongly inhibited in PARP-1-deficient mice and by DPQ. In summary, this study shows that inhibition of PARP on itself is able to control tumor growth, and PARP inhibition or genetic deletion of PARP-1 prevents from tumor promotion through their ability to cooperate with the activation AP-1, NF-kappaB, and HIF-1alpha.

PARP inhibition sensitizes p53-deficient breast cancer cells to doxorubicin-induced apoptosis

p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.

Crosstalk between PARP-1 and NF-jB modulates the promotion of skin neoplasia

Poly (ADP-ribose) polymerase-1 (PARP-1)-deficient mice are protected against septic shock, type I diabetes, stroke and inflammation. It is now accepted that inflammation and related events, such as activation of NF-κB, are key components in the initiation and progression of epithelial cancer and in particular in the neoplastic transformation of keratinocytes and skin carcinogenesis. Here, we report that PARP-1-deficient mice display a strikingly reduced susceptibility to skin carcinogenesis. In parp-1−/− mice, development of papilloma-like premalignant lesions induced with DMBA and TPA, is strongly delayed and the final number of tumor-bearing mice and total tumor number were significantly reduced. In addition, epidermis of parp-1−/− mice did not show increased proliferation rates after treatment with carcinogen. Deregulated NF-κB is a hallmark for tumorigenesis together with the concomitant release of early inflammatory mediators. In the absence of PARP-1, NF-κB activation and induction κB-target genes did not take place during the promotion of tumor development. These results suggest that PARP-1 abolition impairs the promotion of skin carcinogenesis interfering with the activation of NF-κB and might have an important implication in targeting PARP-1 as a new antineoplastic therapeutic approach.

Therapeutic effect of a poly(ADP-ribose) polymerase-1 inhibitor on experimental arthritis by downregulating inflammation and Th1 response.

Poly(ADP-ribose) polymerase-1 (PARP-1) synthesizes and transfers ADP ribose polymers to target proteins, and regulates DNA repair and genomic integrity maintenance. PARP-1 also plays a crucial role in the progression of the inflammatory response, and its inhibition confers protection in several models of inflammatory disorders. Here, we investigate the impact of a selective PARP-1 inhibitor in experimental arthritis. PARP-1 inhibition with 5-aminoisoquinolinone (AIQ) significantly reduces incidence and severity of established collagen-induced arthritis, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of AIQ is associated with a striking reduction of the two deleterious components of the disease, i.e. the Th1-driven autoimmune and inflammatory responses. AIQ downregulates the production of various inflammatory cytokines and chemokines, decreases the antigen-specific Th1-cell expansion, and induces the production of the anti-inflammatory cytokine IL-10. Our results provide evidence of the contribution of PARP-1 to the progression of arthritis and identify this protein as a potential therapeutic target for the treatment of rheumatoid arthritis.