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.     

Poly(ADP-ribose) polymerase-1 is a positive regulator of the p53-mediated G1 arrest response following ionizing radiation

Poly(ADP-ribose) polymerase-1 (PARP-1) and the p53 tumor suppressor protein are both involved in the cellular response to genotoxic stress. Upon binding to the site of DNA strand breakage, PARP-1 is activated, leading to rapid and transient poly(ADP-ribosyl)ation of nuclear proteins using NAD+ as substrate. To investigate the role of PARP-1 in the p53 response to ionizing radiation in human cells, PARP-1 function was disrupted in wild-type p53 expressing MCF-7 and BJ/TERT cells using two strategies: chemical inhibition with 1,5-dihydroxyisoquinoline, and trans-dominant inhibition by overexpression of the PARP-1 DNA-binding domain. Although a number of proteins can catalyze poly(ADP-ribosyl)ation in addition to PARP-1, we show that PARP-1 is the only detectable active species in BJ/TERT and MCF-7 cells. 1,5-Dihydroxyisoquinoline treatment prior to ionizing radiation delayed and attenuated the induction of two p53-responsive genes, p21 and mdm-2, and led to suppression of the p53-mediated G1-arrest response in MCF-7 and BJ/TERT cells. Trans-dominant inhibition of PARP-1 by overexpression of the PARP-1 DNA-binding domain in MCF-7 cells also led to a delay and attenuation in p21 induction and suppression of the p53-mediated G1 arrest response to ionizing radiation. Hence, inhibition of endogenous PARP-1 function suppresses the transactivation function of p53 in response to ionizing radiation. This study establishes PARP-1 as a critical regulator of the p53 response to DNA damage.     

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.     

Poly(ADP-ribosyl)ation of FOXP3 Protein Mediated by PARP-1 Protein Regulates the Function of Regulatory T Cells

Poly(ADP-ribose) polymerase 1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes but also enhanced suppressive function of regulatory T cells. Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the posttranslational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications for developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.     

CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation

The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.     

Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to DNA-damaging agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

The emerging role of poly(ADP-ribose) polymerase-1 in longevity

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.     

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells

The underlying mechanisms of arsenic carcinogenicity are still not fully understood. Mechanisms currently discussed include the induction of oxidative DNA damage and the interference with DNA repair pathways. Still unclear is the role of biomethylation, which has long been considered to be one major detoxification process. Methylated arsenicals have recently been shown to interfere with DNA repair in cellular and subcellular systems, but up to now no DNA repair protein has been identified being particular sensitive towards methylated arsenicals in cultured cells. Here we report that the trivalent methylated metabolites MMA(III) and DMA(III) inhibit poly(ADP-ribosyl)ation in cultured human HeLa S3 cells at concentrations as low as 1nM, thereby showing for the first time an inactivation of an enzymatic reaction related to DNA repair by the trivalent methylated arsenicals at very low environmentally relevant concentrations. In contrast the pentavalent metabolites MMA(V) and DMA(V) showed no such effects up to high micromolar concentrations. All investigated arsenicals did not alter gene expression of PARP-1. However, all trivalent arsenicals were able to inhibit the activity of isolated PARP-1, indicating that the observed decrease in poly(ADP-ribosyl)ation in cultures human cells, predominantly mediated by PARP-1, is likely due to changes in the activity of PARP-1. Since poly(ADP-ribosyl)ation plays a major role in DNA repair, cell cycle control and thus in the maintenance of genomic stability, these findings could in part explain DNA repair inhibition and the genotoxic and carcinogenic effects of arsenic.