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Poly (ADP) Ribose Polymerase-1 (PARP-1), a fundamental DNA repair enzyme is known to regulate β cell death, replication, and insulin secretion. PARP-1 knockout (KO) mice are resistant to diabetes while PARP-1 overactivation contributes to β cell death. Additionally, PARP-1 inhibition improves diabetes complications in patients with type-2 diabetes. Despite these beneficial effects, the use of PARP-1 modulating agents in diabetes treatment are largely neglected, primarily due to the poorly studied mechanistic action of PARP-1 catalytic function in human β cell development. In the present study, we evaluated PARP-1 regulatory action in human β cell differentiation using the human pancreatic stem cell line, PANC-1. We surveyed islet census and histology from PARP-1 wild-type versus KO mice pancreas in head-to-head comparison with PARP-1 regulatory action for in-vitro β cell differentiation following either PARP-1 depletion or its pharmacological inhibition in PANC-1-differentiated islet cells. shRNA mediated PARP-1 depleted (SiP), or control replete (U6) PANC-1 cells were differentiated into islet-like clusters using established protocols. We observed complete abrogation of new β cell formation with absolute PARP-1 depletion while its catalytic inactivation using the strong inhibitor, PJ34, promoted the endocrine β cell differentiation and maturation. Immunohistochemistry and proteomics for key endocrine differentiation factors in addition to β cell maturation markers highlighted potential regulatory and augmented β cell differentiation due to non-catalytic function of PARP-1 elicited p-38 MAPK phosphorylation and Neurogenin-3 (Ngn3) re-activation. In summary, the study reestablishes a novel ability of catalytic independent action of PARP-1 in regulating islet cell differentiation and potential avenues for pilot clinical testing of PARP-1 inhibitors for β cell replacement therapies for the treatment of diabetes.

DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities.

Mutations in ZBTB24 cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. Helfricht et al. demonstrate that the loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination, proving a molecular basis for the immunodeficiency in ICF patients., The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome., Graphical Abstract

For patients with inoperable neuroendocrine tumors (NETs) expressing somatostatin receptors, peptide receptor radionuclide therapy (PRRT) with 177Lu-[DOTA0-Tyr3]-octreotate (177Lu-octreotate) is one of the most promising targeted therapeutic options but it rarely achieves cure. Therefore, different approaches are being tested to increase the efficacy of 177Lu-octreotate PRRT in NET patients. Using the gastroenteropancreatic BON-1 and the bronchopulmonary NCI-H727 as NET cell models, here we report that pharmacological inhibitors of DNA repair-associated enzyme poly(ADP-ribose) polymerase-1 (PARPi) potentiate the cytotoxic effect of 177Lu-octreotate on 2D monolayer and 3D spheroid models of these two types of NET cells. PARPi mediates this effect by enhancing 177Lu-octreotate-induced cell cycle arrest and cell death. Thus, the use of PARPi may offer a novel option for improving the therapeutic efficacy of 177Lu-octreotate PRRT of NETs.

The peptide receptor radionuclide therapy (PRRT) with 177Lu-DOTA-octreotate (LuTate) is recommended for different types of neuroendocrine tumors (NETs) which overexpress somatostatin receptors (SSTR). A combination with chemotherapy improves objective response to LuTate in NET patients and here we characterized chemotherapy-induced upregulation of SSTR2 receptors as a cause for this improved response to LuTate. The NET cell lines with low (BON-1) or relatively high (NCI-H727) SSTR2-expression levels, and non-NET cancer and normal cells were treated with chemotherapeutic drugs and assessed for upregulation of SSTR2. We report that an exposure to low or high doses of drugs, such as temozolomide for 24 h or 5 day results in upregulation of SSTR2 between 3&ndash, 7 days, increased LuTate uptake and decreased rate of cell proliferation. This effect is at the level of SSTR2-mRNA and is more pronounced in low SSTR2 expressing BON-1 than in high SSTR2 expressing NCI-H727 or non-NET cancer or normal cells. Thus, a properly timed pre-treatment with low-dose chemotherapy could not only improve therapeutic efficacy of LuTate in NET patients who are presently eligible for PRRT, but also allow PRRT to be administered to patients with low SSTR-expressing NETs, who would otherwise not respond to this modality because of insufficient radiation delivery.

For patients with inoperable neuroendocrine tumors (NETs) expressing somatostatin receptors, peptide receptor radionuclide therapy (PRRT) with 177Lu-[DOTA0-Tyr3]-octreotate (177Lu-octreotate) is one of the most promising targeted therapeutic options but it rarely achieves cure. Therefore, different approaches are being tested to increase the efficacy of 177Lu-octreotate PRRT in NET patients. Using the gastroenteropancreatic BON-1 and the bronchopulmonary NCI-H727 as NET cell models, here we report that pharmacological inhibitors of DNA repair-associated enzyme poly(ADP-ribose) polymerase-1 (PARPi) potentiate the cytotoxic effect of 177Lu-octreotate on 2D monolayer and 3D spheroid models of these two types of NET cells. PARPi mediates this effect by enhancing 177Lu-octreotate-induced cell cycle arrest and cell death. Thus, the use of PARPi may offer a novel option for improving the therapeutic efficacy of 177Lu-octreotate PRRT of NETs.

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Summary The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability., Graphical Abstract, Highlights • PARP1 recruits the chromatin remodeler CHD2 to DNA damage • CHD2 promotes chromatin expansion and H3.3 deposition at DNA breaks • CHD2 promotes the assembly of NHEJ repair complexes at DNA breaks • PARP1 drives CHD2- and H3.3-dependent DNA repair by NHEJ, Luijsterburg et al. define a PARP1-dependent mechanism that regulates NHEJ through localized chromatin expansion and deposition of the histone variant H3.3 by the nucleosome remodeler CHD2 at DNA breaks. Their data also show that these CHD2-mediated events promote DNA repair by facilitating the assembly of NHEJ complexes in chromatin.

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.

OBJECTIVE: Carcinoid cancer patients often have elevated levels of serotonin or its precursor 5-hydroxytryptophan. Normally, serotonin synthesis accounts for a small fraction of tryptophan catabolism, which should be directed along a pathway that allows partial conversion to niacin; hence, increased diversion of tryptophan toward serotonin could cause variable degrees of niacin deficiency in carcinoid patients. Therefore, the prevalence of niacin deficiency among carcinoid patients was investigated by clinical assessment of pellagra and biochemical assessment of whole blood niacin number, a ratio derived from two biologically active forms of niacin (NAD/NADP × 100). METHODS: Clinical and biochemical niacin status were assessed in a cohort of newly diagnosed carcinoid patients with carcinoid syndrome (CCS, n = 36), carcinoid patients without carcinoid syndrome (CWCS, n = 32) and noncarcinoid controls (n = 24) recruited at two primary care clinics. Other aspects of serotonin metabolism were measured by analyses of plasma serotonin and tryptophan and urinary excretion of 5-hydroxyindoleacetic acid. RESULTS: Biochemical niacin deficiency (niacin number < 130) was significantly more common in CCS patients (10 out of 36) compared to controls (p < 0.05, Fisher’s exact test), while CWCS patients displayed an incidence that was not significantly elevated (4 out of 32). Only one CCS patient, who was also identified biochemically as niacin deficient, was clinically diagnosed with pellagra. CONCLUSION: Biochemical niacin deficiency is more prevalent among newly diagnosed CCS patients than in controls. Manifestation of pellagra is a less sensitive indicator, and dependence on this endpoint could lead to a lack of appropriate nutritional support for this group of patients. (Am J Gastroenterol 2005;100:2307‐2314)

The damage to DNA caused by ultraviolet B radiation (280-320 nm) contributes significantly to development of sunlight-induced skin cancers. The susceptibility of mice to ultraviolet B-induced skin carcinogenesis is increased by an inhibitor of the DNA damage-activated nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), hence PARP activation is likely to be associated with cellular responses that suppress carcinogenesis. To understand the role of activated PARP in these cellular functions, we need to first clearly identify the cause of PARP activation in ultraviolet B-irradiated cells. Ultraviolet B, like ultraviolet C, causes direct DNA damage of cyclobutane pyrimidine dimer and 6, 4-photoproduct types, which are subjected to the nucleotide excision repair. Moreover, ultraviolet B also causes oxidative DNA damage, which is subjected to base excision repair. To identify which of these two types of DNA damage activates PARP, we examined mechanism of early PARP activation in mouse fibroblasts exposed to ultraviolet B and C radiations. The ultraviolet B-irradiated cells rapidly activated PARP in two distinct phases, initially within the first 5 minutes and later between 60-120 minutes, whereas ultraviolet C-irradiated cells showed only the immediate PARP activation. Using antioxidants, local irradiation, chromatin immunoprecipitation and in vitro PARP assays, we identified that ultraviolet radiation-induced direct DNA damage, such as thymine dimers, cause the initial PARP activation, whereas ultraviolet B-induced oxidative damage cause the second PARP activation. Our results suggest that cells can selectively activate PARP for participation in different cellular responses associated with different DNA lesions.

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Commercial formulations of the pesticides: Guthion (azinphos methyl), Sencor (metribuzin), Lorox (linuron), Reglone (diquat), Daconil (chlorothalonil) and Admire (imidacloprid) were studied for their genotoxicity by 32P-postlabeling. Metabolites of the pesticides were obtained enzymatically using arochlor induced rat liver S9 fraction, in an NADPH generating system. The resulting metabolites were reacted with calf thymus DNA and the DNA was analyzed for presence of adducts by either the nuclease P1 or butanol enrichment. Nuclease P1 enrichment resulted in adducts for all the pesticides. Compared to the level of adducts in control DNA, the levels in pesticide-treated DNA were higher for all the pesticides, except Daconil. The increase in adduct numbers for pesticide-treated DNAs ranged from 4.9-12.4 times the control-DNA indicating pesticide genotoxicity in this in vitro system. Enrichment using butanol extraction gave three adducts unique to Sencor-DNA. These adducts were different from those obtained with nuclease P1 enrichment of the same. B(α)P was the positive control for the in vitro metabolism, and two adduct enrichment procedures: nuclease P1 digestion and butanol extraction.

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair–related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

Human promyelomonocytic leukemia cells HL-60 were treated with etoposide or cytochalasin B to induce apoptosis or necrosis, respectively. We report here that during necrosis, the DNA-repair associated nuclear enzyme poly(ADP-ribose) polymerase (PARP) was degraded differently from that observed during apoptosis. While apoptotic HL-60 cells exhibit only the signature 89 kDa fragment of PARP, necrosis of these cells was accompanied by formation of major fragments at MWr approximately 89 and 50 kDa and minor fragments at approximately 40 and 35 kDa. The necrosis-specific degradation of PARP was coincident with other changes detected by flow cytometric analysis, but earlier than the extensive degradation of DNA. Therefore, the unique necrotic degradation of PARP could be used as a sensitive indicator for necrotic death of cells.

An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1 gene is expressed constitutively, its activation cannot be surmised from increased expression of its mRNA or protein, but by demonstrating the consequences of its catalytic -reaction which results in consumption of the substrate nicotinamide adenine dinucleotide (NAD(+)) and formation of three products, namely, polymer of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Here, we describe various approaches commonly used in our laboratory for detection of PARP-1 activation in vivo (cells, tissues, and tumors), in situ, and in vitro via assessment of formation of pADPr, depletion of the substrate NAD, or formation of protons resulting in rapid and reversible intracellular acidification. It is important to note that although some other members of the PARP family can carry out the same catalytic reaction, many of these assays largely reflect PARP-1 activation in a vast majority of the experimental circumstances and more specifically in DNA damage responses. However, if required, PARP-1-specific action should be confirmed by use of PARP-1 knockout or RNAi-mediated knockdown approaches.

An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1 gene is expressed constitutively, its activation cannot be surmised from increased expression of its mRNA or protein, but by demonstrating the consequences of its catalytic -reaction which results in consumption of the substrate nicotinamide adenine dinucleotide (NAD(+)) and formation of three products, namely, polymer of ADP-ribose (pADPr or PAR), nicotinamide, and protons. Here, we describe various approaches commonly used in our laboratory for detection of PARP-1 activation in vivo (cells, tissues, and tumors), in situ, and in vitro via assessment of formation of pADPr, depletion of the substrate NAD, or formation of protons resulting in rapid and reversible intracellular acidification. It is important to note that although some other members of the PARP family can carry out the same catalytic reaction, many of these assays largely reflect PARP-1 activation in a vast majority of the experimental circumstances and more specifically in DNA damage responses. However, if required, PARP-1-specific action should be confirmed by use of PARP-1 knockout or RNAi-mediated knockdown approaches.

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG [1] or physical stimulation of cells by γ-rays [2] are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP [3]. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms. Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro [3]. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots [4] showed that about 60–70% of the 6–8 mers pADPr were lost during fixation and that the very short pADPr (2–5 mers) were very weakly bound to the membrane [5]. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical. We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2–3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.

RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD(1476) and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA.

RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO 4 protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2.

In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate β-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of β-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 μM MNNG is reversed within 1 h, 100 μM MNNG-induced acidification by 0.5–0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H 2 O 2 exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP +/+ fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP −/− fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na + /H + exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5–0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.

Poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for the catabolism of poly(ADP-ribose), a reversible covalent-modifier of chromosomal proteins. Purification of PARG from many tissues revealed heterogeneity in activity and structure of this enzyme. To investigate PARG structure and localization, we developed a highly sensitive one-dimensional zymogram allowing us to analyze PARG activity in crude extracts of Cos-7, Jurkat, HL-60, and Molt-3 cells. In all extracts, a single PARG activity band corresponding to a protein of about 110 kDa was detected. This 110-kDa PARG activity was found mainly in cytoplasmic rather than in nuclear extracts of Cos-7 cells.

The concerted action of poly(ADP-ribose) polymerase (PARP) which synthesizes the poly(ADP-ribose) (pADPr) in response to DNA strand breaks and the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG) determine the level of polymer and the rate of its turnover. In the present study, we have shown that the quail myoblast cells have high levels of basal polymer as compared to the murine C3H10T1/2 fibroblasts. We have conducted this study to investigate how such differences influence polymer synthesis and its catabolism in the cells in response to DNA damage by alkylating agent. In quail myoblast cells, the presence of high MNNG concentration such as 200 μM for 30 min induced a marginal decrease of 15% in the NAD content. For C3H10T1/2 cell line, 64 eM MNNG provoked a depletion of NAD content by approximately 50%. The induction of the polymer synthesis in response to MNNG treatment was 6-fold higher in C3H10T1/2 cells than in quail myoblast cells notwithstanding the fact that 3-fold higher MNNG concentration was used for quail cells. The polymer synthesis thus induced in quail myoblast cells had a 4–5 fold longer half life than those induced in C3H10T1/2 cells. To account for the slow turnover of the polymer in the quail myoblast cells, we compared the activities of the polymer catabolizing enzyme (PARG) in the two cell types. The quail myoblast cells had about 25% less activity of PARG than the murine cells. This difference in activity is not sufficient to explain the large difference of the rate of catabolism between the two cell types implicating other cellular mechanisms in the regulation of pADPr turnover.