Multiple roles for poly(ADP-ribose)polymerase-1 in neurological disease

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear protein activated by DNA damage. PARP-1 activation is associated in DNA repair, cell death and inflammation. Since oxidative stress induced robust DNA damage and wide spread inflammatory responses are common pathologies of various CNS diseases, the interest toward PARP-1 as a therapeutic target has peaked. This review introduces mechanism of PARP-1 activation, the role of PARP-1 in cell physiology and pathology, and discusses the potential of PARP-1 inhibition as a therapy in acute and chronic CNS diseases.     

Induction of ROS formation, poly(ADP-ribose) polymerase-1 activation, and cell death by PCB126 and PCB153 in human T47D and MDA-MB-231 breast cancer cells

The primary purpose of this research is to investigate whether exposure to polychlorinated biphenyls (PCBs), i.e. PCB153 and PCB126, is associated with induction of reactive oxygen species (ROS), poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cell death in human T47D and MDA-MB-231 breast cancer cells. Results indicated that PCB153 and PCB126 induced concentration- and time-dependent increases in cytotoxic response and ROS formation in both T47D and MDA-MB-231 cells. At non-cytotoxic concentrations both PCB153 and PCB126 induced decreases in intracellular NAD(P)H and NAD+ in T47D and MDA-MB-231 cells where T47D cells were more resistant to PCB-induced reduction in intracellular NAD(P)H than MDA-MB-231 cells. Further investigation indicated that three specific PARP inhibitors completely blocked PCB-induced decreases in intracellular NAD(P)H in both T47D and MDA-MB-231 cells. These results imply that decreases in intracellular NAD(P)H in PCB-treated cells may be, in part, due to depletion of intracellular NAD+ pool mediated by PARP-1 activation through formation of DNA strand breaks. Overall, the extent of cytotoxic response, ROS formation, and PARP-1 activation generated in T47D and MDA-MB-231 cells was greater for PCB153 than for PCB126. In addition, the cytotoxicity induced by PCB153 and PCB126 in both T47D and MDA-MB-231 cells was completely blocked by co-treatment of catalase, dimethylsulfoxide, cupper (I)-/iron (II)-specific chelators, and CYP1A/2B inhibitors. This evidence suggests the involvement of ROS, Cu(I), Fe(II), and CYP1A/2B enzymes in mediating the induction of cell death by PCB153 and PCB126. Further, antagonism was observed between PCB126 and PCB153 for effects on cytotoxic response and ROS formation in T47D and MDA-MB-231 cells. Antagonism was also observed between PCB153 and PCB126 in the induction of NAD(P)H depletion at lower concentration (<10 microM) in T47D cells, but not in MDA-MB-231 cells. In conclusions, results from our investigation suggest that ROS formation induced by PCBs is a significant determinant factor in mediating the DNA damage and cell death in human breast cancer cells. The data also suggests that the status of estrogen receptor alpha may play a role in modulating the PCB-induced oxidative DNA damage and cell death in human breast cancer cells.     

Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.     

Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice

To study the effect of continued telomere shortening on chromosome stability, we have analyzed the telomere length of two individual chromosomes (chromosomes 2 and 11) in fibroblasts derived from wild-type mice and from mice lacking the mouse telomerase RNA (mTER) gene using quantitative fluorescence in situ hybridization. Telomere length at both chromosomes decreased with increasing generations of mTER-/- mice. At the 6th mouse generation, this telomere shortening resulted in significantly shorter chromosome 2 telomeres than the average telomere length of all chromosomes. Interestingly, the most frequent fusions found in mTER-/- cells were homologous fusions involving chromosome 2. Immortal cultures derived from the primary mTER-/- cells showed a dramatic accumulation of fusions and translocations, revealing that continued growth in the absence of telomerase is a potent inducer of chromosomal instability. Chromosomes 2 and 11 were frequently involved in these abnormalities suggesting that, in the absence of telomerase, chromosomal instability is determined in part by chromosome-specific telomere length. At various points during the growth of the immortal mTER-/- cells, telomere length was stabilized in a chromosome-specific man-ner. This telomere-maintenance in the absence of telomerase could provide the basis for the ability of mTER-/- cells to grow indefinitely and form tumors.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis.

DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.     

Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.     

Genetic and pharmacological inhibition of poly(ADP-ribose) polymerase-1 interferes in the chlamydial life cycle

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1-/- and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1-/- than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1-/- fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.     

Synthesis, [18F] radiolabeling,and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be ¹⁸F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC₅₀ = 6.3 nM). [¹⁸F]12 was synthesized under conventional conditions in high specific activity with 40–50% decay-corrected yield. MicroPET studies using [¹⁸F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [¹⁸F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [¹⁸F]12 in the tumor is specific to PARP-1 expression.     

A dual approach in the study of poly (ADP-ribose) polymerase: In vitro random mutagenesis and generation of deficient mice

A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory. Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity.In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting. We showed that: (i) they are exquisitely sensitive to -irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to -irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival.     

Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

Unscheduled DNA synthesis has been measured in human fibroblasts under conditons of reduced rates of conversion of NAD to poly(ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of UV induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with UV or N-methyl-N′-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.     

Restoration of the endothelial function in the aortic rings of apolipoprotein E deficient mice by pharmacological inhibition of the nuclear enzyme poly(ADP-ribose) polymerase

Oxidant-mediated activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. The aim of the current study was to investigate whether activation of PARP contributes to the development of endothelial dysfunction in the apolipoprotein E (ApoE) deficient mice. We tested whether PARP inhibition prevents the development of endothelial dysfunction and whether it restores function in vessels with established endothelial dysfunction. ApoE deficient mice were kept on high-fat diet for 12 weeks with and without INO-1001 treatment. Chronic treatment with the PARP inhibitor INO-100 reduced the degree of the endothelial dysfunction (the ability of the vessel to relax to acetylcholine) in the thoracic aortae of ApoE deficient mice. In addition, in vitro incubation of vessels from ApoE deficient mice with established endothelial dysfunction with the PARP inhibitor acutely improved the ability of the rings to relax to acetylcholine. We conclude that the early atherosclerotic functional alterations that develop in the endothelium of the ApoE deficient mice are, at least in part, reversible, and are dependent on the activation of the nuclear enzyme PARP in the endothelial cells.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^- / -  and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in Ogg1^- / -  mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^- / -  mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^- / -  mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^- / -  mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^{? / ?} and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in Ogg1^{? / ?} mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^{? / ?} mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^{? / ?} mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^{? / ?} mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.