Differential regulation of breast cancer bone metastasis by PARP1 and PARP2.

PARP1 and PARP2 dual inhibitors, such as olaparib, have been recently FDA approved for the treatment of advanced breast and ovarian cancers. However, their effects on bone mass and bone metastasis are unknown. Here we show that olaparib increases breast cancer bone metastasis through PARP2, but not PARP1, specifically in the myeloid lineage, but not in the cancer cells. Olaparib treatment or PARP1/2 deletion promotes osteoclast differentiation and bone loss. Intriguingly, myeloid deletion of PARP2, but not PARP1, increases the population of immature myeloid cells in bone marrow, and impairs the expression of chemokines such as CCL3 through enhancing the transcriptional repression by ß-catenin. Compromised CCL3 production in turn creates an immune-suppressive milieu by altering T cell subpopulations. Our findings warrant careful examination of current PARP inhibitors on bone metastasis and bone loss, and suggest cotreatment with CCL3, ß-catenin inhibitors, anti-RANKL or bisphosphonates as potential combination therapy for PARP inhibitors.

Coordinated signals from the DNA repair enzymes PARP-1 and PARP-2 promotes B-cell development and function.

Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 regulate the function of various DNA-interacting proteins by transferring ADP-ribose emerging from catalytic cleavage of cellular ß-NAD+. Hence, mice lacking PARP-1 or PARP-2 show DNA perturbations ranging from altered DNA integrity to impaired DNA repair. These effects stem from the central role that PARP-1 and PARP-2 have on the cellular response to DNA damage. Failure to mount a proper response culminates in cell death. Accordingly, PARP inhibitors are emerging as promising drugs in cancer therapy. However, the full impact of these inhibitors on immunity, including B-cell antibody production, remains elusive. Given that mice carrying dual PARP-1 and PARP-2 deficiency develop early embryonic lethality, we crossed PARP-1-deficient mice with mice carrying a B-cell-conditional PARP-2 gene deletion. We found that the resulting dually PARP-1 and PARP-2-deficient mice had perturbed bone-marrow B-cell development as well as profound peripheral depletion of transitional and follicular but not marginal zone B-cells. Of note, bone-marrow B-cell progenitors and peripheral mature B-cells were conserved in mice carrying either PARP-1 or PARP-2 deficiency. In dually PARP-1 and PARP-2-deficient mice, B-cell lymphopenia was associated with increased DNA damage and accentuated death in actively proliferating B-cells. Moreover, dual PARP-1 and PARP-2 deficiency impaired antibody responses to T-independent carbohydrate but not to T-dependent protein antigens. Notwithstanding the pivotal role of PARP-1 and PARP-2 in DNA repair, combined PARP-1 and PARP-2 deficiency did not perturb the DNA-editing processes required for the generation of a protective antibody repertoire, including Ig V(D)J gene recombination and IgM-to-IgG class switching. These findings provide key information as to the potential impact of PARP inhibitors on humoral immunity, which will facilitate the development of safer PARP-targeting regimens against cancer.

PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress.

During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.

PARP10 suppresses tumor metastasis through regulation of Aurora A activity.

ADP-ribosylation, including poly-ADP-ribosylation (PARylation) and mono-ADP-ribosylation (MARylation), is a multifunctional post-translational modification catalyzed by intracellular ADP-ribosyltransferases (ARTDs or PARPs). Although PARylation has been investigated most thoroughly, the function of MARylation is currently largely undefined. Here, we provide evidences that deficiency of PARP10, a mono-ADP-ribosyltransferase, markedly increased the migration and invasion of tumor cells through regulation of epithelial-mesenchymal transition (EMT), and PARP10 inhibited tumor metastasis in vivo, which was dependent on its enzyme activity. Mechanistically, we found that PARP10 interacted with and mono-ADP-ribosylated Aurora A, and inhibited its kinase activity, thereby regulating its downstream signaling. Moreover, the expression level of PARP10 was downregulated in intrahepatic metastatic hepatocellular carcinoma (HCC) compared with its corresponding primary HCC and adjacent non-tumorous tissues. Taken together, our results indicated that PARP10 has an important role in tumor metastasis suppression via negatively regulation of Aurora A activity.

Role of PARP activity in lung cancer-induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.

Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1-/- ) and Parp-2-/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1-/- , and Parp-2-/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1-/- and Parp-2-/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention.

PARP-1/PARP-2 double deficiency in mouse T cells results in faulty immune responses and T lymphomas.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways.

In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice.

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.

Cyberbullying and adolescent mental health: systematic review / Cyberbullying e saúde mental dos adolescentes: revisão sistemática / Acoso cibernético y la salud mental de los adolescentes: revisión sistemática

Cyberbullying is a new form of violence that is expressed through electronic media and has given rise to concern for parents, educators and researchers. In this paper, an association between cyberbullying and adolescent mental health will be assessed through a systematic review of two databases: PubMed and Virtual Health Library (BVS). The prevalence of cyberbullying ranged from 6.5% to 35.4%. Previous or current experiences of traditional bullying were associated with victims and perpetrators of cyberbullying. Daily use of three or more hours of Internet, web camera, text messages, posting personal information and harassing others online were associated with cyberbullying. Cybervictims and cyberbullies had more emotional and psychosomatic problems, social difficulties and did not feel safe and cared for in school. Cyberbullying was associated with moderate to severe depressive symptoms, substance use, ideation and suicide attempts. Health professionals should be aware of the violent nature of interactions occurring in the virtual environment and its harm to the mental health of adolescents.

Cyberbullying, uma nova forma de violência expressa por meio da mídia eletrônica, tem preocupado pais, educadores e pesquisadores. A associação entre cyberbullying e a saúde mental dos adolescentes será revisada. Revisão sistemática em duas bases de dados: PubMed e a Biblioteca Virtual em Saúde (BVS). A prevalência do cyberbullying variou entre 6,5% a 35,4%. Bullying tradicional prévio ou atual estava associado às vítimas e agressores do cyberbullying. Uso diário de três ou mais horas de Internet, web câmera, mensagens de texto, postar informações pessoais e assediar outros online estavam associados ao cyberbullying. "Cybervítimas" e cyberbullies tinham mais problemas emocionais, psicossomáticos, dificuldades sociais, e não se sentiam seguros e cuidados na escola. O cyberbullying estava associado à sintomatologia depressiva moderada e grave, uso de substâncias, ideação e tentativas de suicídio. Profissionais de saúde devem conhecer as interações de natureza violenta que ocorrem no ambiente virtual e de seus agravos para a saúde mental dos adolescentes.

Se revisa la asociación entre el acoso cibernético y la salud mental de los adolescentes. Se realiza una revisión sistemática de dos bases de datos: PubMed y la Biblioteca Virtual en Salud (BVS). La prevalencia de ciberacoso varió de un 6,5% a un 35,4%. Los acosos cibernéticos tradicionales -pasados o actuales- se asociaron con las víctimas y los acosadores cibernéticos. El uso diario de tres o más horas de Internet, cámara web, mensajes de texto, la publicación de información personal y acosar a los demás se asociaron con el acoso cibernético. Cibervíctimas y acosadores cibernéticos tenían más problemas emocionales, psicosomáticos, dificultades sociales y no se sentían seguros y cuidados en la escuela. El ciberacoso se asoció con síntomas de moderados a graves de depresión, abuso de sustancias, ideación suicida e intentos de suicidio. Los profesionales de salud deben conocer la naturaleza violenta de las interacciones que se producen en el entorno virtual y sus peligros para la salud mental de los adolescentes.

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Evaluación de la calidad de vida de pacientes bipolares chilenos: propiedades psicométricas y utilidad diagnóstica de la versión chilena del cuestionario Quality of Life Bipolar Disorder (QoL.BD-CL) / Assessment of a version adapted and translated into Spanish of the Quality of Life Bipolar Disorder Questionnaire

Background: The Quality of life Bipolar Disorder (QoL.BD) Questionnaire specifically measures quality of life in patients with bipolar disorder. Aim: To adapt a version translated into Spanish of the questionnaire and assess its validity in Chilean patients. Material and Methods: The QoL. BD was adapted to the Chilean population through the back-translation method and then administered to 32 adult patients with a bipolar disorder and 31 subjects without the disease, both groups with similar socioeconomic status. To confirm the diagnosis, the International Neuropsychiatric Interview (MINI), Young (YMRS) and Hamilton (HAM-D) scales were applied. Quality of life was assessed using the SF-36v.2 survey. We determined internal consistency, reliability, convergent validity, the cut-off point, and the sensibility and specificity of the scale. Results: The Chilean version of the Questionnaire [QoL. BD-CL] had a high reliability (α = 0.95) and a high validity in reference to external criteria (correlation coefficients with SF-36 ranging from 0.453 and 0.819; p < 0.01). A cut-off point of 170, with sensitivity of 87.9% and specificity of 80% was determined. Conclusions: QoL.BD-CL has adequate psychometric properties, as well as an adequate sensitivity and specificity to distinguish between negative and positive perceptions of life quality in Chilean patients with bipolar disorders.

The antimitotic potential of PARP inhibitors, an unexplored therapeutic alternative.

ADP-ribosylation or PARsylation is one of the most abundant modifications of proteins and DNA. Although the usual context for PARsylation involves the detection and repair of DNA damage in the cell, poly(ADP-ribose) polymerases are known to regulate a number of biological processes besides maintaining genome integrity. One of these processes is the assembly and maintenance of the mitotic spindle where the presence of PARP-1 and tankyrase 1 (TNKS1), two of the best-characterized members of the PARP superfamily, is of critical importance. Here, we recapitulate the biological implications of the absence of poly(ADP-ribose) polymerases and depletion of PARsylation occurrence in mitosis in order to better understand the antimitotic effects of PARP inhibitors. In this regard, we also present an overview of the existing and more relevant molecules, with a special attention to the historical development of their pharmacological properties and structures, as well as a brief summary of clinical trials involving PARP inhibitors.

PARP inhibition restores extrinsic apoptotic sensitivity in glioblastoma.

BACKGROUND: Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM). We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281) or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. METHODS: The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. RESULTS: The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with up-regulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP) homology protein (CHOP). Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. CONCLUSIONS: PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM.

Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors.

Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD.

The H19 induction triggers trophoblast lineage commitment in mouse ES cells.

Trophoblast lineage differentiation is properly regulated to support embryogenesis. Besides normal developmental process, during germ cell tumor formation or development of other reproductive system diseases, unregulated trophoblast differentiation is also observed and affects the pathogenesis of the diseases. During normal embryogenesis, cell fate of late-stage blastcyst is regulated by a reciprocal repression of the key transcriptional factors; Oct3/4 dominancy inhibits Cdx2 expression in inner cell mass (ICM) and leads them to epiblast/primitive ectoderm but Cdx2 dominancy in trophectoderm (TE) leads them to trophoblast lineage. In contrast during early blastcyst stage, the Cdx2 expression is restricted in TE and not present in ICM, although Oct3/4 signaling does not inhibit the Cdx2 expression in ICM, implying that some factors could be inactivated leading to the suppressed Cdx2 expression in ICM of early blastcyst. ES cells (ESCs), which are derived from ICM, could be a unique model to study trophoblast differentiation in an ectopic context. We previously showed that poly(ADP-ribose) polymerase-1 (Parp-1) deficient ESCs highly expressed non-coding RNA H19 and could differentiate into trophoblast lineage. The expression of H19 is known to start at pre-blastcyst stage during mouse development, and the gene shows high expression only in trophoectoderm (TE) at blastcyst stage. However, its role in trophoblast differentiation has not been clarified yet. Thus, we hypothesized that the H19 activation may act as a trigger for induction of trophoblast differentiation cascade in mouse ESCs. To investigate this issue, we asked whether a forced H19 expression drives ESCs into trophoblast lineage or not. We demonstrated that the H19 induction leads to trophoblast lineage commitment through induction of the Cdx2 expression. We also showed that the expression of Cdx2 is induced in ESCs by forced H19 expression even under a high level of Oct3/4, which could act as a suppressor for Cdx2 expression. It is thus suggested that the H19 induction promotes trophoblast lineage commitment against the repression pressure by Oct3/4 in differentiating ESCs. Taken together, this study suggests that the H19 expression is able to function as a cascade activator of trophoblast lineage commitment possibly by overriding the Oct3/4 action in ESCs.

Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/µm) and Fe-ion irradiation (200 keV/µm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/µm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.

Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos.

We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

Poly (ADP-ribose) transferase/polymerase-1-deficient mice resistant to age-dependent decrease in ß-cell proliferation.

Basal and adaptive ß-cell regeneration capacity declines with old age, but the underlying molecular mechanisms remain incompletely understood. Poly (adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP-1) is considered a multifunctional enzyme and transcription factor that regulates pancreatic ß-cell death, regeneration and insulin secretion. We analyzed the capacity of ß-cell regeneration in 2-month-old (young) and 12-month-old (old) wild-type (WT) and PARP-1⁻/⁻ mice before and after low-dose streptozotocin (STZ), a stimulus of ß-cell regeneration and the underlying mechanism. Before STZ administration, young WT and PARP-1⁻/⁻ mice showed similar ß-cell proliferation. By contrast, old WT but not old PARP-1⁻/⁻ mice showed severely restricted ß-cell proliferation. In further assessment of the adaptive ß-cell regeneration capacity with age, we observed that with a single low dose of STZ, young WT and PARP-1⁻/⁻ mice showed a similar increase in ß-cell proliferation, with few changes in old WT mice. Surprisingly, adaptive ß-cell proliferation capacity was significantly higher in old PARP-1⁻/⁻ mice than old WT mice after STZ administration. The ability of ß-cell mass to expand was associated with increased levels of the regenerating (Reg) genes RegI and RegII but not RegIV. Therefore, PARP-1 is a key regulator in ß-cell regeneration with advancing age in mice.

Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.

Apoptosis, necrosis, and inflammation are hallmarks of cisplatin nephrotoxicity; however, the role and mechanisms of necrosis and inflammation remains undefined. As poly(ADP-ribose) polymerase 1 (PARP1) inhibition or its gene deletion is renoprotective in several renal disease models, we tested whether its activation may be involved in cisplatin nephrotoxicity. Parp1 deficiency was found to reduce cisplatin-induced kidney dysfunction, oxidative stress, and tubular necrosis, but not apoptosis. Moreover, neutrophil infiltration, activation of nuclear factor-κB, c-Jun N-terminal kinases, p38 mitogen-activated protein kinase, and upregulation of proinflammatory genes were all abrogated by Parp1 deficiency. Using proximal tubule epithelial cells isolated from Parp1-deficient and wild-type mice and pharmacological inhibitors, we found evidence for a PARP1/Toll-like receptor 4/p38/tumor necrosis factor-α axis following cisplatin injury. Furthermore, pharmacological inhibition of PARP1 protected against cisplatin-induced kidney structural/functional damage and inflammation. Thus, our findings suggest that PARP1 activation is a primary signal and its inhibition/loss protects against cisplatin-induced nephrotoxicity. Targeting PARP1 may offer a potential therapeutic strategy for cisplatin nephrotoxicity.