Oxidative stress and inflammation are often linked to the prognosis of diseases caused by chronic alcohol consumption. Chronic alcohol consumption plays a key role in brain tissue damage, often leading to the development of cognitive disorders and loss of brain function. In addition to the direct effects of alcohol on brain function, consumption of alcohol can lead to psychosocial stressors such as legal, financial, and interpersonal problems. It has been found that mice that overexpress C1q/Tumor Necrosis Factor-related protein-3 (CTRP3) and exposed to ethanol daily do not die like the mice who did not overexpress CTRP3 and fed the same diet. Although the specific physiological functions regulated by the CTRP family are largely unknown, there is evidence showing that they have diverse biological effects on inflammation, metabolism, and survival signaling in several different types of tissue. Postmortem brain tissue samples were collected from mice that were exposed to ethanol with transgenic overexpression of CTRP3 and from wild type mice that were only exposed to ethanol. Interestingly, previous immunoblotting of the cerebellum and the hippocampus using collected tissue demonstrated that glia activation was present in the CTRP3 overexpressing mice but not in the wild-type ethanol fed mice. This finding suggests that glia cells are either dying in the ethanol fed wild type mice or that CTRP3 protects and prolongs activated glia cells. The current study will determine if markers of oxidative stress and cell viability are altered in the CTRP3 overexpressing mice when compared to wild-type mice at the molecular level. RNA isolation using the Directzol system and cDNA synthesis using punch dissected homogenate tissue collected from the hippocampus was used for this investigation. Gene expression of BDNF, SOD1 and PARP1 in mouse tissue was determined using quantitative PCR. Immunoblotting of a small number of hippocampal tissue using PARP1 was performed. The mice that were CTRP3 overexpressed and fed ethanol will likely exhibit altered gene expression of cytokines and increased oxidative stress gene expression in postmortem hippocampal brain tissue when compared to wild-type ethanol fed mice. The current studies could contribute to the body of knowledge for the development of novel therapies that may alleviate the neuro-inflammatory effects of alcohol use.

Epigenetic deregulation contributes significantly to the development of multiple human diseases, including cancer. While great effort has been made to elucidate the underlying mechanism, our knowledge on epigenetic regulation is still fragmentary, an important gap being how the diverse epigenetic events coordinate to control gene transcription. In the first part of our study, we demonstrated an important link between Snail-mediated transcriptional control and epigenetic regulation during cancer development. Specifically, we found that the highly conserved SNAG domain of Snail sequentially and structurally mimics the N-terminal tail of histone H3, thereby functions as a molecular “hook”, or pseudo substrate, for recruiting histone lysine specific demethylase 1 (LSD1) repressor complex to the E-cadherin promoter. Furthermore, we showed that Snail and LSD1 are both required for E-cadherin repression and EMT induction, and their expression is highly correlated with each other in multiple human tumor tissues. Our findings have important clinical ramifications in that compounds mimicking the SNAG domain may disrupt Snail-LSD1 interaction and inhibit EMT and metastasis. In the second part of our study, we designed a batch of compounds based on the structure of the SNAG domain and are currently screening for candidates capable of competing with SNAG peptide for LSD1 binding. In addition, we applied a peptide pulldown/mass spectrometry-coupled analysis to identify SNAG-interacting proteins, among which are many chromatin enzymes and modulators. Functional characterization of these proteins will help to elucidate the Snail-mediated epigenetic regulation process. In the third part of our study, we found that Snail interacts with poly(ADP-ribose) polymerase 1 (PARP1) through a potential pADPr-binding motif and is subject to poly(ADP-ribosyl)ation, which can stabilize the Snail-LSD1 complex for enhanced PTEN suppression under DNA damage condition. Our findings added another layer to the delicate Snail transcriptional machinery, and indicated that PARP inhibitors may be applied in combination with conventional chemotherapies to target cancers with high expression of Snail and LSD1. In summary, we demonstrated that Snail cooperates with multiple epigenetic machineries to induce EMT as well as survival of tumor cells. Our findings contribute to a better appreciation of Snail-mediated epigenetic network as well as diversification of therapeutic strategies against cancer.

At present reproductive biotechnology methods are on the rise, but their development and application in the broader management of reproduction is dependent on obtaining a sufficient number of quality oocytes cultured in vitro. The prerequisite for this requirement is the creation of the optimal conditions in the course of culturing oocytes. Understanding and knowledge of the processes that occur in oocyte during maturation is an important and necessary condition for optimizing the process of culturing oocytes in vitro and gaining a sufficient number of good quality oocytes in metaphase II of meiotic division. A large number of mechanisms that affect and control oocyte maturation are known, however it cannot be claimed that this process has been fully explained and studied. One factor which has a potential role in the regulation of meiotic maturation of oocytes is gasotransmitter hydrogen sulfide (H2S), a critical signaling molecule of endogenous origin. The study of H2S led to the hypothesis that H2S actively influences the course of meiotic maturation of pig oocytes by regulating key signaling cascades. The aim of this work was to determine the involvement of H2S in the regulation of the MEK1-MAPK signaling cascade, responsible for the initiation and progress of the meiotic maturation of oocytes and the MEK1-PARP-1 cascade as signaling that supports cell viability. For this purpose, pig oocytes cultured in modified media were used, supplemented with a specific combination of enzyme inhibitors (3Ki) or in a culture medium with donor H2S. The ocytes were then subjected to immunocytochemistry staining, fluorescence microscopy and image analysis. The results show that H2S is involved in the regulation of meiotic maturation. It confirmed the hypothesis of the endogenous production of H2S in the course of the meiotic maturation of pig oocytes and the influence of the MAPK signaling cascade. Based on the results, it is however likely that the MEK1-PARP-1 signaling cascade is not affected by H2S, unlike MAPK signaling, comprising the mentioned MEK1 as superior kinase. MAPK kinase activity is significantly lower in oocytes after treatment 3Ki. Further experiments are for a detailed understanding of these regulatory pathways and for the proper verification of the mechanism of the effects of H2S necessary, in particular for a full understanding of the target control factors by the post-translational modification of S-sulfhydration.

No description available.

Cancer cachexia is a wasting syndrome causing involuntary weight loss and muscle atrophy. PARP1 is a nicotinamide dinucleotide-dependent enzyme that modifies target proteins by PARylation. The reversal process, dePARylation, is mediated by the PARG enzyme. PARP1 inhibitors are potent cancer agents, while PARG inhibitors are in clinical trials for similar cancers. Here we examine the role of PARylation on muscle homeostasis in cancer cachexia. We employed mouse models with inducible muscle specific knockouts of Parp1 (Parp1-IMKO) or Parg (Parg-IMKO) to investigate their implications on skeletal muscle in a cancer cachexia model. We assessed muscle loss, grip strength, and gene expression. Results show that Parp1- IMKO mice had increased muscle wasting, while Parg-IMKO had degradation rates similar to wild-type mice during cancer cachexia. This suggests reduced PARylation might worsen cancer cachexia, while an increase does not. This supports PARG inhibitor development as anticancer alternatives. Our study highlights challenges with PARP1 inhibitors and the need to study PARylation and dePARylation in muscle health during cancer cachexia, impacting clinical strategies using PARP1 or PARG inhibitors.

La famille des GTPases Rho, comprenant 20 membres, contrôle la dynamique du cytosquelette d'actine et différents processus cellulaires comme la migration. En plus de leurs rôles bien établis, certaines GTPases Rho, notamment RhoB et Rac1, ont émergé en tant que gènes de réponse précoce aux dommages à l'ADN. En effet, RhoB est induite en réponse à divers stress génotoxiques, y compris la camptothécine (CPT), les UV et le cisplatine, et protège principalement les cellules de l'apoptose. Le rôle des autres GTPases Rho en réponse précoce aux génotoxiques reste largement méconnu. Dans ce projet, nous avons utilisé la camptothécine, un inhibiteur de la topoisomérase I (TOP1), qui stabilise sélectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de façon précoce par les dommages à l'ADN. En plus de RhoB, nous avons identifié RND1 comme un gène rapidement induit par la CPT. L'induction de RND1 est réversible et étroitement corrélée à la présence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le péroxyde d'hydrogène, qui stabilisent indirectement les TOP1cc, induisent également RND1. La CPT augmente la transcription de RND1 indépendamment de l'activité de son promoteur minimal. De plus, la CPT augmente l'activité de la poly ADP-ribose polymérase (PARP1), dont l'inhibition prévient la transcription de RND1. La surexpression de RND1 augmente également l'expression de PARP1, suggérant une régulation positive entre PARP1 et RND1 en réponse aux TOP1cc. Ainsi, nous proposons qu'en réponse à la CPT, les TOP1cc activent PARP1, qui à son tour favorise la transcription de RND1, initiant ainsi une boucle de rétrocontrôle positive. Enfin, nous avons montré que RND1 protège les cellules contre l'apoptose induite par la CPT et entraîne leur résistance à la CPT. L'ensemble de ces résultats ont permis d'identifier RND1 comme nouvelle GTPase Rho impliquée dans la réponse au stress et proposent un nouveau mécanisme de régulation de la transcription des gènes en réponse aux TOP1cc via l'activation de PARP1. Ces résultats suggèrent par ailleurs qu'inhiber la signalisation de RND1 pourrait sensibiliser les cellules tumorales aux dérivés cliniques de la CPT. / Rho GTPase family comprises 20 members that regulate key cellular functions such as actin cytoskeleton organization and migration. Beside their canonical functions, certain Rho GTPases, including RhoB and Rac1, emerged as early DNA damage-inducible genes. Indeed, RhoB is readily induced in response to various genotoxic stress, including camptothecin (CPT), UV and cisplatin, and primarily protect cells against apoptotic cell death. Whether other Rho GTPases also respond early to genotoxics is largely unknown. In this project, we used camptothecin, a topoisomerase I (TOP1) inhibitor that selectively stabilized TOP1-DNA cleavage complexes (TOP1cc) onto chromatin, to screen for early DNA damage-inducible Rho GTPases. Besides RhoB, we identified RND1 as a gene rapidly induced by CPT. RND1 induction is reversible and closely associated with the presence of TOP1cc induced by CPT. Consistently, UV light and hydrogen peroxide, which indirectly stabilized TOP1cc, induce RND1 as well. CPT increases minimal promoter-independent RND1 transcription. Additionally, CPT increases poly ADP-ribose polymerase (PARP1) activity, whose inhibition prevents RND1 transcription. Overexpression of RND1 also increases PARP1 expression, suggesting a positive regulation between PARP1 and RND1 in response to TOP1cc. Thus, we propose that in response to CPT, TOP1cc activate PARP1, which in turn promotes RND1 transcription resulting in a positive feedback loop. Finally, we found that RND1 protects cells against CPT-induced apoptosis and leads to resistance to CPT. Together, these results highlight RND1 as a new Rho GTPase involved in the response to stress and propose a new mechanism for TOP1cc-induced gene transcription through PARP1 activation. These findings further suggest that inhibiting RND1 signaling could sensitize tumor cells to CPT derivatives.

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+/ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Canada each year. Currently, most therapeutic approaches target rapidly dividing cancer cells by inhibition of normal cellular processes, however these therapies are not selective for cancer cells and unwanted side effects occur. Accordingly, novel cancer-targeted therapeutic strategies and drug targets are urgently needed to diminish the morbidity and mortality rates associated with CRC. Synthetic lethality is a new therapeutic approach that is designed to better target and kill cancer cells by exploiting a cancer-associated mutation (i.e. RAD54B-deficiency) thereby minimizing adverse side effects. We hypothesize that RAD54B-deficient CRC cells will be selectively killed via a synthetic lethal (SL) interaction with PARP1. We have identified and validated a novel drug target, PARP1, within CRC cells harboring RAD54B-deficiencies through a SL paradigm. This study represents the first steps necessary to identify and develop precision medicine based therapeutic strategies to combat CRC. / October 2016

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD+) to produce poly (ADP-ribose) (PAR) polymers on target proteins or itself. As a result, hyper-activity of PARP1 may lead to necrosis by excessively depleting ATP pool which results in mitochondrial energetic collapse. On the other hand, it is known that Ca2+ overload induces necrosis, but much still remains unknown about how Ca2+ overload-induced necrosis is regulated in cells. In this study, we show that ATR, besides its hallmark regulatory role in DDR, also plays a role in NDDR by suppressing ionomycin-induced necrosis. Ionomycin as a Ca2+ ionophore can dramatically raise the intracellular level of Ca2+, leading to necrosis. We found that this Ca2+ overload-induced necrosis occurs without inducing DDR in cells. Instead, the hyper-poly(ADP-ribosyl)ation (PARylation) activity of activated PARP1 could be a reason leading to necrosis, as NAD+ supplied to media can rescue ionomycin-induced necrosis. In vitro PARylation assay also demonstrates that PARP1 hyper-activation is Ca2+ dependent. In cells, ATR-PARP1 interaction happened after ionomycin treatment. Furthermore, ionomycin treatment induces more full-length PAR polymers formed in ATR-deficient cells than in ATR-proficient cells. The interaction of kinase-dead ATR and PARP1 dramatically decreased as compared to wild-type ATR. Therefore, ATR plays a novel role in NDDR wherein it is able to suppress Ca2+ overload-induced PARP1-mediated necrosis. Ca2+ overload-induced cell death is a major cause of many human medical conditions and diseases, such as brain injury, stroke and ischemia et al. Our ongoing studies will help to define the molecular mechanisms of the anti-necrosis activities of ATR, which may support ATR as a new clinical target for therapeutic treatment of those diseases.

PARP inhibitors (PARPi) have been used to induce synthetic lethality in BRCA-deficient tumors in clinical trials with limited success due to the development of resistance to PARPi. BRCA-deficient cells are unable to repair DNA double strand breaks by the accurate homologous recombination repair (HR), and therefore rely on alternative DNA repair pathways for survival. We hypothesized that RAD52-mediated DNA repair mechanisms remain active and are thus protecting some PARPi-treated BRCA-deficient tumor cells from apoptosis, and that targeting RAD52 should enhance the synthetic lethal effect of PARPi. We show here that RAD52 inhibitors (RAD52i) attenuated single-strand annealing (SSA) and residual HR activity in BRCA-deficient cells. Simultaneous targeting of PARP1 and RAD52 with small molecule inhibitors or via expression of dominant-negative mutants induced an accumulation of DSBs and selective eradication of BRCA-deficient solid tumor and leukemia cells, while BRCA-proficient cells were unaffected. Parp1-/-Rad52-/- transgenic mice are healthy and indistinguishable from wild-type mice due to the presence of the BRCA-pathway, and Parp1-/-Rad52-/- mice with inducible BRCA1-deficient leukemia displayed significantly prolonged survival when compared to Parp1-/- and Rad52-/- counterparts. Finally, PARPi + RAD52i selectively targeted BRCA1-deficient solid tumors in immunodeficient mice with minimal toxicity to normal cells and tissues which are protected by the BRCA-pathway, indicating minimal side effects. In conclusion, our data indicate that combination treatment of RAD52i and PARPi will significantly improve therapeutic outcome of BRCA-deficient malignancies compared to treatment with PARPi monotherapy, while leaving healthy cells and tissues unharmed. / Biomedical Sciences