PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome

The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt.Methods: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy.Results: Here we present evidence for a PARP1 mediated, PARylation- dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP- inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection.Conclusions: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt- mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints

PARP1 promoter links cell cycle progression with adaptation to oxidative environment

Although electrophiles are considered as detrimental to cells, accumulating recent evidence indicates that proliferating non-cancerous and particularly cancerous cells utilize these agents for pro-survival and cell cycle promoting signaling. Hence, the redox shift to mild oxidant release must be balanced by multiple defense mechanisms. Our latest findings demonstrate that cell cycle progression, which dictates oxidant level in stress-free conditions, determines PARP1 transcription. Growth modulating factors regulate CDK4/6-RBs-E2Fs axis. In cells arrested in G1 and G0, RB1-E2F1 and RBL2-E2F4 dimers recruit chromatin remodelers such as HDAC1, SWI/SNF and PRC2 to condense chromatin and turn off transcription. Release of retinoblastoma-based repressive complexes from E2F-dependent gene promoters in response to cell transition to S phase enables transcription of PARP1. This enzyme contributes to repair of oxidative DNA damage by supporting several strand break repair pathways and nucleotide or base excision repair pathways, as well as acting as a co-activator of transcription factors such as NRF2 and HIF1a, which control expression of antioxidant enzymes involved in removal of electrophiles and secondary metabolites. Furthermore, PARP1 is indispensible for transcription of the pro-survival kinases MAP2K6, ERK1/2 and AKT1, and for maintaining MAPK activity by suppressing transcription of the MAPK inhibitor, MPK1. In summary, cell cycle controlled PARP1 transcription helps cells to adapt to a pro-oxidant redox shift

Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC50). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations

PARP Inhibitor PJ34 Protects Mitochondria and Induces DNA-Damage Mediated Apoptosis in Combination With Cisplatin or Temozolomide in B16F10 Melanoma Cells

<p>PARP-1 inhibition has recently been employed in both mono- and combination therapies in various malignancies including melanoma with both promising and contradicting results reported. Although deeper understanding of the underlying molecular mechanisms may help improving clinical modalities, the complex cellular effects of PARP inhibitors make disentangling of the mechanisms involved in combination therapies difficult. Here, we used two cytostatic agents used in melanoma therapies in combination with PARP inhibition to have an insight into cellular events using the B16F10 melanoma model. We found that, when used in combination with cisplatin or temozolomide, pharmacologic blockade of PARP-1 by PJ34 augmented the DNA-damaging and cytotoxic effects of both alkylating compounds. Interestingly, however, this synergism unfolds relatively slowly and is preceded by molecular events that are traditionally believed to support cell survival including the stabilization of mitochondrial membrane potential and morphology. Our data indicate that the PARP inhibitor PJ34 has, apparently, opposing effects on the mitochondrial structure and cell survival. While, initially, it stimulates mitochondrial fusion and hyperpolarization, hallmarks of mitochondrial protection, it enhances the cytotoxic effects of alkylating agents at later stages. These findings may contribute to the optimization of PARP inhibitor-based antineoplastic modalities.</p

Identification of the novel lapine rotavirus genotype P[22] from an outbreak of enteritis in a Hungarian rabbitry

Application of improved molecular techniques in the detection and characterization of rotavirus strains has led to the recent description of several new combinations, specificities, and genetic variants of the outer capsid genes, VP7 and VP4. In spite of the enormous diversity of mammalian rotavirus strains, the few lapine rotaviruses characterized to date, appear to carry a narrow range of such antigen combinations; only P[14], G3 and, based on a more recent study, P[22], G3 rotaviruses have proved to be epidemiologically important in rabbits. In the present study, we characterized a lapine group A rotavirus with a super-short electropherotype detected in an outbreak of fatal enteritis in a Hungarian commercial rabbitry. Based on sequence and phylogenetic analysis of the VP7, VP4, and NSP4 genes, our lapine strain is a P[22], G3 rotavirus that carries the NSP4 genotype shared by most lapine rotaviruses. Although the P[22] VP4 specificity has been newly identified, the relatively high sequence variation between our strain and those identified in Italy (89.1-90.4% nucleotide identity; region VP8*) implies that these strains diversified far before they were described for the first time, strongly suggesting that this genotype may have circulated in rabbitries or in nature without prior detection. We conclude that genotype P[22] lapine rotaviruses show a wider geographical dispersal than previously thought, although understanding their true epidemiological significance needs further investigation

BGP-15 Protects against Oxidative Stress- or Lipopolysaccharide-Induced Mitochondrial Destabilization and Reduces Mitochondrial Production of Reactive Oxygen Species.

Reactive oxygen species (ROS) play a critical role in the progression of mitochondria-related diseases. A novel insulin sensitizer drug candidate, BGP-15, has been shown to have protective effects in several oxidative stress-related diseases in animal and human studies. In this study, we investigated whether the protective effects of BGP-15 are predominantly via preserving mitochondrial integrity and reducing mitochondrial ROS production. BGP-15 was found to accumulate in the mitochondria, protect against ROS-induced mitochondrial depolarization and attenuate ROS-induced mitochondrial ROS production in a cell culture model, and also reduced ROS production predominantly at the complex I-III system in isolated mitochondria. At physiologically relevant concentrations, BGP-15 protected against hydrogen peroxide-induced cell death by reducing both apoptosis and necrosis. Additionally, it attenuated bacterial lipopolysaccharide (LPS)-induced collapse of mitochondrial membrane potential and ROS production in LPS-sensitive U-251 glioma cells, suggesting that BGP-15 may have a protective role in inflammatory diseases. However, BGP-15 did not have any antioxidant effects as shown by in vitro chemical and cell culture systems. These data suggest that BGP-15 could be a novel mitochondrial drug candidate for the prevention of ROS-related and inflammatory disease progression

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

AbstractWe found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT