In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.

• MeSH
• buněčné linie MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny metabolismus   genetika   MeSH
• histony metabolismus   MeSH
• jaderné proteiny metabolismus   genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• oprava DNA * MeSH
• poly-ADP-ribosylace MeSH
• poly(ADP-ribosa)polymerasa 1 * metabolismus   genetika   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   genetika   MeSH
• protein XRCC1 metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.

• MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasa ATP metabolismus   MeSH
• DNA-polymerasa beta metabolismus   MeSH
• DNA genetika   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• jednořetězcové zlomy DNA MeSH
• lidé MeSH
• oprava DNA účinky léků   genetika   MeSH
• PARP inhibitory farmakologie   MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• poškození DNA účinky léků   genetika   MeSH
• protein XRCC1 metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

• MeSH
• 5-methylcytosin metabolismus   MeSH
• buněčné linie MeSH
• DNA biosyntéza   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• metylace MeSH
• neurony metabolismus   MeSH
• oprava DNA * MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• replikace DNA MeSH
• sekvenční analýza DNA MeSH
• zesilovače transkripce genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant-active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis.

• MeSH
• apoptóza * MeSH
• Basidiomycota chemie   MeSH
• HCT116 buňky MeSH
• kolorektální nádory farmakoterapie   metabolismus   patofyziologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• protoonkogenní proteiny c-bcl-2 genetika   MeSH
• regulace genové exprese u nádorů MeSH
• signální dráha Wnt účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Accurate copying of DNA during S phase is essential for genome stability and cell viability. During genome duplication, the progression of the DNA replication machinery is challenged by limitations in nucleotide supply and physical barriers in the DNA template that include naturally occurring DNA lesions and secondary structures that are difficult to replicate. To ensure correct and complete replication of the genome, cells have evolved several mechanisms that protect DNA replication forks and thus maintain genome integrity and stability during S phase. One class of enzymes that have recently emerged as important in this process, and therefore as promising targets in anticancer therapy, are the poly(ADP-ribose) polymerases (PARPs). We review here the roles of these enzymes during DNA replication as well as their impact on genome stability and cellular viability in normal and cancer cells.

• MeSH
• aktivace enzymů MeSH
• cílená molekulární terapie MeSH
• lidé MeSH
• multigenová rodina MeSH
• náchylnost k nemoci MeSH
• nestabilita genomu MeSH
• oprava DNA MeSH
• PARP inhibitory farmakologie   terapeutické užití   MeSH
• poly(ADP-ribosa)polymerasy genetika   metabolismus   MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• replikace DNA MeSH
• S fáze fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

• MeSH
• akrylamidy farmakologie   MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• buňky 3T3-L1 MeSH
• buňky Hep G2 MeSH
• cytoplazma metabolismus   MeSH
• histony metabolismus   MeSH
• kontrolní body buněčného cyklu MeSH
• lidé MeSH
• mutageneze cílená MeSH
• myši MeSH
• NAD metabolismus   MeSH
• nikotinamidfosforibosyltransferasa chemie   genetika   metabolismus   MeSH
• oxidační stres MeSH
• piperidiny farmakologie   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• proliferace buněk MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sirtuiny metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

• MeSH
• "flap" endonukleasy metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasa ATP metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• lidé MeSH
• oprava DNA MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poly(ADP-ribosa)polymerasy genetika   metabolismus   MeSH
• polyadenosindifosfátribosa metabolismus   MeSH
• poškození DNA MeSH
• protein XRCC1 metabolismus   MeSH
• replikace DNA fyziologie   MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Purpose: Cluster I pheochromocytomas and paragangliomas (PCPGs) tend to develop malignant transformation, tumor recurrence, and multiplicity. Transcriptomic profiling suggests that cluster I PCPGs and other related tumors exhibit distinctive changes in the tricarboxylic acid (TCA) cycle, the hypoxia signaling pathway, mitochondrial electron transport chain, and methylation status, suggesting that therapeutic regimen might be optimized by targeting these signature molecular pathways.Experimental Design: In the present study, we investigated the molecular signatures in clinical specimens from cluster I PCPGs in comparison with cluster II PCPGs that are related to kinase signaling and often present as benign tumors.Results: We found that cluster I PCPGs develop a dependency to mitochondrial complex I, evidenced by the upregulation of complex I components and enhanced NADH dehydrogenation. Alteration in mitochondrial function resulted in strengthened NAD+ metabolism, here considered as a key mechanism of chemoresistance, particularly, of succinate dehydrogenase subunit B (SDHB)-mutated cluster I PCPGs via the PARP1/BER DNA repair pathway. Combining a PARP inhibitor with temozolomide, a conventional chemotherapeutic agent, not only improved cytotoxicity but also reduced metastatic lesions, with prolonged overall survival of mice with SDHB knockdown PCPG allograft.Conclusions: In summary, our findings provide novel insights into an effective strategy for targeting cluster I PCPGs, especially those with SDHB mutations. Clin Cancer Res; 24(14); 3423-32. ©2018 AACR.

• MeSH
• apoptóza genetika   MeSH
• biologické modely MeSH
• buněčný cyklus genetika   MeSH
• chemorezistence genetika   MeSH
• cílená molekulární terapie MeSH
• feochromocytom farmakoterapie   genetika   metabolismus   patologie   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši MeSH
• NAD metabolismus   MeSH
• nádorové buněčné linie MeSH
• oprava DNA * MeSH
• paragangliom farmakoterapie   genetika   metabolismus   patologie   MeSH
• PARP inhibitory farmakologie   terapeutické užití   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• signální transdukce účinky léků   MeSH
• sukcinátdehydrogenasa genetika   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

Autophagy and poly(ADP-ribose) polymerase 1 (PARP-1) are activated and involved in a series of cell processes under oxidative stress, which is associated with pathogenesis of atherosclerosis. Research on their relationship under oxidative stress has been limited. In this study, we aimed to investigate the activation, relationship, and role of autophagy and PARP-1 in vascular smooth muscle cell (VSMC) death under oxidative stress. This study explored the signal molecule PARP-1 and autophagy in VSMCs using gene silencing and the hydrogen peroxide (H2O2)-stimulated oxidative stress model. We observed that H2O2 could induce autophagy in VSMCs, and the inhibition of autophagy could protect VSMCs against oxidative stress-mediated cell death. Meanwhile, PARP-1 could also be activated by H2O2. Additionally, we analysed the regulatory role of PARP-1 in oxidative stress-mediated autophagy and found that PARP-1 was a novel factor involved in the H2O2-induced autophagy via the AMPK-mTOR pathway. Finally, PARP-1 inhibition protected VSMCs against caspase-dependent apoptosis. These data suggested that PARP-1 played a critical role in H2O2-mediated autophagy and both of them were involved in apoptosis of VSMCs.

• MeSH
• apoptóza * účinky léků   MeSH
• autofagie * účinky léků   MeSH
• kaspasy metabolismus   MeSH
• myocyty hladké svaloviny účinky léků   metabolismus   patologie   MeSH
• myši inbrední C57BL MeSH
• oxidační stres * účinky léků   MeSH
• peroxid vodíku toxicita   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• svaly hladké cévní patologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116-/- and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified.

• MeSH
• apoptóza genetika   MeSH
• buňky HT-29 MeSH
• Caco-2 buňky MeSH
• cyklin B1 genetika   metabolismus   MeSH
• dospělí MeSH
• HCT116 buňky MeSH
• Kaplanův-Meierův odhad MeSH
• kolorektální nádory genetika   metabolismus   patologie   MeSH
• kontrolní body fáze G1 buněčného cyklu genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• poly(ADP-ribosa)polymerasy genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteinkinasa CDC2 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• RNA dlouhá nekódující genetika   metabolismus   MeSH
• RNA interference MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• vazba proteinů MeSH
• western blotting MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH