Resveratrol protects intestinal epithelial cells against radiation-induced damage by promoting autophagy and inhibiting apoptosis through SIRT1 activation.

Intrinsic autophagy is important for the maintenance of intestinal homeostasis and intestinal regeneration. Ionizing radiation suppresses intrinsic autophagy and reduces damage-induced regeneration in the intestine, resulting in intestinal injury. Resveratrol, a sirtuin 1 (SIRT1) agonist, promotes autophagy and exerts radioprotective effect. In this study, the protective effect of resveratrol against radiation-induced intestinal injury and its potential mechanism were investigated. Intestinal epithelial cells (IEC-6) were exposed to 10 Gy ionizing radiation and resveratrol (0.1-40.0 µM). Cell viability was investigated using Cell Counting Kit 8 (CCK8), apoptosis was observed by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry, and the expression of apoptotic and autophagic proteins was determined by western blotting. Resveratrol exerted a high toxicity against IEC-6 cells, but at low concentrations, it inhibited ionizing radiation-induced apoptosis. Resveratrol increased SIRT1 expression after irradiation and inhibited ionizing radiation-induced p53 acetylation and pro-apoptotic protein, Bax, expression. Furthermore, resveratrol promoted autophagy via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, thereby protecting IEC-6 cells against radiation-induced damage. These results suggest that resveratrol reduces radiation-induced IEC-6 cell damage by inhibiting apoptosis and promoting autophagy via the activation of SIRT1, and that the PI3K/AKT/mTOR signaling pathway is involved in the induction of autophagy.

SIRT7-dependent deacetylation of NPM promotes p53 stabilization following UV-induced genotoxic stress.

Adaptation to different forms of environmental stress is crucial for maintaining essential cellular functions and survival. The nucleolus plays a decisive role as a signaling hub for coordinating cellular responses to various extrinsic and intrinsic cues. p53 levels are normally kept low in unstressed cells, mainly due to E3 ubiquitin ligase MDM2-mediated degradation. Under stress, nucleophosmin (NPM) relocates from the nucleolus to the nucleoplasm and binds MDM2, thereby preventing degradation of p53 and allowing cell-cycle arrest and DNA repair. Here, we demonstrate that the mammalian sirtuin SIRT7 is an essential component for the regulation of p53 stability during stress responses induced by ultraviolet (UV) irradiation. The catalytic activity of SIRT7 is substantially increased upon UV irradiation through ataxia telangiectasia mutated and Rad3 related (ATR)-mediated phosphorylation, which promotes efficient deacetylation of the SIRT7 target NPM. Deacetylation is required for stress-dependent relocation of NPM into the nucleoplasm and MDM2 binding, thereby preventing ubiquitination and degradation of p53. In the absence of SIRT7, stress-dependent stabilization of p53 is abrogated, both in vitro and in vivo, impairing cellular stress responses. The study uncovers an essential SIRT7-dependent mechanism for stabilization of the tumor suppressor p53 in response to genotoxic stress.

The Histone Deacetylase Inhibitor Romidepsin Spares Normal Tissues While Acting as an Effective Radiosensitizer in Bladder Tumors in Vivo.

PURPOSE: Muscle-invasive bladder cancer has a 40% to 60% 5-year survival rate with radical treatment by surgical removal of the bladder or radiation therapy-based bladder preservation techniques, including concurrent chemoradiation. Elderly patients cannot tolerate current chemoradiation therapy regimens and often receive only radiation therapy, which is less effective. We urgently need effective chemotherapy agents for use with radiation therapy combinations that are nontoxic to normal tissues and tolerated by elderly patients. METHODS AND MATERIALS: We have identified histone deacetylase (HDAC) inhibitors as promising agents to study. Pan-HDAC inhibition, using panobinostat, is a good strategy for radiosensitization, but more selective agents may be more useful radiosensitizers in a clinical setting, resulting in fewer systemic side effects. Herein, we study the HDAC class I-selective agent romidepsin, which we predict to have fewer off-target effects than panobinostat while maintaining an effective level of tumor radiosensitization. RESULTS: In vitro effects of romidepsin were assessed by clonogenic assay and showed that romidepsin was effective in the nanomolar range in different bladder cancer cells and radiosensitized these cells. The radiosensitizing effect of romidepsin was confirmed in vivo using superficial xenografts. The drug/irradiation combination treatment resulted in significant tumor growth delay but did not increase the severity of acute (3.75 days) intestinal normal tissue toxicity or late toxicity at 29 weeks. Moreover, we showed that romidepsin treatment impaired both homologous recombination and nonhomologous end joining DNA repair pathways, suggesting that the disruption of DNA repair pathways caused by romidepsin is a key mechanism for its radiosensitizing effect in bladder cancer cells. CONCLUSIONS: This study demonstrates that romidepsin is an effective radiosensitizer in vitro and in vivo and does not increase the acute and late toxicity after ionizing radiation. Romidepsin is already in clinical use for the cutaneous T-cell lymphoma, but a phase 1 clinical trial of romidepsin as a radiosensitizer could be considered in muscle-invasive bladder cancer.

Elevated HDAC activity and altered histone phospho-acetylation confer acquired radio-resistant phenotype to breast cancer cells.

BACKGROUND: Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. RESULTS: This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G0/G1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor-based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. CONCLUSION: The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor-based radio-sensitization.

Radiation biology and oncology in the genomic era.

Radiobiology research is building the foundation for applying genomics in precision radiation oncology. Advances in high-throughput approaches will underpin increased understanding of radiosensitivity and the development of future predictive assays for clinical application. There is an established contribution of genetics as a risk factor for radiotherapy side effects. An individual's radiosensitivity is an inherited polygenic trait with an architecture that includes rare mutations in a few genes that confer large effects and common variants in many genes with small effects. Current thinking is that some will be tissue specific, and future tests will be tailored to the normal tissues at risk. The relationship between normal and tumor cell radiosensitivity is poorly understood. Data are emerging suggesting interplay between germline genetic variation and epigenetic modification with growing evidence that changes in DNA methylation regulate the radiosensitivity of cancer cells and histone acetyltransferase inhibitors have radiosensitizing effects. Changes in histone methylation can also impair DNA damage response signaling and alter radiosensitivity. An important effort to advance radiobiology in the genomic era was establishment of the Radiogenomics Consortium to enable the creation of the large radiotherapy cohorts required to exploit advances in genomics. To address challenges in harmonizing data from multiple cohorts, the consortium established the REQUITE project to collect standardized data and genotyping for ~5,000 patients. The collection of detailed dosimetric data is important to produce validated multivariable models. Continued efforts will identify new genes that impact on radiosensitivity to generate new knowledge on toxicity pathogenesis and tests to incorporate into the clinical decision-making process.

Alterations in histone acetylation following exposure to 60Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells.

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0-2 Gy 60Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic + r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0-1 Gy 60Co γ-rays. SAHA treatment significantly decreased dic + r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy 60Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by 60Co γ-radiation.

Low-dose radiation differentially regulates protein acetylation and histone deacetylase expression in human coronary artery endothelial cells.

PURPOSE: Ionizing radiation induces cardiovascular disease, the endothelium being the main target. The exact mechanism of the damage is unclear but the involvement of multiple signaling pathways is probable. Reversible lysine acetylation is a posttranslational protein modification that regulates activity across a broad range of signaling pathways. The aim of this study was to determine if a low radiation dose results in acetylome alteration in endothelial cells. MATERIALS AND METHODS: Human coronary artery endothelial cell line was irradiated with Cs-137 gamma-rays (0.5 Gy) and proteomics analysis was performed using enriched acetylated peptides and all peptides. Data were validated using immunoblotting, deacetylase activity assay, and RhoA activity assay. RESULTS: Nearly a hundred proteins were found to have an altered acetylation status 24 h after irradiation, primarily due to an overall decrease in acetylation. The expression of specific deacetylases was significantly increased, coinciding with an enhancement in global deacetylase activity. Proteins changed in their acetylation status belonged to several pathways including protein synthesis, cytoskeleton-related processes, protein folding and calcium signaling. The predicted changes in the RhoA/actin cytoskeleton pathway were validated by immunoassay. CONCLUSIONS: This study shows that protein acetylation is an important mediator of radiation responses in human cardiac coronary endothelial cells. Increased knowledge of the endothelial response to radiation is crucial for the development of normal tissue-sparing modalities during radiation therapy.

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization.

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm2, for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.

Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress.

Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions.

Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation.

BACKGROUND: Ultraviolet-B (UVB) exposure attributes to the formation of age-related nuclear cataract (ARNC), which is mediated with DNA damage. DNA damage, an important factor for pathogenesis of ARNC, is induced by UVB, and is generally resolved by the nucleotide excision repair (NER) repair mechanism. Cockayne syndrome complementation group B (CSB) protein coded by ERCC6 is a vital component for NER. However, we found no association between selected ERCC6 polymorphisms and ARNC. In this study, we investigated whether UVB exposure could alter ERCC6 expression and the process could involve epigenetic changes of DNA methylation and/or histone acetylation of ERCC6 in the lens epithelial cells (LECs). We also assessed the involvement of those coordinated changes in lens tissue from ARNC patients. RESULTS: mRNA and protein expression of ERCC6 in lens tissue (LECs) were lower in ARNCs than those in the controls. This reduction corresponded to methylation of a CpG site at the ERCC6 promoter and histone modifications (methylation and acetylation) nearby this site. UVB-treated human lens epithelium B3 (HLE-B3) and 239T cell presented (1) increased apoptosis, suggesting reduced UV-damage repair, (2) hypermethylation of the CpG site located at position -441 (relative to transcription start site) within the binding region for transcriptional factor Sp1 in the ERCC6 promoter, (3) the enhancement of histone H3K9 deacetylation, (4) induction in DNA methyltransferases 3b (DNMT3b) and histone deacetylase1 (HDAC1) associated to the CpG site of ERCC6 by CHIP assay. CONCLUSIONS: These findings suggest an orchestrated mechanism triggered by UVB radiation where the concurrent association of specific hypermethylation CpG site, H3K9 deacetylation of ERCC6, and repression of ERCC6 gene expression. Taken together, with the similar changes in the lens tissue from ARNC patients, our data unveiled a possible mechanism of epigenetic modification of DNA repair gene in the pathogenesis of ARNC.

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

Radiation-induced modulation of immunogenic genes in tumor cells is regulated by both histone deacetylases and DNA methyltransferases.

Radiation treatment is a pivotal therapy for several cancer types, including colorectal cancer. It has been shown that sublethal doses of radiation modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. We have recently shown that low dose radiation enhances expression of multiple death receptors (Fas, DR4 and DR5) and co-stimulatory molecules (4-1BBL and OX-40L) in colorectal cancer (CRC) cells; however, it is unclear how ionizing radiation (IR) enhances expression of these molecules mechanistically. In the present study, we elucidate the molecular mechanisms by which radiation controls expression of these molecules in CRC. Here we report that, enhanced expression of these genes following radiation treatment of CRC cells is due, in part, to changes in DNA methylation and histone acetylation. We observed that radiation (5 Gy) significantly increased histone acetylation at the promoter regions of 4-1BBL, Fas and DR5 but not OX-40L. However, radiation did not induce changes in the global levels of acetylated histone H3 suggesting specificity of IR-induced changes. Furthermore, evaluation of epigenetic controlling enzymes revealed that IR did not alter overall cellular levels of HDACs (HDAC1, HDAC2 or HDAC3) or DNMTs (DNMT1, DNMT3a, or DNMT3b). Instead, radiation decreased binding of HDAC2 and HDAC3 at the promoter regions of Fas and 4-1BBL, respectively. Radiation also resulted in reduced DNMT1 at both the Fas and 4-1BBL promoter regions but not a control gene. We conclude that single dose radiation can influence the expression of immune response relevant genes in colorectal tumor cells by altering the binding of epigenetic enzymes, and modulating histone acetylation, at specific gene promoters.

Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2.

Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines.

Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.

Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells.

Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium.

DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells.

HDAC inhibitors have radiosensitizing effects in established cancer cell lines. This study was conducted to compare the efficacy of SAHA, LBH589, Valproic Acid (VPA), MS275 and Scriptaid in the patient-derived glioblastoma model. In more detail, SAHA and LBH589 were evaluated to determine predictors of response. Acetylated-histone-H3, γH2AX/53BP1, (p)Chek2/ATM, Bcl-2/Bcl-XL, p21(CIP1/WAF1) and caspase-3/7 were studied in relation to response. SAHA sensitized 50% of cultures, LBH589 45%, VPA and Scriptaid 40% and MS275 60%. Differences after treatment with SAHA/RTx or LBH589/RTx in a sensitive and resistant culture were increased acetylated-H3, caspase-3/7 and prolonged DNA damage repair γH2AX/53BP1 foci. pChek2 was found to be associated with both SAHA/RTx and LBH589/RTx response with a positive predictive value (PPV) of 90%. Bcl-XL had a PPV of 100% for LBH589/RTx response. Incubation with HDACi 24 and 48 hours pre-RTx resulted in the best efficacy of combination treatment. In conclusion a subset of patient-derived glioblastoma cultures were sensitive to HDACi/RTx. For SAHA and LBH589 responses were strongly associated with pChek2 and Bcl-XL, which warrant further clinical exploration. Additional information on responsiveness was obtained by DNA damage response markers and apoptosis related proteins.

An efficient enzymatic modification of cordycepin in ionic liquids under ultrasonic irradiation.

A comparative study of the immobilized Candida antarctica lipase B (Novozym 435)-catalyzed acylation of cordycepin with vinyl acetate in ionic liquids (ILs) under ultrasonic irradiation and shaking was conducted. The application of ultrasonic irradiation instead of shaking during acylation resulted in an enhanced reaction rate and a higher level of substrate conversion. Among the various ILs examined, 1-butyl-3-methylimidazolium tetrafluorobrate ([C4MIm][BF4]) was the best medium for the reaction because it produced the highest substrate conversion. In [C4MIm][BF4], the optimal ultrasonic power, water activity, and reaction temperature were 120 W, 0.33, and 50 °C, respectively. The acylation of cordycepin in [C4MIm][BF4] proved to be regioselective under both conditions: the C5'-OH was acylated. Novozym 435 exhibited a much higher operational stability in [C4MIm][BF4], and 58.0% of its original activity was maintained after ten reuse cycles under ultrasonic irradiation. Compared with the cordycepin, the rate of adenosine deaminase-catalyzed deamination was greatly reduced when the 5'-OH was substituted by acetyl group. These results demonstrated that the combined application of ultrasonic irradiation and IL as a medium was an efficient approach for the enzymatic modification of cordycepin.

Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response.

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

CREB-binding protein regulates Ku70 acetylation in response to ionization radiation in neuroblastoma.

Ku70 was originally described as an autoantigen, but it also functions as a DNA repair protein in the nucleus and as an antiapoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70's binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. Although regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here, we showed that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells, suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells.

GCN5 protects vertebrate cells against UV-irradiation via controlling gene expression of DNA polymerase η.

By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to ∼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.