BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage

BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53(Ser-15) following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53(Ser-15) efficiently. Phosphorylation of p53(Ser-15) is necessary for an IR-induced G(1)/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53(Ser-15) phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G(1)/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G(1)/S cell cycle progression after DNA damage.

Old drug, New target:Ellipticines selectively inhibit RNA Polymerase I transcription

Transcription byRNApolymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC50 in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity.Wehave shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.

Dysfunction of Endothelial Progenitor Cells from Smokers and Chronic Obstructive Pulmonary Disease Patients Due to Increased DNA Damage and Senescence

Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention. 

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

Formation mechanism of levoglucosan and formaldehyde during cellulose pyrolysis

Biomass pyrolysis is an efficient way to transform raw biomass or organic waste materials into useable energy, including liquid, solid, and gaseous materials. Levoglucosan (1,6-anhydro-β-d-glucopyranose) and formaldehyde are two important products in biomass pyrolysis. The formation mechanism of these two products was investigated using the density functional theory (DFT) method based on quantum mechanics. It was found that active anhydroglucose can be obtained from a cellulose homolytic reaction during high-temperature steam gasification of the biomass process. Anhydroglucose undergoes a hydrogen-donor reaction and forms an intermediate, which can transform into the products via three pathways, one (path 1) for the formation of levoglucosan and two (paths 2 and 3) for formaldehyde. A total of six elementary reactions are involved. At a pressure of 1 atm, levoglucosan can be formed at all of the temperatures (450-750 K) considered in this simulation, whereas formaldehyde can be formed only when the temperature is higher than 475 K. Moreover, the energy barrier of levoglucosan formation is lower than that of formaldehyde, which is in agreement with the mechanism proposed in the experiments. © 2011 American Chemical Society.

Grazing under experimental hypercapnia and elevated temperature does not affect the radula of a chiton (Mollusca, Polyplacophora, Lepidopleurida)

Chitons (class Polyplacophora) are benthic grazing molluscs with an eight-part aragonitic shell armature. The radula, a serial tooth ribbon that extends internally more than half the length of the body, is mineralised on the active feeding teeth with iron magnetite apparently as an adaptation to constant grazing on rocky substrates. As the anterior feeding teeth are eroded they are shed and replaced with a new row. The efficient mineralisation and function of the radula could hypothetically be affected by changing oceans in two ways: changes in seawater chemistry (pH and pCO(2)) may impact the biomineralisation pathway, potentially leading to a weaker or altered density of the feeding teeth; rising temperatures could increase activity levels in these ectothermic animals, and higher feeding rates could increase wear on the feeding teeth beyond the animals' ability to synthesise, mineralise, and replace radular rows. We therefore examined the effects of pH and temperature on growth and integrity in the radula of the chiton Leptochiton asellus. Our experiment implemented three temperature (similar to 10, 15, 20 degrees C) and two pCO(2) treatments (similar to 400 mu atm, pH 8.0; similar to 2000 mu atm, pH 7.5) for six treatment groups. Animals (n = 50) were acclimated to the treatment conditions for a period of 4 weeks. This is sufficient time for growth of ca. 7-9 new tooth rows or 20% turnover of the mineralised portion. There was no significant difference in the number of new (non-mineralised) teeth or total tooth row count in any treatment. Examination of the radulae via SEM revealed no differences in microwear or breakage on the feeding cusps correlating to treatment groups. The shell valves also showed no signs of dissolution. As a lineage, chitons have survived repeated shifts in Earth's climate through geological time, and at least their radulae may be robust to future perturbations.

NO Oxidation on Platinum Group Metals Oxides:First Principles Calculations Combined with Microkinetic Analysis

By combining density functional theory calculation and microkinetic analysis, NO oxidation on the platinum group metal oxides (PtO(2), IrO(2), OsO(2)) is investigated, aiming at shedding light on the activities of metal oxides and exploring the activity variations of metal oxides compared to their corresponding metals. A microkinetic model, taking into account the possible low diffusion of surface species on metal oxide surfaces, is proposed for NO oxidation. The resultant turnover frequencies of NO oxidation show that under the typical experimental condition, T = 600 K, p(O2) = 0.1 atm, p(NO) = 3 x 10(-4) atm, p(NO2) = 1.7 x 10(-4) atm; (i) IrO(2)(110) exhibits higher activity than PtO(2)(110) and OsO(2)(110), and (ii) compared to the corresponding metallic Pt, Ir, and Os, the activity of PtO(2) to catalyze NO oxidation is lower, but interestingly IrO(2) and OsO(2) exhibit higher activities. The reasons for the activity differences between the metals and oxides are addressed. Moreover, other possible reaction pathways of NO oxidation on PtO(2)(110), involving O(2) molecule (NO + O(2) -> OONO) and lattice bridge-O(2c), are also found to give low activities. The origin of the Pt catalyst deactivation is also discussed.

Direct oxidation of amines to nitriles in the presence of ruthenium-terpyridyl complex immobilized on ILs/SILP

The immobilization of a ruthenium complex (Ru2Cl4(az-tpy)2) within a range of supported ionic liquids ([C4C1im]Cl, [C4C1im][NTf2], [C6C1im]Cl, [C4C1pyrr]Br, [C4C1im]Br, [C4C1pyrr]Cl) dispersed silica (SILP) operates as an efficient heterogeneous catalyst in oxidation of long chain linear primary amines to corresponding nitriles. This reaction follows a “green” route using a cheap and easy to handles oxidant (oxygen or air). The conversion was found to be strongly influenced by the alkyl chain length of the amine substrate and the choice of oxidant. No condensation reaction was observed between the starting amines and the selectivity to nitrile is 100%. Moving from a composition of 20 atm N2/5 atm O2 to 5 atm N2/20 atm O2 led to enhancements in the conversion (n-alkylamines) and selectivity (benzonitrile) which have been correlated with an increase of the solubilized oxygen. This was further supported by using different inert gas (nitrogen, helium, argon)/oxygen mixtures indicating that the O2 solubility in the SILP system, has an important effect on conversions and TON in this reaction using SILP catalysts. Experiments performed in the presence of CO2 led to a different behaviour due to the formation of amine-CO2 adducts. The application of the Weisz–Prater criterion confirmed the absence of any diffusional constraints.

Novel titania sol-gel derived film for luminescence-based oxygen sensing.

A novel method for the preparation of titania sol–gel derived oxygen sensors based on the ruthenium(II) dye, [Ru(bpy)3]2+, is described. A titania sol–gel paste film was cast onto microscope slides, and the dye ion-paired to the deprotonated, hydroxylated groups on the film's surface from an aqueous solution of the dye at pH 11. The resulting sensor film is extremely oxygen sensitive, with a PO2 (S = 1/2) value (i.e. the partial pressure of oxygen required in order to reduce the original, oxygen free, luminescence intensity by 50%) of 0.011 atm. The sensor undergoes 95% response to oxygen in 4 s, and shows 95% recovery of its luminescence in argon within 7 s.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.

METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.

RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.

CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).