CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesDNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer.Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation.The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265).ALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.Icelandic Centre for Researc

Northern lights assay: a versatile method for comprehensive detection of DNA damage.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadDNA damage assays have various limitations in types of lesions detected, sensitivity, specificity and samples that can be analyzed. The Northern Lights Assay (NLA) is based on 2D Strandness-Dependent Electrophoresis (2D-SDE), a technique that separates nucleic acids based on length, strandness, structure and conformation changes induced by damage. NLA is run on a microgel platform in 20-25 min. Each specimen is analyzed in pairs of non-digested DNA to detect single- and double-stranded breaks (DSBs) and Mbo I-digested DNA to detect other lesions. We used NLA to evaluate DNA in solution and isolated from human cells treated with various genotoxic agents. NLA detected and distinguished between single- and DSBs, interstrand and intrastrand DNA crosslinks, and denatured single-stranded DNA. NLA was sufficiently sensitive to detect biologically relevant amount of DNA damage. NLA is a versatile, sensitive and simple method for comprehensive and simultaneous analysis of multiple types of damage, both in purified DNA and in DNA isolated from cells and body fluids. NLA can be used to evaluate DNA quality in biosamples, monitor complex molecular procedures, assess genotoxicity, diagnose genome instability, facilitate cancer theranostics and in basic nucleic acids research.University of Iceland Research Fund Landspitali University Hospital Research Fund Icelandic Center for Research Funds Lifeind ehf. University of Iceland Research Fun

Sviperfðabreytingar á ALKBH3 og áhrif sviperfða á RNA í stjórnun á DNA viðgerð

The DNA damage response is crucial to maintaining the integrity of DNA and the health of a cell. Unrepaired lesions within the DNA can lead to genomic instability and potentially aid in the formation of diseases such as cancer. Some cancers possess dysfunctional DNA repair and chemotherapeutic treatments may aim to exploit this weakness that distinguishes cancer cells from normal healthy cells. ALKBH3 is a DNA repair protein involved in the repair of alkylation damage. Within The Cancer Genome Atlas we found that ALKBH3 contained a hyper-methylated promoter in 20% of breast cancers. This hyper-methylation, a form of epigenetic regulation, lead to a dramatic reduction of ALKBH3 mRNA expression and therefore a decrease in total ALKBH3 protein levels. We looked within a sample of Icelandic breast tumors and found they too possessed hyper-methylated promoters for ALKBH3. Importantly, this methylation occurs only within the tumor tissue, but not the normal tissue of the same patients. Additionally, patients who contained high levels of promoter methylation had statistically significant decreased survival. The project then explored the functional consequences of the absence of ALKBH3. We discovered that the knockdown of ALKBH3 causes a decrease in protein levels of a protein crucial in DNA double-strand break repair. We determined that this protein is regulated by ALKBH3 through a form of regulation called epitranscriptomics. Epitranscriptomics is a new field of study of how RNA fate is determined, largely through the addition and removal of methyl groups on RNA nucleotides. This project helps elucidate a potential contributing factor to cancer development as well as provide a potential target for chemotherapeutic treatment.Eggertssjóður 2015: Hlutverk ALKBH3 í tvíþátta DNA viðgerð Rannis Project Grant 2015-2017: DNA repair defects and drug response in breast and ovarian cancer (152077-051) Rannis Project Grant 2017-2019: The role of ALKBH3 in the regulation of DNA double strand break repair (185242-051

On the Structure of Ultrathin FeO Films on Ag(111)

Ultrathin transition metal oxide films exhibit unique physical and chemical properties not observed for the corresponding bulk oxides. These properties, originating mainly from the limited thickness and the interaction with the support, make those films similar to other supported 2D materials with bulk counterparts, such as transition metal dichalcogenides. Ultrathin iron oxide (FeO) films, for example, were shown to exhibit unique electronic, catalytic and magnetic properties that depend on the type of the used support. Ag(111) has always been considered a promising substrate for FeO growth, as it has the same surface symmetry, only ~5% lattice mismatch, is considered to be weakly-interacting and relatively resistant to oxidation. The reports on the growth and structure of ultrathin FeO films on Ag(111) are scarce and often contradictory to each other. We attempted to shed more light on this system by growing the films using different preparation procedures and studying their structure using scanning tunneling microscopy (STM), low energy electron diffraction (LEED) and X-ray photoelectron spectroscopy (XPS). We observed the formation of a previously unreported Moiré superstructure with 45 Å periodicity, as well as other reconstructed and reconstruction-free surface species. The experimental results obtained by us and other groups indicate that the structure of FeO films on this particular support critically depends on the films’ preparation conditions. We also performed density functional theory (DFT) calculations on the structure and properties of a conceptual reconstruction-free FeO film on Ag(111). The results indicate that such a film, if successfully grown, should exhibit tunable thickness-dependent properties, being substrate-influenced in the monolayer regime and free-standing-FeO-like when in the bilayer form