Synthesis of trimetallic iron-boron core and gold shell nanoparticles for experimental cancer radiotherapy.

Cancer is a significant and constantly growing clinical problem all over the word. For many types of cancer there has been little change in mortality rate of CRC in the past decades and treatment options are limited. A striking example is malignant Glioblastoma (GBM) which exhibits a high degree of infiltration of surrounding healthy brain tissue, extremely high mortality rate, morbidity and most life-years lost of any cancer.

Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration.

DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective.

Ku70 N-terminal lysines acetylation/deacetylation is required for radiation-induced DNA-double strand breaks repair.

Ku70 protein in hetero-trimeric complex with Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) represents a critical component of the nonhomologous-end-joining (NHEJ), the major machinery of DSBs repair in mammalian cells. It has been previously shown that modulation of Ku70 acetylation by histone deacetylases (HDAC) inhibitors induced sensitization of cancer cells to chemotherapeutic agents. However, the effects of such modulation on the repair of Ionizing Radiation (IR)-induced DSBs and the importance of dynamic equilibrium of acetylation/deacetylation have not been studied in details.

Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure.

Intracellular synchrotron nanoimaging and DNA damage/genotoxicity screening of novel lanthanide-coated nanovectors.

Aims: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization.

Materials & Methods: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications.

Issues surrounding standard cytotoxicity testing for assessing activity of non-covalent DNA-binding metallo-drugs.

Investigating the methods commonly used to evaluate in vitro cytotoxicity of novel compounds, specifically non-covalent DNA binders, identifies that these methods may not be appropriate. The level of anticancer activity depends not only on the incubation time but also on the absolute amount (number of moles) of drug compound applied, rather than the concentration.

Association of the thyroid stimulating hormone receptor gene (TSHR) with Graves' disease.

Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease.

Evolutionary and functional conservation of the DNA non-homologous end-joining protein, XLF/Cernunnos.

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks.

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1.

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX.

Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms.

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms.

AHNAK interacts with the DNA ligase IV-XRCC4 complex and stimulates DNA ligase IV-mediated double-stranded ligation.

AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV-XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV-XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction.

Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA.

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells.

Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule.

The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate.

Impact of DNA ligase IV on the fidelity of end joining in human cells.

A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining.

Nonhomologous end joining and V(D)J recombination require an additional factor.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells.

Identification of a DNA nonhomologous end-joining complex in bacteria.

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku.

DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS).

Cellular and biochemical impact of a mutation in DNA ligase IV conferring clinical radiosensitivity.

DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who overresponded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches.

The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact.

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11].

Defects in the kinetics of the repair of DNA double-strand breaks and inhibition of DNA synthesis in the ataxia telangiectasia AT5BI-VA cell line: comparison to a corrected hybrid, atxbc.

The nature of the primary biochemical defect in the human radiosensitive and cancer-prone syndrome, ataxia telangiectasia (AT), has remained obscure despite many efforts to elucidate it. In this study, AT complementation group D cells and a nearly isogenic corrected AT-hamster hybrid derivative have been analyzed for induction and repair of initial double-strand breaks (DSBs) after exposure to ionizing radiation, using a sensitive field-inversion electrophoresis technique. Results suggesting that initial levels of damage are the same in these two cell types, but indicating differences in the fast component of DNA repair, have been compared and correlated with those resulting from a study of the radioresistant DNA synthesis defect and its correction in the same cell lines.

Relative contributions of levels of initial damage and repair of double-strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part II. Neutrons.

We have compared DNA double-strand break (dsb) induction and rejoining, using field-inversion gel electrophoresis, with survival in mutant (XR-V15B) and in wild-type parental (V79B) hamster cell lines after low dose neutron and X-irradiation. We found that neutrons do not appear to induce more dsbs than X-rays and deduce that increased sensitivity to neutrons is therefore not due to a higher initial yield of dsbs. Even with low doses of neutrons, there is a visible increase in the production of a smaller subset of DNA fragments which arise only after very high dose X-irradiation.

Relative contributions of levels of initial DNA damage and repair of double strand breaks to the ionizing radiation-sensitive phenotype of the Chinese hamster cell mutant, XR-V15B. Part I. X-rays.

In order to investigate the relationships between the induction and rejoining of DNA double-strand breaks (dsb) and their biological consequences it is necessary to measure these lesions uniquely and accurately, especially at relevant low doses of ionizing radiation. Differences in radiosensitivity between cell lines could be due to variations in dsb induction or to differences in the efficiency and/or accuracy of enzymatic repair of these lesions. We have used field-inversion gel electrophoresis to investigate dsb induction and rejoining in V79B parental and XR-V15B ionizing radiation-sensitive mutant cell lines.

Field-inversion gel electrophoresis analysis of the induction and rejoining of DNA double-strand breaks in cells embedded in agarose.

Among the techniques available for the measurement of the induction and rejoining of DNA double-strand breaks (DSBs), pulsed-field gel electrophoresis appears to have the greatest potential to improve the sensitivity limits to study these lesions in the dose range closest to that used in cell survival experiments. Encapsulating the cells in agarose during the experimental procedure allows the accurate and reproducible measurement of rejoining kinetics with a very minimal time delay immediately after irradiation. The method allows direct comparison of the amount of initial DNA damage sustained with repair kinetics in experiments designed to elucidate the mechanisms underlying differences in radiosensitivity between cell lines, together with analysis of the effect of different radiation qualities.