Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2 French Canadian families with high risk of breast cancer

<p>Abstract</p> <p>Background</p> <p>The Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer.</p> <p>Methods</p> <p>In a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of the <it>NBN </it>gene in our cohort of 97 high-risk non-<it>BRCA1 </it>and -<it>BRCA2 </it>breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population. <it>In silico </it>programs (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines.</p> <p>Results</p> <p>Twenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone γ-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines.</p> <p>Conclusion</p> <p>Our analysis of <it>NBN </it>sequence variations indicated that potential <it>NBN </it>alterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located in <it>NBN </it>promoter region.</p

A Kinase-Independent Role for the Rad3ATR-Rad26ATRIP Complex in Recruitment of Tel1ATM to Telomeres in Fission Yeast

ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1ATM and Rad3ATR recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1ATM to telomeres independently of the previously established Nbs1 C-terminal Tel1ATM interaction domain. Recruitment of Tel1ATM to telomeres in nbs1-c60Δ cells, which lack the C-terminal 60 amino acid Tel1ATM interaction domain of Nbs1, was dependent on Rad3ATR-Rad26ATRIP, but the kinase domain of Rad3ATR was dispensable. Thus, our results establish that the Rad3ATR-Rad26ATRIP complex contributes to the recruitment of Tel1ATM independently of Rad3ATR kinase activity, by a mechanism redundant with the Tel1ATM interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3ATR-Rad26ATRIP to telomeres. In response to replication stress, mammalian ATR–ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR–ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity

Dimethyl Sulfoxide Promotes the Multiple Functions of the Tumor Suppressor HLJ1 through Activator Protein-1 Activation in NSCLC Cells

Background: Dimethyl sulfoxide (DMSO) is an amphipathic molecule that displays a diversity of antitumor activities. Previous studies have demonstrated that DMSO can modulate AP-1 activity and lead to cell cycle arrest at the G1 phase. HLJ1 is a newly identified tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. Its transcriptional activity is regulated by the transcription factor AP-1. However, the effects of DMSO on HLJ1 are still unknown. In the present study, we investigate the antitumor effects of DMSO through HLJ1 induction and demonstrate the mechanisms involved. Methods and Findings: Low-HLJ1-expressing highly invasive CL1–5 lung adenocarcinoma cells were treated with various concentrations of DMSO. We found that DMSO can significantly inhibit cancer cell invasion, migration, proliferation, and colony formation capabilities through upregulation of HLJ1 in a concentration-dependent manner, whereas ethanol has no effect. In addition, the HLJ1 promoter and enhancer reporter assay revealed that DMSO transcriptionally upregulates HLJ1 expression through an AP-1 site within the HLJ1 enhancer. The AP-1 subfamily members JunD and JunB were significantly upregulated by DMSO in a concentration-dependent manner. Furthermore, pretreatment with DMSO led to a significant increase in the percentage of UV-induced apoptotic cells. Conclusions: Our results suggest that DMSO may be an important stimulator of the tumor suppressor protein HLJ1 throug

Association between the NBS1 E185Q polymorphism and cancer risk: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>NBS1 is a key DNA repair protein in the homologous recombination repair pathway and a signal modifier in the intra-S phase checkpoint that plays important roles in maintaining genomic stability. The <it>NBS1 </it>8360G>C (<it>Glu185Gln</it>) is one of the most commonly studied polymorphisms of the gene for their association with risk of cancers, but the results are conflicting.</p> <p>Methods</p> <p>We performed a meta-analysis using 16 eligible case-control studies (including 17 data sets) with a total of 9,734 patients and 10,325 controls to summarize the data on the association between the <it>NBS1 </it>8360G>C (E185Q) polymorphism and cancer risk.</p> <p>Results</p> <p>Compared with the common 8360GG genotype, the carriers of variant genotypes (i.e., 8360 GC/CC) had a 1.06-fold elevated risk of cancer (95% CI = 1.00–1.12, <it>P </it>= 0.05) in a dominant genetic model as estimated in a fixed effect model. However, the association was not found in an additive genetic model (CC <it>vs </it>GG) (odds ratio, OR = 0.98, 95% CI = 0.85–1.13, <it>P </it>= 0.78) nor in a recessive genetic model (CC <it>vs </it>GC +GG) (OR = 0.94, 95% CI = 0.82–1.07, <it>P </it>= 0.36). The effect of the 8360G>C (E185Q) polymorphism was further evaluated in stratification analysis. It was demonstrated that the increased risk of cancer associated with 8360G>C variant genotypes was more pronounced in the Caucasians (OR = 1.07, 95% CI = 1.01–1.14, <it>P </it>= 0.03).</p> <p>Conclusion</p> <p>Our meta-analysis suggests that the <it>NBS1 </it>E185Q variant genotypes (8360 <it>GC/CC</it>) might be associated with an increased risk of cancer, especially in Caucasians.</p

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress

ATM-dependent phosphorylation of nibrin in response to radiation exposure

Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). Them are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50 (refs 1,2). This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (1R) and is observed in brightly staining nuclear foci after a longer period of time(3). The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. Here we demonstrate that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. We also show that ATM physically interacts with and phosphorylates nibrin on serine 343 both in vivo and in vitro. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. Our data provide a biochemical explanation for the similarity in phenotype between A-T and NBS

Fine localization of the Nijmegen breakage syndrome gene to 8q21: evidence for a common founder haplotype.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88

Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction