Veerle Vandersickel

Polymorphisms in nonhomologous end‐joining genes associated with breast cancer risk and chromosomal radiosensitivity

As enhanced chromosomal radiosensitivity (CRS) results from non‐ or misrepaired double strand breaks (DSBs) and is a hallmark for breast cancer and single nucleotide polymorphisms (SNPs) in DSB repair genes, such as non homologous end‐joining (NHEJ) genes, could be involved in CRS and genetic predisposition to breast cancer. In this study, we investigated the association of five SNPs in three different NHEJ genes with breast cancer in a population‐based case‐control setting. The total patient population composed of a selected group of patients with a family history of the disease and an unselected group, consisting mainly of sporadic cases. SNP analysis showed that the c.2099‐2408G>A SNP (XRCC6) has a significant, positive odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30–6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the selected patient group. For the c.‐1310 C>G SNP (XRCC5) a significant OR of 1.85 (95%CI: 1.01–3.41) was found for the He genotype in the unselected patient group. On the contrary, the HV genotype of c.1781G>T (XRCC5) displays a significant, negative OR of 0.43 (95%CI: 0.18–0.99) in the total patient population. The He+HV genotypes of the c.2099‐2408G>A SNP (XRCC6) also showed high and significant ORs in the group of “radiosensitive,” familial breast cancer patients. In conclusion, our results provide preliminary evidence that the variant allele of c.‐1310C>G (XRCC5) and c.2099‐2408G>A (XRCC6) are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC5) on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.

The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons

BackgroundA better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete.MethodsWe investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay.ResultsRNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons.ConclusionsInterfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.

Is a semi-automated approach indicated in the application of the automated micronucleus assay for triage purposes?

Within the EU MULTIBIODOSE project, the automated micronucleus (MN) assay was optimised for population triage in large-scale radiological emergencies. For MN scoring, two approaches were applied using the Metafer4 platform (MetaSystems, Germany): fully automated scoring and semi-automated scoring with visual inspection of the gallery of MN-positive objects. Dose-response curves were established for acute and protracted whole-body and partial-body exposures. A database of background MN yields was set up, allowing determination of the dose detection threshold in both scoring modes. An analysis of the overdispersion of the MN frequency distribution σ(2)/µ obtained by semi-automated scoring showed that the value of this parameter represents a reliability check of the calculated equivalent total body dose in case the accident overexposure is a partial-body exposure. The elaborated methodology was validated in an accident training exercise. Overall, the semi-automated scoring procedure represents important added value to the automated MN assay.

Variant ataxia telangiectasia: clinical and molecular findings and evaluation of radiosensitive phenotypes in a patient and relatives

Variant ataxia telangiectasia (A-T) may be an underdiagnosed entity. We correlate data from radiosensitivity and kinase assays with clinical and molecular data from a patient with variant A-T and relatives. The coding region of ATM was sequenced. To evaluate the functional effect of the mutations, we performed kinase assays and developed a novel S-G2 micronucleus test. Our patient presented with mild dystonia, moderately dysarthric speech, increased serum α-fetoprotein but no ataxia nor telangiectasias, no nystagmus or oculomotor dyspraxia. She has a severe IgA deficiency, but does not have recurrent infections. She is compound heterozygote for ATM c.8122G>A (p.Asp2708Asn) and c.8851-1G>T, leading to in frame loss of 63 nucleotides at the cDNA level. A trace amount of ATM protein is translated from both alleles. Residual kinase activity is derived only from the p.Asp2708Asn allele. The conventional G0 micronucleus test, based on irradiation of resting lymphocytes, revealed a radiosensitive phenotype for the patient, but not for the heterozygous relatives. As ATM is involved in homologous recombination and G2/M cell cycle checkpoint, we optimized an S-G2 micronucleus assay, allowing to evaluate micronuclei in lymphocytes irradiated in the S and G2 phases. This test showed increased radiosensitivity for both the patient and the heterozygous carriers. Intriguingly, heterozygous carriers of c.8851-1G>T (mutation associated with absence of kinase activity) showed a stronger radiosensitive phenotype with this assay than heterozygous carriers of p.Asp2708Asn (mutation associated with residual kinase activity). The modified S-G2 micronucleus assay provided phenotypic insight into complement the diagnosis of this atypical A-T patient.

Relative biological effectiveness of mammography X-rays at the level of DNA and chromosomes in lymphocytes

Abstract Purpose: In many countries, breast cancer screening programs based on periodic mammography exist, giving a large group of women regularly a small dose of ionizing radiation. In order to assess the benefit/risk ratio of those programs the relative biological effectiveness (RBE) of mammography X-rays needs to be determined. Materials and methods: Blood of five healthy donors was irradiated in vitro with 30 kV X-rays and 60Co γ-rays with doses between 5 and 2000 mGy. The phosphorylated histone subtype H2A isoform X-foci (γH2AX-foci) technique was used to quantify the number of DNA double-strand breaks (DSB) after irradiation. Chromosomal damage resulting from non- or misrepaired DNA DSB was quantified with the micronucleus (MN)-assay and the sensitivity was improved by counting only centromere negative micronuclei (MNCM−). Results: The threshold detection dose obtained with the γH2AX-foci test was 10 mGy for mammography X-rays compared to 50 mGy for γ-rays. With the MN-assay respectively MN-centromere-assay threshold detection doses of 100, respectively, 50 mGy were obtained for mammography X-rays compared to 200 respectively 100 mGy for γ-rays. An RBE of 1.4 was obtained with the γH2AX-foci assay. With the MN-assays low-dose RBE values between 3 and 4 were determined. Conclusion: Our results indicate that exposure to mammography X-rays resulted in a modest increase in the induction of DSB compared to γ-rays. However, due to the higher linear energy transfer (LET) of mammography X-rays more clustered DNA damage is produced that is more difficult to repair and results in a more pronounced increase in micronucleus formation.

Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells

Purpose: To investigate the radiosensitising effect of Ku autoantigen 70 (Ku70) and Ku autoantigen 80 (Ku80) knockdown by lentivirus-mediated RNA interference (RNAi) in the MCF10A immortalised human mammary epithelial cell line. Materials and methods: MCF10A cells were infected with lentiviral vectors for RNAi of Ku70. The Ku70-knockdown cell line (Ku70i) and a mock-infected control cell line (LVTHM) were used to perform radiation experiments. For the in vitro Micronucleus (MN) assay, both cell lines were irradiated with doses of 2 and 4 Gy 60Co γ-rays. For cell survival experiments, doses ranging between 0 and 8 Gy were used. Results: Western blot analysis showed that the Ku70 lentiviral vector was effective in silencing the expression of both Ku70 and Ku80. A significantly higher radiation-induced MN yield was obtained in the Ku70i cell line compared to the control LVTHM cell line. RNAi of Ku70 also resulted in a lower survival yield after irradiation compared to the control cell line. Analysis of cell death mechanisms showed that MCF10A cells (Ku70i and LVTHM) do not undergo apoptosis, but undergo post-irradiation cellular senescence. Conclusion: RNAi of Ku70 resulted in increased chromosomal and cellular radiosensitivity in the MCF10A human mammary cell line after irradiation with 60Co γ-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.

Validation of semi-automatic scoring of dicentric chromosomes after simulation of three different irradiation scenarios.

AbstractLarge scale radiological emergencies require high throughput techniques of biological dosimetry for population triage in order to identify individuals indicated for medical treatment. The dicentric assay is the “gold standard” technique for the performance of biological dosimetry, but it is very time consuming and needs well trained scorers. To increase the throughput of blood samples, semi-automation of dicentric scoring was investigated in the framework of the MULTIBIODOSE EU FP7 project, and dose effect curves were established in six biodosimetry laboratories. To validate these dose effect curves, blood samples from 33 healthy donors (>10 donors/scenario) were irradiated in vitro with 60Co gamma rays simulating three different exposure scenarios: acute whole body, partial body, and protracted exposure, with three different doses for each scenario. All the blood samples were irradiated at Ghent University, Belgium, and then shipped blind coded to the participating laboratories. The blood samples were set up by each lab using their own standard protocols, and metaphase slides were prepared to validate the calibration curves established by semi-automatic dicentric scoring. In order to achieve this, 300 metaphases per sample were captured, and the doses were estimated using the newly formed dose effect curves. After acute uniform exposure, all laboratories were able to distinguish between 0 Gy, 0.5 Gy, 2.0, and 4.0 Gy (p < 0.001), and, in most cases, the dose estimates were within a range of ± 0.5 Gy of the given dose. After protracted exposure, all laboratories were able to distinguish between 1.0 Gy, 2.0 Gy, and 4.0 Gy (p < 0.001), and here also a large number of the dose estimates were within ± 0.5 Gy of the irradiation dose. After simulated partial body exposure, all laboratories were able to distinguish between 2.0 Gy, 4.0 Gy, and 6.0 Gy (p < 0.001). Overdispersion of the dicentric distribution enabled the detection of the partial body samples; however, this result was clearly dose-dependent. For partial body exposures, only a few dose estimates were in the range of ± 0.5 Gy of the given dose, but an improvement could be achieved with higher cell numbers. The new method of semi-automation of the dicentric assay was introduced successfully in a network of six laboratories. It is therefore concluded that this method can be used as a high-throughput screening tool in a large-scale radiation accident.

Induction and disappearance of γH2AX foci and formation of micronuclei after exposure of human lymphocytes to 60Co γ-rays and p(66)+ Be(40) neutrons

Abstract Purpose: To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (γH2AX foci) after in vitro exposure of human lymphocytes to either 60Co γ-rays or p(66)+ Be(40) neutrons. Materials and methods: MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0–4 Gy γ-rays or 0–2 Gy neutrons. Also, γH2AX foci DR curves were obtained following exposure to doses ranging from 0–0.5 Gy of either γ-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy γ-rays or neutrons were studied up to 24 hours post-irradiation. Results: Micronuclei yields following neutron exposure were consistently higher compared to that from 60Co γ-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced γH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from γ-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from γ-rays. Foci formations were more likely to be over-dispersed for neutron irradiations. Conclusion: Although neutrons are more effective to induce MN, the absolute number of induced γH2AX foci are less at first compared to γ-rays. With time neutron-induced foci are more persistent. These findings are helpful for using γH2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

Discrepancies between in silico and in vitro data in the functional analysis of a breast cancer-associated polymorphism in the XRCC6/Ku70 gene

Previous results from our research group have shown that the c.-1310 C↷G single nucleotide polymorphism in the promoter region of the XRCC6/Ku70 gene is significantly associated with breast cancer in a sample human patient population. In an attempt to attribute a functional meaning to this polymorphism, we performed a thorough analysis using a number of established in silico tools that strongly suggested that the c.-1310C↷G transversion would activate a cryptic splicing acceptor located upstream of the canonical promoter of Ku70, but downstream of a putative alternative promoter (PAP) of the same gene. Experimental investigation of alternative transcripts, as well as of the activity of the PAP detected in silico, did not support the initial hypothesis of a functional role of the c.-1310C↷G mutation in alternative splicing. Although a functional role of the SNP has yet to be determined, some evidence points to the linkage disequilibrium of the G variant of the polymorphism, with mutations located at critical sites within the promoter region of Ku70.