Induction and disappearance of γH2AX foci and formation of micronuclei after exposure of human lymphocytes to 6°Co γ-rays and p(66)+ Be(40) neutrons.

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 (60)Co 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 (60)Co 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.

Manual versus automated γ-H2AX foci analysis across five European laboratories: can this assay be used for rapid biodosimetry in a large scale radiation accident?

The identification of severely exposed individuals and reassurance of the 'worried well' are of prime importance for initial triage following a large scale radiation accident. We aim to develop the γ-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo γ-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate γ-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2-4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the γ-H2AX assay. We conclude that the γ-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods.

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.

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 (60)Co 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.

Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity.

A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

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

BACKGROUND: A 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. METHODS: We 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. RESULTS: RNAi 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. CONCLUSIONS: Interfering 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.

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 (60)Co gamma-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 (60)Co gamma-rays. These results further strengthen the role of the Ku protein in correct DNA double strand break (DSB) repair.

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.

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 (XRCC5Ku80) [corrected] 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 (XRCC6Ku70)[corrected] 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 (XRCC6Ku70) [corrected] 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 (XRCC5Ku80) [corrected] 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 (XRCC6Ku70) [corrected]and c.2099-2408G>A (XRCC5Ku80) [corrected] are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC6Ku70) [corrected] on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.

Effects of estradiol and progesterone on the variability of the micronucleus assay.

To investigate chromosomal radiosensitivity of lymphocytes the micronucleus (MN) assay has been used for many years. The results of these studies suggest the use of the MN assay as a biomarker for cancer predisposition. However, the MN assay has still some limitations associated with the reproducibility and sensitivity. Especially a high intra-individual variability has been observed. An explanation for this high intra-individual variability is not yet available. In literature it is suggested that the high variability among females is attributable to hormonal status. In this study we investigated if the high intra-individual variability in micronucleus formation in lymphocytes of females after in vitro exposure to ionising radiation is caused by variations in hormone levels of estradiol (E2) and progesterone (PROG). For this, the MN assay was performed on blood samples of 18 healthy women during 7 consecutive weeks while the estradiol and progesterone levels were determined at the same time. The MN assay was also examined in cultures of isolated blood lymphocytes with estradiol or progesterone levels added in vitro. The results demonstrated that estradiol and progesterone levels have no influence on the variations in radiation-induced MN yields observed in blood samples of healthy women. These conclusions were confirmed by the "in vitro" experiments as no correlation between the MN yields and the concentrations of hormones (estradiol or progesterone) added in vitro to isolated lymphocytes cultures was observed.