The inter-individual variability outperforms the intra-individual variability of differentially expressed proteins prior and post irradiation in lymphoblastoid cell lines.

CONTEXT: Radio-sensitivity in normal tissue is characterized by heterogeneity throughout the population and the absence of pre-diagnostic biomarkers. OBJECTIVE: We conducted a proteomic approach to search for radiation characteristic protein regulation. MATERIALS AND METHODS: Cell lines were 10 Gy irradiated and analysed by 2D-DIGE after 24 h. RESULTS were analysed intra- and inter-individually. The principal component analysis and hierarchical clustering was applied to all datasets. RESULTS: Differences in intra-individual spot abundance prior and post irradiation exactly show the separation of sample classes in two groups: sham-irradiated and irradiated. The inter-individual datasets clustered according to the cell line. The intra-individual differences on protein level after gamma-irradiation are very low, compared with the inter-individual differences among cell lines derived from the same tissue. CONCLUSION: The application of 2-D DIGE may offer a realistic chance for a better molecular characterization of radio-sensitivity and for the discovery of candidate biomarkers.

Subdiffusion supports joining of correct ends during repair of DNA double-strand breaks.

The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.

The effectiveness of 20 mev protons at nanosecond pulse lengths in producing chromosome aberrations in human-hamster hybrid cells.

Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a "pulsed mode" that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, A(L) cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately (P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.

The WST survival assay: an easy and reliable method to screen radiation-sensitive individuals.

An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.

No evidence for a different RBE between pulsed and continuous 20 MeV protons.

To obtain greater insight into the future potential of tumor radiotherapy using proton beams generated from high-intensity lasers, it is important to characterize the ionization quality of the new beams by measuring the relative biological effectiveness (RBE) under conditions where the full dose at one irradiation site will be deposited by a few proton pulses less than 1 ns in duration. HeLa cells attached to a Mylar foil were irradiated with 70 kV X rays to obtain a reference dose-response curve or with 3 Gy of 20 MeV protons at the Munich tandem accelerator (Garching), either using a continuous mode where a cell sample was irradiated within a 100-ms time span or using a pulsed mode where radiation was given in a single proton pulse of about 1 ns. After irradiation cytochalasin B was added; 24 h later cells were fixed and stained with acridine orange and micronuclei were counted. The X-ray dose-response curve for the production of micronuclei in HeLa cells followed a linear-quadratic model. The corresponding RBE values for 20 MeV protons in pulsed and continuous irradiation modes were 1.07 +/- 0.08 and 1.06 +/- 0.10 in the first proton experiment and 1.09 +/- 0.08 and 1.05 +/- 0.11 in the second, respectively. There was no evidence for a difference in the RBE for pulsed and continuous irradiation of HeLa cells with 20 MeV protons.

DNA-repair protein distribution along the tracks of energetic ions.

A simple model of homogenous chromatin distribution in HeLa-cell nuclei suggests that the track of an energetic ion hits 30 nm chromatin fibers with a mean distance of 0.55 mum. To test this assumption, living HeLa-cells were irradiated at the irradiation setup of the ion microprobe SNAKE using the ion beams provided by the Munich 14 MV tandem accelerator. After irradiation, the distribution of 53BP1 protein foci was studied by immunofluorescence. The observed 53BP1 distribution along the tracks of 29 MeV (7)Li ions and 24 MeV (12)C ions differed significantly from the expectations resulting from the simple chromatin model, suggesting that the biological track structure is determined by cell nuclear architecture with higher order organisation of chromatin.

Microirradiation of cells with energetic heavy ions.

The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV (16)O ions, the adapted set-up permits an irradiation accuracy of 0.91 microm (full width at half maximum) in the x-direction and 1.60 microm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.

Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.

DNA integration by Ty integrase in yku70 mutant Saccharomyces cerevisiae cells.

In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

A new X-ray sensitive CHO cell mutant of ionizing radiation group 7,XR-C2, that is defective in DSB repair but has only a mild defect in V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA-PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process.

The yeast TEL1 gene partially substitutes for human ATM in suppressing hyperrecombination, radiation-induced apoptosis and telomere shortening in A-T cells.

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.

Subtelomeric repeat amplification is associated with growth at elevated temperature in yku70 mutants of Saccharomyces cerevisiae.

Inactivation of the Saccharomyces cerevisiae gene YKU70 (HDF1), which encodes one subunit of the Ku heterodimer, confers a DNA double-strand break repair defect, shortening of and structural alterations in the telomeres, and a severe growth defect at 37 degrees. To elucidate the basis of the temperature sensitivity, we analyzed subclones derived from rare yku70 mutant cells that formed a colony when plated at elevated temperature. In all these temperature-resistant subclones, but not in cell populations shifted to 37 degrees, we observed substantial amplification and redistribution of subtelomeric Y' element DNA. Amplification of Y' elements and adjacent telomeric sequences has been described as an alternative pathway for chromosome end stabilization that is used by postsenescence survivors of mutants deficient for the telomerase pathway. Our data suggest that the combination of Ku deficiency and elevated temperature induces a potentially lethal alteration of telomere structure or function. Both in yku70 mutants and in wild type, incubation at 37 degrees results in a slight reduction of the mean length of terminal restriction fragments, but not in a significant loss of telomeric (C(1-3)A/TG(1-3))(n) sequences. We propose that the absence of Ku, which is known to bind to telomeres, affects the telomeric chromatin so that its chromosome end-defining function is lost at 37 degrees.

Immortalization and characterization of Nijmegen Breakage syndrome fibroblasts.

Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.

DNA double-strand breaks in mammalian cells exposed to gamma-rays and very heavy ions. Fragment-size distributions determined by pulsed-field gel electrophoresis.

The spatial distribution of DNA double-strand breaks (DSB) was assessed after treatment of mammalian cells (V79) with densely ionizing radiation. Cells were exposed to beams of heavy charged particles (calcium ions: 6.9 MeV/u, 2.1.10(3) keV/microm; uranium ions: 9.0 MeV/u, 1.4.10(4) keV/microm) at the linear accelerator UNILAC of GSI, Darmstadt. DNA was isolated in agarose plugs and subjected to pulsed-field gel electrophoresis under conditions that separated DNA fragments of size 50 kbp to 5 Mbp. The measured fragment distributions were compared to those obtained after gamma-irradiation and were analyzed by means of a convolution and a deconvolution technique. In contrast to the finding for gamma-radiation, the distributions produced by heavy ions do not correspond to the random breakage model. Their marked overdispersion and the observed excess of short fragments reflect spatial clustering of DSB that extends over large regions of the DNA, up to several mega base pairs (Mbp). At fluences of 0.75 and 1.5/microm2, calcium ions produce nearly the same shape of fragment spectrum, merely with a difference in the amount of DNA entering the gel; this suggests that the DNA is fragmented by individual calcium ions. At a fluence of 0.8/microm2 uranium ions produce a profile that is shifted to smaller fragment sizes in comparison to the profile obtained at a fluence of 0.4/microm2; this suggests cumulative action of two separate ions in the formation of fragments. These observations are not consistent with the expectation that the uranium ions, with their much larger LET, should be more likely to produce single particle action than the calcium ions. However, a consideration of the greater lateral extension of the tracks of the faster uranium ions explains the observed differences; it suggests that the DNA is closely coiled so that even DNA locations several Mbp apart are usually not separated by less than 0. 1 or 0.2 microm.

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation.

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways.

Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.

Radiation-induced cell killing is highly dependent upon buffer treatment (filtration compared to autoclaving) due to metal-catalyzed formation of hypochlorite: a cautionary note.

Buffer solutions used in experiments in radiation biology may be sterilized by either autoclaving or filtration. We show here that for phosphate-buffered saline such differences in buffer treatment may result in widely differing dose-effect curves for cell killing. The temperature-dependent transformation of monophosphate ions into di- or polyphosphate evidently proceeds to an appreciable extent upon autoclaving the buffers at 120 degrees C for 10 to 20 min. This increases the capability of the buffer to chelate spurious metal contaminations and, as a consequence, to reduce the amount of cytotoxic hypochlorite being produced. Depending on conditions of buffer treatment we have observed dose modification factors for the colony-forming ability of yeast cells up to the order of 3. Thus effects due to buffer treatment might easily outweigh the effect which the experiment was originally designed to determine. We strongly advise, therefore, that results of parallel sets of experiments in which different methods of buffer sterilization have been used should not be compared directly.

The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination.

In mammalian cells, all subunits of the DNA-dependent protein kinase (DNA-PK) have been implicated in the repair of DNA double-strand breaks and in V(D)J recombination. In the yeast Saccharomyces cerevisiae, we have examined the phenotype conferred by a deletion of HDF1, the putative homologue of the 70-kD subunit of the DNA-end binding Ku complex of DNA-PK. The yeast gene does not play a role in radiation-induced cell cycle checkpoint arrest in G1 and G2 or in hydroxyurea-induced checkpoint arrest in S. In cells competent for homologous recombination, we could not detect any sensitivity to ionizing radiation or to methyl methanesulfonate (MMS) conferred by a hdf1 deletion and indeed, the repair of DNA double-strand breaks was not impaired. However, if homologous recombination was disabled (rad52 mutant background), inactivation of HDF1 results in additional sensitization toward ionizing radiation and MMS. These results give further support to the notion that, in contrast to higher eukaryotic cells, homologous recombination is the favored pathway of double-strand break repair in yeast whereas other competing mechanisms such as the suggested pathway of DNA-PK-dependent direct break rejoining are only of minor importance.

An electrophoretic approach to the assessment of the spatial distribution of DNA double-strand breaks in mammalian cells.

An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells irradiated with gamma-rays or restriction enzyme-treated human DNA was resolved according to size using pulsed field gel electrophoresis, and the resulting DNA mass distributions were measured in ethidium bromide-stained gels. The DNA profiles thus obtained were compared to the predictions on DNA fragment size distribution which follow from a so-called random breakage model to test whether the experimental outcome is compatible with the assumption of a random localization of breaks. Comparisons of fragment distributions may be performed utilizing two equivalent representations that are linked by an adequate transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in units of molecular length, we show for gamma-irradiated samples that the predictions derived from the employed models agree well with the observed data, thus allowing an immediate quantification of double-strand breaks (DSB). Using restriction enzyme-treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability of our approach to the detection of a non-random distribution of DSB. Therefore, we suppose that our approach may also be useful to reveal a clustering of DSB, which is postulated to occur after damage induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.