Headspace measurements of irradiated in vitro cultured cells using PTR-MS.

A pilot study was performed to evaluate a new concept for a radiation biodosimetry method. Proton transfer reaction-mass spectrometry (PTR-MS) was used to find out whether radiation induces changes in the composition of volatile organic compounds (VOCs) in the headspace of in vitro cultured cells. Two different cell lines, retinal pigment epithelium cells hTERT-RPE1 and lung epithelium cells A-549, were irradiated with gamma radiation at doses of 4 Gy and 8 Gy. For measuring the cell-specific effects, the VOC concentrations in the headspace of flasks containing cells plus medium, as well as of flasks containing pure medium were analyzed for changes before and after irradiation. No significant radiation-induced alterations in VOC concentrations in the headspace could be observed after irradiation.

The influence of x-ray contrast agents in computed tomography on the induction of dicentrics and gamma-H2AX foci in lymphocytes of human blood samples.

The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and gamma-H2AX foci) in human lymphocytes after CT scans in the presence of an iodinated contrast agent. Blood samples from a healthy donor were exposed to CT scans in the absence or presence of iotrolan 300 at iodine concentrations of 5 or 50 mg ml(-1) blood. The samples were exposed to 0.025, 0.05, 0.1 and 1 Gy in a tissue equivalent body phantom. Chromosome aberration scoring and automated microscopic analysis of gamma-H2AX foci were performed in parts of the same samples. The theoretical physical dose enhancement factor (DEF) was calculated on the basis of the mass energy-absorption coefficients of iodine and blood and the photon energy spectrum of the CT tube. No significant differences in the yields of dicentrics and gamma-H2AX foci were observed in the absence or presence of 5 mg iodine ml(-1) blood up to 0.1 Gy, whereas at 1 Gy the yields were elevated for both biomarkers. At an iodine concentration of 50 mg ml(-1) serving as a positive control, a biological DEF of 9.5 +/- 1.4 and 2.3 +/- 0.5 was determined for dicentrics and gamma-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml(-1), respectively. Thus, it can be concluded that in the diagnostic dose range (radiation and contrast dose), no relevant biological dose-enhancing effect could be detected, whereas a clear biological dose-enhancing effect could be found for a contrast dose well outside the diagnostic CT range for the complete radiation dose range with both methods.

TEL1 from Saccharomyces cerevisiae suppresses chromosome aberrations induced by ionizing radiation in ataxia-telangiectasia cells without affecting cell cycle checkpoints.

The TEL1 gene from Saccharomyces cerevisiae has been shown to be the closest sequence homologue to ATM, the gene mutated in ataxia-telangiectasia (A-T) patients. Functional homology shared between the ATM and Tell proteins has recently been demonstrated based on heterologous expression of the TEL1 gene in human cells derived from A-T patients. TEL1 expression complemented specific cellular A-T deficiencies, i.e. increased radiation-induced apoptosis, telomere shortening and spontaneous hyperrecombination. The mechanism of cellular A-T complementation by TEL1 appears to be independent of p53-dependent signaling cascades, since the deficiency of A-T cells to properly induce p53 upon ionizing radiation was not corrected by TEL1. We now find that the basic number of chromosome aberrations is increased and the number of radiation-induced chromosome aberrations is suppressed in A-T cells upon TEL1 expression. In cell cycle analyses, we find no changes in basic cell cycle distribution or in radiation-induced cell cycle checkpoints following TEL1 expression. We conclude that the radioprotective function of the Tel1 protein includes suppression of apoptosis and suppression of chromosome aberrations, and that both cellular end-points can be uncoupled from ionizing radiation-induced cell cycle checkpoints.

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.

Radiation inducible DNA repair processes in eukaryotes.

Eukaryotic cells respond to radiation-induced damage in DNA and other cellular components by turning on cascades of regulatory events which constitute a complex network of pathways of cell cycle checkpoints, DNA repair and damage tolerance mechanisms, recombination and delayed cell death (apoptosis). By virtue of the high homology in structure and function of yeast and mammalian proteins several DNA repair pathways that may be upregulated in response to radiation, and some of their regulatory factors involved in sensing of damage, signal transduction by protein kinase cascades and transcription have been identified. In yeast, genes for DNA synthesis and replicative damage bypass, for base and nucleotide excision repair, in particular global genome repair, and for crucial steps in DNA double strand break repair by homologous recombination show enhanced expression in response to radiation. In mammalian cells, the identification of homologous genes and upregulated homologous DNA repair pathways makes fast progress. It is, however, evident that the regulatory network is considerably more complex than in yeast. The improved understanding on the molecular level of the radiation-inducible cellular responses to radiation is of high public interest. Especially, the response to very low doses may have relevance for the risk estimation for ionising radiation and, possibly as well, ultraviolet light (UV-B), and for the design of suitable dose fractionation schemes for radiotherapy.

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.

Genetic interactions between mutants of the 'error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis.

We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.]. In the present study we characterize the effect of mutations in genes of the 'error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products.

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.

A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae.

We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type.

Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype.

The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains. Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations. Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation. While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g. UVC sensitivity and UVC-induced mutability were restored to a wild-type level. Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype. Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes. This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation. Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele. The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool.

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.

The RAD5 gene product is involved in the avoidance of non-homologous end-joining of DNA double strand breaks in the yeast Saccharomyces cerevisiae.

In wild-type yeast, the repair of a 169 bp double-strand gap induced by the restriction enzymes ApaI and NcoI in the URA3gene of the shuttle vector YpJA18 occurs with high fidelity according to the homologous chromosomal sequence. In contrast, only 25% of the cells of rad5-7 and rad5 Delta mutants perform correct gap repair. As has been proven by sequencing of the junction sites, the remaining cells recircularise the gapped plasmids by joining of the non-compatible, non-homologous ends. Thus, regarding the repair of DNA double-strand breaks, the rad5 mutants behave like mammalian cells rather than budding yeast. The majority of the end joined plasmids miss either one or both of the 3'and 5'protruding single-strands of the restriction ends completely and have undergone blunt-end ligation accompanied by fill-in DNA synthesis. These results imply an important role for the Rad5 protein (Rad5p) in the protection of protruding single-strand ends and for the avoidance of non-homologous end joining during repair of double-strand gaps in budding yeast. Alternatively, the Rad5p may be an accessory factor increasing the efficiency of homologous recombination in yeast, however, the molecular mechanism of Rad5p function requires further investigation.

Application of repetitive sequence-based PCR (inter-LINE PCR) for the analysis of genomic rearrangements and for the genome characterization on different taxonomic levels.

Oligonucleotide primers derived from consensus LINE-sequences generated highly reproducible, species-specific PCR product patterns suitable for the identification of genomic rearrangements and for the discrimination on different taxonomic levels of higher and lower eukaryotes and even prokaryotes.

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.