Rejoining Of Double-strand Breaks Research Articles

Induction and rejoining of DNA double-strand breaks in bladder tumor cells.

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.

Radiation-induced double-strand breaks in mammalian DNA: influence of temperature and DMSO

Purpose : To investigate the effects of subphysiological irradiation temperature (2-28°C) and the influence of the radical scavenger DMSO on the induction of double-strand breaks (DSB) in chromosomal DNA from a human breast cancer cell line (MCF7) as well as in intact cells. The rejoining of DSB in cells irradiated at 2°C or 37°C was also investigated. Materials and methods : Agarose plugs with [ 14 C]thymidine labelled MCF-7 cells were lysed in EDTA-NLS-proteinase-K buffer. The plugs containing chromosomal DNA were irradiated with Xrays under different temperatures and scavenging conditions. Intact MCF-7 cells were irradiated in Petri dishes and plugs were made. The cells were then lysed in EDTA-NLS-proteinaseK buffer. The induction of DSB was studied by constant field gel electrophoresis and expressed as DSB/100Mbp, calculated from the fraction of activity released into the gel. Results : The induction of DSB in chromosomal DNA was reduced by a decrease in temperature. This protective effect of low temperature was inhibited when the DNA was irradiated in the presence of DMSO. No difference was found when intact cells were irradiated at different temperatures. However, the rapid phase of rejoining was slower in cells irradiated at 37°C than at 2°C. Conclusions : The induction of DSB in naked DNA was reduced by hypothermic irradiation. The temperature had no influence on the induction of DSB in the presence of a high concentration of DMSO, indicating that the temperature effect is mediated via the indirect effects of ionizing radiation. Results are difficult to interpret in intact cells. Rejoining during irradiation at the higher temperature may counteract an increased induction. The difference in rejoining may be interpreted in terms of qualitative differences between breaks induced at the two temperatures.

No dose-dependence of DNA double-strand break misrejoining following α-particle irradiation

Purpose : To investigate whether an explanation for the high effectiveness of densely ionizing radiation with regard to complex biological endpoints can be derived from measurements of radiation-induced double-strand break (DSB) misrejoining. Materials and methods : Misrejoining of radiation-induced DSB in normal human fibroblasts was determined by comparing hybridization analysis of large restriction fragments as a measure for correct rejoining, with results from a conventional pulsed-field gel electrophoresis technique (FAR) that measures total DSB rejoining. In order to investigate DSB misrejoining at doses for which chromosome aberration data are available, a dose fractionation protocol was applied so that the number of DSB at any given timepoint was low but the cumulative amount of misrejoined DSB sufficient for detection and precise quantitation. Results and conclusion : After an acute 80 Gy α -particle exposure and a repair incubation period of 24 h, 50% of all initially induced DSB were misrejoined, in agreement with data obtained for X-rays. X-irrradiation with 16 x 5 Gy, 8 x 10 Gy, 4 x 20 Gy, or 2 x 40 Gy and repair incubation of 24 h following each individual dose fraction was recently reported to yield misrejoining frequencies that strongly decrease with increasing fractionation (Löbrich et al. 2000; Genes, Chromosomes and Cancer, 27, 59-68). In the present study, constant misrejoining frequencies of 50% were observed after α -particle exposure with the same fractionation protocol. This difference between α -particles and X-rays is in accordance with the high biological effectiveness of densely ionizing radiation and provides a direct link between misrejoining of DSB and cytologically visible exchange aberrations. Further evidence suggests that if the same dose range is compared, the number of misrejoined DSB exceeds the number of microscopically visible aberrations by an order of magnitude for both radiation types, probably reflecting the high resolution of the hybridization approach compared with cytological techniques.

Evidence for the expression of radiation-induced potentially lethal damage being a p53 -dependent process

Purpose : To test the hypothesis that the expression of potentially lethal damage (PLD) is a p 53 -dependent process. Materials and methods : Previously reported data on radiation sensitivity, DNA double-strand break rejoining, PLD expression and repair (PLDR) were analyzed for a group of 12 human tumor cell lines and three human diploid fibroblast cell lines. Seven of these cell lines had normal p53 gene expression while the other eight were functionally p53 -deficient. None of the cell lines was sensitive to radiation-induced apoptosis. Results : Cell lines with a normal p53 expression were more sensitive to radiation, but only when sensitivity was measured in plateau-phase cultures under conditions where PLDR was minimized. Mutation or functional inactivation of p53 by HPV E6 transformation led to a more radioresistant phenotype under these conditions as well as a significant reduction in PLDR. PLDR was inversely proportional to the percentage of radiationinduced DNA double-strand breaks rejoined in 1h in the p53 normal cell lines. Conclusions : These results suggest that the expression of PLD is primarily a p53 -dependent process. In the absence of functional p53 gene expression, the effects of PLD are minimized. These observations help clarify the role of p53 in tumor response to radiation therapy because they suggest that the effects of alterations in p53 are highly dependent on the microenvironment of the tumor, i.e. whether conditions allow for PLDR.

The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit.

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

Double strand break rejoining by mammalian mitochondrial extracts.

DNA end-joining was measured by incubating linearized plasmid DNA with mitochondrial protein extracts. A spectrum of end-joined molecules ranging from re-circularized monomer to dimer and higher molecular weight forms was observed. The DNA end-joining reaction required ATP and Mg2+, and was inhibited by sodium chloride. Both cohesive- and blunt-ended DNA molecules were end-joined, although the former were more efficient substrates. Molecular analysis of rejoined molecules revealed that >95% of the linearized DNA were precisely end-joined. The few imprecisely end-joined molecules recovered, sustained deletions that spanned direct repeat sequences. The deletions observed are strikingly similar to those present in mitochondrial genomes of patients with Kearns-Sayre or Pearson syndromes, certain ophthalmic myopathies and the aged. These results suggest that mammalian mitochondria possess a DNA double strand break repair activity similar to that seen in the nucleus, and that this repair pathway may play a role in the generation of mitochondrial DNA deletions associated with a number of human pathologies.

Wortmannin Sensitizes Mammalian Cells to Radiation by Inhibiting the DNA-Dependent Protein Kinase-Mediated Rejoining of Double-Strand Breaks

Wortmannin has been shown to be an efficient radiosensitizer. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and double-strand break (DSB) rejoining, it is believed that its mechanism of radiation sensitization is through the inhibition of DNA-PK-mediated repair of DSBs. However, since wortmannin is not a specific inhibitor, the possibility that other kinases are inhibited and thereby may contribute to radiosensitization cannot be ruled out. Here we present data confirming the radiosensitizing effect of wortmannin on cells of different cell lines. In the same range of wortmannin concentrations, survival after exposure to ionizing radiation correlated well with DSB rejoining and the induction of micronuclei, suggesting that the inhibition of the processing of DSBs is involved in the sensitizing effect. Pretreatment with wortmannin enhanced the radiosensitivity of ataxia telangiectasia (AT) cells, thereby precluding the participation of ATM protein in the radiation sensitization by wortmannin. At the same time, irradiated DNA-PK-deficient cells were not significantly affected by pretreatment with wortmannin. These observations support a likely mechanism; that is, wortmannin sensitizes cells to radiation through inhibition of the DNA-PK-mediated rejoining of DSBs.

Double strand break rejoining by the Ku-dependent mechanism of non-homologous end-joining

The DNA-dependent protein kinase functions in the repair of DNA double strand breaks (DSBs [ppand in V(D)J recombination. To gain insight into the function of DNA-PK in this process we have carried out a mutation analysis of Ku80 and DNA-PKcs. Mutations at multiple sites within the N-terminal two thirds of Ku80 result in loss of Ku70/80 interaction, loss of DNA end-binding activity and inability to complement Ku80 defective cell lines. In contrast, mutations in the carboxy terminal region of the protein do not impair DNA end-binding activity but decrease the ability of Ku to activate DNA-PK. To gain insight into important functional domains within DNA-PKcs, we have analysed defective mutants, including the mouse scid cell line, and the rodent mutants, irs-20 and V-3. Mutational changes in the carboxy terminal region have been identified in all cases. Our results strongly suggest that the C-terminus of DNA-PKcs is required for kinase activity.

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells.

The induction of collateral radioresistance after the development of cisplatin resistance is a well-documented phenomenon; however, the exact processes that are responsible for the cisplatin-induced radioresistance remain to be elucidated. There was no obvious difference in the level of radiation-induced DNA double strand breaks (DSBs), in DSB rejoining rates, or the level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in the cisplatin- and radiation-sensitive 2780/WT and cisplatin-resistant 2780/CP cell lines. However, there was a significantly (P< 0.01) lower level of DSB misrejoining activity within nuclear protein extracts derived from the cisplatin-and radiation-sensitive 2780/WT and OAW42/WT tumour cell lines than in similar extracts from their cisplatin- (and radiation-) resistant 2780/CP and OAW42/CP counterparts. All of the DSB misrejoining events involved deletions of between 134 and 444 bp that arose through illegitimate recombination at short repetitive sequences, such as those that arise through non-homologous repair (NHR). These data further support the notion that the radiosensitivity of DSB repair proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. Furthermore, our data suggest that the induction of acquired cisplatin resistance is associated with a two- to threefold decrease in the activity of a non-conservative DSB rejoining mechanism that appears to be a manifestation of NHR. © 1999 Cancer Research Campaign

Induction of DNA double-strand breaks by electroporation of restriction enzymes into mammalian cells.

The introduction of restriction endonucleases into mammalian cells in culture provides a unique method for introducing double-strand breaks (DSBs) into the DNA of the host cell. Restriction enzymes recognize, bind, and cleave specific DNA sequences to produce a DNA DSB in the absence of other types of DNA damage. Not only is the DSB the only lesion produced in DNA, but the type of break (i.e., whether it has blunt ends or cohesive ends with 3′- or 5′-overhangs), the nucleotide sequences at the break site, and in certain instances, the precise chromosomal location of the induced break are also known. Once inside a cell, restriction enzymes are extremely efficient inducers of DSBs as measured by pulsed-field gel electrophoresis () and neutral filter elution (). Consequently, the introduction of restriction enzymes into cells has been used to investigate the biological consequences of DSBs in the genome at the molecular, cytogenetic, and cellular level. At the molecular level, the rejoining of DSBs with known end structure has been investigated (), and although it is not possible to determine the frequency of faithfully repaired DSBs, Phillips and Morgan () were able to describe unambiguously illegitimate recombination events occurring within the chromosome. At the cellular level, restriction enzyme-induced DSBs lead to mutations (,), chromosome rearrangements (, , , ), gene amplification () and cell killing (,). Interestingly, restriction enzymes do not induce delayed chromosomal instability, suggesting that DSBs are not the signal responsible for initiating this process ().

Defective Modulation of Double-Strand Break Repair in Ataxia Telangiectasia Cells by Gamma Radiation

Ataxia telangiectasia (AT) cells are defective in responding to damage induced by ionizing radiation. To study the modulation of double-strand break (DSB) repair by ionizing radiation and a defect in such modulation in AT cells, we compared processing of linearized shuttle vector pZ189 (linear DNA) by unirradiated or gamma-irradiated normal and AT lymphoblast hosts. The linear DNA processed in unirradiated AT and normal host cells yielded similar mutation frequencies in the supF-tRNA target gene. Irradiation of normal but not AT host cells decreased plasmid mutation frequency 2-fold if transfection occurred immediately. However, if transfection occurred 2 h after host cell irradiation, mutation frequencies increased 2-fold above those in unirradiated controls in both normal and AT hosts. DSB rejoining capability, based on the ratio of the number of progeny arising from equal amounts of linear DNA and supercoiled, undamaged pZ189, was 25- to 50-fold higher in normal than in AT hosts when both were unirradiated. Irradiation decreased DSB rejoining capability 2- to 5-fold in normal hosts but did not alter that of AT hosts. These findings demonstrate that AT cells normally rejoin DSBs as accurately as normal cells but do so less often, and that AT cells are defective in modulation of DSB rejoining by ionizing radiation.

Individual radiosensitivity and DNA repair proficiency: the value of the comet assay

Some rare hereditary syndromes demonstrate high cancer risk and hypersensitivity in response to exposures to agents such as ultraviolet or ionising radiation, and are characterised by a defective processing of DNA damage. They highlight the importance of the individual capacity of restoring the genomic integrity in the individual risk associated to exposures. The comet assay, a simple technique that detects DNA strand breaks, requires few cells and allows examination of DNA repair capacities in established cell lines, in blood samples or biopsies. The assay has been validated on cellular systems with known repair defects such as xeroderma pigmentosum defective in nucleotide excision repair, on mutant rodent cell lines defective in DNA single strand break rejoining (XRCC1) (alkaline version) or DNA double strand breaks rejoining (XRCC5/Ku80 and XRCC7/DNAPKcs) (neutral conditions). This assay does not allow to distinguish a defective phenotype in ataxia telangiectasia cells. It shows in homozygous mouse embryo fibroblasts Brca2-/- an impaired DNA double strand break rejoining. Simplicity, rapidity and sensitivity of the alkaline comet assay allow to examine the response of lymphocytes. It has been applied to the analysis of the role of DNA repair in the pathogenesis of collagen diseases, and the involvement of individual DNA repair proficiency in the thyroid tumorigenesis induced in some patients after therapeutic irradiation at childhood has been questioned. Preliminary results of these studies suggest that this type of approach could help for adapting treatment modalities and surveillance in subgroups of patients defective in DNA repair process. It could also have some incidence in the radioprotection field.

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

G2 chromatid breaks in murine SCID cells.

The G2 chromosomal radiosensitivity of murine SCID (severe combined immunodeficient) and normal fibroblasts has been investigated. We have also investigated the G2 response of these cell lines to the restriction endonuclease PvuII. We show that chromatid breaks are induced linearly with radiation dose in both cell lines and SCID cells are approximately 1.6 times as radiosensitive as normal murine fibroblasts when tested using a G2 assay with a 2 h sampling time. The disappearance of chromatid breaks with time after irradiation was first order with a half-time of approximately 1.5 h in both cell lines. Thus, although SCID cells are deficient in the rejoining of double-strand breaks (dsb), they show similar kinetics of disappearance of chromatid breaks with time as normal CB17 cells, indicating that the 'rejoining' of chromatid breaks does not reflect dsb repair. When CB17 and SCID cells were treated with PvuII, which generates dsb in cellular DNA in the presence of streptolysin O (as a porating agent), approximately 3 times more chromatid breaks were observed in SCID than CB17 cells. We conclude that SCID cells convert a higher number of dsb into chromatid breaks than do CB17 cells. The conversion process is interpreted in terms of the recently proposed 'signal' model, whereby a signal, resulting from a single dsb, triggers the cell to make a recombinational exchange which, if incomplete, gives rise to a visible chromatid break. In terms of the signal model, elevated conversion of dsb into chromatid breaks results from altered signalling and the disappearance of chromatid breaks with time following irradiation represents the completion of recombinational exchanges rather than repair of dsb.

Rejoining of DNA double-strand breaks after the introduction of chromosome 11 into a radiosensitive bladder carcinoma cell line

Insertion of a normal chromosome 11 into tumour cell lines can protect against a sensitivity to irradiation and oxidative stress. A possible mechanism underlying this effect is that there is a correction of a defect in the rejoining of double-strand breaks (dsb) by the chromosome insertion. In order to explore this hypothesis, three cell lines were evaluated for their ability to rejoin dsb: (1) a bladder carcinoma cell line (`parent') previously shown to be sensitive to irradiation and radical generating species; (2) a derivative of this cell line into which a normal chromosome 11 had been inserted by microcell fusion (`hybrid') showing corrected radiosensitivity; and (3) a `revertant' cell line that had spontaneously lost the insert and reverted to the radiosensitive phenotype. Nuclear extracts from the 3 lines were isolated and evaluated for their capacity to rejoin plasmid (pUC18) DNA broken at defined restriction sites (SalI, EcoRI, KpnI, SmaI) in the lacZ gene. The extent of rejoining was determined by gel electrophoresis and the fidelity of rejoining determined by expression of the lacZ gene in E. coli DH5α bacteria. Results suggest there is no difference between the `parent', `hybrid' and `revertant' nuclear extracts in the fidelity and the total extent of rejoining, regardless of the type of break. However, there is an alteration in the distribution of rejoined products. Nuclear extracts from `hybrid' cells tend to rejoin linear DNA into circular monomers with a greater efficiency than extracts from both `parent' and `revertant' cells. This alteration in distribution is observed when 3′- or 5′-protruding ends are rejoined but not in the rejoining of blunt ends. The results suggest that loci on chromosome 11 are involved in the rejoining of dsb, affecting the relative amount of the different rejoined products. Whether this alteration plays a role in the `parent' cell's radiosensitivity is yet to be determined.

In vitro rejoining of double strand breaks induced in cellular DNA by bleomycin and restriction endonucleases

We have previously described a cell-free assay that can be employed to study rejoining of radiation-induced DNA double-stranded breaks (dsb) in 'naked' DNA prepared from agarose-embedded cells using an extract of HeLa cells as a source of enzymes. Rejoining of dsb in this assay is absolutely dependent on cell extract and proceeds, under optimal reaction conditions, to an extent and with kinetics similar to those observed in intact cells. Here, we extend these experiments and demonstrate that the assay also supports rejoining of bleomycin and restriction endonuclease-induced dsb, agents that generate dsb with known ends. Rejoining of bleomycin-induced dsb proceeds to an extent and with kinetics similar to those observed with radiation-induced dsb. The kinetics of rejoining of restriction endonuclease-induced dsb are also similar to those of radiationinduced dsb. However, more and more dsb remain unrejoined as the extent of DNA fragmentation increases when enzymes cutting the DNA at frequent intervals are used. Dsb with blunt ends are rejoined with a similar efficiency to dsb with cohesive ends. Rejoining of restriction endonuclease-induced dsb is, in the presence of cell extract, more efficient than in the presence of T4 DNA ligase, suggesting the action in the overall reaction of activities in addition to DNA ligases. The experiments presented generalize the utility of the assay in studying the enzymology of dsb rejoining after treatment with radiomimetic drugs and restriction endonucleases and should be useful in the elucidation of the enzymatic requirements of dsb repair.

In vitro rejoining of double-strand breaks in cellular DNA by factors present in extracts of HeLa cells.

We described previously a cell-free assay, that could be employed to study the rejoining of radiation-induced DNA double-strand breaks (dsb) in agarose embedded nuclei by activities present in an extract prepared from exponentially growing HeLa cells. Here, we extend the study and present an in vitro assay for rejoining of radiation-induced DNA dsb that employs 'naked' DNA prepared from agarose-embedded cells as a substrate and extract of HeLa cells as an enzyme source. There is no detectable residual protein on substrate DNA after extensive lysis with ionic detergents and treatment with proteases, as determined by SDS-PAGE and silver staining. We demonstrate that rejoining of dsb is absolutely dependent on cell extract and that, under optimal reaction conditions, it proceeds to an extent and with kinetics similar to those observed in intact cells. Dsb rejoining in this assay requires Mg 2+ and is inhibited by high concentrations of either K+ or Na+. This assay complements the nuclei assay for DNA dsb repair previously developed, and may be preferable to the latter in the purification of factors involved in DNA dsb repair, as it employs as substrate DNA deprived of proteins.

A YAC Contig Encompassing the XRCC5 (Ku80) DNA Repair Gene and Complementation of Defective Cells by YAC Protoplast Fusion

The Chinese hamster ovaryxrsmutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33–q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred toxrscells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.

Induction and Rejoining of DNA Double-Strand Breaks and Interphase Chromosome Breaks after Exposure to X Rays in One Normal and Two Hypersensitive Human Fibroblast Cell Lines

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells. The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology. The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 ‘revertant’ xrs-5 clones isolated following treatment with 5-azacytidine. Nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity. Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity. Radiation sensitivity did correlate with DNA double-strand break rejoining ability. The radioresistant clones showing the over-condensed xrs-5-like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins.