Rejoining Of DNA 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 heat-labile sites that convert into DNA double-strand breaks.

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells.

Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs. Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay. The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay. Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line. The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells. Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma. These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs. Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.

Hyperthermia does not affect rejoining of DNA double-strand breaks in a cell-free assay

Purpose : Heat radiosensitization is poorly understood but is believed to be caused by an inhibition in the repair of radiation-induced DNA lesions. This inhibition in DNA repair may be caused either by direct heat inactivation of repair enzymes, or by heat-induced protein denaturation that leads to their precipitation onto nuclear chromatin structures, generating a barrier that prevents repair enzymes from reaching the damage sites. Material and methods : A previously described (Ganguly and Iliakis, Int J Radiat Biol 1995, 68, 447-457) cell-free assay was introduced to evaluate rejoining of radiation-induced DNA double-strand breaks (dsb) in heated (45.5°C, 20 min) nuclei prepared from A549 cells, in reactions assembled with extracts of non-heated and non-irradiated HeLa cells. The assay allowed the functional evaluation of the effect of precipitated nuclear protein on dsb rejoining. By combining heated nuclei with extracts of non-heated cells the assay avoided complications that would otherwise arise when intact cells are studied, where both nuclear structures and repair factors are heated and therefore potentially altered. Results : It was observed that exposure of A549 cells to 45.5°C for 20 min caused a 50% increase in the relative protein content of isolated nuclei but had no effect on the in vitro rejoining of dsb. In agreement with earlier reports, a greatly reduced rate of dsb rejoining was observed either in intact A549 or HeLa cells after exposure to heat. Conclusions : The results indicate that an increased retention of proteins in heated nuclei is not necessarily associated with an inhibition of dsb rejoining. While the in vitro system may only reproduce certain aspects of the in vivo conditions, the results suggest that protein accretion as a mechanism of heat radiosensitization requires further testing using functional assays.

Rejoining of DNA fragments produced by radiations of different linear energy transfer

Purpose : To analyse the rejoining of DNA double-strand breaks (dsb) produced by high-linear energy transfer (LET) ions, with the specific focus on the influence, on the rejoining estimates, of the way dsb are distributed along chromosomes. Material and methods : Low passages of normal human fibroblasts (GM5758) were irradiated with 60 Co photons, 40 keV/mum helium ions or nitrogen ions with LETs of 80, 125, 175 and 225 keV/mum. The double-stranded DNA fragment distributions, ranging from 5 kbp to 5.7 Mbp, were assayed by pulsed-field gel electrophoresis after repair incubation for 0–22 h. Results : The rejoining was biphasic and the half-times of the two phases were 15 min and 2–3 h, respectively, and were independent of LET. Although the majority of breaks were rejoined by the fast phase, both the fraction of dsb rejoined by the slow phase and the fraction of unrejoined dsb at 20–22 h increased with increasing LET. Conclusions : DNA fragment analysis detected LET-dependent differences in the amount of rejoining while the half-times were independent of LET. The majority of dsb were rapidly rejoined even after high-LET irradiation. If fragment-size distribution is not taken into account, both the fraction of breaks rejoined by slow kinetics, and the fraction of unrejoined breaks, can be overestimated when the LET is increased.

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Purpose : We investigated the effect of protein- and non proteinthiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. Materials and methods : We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. Results : Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. Conclusions : These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

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.

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

Purpose : To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. Methods : Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. Results : When mediated by a DSB-repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose- responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear-quadratic model. Conclusions : Using a minimal number of assumptions, the kinetics and dose-response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose-response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

Joining of correct and incorrect DNA double‐strand break ends in normal human and ataxia telangiectasia fibroblasts

Chromosomal aberrations are believed to result from the incorrect joining of DNA double-strand breaks (DSBs). In an attempt to investigate induction and rejoining quality of DSBs following ionizing radiation exposure in specific genomic locations of mammalian DNA, an experimental approach based on Southern hybridization of single-copy probes to NotI restriction fragments was developed. Induction of DSBs is measured from the decrease of the band intensity representing the unbroken restriction fragment. An increase in intensity of the hybridization band following repair incubation determines reconstitution of the original restriction fragment and thus rejoining of correct DNA ends. We investigated the dose dependence of DSB misrejoining using X-ray doses of 5, 10, 20, 40, and 80 Gy and provide evidence that the number of misrejoined DSBs exceeds, for the same doses used, the number of cytogenetically visible aberrations by an order of magnitude, reflecting the higher resolution of our assay. Induction of DSBs and joining of correct and incorrect break ends were further investigated in cells from a patient with the cancer-prone disease ataxia telangiectasia (AT) and in heterozygous AT cells. We found, compared to normal cells, identical induction rates and identical kinetics for joining correct ends following an 80-Gy X-ray exposure. After 5 and 10 Gy, however, AT homozygotes showed a 50% elevation in the proportion of breaks that are not correctly rejoined. These data indicate a defect in the accuracy of DSB rejoining in AT cells that may account for radiation sensitivity and the occurrence of the high level of chromosomal aberrations observed in AT cells. Genes Chromosomes Cancer 27:59–68, 2000. © 2000 Wiley-Liss, Inc.

Joining of correct and incorrect DNA double-strand break ends in normal human and ataxia telangiectasia fibroblasts

Chromosomal aberrations are believed to result from the incorrect joining of DNA double-strand breaks (DSBs). In an attempt to investigate induction and rejoining quality of DSBs following ionizing radiation exposure in specific genomic locations of mammalian DNA, an experimental approach based on Southern hybridization of single-copy probes to NotI restriction fragments was developed. Induction of DSBs is measured from the decrease of the band intensity representing the unbroken restriction fragment. An increase in intensity of the hybridization band following repair incubation determines reconstitution of the original restriction fragment and thus rejoining of correct DNA ends. We investigated the dose dependence of DSB misrejoining using X-ray doses of 5, 10, 20, 40, and 80 Gy and provide evidence that the number of misrejoined DSBs exceeds, for the same doses used, the number of cytogenetically visible aberrations by an order of magnitude, reflecting the higher resolution of our assay. Induction of DSBs and joining of correct and incorrect break ends were further investigated in cells from a patient with the cancer-prone disease ataxia telangiectasia (AT) and in heterozygous AT cells. We found, compared to normal cells, identical induction rates and identical kinetics for joining correct ends following an 80-Gy X-ray exposure. After 5 and 10 Gy, however, AT homozygotes showed a 50% elevation in the proportion of breaks that are not correctly rejoined. These data indicate a defect in the accuracy of DSB rejoining in AT cells that may account for radiation sensitivity and the occurrence of the high level of chromosomal aberrations observed in AT cells. Genes Chromosomes Cancer 27:59-68, 2000.

Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

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.

Rejoining of DNA Double-Strand Breaks in Ku80-Deficient Mouse Fibroblasts

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

Differential Antimutagenicity of WR-1065 Added after Irradiation in L5178Y Cell Lines

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5′- 33P] Poly (dA)·(oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e -α D-β D 2 , the two cell lines roughly have the same α value (∼0.40 Gy -1) whereas the β value was considerably higher in UM-SCC-14A (0.067 Gy -2 ± 0.007 Gy -2 [SEM]) as compared to UM-SCC-1 (0.013 Gy -2 ± 0.004 Gy -2 [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( p < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro

Purpose: To investigate the role of DNA-dependent protein kinase (DNA-PK) in the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb). Materials and methods: This study employed previously described in vitro assays that utilize nuclei or 'naked' DNA prepared from agarose-embedded cells as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb. Results: It is shown that concentrations of wortmannin completely inhibiting DNA-PK activity profoundly a ect the rejoining of dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell extracts using ammonium sulphate precipitation, generates protein fractions that are able to support dsb rejoining, despite the fact that they do not contain detectable amounts of either DNA-PKcs or Ku80. E cient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains una ected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells. Conclusions: These findings are in contrast with genetic studies demonstrating a requirement for DNA-PK activity for efficient rejoining of dsb in vivo. The difference between in vitro and in vivo results may not be attributed to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in vivo and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, or by postulating a purely regulatory/damagesensing role for DNA-PK in intact cells but no direct involvement in dsb rejoining.

DNA-dependent protein kinase content and activity in lung carcinoma cell lines: correlation with intrinsic radiosensitivity

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min ( r 2=0.818) and the late phase, at 120 min postirradiation ( r 2=0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS ( r 2=0.941 and r 2=0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.

Rejoining of DNA double-strand breaks in vitro by single-strand annealing.

Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error-prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End-joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single-strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase epsilon, FEN-1 endonuclease, and exonuclease activities of 5'-3' and 3'-5' directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA-dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error-prone NHEJ pathway that creates deletions by microhomology-driven SSA.

Joining of Correct and Incorrect DNA Ends at Double-Strand Breaks Produced by High-Linear Energy Transfer Radiation in Human Fibroblasts

DNA double-strand breaks (DSBs) were measured within a 3.2-Mbp NotI fragment on chromosome 21 of cells of a normal human fibroblast cell line. Correct rejoining of DSBs was followed by measuring reconstitution of the original-size NotI fragment, and this was compared to total rejoining as measured by a conventional pulsed-field gel electrophoresis technique (FAR assay). After 80 Gy of particle irradiations with LETs in the range of 7-150 keV/microm, it was found that the repair kinetics was generally slower after irradiation with high-LET particles compared to X irradiation and that a larger proportion of the breaks remained unrepaired after 24 h. On the other hand, the misrejoining frequency as measured by the difference between correct and total rejoining after 24 h did not change with LET, but was approximately the same for all radiations at this dose, equal to 25-30% of the initial breaks. This result is discussed in relation to formation of chromosomal aberrations, deletion mutations and other biological end points.

Radiation rendered more cytotoxic by fludarabine monophosphate in a human oropharynx carcinoma cell-line than in fetal lung fibroblasts.

Fludarabine monophosphate (fludarabine-P) is a relatively new drug in the treatment of different haematological diseases. The mechanism of action also implies a possible role of this drug as a radiosensitizer. Up to now no in vitro investigations dealing with radiosensitizing effects of fludarabine-P in carcinoma cell lines and fibroblasts have been published. The aim of our studies was to analyse the cytotoxic and radiosensitizing effects of different dosages and application schedules of fludarabine-P in a human squamous carcinoma cell line of the oropharynx (ZMK-1) and of fetal lung fibroblasts (MRC-5) in vitro. Possible mechanisms of interaction of fludarabine-P and radiation were investigated. ZMK-1 and MRC-5 cells were cultured under standard conditions with different concentrations of fludarabine-P in combination with escalating doses of radiation. Cytotoxic effects were measured by colony-forming assays. Induction and rejoining of radiation-induced DNA double-strand breaks after incubation with fludarabine-P were measured using constant-field gel electrophoresis. Incubation times for rejoining varied from 0 h to 24 h. Fludarabine-P showed a radiosensitizing activity in ZMK-1 tumour cells and MRC-5 fibroblasts. The observed effects depended on the concentration and the incubation time. The largest effect was demonstrable for an incubation of 5 days, which started shortly before irradiation, whereas an incubation solely before irradiation did not have a clear effect on the cellular survival. The sensitizer enhancement ratio, at the 10% survival level, in the ZMK-1 cells was 2.2 in comparison to 1.6 in MRC-5 cells. The analysis of the interaction of fludarabine-P and ionising radiation by means of the isobologram approach, revealed an overadditive effect in the tumour cell line and an additive effect in the lung fibroblasts. Fludarabine-P did not modify the rejoining of radiation-induced DNA double-strand breaks in either cell line. We conclude that fludarabine-P in clinically attainable doses is a strong radiosensitizer in ZMK-1 cells and has a lower activity in the MRC-5 fibroblasts in vitro. The radiosensitization of fludarabine-P seems to be over additive in the malignant cells and additive in normal fetal fibroblasts. This would indicate that fludarabine-P might enhance the therapeutic ratio of radiation. Further investigations are warranted to identify the potential of this drug as a radiosensitizer in vivo and to elucidate the mechanism of interaction of the drug and radiation.