Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells

We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T). The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells. This permeabilization can be reversed by diluting the cells in growth medium. Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation. Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells. A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells. Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type. It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion. It has a molecular weight (MW) in the range 20-30 000 D. The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible.     

DNA replication in mammalian cells after exposure to factors of a physical, chemical or biological nature. III. 5-Fluorodeoxyuridine induces DNA synthesis in cells not suppressed by gamma irradiation

The influence of preincubation of HeLa and Chinese hamster V79 cells with fluorodeoxyuridine (FUdR, 10(-6) M) on DNA replication and molecular weight of nascent DNA was studied after gamma-irradiation with a dose as much as 10 Gy. The 60Co-radiation inhibits DNA synthesis in both HeLa and V79 cells by 30-40 per cent. The incubation with FUdR before irradiation suppresses the inhibitory effect of irradiation on DNA synthesis. It is suggested that differences in gamma-radiation inhibition of DNA synthesis may result from the FUdR-induced changes in chromatin structure, rather than from synchronization of cell growth. This suggestion is based on the observation that the radioresistant mode of DNA synthesis occurred 18 hours following the short-term (6 hours) incubation with FUdR in cell cultures differing from each other in almost 2-fold their cell longevity.     

DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells

Induction and rejoining of DNA single-strand breaks (ssb) and double-strand breaks (dsb) after gamma-irradiation were measured, respectively, by alkaline and neutral sucrose gradient sedimentation methods. The radiosensitive mutants irs1, irs2, and irs3 showed no significant difference from wild-type V79 hamster cells in ability to rejoin either ssb or dsb, while the previously-described xrs-1 mutant showed the expected defect in rejoining dsb. The resistance of DNA synthesis to gamma-irradiation was measured in the 3 irs mutants and, for comparative purposes, in transformed human cell lines from normal and ataxia-telangiectasia (A-T) individuals. The irs2 mutant was found to be very similar in response to the A-T lines, showing a marked decrease in inhibition of DNA synthesis, compared to V79 cells, in both time-course and dose-response experiments. However, irs1 also had some decrease in inhibition at the higher doses used, while irs3 was similar to the wild-type V79 cells. Both irs1 and irs2 were found to be considerably more sensitive to the DNA topoisomerase I-inhibitor camptothecin, while irs3 was only slightly more sensitive than the parent V79 line. These data place the irs mutants in a similar category of radiosensitive phenotype to A-T cells, but we view this as only the beginning of a useful classification of this type of mutant. The irs2 mutant has the strongest links to A-T cells, through its sensitivity profile to DNA-damaging agents and radioresistant DNA synthesis, but irs1 in particular has other similarities to A-T.     

A derivative of an ataxia-telangiectasia (A-T) cell line with normal radiosensitivity but A-T-like inhibition of DNA synthesis

Ataxia-telangiectasia (A-T) cells are hypersensitive to the lethal effects of ionizing radiation and fail to inhibit DNA synthesis following radiation exposure. A cell line derived from an A-T line following DNA-mediated gene transfer has normal radiation sensitivity, but the kinetics of DNA synthesis after gamma-irradiation are similar to those of A-T cells.     

Human chromosome 11 complements ataxia-telangiectasia cells but does not complement the defect in AT-like Chinese hamster cell mutants

It has been shown that the X-ray-sensitive Chinese hamster V79 mutants (V-E5, V-C4 and V-G8) are similar to ataxia-telangiectasia (A-T) cells. To determine whether the AT-like rodent cell mutants are defective in the gene homologous to A-T (group A, C or D), human chromosome 11 was introduced to the V-E5 and V-G8 mutant cells by microcell-mediated chromosome transfer. Forty independent hybrid clones were obtained in which the presence of chromosome 11 was determined by in situ hybridization. The presence of the region of chromosome 11q22–23 was shown by molecular analysis using polymorphic DNA markers specific for the ATA, ATC and ATD loci. Seventeen of the obtained monochromosomal Chinese hamster hybrids contained a cytogenetically normal human chromosome 11, but only twelve hybrid cell lines were shown to contain an intact 11q22–23 region. Despite the complementation of the X-ray sensitivity by a normal chromosome 11 introduced to A-T cells (complementation group D), these twelve Chinese hamster hybrid clones showed lack of complementation of X-ray and streptonigrin hypersensitivity. The observed lack of complementation does not seem to be attributable to hypermethylation of the human chromosome 11 in the rodent cell background, since 5-azacytidine treatment had no effect on the streptonigrin hypersensitivity of the hybrid cell lines. These results indicate that the gene defective in the AT-like rodent cell mutants is not homologous to the ATA, ATC or ATD genes and that the human gene complementing the defect in the AT-like mutants seems not to be located on human chromosome 11.     

Densely methylated DNA islands in mammalian chromosomal replication origins.

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.     

3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells

3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADP-ribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.     

Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation. Variation in the rate of movement of the replication fork

Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. This intermediate-density DNA has been previously shown to correspond to fork structures, and thus it seems that inhibition of DNA synthesis after irradiation is to a great extent caused by the forks pausing at the lesions. Later on, when recovery in the rate of DNA synthesis occurs, the accumulation of intermediate-density DNA is no longer observed. The density distribution of DNA along the gradient can thus provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine.

The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation can be postponed or diminished by a post-irradiation treatment with 1.0 to 1.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibit depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis have no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiates X-ray-induced cell killing, this reduction in survival is due primarily to effects on cells in S-phase.     

Delayed chromosomal instability induced by DNA damage.

DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability.     

Tn10 transposition does not respond to environmental stress

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Hypersensitivity of cells from a new chromosomal-breakage syndrome to DNA-damaging agents

Cells derived from a patient with severe chromosomal breakage, immunodeficiency, and growth retardation were found to resemble those from individuals with ataxia telangiectasia (A-T) in terms of their sensitivity to cell killing and the induction of cytogenic abnormalities by X-rays. Their response to other DNA-damaging agents, including 254-nm UV light, mitomycin C, MNNG, and bleomycin was also A-T-like. In contrast to classical A-T, however, X-irradiated cells exhibited a G₁ block after release from density inhibition of growth that was not significantly different from that of normal controls.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.     

The units of DNA replication in the mammalian chromosomes: Evidence for a large size of replication units

The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 m/min, the average being 0.6 m/min. In the Chinese hamster cells the rate of DNA replication is greater, varying from 0.3 to 1.2 m/min, the average being 0.8 m/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 m, the average value being 130 m. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 m (the average value being 100 m) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 m (the average being about 140 m) are completely replicated. Provided that the replication is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.     

Effect of methylnitronitrosoguanidine on DNA synthesis in animal cells: decrease in the rate of replication fork movement

The effect of methylnitronitrosoguanidine (MNNG) on the rate of DNA replication fork (RF) progress has been studied by DNA fiber autoradiography in asynchronous Microtus agrestis and Chinese hamster cells. The rate of RF progress has been shown to be decreased by 14% and 36% at MNNG concentrations of 50 and 100 microM in M. agrestis cells; the rate of DNA synthesis being reduced by 50 and 75% respectively. In Chinese hamster cells the MNNG concentration of 5 microM does not affect the rate of RF and that of 10 microM decreases the latter by 11%, the respective fall in the synthesis of DNA rate being 13 and 57%. It has been concluded that the decreased RF rate contributes only partially to the overall DNA synthesis inhibition following the MNNG administration. Inhibition of DNA synthesis at the MNNG concentrations reducing the DNA synthesis by less then 40-50% is mainly caused by the inhibition of the initiation points and, possibly, by the stopping of operating RF. Further DNA synthesis inhibition (at the MNNG concentrations leading to DNA synthesis decrease exceeding 50%) is mostly due to the reduced RF progress rate.     

Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.     

The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication

Exposure of cells to ionizing radiation inhibits DNA replication in a dose-dependent manner. The dose response is biphasic and the initial steep component reflects inhibition of replicon initiation thought to be mediated by activation of the S-phase checkpoint. In mammalian cells, inhibition of replicon initiation requires the ataxia telagiectasia mutated (ATM) gene, a member of the phosphatidyl inositol kinase-like (PIKL) family of protein kinases. We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation. Exposure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibited replicon initiation in a time-dependent manner. Inhibition was at a maximum 1 h after irradiation and recovered at later times. Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for recovery, or only limited recovery, was observed for up to 8 h after irradiation. In addition a defect was observed in the maturation of nascent DNA. Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h. Normal kinetics of recovery were observed in xrs-5 cells, deficient in Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these cell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells may not be attributed entirely to the prolonged presence of unrepaired DNA dsb. We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in normal cells facilitates recovery of DNA replication after irradiation.