G2 phase repair of X-ray-induced chromosomal DNA damage in trichothiodystrophy cells

The repair of X-ray-induced DNA damage during G₂ cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5–1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.     

Transformation and repair replication in lymphocytes from ataxia telangiectasia

Peripheral blood leukocytes (PBL) isolated from five patients with ataxia telangiectasia (AT) proved more difficult to transform following addition of exogenous Epstein-Barr virus than PBL isolated from AT heterozygotes or normal adults. PBL isolated from one AT patient transformed within the range expected for normal PBL. Once established in culture, the resulting lymphoblastoid cell lines (LCLs) were immortal and, though they grew slower than normal control LCLs, provided useful material for studying cellular phenotypes associated with AT lymphoid cell lines. All the resulting LCLs established from ataxia were more sensitive to X-irradiation than were LCLs established from controls as measured by colony formation in microtiter plates. Furthermore, X-ray-induced inhibition of semiconservative DNA synthesis in ataxia LCLs was less than that seen in normal LCLs. These results are in agreement with those obtained using cultured AT fibroblasts, indicating that in vitro transformation by exogenously added Epstein-Barr virus does not alter the phenotype of the ataxia cell as measured by these two parameters. However, no deficiency in X-ray-induced excision repair of DNA was demonstrable in LCLs established from four AT patients. Nor was there a deficiency in AT LCL host cell reactivation of herpes simplex virus X-irradiated under anoxic conditions. Taken together, these data point toward a defect in ataxia lymphoblasts other than repair enzyme(s) per se, one possibly associated with chromosomal structure, function, or modification.     

The effects of ionizing radiation on cell cycle progression in ataxia telangiectasia

Although ataxia telangiectasia (AT) cells are more sensitive than normal cells to killing by ionizing radiation, their DNA synthesis is more resistant to inhibition by radiation. It was thought that this anomaly in DNA synthesis was likely to perturb cell cycle progression. Flow cytometry and the fraction of labelled mitoses (FLM) were used to investigate effects of irradiation in normal and AT cell lines. The FLM indicated that radiation apparently induced a longer G2 delay in normal cells than in AT cells. However, flow cytometry showed that radiation induced much larger and more prolonged increases in the proportion of G2 cells in AT than in normals. AT populations also showed much larger postirradiation decreases in viable cell numbers. These data suggest that a large proportion of the radiosensitive AT cells are not reversibly blocked in G2 but die there, and never proceed through mitosis. The less radiosensitive normal cells are delayed in G2 and then proceed through mitosis. We suggest that the apparently shorter radiation-induced mitotic delay seen in AT cells by FLM is not real but is an artifact arising from perturbation of steady state conditions by selective elimination of a particular cohort of AT cells. Accumulation of AT cells in G2 is compatible with radiosensitivity of these cells and may arise from a defect in DNA repair or an anomaly in DNA replication.     

Rapid rejoining of X-ray-induced DNA single-strand breaks in tuberous sclerosis fibroblasts

The formation and rejoining rates of X-ray-induced DNA single-strand breaks (SSBs) were examined in radiosensitive and non-radiosensitive fibroblast lines from patients with tuberous sclerosis (TS), and fibroblasts from normal individuals, using the sensitive and quantitative alkaline elution method. No difference was found between these cell lines in the frequency of DNA SSBs directly produced by X-irradiation at any dose up to 750 rad. Kinetic analysis of the rate of rejoining of DNA SSBs after X-irradiation at 500 rad indicated that the rate of rejoining involved at least two components, an initial fast component and a slower component. TS fibroblast lines, either radiosensitive or nonsensitive, were proficient as to DNA SSB repair, but they showed an increased rate of rejoining in the initial fast repair process, when compared to normal fibroblast lines. Although the molecular basis for the accelerated rejoining of DNA SSBs remains unknown, it is possible that the abnormality may be related to a basic defect in TS.     

Radioresistant DNA synthesis and human genetic disease

Summary Sixty-eight human fibroblast cell strains were assayed for radioresistant DNA synthesis (RDS), which is defined here as the absence of a steep component of inhibition of DNA synthesis in a dose-response curve when rate of DNA synthesis is plotted against radation doses from 0 to 20 Gy or more. Twenty-seven strains from patients who were previously diagnosed to have ataxia-telangiectasia (AT) were positive for this feature. Among the cell strains that did not show RDS were two from AT obligate heterozygotes (i.e., the parents of AT patients), two from patients with Alzheimer disease, two from patients with Friedreich ataxia, one from a patient with Bloom syndrome, one from a patient with Down syndrome, and six from patients with various immunodeficiencies. Four strains demonstrated RDS that was less pronounced than in most AT cells: one was from a patient with Nijmegen breakage syndrome, one was from a patient without ataxia but with choreiform movement disorder, telangiectasia, and elevated concentrations of -fetoprotein in the blood, and two were from AT patients. RDS therefore is not a necessary trait of human genetic diseases that involve radiosensitivity or immunodeficiency. Although recent reports suggest that some AT patients do not exhibit RDS, we found RDS in all the AT cell we tested.     

Patients with an inherited syndrome characterized by immunodeficiency, microcephaly, and chromosomal instability: genetic relationship to ataxia telangiectasia.

Fibroblast cultures from six unrelated patients having a familial type of immunodeficiency combined with microcephaly, developmental delay, and chromosomal instability were studied with respect to their response to ionizing radiation. The cells from five of them resembled those from individuals with ataxia telangiectasia (AT) in that they were two to three times more radiosensitive on the basis of clonogenic cell survival. In addition, after exposure to either X-rays or bleomycin, they showed an inhibition of DNA replication that was less pronounced than that in normal cells and characteristic of AT fibroblasts. However, the patients are clinically very different from AT patients, not showing any signs of neurocutaneous symptoms. Genetic complementation studies in fused cells, with the radioresistant DNA synthesis used as a marker, showed that the patients' cells could complement representatives of all presently known AT complementation groups. Furthermore, they were shown to constitute a genetically heterogeneous group as well. It is concluded that these patients are similar to AT patients with respect to cytological parameters. The clinical differences between these patients and AT patients are a reflection of genetic heterogeneity. The data indicate that the patients suffer from a chromosome-instability syndrome that is distinct from AT.     

Radioresistant DNA synthesis: an intrinsic feature of ataxia telangiectasia

DNA synthesis in 6 ataxia langiectasia (AT) cell strains was much more resistant to X-irradiation than was DNA synthesis in normal human diploid cells. 3 of the cell strains tested have been classified as proficient in repair replication. These data, along with those reported elsewhere, strongly suggest that radioresistant DNA synthesis is an intrinsic feature of this disease.The radioresistance of DNA synthesis in AT cells is primarily due to a reduced inhibition of replicon initiation compared to that occuring in normal cells, but DNA chain elongation is also more radioresistant in AT cells. The small inhibition of DNA synthesis that does occur in AT cells at doses up to 2000 rad is almost exclusively due to inhibition of replicon initiation and not to inhibition of chain elongation, as would be expected from results with normal human cells or from previous studies with established cell lines.     

Complementation of chromosomal aberrations in AT/NBS hybrids: inadequacy of RDS as an endpoint in complementation studies with immortal NBS cells

Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare autosomal recessive hereditary disorders characterized by radiosensitivity, chromosomal instability, immunodeficiency and proneness to cancer. Although the clinical features of both syndromes are quite distinct, the cellular characteristics are very similar. Cells from both NBS and AT patients are hypersensitive to ionizing radiation (IR), show elevated levels of chromosomal aberrations and display radioresistant DNA synthesis (RDS). The proteins defective in NBS and AT, NBS1 and ATM, respectively, are involved in the same pathway, but their exact relationship is not yet fully understood. Stumm et al. (Am. J. Hum. Genet. 60 (1997) 1246) have reported that hybrids of AT and NBS lymphoblasts were not complemented for chromosomal aberrations. In contrast, we found that X-ray-induced cell killing as well as chromosomal aberrations were complemented in proliferating NBS-1LBI/AT5BIVA hybrids, comparable to that in NBS-1LBI cells after transfer of a single human chromosome 8 providing the NBS1 gene. RDS observed in AT5BIVA cells was reduced in these hybrids to the level of that seen in immortal NBS-1LBI cells. However, the level of DNA synthesis, following ionizing radiation, in SV40 transformed wild-type cell lines was the same as in NBS-1LBI cells. Only primary wild-type cells showed stronger inhibition of DNA synthesis. In summary, these results clearly indicate that RDS cannot be used as an endpoint in functional complementation studies with immortal NBS-1LBI cells, whereas the cytogenetic assay is suitable for complementation studies with immortal AT and NBS cells.     

The effect of 3-aminobenzamide on X-ray induction of chromosome aberrations in Down syndrome lymphocytes

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.     

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects

In the present study, both post-irradiation DNA synthesis and G₁ phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by ³H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G₂ phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G₂ phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G₂ accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cyle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G₂ phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomally in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

An evaluation of the Drosophila zeste somatic eye mutation test for the detection of genotoxic azo dyes and an aromatic amine

The rate of DNA synthesis was studied in normal cell strain and in strains from patients suffering from inherited disorder ataxia telangiectasia (AT). After exposure to reactively low doses of oxic X-rays (0–4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an “excision-deficient” and an “excision-proficient” strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad.These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis.     

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.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

The effect of methylnitronitrosoguanidine on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in asynchronous and synchronous cultured cells

Methylnitronitrosoguanidine (MNNG) is reported to inhibit DNA synthesis in intact human cells, in the cells from patients with ataxia telangiectasia (AT) or the cells from two rodent species. DNA synthesis in different cell lines exhibits varying sensitivity to MNNG inhibitory effect. 4-5-fold higher concentrations of MNNG are required for 50% inhibition of DNA synthesis in AT cells or in field vole cells as compared with the concentration required for human cells or Chinese hamster. The different compactness of two chromatin fractions might possibly result in lower sensitivity of DNA synthesis in heterochromatin to MNNG-induced inhibition as compared with the sensitivity of euchromatin. The genetic expression of AT defect on the cellular level is supposed to be connected with changes in supramolecular packaging of chromatin in interphase nuclei.     

Normal inhibition of DNA synthesis following gamma-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

Inhibition of DNA synthesis was studied in gamma-irradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of gamma-rays in all of the cell lines. 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to gamma-rays. Contrary to other data in the literature these results demonstrate that radioresistant DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivity.     

The effect of mitotic inhibitors on DNA strand size and radiation-associated break repair in Down syndrome fibroblasts

The effect of mitotic inhibitors on formation and repair of DNA breaks was studied in cultured fibroblasts from patients with Down syndrome in order to investigate the hypothesis that the karyotyping procedure itself may play a role in the increased chromosome breakage seen in these cells after gamma radiation exposure. Using the nondenaturing elution and alkaline elution techniques to examine fibroblasts from Down syndrome patients and from controls, no specific abnormalities in Down syndrome cells could be detected after exposure to mitotic inhibitors, including rate and extent of elution of DNA from filters as well as repair of radiation-induced DNA breaks. In both normal and Down syndrome cell strains, however, exposure to mitotic inhibitors was associated with a decrease in cellular DNA strand size, suggesting the presence of drug-induced DNA strand breaks. The mechanism of increased chromosome sensitivity of Down syndrome cells to gamma radiation remains unknown.     

Variant of ataxia-telangiectasia with low-level radiosensitivity

Summary In the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or blcomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.     

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.     

Responses of Huntington's disease and ataxia telangiectasia lymphoblastoid cells to bleomycin

Ionizing radiation sensitive, mutant human lymphoblastoid cell lines derived from patients with Huntington's disease (HD), or ataxia telangiectasia (AT) both showed cross sensitivity to bleomycin, as assayed by reduced cell viability and increased frequency of chromosome aberrations compared to normal controls. In contrast to AT cells which failed to show inhibition of DNA synthesis after exposure to ionizing radiation, or bleomycin treatment, the sensitive cells from HD patients had depressed rates of DNA synthesis after damage with these agents, similar to that seen in normal cells. In terms of progression through the cell cycle bleomycin damaged AT cells moved from G1 into S and from S to G2 + M at almost the same rate as untreated cells. Bleomycin treated HD cells showed a large proportion of cells blocked in G1, cells were slowed down in S, the rate of entry to G2 + M was reduced and only 5% of cycling cells reached G2. Progress through the cell cycle in normal cells exposed to bleomycin showed a partial block in G1 and the rate of entry to G2 + M was reduced. These differences in response of normal, AT and HD cells to ionizing radiation and bleomycin treatment indicates that the defect underlying the sensitivity is different in HD cells from that in AT cells.