Gamma-ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines

Well characterized gamma-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Normal human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline sucrose gradient centrifugation, repression of DNA synthesis by low doses of gamma-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression in synthesis of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW DNA synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

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.     

Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radiosensitivity from radioresistant DNA synthesis.     

γ-Ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telagiectasia cell lines

Well characterized γ-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telagiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characterteristics of γ-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Norman human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline gradient centrifugation, repression of DNA synthesis by low doses of γ-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.     

DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair γ-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to γ-irradiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrateed to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10–40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.     

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.     

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.     

DNA replication and repair in ataxia telangiectasia cells exposed to bleomycin

A marked increase in sensitivity to bleomycin was observed in two ataxia telangiectasia (AT) lymphoblastoid cell lines compared to that in cell lines from two normal individuals. This sensitivity was obtained at two different concentrations of bleomycin. While normal cells showed a rapid recovery of ability to divide, there was no indication of such a recovery in AT cells up to 120 h after bleomycin treatment. A similar level of breakage of DNA occurred in both cell types after incubation with bleomycin. The rate of repair of these breaks was also the same. DNA synthesis was found to be more resistant to bleomycin in AT cells than in control cells. The latter data are in keeping with results previously obtained using ionizing radiation.     

Comparative od adaptive response to gamma-radiation and nickel sulfate treatment in human cells

Increased viability of human cells (line rhabdomyosarcoma) to challenge doses of NiSO4 (10(-5)-10(-3) M) was formed when cells were preirradiated with low doses of gamma-radiation (10-14 cGy). Observed adaptive response was similar to radioadaptive response in human fibroblasts, pretreated with low doses of gamma-radiation and challenge dose of the same mutagen. Pretreatment with low concentration of NiSO4 induced in human fibroblasts increased resistance of DNA to the treatment with challenge doses of gamma-radiation and stimulated DNA repair synthesis after treatment with NiSO4 and 4-nitroquinoline-1-oxide. These data confirm the existence of cross-adaptation in the experiments with NiSO4.     

Comparison of gamma-radiation-induced accumulation of ataxia telangiectasia and control cells in G2 phase

Recent reports from a number of laboratories have linked radiosensitivity in ataxia telangiectasia (A-T) to a large and prolonged block of some cells in G2 phase. Previous results from this laboratory, largely with one Epstein-Barr virus-transformed A-T lymphoblastoid cell line, presented evidence for a dramatic increase in the number of cells in G2 phase over controls during a 24-h period post irradiation. We describe here a study of the effect of gamma-radiation on G2 phase delay in several A-T cell lines. Based on previous results with several cell lines 24 h post irradiation was selected as the optimum time to discriminate between G2 phase delay in control and A-T cells. All A-T homozygotes showed a significantly greater number of cells in G2 phase, 24 h post irradiation, than observed in controls. A more prolonged delay in G2 phase after irradiation was seen in different A-T cell types that included lymphoblastoid cells, fibroblasts and SV40-transformed fibroblasts. At the radiation dose used it was not possible to distinguish A-T heterozygotes from controls.     

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.     

Nascent DNA Synthesis in Ultraviolet Light-Irradiated Mouse, Human, and Chinese Hamster Cells

The technique of alkaline sucrose gradient centrifugation was used to study newly synthesized DNA in control and ultraviolet light-irradiated mouse L, human HeLa, and Chinese hamster ovary cells. Nascent DNA molecular weight distributions did not appear to differ among the three cell lines for unirradiated cells. However, at short times after ultraviolet light irradiation, human HeLa cells appeared to synthesize more low molecular weight DNA than either mouse L or Chinese hamster ovary cells. Since this difference was not related to differences in either the rate of DNA synthesis or amount of ultraviolet damage in the irradiated cells it appeared to be a phenotypic characteristic of the cell lines tested. A parallel was noted for these three cell lines between an increase in the synthesis of low molecular weight DNA, detected on alkaline sucrose gradients, and cell killing as measured by the ability of irradiated cells to form colonies.     

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.     

Study of DNA synthesis in short-term cultures of mammalian cells in the evaluation of the reaction of tumor and normal tissues to irradiation and hyperthermia

The effect of radiation of hyperthermia was estimated with a reference to the degree and duration of inhibition of DNA synthesis in the primary suspension cell cultures of Lewis lung carcinoma and bone marrow carcinoma of mice in vitro. The optimum conditions were chosen for cultivation of the primary suspension cell cultures according to the DNA synthesis. A study was made of the peculiarities of suppression and recovery of DNA synthesis in cells of Lewis lung carcinoma and bone marrow carcinoma of mice exposed to different gamma-radiation doses and hyperthermia.     

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.     

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.     

Inhibition and recovery of DNA synthesis in human tumor cell lines following radiation exposure

Eight human tumor cell lines with radiosensitivities (D0) ranging from 1 to 3 Gy were analyzed for their response to radiation-induced inhibition of DNA synthesis. These cell lines differ in their sensitivity to induction of DNA double-strand breaks and in the rate at which they rejoin DNA double-strand breaks. Fifty-gray doses of gamma rays induced between 35 and 75% inhibition in rates of DNA synthesis. The magnitude of the inhibition was not related to cellular radiosensitivity, frequency of initial DNA double-strand breaks, or the rate of rejoining of DNA double-strand breaks. All the cell lines studied had similar kinetics of recovery from inhibition of DNA synthesis following radiation exposure. These results suggest that factors other than or in addition to frequency of DNA double-strand breaks are important in the control of DNA synthesis following exposure to ionizing radiation in human tumor cell lines.     

Caffeine inhibition of postreplication repair of UV-damaged DNA in mouse epidermal cells

The effect of caffeine (0.25–1.5 mM) on UV-irradiated (5 and 10 J/m²) primary cultures of mouse epidermal cells (EPD) and an in vitro transformed cell line (PDV) was studied at the cellular and molecular levels. A synergistic reduction in cell survival induced by caffeine with UV-irradiation was found in the PDV cells at 10 J/m² but not at 5 J/m². When conversion of low molecular weight newly-synthesized DNA to high molecular weight DNA was studied in both cell types, caffeine at 1.5 mM had no effect on this conversion in unirradiated cultures. At 5 J/m², caffeine had a transitory inhibitory effect on this conversion. However, at 10 J/m² caffeine had a strong permanent inhibitory effect on this conversion at doses higher than 0.5 mM in PDV cells and higher than 0.25 mM in EPD cells. This apparent inhibition of elongation by caffeine in irradiated cells could not be accounted for by an effect on the rate of DNA synthesis. In PDV cells there was a direct correlation in terms of effective caffeine dose level between synergistic reduction in cell survival after UV and the effect on DNA elongation. Irradiated EPD cells were more sensitive to the inhibitory effect of caffeine on DNA elongation.     

Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patients

The repair of mitomycin C (MMC)-induced DNA crosslinking was analyzed by denaturation-renaturation gel electrophoresis in ribosomal RNA genes in lymphoblastoid cell lines from 4 patients with Fanconi's anemia (FA). In comparison to normal lymphoblastoid cell lines, 2 lines of FA cells belonging to complementation group A clearly exhibited higher sensitivity to MMC and an almost identical deficiency in the removal of DNA crosslinking. A complementation group B cell line, HSC 62, exhibited a lower sensitivity than group A cells and a lesser deficiency in crosslink repair. Another 'non-A' group cell line, HSC 230, reproducibly exhibited even higher sensitivity to MMC than group A cells. The results on MMC crosslinkage removal at the molecular level correlated well with cell survival. The observed subtle differences of repair among the 4 FA cell lines might represent possible genetic differences in the respective FA complementation groups.     

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.