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.     

Hypersensitivity and reduced inhibition of DNA synthesis in ataxia telangiectasia lymphoblasts treated with low levels of neocarzinostatin

The effects of neocarzinostatin (NCS) on lymphoblastoid cell lines (LCLs) established from ataxia telangiectasia (A-T) were determined. A-T lymphoblasts were found to be hypersensitive to low levels of NCS as measured by cell growth and cell survival. On the other hand, A-T lymphoblasts failed to postpone DNA synthesis to the same degree as normal lymphoblasts following treatment with NCS. LCLs established from Nijmegen breakage syndrome (NBS) could be distinguished from ataxia and normal cell lines by their intermediate level of survival following exposure to NCS.     

DNA repair in lymphoblastoid cell lines established from human genetic disorders

Lymphoblastoid cell lines (LCLs) established from chromosomal breakage syndromes or related genetic disorders have been used to study the effects of mutagens on human lymphoid cells. The disorders studied include xeroderma pigmentosum, ataxia telangiectasia, Fanconi's anemia, Bloom's syndrome and Cockayne's syndrome. Three approaches were used to assess the cells' ability to cope with a particular mutagen: (1) assaying recovery of DNA snythetic capabilities as measured by [³H]thymidine (dT) incorporation; (2) measurements of classical excision DNA repair by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (3) determining cell survival by colony formation in microtiter plates. LCLs established from xeroderma pigmentosum showed increased sensitivities to ultraviolet (354 nm) light and N-acetoxy-2-acetylaminofluorene (AAAF) as determined by DNA synthesis or colony formation and had diminished levels of excision-repair. Cockayne's syndrome LCLs, on the other hand, had increased sensitivities to ultraviolet (UV) light, AAAF and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while showing near normal levels of DNA-repair after treatment with each agent. An LCL established from ataxia telangiectasia had decreased DNA repair synthesis and defective colony-forming ability following treatment with MNNG. LCLs, in addition to ease of establishment, appear likely to provide useful material for the study of DNA repair replication and its relationship to carcinogenesis.     

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.     

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells

Our previous results indicated a close relationship between the presence of a BRCA1 mutation in lymphocytes and hypersensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H(2)O(2)). Comparative investigations with the comet assay (single-cell gel electrophoresis) suggested a normal rate of damage removal and pointed to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. We now wanted to see whether similar results could be obtained with lymphoblastoid cell lines (LCLs) and whether such permanent cells are suitable as a model for the investigation of mechanisms involved in mutagen sensitivity. Our results show that LCLs with a BRCA1 mutation are also hypersensitive to the chromosome-damaging effects of gamma irradiation or H(2)O(2), as revealed by the micronucleus test. Interestingly, LCLs heterozygous for an ataxia telangiectasia (AT) mutation have similar characteristics as BRCA1 cells with respect to the induction and repair of DNA damage induced by either gamma irradiation or H(2)O(2). However, caffeine enhanced the induction of micronuclei by gamma irradiation only in normal and heterozygous AT cells but not in BRCA1 cells, thus indicating a difference in the pathways leading to mutagen sensitivity in cells with a BRCA1 or an AT mutation. Our results suggest that caffeine could be useful in discriminating AT heterozygotes from carriers of a BRCA1 mutation, as well as BRCA1 mutation carriers from normal individuals.     

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.     

Superoxide dismutase activity and chromosome damage in cultured chromosome instability syndrome cells

The basal levels of superoxide dismutase (SOD) activity and chromosome aberration (CA) and sister-chromatid exchange (SCE) frequencies were examined in cultured fibroblasts or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs). These cells were derived from patients with chromosome instability syndromes (CISs) including Bloom's syndrome (BS), Fanconi's anemia (FA) and ataxia telangiectasia (AT). Embryonal fibroblasts and LCLs from normal subjects served as controls. Although LCLs tended to exhibit a higher SOD level than fibroblasts due to an elevation of Cu/Zn-SOD activity, BS and FA fibroblasts with increased frequencies of CAs and/or SCEs showed abnormally elevated SOD activity due to the manifold increase of Mn-SOD levels compared with control cells. However, BS and AT LCLs with almost control levels of CA and SCE frequencies showed no, or a slightly elevated, SOD activity, suggesting a possible selection of such cells during EBV transformation. The observed parallelism between the SOD activity and the cytogenetic manifestation may imply an involvement of active oxygen species, especially superoxide radicals, in the increased chromosome damage of CIS cells.     

X-ray-induced G2 arrest in ataxia telangiectasia lymphoblastoid cells

Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiectasia (AT) lymphoblastoid cells and normal human cells. Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in AT cells than in normal cells as previously reported by other investigators. However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistinguishable from that in normal cells. Moreover, no significant difference in cell viability was noted between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar. These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to more stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there. The possible reasons for this greater accumulation are discussed.     

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.     

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

Summary Lymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic -irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic -irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.     

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 analysis of DNA methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

Previous reports have shown that DNA methylation profiles within primary human malignant tissues are altered when these cells are transformed into cancer cell lines. However, it is unclear if similar differences in DNA methylation profiles exist between DNA derived from peripheral blood leukocytes (PBLs) and corresponding Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs). To assess the utility of LCLs as a resource for methylation studies we have compared DNA methylation profiles in promoter and 5/ regions of 318 genes in PBL and LCL sample pairs from patients with type 1 diabetes with or without nephropathy. We identified a total of 27 (~8%) genes that revealed different DNA methylation profiles in PBL compared with LCL-derived DNA samples. In conclusion, although the profiles for most promoter regions were similar between PBL-LCL pairs, our results indicate that LCL-derived DNA may not be suitable for DNA methylation studies at least in diabetic nephropathy.     

Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro.

Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.     

Measurement of DNA polymerase beta in skin fibroblast cell lines from patients with ataxia telangiectasia

DNA polymerase beta levels were measured in 4 cell lines of normal human skin fibroblasts and in 5 cell lines of skin fibroblasts from patients with ataxia telangiectasia, an autosomal recessive disease exhibiting marked X-ray sensitivity. The enzyme specific activities for the normal lines were similar and the mean value was 2-fold lower than the mean value for the ataxia lines. With both kinds of cells, the enzyme level did not change as the cultures progressed from logarithmic to stationary phase of growth. Thus, this putative DNA repair enzyme appears to be 'constitutive' in human skin fibroblast lines, and a modest elevation of beta-polymerase activity is associated with ataxia telangiectasia. These results are discussed in the context to current views about DNA-repair enzymes in X-ray-sensitive cultured mammalian cells.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

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.     

Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks

Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium. While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain. In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.     

The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: a pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O⁶-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.