The thermal sensitivity of normal and ataxia telangiectasia human fibroblasts

Human normal and ataxia telangiectasia (AT) heterozygote and homozygote cell strains were heated at 42.0 and 45.0°C to determine their thermal responses. All cell strains had approximately the same thermal sensitivity and were less thermally sensitive than Chinese hamster cells or many other rodent cell lines reported in the literature. No shoulders were observed on the survival curves for heating at 42.0 or 45.0°C. Thermal tolerance developed in both the normal and AT cell strains with heating for prolonged intervals at 42.0°C.     

Unscheduled DNA synthesis induced by streptonigrin in ataxia telangiectasia fibroblasts

Streptonigrin is an antitumour antibiotic, which at low dosesproduces DNA strand breaks in cultured cells leading, e. g.,to decreased colony-forming ability and decreased rates of DNAsynthesis. At higher doses the drug can induce unscheduled DNAsynthesis (UDS) presumably as a consequence of excision of largeDNA adducts. Ataxia telangiec-tasia (A-T) cells are unusuallysensitive to streptonigrin, but we show here that they can performexcision repair, as demonstrated by UDS, at the same level asnormal cells following exposure to the drug. This result suggeststhat of the apparent two modes of action of streptonigrin itis the DNA strand-breaking capacity to which A-T cells are unusuallysensitive. This is consistent with previous reports suggestingsome form of DNA strand break in A-T cells is deficiently repaired.     

Increased level of bleomycin-induced chromosome breakage in ataxia telangiectasia skin fibroblasts

Ataxia telangiectasia (AT) is an autosomal recessive disorder in which increased level of chromosome breakage and specific sensitivity to radiation and carcinogens have been reported. The effect of the radiomimetic drug bleomycin on chromosome breakage has been tested in skin fibroblasts of three patients with AT, two AT obligate heterozygotes, two normal human controls, and one normal amniotic fluid cell culture. Bleomycin in two concentrations (1 and 5 micrograms/ml) was added for 1 hr and cultures were harvested 4 hr later. A significant increase in chromosome damage was found in AT fibroblasts: a higher number of total breaks per cell, affected cells, and breaks per affected cell was found. The heterozygotes did not differ significantly from the controls. Chromosome breakage in skin fibroblasts of AT patients after bleomycin treatment has not been reported before.     

An aberration in gamma-ray-enhanced reactivation of irradiated adenovirus in ataxia telangiectasia fibroblasts

Ataxia telangiectasia (AT) is a rare human genetic disorder,whose numerous clinical hallmarks include a predisposition tolymphoreticular cancers and a hypersensitivity to conventionalradiotherapy. Furthermore, AT cells in vitro exhibit a hypersensitivityto ionising radiation that appears to be correlated with anincreased frequency of chromosomal aberrations, a resistanceof de novo DNA synthesis to inhibition by radiation-inducedDNA damage, a reduced mitotic delay and possible defects inDNA repair. In this study, a sensitive viral assay has beenused to investigate the capacity of gamma-irradiated AT cellsto support the replication of undamaged virus, as well as theextent to which the survival of radiation-damaged virus wasaffected by gamma-irradiation of these host cells prior to infection.The expression of such enhanced reactivation (ER) of both u.v.-irradiated(u.v. dose = 1.2 x 10³ J/m²) and gamma-irradiated (dose = 2Mrad) adenovirus type 2 (Ad2) was examined in a variety of normaland AT human fibroblast strains. Unirradiated and gamma-irradiatedfibroblasts were infected with unirradiated or irradiated Ad2,either immediately or at different times after cell monolayerirradiation, and at 48 h after infection cultures were examinedby indirect immunofluorescence to determine the number of cellsin which Ad2 viral structural antigen (Vag) was expressed. Forimmediate infection of normal human fibroblasts, both a decreasein unirradiated virus expression and an increase in ER wereobserved with increasing gamma-ray dose to the cells. In contrast,AT fibroblasts were found to be deficient in gamma-ray ER ofirradiated Ad2, and this defect appeared to be related to amarked relative radioresistance of unirradiated virus expressionin AT compared to normal cells. The potential significance ofthese results is discussed in the context of mammalian ER, whichis believed to be, at least in part, an expression of a mutagen-inducible(and possibly error-prone) DNA repair mechanism.     

DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection

During selection for methotrexate resistance, SV40-transformedhuman skin fibroblasts from patients with ataxia telangiectasia(A-T) underwent amplification of the dihydrofolate reductase(DHFR) gene, experienced nearly complete loss of the integratedSV40 sequences and showed a 3.6-fold increase in Ki-ras genecopy number. Over a period of months methotrexate-resistant(MTX^r) A-T subclones were obtained, which were able to growin progressively increasing MTX concentrations up to 100 µM.The ED₅₀ values determined as the effective dose of MTX causing50% growth inhibition in comparison to control cells increasedfrom 3x10^–2 µM for MTX^s AT5BI-VA cells to 250 µMMTX for the MTX^r AX100 subclone. In contrast, human skin fibroblastsof healthy individuals did not show DHFR gene amplificationand loss of SV40 sequences under comparable conditions and wereunable to grow in MTX concentrations >1 µM. Gene amplificationand loss of DNA sequences are features underlying the genomicinstability known to be a characteristic property of A-T cellsand being probably responsible for the high cancer incidencein these patients.     

Gefitinib radiosensitizes non-small cell lung cancer cells through inhibition of ataxia telangiectasia mutated

Purpose  

Inhibitors of epidermal growth factor receptor (EGFR) have shown dramatic results in a subset of patients with non-small cell lung cancer (NSCLC), and have also been shown to enhance the effect of ionizing radiation (IR). We investigated how gefitinib, an orally given EGFR inhibitor for NSCLC patients, can radiosensitize NSCLC cells.     

Radiotherapeutic management of medulloblastoma in a pediatric patient with ataxia telangiectasia

Ataxia telangiectasia (AT) is a genetic disorder with a predisposition to malignancy. Cells from patients with AT demonstrate an increased sensitivity to ionizing radiation which creates a problem when these patients require treatment for their malignant disease. An eleven-year-old boy with a previous diagnosis of AT was seen in consultation following partial resection of medulloblastoma in the posterior fossa. To estimate how much the conventional radiation dose might have to be reduced, we compared the radiosensitivity of bone marrow myeloid progenitor cells from this patient to that of cells from the marrow of normal individuals, using colony formation in an agar culture assay system as the endpoint (CFU-Cs). Neither radiation dose-survival curve exhibited a shoulder--each displayed an extrapolation number of 0.99. The survival curve of normal cells displayed a steep slope with a D0 of 0.98 Gy (0.83-1.19 Gy, 95% confidence limits); the slope for the AT cells was considerably steeper with a value for D0 of 0.32 Gy (0.29-0.35 Gy). The ratio of D0's indicated that these AT cells were approximately 3X more radiosensitive than normal cells. Based on this, the daily dose was reduced from 1.8 to 0.6 Gy and the radiation was restricted to 25 treatments to the posterior fossa rather than the conventional cranio-spinal treatment. An additional 5 treatments at 1.0 Gy per day were given to the whole brain. The patient's skin responded to these reduced fraction sizes and doses to a similar degree as normal patients' skin following a standard schedule and the patient is doing well nine months after initiation of treatment.     

Unusual sensitivity of ataxia telangiectasia cells to bleomycin.

Peripheral blood lymphocytes from four patients with ataxia telangiectasia (AT), an inherited disorder showing, among other features, radiosensitivity and a high frequency of cancers, were shown to be cytogenetically more sensitive to bleomycin than were lymphocytes from both normal individuals and a single patient with xeroderma pigmentosum. With cell survival techniques, a biphasic dose-response curve was seen for both normal and AT fibroblasts, although the AT cells showed a much lower survival. The increased sensitivity to bleomycin in AT cells might be expected since it is a radiomimetic drug, but more importantly the known action of bleomycin in producing DNA strand scission suggests that AT cells might be defective in rejoining a proportion of DNA strand breaks.     

Gene amplification in fibroblasts from ataxia telangiectasia (AT) patients and in X-ray hypersensitive AT-like Chinese hamster mutants

In search of functions involved in the regulation of gene amplification, and given the relevance of chromosome breakage in initiating the process, we analyzed the gene amplification ability of cells hypersensitive to inducers of DNA double-strand breaks and defective in cell cycle control: two human fibroblast strains derived from patients affected by ataxia telangiectasia (AT) and two hamster mutant cell lines belonging to complementation group XRCC8 of the rodent X-ray-sensitive mutants. These mutants are considered hamster models of AT cells. To measure gene amplification, the frequency and the rate of occurrence of N-(phosphonacetyl)-L-aspartate resistant cells were determined. In both hamster mutants, these two parameters were increased by about an order of magnitude compared with parental cells, suggesting that amplification ability was increased by the genetic defect. In primary AT fibroblasts, as in normal human fibroblasts, gene amplification was undetectable and a block in the G(1) phase of the cell cycle was induced upon PALA treatment. These results suggest that in AT fibroblasts, where only the ATM gene is mutated, ATM-independent mechanisms prevent gene amplification, while, in the immortalized hamster cell lines, which are already permissive for gene amplification, the AT-like defect increases the probability of gene amplification.     

Clonal evolution of T-cell chronic lymphocytic leukaemia in a patient with ataxia telangiectasia

Ataxia telangiectasia (A-T) is an autosomal recessive disorder in which patients show an unusual predisposition to malignant disease, including T-cell chronic lymphocytic leukaemia. We report here the steady growth over 5 years of a complex, cytogenetically abnormal clone of T lymphocytes in an A-T patient who was subsequently found to have an OKT3/OKT8 chronic lymphocytic leukaemia. The tumour cells at diagnosis had clearly evolved from a pre-existing, cytogenetically abnormal T-cell clone which contained an inv(14) chromosomal inversion alone.     

Lower ataxia telangiectasia mutated (ATM) mRNA expression is correlated with poor outcome of laryngeal and pharyngeal cancer patients

BackgroundAtaxia telangiectasia mutated (ATM) kinase is a critical regulator in initiating DNA damage response and activating DNA repair. However, the correlation between ATM expression and the outcome of laryngopharyngeal cancer patients is unknown. We hypothesize that ATM expression is correlated with a worse outcome in laryngopharyngeal cancer patients.Patients and methodsThe ATM messenger RNA (mRNA) expression of 80 tumors of laryngeal and pharyngeal cancer was examined by real-time quantitative RT-PCR. Overall survival rates were measured using Kaplan–Meier estimates and the log-rank tests. The adjusted hazard rate ratios (HRRs) were computed by multivariate Cox regressions.ResultsReduced ATM mRNA was found in 65 of 80 studied cases. Lower ATM expression [tumor/normal <0.3, HRR = 2.49; 95% confidence interval (CI) 1.27–4.88], younger age (<55 years, HRR = 2.71; 95% CI 1.16–6.32), and larger tumor (T₃/T₄, HRR = 2.21; 95% CI 1.10–4.44) were independent risk factors for survival. Patients with lower ATM and younger age (HRR = 6.51; 95% CI 2.05–20.66) or with lower ATM and T₃/T₄ tumor (HRR = 5.23; 95% CI 2.04–13.40) exhibited the poorest outcome.ConclusionThe expression of ATM mRNA, which is frequently downregulated in laryngeal and pharyngeal cancers, could be a valuable prognostic marker.     

Radiosensitive melanoma cell line with mutation of the gene for ataxia telangiectasia.

The human melanoma cell lines MM96L, A2058 and HT144 were examined for sensitivity to ionizing radiation and UVB radiation. HT144 demonstrated a significant increase in sensitivity to ionizing and UVB radiation compared with the MM96L and A2058 cells. Sensitivity to both agents was associated with susceptibility to apoptosis. Using a protein truncation assay, a mutation for the gene for ataxia telangiectasia (ATM) was identified in HT144 cells. This was confirmed to be a homozygous mutation by subsequent sequencing of the abnormal region. Protein truncation assay of the other two cell lines showed no abnormality. The results suggest that somatic mutation of the A-T gene may be important in determining tumour radiosensitivity.     

Lack of correlation between hypersensitivity to cell killing and impaired inhibition of DNA synthesis in ataxia telangiectasia fibroblasts treated with 4-nitroquinoline 1-oxide

Compared to normal controls from healthy subjects, cells cultured from patients with the autosomal recessive, cancer-prone disorder ataxia telangiectasia (AT) uniformly display impaired clonogenic survival, in concert with decreased inhibition of DNA synthesis, on exposure to ionizing radiation. In this study we have determined the effects of 4-nitroquinoline 1-oxide (4NQO), a partially radiomimetic chemical carcinogen, on colony-forming ability and rate of DNA synthesis in non-transformed skin fibroblasts strains derived from clinically normal volunteers and AT patients. Strain AT3BI, belonging to AT complementation group A, displayed substantial hypersensitivity to the lethal action of 4NQO, whereas the survival response of strain AT5BI (group D) did not differ significantly from that of the three normal controls. The 4NQO-hypersensitive AT3BI cells exhibited normal inhibition of DNA synthesis when treated with the chemical, as manifested by both dose-response and time-course measurements. However, 4NQO treatment decreased the rate of DNA synthesis to a much lesser degree in AT5BI than in normal strains. Hence, our data demonstrate unequivocally that, unlike that universally observed following exposure of AT cells to ionizing radiation, carcinogen-resistant DNA synthesis does not segregate with elevated cytotoxicity when cultured fibroblasts representing at least some genetic forms of AT (i.e. groups A and D) are damaged by 4NQO.     

Radioresistant malignant myoepithelioma of the breast with high level of ataxia telangiectasia mutated protein

Malignant myoepithelioma of the breast (MMB) is a rare and often aggressive disease with poor prognosis. Little is known regarding its optimal treatment and progression. We describe the clinical history of a woman following excision of a benign adenomyoepithelioma which recurred years later as a radioresistant malignant myoepithelioma with high levels of ataxia telangiectasia mutated protein and mutant p53 (Cys135Phe). MMB requires close follow‐up and aggressive treatment. If adjuvant radiotherapy is adopted to improve local control, minimal postoperative delay and higher doses than for standard post‐mastectomy radiation are recommended.     

Thermal enhancement of radiosensitivity in normal and ataxia telangiectasia human cells

Normal human and ataxia telangiectasia (AT) fibroblasts were tested for enhancement of radiosensitivity by hyperthermia. In normal fibroblasts, thermal enhancement of radiosensitivity occurred at 42.0 degrees C and 45.0 degrees C and was greatest for simultaneous treatments of heat and radiation. This thermal enhancement decreased, as an incubation time at 37.0 degrees C was introduced either between heat and X-ray, or X-ray and heat, treatments. AT cells were more radiosensitive (D0 = 0.67 Gy) than normal cells (D0 = 1.4 Gy). Heating at 42.0 degrees or 45.0 degrees C resulted in enhanced radiosensitivity, which was equal for heating before, during or after irradiation. These data show that normal human fibroblasts can recover from heat and radiation treatments, while AT fibroblasts lack this ability.     

Hypoxia-induced phosphorylation of Chk2 in an ataxia telangiectasia mutated-dependent manner

Chk2 is a serine/threonine kinase that signals to cell cycle arrest, DNA repair, and apoptotic pathways following DNA damage. It is activated by phosphorylation in response to ionizing radiation, UV light, stalled replication forks, and other types of DNA damage. Hypoxia is a common feature of solid tumors and has been shown to affect the regulation of many genes, including several DNA repair factors. We show here that Chk2 is phosphorylated on Thr68 and thereby activated in cells in response to hypoxia, and that this phosphorylation is dependent on the damage response kinase ataxia telangiectasia mutated (ATM) but not on the related kinase ATM and Rad3-related. Moreover, phosphorylation of Chk2 under hypoxia was attenuated in cells deficient in the repair factors MLH1 or NBS1. Finally, Chk2 serves to protect cells from apoptosis under hypoxic growth conditions. These results identify hypoxia as a new stimulus for Chk2 activation in an ATM-, MLH1-, and NBS1-dependent manner, and they suggest a novel pathway by which tumor hypoxia may influence cell survival and DNA repair.     

Deficient expression of enhanced reactivation of parvovirus H-1 in ataxia telangiectasia cells irradiated with X-rays or u.v. light

Cells of patients with ataxia telangiectasia (AT), an inheriteddisease characterized by a high propensity to cancer, are hypersensitiveto ionizing radiation. We investigated whether the hyper-radiosensitivityof AT cells correlated with a defect in their constitutive and/orconditional ability to rescue a damaged exogenous virus. Forthat purpose, parvovirus H-1, a single-stranded DNA virus whoseintranuclear replication mostly relies on host cell functions,was used as a probe. The survival of u.v.-or -irradiated H-1was measured in X-, u.v.- or mock-irradiated human cells ofnormal (NB-E) or AT (AT5BIVA) origin. -irradiated H-1 survivedto similar extents in untreated normal and AT cell lines. BothX- and u.v.-irradiatlon induced normal cells to achieve an enhancedreactivation (ER) of -- or u.v.-damaged H-1. In contrast, neitherdose-effect curves nor time course revealed significant levelsof ER expression after X- or u.v.-irradiation in AT5BIVA cells.Our results suggest that the impairment of ER of damaged parvovirusesmay constitute a marker of the AT cell phenotype and be relatedto the radiosensitivity of AT cells.