Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients.

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.

Individual Radiosensitivity Assessment of the Families of Ataxia-Telangiectasia Patients by G2-Checkpoint Abrogation.

OBJECTIVES: Ataxia-telangiectasia (A-T) is an autosomal recessive multisystem disorder characterised by cerebellar degeneration, telangiectasia, radiation sensitivity, immunodeficiency, oxidative stress and cancer susceptibility. Epidemiological research has shown that carriers of the heterozygous ataxia-telangiectasia mutated (ATM) gene mutation are radiosensitive to ionising irradiation and have a higher risk of cancers, type 2 diabetes and atherosclerosis. However, there is currently no fast and reliable laboratory-based method to detect heterozygous ATM carriers for family screening and planning purposes. This study therefore aimed to evaluate the ability of a modified G2-assay to identify heterozygous ATM carriers in the families of A-T patients. METHODS: This study took place at the Tehran University of Medical Sciences, Tehran, Iran, between February and December 2017 and included 16 A-T patients, their parents (obligate heterozygotes) and 30 healthy controls. All of the subjects underwent individual radiosensitivity (IRS) assessment using a modified caffeine-treated G2-assay with G2-checkpoint abrogation. RESULTS: The mean IRS of the obligate ATM heterozygotes was significantly higher than the healthy controls (55.13% ± 5.84% versus 39.03% ± 6.95%; P <0.001), but significantly lower than the A-T patients (55.13% ± 5.84% versus 87.39% ± 8.29%; P = 0.001). A receiver operating characteristic (ROC) curve analysis of the G2-assay values indicated high sensitivity and specificity, with an area under the ROC curve of 0.97 (95% confidence interval: 0.95-1.00). CONCLUSION: The modified G2-assay demonstrated adequate precision and relatively high sensitivity and specificity in detecting heterozygous ATM carriers.

A flow cytometry assay that measures cellular sensitivity to DNA-damaging agents, customized for clinical routine laboratories.

OBJECTIVES: The clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay. DESIGN AND METHODS: We have developed a faster and less labor-intensive cell division assay (CD assay) using flow cytometry and incorporation of a fluorescent thymidine analogue. The CD assay was calibrated to the clonogenic assay and optimized for peripheral blood lymphocytes. RESULTS: Following ionizing radiation of primary human skin fibroblasts, the four-day CD assay gave similar results as the 14-day clonogenic survival assay. In lymphocytes isolated from patient blood samples, the CD assay was able to detect increased radiosensitivity in ataxia telangiectasia patients and increased radiosensitivity after in vitro treatment with DNA-PK and ATM inhibitors. The CD assay found a variation in the intrinsic radiosensitivity of lymphocytes isolated from healthy control samples. The CD assay was able to measure the anti-proliferation effect of different chemotherapeutic drugs in lymphocytes. CONCLUSIONS: Our results indicate that the CD assay is a fast and reliable method to measure the anti-proliferation effect of DNA-damaging agents with a potential to find the most sensitive patients in the work-up before cancer treatment.

Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability.

Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a comparable nucleotide substitution rate in AT-iPS cells. Taken together, these data show that ATM is involved in protection from irradiation-induced cell death.

Ataxia-telangiectasia and wilms tumor: reduced treatment but early relapse.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, a high incidence of lymphoreticular tumors, and an increased sensitivity to chemoradiotherapy-induced DNA damage. The appropriate cancer therapy remains unknown because of high toxicity rates with full-dose conventional protocols. We present a patient with A-T and nephroblastoma, who received an adapted treatment regimen. To our knowledge this is the second report on nephroblastoma in a patient with A-T but the first with confirmed premortem studies. Although the patient tolerated the chemotherapy regimen well, the patient relapsed and died a year after initial diagnosis.

Ataxia-telangiectasia mutated and the Mre11-Rad50-NBS1 complex: promising targets for radiosensitization.

Radiotherapy plays a central part in cancer treatment, and use of radiosensitizing agents can greatly enhance this modality. Although studies have shown that several chemotherapeutic agents have the potential to increase the radiosensitivity of tumor cells, investigators have also studied a number of molecularly targeted agents as radiosensitizers in clinical trials based on reasonably promising preclinical data. Recent intense research into the DNA damage-signaling pathway revealed that ataxia-telangiectasia mutated (ATM) and the Mre11-Rad50-NBS1 (MRN) complex play central roles in DNA repair and cell cycle checkpoints and that these molecules are promising targets for radiosensitization. Researchers recently developed three ATM inhibitors (KU-55933, CGK733, and CP466722) and an MRN complex inhibitor (mirin) and showed that they have great potential as radiosensitizers of tumors in preclinical studies. Additionally, we showed that a telomerase-dependent oncolytic adenovirus that we developed (OBP-301 [telomelysin]) produces profound radiosensitizing effects by inhibiting the MRN complex via the adenoviral E1B55kDa protein. A recent Phase I trial in the United States determined that telomelysin was safe and well tolerated in humans, and this agent is about to be tested in combination with radiotherapy in a clinical trial based on intriguing preclinical data demonstrating that telomelysin and ionizing radiation can potentiate each other. In this review, we highlight the great potential of ATM and MRN complex inhibitors, including telomelysin, as radiosensitizing agents.

Telangiectasia esencial generalizada: descripción de tres casos tratados con un sistema de luz intensa pulsada / Generalized Essential Telangiectasia: a Report of 3 Cases Treated Using an Intense Pulsed-Light System

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Telomere length in lymphoblast cell lines derived from clinically radiosensitive cancer patients.

Approximately 1-5 percent of cancer patients suffer from significant side effects in normal tissue after radiotherapy (RT). Although RT is an effective cancer therapy, treatment dose intensities are restricted to minimize the incidence of such normal tissue reactions. Therefore, most patients receive lower dose intensities than can be tolerated in normal tissue. A primary aim for radiation oncology is to identify radiosensitive (RS) individuals prior to treatment. Such predictive ability should result in an improvement in tumor control rates and/or a reduction in the incidence of RT side effects. Recent evidence suggests a link between RS and telomere length. A positive correlation between cellular RS and telomere length in a cohort of breast cancer patients has been reported. Furthermore,individuals with cancer-prone recessive RS syndromes, such as ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome(NBS), have shortened telomeres. To determine whether the association between telomere length and RS could be used as a predictive assay to prospectively identify RS cancer patients, we utilized a bank of lymphoblastoid cell lines (LCLs) derived from 33 RS patients, along with 18 LCL samples from RT patients who did not have severe reactions, to assess the link between RS and telomere length. We found a subset of RS patient LCLs had abnormally long telomere lengths, so these data suggest that RS could potentially be predicted for a subset of RS patients based on telomere length in LCLs, and contribute to therapy individualization.

PKB/Akt media la radio sensibilidad asociada a Ataxia Telangiectasia / PKB/Akt mediates radiosensitivity associated to ataxia telangiectasia

El gen mutado en el síndrome de Ataxia Telangiectasia está implicado en múltiples funciones celulares queabarcan desde la respuesta al daño genotóxico causado por la radiación ionizante hasta la respuesta a insulina.Curiosamente la ruta de señalización mediada por Akt esta relacionada con los mismos estímulos. En el presenteestudio demostramos como el gen mutado en Ataxia Telangiectasia (ATM) controla la fosforilación deAkt, en respuesta a estímulos como la radiación ionizante o la insulina. Estos datos han sido obtenidos usandomodelos experimentales como células derivadas de enfermos de Ataxia o de ratones nulos para la expresión deATM. Nuestro estudio propone nuevas explicaciones para entender la radio sensibilidad característica de estosenfermos y destaca el papel primordial de Akt en la respuesta a la radiación ionizante

The gene mutated in ataxia telangiectasia (ATM) has been implicated in several functions such as cell cycle, response to DNA damage, and insulin. Curiously, the PKB/Akt-mediated signaling route is related to the same cellular responses. We show in this work that ATM is a major determinant of full PKB/Akt activation in response to insulin or gamma-radiation. This conclusion was inferred from the results obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells, and also in cell lines derived from ataxia telangiectasia patients or KO mice. Our study proposes new clues to understand the radiosensitivity associated to ataxia telangiectasia and supports a critical role for PKB/Akt in the cellular response to ionizing radiation

[Specific features of p53 protein induction after ionizing radiation in cells of patients with Nijmegen breakage syndrome]. / Osobennosti radiatsionnoi induktsii belka p53 v kletkakh cheloveka pri Nijmegen breakage syndrome.

Synthesis of p53 and WAF1 (p21) proteins was studied in cells of patients with Nijmegen breakage syndrome (NBS) and of patients with ataxia telangiectasia (AT), as well as in normal cells with respect to their response to ionizing radiation (IR). In the NBS cells, the p53 protein was progressively accumulated with increasing radiation dose and reached the maximum 2 h after exposure to radiation at a dose of 5 Gy. The amount of p53 protein was consistently lower than that in normal cells, which was correlated with low content of the WAF1, the protein regulated by p53 at the level of transcription. Suboptimal induction of p53 observed in NBS cells was also characteristic of the AT cells, though the quantitative parameters of the protein synthesis in AT cells were intermediate relative to those in normal and NBS cells. In four NBS lines, the time schedule of p53 synthesis was similar to that observed in normal cells, whereas in AT cells, induction of p53 was significantly delayed as compared to control. In response to irradiation, the amount of p53 protein synthesized in patients with AT and NBS was significantly lower than that in normal cells. The results obtained, as well as the previously published medical and genetic evidence, suggest that the two diseases are of different origin and different genes are responsible for their development.

Roles of DNA-dependent protein kinase and ATM in cell-cycle-dependent radiation sensitivity in human cells.

PURPOSE: The roles of DNA-dependent protein kinase (DNA-PK) and ATM in the cell-cycle-dependent radiosensitivity in human cells were investigated. METHODS AND MATERIALS: A DNA-PK activity-deficient human glioblastoma cell line M059J, ataxia telangiectasia cell lines AT3BISV and AT5BIVA, and control cell lines were used. Wortmannin inhibited DNA-PK and ATM activities. Cells were synchronized by hydroxyurea. Progression through the cell cycle was analysed by flow cytometry. RESULTS: M059J exhibited hyper-radiosensitivity throughout the cell cycle, with extreme hyper-radiosensitivity in G to early S-phase compared with the control cell line M059K. AT3BISV and AT5BIVA exhibited hyper-radiosensitivity throughout the cell cycle but showed a similar pattern of cell-cycle-dependent radiosensitivity to that observed in LM217 or HeLa cells. In AT3BISV and AT5BIVA, radiosensitization by wortmannin was observed throughout the cell cycle and was most prominent in G1 to early S-phase. Wortmannin did not sensitize M059J to ionizing radiation in any cell-cycle phase. DNA-PK activities were not different throughout the cell cycle. CONCLUSION: The results suggest that (1) non-homologous endjoining plays a dominant role in G1 to early S-phase and a minor role in late S to G2-phase in repairing DNA double-strand breaks, (2) the role of ATM in repairing double-strand breaks may be almost cell-cycle-independent and (3) the dominant role of non-homologous end-joining during G1 to early S-phase is not due to cell-cycle-dependent fluctuations in DNA-PK activity.

Chemo- and radiosensitivity testing in a patient with ataxia telangiectasia and Hodgkin disease.

Treatment of Hodgkin disease (HD) in ataxia telangiectasia (AT) patients is hampered by hypersensitivity to radiation and chemotherapy. Most patients die, due to toxicity or, rarely, to progressive disease. The authors report on a 9-year-old girl with stage IIA HD and AT She was treated with a tailored combined modality approach. No unacceptable toxicity was found, but the girl died of a relapse outside the irradiation field. In comparison with fibroblasts of non-AT patients, the fibroblasts of the patient were 3 times as sensitive for radiotherapy but just 1.2 times as sensitive for doxorubicin. A good correlation was shown between in vitro radio- and chemosensitivity testing and the observed clinical toxicity. The authors suggest, therefore, treating AT patients as much as possible according to standard protocols by adjusting the radiotherapy delivery and the chemotherapy regimen to individual doses derived from in vitro radio- and chemosensitivity testing.

ATM, a central controller of cellular responses to DNA damage.

Mutations in the ATM gene lead to the genetic disorder ataxia-telangiectasia. ATM encodes a protein kinase that is mainly distributed in the nucleus of proliferating cells. Recent studies reveal that ATM regulates multiple cell cycle checkpoints by phosphorylating different targets at different stages of the cell cycle. ATM also functions in the regulation of DNA repair and apoptosis, suggesting that it is a central regulator of responses to DNA double-strand breaks.

Normal tissue radiobiology: from the laboratory to the clinic.

This manuscript is in four parts, presenting the four talks given in a symposium on normal tissue radiobiology. The first part addresses the general concept of the role of parenchymal cell radiosensitivity vs. other factors, highlighting research over the last decade that has altered our understanding of factors underlying normal tissue response. The other three parts expand on specific themes raised in the first part dealing in particular with (1) modifications of fibroblast response to irradiation in relation to the induction of tissue fibrosis, (2) the use of the linear-quadratic equation to model the potential benefits of using different means (both physical and biologic) of modifying normal tissue response, and (3) the specific role of the growth factor TFG-beta1 in normal tissue response to irradiation. The symposium highlights the complexities of the radiobiology of late normal tissue responses, yet provides evidence and ideas about how the clinical problem of such responses may be modified or alleviated.

Effect of protein kinase C inhibitor (PKCI) on radiation sensitivity and c-fos transcription.

PURPOSE: The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. Although ionizing radiation was known to induce c-fos transcription and cellular protein kinase C (PKC) induces the expression of this immediate response gene, little is known about how mutated AT (ATM) or PKC-mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKC inhibitor (PKCI) on radiation sensitivity and c-fos transcription in normal and AT cells, and also studied whether PKCI effect on c-fos occurs in Ras-dependent pathway. METHODS AND MATERIALS: Normal (LM217) and AT (AT5BIVA) cells were transfected with PKCI expression plasmid and integration and overexpression of PKCI was evaluated by polymerase chain reaction and northern blotting, respectively. Cells were irradiated at a dose of 5 Gy/min with 137Cs irradiator and harvested 48 h after irradiation and investigated apoptosis with TUNEL method. The c-fos transcription activity was studied by performing compute assisted tomography (CAT) assay of reporter gene after transfection of c-fos CAT plasmid into LM and AT cells. Overexpression of Ras protein in transfected cells was shown by western blotting. RESULTS: Our results demonstrated for the first time a role of PKCI on the radiation sensitivity and c-fos transcription in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells (5% to 20%) but reduced apoptosis slightly in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than in LM cells. This c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells. CONCLUSIONS: Overexpression of PKCI increased radiation sensitivity and repressed c-fos transcription in LM cells but not in AT cells, and this is related with Ras. These results suggest that the effect of PKCI on c-fos transcription activity is related with Ras dependent signal transduction pathways and these mechanisms are different between normal fibroblasts, LM and ATM mutated, AT cells. The data obtained by this study provided evidence for novel transcriptional difference between LM and AT cells and this may be a reason for increased radiation sensitivity of AT cells.

High levels of delayed radiation-induced apoptosis observed in lymphoblastoid cell lines from ataxia-telangiectasia patients.

PURPOSE: Cells from ataxia-telangiectasia (A-T) patients are extremely sensitive to radiation but display decreased apoptosis, as measured during the first 3 days following radiation. To explain this apparent contradiction, we examined apoptosis in normal and A-T cells at late time points following radiation, under the assumption that radiation-induced apoptosis is delayed in the A-T cells. METHODS AND MATERIALS: Blood cells and lymphoblastoid cell lines from A-T patients, as well as healthy donors, were irradiated with X-rays. Apoptosis was measured at different time points (up to 7 and 30 days for lymphocytes and lymphoblastoid cells, respectively) using a flow cytometric method based on the reduction of intracellular DNA content (sub-G1 population). RESULTS: Compared to normal cells, CD4 and CD8 A-T lymphocytes displayed constantly reduced levels of radiation-induced apoptosis for up to 7 days after treatment. A-T lymphoblastoid cells, however, displayed a delayed and prolonged apoptosis. CONCLUSION: A-T lymphoblastoid cells show high levels of delayed radiation-induced apoptosis, which may contribute to the high cellular radiosensitivity displayed by the A-T phenotype. ATM (the gene mutated in A-T) plays different roles in the apoptotic response to ionizing radiation in quiescent lymphocytes and proliferative lymphoblastoid cells.

ATM--a key determinant of multiple cellular responses to irradiation.

Ataxia-telangiectasia is a rare clinical disorder manifesting a variety of different abnormalities, including progressive neurodegeneration, increased cancer incidence, immune deficiency, sterility, and extreme radiosensitivity. Recent studies have demonstrated that the defective gene product in this disease, ATM, is a protein kinase. The identification of several different substrates for this kinase is beginning to explain the wide array of different physiologic abnormalities that occur when this gene product is dysfunctional. Since the ATM protein is a critical signaling molecule in the cellular response to ionizing irradiation, the identification of these substrates also results in elucidation of the steps involved in a number of different cellular signaling pathways initiated by irradiation. Such insights also result in the identification of potential new targets for enhancing the efficacy of radiation therapy.

The inherited basis of human radiosensitivity.

Certain individuals cannot tolerate 'conventional' doses of radiation therapy. This is known to be true of patients with ataxia-telangiectasia and ligase IV deficiency. Although in vitro testing may not correlate completely with clinical radiosensitivity, fibroblasts and lymphoblasts from patients with both of these disorders have been clearly shown to be radiosensitive. Using a colony survival assay (CSA) to test lymphoblastoid cells after irradiation with 1 Gy, a variety of other genetic disorders have been identified as strong candidates for clinical radiosensitivity, such as Nijmegen breakage syndrome, Mre 11 deficiency, and Fanconi's anemia. These data are presented and considered as a starting-point for the inherited basis of human radiosensitivity.

Radiosensitivity and oxidative signalling in ataxia telangiectasia: an update.

Radiosensitivity is a major hallmark of the human genetic disorder ataxia telangiectasia. This hypersensitivity to ionizing radiation has been demonstrated in vivo after exposure of patients to therapeutic doses of radiation and in cells in culture. Clearly an understanding of the nature of the molecular defect in ataxia telangiectasia will be of considerable assistance in delineating additional pathways that determine cellular radiosensitivity/radioresistance. Furthermore, since patients with this syndrome are also predisposed to developing a number of leukaemias and lymphomas, the possible connection between radiosensitivity and cancer predisposition is of interest. Now that the gene (ATM) responsible for this genetic disease has been cloned and identified, progress is being made in determining the role of the ATM protein in mediating the effects of cellular exposure to ionizing radiation and other forms of redox stress. Proteins such as the product of the tumour suppressor gene p53 and the proto-oncogene c-Abl (a protein tyrosine kinase) have been shown to interact with ATM. Since several intermediate steps in both the p53 and c-Abl pathways, activated by ionizing radiation, are known it will be possible to map the position of ATM in these pathways and describe its mechanism of action. What are the clinical implications of understanding the molecular basis of the defect in ataxia telangiectasia (A-T)? As outlined above, since radiosensitivity is a universal characteristic of A-T, understanding the mechanism of action of ATM will provide additional information on radiation signalling in human cells. With this information it may be possible to sensitize tumour cells to radiation and thus increase the therapeutic benefit of radiotherapy. This might involve the use of small molecules that would interfere with the normal ATM-controlled pathways and thus sensitize cells to radiation or alternatively it might involve the efficient introduction of ATM anti-sense cDNA constructs into tumours to achieve the same end-point.

[Genetic predisposition and radiation sensitivity of normal tissue]. / Genetische Prädisposition und Strahlenempfindlichkeit bei normalen Geweben.

BACKGROUND: After radiotherapy there are always some patients who develop strong acute and late reactions in normal tissues. In these patients frequently a genetic predisposition is observed. There are found DNA-repair deficiencies and changes in the regulation of the cell cycle which are responsible for the increased radiosensitivity with enhanced cell killing. METHODS: The micronucleus test and the comet assay appear to be appropriate tests in order to measure this increased radiosensitivity. Both tests are characterized by being relatively quick and simple and can be performed with small cell numbers. It is possible to study blood lymphocytes and fibroblasts with these tests. RESULTS: Both tests can predict the radiosensitivity of normal tissues especially if they are applied in combination. CONCLUSIONS: Epidemiological studies with patients after radiotherapy show evidence that the increased radiosensitivity also causes an enhanced induction of secondary tumors by ionizing radiation. This is supported by corresponding animal models.