Enhanced Frequency of Sister Chromatid Exchanges Induced By Diepoxybutane Is Specific Characteristic of Fanconi Anemia Cellular Phenotype

Fanconi anemia (FA) is a rare genetically heterogeneous disease characterized by developmental abnormalities, progressive bone marrow failure, and cancer susceptibility. We examined spontaneous, diepoxybutane (DEB)-induced and radiation-induced sister chromatid exchanges (SCEs) in wholeblood lymphocyte cultures of bone marrow failure (BMF) patients including Fanconi anemia, mothers of affected individuals, and healthy controls. The baseline frequency of SCE in FA cells was similar to that observed in controls. However, in response to DEB SCE frequencies in FA patients and their mothers were significantly increased compared to both non-FA BMF families and healthy controls. In response to ionizing radiation, cells displayed increased frequency of SCE, but no differences between FA patients and non-FA BMF patients were seen. Our data confirm and expand previous findings by showing that SCE induced by DEB can be used as an adjunct diagnostic test not only for FA patients, but also for female heterozygous carriers, at least for complementation groups FANCA and FANCD2

Radiation-induced mitotic catastrophe in FANCD2 primary fibroblasts

Purpose: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells. Materials and methods: Analyses of phosphorylated histone H2AX (gamma-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h). Results: The accumulation of gamma-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were gamma-H2AX foci-positive, significantly higher than in the control (p LT 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were gamma-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as gamma-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis. Conclusion: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation

Influence of Catalase on the Radiosensitivity of Fanconi Anemia Lymphocytes in Vitro

Fanconi anemia (FA) is a genetic disease characterized by progressive pancytopenia and cancer susceptibility. The clinical and cellular phenotypes of Fanconi anemia are associated with a set of redox abnormalities, indicating that FA is an oxidative stress-related disorder. Fanconi anemia cells are highly sensitive to DNA clastogen agents, but their response to ionizing radiation is still unclear. The aim of this study was to evaluate the in vitro radiosensitivity of Fanconi anemia homozygotes and heterozygotes, and to assess the contribution of catalase and superoxide dismutase (SOD) to the overall radiobiological response of the cells. The incidence of radiation-induced lymphocyte micronuclei was used as the indicator of radiation sensitivity in vitro, whereas the activity of antioxidant enzymes was determined in erythrocytes. Patients with FA exhibited a two-fold decrease in catalase activity, accompanied by lowered activity of SOD, and increased incidence of baseline micronuclei. In the entire group of patients (with one exception), a reduced yield of radiation-induced micronuclei in lymphocytes was observed, and this was categorized as a radioresistant response. A mild radioresistant in vitro response was also observed in carrier-mothers, accompanied by reduced activity of catalase. The radiosensitivity of carrier-fathers was normal. The results of this study suggest that reduced activity of catalase is an important contributor to the radiobiological response of cells

Comet assay and cytogenetic findings in differential diagnosis of fanconi anemia

Fanconi anemia (FA) is a complex genetic disease with a variety of congenital and hematological symptoms, including the predisposition for cancer development. The main hallmark of FA cells, an increased chromosomal fragility, in the presence of the DNA-interstrand cross-linking chemicals, mitomycin C or diepoxybutane (DEB), makes the diagnosis of FA much easier. Cytogenetic method can detect the FA patients with highly elevated chromosomal breakage, but also some of the patients with borderline sensitivity to DEB no matter if they have FA or not. These particular circumstances lead us to introduce comet assay along with cytogenetic analysis, in order to determine DNA lesions and chromosomal fragility in untreated and DEB-treated lymphocytes of full blood from seven patients with clinical features of FA. Highly elevated DEB induced chromosomal sensitivity confirmed the diagnosis in five patients (FA group: 0.48-4.47 breaks/cell vs control group: 0.00-0.08 breaks/cell). Borderline DEB sensitivity (FA* group: 0.15-0.44 breaks/cell) was found in the remaining two patients. Results of the comet assay showed higher baseline and DEB-induced DNA damage values (Olive tail moment and tail intensity) in all five FA and one FA* patient, when compared to the control group. These findings could provide a new model of FA screening test algorithm, including comet assay as additional and very useful accurate tool, beside the DEB test, in differential diagnosis of FA

Fanconi Anemia Is Characterized by Delayed Repair Kinetics of DNA Double-Strand Breaks

Among patients with bone marrow failure (BMF) syndrome, some are happened to have underlying Fanconi anemia (FA), a genetically heterogeneous disease, which is characterized by progressive pancytopenia and cancer susceptibility. Due to heterogeneous nature of the disease, a single genetic test, as in vitro response to DNA cross-linking agents, usually is not enough to make correct diagnosis. The aim of this study was to evaluate whether measuring repair kinetics of radiation-induced DNA double-strand breaks (DSBs) can distinguish Fanconi anemia from other BMF patients. An early step in repair of DSBs is phosphorylation of the histone H2AX, generating gamma-H2AX histone, which extends over mega base-pair regions of DNA from the break site and is visualised as foci (gamma-H2AX foci) with specific antibodies. The primary fibroblasts, established from FA patients, were exposed to gamma-rays, a dose of 2 Gy (Co-60), incubated for up to 24 hours under repair-permissive conditions, and assayed for the level of gamma-H2AX foci and apoptosis at different recovery times after the treatment. Cell lines originating from FA patients displayed a significant delay in the repair of radiation-induced DNA DSBs relative to non-FA bone marrow failure (non-FA BMF) and control cell lines. The delay is especially evident at recovery time of 24 hours, and is seen as about 8-fold increase of residual gamma-H2AX foci compared to self-state before irradiation. The delay in repair kinetics of FA cells represents the unique feature of FA cellular phenotype, which should be exploited to distinguish FA cellular phenotype

Leukocyte apoptosis as a predictor of radiosensitivity in Fanconi anemia

Fanconi anemia (FA) is a rare cancer-prone genetic disease characterized by impaired oxygen metabolism and defects in DNA damage repair. Response of FA cells to ionizing radiation has been an issue intensively debated in the literature. To study in vitro radiosensitivity in patients suffering from FA and their parents (heterozygous carriers), we determined radiation-induced leukocyte apoptosis using flow cytometry. As TP53 gene is involved in the control of apoptosis, we studied its status in FA lymphocytes using dual colour fluorescence in situ hybridization (FISH). FA patients and female heterozygous carriers display radiosensitive response to ionizing radiation seen as abnormal elimination of cells via apoptosis. By employment of FISH, the TP53 allele loss in FA lymphocytes was not observed. In diseases related to oxidative stress, determination of radiation-induced apoptosis is the method of choice for testing the radiosensitivity