Browsing by Author "Leskovac, Andreja (9133794800)"

Background: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. Results: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. Conclusion: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further. © 2012 joksic et al.; licensee BioMed Central Ltd.

Background: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. Results: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. Conclusion: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further. © 2012 joksic et al.; licensee BioMed Central Ltd.

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 (γ-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 γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 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 γ-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 γ-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. © 2014 Informa UK, Ltd.

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 (γ-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 γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 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 γ-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 γ-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. © 2014 Informa UK, Ltd.