RESUMEN
Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.
Asunto(s)
Estructuras Cromosómicas/ultraestructura , Cromosomas de los Mamíferos/ultraestructura , Mitosis , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Cafeína/toxicidad , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de la radiación , Núcleo Celular/ultraestructura , Centrómero/efectos de los fármacos , Centrómero/efectos de la radiación , Centrómero/ultraestructura , Cromatina/efectos de los fármacos , Cromatina/efectos de la radiación , Cromatina/ultraestructura , Aberraciones Cromosómicas , Estructuras Cromosómicas/efectos de los fármacos , Estructuras Cromosómicas/efectos de la radiación , Cromosomas de los Mamíferos/efectos de los fármacos , Cromosomas de los Mamíferos/efectos de la radiación , Cricetinae , Cricetulus , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Fijadores/farmacología , Proteínas Luminiscentes/genética , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Fenotipo , Huso Acromático/efectos de los fármacos , Huso Acromático/efectos de la radiación , Huso Acromático/ultraestructura , Transfección , Rayos Ultravioleta , Proteína Fluorescente RojaRESUMEN
A simple model of homogenous chromatin distribution in HeLa-cell nuclei suggests that the track of an energetic ion hits 30 nm chromatin fibers with a mean distance of 0.55 mum. To test this assumption, living HeLa-cells were irradiated at the irradiation setup of the ion microprobe SNAKE using the ion beams provided by the Munich 14 MV tandem accelerator. After irradiation, the distribution of 53BP1 protein foci was studied by immunofluorescence. The observed 53BP1 distribution along the tracks of 29 MeV (7)Li ions and 24 MeV (12)C ions differed significantly from the expectations resulting from the simple chromatin model, suggesting that the biological track structure is determined by cell nuclear architecture with higher order organisation of chromatin.