Abnormal Nuclear Structures are a Major Determinant of Proliferation Capacity Following Irradiation

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Ionizing radiation is effective at stopping cancer growth, but what ultimately kills or permanently arrests a cell has remained unknown. In addition to causing direct DNA damage, ionizing radiation results in formation of abnormal nuclear structures (micronuclei, chromosome bridges and primary nuclear abnormalities) following progression through mitosis. We and others had shown that formation of these abnormal nuclear structures leads to a large number of chromosome rearrangements specifically on the involved chromosome within a single cell cycle. We hypothesized that rather than direct DNA damage from radiation itself, rapid karyotype evolution following formation of abnormal nuclear structures may be a major determinant of cell’s proliferation capacity following exposure to ionizing radiation. We identified a marker called BAF that allows simultaneous detection of multiple abnormal nuclear structures (micronuclei, chromosome bridges and primary nuclear ruptures) by live cell imaging. Single cells were treated with a 2, 4, 6, 8 and 12 Gy fractions of ionizing radiation using gamma irradiator and followed by live cell imaging over several days monitoring for formation of abnormal nuclear structures. Their proliferation capacity was then determined by monitoring clone formation over 2 weeks. Individual cells from permanently arrested colonies were isolated and subjected to single-cell whole genome sequencing to assess for genetic causes of the arrest. As expected, abnormal nuclear structures formed at high frequency following progression through mitosis. There was a strong correlation between increasing dose and increased frequency of all abnormal nuclear structure types. Mitotic slippage resulting in genome fragmentation into multiple nuclei (“bunch-of-grapes” phenotype) was observed only starting at 4 Gy. p53 status was a major factor influencing the frequency of abnormal nuclear structure formation, while cell cycle stage at the time of irradiation was less important. Among cells that formed abnormal nuclear structures, none grew into colonies above 80 cells. 10/12 (83%) underwent several divisions but then arrested < 10 cells. Among irradiated cells that did not form nuclear structures after mitosis, 9/15 (60%) grew into large colonies (>1000 cells) and 6/15 (60%) arrested at 10-50 cell stage. Single-cell sequencing allowed us to determine specific genetic features responsible for the loss of proliferation capacity. Our results show that formation of abnormal nuclear structures is a major determinant of cells proliferation capacity following exposure to ionizing radiation. Combinatorial treatments aimed at enhancing formation of abnormal nuclear structures may thus be an effective therapeutic strategy.