DNA Damage Follows Repair Factor Depletion and Portends Genome Variation in Cancer Cells after Pore Migration

Abstract

Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1 – a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSC) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic ‘comet’ measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes.

Mitotic progression following DNA damage enables pattern recognition within micronuclei

“…When MCF10A cells were subjected to migration through 3µm pores we observed cGAS localization to both micronuclei and nuclear blebs consistent with nuclear envelop disruption and transient DSB production (Extended data 4d and 4e) 20 . cGAS positivity of these subcellular structures still occurred in the presence of CDKi, demonstrating that mechanical nuclear rupture bypasses the need for mitotic progression.…”

Rebuilding Chromosomes After Catastrophe: Emerging Mechanisms of Chromothripsis

“…During interphase, cGAS can be rapidly detected at sites of NE rupture at the nuclear periphery, which is subsequently followed by the focal acquisition of DNA damage [43, 44]. Similar rupturing events were reported to promote genomic copy number aberrations [48], and cGAS has been shown to associate with condensed chromosomes after NE breakdown during mitosis [49]. Consistent with this, recent work confirmed that cGAS could indeed sense micronuclear DNAs exposed to the cytoplasm following micro-NE disruption as cytosolic self-DNA [50], although the downstream consequences on the micronuclear chromosome itself are unknown.…”

Mathematically universal and biologically consistent astrocytoma genotype encodes for transformation and predicts survival phenotype

“…37–40 Note that in the absence of CDK inhibitors, DNA-damaged cells acquire deformed polyploid nuclei. 41,42 In response to Shh-mediated developmental signals, NOTCH1/2 facilitate the clearance of the tumor suppressor Ptch1, the concurrent accumulation of the Shh signal-transducing protein encoded by SMO , and the increased downstream conversion of the proteins encoded by the oncogenes GLI1/3 into cell cycle transcriptional activators. 43,44 Note that Notch is critical for an Shh-induced medulloblastoma brain cancer tumor’s development.…”