Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells

Abstract

Following prolonged mitotic spindle disruption by microtubule poisons, mammalian cells delay their entry into anaphase, then progressively slip out of mitosis and become tetraploid. Normal cells then stop cycling before S-phase onset, but the mechanisms underlying this arrest are still unclear. Here we show that a double block prevents endo-reduplication. First, cells that exit mitosis without a functional microtubule network are driven toward G0. Reconstitution of the network unmasks a second block that relies on DNA double-strand breaks occurring early in the G1 phase that follows the mitotic block. We propose that a stress signal elicited upon mitotic impairment triggers breakage, which couples the leaky spindle checkpoint to the stringent DNA damage response. Consistent with this finding, cells defective for the damage response continue cycling and acquire, within a single cell cycle, both chromosome rearrangements and abnormal chromosome numbers that remarkably mimic the complex genetic hallmark of tumorigenesis.

Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle

“…However, γH2A.X was never found in cells with high cyclin B1, showing absence of DSBs in G2 phase. These data indicate that DNA damage is generated during mitosis, in agreement with break formation after spindle disruption (10,11) and the mitotic origin of micronuclei (21). The lack of γH2A.X in G2 cells suggests that damage is eliminated during S phase.…”

“…rarely found in sporadic tumors (5,7,8). In contrast, several lines of evidence implicate the mitotic spindle in DSB generation; genetic or drug-induced spindle abnormalities cause γH2A.X accumulation (10,11), γH2A.X levels in untreated cells correlate with numerical chromosome instability (9), and cancer cell lines with a CIN phenotype show numerous translocations close to centromeric regions (34).…”

“…Spindle disruption not only generates micronuclei (21), but also causes kinetochore distortion and DNA damage (10,11,29), suggesting a common mechanism. The individual micronuclei in Dido mutant MEFs, positive for γH2A.X and distorted centromeres, corroborated this hypothesis.…”

“…Spindle defects, an important cause of aneuploidy (1), are frequent in solid tumors, and background γH2A.X levels correlate well with aneuploidy (9). γH2A.X has also been observed after the induction of mitotic arrest by spindle disruption (10,11). When combined with chromosome end-to-end fusions, the mitotic spindle can damage chromosomes by a mechanism termed the breakage-fusion-bridge (BFB) cycle (12,13).…”

Consequences of mitotic slippage for antimicrotubule drug therapy

“…This was attributed to partial activation of caspase activity during a prolonged mitotic arrest. This rendered DNA fragmentation and a p53-mediated DNA damage response in post-slippage cells (Quignon et al 2007, Orth et al 2012. Work from the Shi Lab further showed that the DNA damage response pathway was primarily responsible for post-slippage apoptosis in a p53-dependent manner (Zhu et al 2014).…”