During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication‐mediated DNA double‐strand breaks (DSBs). Ataxia‐telangiectasia mutated and Rad3‐related kinase (ATR) and DNA‐dependent protein kinase (DNA‐PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA‐PK‐dependent manner. To distinguish the roles of phosphatidylinositol 3‐kinase‐related protein kinases including DNA‐PK, ataxia‐telangiectasia mutated (ATM), and ATR, in the response to replication‐mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock‐down with siRNA suppressed CPT‐induced RPA2 hyperphosphorylation and focus formation. CPT‐induced RPA2 focus formation was normally observed in DNA‐PK‐ or ATM‐deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA‐PK‐dependent in CPT‐treated cells and DNA‐PK‐independent in HU‐treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU‐treated cells occurred later than in the CPT‐treated cells, indicating that the DNA‐PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA‐PK is responsible for the RPA2 hyperphosphorylation following ATR‐dependent RPA2 focus formation in response to replication‐mediated DSBs directly induced by CPT.