Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells

Abstract

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

Cytokinesis Failure Triggers Hippo Tumor Suppressor Pathway Activation

“…Indeed, the restrained growth of tetraploid cells has long been recognized: It was first demonstrated in 1967 that inhibition of cytokinesis in non-transformed cells severely impairs the proliferation of the resulting binucleated tetraploids (Carter, 1967). Subsequently, it became clear that p53 is the key mediator of this arrest (Andreassen et al, 2001; Ganem and Pellman, 2007; Kuffer et al, 2013; Wright and Hayflick, 1972). However, the defect(s) that triggers this stress response, and the downstream signaling pathways that activate p53, remain key unresolved questions in cancer biology.…”

Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication

“…This pathway of proliferation restriction seems to be specific for loss of adhesion-dependent cytokinesis failure that may involve perturbation of integrin signaling. 23,37,38 Conversely, binucleated cells induced by the actin inhibitors cytochalasins or the myosin inhibitor blebbistatin have been found to arrest at the first or second mitosis from drug treatment through accumulation of p53/p21, 16,19,20 demonstrating that the pathways activated to arrest cell cycle progression after loss of substrate adhesion or inhibition of cytokinesis in adherence conditions are fundamentally distinct. Interestingly, recent work on primary rat embryonic and human fibroblasts demonstrates that non-transformed cells respond to drug-induced tetraploidy by entering senescence from a tetraploid G1 stage.…”

“…14,15 Nevertheless, tetraploids arising in untransformed cultures from mitotic slippage, cell fusion or cytokinesis failure induced by chemical treatment or depletion of proteins required for cytokinesis are usually limited in their proliferation by a p53-mediated pathway. 6,[16][17][18] Recent studies have linked p53 activation in tetraploids to the induction of oxidative stress leading to ATM activation at the first tetraploid mitosis 19 or to the activation of the tumor suppressor Hippo pathway. 20 However, it is still unclear whether the p53-dependent pathway restricting binucleated cell proliferation is inherent to the binucleation condition or other pathways may intervene, depending on the origin of cell binucleation.…”

The deregulated microRNAome contributes to the cellular response to aneuploidy

“…CCL-247). HCT116 H2B-GFP was generated previously by lipofection (FugeneHD, Roche) transfection of pBOS-H2B-GFP (BD Pharmingen) according to manufacturer's protocols [53]. The tetrasomic cell line HCT116 5/4 were a kind gift of Minoru Koi (Baylor University Medical Centre, Dallas, Texas, USA).…”