p53-dependent polyploidisation after DNA damage in G2 phase

Abstract

9Cell cycle progression in the presence of damaged DNA can lead to accumulation of mutations 10 and pose a risk for tumour development. In response to DNA damage in G2 phase, human 11 cells can be forced to exit the cell cycle in a p53-p21-and APC/C Cdh1 -dependent manner. Cells 12 that exit the cell cycle in G2 phase become senescent, but it is unclear what determines this 13 commitment and whether other cell fates occur. We find that a subset of immortalised RPE-1 14 cells and primary human fibroblasts spontaneously initiate DNA re-replication several days 15 after forced cell cycle exit in G2 phase. By combining single cell tracking for more than a week 16 with quantitative immunofluorescence, we find that the resulting polyploid cells contain 17 increased levels of damaged DNA and frequently exit the cell cycle again in the next G2 phase. 18Subsequently, these cells either enter senescence or commit to another round of DNA re-19 replication, further increasing the ploidy. At least a subset of the polyploid cells show abnormal 20 centrosome numbers or localisation. In conclusion, cells that are forced to exit the cell cycle in 21 G2 phase face multiple choices that lead to various phenotypes, including propagation of cells 22 with different ploidies. Our findings suggest a mechanism by which p53-positive cells can 23 evade senescence that risks genome integrity. 24 25 Main points 26 -Cell cycle exit from G2 phase does not necessarily lead to senescence 27 28 -Resumption of proliferation after G2 phase cell cycle exit starts with DNA replication 29 30 -Successive cell cycle exits lead to propagation of cells with different ploidies 31 Introduction 35Cell cycle progression in the presence of damaged DNA can lead to propagation of mutations. 36In response to damaged DNA, cells pause the cell cycle, in particular prior to initiating DNA 37 replication or cell division. This pause, termed a checkpoint, is poorly sustained over time. In 38 case of excessive or sustained DNA damage, cells therefore permanently block proliferation, 39 either by regulated cell death, or by entering into senescence. In G2 phase, the road to 40 senescence is initiated by p53-and p21-dependent activation of APC/C Cdh1 1-5 . Activation of 41 APC/C Cdh1 leads to degradation of many key regulators of G2 phase and mitosis, thereby re-42 setting the cell cycle. Whereas we have referred to this phenomenon as cell cycle exit 6 , others 43 described it as cell cycle withdrawal 7 or mitosis skip 8 . Common to all is the description that this 44 is the first irreversible step leading to senescence from G2 phase. While it can be modulated 45 by checkpoint duration 8 , efficiency of DNA repair 9 , and CDK activity in G2 phase 10 , the 46 irreversible step rendering cell cycle exit independent of upstream checkpoint kinase signalling 47 is marked by translocation of Cyclin B1 to the nucleus 6-8 . 48 49 While cell cycle exit occurs rapidly in single cells, establishment of senescence is a gradual 50 process involving large changes in gene expression, c...

AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest and Polyploidization

“…Here, we provide evidence that AXL knock-out alone is sufficient to block the survival of cancer cells by a mechanism associating G2 arrest with polyploidization (Figures 3, 5 and 6). G2 arrest and polyploidization have been observed in p53-deficient cancer cells following exposure to DNA damaging agents [25], although this has also been reported for p53-positive cells [40]. As SNU475 cell line displays multiple mutations, including p53 mutations [20,26], it will be interesting to further explore the mechanisms of polyploidization in these cells.…”