Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

Abstract

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

“…10 During checkpoint adaptation, cells enter mitosis despite having damaged DNA. 11,12 In these cells, the activity of Chk1 decreases, thus permitting the activation of Cdk1 followed by entry into mitosis damaged DNA.…”

“…Although most cells that undergo checkpoint adaptation die, a small yet biologically significant number of cells survive. 10,12 Because these cells have undergone mitosis with damaged DNA, it is likely they acquire changes to their genome. 13 One genomic change that characterizes cancer cells is the presence of micronuclei.…”

When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage

“…Since the process of adaptation has been predominantly studied in the context of DNA doublestrand breaks (DSBs) (see below), we will limit our overview to the cellular responses induced by this type of DNA lesion. However, it is important to keep in mind that adaptation can occur in response to a wide variety of genotoxic lesions (Kubara et al 2012;Syljuasen et al 2006;Yoo et al 2004). For a detailed discussion on checkpoint responses and DNA repair pathways, we direct readers to several recent reviews (Chapman et al 2012;Huertas 2010;Jackson and Bartek 2009;Moynahan and Jasin 2010;Panier and Durocher 2013).…”

“…Typical agents used in adaptation studies include DNA replication-inhibiting drugs, such as aphidicolin (Yoo et al 2004), topoisomerase I inhibitors, such as camptothecin (Kubara et al 2012), or non-specific DNA-damaging agents, such ionizing radiation (Syljuasen et al 2006). These agents are used at doses that would induce enough DNA damage to exceed (at least momentarily) the DNA repair capacity of cells, but not grossly so, in order to avoid the induction of immediate cell death.…”

“…The goal of both systems described above is to create a situation whereby cells could either maintain a checkpoint-induced cell cycle arrest, or take the decision to re-enter a proliferative state despite the presence of significant levels of DNA damage in their genome. This is usually detected by monitoring cell morphology (e.g., yeast budding or microcolony formation on solid medium; Lee et al 1998;Sandell and Zakian 1993;Toczyski et al 1997) or the appearance of changes in cell cycle status (e.g., passage from interphase to mitosis, completion of DNA replication, appearance of chromosome condensation; Kubara et al 2012;Syljuasen et al 2006;Yoo et al 2004). At the molecular level, adaptation is also associated with silencing of the DNA damage signaling machinery (e.g., dephosphorylation of checkpoint effectors; Donnianni et al 2010;Pellicioli et al 2001;Vidanes et al 2010), while markers of DNA damage persist.…”