Distinct mechanisms act in concert to mediate cell cycle arrest

Abstract

In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.DNA damage ͉ dynamics ͉ mathematical model ͉ p53 ͉ cyclins O ne goal of systems biology is to quantitatively understand the dynamics of signaling pathways. As mathematical models of individual pathways emerge, we are challenged to interconnect them into a detailed understanding of how different pathways control the processing of information within the cell. The networks controlling cell cycle progression and the response to DNA damage are natural choices for such an integrative study. Each has been individually modeled successfully, and a great deal is understood about how specific interactions and regulation affect the dynamics of each network. However, in the absence of an extended model bridging these two pathways, the quantitative interaction between them remains undescribed. Here we develop a computational model of the combined networks and use it together with experimental measurements to determine the relative contribution and specific function of different cell cycle arrest mechanisms in response to DNA damage.During the cell cycle, mammalian cells coordinate cell growth, genome replication, and division. Two irreversible events subdivide the cell cycle into distinct phases: the onset of DNA replication defines S phase; and cell division defines M phase. Cells grow and carry out additional functions during the gap phases G1 and G2. The changing activity states of cyclindependent kinases (Cdks) regulate the transition between different stages of the cell cycle (1). Cyclin D/Cdk4 and -6 and cycli...

Chk1 is dispensable for G2 arrest in response to sustained DNA damage when the ATM/p53/p21 pathway is functional

“…According to the currently accepted model (Lobrich and Jeggo, 2007;Chen and Poon, 2008;Reinhardt and Yaffe, 2009;Toettcher et al, 2009), DNA damageinduced G2 arrest and inactivation of the key mitotic regulator, cyclin B1-Cdk1, requires action of two checkpoint kinases, Chk1 and Chk2. Although the role of Chk2 in G2 arrest has been contested (Ahn et al, 2003;Jallepalli et al, 2003;Jin et al, 2008), an everincreasing family of Chk1 targets (Dai and Grant, 2010) reinforces the notion that Chk1 is the major player of the DNA damage checkpoint and hence a promising target in combined anticancer therapy (Merry et al, 2010).…”

“…By associating with cyclin B1-Cdk1 and sequestering it in the nucleus, p21 prevents Cdk1 activation either by CAK (Smits et al, 2000) or Cdc25 (Charrier-Savournin et al, 2004) in the cytoplasm (Lindqvist et al, 2005;Gavet and Pines, 2010), thus participating directly in G2 arrest (Figure 8). Moreover, as in the case of sustained or unrepaired DNA damage, permanent Cdk1 inactivation by p21 would preclude either the resumption of cell cycle progression into mitosis, if the signaling is turned off (checkpoint silencing or adaptation; (Deckbar et al, 2007;Jurvansuu et al, 2007;van Vugt and Yaffe, 2010)), or endoreplication, if the checkpoint signaling persists (Toettcher et al, 2009;Davoli et al, 2010). Finally, in addition to contributing to the maintenance of G2 arrest (Bunz et al, 1998;Chan et al, 2000), we propose that p21-mediated Cdk1 inhibition together with pRb phosphorylation block, may be a prerequisite for senescent-like cell cycle exit in G2 (Baus et al, 2003).…”

“…Its inactivation or, rather, prevention of its activation is a conditio sine qua non to delay or block G2/M progression in the case of genotoxic stress. According to the current paradigm, this is achieved by ATM/ATR-activated Chk1 and Chk2 kinases that, by inhibiting the Cdc25 phosphatase family, maintain cyclin B1-Cdk1 in an inactive cytoplasmic state (Bartek and Lukas, 2003;Lobrich and Jeggo, 2007;Chen and Poon, 2008;Toettcher et al, 2009). However, several studies have questioned the role of Chk2, implying that Chk1 might be the major G2/M checkpoint regulator (Ahn et al, 2003;Jallepalli et al, 2003;Syljuasen et al, 2006;Jin et al, 2008;Zhang et al, 2008).…”

Senescence from G2 arrest, revisited

“…A model originally proposed by the Lahav group suggests that cells stably blocked in G2 eventually undergo mitotic bypass -a passage from G2 to G1 without chromosome segregation -and enter a tetraploid G1 phase where they cannot replicate due to p21-mediated inhibition of cyclin E1-Cdk2. 89 The absence of mitotic regulators in these 4N cells could be explained by both their degradation and p53/pRbmediated repression (Fig. 2).…”

Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression

“…As a consequence, cells arrest in G2, with high levels of p53 and inactive Cyclin B-Cdk1 and Cyclin A-Cdk1/2 complexes (Smits et al, 2000;Bassermann et al, 2008;Toettcher et al, 2009). In addition, the checkpoint can reduce the transcription of a large number of G2 genes through the actions of p53 and Chk1 (Taylor and Stark, 2001;Tabach et al, 2005;Spurgers et al, 2006;Jurvansuu et al, 2007;Shimada et al, 2008).…”