Please cite this article in press as: Krenning et al., Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.05.007
Molecular Cell
Article Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence Lenno Krenning,1 Femke M. Feringa,1 Indra A. Shaltiel,1 Jeroen van den Berg,1 and Rene´ H. Medema1,* 1Division of Cell Biology I and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.05.007
SUMMARY
DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/ CCdh1 (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/CCdh1 activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.
INTRODUCTION The cellular response to DNA double strand breaks involves the activation of the ATM and ATR checkpoint kinases, which promote DNA repair, transient cell-cycle arrest, as well as the induction of apoptosis or senescence (Abraham, 2001; Harper and Elledge, 2007). A central player in the induction and maintenance of cell-cycle arrest in response to DNA damage is the tumor suppressor protein p53. p53-dependent upregulation of the Cdk inhibitor p21 is essential to arrest cells in G1 after DNA et al., 1994; Waldman et al., 1995). In addition, damage (Dulic both p53 and p21 are required to maintain a G2 arrest (Bunz et al., 1998). In part, this is achieved through p53-mediated transcriptional repression of promitotic genes (Jackson et al., 2005; Taylor and Stark, 2001). Live-cell microscopy has shown that p53 levels oscillate in response to DNA damage and that the amount of oscillations is dependent on the amount of DNA damage (Lahav et al., 2004). The oscillatory pattern of p53 is dependent on the type of DNA damage present in the cell (Batchelor et al., 2011), and changing the oscillatory dynamics of p53 can readily influence
cell fate decisions (Purvis et al., 2012). p53 can promote a permanent cell-cycle arrest in response to prolonged exposure to DNA damage in G2 (Baus et al., 2003) and is required for the induction of senescence in response to continuous stimuli such as critically short telomeres or oncogene activation (Rufini et al., 2013). Increasing overall p53 activation results in a failure to sustain sufficient levels of promitotic genes, resulting in a loss of recovery competence in G2 cells (Lindqvist et al., 2009). This could set the stage for a permanent cell-cycle withdrawal. However, it remains unclear how cellular fate is controlled in response to DNA damage in individual cells at different stages of the cell cycle (Campisi and d’Adda di Fagagna, 2007). We therefore set out to study the effects of DNA damage on cell fate decisions in single cycling cells. RESULTS Limited Reversibility of a G2 Checkpoint Arrest To visualize cell fate decisions in individual cells following a DNA damaging insult, we took advantage of the Fucci system, which uses fluorescent proteins fused to the degradation motifs of Cdt1 and Geminin, to mark G1 and S/G2 cells, respectively (Figure S1A available online) (Sakaue-Sawano et al., 2008). As mutations in checkpoint components are common in transformed cells, we made use of nontransformed, immortalized retinal pigment epithelial cells (RPE-1), which have a functional G1 and G2 checkpoint in response to DNA damage (Figure S1B). Irradiation (IR) of RPE-Fucci cells with 4 Gy resulted in spontaneous recovery of approximately 50% of the G1 cells in the 70 hr following the damage, determined by S phase entry (Figure 1A, yellow line). In contrast, only 25% of the G2 cells spontaneously recovered from this insult (Figure 1B, yellow line). The other 75% of the G2 cells did not recover, but lost expression of Geminin and acquired a G1-like state without proceeding through mitosis (Figure S1C). Interestingly, all of the G2 cells that recovered during the course of the experiment did so within the first 24 hr after DNA damage, whereas damaged G1 cells continued to recover throughout the experiment (Figures 1A and 1B, yellow lines). This implies that cell fate in the G2 cell is determined within 1 day after DNA damage, while the damageinduced cell-cycle arrest remains reversible for much longer in G1 cells. Indeed, we found that checkpoint silencing at 24 hr after the damaging insult failed to promote cell-cycle reentry of G2-arrested cells (Figure 1B, blue line), while it did cause all of the cells arrested in G1 to reenter the cell cycle (Figure 1A, blue line). Addition of checkpoint inhibitors immediately following Molecular Cell 55, 1–14, July 3, 2014 ª2014 Elsevier Inc. 1
Please cite this article in press as: Krenning et al., Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.05.007
Molecular Cell Transient p53 Activation Induces Senescence in G2
A
B
C
E
D
F
G
Figure 1. Limited Reversibility of a G2 Checkpoint Arrest (A) RPE-Fucci cells were subjected to 4 Gy IR and subsequently imaged every 30 min for 70 hr. Checkpoint inhibitors were added at different time-points following IR, and progression from G1 into S phase was monitored. Cells entering S phase within the first 4 hr after IR were excluded from analysis, as they have already passed the G1 restriction point at the time of IR. A representative experiment is shown. (B) Same as in (A), except progression from G2 into mitosis was analyzed. Mitotic cells at the moment of IR were excluded. A representative experiment is shown. (C) Irradiated RPE-Fucci cells in G2 were isolated using FACS. The G2 cells were replated, and colony formation and b-gal were analyzed 6 days later. n = 3; representative experiment is shown. (D–G) RPE-Fucci cells were subjected to 10 Gy IR, imaged every hour for 6 days, and subsequently stained for b-gal. Numbered cells in (D) were in G2 at the moment of IR. (E) Representative images of cells indicated in (D). (F) Quantification of irradiated G2 cells that arrest for 6 days and become b-gal positive. Means ±SD; n = 2. (G) Representative images of control and 10 Gy irradiated cells at 6 days after IR stained for b-gal.
IR caused all G1 and G2 cells to enter the next cell-cycle phase, indicative of efficient checkpoint silencing upon the addition of inhibitors (Figures 1A and 1B, red lines). Similar results were obtained when damaging RPE-Fucci cells with a lower dose of IR (Figures S1D and S1E) and in BJ-Tert cells (another nontransformed, immortalized cell line) expressing the Fucci-system (BJFucci) (Figures S1F and S1G). Collectively, these results show 2 Molecular Cell 55, 1–14, July 3, 2014 ª2014 Elsevier Inc.
that reversibility of the checkpoint response in G2 cells is restricted to a much shorter time period following DNA damage when compared to G1 cells. To investigate if the prolonged cell-cycle arrest observed in the G2 cells results in a permanent cell-cycle exit, we analyzed the cell-cycle distribution of RPE-1 cells up to 10 days post IR. IR resulted in a marked increase in the 4N fraction, and this fraction
Please cite this article in press as: Krenning et al., Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.05.007
Molecular Cell Transient p53 Activation Induces Senescence in G2
remained constant over time (Figure S1H). To investigate if these cells undergo senescence, we irradiated RPE-Fucci cells using 2 or 4 Gy and subsequently isolated the cells in G2 (expressing green fluorescent mAG-Geminin) by fluorescence-activated cell sorting (FACS). Six days later, we determined the survival of these cells using a colony formation and additionally stained for senescence-associated b-galactosidase (b-gal) (Figure 1C), a widely used marker for cellular senescence (Campisi and d’Adda di Fagagna, 2007). IR of G2 cells with 2 or 4 Gy severely reduced cell growth compared to nonirradiated G2 cells (Figure 1C). Strikingly, irradiated G2 cells show a marked increase in the amount of b-gal-positive cells 6 days after IR (Figure 1C). Using time-lapse analysis, we could confirm that the majority of irradiated G2 cells failed to enter mitosis for up to 6 days and entered senescence from their G2-arrested state (Figures 1D–1G). Collectively, these data show that the decision to enter senescence is established within 24 hr in a damaged G2 cell. To determine the exact timing of this decision, we damaged asynchronously growing RPE-1 cells by IR or treated cells with the topoisomerase-II inhibitor etoposide. To determine the fate of cells damaged in G2, BrdU was added to be able to exclude cells damaged in S phase. Subsequently, the checkpoint was silenced at different time points following DNA damage by the addition of caffeine (Figure 2A). G2 recovery was analyzed as the percentage of 4N BrdU-negative cells that were positive for the mitosis-specific phosphorylation of histone H3 on serine 10 (Figure 2B). We observed that, relative to nonirradiated control cells, approximately 15% of the G2 cells recover spontaneously from 5 Gy IR (Figure 2C, no caffeine). Checkpoint silencing at 1 hr after IR results in near complete recovery, but the ability to induce checkpoint recovery in G2 cells decreases rapidly over time and is almost completely lost 5 hr after IR (Figure 2C). We observed a similar decrease in checkpoint recovery over time in both RPE-1 and BJ-Tert cells after treatment with etoposide (Figures 2D and S2), although the time at which the response becomes irreversible is somewhat delayed in BJ-Tert cells. Taken together, these results show that nontransformed cells progressively lose the ability to recover from a DNA-damageinduced G2 arrest within the first hours after the damage, causing them to enter a state of senescence. In contrast, G1 cells retain the capacity to reenter the cell cycle after DNA damage for a much longer period of time. Irreversible Withdrawal from the Cell Cycle in G2 Depends on p53 p53-mediated repression of mitotic regulators can result in the loss of recovery competence in transformed cell lines (Lindqvist et al., 2009). In addition, p53 is required for DNA-damageinduced senescence (Campisi and d’Adda di Fagagna, 2007). Therefore, we investigated whether the establishment of the irreversible G2 arrest observed in the RPE-1 cells is dependent on p53. We observed that recovery competence was fully sustained in the RPE-1 cells after p53 knockdown (kd) (Figure 2E, red bars). In fact, depletion of p53 caused the majority of G2 cells to spontaneously reenter the cell cycle after 5 Gy of IR, even without the addition of caffeine (Figure 2E). Although spontaneous recovery of p53 kd RPE-1 cells was hardly observed following 10 Gy of IR, the addition of caffeine, even at 4 hr after IR, was sufficient to
drive most of these cells back into the cell cycle (Figure 2E). Similarly, RPE-1 cells depleted of p53 fully retained their ability to reenter the cell cycle when damaged with etoposide (Figure 2F). Taken together, these results show that cell fate decisions after DNA damage in G2 are largely determined by p53. Nuclear Translocation and Degradation of Cyclin B1 Precedes Cell-Cycle Exit in G2 The observation that G2 recovery competence is tightly regulated by p53 prompted us to investigate how endogenous Cyclin B1 levels are controlled in response to DNA damage in G2. We therefore made use of RPE-1 cells in which a fluorescent tag was introduced in one allele of Cyclin B1 (RPE CCNB1YFP). Introduction of this fluorescent tag does not perturb Cyclin B1 function (Shaltiel et al., 2014). Similarly to G2 RPE-Fucci cells, IR of G2 RPE CCNB1YFP cells, isolated by FACS sorting, resulted in impaired outgrowth and the induction of senescence (Figure 3A). Additionally, we find that the RPE CCNB1YFP cells lose the ability to recover from a G2 arrest over time in a similar manner to wildtype RPE-1 cells (Figure S3A). Analysis of Cyclin B1 by timelapse fluorescence microscopy confirmed that Cyclin B1-YFP accumulates during G2, is degraded at the metaphase-toanaphase transition, and is predominantly localized in the cytoplasm until the beginning of mitosis, when it rapidly translocates to the nucleus just prior to nuclear envelope breakdown (Pines and Hunter, 1991) (Figures S3B and S3C). Interestingly, in response to DNA damage we could discriminate different patterns of Cyclin B1 behavior. In cells that failed to recover from the G2 arrest, we observed a near-complete translocation of Cyclin B1 to the nucleus 2–4 hr after IR followed by a rapid decrease in Cyclin B1 between 5–10 hr after IR (Figure 3B, upper panels). In contrast, cells that eventually reentered the cell cycle retained Cyclin B1 in the cytoplasm and maintained Cyclin B1 at high levels, only to translocate it to the nucleus immediately prior to the onset of mitosis (Figure 3B, lower panels). A minority (
Molecular Cell
Article Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence Lenno Krenning,1 Femke M. Feringa,1 Indra A. Shaltiel,1 Jeroen van den Berg,1 and Rene´ H. Medema1,* 1Division of Cell Biology I and Cancer Genomics Center, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.05.007
SUMMARY
DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/ CCdh1 (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/CCdh1 activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.
INTRODUCTION The cellular response to DNA double strand breaks involves the activation of the ATM and ATR checkpoint kinases, which promote DNA repair, transient cell-cycle arrest, as well as the induction of apoptosis or senescence (Abraham, 2001; Harper and Elledge, 2007). A central player in the induction and maintenance of cell-cycle arrest in response to DNA damage is the tumor suppressor protein p53. p53-dependent upregulation of the Cdk inhibitor p21 is essential to arrest cells in G1 after DNA et al., 1994; Waldman et al., 1995). In addition, damage (Dulic both p53 and p21 are required to maintain a G2 arrest (Bunz et al., 1998). In part, this is achieved through p53-mediated transcriptional repression of promitotic genes (Jackson et al., 2005; Taylor and Stark, 2001). Live-cell microscopy has shown that p53 levels oscillate in response to DNA damage and that the amount of oscillations is dependent on the amount of DNA damage (Lahav et al., 2004). The oscillatory pattern of p53 is dependent on the type of DNA damage present in the cell (Batchelor et al., 2011), and changing the oscillatory dynamics of p53 can readily influence
cell fate decisions (Purvis et al., 2012). p53 can promote a permanent cell-cycle arrest in response to prolonged exposure to DNA damage in G2 (Baus et al., 2003) and is required for the induction of senescence in response to continuous stimuli such as critically short telomeres or oncogene activation (Rufini et al., 2013). Increasing overall p53 activation results in a failure to sustain sufficient levels of promitotic genes, resulting in a loss of recovery competence in G2 cells (Lindqvist et al., 2009). This could set the stage for a permanent cell-cycle withdrawal. However, it remains unclear how cellular fate is controlled in response to DNA damage in individual cells at different stages of the cell cycle (Campisi and d’Adda di Fagagna, 2007). We therefore set out to study the effects of DNA damage on cell fate decisions in single cycling cells. RESULTS Limited Reversibility of a G2 Checkpoint Arrest To visualize cell fate decisions in individual cells following a DNA damaging insult, we took advantage of the Fucci system, which uses fluorescent proteins fused to the degradation motifs of Cdt1 and Geminin, to mark G1 and S/G2 cells, respectively (Figure S1A available online) (Sakaue-Sawano et al., 2008). As mutations in checkpoint components are common in transformed cells, we made use of nontransformed, immortalized retinal pigment epithelial cells (RPE-1), which have a functional G1 and G2 checkpoint in response to DNA damage (Figure S1B). Irradiation (IR) of RPE-Fucci cells with 4 Gy resulted in spontaneous recovery of approximately 50% of the G1 cells in the 70 hr following the damage, determined by S phase entry (Figure 1A, yellow line). In contrast, only 25% of the G2 cells spontaneously recovered from this insult (Figure 1B, yellow line). The other 75% of the G2 cells did not recover, but lost expression of Geminin and acquired a G1-like state without proceeding through mitosis (Figure S1C). Interestingly, all of the G2 cells that recovered during the course of the experiment did so within the first 24 hr after DNA damage, whereas damaged G1 cells continued to recover throughout the experiment (Figures 1A and 1B, yellow lines). This implies that cell fate in the G2 cell is determined within 1 day after DNA damage, while the damageinduced cell-cycle arrest remains reversible for much longer in G1 cells. Indeed, we found that checkpoint silencing at 24 hr after the damaging insult failed to promote cell-cycle reentry of G2-arrested cells (Figure 1B, blue line), while it did cause all of the cells arrested in G1 to reenter the cell cycle (Figure 1A, blue line). Addition of checkpoint inhibitors immediately following Molecular Cell 55, 1–14, July 3, 2014 ª2014 Elsevier Inc. 1
Please cite this article in press as: Krenning et al., Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.05.007
Molecular Cell Transient p53 Activation Induces Senescence in G2
A
B
C
E
D
F
G
Figure 1. Limited Reversibility of a G2 Checkpoint Arrest (A) RPE-Fucci cells were subjected to 4 Gy IR and subsequently imaged every 30 min for 70 hr. Checkpoint inhibitors were added at different time-points following IR, and progression from G1 into S phase was monitored. Cells entering S phase within the first 4 hr after IR were excluded from analysis, as they have already passed the G1 restriction point at the time of IR. A representative experiment is shown. (B) Same as in (A), except progression from G2 into mitosis was analyzed. Mitotic cells at the moment of IR were excluded. A representative experiment is shown. (C) Irradiated RPE-Fucci cells in G2 were isolated using FACS. The G2 cells were replated, and colony formation and b-gal were analyzed 6 days later. n = 3; representative experiment is shown. (D–G) RPE-Fucci cells were subjected to 10 Gy IR, imaged every hour for 6 days, and subsequently stained for b-gal. Numbered cells in (D) were in G2 at the moment of IR. (E) Representative images of cells indicated in (D). (F) Quantification of irradiated G2 cells that arrest for 6 days and become b-gal positive. Means ±SD; n = 2. (G) Representative images of control and 10 Gy irradiated cells at 6 days after IR stained for b-gal.
IR caused all G1 and G2 cells to enter the next cell-cycle phase, indicative of efficient checkpoint silencing upon the addition of inhibitors (Figures 1A and 1B, red lines). Similar results were obtained when damaging RPE-Fucci cells with a lower dose of IR (Figures S1D and S1E) and in BJ-Tert cells (another nontransformed, immortalized cell line) expressing the Fucci-system (BJFucci) (Figures S1F and S1G). Collectively, these results show 2 Molecular Cell 55, 1–14, July 3, 2014 ª2014 Elsevier Inc.
that reversibility of the checkpoint response in G2 cells is restricted to a much shorter time period following DNA damage when compared to G1 cells. To investigate if the prolonged cell-cycle arrest observed in the G2 cells results in a permanent cell-cycle exit, we analyzed the cell-cycle distribution of RPE-1 cells up to 10 days post IR. IR resulted in a marked increase in the 4N fraction, and this fraction
Please cite this article in press as: Krenning et al., Transient Activation of p53 in G2 Phase Is Sufficient to Induce Senescence, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.05.007
Molecular Cell Transient p53 Activation Induces Senescence in G2
remained constant over time (Figure S1H). To investigate if these cells undergo senescence, we irradiated RPE-Fucci cells using 2 or 4 Gy and subsequently isolated the cells in G2 (expressing green fluorescent mAG-Geminin) by fluorescence-activated cell sorting (FACS). Six days later, we determined the survival of these cells using a colony formation and additionally stained for senescence-associated b-galactosidase (b-gal) (Figure 1C), a widely used marker for cellular senescence (Campisi and d’Adda di Fagagna, 2007). IR of G2 cells with 2 or 4 Gy severely reduced cell growth compared to nonirradiated G2 cells (Figure 1C). Strikingly, irradiated G2 cells show a marked increase in the amount of b-gal-positive cells 6 days after IR (Figure 1C). Using time-lapse analysis, we could confirm that the majority of irradiated G2 cells failed to enter mitosis for up to 6 days and entered senescence from their G2-arrested state (Figures 1D–1G). Collectively, these data show that the decision to enter senescence is established within 24 hr in a damaged G2 cell. To determine the exact timing of this decision, we damaged asynchronously growing RPE-1 cells by IR or treated cells with the topoisomerase-II inhibitor etoposide. To determine the fate of cells damaged in G2, BrdU was added to be able to exclude cells damaged in S phase. Subsequently, the checkpoint was silenced at different time points following DNA damage by the addition of caffeine (Figure 2A). G2 recovery was analyzed as the percentage of 4N BrdU-negative cells that were positive for the mitosis-specific phosphorylation of histone H3 on serine 10 (Figure 2B). We observed that, relative to nonirradiated control cells, approximately 15% of the G2 cells recover spontaneously from 5 Gy IR (Figure 2C, no caffeine). Checkpoint silencing at 1 hr after IR results in near complete recovery, but the ability to induce checkpoint recovery in G2 cells decreases rapidly over time and is almost completely lost 5 hr after IR (Figure 2C). We observed a similar decrease in checkpoint recovery over time in both RPE-1 and BJ-Tert cells after treatment with etoposide (Figures 2D and S2), although the time at which the response becomes irreversible is somewhat delayed in BJ-Tert cells. Taken together, these results show that nontransformed cells progressively lose the ability to recover from a DNA-damageinduced G2 arrest within the first hours after the damage, causing them to enter a state of senescence. In contrast, G1 cells retain the capacity to reenter the cell cycle after DNA damage for a much longer period of time. Irreversible Withdrawal from the Cell Cycle in G2 Depends on p53 p53-mediated repression of mitotic regulators can result in the loss of recovery competence in transformed cell lines (Lindqvist et al., 2009). In addition, p53 is required for DNA-damageinduced senescence (Campisi and d’Adda di Fagagna, 2007). Therefore, we investigated whether the establishment of the irreversible G2 arrest observed in the RPE-1 cells is dependent on p53. We observed that recovery competence was fully sustained in the RPE-1 cells after p53 knockdown (kd) (Figure 2E, red bars). In fact, depletion of p53 caused the majority of G2 cells to spontaneously reenter the cell cycle after 5 Gy of IR, even without the addition of caffeine (Figure 2E). Although spontaneous recovery of p53 kd RPE-1 cells was hardly observed following 10 Gy of IR, the addition of caffeine, even at 4 hr after IR, was sufficient to
drive most of these cells back into the cell cycle (Figure 2E). Similarly, RPE-1 cells depleted of p53 fully retained their ability to reenter the cell cycle when damaged with etoposide (Figure 2F). Taken together, these results show that cell fate decisions after DNA damage in G2 are largely determined by p53. Nuclear Translocation and Degradation of Cyclin B1 Precedes Cell-Cycle Exit in G2 The observation that G2 recovery competence is tightly regulated by p53 prompted us to investigate how endogenous Cyclin B1 levels are controlled in response to DNA damage in G2. We therefore made use of RPE-1 cells in which a fluorescent tag was introduced in one allele of Cyclin B1 (RPE CCNB1YFP). Introduction of this fluorescent tag does not perturb Cyclin B1 function (Shaltiel et al., 2014). Similarly to G2 RPE-Fucci cells, IR of G2 RPE CCNB1YFP cells, isolated by FACS sorting, resulted in impaired outgrowth and the induction of senescence (Figure 3A). Additionally, we find that the RPE CCNB1YFP cells lose the ability to recover from a G2 arrest over time in a similar manner to wildtype RPE-1 cells (Figure S3A). Analysis of Cyclin B1 by timelapse fluorescence microscopy confirmed that Cyclin B1-YFP accumulates during G2, is degraded at the metaphase-toanaphase transition, and is predominantly localized in the cytoplasm until the beginning of mitosis, when it rapidly translocates to the nucleus just prior to nuclear envelope breakdown (Pines and Hunter, 1991) (Figures S3B and S3C). Interestingly, in response to DNA damage we could discriminate different patterns of Cyclin B1 behavior. In cells that failed to recover from the G2 arrest, we observed a near-complete translocation of Cyclin B1 to the nucleus 2–4 hr after IR followed by a rapid decrease in Cyclin B1 between 5–10 hr after IR (Figure 3B, upper panels). In contrast, cells that eventually reentered the cell cycle retained Cyclin B1 in the cytoplasm and maintained Cyclin B1 at high levels, only to translocate it to the nucleus immediately prior to the onset of mitosis (Figure 3B, lower panels). A minority (
