After DNA damage, p21+/+ cells stably arrest in G2, whereas p21−/− cells ultimately progress into mitosis. We show here that DNA damage induces stable G2 arrest through down-regulation of Emi1 in p21+/+ cells but not in p21−/− cells. Down-regulation of Emi1 activates APC, which in turn leads to the degradation of APC substrates, including cyclins, in γ-irradiated p21+/+ cells. The loss of critical mitotic proteins blocks cells from proceeding into mitosis. By contrast, Emi1 protein levels are unchanged in γ-irradiated p21−/− cells, and APC remains inactive, causing the retention of APC substrates. Thus, irradiated p21−/− cells fail to sustain G2 arrest and progress into mitosis. We conclude that stable p21-dependent G2 arrest after DNA damage requires Emi1 down-regulation.
We confirmed the involvement of Emi1 down-regulation and APC activation in G2 arrest after DNA damage in two ways. First, we demonstrated that down-regulation of Cdh1 in irradiated p21+/+ cells overcame the G2 arrest and led to the progression of cells into mitosis. Second, we showed that Emi1 siRNA inhibits the entry of irradiated p21−/− cells into mitosis and that this inhibition can be overcome if Cdh1 is also down-regulated in the cells. Treatment of γ-irradiated p21−/− cells with the CDK inhibitor roscovitine, as well as down-regulation of CDKs with siRNA, leads to a decrease in Emi1 levels, activates the APC, and arrests cells in G2 phase.
We have examined whether APC-dependent substrate degradation in irradiated p21+/+
cells occurs in G2-arrested cells. Our criteria for G2 arrest were the near absence of the following: nuclear envelope breakdown, MPM2 staining, histone H3 phosphorylation, and condensed chromosomes. Although it is possible that a small fraction of irradiated p21+/+
cells proceed to mitosis, this fraction cannot account for the nearly complete degradation of APC substrates observed in irradiated p21+/+
cells. We therefore favor the conclusion that APC substrate degradation in irradiated p21+/+
cells occurs predominantly in G2-arrested cells. The entry of irradiated p21−/−
cells into and out of mitosis has been demonstrated convincingly by Bunz et al. (1998)
, and we have confirmed this finding.
There is a delay between Emi1 down-regulation and the degradation of APC substrates. It is possible that this delay in degradation of substrates may originate from the competing effects of deubiquitinating enzymes (DUBs) that rapidly remove ubiquitin from substrates and delay degradation (Amerik and Hochstrasser, 2004
) and activation of degradation through APC activity. For example, USP28 is a DUB that controls the abundance of several checkpoint proteins that otherwise become unstable in response to irradiation (Zhang et al., 2006
). DUBs might balance opposing APC E3 activity in DNA-damaged cells for a period of time until the balance of these opposing pathways changes in favor of APC. It is possible that this shift of balance does not occur in some cells (see Supplemental Figure S4E).
We have shown that Emi1 protein has a short half-life in cells released from S phase synchrony. This prompted us to test whether CDK inhibition in G2 might act to repress Emi1 mRNA after DNA damage. Indeed, Emi1 mRNA levels are dramatically reduced in γ-irradiated p21+/+
cells, consistent with the reduction of Emi1 protein after DNA damage. Although Emi1 mRNA levels remain relatively unchanged after irradiation of p21−/−
cells, CDK inhibition by roscovitine leads to the reduction of Emi1 mRNA to a level <0.2% of control irradiated p21−/−
cells. A role for Rb in regulating Emi1 transcript levels has been suggested from work in Rb, p107, p130 triple knockout mouse embryonic fibroblasts (Balciunaite et al., 2005
). Exogenous E2F1 or E2F3 were shown to induce Emi1 gene expression, whereas transfection with constitutively active Rb represses Emi1 gene expression (Hsu et al., 2002
). We observe significant stabilization of Emi1 mRNA after down-regulation of Rb in irradiated p21+/+
cells, thereby suggesting that DNA damage induced transcriptional repression of Emi1 in p21+/+
cells may be mediated in part through Rb. Further investigation using chromatin immunoprecipitation- and Emi1-specific PCR is in progress to address how the Emi1 promoter is regulated after DNA damage.
It is interesting that Emi1 decrease over the long term in nonirradiated and in irradiated cells has different consequences. Emi1 decrease in nonirradiated cells leads to rereplication (Di Fiore and Pines, 2007
), whereas we show that decrease in Emi1 in G2-irradiated cells results in long-term G2 arrest. Di Fiore and Pines show that although cyclins A2 and B are reduced, cyclin E is increased in Emi1 down-regulated cells, and this may be sufficient to drive rereplication. Our results show that cells respond differently to DNA damage. We have shown that Cdk2-associated kinase activity is decreased in irradiated p21+/+
cells. We also have found that cyclin E-associated kinase activity is inhibited after DNA damage in p21+/+
cells (data not shown). Most importantly, p21+/+
cells do not progress toward rereplication because the degradation of critical mitotic proteins prevents their progression to mitosis.
Normal cell division is critical for maintaining genome ploidy. p21−/−
HCT116 cells become polyploid after DNA damage because the cells enter mitosis, but fail to execute mitosis properly (Bunz et al., 1998
; Andreassen et al., 2001
). As a consequence, the cells exit mitosis and enter interphase with 4N DNA content. Our finding that APC substrates, such as securin and cyclin B, are not degraded in irradiated p21−/−
cells even after mitotic exit further explains the failure of the cells that enter M phase to execute mitosis properly, as degradation of APC substrates is necessary for separation of sister chromatids in anaphase and for correct mitotic exit to form two daughter cells (reviewed in Peters, 2002
The role of APC in the DNA damage checkpoint has not been extensively studied in mammalian cells. APC activity is not high in G2 phase (Fang et al., 1998
; Kramer et al., 1998
). Here we show that APC activation in G2 phase leads to stable arrest in irradiated p21+/+
cells. We show that down-regulation of Emi1 after DNA damage leads to APC activation. Emi1 can competitively inhibit the binding of substrates to Cdc20 and Cdh1 (Reimann et al., 2001b
). In addition, low CDK activity after DNA damage may facilitate activation of APCCdh1
, as CDK phosphorylated Cdh1 does not activate APC (Zachariae et al., 1998
; Jaspersen et al., 1999
; Lukas et al., 1999
; Kramer et al., 2000
). The ability of Cdh1-depleted cells to enter mitosis after DNA damage as reported here and by Sudo et al. (2001)
is intriguing given that CDK activity is inhibited after irradiation.
We further show that APC activation, which leads to degradation of APC substrates including cyclins, is p21-dependent. Sudo et al. (2001)
have shown APCCdh1
activation after DNA damage, but neither the mechanism of APC activation nor its physiological relevance were addressed. In their study, APC activation did not alter the levels of APC substrates examined. While this manuscript was in preparation, Bassermann et al. (2008)
reported that APCCdh1
targets Plk1 after DNA damage but not 13 other APC substrates tested, including cyclins. They proposed that there are two pools of APCCdh1
in G2 cells. One pool that targets substrates other than Plk1 is inactive both in the presence and absence of DNA damage. The other pool of APCCdh1
, which targets Plk1, is activated after DNA damage and controls the G2 checkpoint. In contrast to Sudo et al. (2001)
and Bassermann et al. (2008)
, we find that APC activation after DNA damage leads to the degradation of APC substrates, including cyclin A2 and B1. Our results further show that APC activation is observed late, between 15 and 24 h after irradiation, in contrast to the activation of APCcdh1
within hours of DNA damage, as reported in HeLa cells, and the early activation of a Plk1-specific APCCdh1
pool in U2OS cells. Although we also find that APC activation after DNA damage leads to Plk1 degradation (C, Supplemental Figure S8), this degradation occurs late, in keeping with the timing of APC activation in our study. In contrast to Basserman et al.
, we observe that the Plk1 protein levels and Plk1 activity are maintained up to 24 h after irradiation (Supplemental Figure S8B). A decrease in Emi1 and in APC substrates upon DNA damage is not cell type-specific, as we observed these effects in several human cell lines, depending on their p53 or p21 status. Additional work is required before issues, such as whether there are two pools of active APCCdh1
after DNA damage and whether Plk1 activity is a target of the early active APC, are resolved.
In summary, p21-dependent down-regulation of Emi1 in G2 phase after DNA damage plays an essential role in sustained G2 arrest and our work thus offers a new insight into the mechanism of p21-dependent cell cycle arrest after DNA damage.