Overexpression of Wild-Type Xenopus Chk2 (wt-Chk2) Triggers a Cell Cycle Checkpoint prior to the MBT
Prior to the MBT, Xenopus embryos do not arrest cell cycles in response to damaged or unreplicated DNA (Newport and Dasso, 1989; Anderson et al., 1997; Hensey and Gautier, 1997) even though Chk1 and Chk2 kinases are expressed (Nakajo et al., 1999; Kappas et al., 2000; Gotoh et al., 2001). Chk1 does not become activated by unreplicated DNA until the MBT or by ionizing radiation (IR) until early gastrulation (Kappas et al., 2000), correlating with the time at which embryos arrest cell cycles in response to unreplicated or damaged DNA. However, overexpression of Chk1 triggers degradation of Cdc25A, tyrosine phosphorylation of Cdks, and cell cycle arrest in pre-MBT embryos (Kappas et al., 2000; Shimuta et al., 2002; Petrus et al., 2004). When embryos are treated at the MBT with agents that block DNA replication, Chk2 likewise is activated (Gotoh et al., 2001).
To determine whether pre-MBT cell cycles respond to exogenous Chk2, mRNA encoding FLAG-tagged wild-type Chk2 (wt-Chk2) or FLAG-tagged luciferase (as a control) was microinjected into one-cell stage embryos. Western analysis with a FLAG antibody confirmed expression of exogenous proteins (Fig. 1A). Embryos expressing exogenous wt-Chk2 displayed a dose-dependent delay of cleavage cycles. Embryos injected with 2 ng wt-Chk2 mRNA exhibited slower cleavage cycles compared to controls. At 4.5 hr post-fertilization, control embryos had reached developmental Stage 6.5, corresponding to 64 cells (Nieuwkoop and Faber, 1975), whereas embryos expressing wt-Chk2 were approximately one cycle delayed (Stage 6 = 32 cells; Fig. 1B). At 8 hr pf, embryos expressing wt-Chk2 completed the MBT and remained delayed by approximately one cell cycle compared to controls (Fig. 1B). These embryos proceeded to gastrulate and survived through neurulation (not shown; see Fig. 3B). Embryos injected with a higher dose of 8–10 ng wt-Chk2 mRNA arrested cell cycles at the 2–4 cell stage, whereas control embryos injected with the same dose of luciferase mRNA divided normally (Fig. 1C).
Figure 1. Overexpression of wt-Chk2 leads to cell cycle lengthening prior to the MBT. One-cell stage embryos were microinjected with FLAG-tagged wt-Chk2 or luciferase (control) mRNA. A: Embryos injected with 2 ng mRNA were collected at indicated times post-fertilization (hr pf) and analyzed for the FLAG tag by Western blotting. Duplicate collections were made of embryos expressing Chk2 to verify consistent expression. B: Xenopus embryos injected with either 2 ng luciferase or wt-Chk2 mRNA were photographed at 4.5 hr pf, when embryos expressing exogenous wt-Chk2 were delayed approximately one cell cycle compared to luciferase controls. Embryos were photographed again at 8 hr. C: Xenopus embryos were injected with either 8 ng luciferase or wt-Chk2 mRNA. Embryos expressing exogenous wt-Chk2 arrested at the 2–4 cell stage and were photographed at 5 hr pf, when control embryos possessed approximately 64 cells. D,E: Overexpression of wt-Chk2 results in premature inhibitory phosphorylation of Cdks on tyrosine and premature degradation of Cdc25A. One-cell stage Xenopus embryos were microinjected with either 2 ng luciferase (control) or wt-Chk2 mRNA. Embryos were collected at indicated times post-fertilization (hr pf), and subjected to Western blotting with an antibodies against tyrosine-phosphorylated Cdks (D) and against Cdc25A (E).
Download figure to PowerPoint
Figure 3. Expression of exogenous wt-Chk2 inhibits apoptosis following IR. A: Endogenous Chk2 becomes activated after the MBT in response to IR. Embryos were treated at 3 hr (pre-MBT; top) or 14 hr (post-MBT; bottom) with the indicated dose of IR (Gy), then collected at the times indicated, and subjected to Western analysis with a Chk2 antibody. The phosphorylated form of Chk2 is retarded in electrophoretic mobility (McSherry and Mueller, 2004). B: One-cell stage Xenopus embryos were microinjected with either 2 ng of luciferase (control) or wt-Chk2 mRNA. Embryos were subjected to 30 Gy IR prior to the MBT, at ∼3.5 hr pf. One hundred percent of embryos expressing luciferase exhibited abnormal morphology, consistent with apoptosis, at gastrulation (11 hr pf). In contrast, only 10% of embryos expressing exogenous wt-Chk2 exhibited abnormal morphology at gastrulation. Furthermore, 25–30% of IR-treated embryos expressing exogenous wt-Chk2 completed gastrulation and proceeded to neurulate at 15 hr pf (as shown).
Download figure to PowerPoint
To determine whether the cell cycle arrest induced by wt-Chk2 resulted from engagement of known checkpoint events, embryos expressing luciferase or wt-Chk2 were assayed for tyrosine phosphorylation of Cdks by Western analysis with a phosphoCdk antibody. In metazoans, Chk2 inactivates Cdc25 phosphatases, resulting in Cdks in their tyrosine-phosphorylated, inactive state (Bartek et al., 2001; McGowan, 2002). In untreated embryos, periodic tyrosine phosphorylation of Cdks occurs but is low until after the MBT (Ferrell et al., 1991; Hartley et al., 1996; Kim et al., 1999). Likewise, the level of tyrosine phosphorylation in control embryos was low until after the MBT (Fig. 1D; 7–9 hr pf). In contrast, Cdks were phosphorylated on tyrosine well before the MBT, by 3 hr pf in embryos expressing exogenous wt-Chk2, indicating that the cell cycle delay resulted from reduced activity of Cdks. Therefore, pre-MBT embryos, which do not arrest cleavage cycles in response to damaged DNA (Anderson et al., 1997; Hensey and Gautier, 1997), can arrest the cell cycle in response to exogenous wt-Chk2. These data fit an emerging model that embryos possess the necessary components of checkpoint signaling pathways, but require threshold amounts of DNA to effectively transduce a signal resulting in cell cycle arrest (Kappas et al., 2000; Conn et al., 2004).
The extent of tyrosine phosphorylation in these embryos expressing wt-Chk2 was approximately the same as in control embryos at 9 hr post-fertilization (Fig. 1D), when cell cycles are asynchronous, averaging >200 min in length (Howe et al., 1995). Since the embryos expressing this dose of wt-Chk2 exhibited only modestly delayed cleavage cycles (Fig. 1B), additional changes at the MBT (such as changes in the pool of cyclin mRNAs (Audic et al., 2002) and the expression of the Cdk inhibitor, Xic1 (Su et al., 1995), are likely to contribute to the longer cell cycles at the MBT.
Another event of the MBT that may contribute to cell cycle lengthening is the degradation of Cdc25A, leading to enhanced tyrosine phosphorylation of Cdks (Kim et al., 1999). To determine whether expression of exogenous wt-Chk2 triggered premature degradation of Cdc25A, embryos were assayed for steady-state levels of Cdc25A (Fig. 1E). As shown in Figure 1D, Cdc25A content was decreased in embryos expressing exogenous wt-Chk2 compared to control embryos. These data provide further evidence that exogenous Chk2 disrupts cleavage cycles by perturbing known cell cycle checkpoint signaling pathways, leading to decreased activity of Cdks.
Expression of Dominant-Negative Chk2 (DN-Chk2) Triggers a Post-MBT Apoptotic Death
Although pre-MBT embryos do not engage checkpoints in response to damaged DNA, Chk1 and Chk2 are expressed throughout early development (Nakajo et al., 1999; Kappas et al., 2000; Gotoh et al., 2001). Chk1 is transiently activated at the MBT (Shimuta et al., 2002) and is required for embryonic survival beyond gastrulation (Shimuta et al., 2002; Carter and Sible, 2003). To determine whether Chk2 is required for early development, one-cell stage embryos were microinjected with either 8 ng luciferase or catalytically inactive DN-Chk2 mRNA, and development was observed. Both embryos expressing luciferase or DN-Chk2 developed normally through the MBT. During gastrulation, ∼90% of embryos (52/58) expressing DN-Chk2 appeared abnormal with loss of organization and cell-cell attachment, whereas all embryos expressing luciferase appeared normal. At neurulation, embryos expressing luciferase continued to develop normally, whereas the embryos expressing DN-Chk2 had lost attachments and appeared abnormal (Fig. 2A). The morphology of embryos expressing Chk2 resembled that of embryos that had undergone apoptosis in response to IR (Anderson et al., 1997; Hensey and Gautier, 1997), unreplicated DNA (Carter and Sible, 2003), and expression of DN-Chk1 (Carter and Sible, 2003).
Figure 2. Expression of DN-Chk2 results in apoptosis after the MBT. One-cell stage Xenopus embryos were microinjected with either 8 ng luciferase (control) or dominant-negative Chk2 (DN-Chk2) mRNA. A: Embryos expressing DN-Chk2 and luciferase developed normally until the early gastrula stage. Embryos shown were photographed when controls were neurulating (∼17 hr pf). B: Whole-mount TUNEL assays were performed on albino embryos expressing luciferase or DN-Chk2. When abnormal morphology was evident at the gastrulation (13 hr pf), embryos were collected and processed. A positive TUNEL reaction is indicated by deep purple staining. Control (luciferase) embryos exhibit normal morphology but are white because they are albino. C: Embryo extracts were collected at the times indicated and incubated with recombinant PARP, a substrate for caspase 3 (Carter and Sible, 2003). The presence of a cleaved PARP fragment indicates caspase activity. D: Embryo extracts were collected at the times indicated. Western blotting was performed with antibodies against tyrosine-phosphorylated Cdks (PCdk) and Cdc25A.
Download figure to PowerPoint
To determine whether DN-Chk2 induced apoptosis, albino embryos expressing luciferase or DN-Chk2 were collected at gastrulation and subjected to whole-mount TUNEL assays, which detect fragmented DNA, characteristic of apoptotic cells (Sible et al., 1997). Approximately 90% (50/55) of embryos expressing DN-Chk2 exhibited a positive TUNEL reaction indicated by punctate, deep purple staining. By comparison, ∼2% (1/52) of embryos expressing luciferase exhibited positive TUNEL staining (Fig. 2B).
As a biochemical assay of apoptosis, embryos expressing luciferase or DN-Chk2 were collected at the times indicated and assayed for caspase activity by the cleavage of PARP protein (Carter and Sible, 2003). Embryos expressing DN-Chk2 were positive for caspase activity beginning at gastrulation (Fig. 2C), indicating that these embryos had undergone apoptosis. Therefore, Xenopus embryos may require Chk2 function in order to survive beyond the MBT.
Previously, Shimuta et al. (2002) observed death in embryos expressing DN-Chk1 but not DN-Chk2. The death of embryos expressing DN-Chk1 corresponded with a delayed increase in tyrosine phosphorylation of Cdks and degradation of Cdc25A and could be mimicked by expressing nondegradable Cdc25A (Shimuta et al., 2002). Because different mutations were generated to create the dominant-negative constructs, the proteins expressed by their group may have functioned differently in the embryo or may have been expressed at lower levels. To test whether our DN-Chk1 and DN-Chk2 created the same effects on cell cycle remodeling at the MBT, we compared the effects of each (Fig. 2D). Our data indicate that both DN-Chk1 and DN-Chk2 delay the increase in tyrosine phosphorylation of Cdks and degradation of Cdc25A, which normally begins at the MBT. Therefore, expression of our DN-Chk2 construct disrupts cell cycle remodeling at the MBT and triggers a program of apoptosis.
Exogenous wt-Chk2 Inhibits Apoptosis following Ionizing Radiation (IR)
Embryos exposed before the MBT to a variety of assaults to the genome, including IR, develop normally through the MBT and then die by apoptosis during early gastrulation (Anderson et al., 1997; Hensey and Gautier, 1997; Sible et al., 1997; Stack and Newport, 1997). When embryos are exposed to these same assaults after the MBT, they are resistant to apoptosis (Anderson et al., 1997; Hensey and Gautier, 1997). To see how susceptibility to apoptosis correlated with activation of Chk2, embryos were exposed to IR before (3 hr pf) or after (14 hr pf) the MBT, then collected at several times points and analyzed by Western blotting for activation of Chk2, as determined by a shift in electrophoretic mobility, indicative of the activating phosphorylation. When exposed to 30 or 60 Gy IR before the MBT, no shift in Chk2 mobility was observed after 1 or 4 hr, suggesting that little or no Chk2 was activated in these embryos, which ultimately died by apoptosis during early gastrulation (Fig. 3A). In contrast, when embryos were irradiated after the MBT, a fraction of the Chk2 was shifted 1 or 2 hr later. The amount of shifted Chk2 was greater in embryos treated with 60 Gy versus 30 Gy IR, indicating a dose-dependent effect (Fig. 3A). These experiments indicate an inverse correlation between activation of Chk2 and susceptibility to apoptosis in embryos.
Based on these results as well as our observation that DN-Chk2 triggers apoptosis during gastrulation of Xenopus embryos (Fig. 2), we hypothesized that wt-Chk2 could play a dominant role in blocking apoptosis. To test this hypothesis, one-cell stage embryos were microinjected with 2 ng luciferase or wt-Chk2 mRNA, then exposed to 30 Gy IR before the MBT, at 3.5 hr pf. All embryos (20/20) expressing luciferase exhibited abnormal morphology, consistent with apoptosis, at gastrulation (Fig. 3B). In contrast, only 10% of embryos (2/20) expressing exogenous wt-Chk2 exhibited abnormal morphology at gastrulation, and 25–30% completed gastrulation and formed a neural plate (Fig. 3B). Therefore, irradiated embryos expressing exogenous Chk2 developed normally, well beyond the developmental stage at which irradiated embryos usually, die by apoptosis. These data indicate that Chk2 inhibits IR-induced apoptosis in the early Xenopus embryo.
Does Chk2 Inhibit or Promote Apoptosis?
We provide evidence that Chk2 inhibits apoptosis during early development of Xenopus laevis. However, other studies have indicated that Chk2 promotes apoptosis (Ahn et al., 2004). Mice lacking the Chk2 gene are viable, but deficient in IR-induced apoptosis (Hirao et al., 2002; Jack et al., 2002, 2004; Takai et al., 2002). In the Drosophila eye, Chk2 promotes the induction of apoptosis in response to DNA damage (Peters et al., 2002). In both cases, Chk2 promotes apoptosis via phosphorylation of p53. In human breast carcinoma cells, Chk2 promotes DNA damage-induced apoptosis by phosphorylating the transcription factor E2F-1 (Stevens et al., 2003), and in lymphoma cells by phosphorylating the scaffolding protein PML (Yang et al., 2002). Thus, in response to DNA damage, Chk2 can phosphorylate at least three targets to promote apoptosis (Ahn et al., 2004).
In contrast, other studies have implicated Chk2 as an inhibitor of apoptosis. Mouse embryonic stem cells are deficient in a G1 checkpoint and highly susceptible to IR-induced apoptosis. Ectopic expression of Chk2 in these cells confers protection from IR-induced apoptosis. This protection may be p53-independent, since the p53 target, p21, is not induced (Hong and Stambrook, 2004). Expression of DN-Chk2 in human heterokarya and HCT116 colon carcinoma cells promotes apoptosis in response to mitotic catastrophe (Castedo et al., 2004). Likewise, we show both that ectopic wt-Chk2 blocked IR-induced apoptosis (Fig. 3) and DN-Chk2 promoted apoptosis (Fig. 2) in the early Xenopus embryo, implicating Chk2 as an inhibitor of apoptosis.
Our studies may shed light upon the apparent paradox regarding the relationship between Chk2 and apoptosis. Early embryos contain p53, but development is not noticeably affected by expression of either wt- or DN-p53 until well past gastrulation (Wallingford et al., 1997). Furthermore, p53 cannot function as a transcription factor before the MBT because transcription is silent (Newport and Kirschner, 1982b). Therefore, early embryos provide a context in which the p53 signaling pathway is not fully functional, and may explain why Chk2 does not promote apoptosis. On the other hand, we have shown that Chk2 and Chk1 are capable of activating cell cycle checkpoints in the early embryos by triggering tyrosine phosphorylation of cyclin-dependent kinases (Fig. 1; Kappas et al., 2000). By causing cell cycle delay in response to IR, Chk2 may allow for repair and may also promote repair pathways through its interaction with proteins such as BRCA1 and Mus81 (McGowan, 2002). Since many tumors lack functional p53, it will be important to determine whether Chk2 truly blocks apoptosis in a p53-negative background, in order to decide when drugs that target Chk2 are appropriate chemotherapeutics.
Interestingly, induction of apoptosis by DN-Chk2 did not require exposure to IR or any other overt assault to the embryo. At this stage, we do not know why Xenopus embryos require Chk2 and Chk1 (Shimuta et al., 2002; Carter and Sible, 2003) during early development, but we suggest two possibilities: (1) inhibition of Chk2 during the 12 rapid cycles, which lack checkpoints before the MBT inevitably results in DNA damage that triggers apoptosis post-MBT, or (2) the lengthening of cell cycles at the MBT requires functional Chk2 and/or Chk1 (Shimuta et al., 2002; Carter and Sible, 2003). As we explore these possibilities in the Xenopus embryo, we will build a better understanding of how checkpoint controls are coordinated with other aspects of metazoan development.