The results presented here show that Chk1 plays an important role during normal S-phase progression by minimizing the occurrence of aberrant replication-associated events. These results are consistent with a recent study where conditional Chk1 heterozygosity caused accumulation of DNA damage during DNA replication in mice (14
). Our study shows that as a consequence of Chk1 inhibition in human S-phase cells, ATR is rapidly activated (within 1 h). This is likely due to stabilization of Cdc25A and the accompanying hyperactivation of Cdk2, resulting in increased loading onto chromatin of the key replication factor Cdc45, increased activation of replication origins, and subsequent increased association of RPA to ssDNA (Fig. ). As a further consequence of this process, the genome is destabilized, as evidenced by massive accumulation of DNA strand breaks. Based on our results we propose that Chk1-mediated control of the initiation of DNA synthesis is required for normal S-phase progression of human cells. These data are consistent with and mechanistically further extend the recent findings in Xenopus
cell-free system demonstrating the critical involvement of ATR in preventing unscheduled replication (19
FIG. 6. Proposed models for how Chk1 inhibition causes activation of ATR and massive DNA lesions in S-phase cells. (A) In response to Chk1 inhibition, Cdc25A is stabilized and the Cdk activity is rapidly increased, which leads to increased initiation of DNA replication (more ...)
An alternative, but not mutually exclusive, possibility is that Chk1 inhibition may cause DNA breaks and activation of ATR due to lack of Chk1-mediated maintenance of stalled replication forks during normal S phase (Fig. ). Chk1, as well as ATR, are required to prevent collapse of stalled replication forks after treatment with replication inhibitors such as aphidicolin (3
). ATR also likely prevents replication fork collapse during normal S-phase progression, as ATR is required to avoid chromosome breaks in mitosis in the absence of exogenous treatment (3
). It is possible that Chk1 is also needed to prevent fork collapse during normal S-phase progression and that this may contribute to the responses reported here. Although the pattern of BrdU incorporation appeared normal in cells with massive γ-H2AX after Chk1 inhibition, clearly demonstrating that a massive collapse of replication forks did not occur (Fig. ), we cannot exclude that the integrity of a small portion of replication forks was perturbed. However, taken into account our observations that Cdk2 and Cdc45 are required for the responses (Fig. ), and that transient transfections with Cdk2AF or Cdc25A caused massive γ-H2AX (data not shown), the model outlined in Fig. seems more plausible.
In response to Chk1 inhibition by drugs, measurements of both cyclin A-, E-, and B-associated Cdk activities showed an ca. two- to threefold transient and rapid increase, which occurs due to lack of Chk1-mediated phosphorylation of the Cdc25s (29
). The results of our present study clearly indicate that increased Cdk activity in response to Chk1 inhibition generates increased initiation of DNA replication, which ultimately leads to induction of DNA strand breaks. In this context, it is interesting that constitutive overexpression of cyclin E causes chromosomal instability by an unknown mechanism likely associated with defective DNA replication (23
). One possibility is that high levels of cyclin E may cause DNA damage and chromosomal instability due to increased initiation of DNA replication in a similar way as described here, although a recent study (6
) proposed that cyclin E-mediated impairment of prereplication complex assembly in early G1
phase may be involved. On the other hand, more studies are required to explore whether increased Cdk activity is sufficient to trigger the whole cascade of responses that we have observed after Chk1 inhibition in S-phase cells. At the present stage we cannot exclude that Chk1 may target additional factors involved in control of replication initiation. For example, it was suggested that ATR-Chk1, together with Cdk2, may regulate phosphorylation of Mcm4 in a complex manner after treatment with hydroxyurea (12
), and Chk1-mediated control of Mcm4 or other origin-binding proteins could likely be involved.
When cells treated with CEP-3891 or transfected with Chk1 siRNA were assayed simultaneously for γ-H2AX and DSB induction by the TUNEL assay, we consistently observed that only a subset of the γ-H2AX-positive cells were TUNEL positive (Fig. ). These results indicate that the induction of massive DNA breaks is a late event likely occurring after the onset of massive phosphorylation of γ-H2AX. Consistent with previous studies (37
), the signal that first activates ATR in response to Chk1 inhibition may therefore be ssDNA rather than DSBs. However, we cannot exclude that a small number of DSBs not detectable by the TUNEL assay may be involved.
Interestingly, we observed significantly less phosphorylation of H2AX in response to Chk1 inhibition when Cdk2 was downregulated by conditionally expressed shRNA (Fig. ). In agreement with several recent studies reporting that Cdk2 is dispensable for cell cycle progression (22
), we have not observed any effects on cell cycle progression by depleting Cdk2 in U-2-OS cells in the absence of UCN-01 or CEP-3891 treatment (data not shown). Taken together, our results demonstrate for the first time that although Cdk2 may be dispensable for normal cell cycle progression, Cdk2 is not dispensable in the situation when cells are exposed to external stress such as Chk1-inhibitors.
It was previously suggested that Chk1 is essential for early embryonic viability due to its control of entry into mitosis (33
). Defective Chk1 function was predicted to cause premature activation of cyclin B-Cdk1 and mitotic catastrophe (33
). Our results demonstrating that Chk1 is required during normal S-phase progression to avoid replication-associated defects, together with the recent report where conditional Chk1 heterozygosity caused DNA damage during DNA replication in mice (14
), add a new possible explanation of this phenomenon. We propose that lack of Chk1-mediated control of the initiation of DNA replication might contribute to lethality of Chk1−/−
cells. It is plausible to assume that induction of lethal DNA breaks occurs during S phase of early embryonic cells from Chk1−/−
mice through a similar mechanism to the one described here in human cells. On the other hand, Chk1 was previously shown to be dispensable for the viability of somatic cells derived from the chicken cell line DT40 under normal growth conditions in culture (39
). However, such Chk1-deficient cells did proliferate significantly more slowly in the absence of any externally induced DNA damage or replication stress, which was attributed to an increase in the incidence of spontaneous cell death (39
), and which could possibly be due to similar replication defects as we have described in the present study. Alternatively, these highly genetically unstable chicken cells may behave differently than the human cell lines used in our present study.
Due to its critical role in regulating the DNA damage-induced checkpoints, Chk1 has been proposed as a potential target for cancer treatment (reviewed in reference 42
). The principle idea of this strategy is to combine standard chemo- or radiotherapy with drugs that inhibit Chk1 kinase in order to inhibit the S and G2
checkpoints. Cancer cells could likely be particularly sensitive to such treatment since they commonly lack normal G1
checkpoint control and may rely more on the S and G2
checkpoints compared to normal cells (5
). The results presented here, showing that Chk1 inhibition per se leads to massive induction of DNA breaks in human S-phase cells, demonstrate that much remains to be understood in terms of the role of Chk1 kinase in the absence or presence of genotoxic agents. In particular, it will be important to determine to which extent these S-phase responses may vary between different cancer and normal cell types. Deeper insight into this issue will be important before pharmacological manipulation of Chk1 can be utilized in the clinic to enhance the efficacy of anticancer therapies.