Our study focused on the involvement of Chk2 and histone H2AX in response to DR activation by TRAIL. We show that Chk2, following its activation by DNA-PK and ATM, can amplify the apoptotic response elicited by TRAIL by a feedback loop independently of p53. The Chk2-mediated positive feedback extends outside the nucleus, as it involves upstream caspases 8 and 9. Caspase 8 forms a complex with the TRAIL membrane receptors (DR4 and DR5) within the DISC, and caspase 9 is a key effector of the apoptosome. Another TRAIL-induced feedback loop implicating caspase 8 has recently been described, which involves the nuclear translocation of a self-cleaved death effector domain segment of caspase 8 and p53-dependent upregulation of procaspase 8 gene expression (62
). Our feedback mechanism would act more directly on caspases than the p53-dependent transcriptional activation of procaspase 8 (62
). Figure gives an outline of the TRAIL-induced DDR pathways and schematizes the feedback loops that are revealed in our study.
Schematic representation of the caspase- and Bax-dependent activation of DNA-PK, ATM, H2AX, and Chk2 in response to TRAIL. Open arrows correspond to activation.
The functional impact of Chk2 on TRAIL-induced apoptosis was detectable as an enhancement of cell detachment, an increased cell death measured by clonogenic assays, an increased γ-H2AX induction, and a faster activation of caspases (Fig. ). In fact, TRAIL was remarkably potent in HCT116 cells. Even after only 1 h of TRAIL exposure, only ~1% of the cells were able to form colonies (Fig. ). Therefore, the cells progress to apoptosis once they have been committed upon TRAIL exposure. Inactivation of Chk2 increased the number of surviving colonies by 40 to 60% (Fig. ), demonstrating the importance of Chk2 in eliminating residual cells. The functional impact of Chk2 may be quite significant because of the potentially severe physiological consequences of abortive apoptosis. Apoptosis has been viewed as an important mechanism to eliminate oncogenes from dying tumor cells or virus-infected cells. Also, even when cells die, it is important that apoptosis proceed to completion to avoid autoimmune diseases, which have been linked to circulating DNA fragments (39
). Thus, it is possible that Chk2 promotes the completion of the apoptosis program, thereby avoiding the survival of abnormal cells and the release of toxic metabolic intermediates.
To our knowledge, our study is the first to implicate Chk2 in TRAIL-induced apoptosis independently of p53. Prior studies had provided functional evidence for the proapoptotic role of Chk2 in the context of DNA damage produced by IR, radiomimetic agents, and arsenic trioxide (20
). In those models, the proapoptotic function of Chk2 was linked to p53 activation. In contrast, in our study, the proapoptotic function of Chk2 is p53 independent. p53 was phosphorylated on serine 15 but neither phosphorylated on serine 20 nor stabilized as determined by lack of p53 protein accumulation in TRAIL-treated cells (Fig. ). Also, p21WAF1/CIP1
was not upregulated by TRAIL (Fig. ). These results are in accordance with the literature showing that phosphorylation on serine 20 is required for stabilization of p53 (9
) and enhancement of the transcriptional activation of p21WAF1/CIP1
by p53 (22
was rapidly cleaved in response to TRAIL (Fig. ), probably due to caspase 3 activity (40
). The degradation of p21WAF1/CIP1
could facilitate apoptosis (36
). We also found that the proapoptotic role of Chk2 was unaffected in the absence of p53 (Fig. ). Thus, our findings are consistent with other studies showing that p53 is not essential for TRAIL-induced apoptosis (45
A main focus of our study is the H2AX response to TRAIL. We show that histone H2AX is rapidly phosphorylated on serine 139 (γ-H2AX) and that γ-H2AX tends to form a confluent staining that initiates in peripheral nuclear regions (γ-H2AX ring staining) before gross changes in nuclear morphology that characterize apoptosis (Fig. , , , , and ). We had previously shown γ-H2AX formation in response to Fas antibody, staurosporine, and DNA-damaging agents (50
) soon after the discovery of γ-H2AX (51
). But at that time, we did not investigate the nuclear distribution of γ-H2AX by immunofluorescence microscopy. In the present study, several novel points are noteworthy regarding TRAIL-induced γ-H2AX activation. It is remarkably rapid (starting within 1 h), while the cells retain an overall normal morphology. Only a fraction of the cells initially show γ-H2AX staining, and those cells tend to be in S phase, although not exclusively (Fig. ). The initial γ-H2AX staining initiates as a confluent pattern at the periphery of the nucleus (γ-H2AX ring staining) before diffusing to form a panstaining pattern covering the entire nucleus and, later on, the nuclear bodies (Fig. , , and ). This confluent pattern was also observed in normal epithelial cells treated with TRAIL, although in much fewer cells than in the cancer cells examined here (Fig. ), which suggests conservation of the γ-H2AX induction process in both cancer and normal cells. The γ-H2AX peripheral distribution (γ-H2AX ring staining) occurred within the peripheral heterochromatic regions at the contact of the lamin B1 nuclear boundary (Fig. ). The TRAIL-induced γ-H2AX patterns never formed detectable nuclear foci and were not accompanied by the recruitment/colocalization of 53BP1, another ubiquitous DDR factor, which sets apart the TRAIL-induced γ-H2AX response from the focal response(s) characteristic of DNA-damaging agents (49
). The ring staining for γ-H2AX also contained activated ATM phosphorylated on S1981, activated DNA-PK phosphorylated on T2609, and activated Chk2 phosphorylated on T68 (Fig. ). Further studies are under way to determine whether this novel γ-H2AX response is induced during other apoptotic processes besides TRAIL-induced DR pathway activation.
We found that Chk2 has an impact on the progressive induction of γ-H2AX staining in response to TRAIL. Chk2 siRNA slowed down the formation of γ-H2AX-positive cells with nuclear segmentation (type III staining) (Fig. ). This reduction could result from delayed caspase activation (Fig. ). However, it is also plausible that Chk2 acts directly on chromatin. In fact, histone H1 is a substrate for Chk2 (67
). Thus, H1 phosphorylation by Chk2 might be part of the chromatin modifications controlled by Chk2. This possibility is attractive in the context of recent studies showing that Chk2 activation by DNA damage induces the release of H1.2 from chromatin and its translocation to the mitochondria, where H1.2 can act as a positive regulator of apoptosome formation (11
Our experiments demonstrate that the kinase responsible for the induction of γ-H2AX by TRAIL is DNA-PK (Fig. ). In contrast, Chk2 is phosphorylated by both ATM and DNA-PK in response to TRAIL. Thus, the ATM and DNA-PK pathways appear to branch out upstream from Chk2 and H2AX (Fig. ). Our finding that DNA-PK is the primary γ-H2AX kinase during TRAIL-induced apoptosis is consistent with another study showing that DNA-PK is responsible for γ-H2AX formation during staurosporine-induced apoptosis (38
). However, contrary to a study of UVA-induced apoptosis (32
), we found that JNK kinase is not involved in γ-H2AX induction by TRAIL and that knocking out H2AX by siRNA had no effect on TRAIL-induced apoptosis (data not shown). These differences suggest the existence of various nuclear pathways activated during apoptosis. The general dispensability of H2AX for the execution of apoptosis is actually consistent with the viability and normal embryonic development of H2AX knockout mice (7
To investigate the potential involvement of ATR in the TRAIL pathway, we looked at the activation of Chk1, which is a preferential ATR substrate. Activation of Chk1 following its cleavage by caspases has been reported to contribute to Chk1-dependent apoptosis in response to DNA-damaging agents (34
). Our present study provides no evidence for Chk1 implication in TRAIL-induced apoptosis (Fig. ). Also, inhibition of ATM and DNA-PK was sufficient to suppress completely the activation of Chk2 and H2AX (Fig. ). Together, these data suggest that ATM, DNA-PK, and Chk2 are the main kinases involved in the TRAIL-induced response, contrary to ATR and Chk1. Nevertheless, we cannot exclude a potential involvement of ATR. Even if Chk1 was not phosphorylated, other proteins could be targeted by ATR.
The observed cross talk between ATM and DNA-PK following TRAIL treatment is worth noting because it appears to be mutual rather than limited to only one direction in which DNA-PK is phosphorylated by ATM (10
). Our present finding that ATM can be phosphorylated on S1981 in a DNA-PK-dependent manner (Fig. ) is novel. An effect of DNA-PK on ATM has nevertheless been suggested by some prior studies. In glioblastoma cells deficient for DNA-PK (M059J), the ATM protein levels are low (8
). In addition, the V3 radiosensitive CHO cell line presents low ATM protein levels, and when the amount of DNA-PK is restored by transfection, the levels of ATM protein are also restored (41
). In murine cells with different degrees of DNA-PK deficiency, ATM protein is reduced (41
From a therapeutic standpoint, our findings suggest that the levels of Chk2 and activated P-Chk2-T68 in tumor tissues could have a prognostic value for predicting the efficiency of a TRAIL therapy. Moreover, our results provide a rationale for combining TRAIL with DNA-damaging agents. Indeed, DNA damage (IR or DNA-targeted drugs) might sensitize tumor cells to TRAIL by preactivating Chk2 and the positive feedback loop that amplifies TRAIL-induced apoptosis. Such a mechanism may partly account for the known synergism between TRAIL and DNA-damaging therapies (47
). Chk2 activation in some human tumors and precancerous lesions (3
) could also delay or prevent cancer development, reinforcing the importance of an efficient DDR.