Reactive oxygen species (ROS), including peroxide, superoxide, singlet O2
, and the highly reactive hydroxyl radical, cause oxidative damage and cellular stress. The balance between cellular life and death is, in part, a function of the ability of a cell to control oxidant insult (3
). Cellular antioxidants and stress responses defend against the modification of macromolecular targets exerted by ROS (32
). Accumulation of oxidative damage also has been implicated in the degenerative pathologies associated with organismal aging (41
) and in the development of diseases such as atherosclerosis and cancer (reviewed in reference 21
). The role of ROS as a trigger for apoptosis, in particular, has been suggested by a large body of work (reviewed in reference 29
). Cytotoxic stresses often are accompanied by increases in intracellular ROS levels, and the generation of ROS within the cell may serve as a second messenger in the initiation of death responses triggered by other stimuli. Many antioxidants delay apoptotic responses triggered, for example, by anticancer drugs and gamma irradiation.
Activation of ASK1 is a critical cellular response to ROS, affecting the balance of death and survival (1
). Our finding that CDC25A inhibits ASK1 suggests that CDC25A may play an important role in the cross talk between the cell cycle machinery and oxidative stress responsive pathways. That the inhibitory effect of CDC25A is manifest with respect to oxidant-triggered cell death, but not all death responses, is consistent with a specific role in the signaling phase (as distinct from the effector phase) of cell death. Significantly, this role of CDC25A is independent of its function as a CDK2 phosphatase in promoting cell cycle progression, suggesting that the physical association, but not the catalytic action, of CDC25A is important for the inhibition of ASK1. We have shown that overexpression of CDC25A diminishes homo-oligomerization of ASK1. The binding of CDC25A to ASK1 at the region adjacent to the kinase domain could inhibit oligomerization of ASK1. Homo-oligomerization is an important process for ASK1 activation (20
). ASK1 interacts physically with the reduced form of thioredoxin, a redox-sensitive protein, and is sequestered in an inactive form (53
). Oxidation of thioredoxin by intracellular ROS results in the activation of ASK1, involving homo-oligomerization of ASK1 and its association with death receptor-associated proteins such as TRAF2 (20
). Thus, physical association of CDC25A is inhibitory to this series of events leading to full activation of ASK1.
Our data also imply that CDC25A is involved in the control of oxidative stress responses by mitogenic and oncogenic signals. The expression of CDC25A is regulated by mitogenic signals via the E2F transcription factors (61
). A number of cancers, including breast and head or neck cancers, display overexpression of CDC25A (6
). Increased expression of CDC25A under these mitogenic or oncogenic conditions may be related to reduced responsiveness of immortalized or transformed cells to oxidative stress (21
). Ectopic expression of CDC25A has been reported to trigger cell death under conditions of growth factor deprivation (16
). However, our data, together with those from another work (34
), suggest that CDC25A may not be involved in the induction of apoptosis generally. The transforming ability of CDC25A in rodent fibroblasts (18
) rather implies that overexpression of CDC25A, together with that of Ras, facilitates survival of cells with unrestricted cell cycle progression. During oncogenic transformation, cells are thought to undergo various stresses. CDC25A could function simultaneously to inhibit stress-responsive pathways that normally lead to apoptosis, while it promotes cell cycle progression in mitogenically stimulated cells. The modest reduction in cellular susceptibility to oxidant-induced death afforded by upregulation of CDC25A may serve, under conditions of oncogenic transformation, to enhance the frequency of productively transformed cells that escape apoptosis. The expression of CDC25A also could affect the responsiveness of cancer cells to oxidative and/or genotoxic stresses caused by cancer therapies. In a recent study (6
), overexpression of CDC25A was found in 47% of patients with small (<1 cm) breast carcinoma and was associated with poor prognosis. In addition, we have recently observed that ovarian cancer cell lines expressing high levels of CDC25A tend to display diminished activation of JNK1 and p38 in response to oxidative stress, compared with those lines expressing low levels of CDC25A (X. Zou and H. Kiyokawa, unpublished observations).
Persistent accumulation of intracellular ROS could result in DNA damage, which triggers checkpoint signals to inhibit cell cycle progression. The G1
checkpoint largely relies on the p53-mediated transcription of the CDK inhibitor p21 (5
), but also involves degradation of CDC25A protein triggered by the checkpoint kinase Chk1 (39
). When ROS accumulation causes DNA damage, activated Chk1 facilitates degradation of CDC25A, and ASK1 could activate the downstream stress kinases without the inhibitory interference from CDC25A. This presents a feedback loop from the cell cycle checkpoint to the stress-responsive pathways. Chk1 has been demonstrated to phosphorylate G2
/M-regulatory CDC25C, facilitating cytoplasmic sequestration of CDC25C by some of the 14–3-3 proteins (38
). Thus, CDC25 phosphatases are critical components of both G1
checkpoints. In this study, we have observed that CDC25A and ASK1 colocalize primarily in the cytoplasm, while CDC25A exists as well within the nucleus. Nuclear CDC25A presumably functions as a CDK activator. At present, it is unclear whether the subcellular localization of CDC25A is regulated by some kind of checkpoint mechanisms under various cellular conditions. We have observed no gross change in the localization of CDC25A and ASK1 in H2
-treated OVCAR-8 cells or tet
-CDC25A-293 cells (unpublished observations). It remains to be clarified whether other stressful conditions alter the subcellular localization of these two proteins.
An increase in CDC25A expression, associated with oncogenic transformation, could have diverse effects on the fine-tuned network of the cell cycle checkpoint and stress responses. Increased expression of CDC25A diminishes activation of the stress kinase cascades upon oxidative stress and suppresses the acute death response, as shown in this study. Disruption of the G1
checkpoint by transient overexpression of CDC25A has been shown to increase the amount of DNA breaks after UV irradiation (39
). Many factors, including the extracellular environment, intracellular redox state, and expression of various genes, contribute to the determination of cell fate (i.e., cell death, survival with restored function, and transformation). We propose that the expression of CDC25A is one important factor that pertains particularly to the interplay of oxidative stresses, apoptotic stimuli, and mitogenic signaling. The detailed mechanism of the role of CDC25A at this interface awaits further investigation.