A positive role for c-Abl in Atm and Atr activation in DNA damage response

DNA damage triggers Atm- and/or Atr-dependent signaling pathways to control cell cycle progression, apoptosis, and DNA repair. However, how Atm and Atr are activated is not fully understood. One of the downstream targets of Atm is non-receptor tyrosine kinase c-Abl, which is phosphorylated and activated by Atm. The current view is that c-Abl relays pro-apoptotic signals from Atm to p73 and p53. Here we show that c-Abl deficiency resulted in a broad spectrum of defects in cell response to genotoxic stress, including activation of Chk1 and Chk2, activation of p53, nuclear foci formation, apoptosis, and DNA repair, suggesting that c-Abl might also act upstream of the DNA damage-activated signaling cascades in addition to its role in p73 and p53 regulation. Indeed, we found that c-Abl is required for proper activation of both Atm and Atr. c-Abl is bound to the chromatin and shows enhanced interaction with Atm and Atr in response to DNA damage. c-Abl can phosphorylate Atr on Y291 and Y310 and this phosphorylation appears to have a positive role in Atr activation under genotoxic stress. These findings suggest that Atm-mediated c-Abl activation in cell response to double-stranded DNA breaks might facilitate the activation of both Atm and Atr to regulate their downstream cellular events.     

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.     

c-Abl tyrosine kinase selectively regulates p73 nuclear matrix association

p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73 alpha nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73 alpha but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73 alpha is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation.     

An essential role of human Ada3 in p53 acetylation

The p53 tumor suppressor protein functions as a critical component of genotoxic stress response by regulating the expression of effector gene products that control the fate of a cell following DNA damage. Unstressed cells maintain p53 at low levels through regulated degradation, and p53 levels and activity are rapidly elevated upon genotoxic stress. Biochemical mechanisms that control the levels and activity of p53 are therefore of great interest. We and others have recently identified hAda3 (human homologue of yeast alteration/deficiency in activation 3) as a p53-interacting protein and enhancer of p53 activity. Here, we show that endogenous levels of p53 and Ada3 interact with each other, and by using inducible overexpression and short hairpin RNA-mediated knockdown strategies we demonstrate that hAda3 stabilizes p53 protein by promoting its acetylation. Use of a p53 mutant with mutations of known p300/CREB-binding protein acetylation sites demonstrated that hAda3-dependent acetylation is required for increase in p53 stability and target gene induction. Importantly, we demonstrate that endogenous hAda3 is essential for DNA damage-induced acetylation and stabilization of p53 as well as p53 target gene induction. Overall, our results establish hAda3, a component of coactivator complexes that include histone acetyltransferase p300/CREB-binding protein, as a critical mediator of acetylation-dependent stabilization and activation of p53 upon genotoxic stress in mammalian cells.     

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Exclusion of c-Abl from the nucleus restrains the p73 tumor suppression function

The p73alpha protein is a functional homolog of the p53 tumor suppressor. Although the TP53 gene is frequently mutated in human cancers, the TP73 gene is rarely inactivated. We have found that p73alpha is highly expressed in a significant fraction of anaplastic thyroid cancer, whereas it is not detectable in normal thyroid epithelial cells or in papillary and follicular thyroid cancer cells. Interestingly, the tumor suppression function of p73alpha is actively restrained in anaplastic thyroid cancer cells. We have also found that c-Abl tyrosine kinase, an activator of p73, is excluded from the nucleus of p73alpha-positive thyroid cancer cells; whereas c-Abl undergoes nuclear-cytoplasmic shuttling in normal thyroid and p73-negative thyroid cancer cells. We constructed an AblNuk-FK506-binding protein (FKBP) fusion protein to enforce the nuclear accumulation of an inducible Abl kinase. Activation of this nuclear AblNuk-FKBP by dimerization with AP20187 in anaplastic thyroid cancer cells increased the levels of p73alpha and p21Cip1 and caused p73-dependent apoptosis. These results suggest subcellular segregation of c-Abl from p73 to be a strategy for disrupting the tumor suppression function of p73alpha.     

Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization

The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G(1) growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53Delta363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinase-independent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.     

MMR/c-Abl-dependent activation of ING2/p73α signaling regulates the cell death response to N-methyl-N′-nitro-N-nitrosoguanidine

Agents inducing O⁶-methylguanine (O⁶MeG) in DNA such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are cytotoxic and a deficiency in mismatch repair (MMR) results in lack of sensitivity to this genotoxin (termed alkylation tolerance). Here, we show that ING2, a member of the inhibitor of growth family, is required for cell death induced by MNNG. We further observe that MNNG treatment increases cellular protein levels of ING2 that is dependent on intact MMR function and that MNNG-induced ING2 localizes and associates with p73α in the nucleus. Suppression of ING2 by short hairpin RNA (shRNA) in MMR-proficient colorectal cancer cells decreased its sensitivity to MNNG and, in addition, abrogated MNNG-induced stabilization and acetylation of p73α. Interestingly, suppression of p73α had a greater impact on MNNG-induced cell death than ING2 leading us to conclude that ING2 regulates the cell death response, in part, through p73α. Inhibition of c-Abl by STI571 or suppression of c-Abl expression by shRNA blocked ING2 induction and p73α acetylation induced by this alkylator. Similarly, suppression of MMR (MLH1) by shRNA abrogated ING2 induction/p73α acetylation. Taken together, these results demonstrate that MLH1/c-Abl-dependent activation of ING2 > p73α signaling regulates cell death triggered by MNNG and further suggests that dysregulation of this event may, in part, be responsible for alkylation tolerance observed in MMR compromised cells.     

Rescue of platinum-damaged oocytes from programmed cell death through inactivation of the p53 family signaling network

Non-proliferating oocytes within avascular regions of the ovary are exquisitely susceptible to chemotherapy. Early menopause and sterility are unintended consequences of chemotherapy, and efforts to understand the oocyte apoptotic pathway may provide new targets for mitigating this outcome. Recently, the c-Abl kinase inhibitor imatinib mesylate (imatinib) has become the focus of research as a fertoprotective drug against cisplatin. However, the mechanism by which imatinib protects oocytes is not fully understood, and reports of the drug''s efficacy have been contradictory. Using in vitro culture and subrenal grafting of mouse ovaries, we demonstrated that imatinib inhibits the cisplatin-induced apoptosis of oocytes within primordial follicles. We found that, before apoptosis, cisplatin induces c-Abl and TAp73 expression in the oocyte. Oocytes undergoing apoptosis showed downregulation of TAp63 and upregulation of Bax. While imatinib was unable to block cisplatin-induced DNA damage and damage response, such as the upregulation of p53, imatinib inhibited the cisplatin-induced nuclear accumulation of c-Abl/TAp73 and the subsequent downregulation of TAp63 and upregulation of Bax, thereby abrogating oocyte cell death. Surprisingly, the conditional deletion of Trp63, but not ΔNp63, in oocytes inhibited apoptosis, as well as the accumulation of c-Abl and TAp73 caused by cisplatin. These data suggest that TAp63 is the master regulator of cisplatin-induced oocyte death. The expression kinetics of TAp63, c-Abl and TAp73 suggest that cisplatin activates TAp63-dependent expression of c-Abl and TAp73 and, in turn, the activation of TAp73 by c-Abl-induced BAX expression. Our findings indicate that imatinib protects oocytes from cisplatin-induced cell death by inhibiting c-Abl kinase, which would otherwise activate TAp73-BAX-mediated apoptosis. Thus, imatinib and other c-Abl kinase inhibitors provide an intriguing new way to halt cisplatin-induced oocyte death in early follicles and perhaps conserve the endocrine function of the ovary against chemotherapy.