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.     

A novel role for DNA mismatch repair and the autophagic processing of chemotherapy drugs in human tumor cells

DNA Mismatch repair (MMR) maintains genome integrity by correcting DNA replication errors and blocking homologous recombination between divergent DNA sequences. The MMR system also activates both checkpoint and apoptotic responses following certain types of DNA damage. In a recent study, we describe a novel role for MMR in mediating an autophagic response to 6?thioguanine (6-TG), a DNA modifying chemical. Our results show that MMR proteins (MLH1 or MSH2) are required for signaling to the autophagic pathway after exposure to 6-TG. Using PFT-alpha, a p53 inhibitor, and shRNA-mediated silencing of p53 expression, we also show that p53 plays an essential role in the autophagic pathway downstream of the MMR system. This study suggests a novel function of MMR in mediating autophagy following chemical (6-TG) DNA mismatch damage through p53 activation. Here, we present the model and the clinical implications of the role of MMR in autophagy.     

Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification.

We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53mediated apoptosis.     

Family Feud in Chemosensitvity: p73 and Mutant p53

The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 (“gain of function”) are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.     

Family feud in chemosensitvity: p73 and mutant p53

The importance of p53 in chemotherapy-induced apoptosis of cancer cells is well established. p53 plays a critical role in the cellular response to DNA damage by regulating genes involved in cell cycle progression, apoptosis, and genomic stability. As a result, p53 tumor status is a critical determinant of both responses to anti-cancer treatment and clinical prognosis. Interestingly, tumors expressing certain mutant forms of p53 ("gain of function") are particularly resistant to chemotherapy, even when compared to cells that lack any detectable p53. Until recently, the explanation for this enhanced chemoresistance was not clear. Recent studies have shown that the p53 homologues, p73 and p63, are also activated by chemotherapies, leading to tumor cell death. Now the discovery that mutant p53 interacts with p73, and that regulation of this interaction by a p53 polymorphism can modulate chemosensitvity provide a new model for how p53-family interactions can influence the response of tumors to anti-cancer therapies. Since p53 mutations are found in more than 50% of human tumors, strategies aimed at manipulating these interactions may prove useful in enhancing the chemotherapy response, and perhaps, overcoming chemoresistance.     

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DeltaNp63alpha associated with its increased phosphorylated status. Since DeltaNp63alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DeltaNp63alpha in HNSCC cells upon cisplatin exposure. We found that DeltaNp63alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397, and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DeltaNp63alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DeltaNp63alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DeltaNp63alpha degradation. Finally, we demonstrated that the DeltaNp63alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DeltaNp63alpha following DNA damage.     

p63 and p73: roles in development and tumor formation

The tumor suppressor p53 is critically important in the cellular damage response and is the founding member of a family of proteins. All three genes regulate cell cycle and apoptosis after DNA damage. However, despite a remarkable structural and partly functional similarity among p53, p63, and p73, mouse knockout studies revealed an unexpected functional diversity among them. p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. Neither p63 nor p73 is the target of inactivating mutations in human cancers. Genomic organization is more complex in p63 and p73, largely the result of an alternative internal promoter generating NH2-terminally deleted dominant-negative proteins that engage in inhibitory circuits within the family. Deregulated dominant-negative p73 isoforms might play an active oncogenic role in some human cancers. Moreover, COOH-terminal extensions specific for p63 and p73 enable further unique protein-protein interactions with regulatory pathways involved in development, differentiation, proliferation, and damage response. Thus, p53 family proteins take on functions within a wide biological spectrum stretching from development (p63 and p73), DNA damage response via apoptosis and cell cycle arrest (p53, TAp63, and TAp73), chemosensitivity of tumors (p53 and TAp73), and immortalization and oncogenesis (DeltaNp73).     

Compensatory expression of p73 in PARP-deficient mouse fibroblasts as response to a reduced level of regularly spliced wild-type p53 protein

We have investigated the effect of PARP gene inactivation on the expression of wild-type (wt) p53 protein. Using immortalized fibroblasts from control and PARP knock-out mice we have found by immunoblotting with the PAb421 antibody a profound decrease of the p53 expression to a barely detectable level in PARP knock-out cells. Surprisingly, longer exposure of immunoblots revealed an immunoreactive band at about 75 kD which was stronger in PARP-deficient cells than in wt cells and was not affected upon doxorubicin treatment. The size of the PAb421 immunoreactive protein and the lack of its inducibility in response to DNA damage resembled those of p73, the first described p53 homologue. Therefore, we examined the reactivity of anti-p53 antibodies with in vitro translated p73 protein. Interestingly, p73 was efficiently immunoprecipitated with distinct antibodies recognizing the carboxy-terminus of p53. In Northern blots we observed p73 signals of comparable intensity in controls and PARP-deficient cells. We conclude that elevated expression of p73 may compensate the reduced level of p53 in PARP-deficient cells.