The serine/threonine kinase CHK2 is a key component of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates >20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In particular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.

The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G 1 arrest. Here we show that the ATMdependent activation of Chk2 by ␥-radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxyterminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.The integrity of genetic information is essential for the life and survival of cells. Genomic lesions arising spontaneously during DNA replication or in response to oxidative metabolism or exposure to radiation or chemical mutagens need to be recognized and repaired. Delay of cell cycle progression at specific checkpoints provides the time necessary to prevent replication and segregation of damaged DNA and to process lesions (reviewed in references 52 and 57). A defective or incorrect activation of the surveillance and repair systems can lead to increased mutagenesis, genomic instability, and ultimately cancer (for a review, see reference 13).The Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT) are rare human autosomal recessive diseases (22, 51) exhibiting hypersensitivity to ionizing radiation (IR), immunodeficiency, and increased predisposition to develop cancer. NBS patients, however, do not manifest the hallmarks of AT, i.e., cerebellar ataxia and oculocutaneous telangiectasia. At the cellular level, NBS and AT patients show chromosome instability, hypersensitivity to genotoxic agents, and cell cycle checkpoints defects (1,29,30). These similarities su...

Human DBC1 (deleted in breast cancer-1; KIAA1967) is a nuclear protein that, in response to DNA damage, competitively inhibits the NAD(+)-dependent deacetylase SIRT1, a regulator of p53 apoptotic functions in response to genotoxic stress. DBC1 depletion in human cells increases SIRT1 activity, resulting in the deacetylation of p53 and protection from apoptosis. However, the mechanisms regulating this process have not yet been determined. Here, we report that, in human cell lines, DNA damage triggered the phosphorylation of DBC1 on Thr454 by ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3-related) kinases. Phosphorylated DBC1 bound to and inhibited SIRT1, resulting in the dissociation of the SIRT1-p53 complex and stimulating p53 acetylation and p53-dependent cell death. Indeed, DBC1-mediated genotoxicity, which was shown in knockdown experiments to be dependent on SIRT1 and p53 expression, was defective in cells expressing the phospho-mutant DBC1(T454A). This study describes the first post-translational modification of DBC1 and provides new mechanistic insight linking ATM/ATR to the DBC1-SIRT1-p53 apoptotic axis triggered by DNA damage.

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM-and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

Hypomorphic mutations of the MRE11 gene are the hallmark of the radiosensitive ataxia-telangiectasia-like disorder (ATLD). Here, we describe a new family with two affected siblings, ATLD5 and ATLD6, now aged 37 and 36, respectively. They presented with late onset cerebellar degeneration slowly progressing until puberty and absence of telangiectasias, and were cancer-free. Both patients were wild-type for ATM and NBS1, but compound heterozygotes for MRE11 gene mutations [1422C-->A, T481K; 1714C-->T, R571X]. The 1422C-->A allele was inherited from the mother, whereas the 1714C-->T, allele paternally inherited, was apparently null as a result of nonsense-mediated mRNA decay (NMD). Interestingly, the 1714C-->T mutation is the same as previously identified in an unrelated English ATLD family (probands ATLD3 and ATLD4), suggesting an important role for NMD in saving potentially lethal mutations. Lymphoblastoid cell lines (LCLs) derived from ATLD5 and ATLD6 were normal for ATM, but defective for Mre11, Rad50 and Nbs1 (the MRN complex) protein expression. Their response to gamma-radiation was abnormal, as evidenced by the enhanced radiosensitivity, attenuated autophosphorylation of ATM-S1981 and phosphorylation of the ATM targets p53-S15 and Smc1-S966, failure to form Mre11 nuclear foci and defective G1 checkpoint arrest. The fibroblasts, but not LCLs, from ATLD5 and ATLD6 showed an impaired ATM-dependent Chk2 phosphorylation. These findings further underscore the interconnection between ATM activity and MRN function, which rationalizes the clinical similarity between ataxia-telangiectasia (A-T) and ATLD.

GTSE-1 (G 2 and S phase-expressed-1) protein is specifically expressed during S and G 2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G 2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycledependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G 2 -specific increase of p53 levels as well as cell sensitization to DNA damageinduced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G 2 phases through regulation of p53 function.

VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR) -induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 Mmol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser 19 and Ser 33 -35 , but not of Chk2 Thr 68 , which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr 68 even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways. [Mol Cancer Ther 2007;6(3):935 -44]

The diverse checkpoint responses to DNA damage may reflect differential sensitivities by molecular components of the damage-signalling network to the type and amount of lesions. Here, we determined the kinetics of activation of the checkpoint kinases ATM and Chk2 (the latter substrate of ATM) in relation to the initial yield of genomic DNA single-strand (SSBs) and double-strand breaks (DSBs). We show that doses of c-radiation (IR) as low as 0.25 Gy, which generate vast numbers of SSBs but only a few DSBs per cell (o8), promptly activate ATM kinase and induce the phosphorylation of the ATM substrates p53-Ser15, Nbs1-Ser343 and Chk2-Thr68. The full activation of Chk2 kinase, however, is triggered by treatments inflicting 419 DSBs per cell (e.g. 1 Gy), which cause Chk2 autophosphorylation on Thr387, Chk2-dependent accumulation of p21 waf1 and checkpoint arrest in the S phase. Our results indicate that, in contrast to ATM, Chk2 activity is triggered by a greater number of DSBs, implying that, below a certain threshold level of lesions (o19 DSBs), DNA repair can occur through ATM, without enforcing Chk2-dependent checkpoints.