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PubMed2003

Chk1 and Chk2 kinases in checkpoint control and cancer.

Accumulation of mutations and chromosomal aberrations is one of the hallmarks of cancer cells. This enhanced genetic instability is fueled by defects in the genome maintenance mechanisms including DNA repair and cell cycle checkpoint pathways. Here, we discuss the emerging roles of the mammalian Chk1 and Chk2 kinases as key signal transducers within the complex network of genome integrity checkpoints, as candidate tumor suppressors disrupted in sporadic as well as some hereditary malignancies and as potential targets of new anticancer therapies.

Animals↗
PubMed2004

CHK1 kinase activity assay.

Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells; (2) preparing substrate; (3) immunoprecipitating CHK1 protein from the cells; (4) assembling the kinase assay; and (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for CHK2 activity assay, except that the CHK2 antibody will replace the CHK1 antibody.

Antibodies, Monoclonal↗
PubMed1996

Signal transduction in the cell cycle regulation of Caulobacter differentiation.

Caulobacter crescentus differentiates to form a new cell type during asymmetric cell division. Recent results indicate that signal transduction pathways mediated by protein kinases and essential response regulators play a central role in the regulation of development and cell division in response to cell cycle checkpoints.

Caulobacter crescentus↗
PubMed1995

Misregulated expression of the cyclin dependent kinase 2 protein in human fibroblasts is accompanied by the inability to maintain a G2 arrest following DNA damage.

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in checkpoint regulation between normal and transformed cells, we have compared the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G1 and G2 for in excess of 48 h after DNA damage. Surprisingly, both cyclin-dependent kinase 2 (CDK2) and cyclin A proteins were specifically down-regulated within 24 h of DNA damage. In contrast, TAg transformed cells did not down-regulate either cyclin A or CDK2 after DNA damage and showed a significantly shortened G2 arrest. To investigate the role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells fail to down-regulate both CDK2 protein and CDK2 protein kinase activity after DNA damage and also show a G2 checkpoint defect; although the cells are able to normally initiate both a G1 and a G2 arrest, they prematurely escape the G2-M arrest after DNA damage. The escape from G2 correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that the misregulation of CDK2 contributes to the failure of checkpoint control by allowing cells to enter mitosis prematurely.

CDC2-CDC28 Kinases↗
PubMed2004

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a targeted mutation of the Chk2 phosphorylation site in Brca1.

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

Aging↗
PubMed2005

ATRIP oligomerization is required for ATR-dependent checkpoint signaling.

The ATM and ATR kinases signal cell cycle checkpoint responses to DNA damage. Inactive ATM is an oligomer that is disrupted to form active monomers in response to ionizing radiation. We examined whether ATR is activated by a similar mechanism. We found that the ATRIP subunit of the ATR kinase and ATR itself exist as homooligomers in cells. We did not detect regulation of ATR or ATRIP oligomerization after DNA damage. The predicted coiled-coil domain of ATRIP is essential for ATRIP oligomerization, stable ATR binding, and accumulation of ATRIP at DNA lesions. Additionally, the ATRIP coiled-coil is also required for ATRIP to support ATR-dependent checkpoint signaling to Chk1. Replacing the ATRIP coiled-coil domain with a heterologous dimerization domain restored stable binding to ATR and localization to damage-induced intranuclear foci. Thus, the ATR-ATRIP complex exists in higher order oligomeric states within cells and ATRIP oligomerization is essential for its function.

Adaptor Proteins, Signal Transducing↗
PubMed1996

5-Azacytidine treatment of the fission yeast leads to cytotoxicity and cell cycle arrest.

A fission yeast gene which shares considerable sequence homology with cytosine-specific DNA methyltransferases has recently been identified. This discovery has led us to investigate the effects of the treatment of fission yeast with the nucleoside analogue 5-azacytidine (5-azaC). 5-AzaC is known to inhibit cytosine methylation as a result of the formation of stable covalent complexes between DNA (cytosine-5) methyltransferases (C5 Mtases) and 5-azaC containing DNA. Here we demonstrate that 5-azaC treatment of Schizosaccharomyces pombe leads to reversible cell cycle arrest at the G2/M transition. This reversible arrest is dependent on the cell cycle checkpoint mechanisms which act to prevent the onset of mitosis in the presence of either damaged or unreplicated DNA. Treatment of S. pombe cell division cycle and checkpoint mutants indicates that 5-azaC causes DNA damage and is likely to inhibit a late stage in DNA replication. The data show that viability in the presence of the drug requires both the DNA damage and the replication checkpoint pathways to be functional. 5-AzaC also elicits a transcriptional response which is associated with DNA damage and the inhibition of DNA replication in fission yeast, and this response is absent in cells carrying G2 checkpoint mutations. The implications of these observations for both the use of 5-azaC in cancer chemotherapy and the existence of cytosine methylation in fission yeast are discussed.

Azacitidine↗
PubMed2003

NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage.

Signaling pathways in response to DNA double strand breaks involve molecular cascades consisting of sensors, transducers, and effector proteins that activate cell cycle checkpoints and recruit repair machinery proteins. NFBD1 (a nuclear factor with BRCT domains protein 1) contains FHA (forkhead-associated), BRCT (breast cancer susceptibility gene 1 carboxyl terminus) domains, and internal repeats and is an early participant in nuclear foci in response to IR. To elucidate its role in the response pathways, small interfering RNA (siRNA) directed against NFDB1 in human cells demonstrated that its absence is associated with increased radio-sensitivity and delayed G(2)/M transition, but not G(1) to S. NFBD1 associates with nuclear foci within minutes following IR, a property similar to histone H2AX, 53BP1, and Chk2, which are all early participants in the DNA damage signaling cascade. Temporal studies show that H2AX is required for the foci positive for NFBD1, but NFBD1 is not needed for 53BP1- and H2AX-positive foci. NFBD1, together with 53BP1, plays a partially redundant role in regulating phosphorylation of the downstream effector protein, Chk2, since abrogation of both diminishes phosphorylated Chk2 in IR-induced foci. These results place NFBD1 parallel to 53BP1 in regulating Chk2 and downstream of H2AX in the recruitment of repair and signaling proteins to sites of DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

DNA damage responses in cell cycle G2 phase and mitosis--tracking and targeting.

BACKGROUND: In order to determine temporal responses of cell cycle populations to DNA damage, a rational combination of cell cycle analyses is critical. Moreover, the targeting of cell cycle checkpoint responses may modify the cytotoxic effect of DNA damage. MATERIALS AND METHODS: The characteristics of cell cycle populations (DNA content, cell cycle transitioning of S phase cells and size of mitotic cell fraction within the total G2/M phase population) in HeLa cells exposed to ionizing radiation were analyzed using three individual flow cytometry-based assays. The potential radiosensitization from inhibiting DNA damage responses was assessed by the colony formation assay. RESULTS: Irradiation resulted in an initial accumulation of S phase cells in G2 phase, from which the arrested cells were subsequently released to enter mitosis. Upon drug inhibition of G2 checkpoint signaling or mitotic progression, the cytotoxic effect of ionizing radiation on the HeLa cells was amplified. CONCLUSION: DNA damage-induced cell cycle responses, analyzed by selected cytometry assays and modified by specific targeting, might contribute to an understanding of how to improve radiotherapy outcome.

Cell Division↗
PubMed1997

The ATM gene and protein: possible roles in genome surveillance, checkpoint controls and cellular defence against oxidative stress.

The autosomal recessive disorder ataxia-telangiectasia (AT) is highly pleiotropic. It is characterized by gradual loss of Purkinje cells in the cerebellum, leading to progressive neuromotor deterioration, immunodeficiency, developmental defects in specific tissues, profound predisposition to malignancy and acute sensitivity to ionizing radiation. AT cells show chromosomal instability, premature senesence, radiosensitivity and defects in cell cycle checkpoints activated by ionizing radiation. Several radiation induced pathways that regulate the cell cycle seem to be defective in AT cells, at least one of which is mediated by TP53. Extensive characterization of the cellular defects of AT cells, together with the recent isolation of the ATM gene, has provided some insight into the possible physiological roles of the ATM protein. Several lines of evidence, including the nature of the agents that elicit the hypersensitivity of AT cells, point to the possibility of a defect in the response to damage induced by oxidative stress, which affects various cellular macromolecules. The ATM protein might have a role in activating defence mechanisms against oxidative stress. This hypothesis broadens the previous concept of the AT defect and explains several aspects of the AT phenotype that cannot be accounted for by defective processing of DNA damage.

Ataxia Telangiectasia↗
PubMed2003

A mutation in protein phosphatase type 2A as a cause of melanoma progression.

The BL6 subline was derived from the F10 line, which was derived from the B16 mouse melanoma cell line. BL6 cells are more invasive than F10 cells and differ genetically from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A). This alteration results in the transcription of mRNA encoding a truncated variant of the B56gamma1 isoform (Delta-gamma-1). Delta-gamma-1 is capable of targeting PP2A to the specific subcellular sites but incapable of promoting the dephosphorylation of specific substrates that is normally mediated by the B56gamma subunit-containing PP2A holoenzyme. It thus appears that activities of this type of holoenzymes decrease in cells expressing Delta-gamma-1. Recently, we found two possible ways how Delta-gamma-1 contributes to the enhanced metastatic potential of BL6 cells. The two ways seemed far away from each other: Delta-gamma-1 influenced both the nuclear and cytoplasmic functions of the cell. In the cytoplasm, Delta-gamma-1 localized at the Golgi complex and accelerated Golgi-mediated vesicle transport. On the other hand, Delta-gamma-1 disturbed the cell-cycle regulation. In response to gamma-irradiation, protein levels of Delta-gamma-1 were markedly increased in BL6 cells. Subsequently the integrity of cell-cycle checkpoint became more aberrant in BL6 cells than that in F10 cells. These two actions of Delta-gamma-1 could results in the enhancement of the malignant phenotypes of melanoma cells, as discussed in this review.

Animals↗
PubMed2002

Checking in on Cds1 (Chk2): A checkpoint kinase and tumor suppressor.

Together, DNA repair and checkpoint responses ensure the integrity of the genome. Coordination of cell cycle checkpoints and DNA repair are especially important following genotoxic radiation or chemotherapy, during which unusually high loads of DNA damage are sustained. In mammalian cells, the checkpoint kinase, Cds1 (also known as Chk2) is activated by ATM in response to DNA damage. The role of Cds1 as a checkpoint kinase depends on its ability to phosphorylate cell cycle regulators such p53, Cdc25 and Brca1. A role for Cds1 in repair is suggested by the finding that it interacts with the Holliday junction resolving activity Mus81. This review focuses on the many questions generated by recent progress in understanding the function and regulation of human Cds1.

Animals↗
PubMed2005

Cross-talk between DNA damage and cell survival checkpoints during G2 and mitosis: pharmacologic implications.

In this study, we wanted to clarify the role of survivin-mediated survival signaling during G2 and M in tumor cells treated with DNA-damaging agents. As a cellular model, we selected MOLT-4 human T-cell lymphoblastic leukemia cells that overexpress survivin and nonfunctional p53. Treatment with melphalan, a classic DNA-damaging agent, led to the induction of the DNA damage checkpoint and growth arrest in the G2 phase of the cell cycle. Checkpoint abrogation by caffeine was accompanied by mitotic entry and rapid apoptotic cell death, whereas cells remaining in G2 remained viable during the same time interval. Unexpectedly, when the spindle checkpoint was activated following G2 abrogation, two different effects could be observed. If the microtubules of the melphalan-treated cells were destabilized by nocodazole, cells became arrested in prometaphase with low survivin levels and entered apoptosis. In contrast, if the microtubules of the melphalan-treated cells were stabilized by taxol, cells were still arrested in prometaphase, but apoptotic execution was inhibited. This effect is, most likely, directly mediated by survivin itself given its well-established antiapoptotic functions. In conclusion, depending on the way the spindle checkpoint was activated in cells with damaged DNA, cells could be either protected by survivin or die during mitosis. We suggest that the efficacy of DNA damage checkpoint abrogators used in combination with DNA-damaging agents may critically depend on whether DNA damage is able to invoke spindle checkpoint response and to activate survivin-associated survival signaling during mitosis.

Antineoplastic Agents, Alkylating↗
PubMed2006

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage.

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

Ataxia Telangiectasia↗
PubMedDate not supplied

Claspin, a regulator of Chk1 in DNA replication stress pathway.

Regulation of the vertebrate checkpoint kinase Chk1 involves several protein complexes including the recently identified protein Claspin. Claspin associates with Chk1 upon replication stress and DNA damage and is required for Chk1 activation in both Xenopus and human systems. More importantly, Claspin is involved in regulation of cell cycle checkpoints. Here, we discuss the emerging roles of Claspin in the Chk1 pathway and its functions in checkpoint control.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ribosomal proteins in cell proliferation and apoptosis.

Ribosomal proteins have the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival. Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. A number of these proteins function as cell proliferation regulators and in some instances as inducers of cell death. Specifically, expression of human ribosomal protein L13a has been shown to induce apoptosis, presumably by arresting cell growth in the G2/M phase of the cell cycle. In addition, inhibition of expression of L13a induces apoptosis in target cells, suggesting that this protein is necessary for cell survival. Similar results have been obtained in the yeast Saccharomyces cerevisiae, where inactivation of the yeast homologues of L13a, rp22 and rp23, by homologous recombination results in severe growth retardation and death. In addition, a closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis. Thus, it appears that a group of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.

Animals↗
PubMed2003

Expression deconvolution: a reinterpretation of DNA microarray data reveals dynamic changes in cell populations.

Cells grow in dynamically evolving populations, yet this aspect of experiments often goes unmeasured. A method is proposed for measuring the population dynamics of cells on the basis of their mRNA expression patterns. The population's expression pattern is modeled as the linear combination of mRNA expression from pure samples of cells, allowing reconstruction of the relative proportions of pure cell types in the population. Application of the method, termed expression deconvolution, to yeast grown under varying conditions reveals the population dynamics of the cells during the cell cycle, during the arrest of cells induced by DNA damage and the release of arrest in a cell cycle checkpoint mutant, during sporulation, and following environmental stress. Using expression deconvolution, cell cycle defects are detected and temporally ordered in 146 yeast deletion mutants; six of these defects are independently experimentally validated. Expression deconvolution allows a reinterpretation of the cell cycle dynamics underlying all previous microarray experiments and can be more generally applied to study most forms of cell population dynamics.

Cell Cycle↗
PubMed1997

Interaction of MAD2 with the carboxyl terminus of the insulin receptor but not with the IGFIR. Evidence for release from the insulin receptor after activation.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor (IR). We identified a human cDNA encoding a protein that appears to be the human homolog of the yeast MAD2 protein, which we term hMAD2. The yeast MAD2 protein was first identified in a genetic screen to identify cell cycle checkpoint regulatory proteins, yet the mechanism by which MAD2 functions in cell cycle control is currently unclear. Here we show that hMAD2 requires the COOH-terminal 30 amino acids of the IR for interaction and that hMAD2 does not interact with the related insulin-like growth factor I receptor. Interestingly, hMAD2 does not require IR tyrosine autophosphorylation for interaction because it interacts with a kinase-dead IR in the yeast two-hybrid system. In support of this finding, hMAD2-GST fusions were found to interact strongly in vitro with receptors derived from noninsulin-stimulated cells. Furthermore, using two independent in vitro assays, IR activation was found to significantly reduce the interaction of hMAD2 with the IR. Lastly, we show that hMAD2 can be coimmunoprecipitated with the IR from Chinese hamster ovary IR cell lysates, suggesting that this interaction occurs in vivo in cells of mammalian origin. Our results suggest that hMAD2 represents a novel class of proteins that is specific for interaction with the IR as compared with the insulin-like growth factor I receptor and that interacts best with the inactive IR and is released upon receptor autophosphorylation. The function of hMAD2 and its potential role in insulin signaling remain to be elucidated.

Amino Acid Sequence↗