DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.     

Regulation of Rad17 Protein Turnover Unveils an Impact of Rad17-APC Cascade in Breast Carcinogenesis and Treatment

Aberrant regulation of DNA damage checkpoint function leads to genome instability that in turn can predispose cellular tissues to become cancerous. Previous works from us and others demonstrated the role of Rad17 in either activation or termination of DNA damage checkpoint function. In the current study, we have revealed the unexpected accumulation of Rad17 in various types of breast cancer cell lines as well as human breast cancer tissues. We observed that Rad17 protein turnover rate in breast epithelial cells is much faster than in breast cancer cells, where the turnover of Rad17 is regulated by the Cdh1/APC pathway. We further observed that Rad17-mediated checkpoint function is modulated by proteolysis. Stabilization of Rad17 disrupts cellular response to chemotherapeutic drug-induced DNA damage and enhances cellular transformation. In addition, manipulation of Rad17 by RNA interference or stabilization of Rad17 significantly sensitize breast cancer cell to various chemotherapeutic drugs. Our present results indicate the manipulation of Rad17 proteolysis could be a valuable approach to sensitize breast cancer cell to the chemotherapeutic treatment despite of the critical role in governing DNA damage response and cellular recovery from genotoxic stress.     

Oxidative stress and cell cycle checkpoint function

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.     

Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit-/- cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit-/- cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression.     

UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.     

Genomic instability and cancer: networks involved in response to DNA damage

A new approach to cancer and new methods in examining rare human chromosome breakage syndromes have brought to light complex interactions between different pathways involved in damage response, cell cycle checkpoint control and DNA repair. The genes affected in these different syndromes are involved in networks of processes that respond to DNA damage and prevent chromosomal aberrations during the cell cycle. The genes involved include the ATM, ATR, FA-associated genes, NBS1 and the cancer susceptibility genes BRCA1 and BRCA2. Chromosomal instability is a common feature of many human cancers and most of the instability syndromes, characterized by sensitivity to different types of DNA damage, also show increased cancer susceptibility. Better understanding of these syndromes and their links with familial cancer provide new insight into associations between defects in DNA damage response, cell cycle control, DNA repair and cancer. Understanding the damage response repair networks that these studies are revealing will have important implications for the development of cancer management and treatment.     

WEE1 inhibition and genomic instability in cancer

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G₁ and G₂ checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G₁ arrest and rely on G₂ arrest for DNA damage responses. WEE1 kinase is an important regulator of the G₂ checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G₂ arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.     

DNA damage-dependent cyclin D1 proteolysis: GSK3β holds the smoking gun

Ubiquitin mediated degradation of cyclin D1 following the G1/S transition counters its mitogen-dependent accumulation during G1 phase of the cell cycle. Although the cellular machinery responsible for this process has been identified, how this regulatory pathway interfaces to cellular stress responses, often referred to as checkpoints, remains to be established. One intensely investigated checkpoint is the cellular response to DNA damage. When DNA damage is sensed, the corresponding DNA damage checkpoint triggers the inhibition of CDK-dependent cell cycle progression, with arrest coordinated by induction of CDK inhibitors and rapid degradation of specific cyclins, such as cyclin D1. In recent work, we identified a phosphorylation- and Fbx4-dependent cyclin D1 degradation mechanism in response to genotoxic stress.18 This work revealed that loss of cyclin D1 regulation compromises the intra-S-phase response to DNA damage, promoting genomic instability and sensitization of cells to S-phase chemotherapy, highlighting a potential therapeutic strategy for cancers exhibiting cyclin D1 accumulation.     

DNA Damage Checkpoints and Cancer

DNA damage checkpoint is one of the surveillance systems to maintain genomic integrity. Checkpoint systems sense the DNA damage and execute cell cycle arrest through inhibiting the activity of cell cycle regulators. This pathway is essential for the maintenance of genome stability and prevention of tumor development. Recent studies have showed that the cellular responses towards DNA damage, such as cell cycle arrest, DNA repair, chromatin remodeling, and apoptosis are well coordinated. Here we describe the molecular mechanisms of checkpoint activation in response to DNA damage and the correlation between checkpoint gene mutation and genomic instability.     

A method for assaying the sensitivity of Drosophila replication checkpoint mutants to anti-cancer and DNA-damaging drugs.

In multi-cellular organisms, failure to properly regulate cell-cycle progression can result in inappropriate cell death or uncontrolled cell division leading to tumor formation. To guard against such events, conserved regulatory mechanisms called "checkpoints" block progression into mitosis in response to DNA damage and incomplete replication, as well as in response to other signals. Checkpoint mutants in organisms as diverse as yeast and humans are sensitive to various chemical agents that inhibit DNA replication or cause DNA damage. This phenomenon is the primary rationale for chemotherapy, which uses drugs that preferentially target tumor cells with compromised checkpoints. In this study, we demonstrate the use of Drosophila checkpoint mutants as a system for assaying the effects of various DNA-damaging and anti-cancer agents in a developing multicellular organism. Dwee1, grp and mei-41 are genes that encode kinases that function in the DNA replication checkpoint. We tested zygotic mutants of each gene for sensitivity to the DNA replication inhibitor hydroxyurea (HU), methyl methanosulfonate (MMS), ara-C, cisplatin, and the oxygen radical generating compound paraquat. The mutants show distinct differences in their sensitivity to each of the drugs tested, suggesting an underlying complexity in the responses of individual checkpoint genes to genotoxic stress.     

Resisting Arrest: Recovery from Checkpoint Arrest Through Dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signalling, and speculate on models for checkpoint maintenance and release.     

Resisting arrest: recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release.     

chk-1 is an Essential Gene and is Required for an S-M Checkpoint During Early Embryogenesis

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe [1]. Genetic analysis revealed that chk1 is involved in a DNA damage G2-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G2-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide [2]. In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles [3, 4]. What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M hence causing premature entry into M-phase.     

Cell cycle checkpoints and their inactivation in human cancer

Checkpoints are mechanisms that regulate progression through the cell cycle insuring that each step takes place only once and in the right sequence. Mutations of checkpoint proteins are frequent in all types of cancer as defects in cell cycle control can lead to genetic instability. This review will focus on three major areas of cell cycle transition control, with particular attention to the alterations found in human cancer. These areas include the G1/S transition, where most cancer-related defects occur, the G2/M checkpoint and its activation in response to DNA damage, and the spindle checkpoint.     

Functional characterization of a novel BRCA1-null ovarian cancer cell line in response to ionizing radiation

The breast and ovarian cancer susceptibility gene BRCA1 plays a major role in the DNA damage response pathway. The lack of well-characterized human BRCA1-null cell lines has limited the investigation of BRCA1 function, particularly with regard to its role in ovarian cancer. We propagated a novel BRCA1-null human ovarian cancer cell line UWB1.289 from a tumor of papillary serous histology, the most common form of ovarian carcinoma. UWB1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele. UWB1.289 is estrogen and progesterone receptor negative and has an acquired somatic mutation in p53, similar to the commonly used BRCA1-null breast cancer cell line HCC1937. We used ionizing radiation to induce DNA damage in both UWB1.289 and in a stable UWB1.289 line in which wild-type BRCA1 was restored. We examined several responses to DNA damage in these cell lines, including sensitivity to radiation, cell cycle checkpoint function, and changes in gene expression using microarray analysis. We observed that UWB1.289 is sensitive to ionizing radiation and lacks cell cycle checkpoint functions that are a normal part of the DNA damage response. Restoration of wild-type BRCA1 function in these cells partially restores DNA damage responses. Expression array analysis not only supports this partial functional correction but also reveals interesting new information regarding BRCA1-positive regulation of the expression of claudin 6 and other metastasis-associated genes and negative regulation of multiple IFN-inducible genes.     

Claspin functions in cell homeostasis—A link to cancer?

Cancer remains one of the leading causes of mortality worldwide. Most cancers present high degrees of genomic instability. DNA damage and replication checkpoints function as barriers to halt cell cycle progression until damage is resolved, preventing the perpetuation of errors. Activation of these checkpoints is critically dependent on Claspin, an adaptor protein that mediates the phosphorylation of the effector kinase Chk1 by ATR. However, Claspin also performs other roles related to the protection and maintenance of cell and genome integrity. For instance, following DNA damage and checkpoint activation, Claspin bridges checkpoint responses to DNA repair or to apoptosis. During DNA replication, Claspin acts a sensor and couples DNA unwinding to strand polymerization, and may also indirectly regulate replication initiation at firing origins. As Claspin participates in several processes that are vital to maintenance of cell homeostasis, its function is tightly regulated at multiple levels. Nevertheless, little is known about its role in cancer. Accumulating evidence suggests that Claspin inactivation could be an essential event during carcinogenesis, indicating that Claspin may function as a tumour suppressor. In this review, we will examine the functions of Claspin and how its deregulation may contribute to cancer initiation and progression. To conclude, we will discuss means by which Claspin can be targeted for cancer therapy.     

Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response

DNA damage that is not properly repaired during genomic replication is a major source of gross chromosomal rearrangements and sequence loss during cell proliferation. In higher eukaryotes such mutations increase the risk of cancer. Eukaryotic cells have multiple checkpoint responses activated by DNA damage and stalled replication forks. We focus here on fork-associated events that activate and respond to S-phase checkpoint kinases.     

Selective Induction of Necrotic Cell Death in Cancer Cells by β-Lapachone through Activation of DNA Damage Response Pathway

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells. We found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to β-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked β-lapachone-induced necrosis. Furthermore, necrotic cell death induced by β-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy     

Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication

Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.