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PubMed2006

Signals from within: the DNA-damage-induced NF-kappaB response.

An appropriate response to genotoxic stress is essential for maintenance of genome stability and avoiding the passage to neoplasia. Nuclear factor kappaB (NF-kappaB) is activated as part of the DNA damage response and is thought to orchestrate a cell survival pathway, which, together with the activation of cell cycle checkpoints and DNA repair, allows the cell in cases of limited damage to restore a normal life cycle, unharmed. In this respect, NF-kappaB is one of the main factors accounting for chemotherapy resistance and as such impedes effective cancer treatment, representing an important drug target. Despite this high clinical relevance, signalling cascades leading to DNA damage-induced NF-kappaB activation are poorly understood and the use of highly divergent experimental set-ups in the past led to many controversies in the field. Therefore, in this review, we will try to summarize the current knowledge of distinct DNA damage-induced NF-kappaB signalling pathways.

Apoptosis↗
PubMed2005

The yeast ubiquitin ligase SCFMet30 regulates heavy metal response.

Cells have developed a variety of mechanisms to respond to heavy metal exposure. Here, we show that the yeast ubiquitin ligase SCF(Met30) plays a central role in the response to two of the most toxic environmental heavy metal contaminants, namely, cadmium and arsenic. SCF(Met30) inactivates the transcription factor Met4 by proteolysis-independent polyubiquitination. Exposure of yeast cells to heavy metals led to activation of Met4 as indicated by a complete loss of ubiquitinated Met4 species. The association of Met30 with Skp1 but not with its substrate Met4 was inhibited in cells treated with cadmium. Cadmium-activated Met4 induced glutathione biosynthesis as well as genes involved in sulfuramino acid synthesis. Met4 activation was important for the cellular response to cadmium because mutations in various components of the Met4-transcription complex were hypersensitive to cadmium. In addition, cell cycle analyses revealed that cadmium induced a delay in the transition from G(1) to S phase of the cell cycle and slow progression through S phase. Both cadmium and arsenic induced phosphorylation of the cell cycle checkpoint protein Rad53. Genetic analyses demonstrated a complex effect of cadmium on cell cycle regulation that might be important to safeguard cellular and genetic integrity when cells are exposed to heavy metals.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae.

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.

Cell Cycle↗
PubMed2004

Down-regulation of nuclear protein ICBP90 by p53/p21Cip1/WAF1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition.

Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21Cip1/WAF1, resulting in cell cycle arrest at G1/S transition by inhibition of cdk activity and DNA replication. ICBP90 was identified as a nuclear protein that binds to the TopoII alpha gene promoter and is speculated to be involved in DNA replication. ICBP90 expression is cell cycle regulated in normal cells but stably high throughout cell cycle in various cancer cell lines. We here demonstrate that ICBP90 expression is down-regulated by the p53/p21Cip1/WAF1-dependent DNA damage checkpoint signals. The reduction of ICBP90 appeared to be caused by both transcriptional suppression and protein degradation. Adenoviral expression of p21Cip1/WAF1 directly led to ICBP90 reduction in p53-/- HCT116 cells without DNA damage. Furthermore, ICPB90 depletion by RNA interference significantly blocked G1/S transition after DNA damage in HeLa cells. The down-regulation of ICBP90 is an important mechanism for cell cycle arrest at G1/S transition, which is induced by the activation of a p53/p21Cip1/WAF1-dependent DNA-damage checkpoint. Deregulation of ICBP90 may impair the control of G1/S transition during checkpoint activation and lead to genomic instability.

Amanitins↗
PubMed2000

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells.

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.

Blotting, Northern↗
PubMed1991

Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle.

Two thioredoxin genes from the yeast Saccharomyces cerevisiae were cloned using synthetic oligonucleotide probes. The DNA sequences of the two genes were found to be 74% identical. The two genes, designated TRX1 and TRX2, were mutagenized in vitro and used to construct a set of thioredoxin deletion mutants. The loss of either thioredoxin gene alone has no effect on cell growth or morphology. However, the simultaneous deletion of both thioredoxin genes profoundly affects the cell cycle. S phase is 3-fold longer, and G1 is virtually absent. In addition, the thioredoxin double mutant shows a 33% increase in generation time, a significant increase in cell size, and a greater proportion of large budded cells. The results suggest that in the absence of TRX1 and TRX2, a slow rate of DNA replication inhibits the normal progress of cellular reproduction. Surprisingly, the loss of both thioredoxins also leads to methionine auxotrophy. Thus yeast glutaredoxin is unable to substitute for thioredoxin in sulfate assimilation. As a first step in studying the cell cycle control mechanisms that respond to the thioredoxin deficiency, it was shown that cell viability does not require the function of RAD9, a known cell cycle checkpoint.

Base Sequence↗
PubMed2004

Characterization of cell-cycle progression and growth of WB-F344 normal rat liver epithelial cells following gamma-ray exposure.

BACKGROUND: Apparently normal rat liver epithelial cells (WB-F344) have been widely used in studies pertaining to carcinogenesis. Ionizing radiation, a well known carcinogen, is known to perturb cell-cycle progression in a dose-dependent manner, thereby causing delay in cell proliferation. However, for WB-F344 cells, there is a paucity of such data, which are of substantial importance in understanding their radiation response. Here, the distribution of phases in the cell-cycle and the proliferation ability of WB-F344 cells are characterized at various time points after the cells have been irradiated with different doses of gamma-rays. METHODS: After WB-F344 cells reached 100% confluence, they were trypsinized and suspended at 3.5 x 10(5) cells/ml in culture medium. Cells were irradiated in suspension with (137)Cs gamma-rays at doses from 1-10 Gy. After irradiation, 1 x 10(5) cells were plated into 60 x 15-mm culture dishes and incubated at 37 degrees C, with 2% CO(2) and 98% air. At 12, 24, 36, 48, and 60 h postirradiation, cells were harvested, counted, and subjected to flow cytometric cell-cycle analysis. RESULTS: Growth curves of WB-F344 cells irradiated with gamma-rays started to separate at 36 h postirradiation. By 60 h postirradiation, the growth curves for each of the 10 absorbed doses were distinctly separated. Drastic redistributions of control and irradiated cells within G(0)/G(1)-, S-, and G(2)/M-phases of the cell cycle were observed during the first 36 h of cell growth. At each time point postirradiation, cell-cycle phase profiles of irradiated cells were altered in a dose-dependent manner. In general, there was a strong correlation between the percentage of G(2)/M-phase cells and absorbed dose, with the exception of 24 h postirradiation. The percentage of G(2)/M-phase cells increased as a function of time postirradiation, suggestive of delays in the passage of cells through the G(2) cell-cycle checkpoint. CONCLUSIONS: This work provides a general description of cell cycle redistribution and repopulation kinetics of WB-F344 cells at various times postirradiation of quiescent cells that were subsequently allowed to proliferate. In general, growth inhibition and delays in progression through G(2)/M-phase correlated well with radiation dose. These data should be of considerable significance in the design of experiments that examine the radiation response of these cells.

Animals↗
PubMed2004

[Gadd45 mediated G2/M cell cycle arrest induced by BRCA1].

BACKGROUND & OBJECTIVE: It is considered that tumor suppressor gene BRCA1 is an important factor in the regulation of cell cycle checkpoint, but the molecular mechanism by which BRCA1 regulates cell cycle G(2)/M arrest is less known. The objective of this study was to investigate the effects of Gadd45 on the BRCA1-induced cell growth suppression. METHODS: BRCA1 induction of Gadd45 protein was analyzed using Western-blot assay following cells transfection with BRCA1 expression vector and cell sorting. Activation of the Gadd45 promoter by BRCA1 was determined by CAT assay. Effect of antisense Gadd45 on the BRCA1-induced cell cycle G(2)-M arrest was examined by flow cytometry analysis. And the effects of antisense Gadd45 on BRCA1-mediated growth suppression in HeLa and HCT116 cell lines was determined by colony formation assay. RESULTS: Gadd45 protein was highly induced after expression of BRCA1. BRCA1 strongly activated the Gadd45 promoter. Antisense Gadd45 substantially abrogated BRCA1-activated cell cycle G(2)-M arrest and BRCA1-induced cell growth suppression on HeLa and HCT116 cell lines. CONCLUSION: Gadd45 is a BRCA1-regulated downstream gene and mediates the role of BRCA1 in the control of cell cycle G(2)/M arrest and growth suppression.

BRCA1 Protein↗
PubMed2003

DNA damage and expression of checkpoint genes p21(WAF1/CIP1) and 14-3-3 sigma in taurine-deficient cardiomyocytes.

OBJECTIVE: Taurine depletion is associated with development of cardiomyopathy. Further, oxidative stress is advanced as a critical factor mediating the effect of taurine deficiency on target organs. However, the molecular mechanism(s) linking taurine deficiency with the development of cardiomyopathy remains elusive. Since transition between apoptotic degeneration and cell proliferation in stress conditions is regulated at cell cycle checkpoints, we determined the expression of two such genes, namely p21(WAF1/CIP1) and 14-3-3 sigma as well as p53 that are responsible for oxidative stress and DNA damage. We also carried out quantitative determination of DNA damage. METHODS: Cardiomyocytes from beta-alanine-induced taurine-depleted (TD) rats were used for this investigation. Single- and double-stranded DNA damage was quantified using comet assay analysis. Western blot and two-dimensional polyacrylamide gel electrophoresis with immunoblotting analysis were applied for protein analysis. RESULTS: Comet assay analysis indicated that the extent of double-stranded DNA damage was greater in TD than in control cardiomyocytes. Whereas only traces of both p53 and p21(WAF1/CIP1) and no detectable expression of 14-3-3 sigma were found in cardiomyocytes of control animals, the TD cardiomyocytes expressed all three genes. CONCLUSIONS: DNA damage and the consequent up-regulation of checkpoint proteins observed in TD cardiomyocytes indicate the involvement of cell cycle control mechanisms in the effect of taurine deficiency on cardiomyocytes. Single- and double-stranded DNA damage and the consequent arrest of cell proliferation in both G(1) and G(2) phases of the cell cycle induced by checkpoint proteins may trigger the cardiomyopathy that is associated with taurine deficiency.

14-3-3 Proteins↗
PubMed2004

The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation.

The centromere defines where on a chromosome the kinetochores assemble. Kinetochores, large protein structures, mediate chromosome segregation during mitosis and meiosis by performing three key functions. Firstly, kinetochores attach chromosomes to the microtubule spindle apparatus. Secondly, kinetochores co-ordinate microtubule dynamics to allow chromosomes to move along the spindle. Lastly, kinetochores generate the 'wait' signal which prevents anaphase onset until all the chromosomes are correctly aligned on the spindle. This signal forms part of the spindle checkpoint mechanism, a highly conserved cell cycle checkpoint which maintains the accuracy of the chromosome segregation process. This article provides a brief historical overview before focusing on some of the outstanding issues and more recent developments in the field.

Amino Acid Sequence↗
PubMed1999

[Effect of inactivating various components of the signal pathways of the tumor suppressor p53 on genomic stability].

To evaluate the role of different p53-regulated signaling pathways in the control of genomic integrity, we studied the frequency of changes in chromosome number and structure of cells of the sublines of mouse primary embryonic fibroblasts with the "knocked-out" genes for proteins p53, p21WAF, pRb, and p19ARF. Protein p21WAF is transactivated by p53 and is responsible for the cell block in the G1 phase of the damaged cells; protein pRb is a target for p21WAF which controls the G1-S-phase transition; and p19ARF protein is responsible for p53 activation in cells with certain anomalies. Inactivation of either of the studied genes proved to increase significantly the frequency of changes in the karyotype. However, the resultant chromosome instability differed: the frequency of the chromosome breaks, both spontaneous and induced with ethylmethane sulfonate (EMS), was in cells with inactivated p53 and lowest in cells with inactivated pRb. These distinctions were not caused by a different effect of various gene inactivation on the cell cycle progression: in all sublines, the cell block in G1 was abolished and the checkpoint function in G2 remained normal. However, the induction of apoptosis in EMS-treated cells differed in the studied sublines. The lowest number of apoptotic nuclei were determined in p53-/- cultures, whereas the highest were in the Rb-/- cultures. It is apparent that the degree of genetic instability is determined by a combined effect of apoptosis and abnormal regulation of the cell-cycle checkpoints.

Animals↗
PubMed1995

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'.

BACKGROUND: The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. RESULTS: We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. CONCLUSIONS: Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Cell Cycle↗
PubMed1995

Checkpoints in the cell cycle of fission yeast.

When cell cycle progression in fission yeast is disrupted, checkpoint controls ensure that the normal sequence of cell cycle events is maintained. Activation of a checkpoint relies on monitoring signals that might involve assembly of macromolecular structures essential for specific cell cycle processes. The past year has seen further elucidation of two new checkpoints operating during the cell cycle of Schizosaccharomyces pombe. One involves the product of the rum1 gene and prevents cells from entering mitosis from the pre-Start G1 interval. The second checkpoint operates during the later stages of the cell cycle and is essential for coupling the events of mitosis and cell division.

Cell Cycle↗
PubMed2005

Docetaxel induces cell death through mitotic catastrophe in human breast cancer cells.

Apoptosis has long been considered to be the prevailing mechanism of cell death in response to chemotherapy. Currently, a more heterogeneous model of tumor response to therapy is acknowledged wherein multiple modes of death combine to generate the overall tumor response. The resulting mechanisms of cell death are likely determined by the mechanism of action of the drug, the dosing regimen used, and the genetic background of the cells within the tumor. This study describes a nonapoptotic response to docetaxel therapy in human breast cancer cells of increasing cancer progression (MCF-10A, MCF-7, and MDA-mb-231). Docetaxel is a microtubule-stabilizing taxane that is being used in the clinic for the treatment of breast and prostate cancers and small cell carcinoma of the lung. The genetic backgrounds of these cells were characterized for the status of key pathways and gene products involved in drug response and cell death. Cellular responses to docetaxel were assessed by characterizing cell viability, cell cycle checkpoint arrest, and mechanisms of cell death. Mechanisms of cell death were determined by Annexin V binding and scoring of cytology-stained cells by morphology and transmission electron microscopy. The primary mechanism of death was determined to be mitotic catastrophe by scoring of micronucleated cells and cells undergoing aberrant mitosis. Other, nonapoptotic modes of death were also determined. No significant changes in levels of apoptosis were observed in response to docetaxel.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Protein kinase C delta stimulates apoptosis by initiating G1 phase cell cycle progression and S phase arrest.

Overexpression of protein kinase C delta (PKCdelta) stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood. PKCdelta-deficient cells are impaired in their response to DNA damage-induced apoptosis, suggesting that PKCdelta is required to mount an appropriate apoptotic response under conditions of stress. The mechanism through which it does so remains elusive. In addition to effects on cell survival, PKCdelta elicits pleiotropic effects on cellular proliferation. We now provide the first evidence that the ability of PKCdelta to stimulate apoptosis is intimately linked to its ability to stimulate G(1) phase cell cycle progression. Using an adenoviral-based expression system to express PKCalpha,-delta, and -epsilon in epithelial cells, we demonstrate that a modest increase in PKCdelta activity selectively stimulates quiescent cells to initiate G(1) phase cell cycle progression. Rather than completing the cell cycle, PKCdelta-infected cells arrest in S phase, an event that triggers caspase-dependent apoptotic cell death. Apoptosis was preceded by the activation of cell cycle checkpoints, culminating in the phosphorylation of Chk-1 and p53. Strikingly, blockade of S phase entry using the phosphatidylinositol 3-kinase inhibitor LY294002 prevented checkpoint activation and apoptosis. In contrast, inhibitors of mitogen-activated protein kinase cascades failed to prevent apoptosis. These findings demonstrate that the biological effects of PKCdelta can be extended to include positive regulation of G(1) phase cell cycle progression. Importantly, they reveal the existence of a novel, cell cycle-dependent mechanism through which PKCdelta stimulates cell death.

Adenoviridae↗
PubMed2004

Role of cell cycle in mediating sensitivity to radiotherapy.

Multiple pathways are involved in maintaining the genetic integrity of a cell after its exposure to ionizing radiation. Although repair mechanisms such as homologous recombination and nonhomologous end-joining are important mammalian responses to double-strand DNA damage, cell cycle regulation is perhaps the most important determinant of ionizing radiation sensitivity. A common cellular response to DNA-damaging agents is the activation of cell cycle checkpoints. The DNA damage induced by ionizing radiation initiates signals that can ultimately activate either temporary checkpoints that permit time for genetic repair or irreversible growth arrest that results in cell death (necrosis or apoptosis). Such checkpoint activation constitutes an integrated response that involves sensor (RAD, BRCA, NBS1), transducer (ATM, CHK), and effector (p53, p21, CDK) genes. One of the key proteins in the checkpoint pathways is the tumor suppressor gene p53, which coordinates DNA repair with cell cycle progression and apoptosis. Specifically, in addition to other mediators of the checkpoint response (CHK kinases, p21), p53 mediates the two major DNA damage-dependent cellular checkpoints, one at the G(1)-S transition and the other at the G(2)-M transition, although the influence on the former process is more direct and significant. The cell cycle phase also determines a cell's relative radiosensitivity, with cells being most radiosensitive in the G(2)-M phase, less sensitive in the G(1) phase, and least sensitive during the latter part of the S phase. This understanding has, therefore, led to the realization that one way in which chemotherapy and fractionated radiotherapy may work better is by partial synchronization of cells in the most radiosensitive phase of the cell cycle. We describe how cell cycle and DNA damage checkpoint control relates to exposure to ionizing radiation.

Antineoplastic Agents↗
PubMed2006

Expression of checkpoint kinase 2 in breast carcinomas: correlation with key regulators of tumor cell proliferation, angiogenesis, and survival.

Checkpoint kinase 2 (Chk2) is a cell-cycle-checkpoint kinase that may act as a tumor suppressor gene due to its important role in DNA damage signaling and cell cycle regulation. The role of Chk2 expression in mammary tumorigenesis, however, is still poorly understood. This study was designed to assess the relationship between the expression of Chk2 and well-established prognostic factors, including disease-free-survival and overall survival; and several regulators of cell proliferation and invasiveness in breast carcinomas, including oncogenes, tumor suppressor genes, apoptosis-related proteins, and angiogenesis-related markers. Immunohistochemistry with 27 primary antibodies was performed in 100 formalin-fixed paraffin-embedded samples of not otherwise specified invasive ductal carcinomas. Clinical data were retrieved from medical files. In normal mammary parenchyma adjacent to the tumors Chk2 stained the nuclei of epithelial cells. Downexpression of Chk2 protein was observed in 23 carcinomas and correlated with advanced disease. Among the regulators of tumor cell proliferation and invasiveness analyzed, the downexpression of Chk2 correlated only with reduced expression of p27 and telomerase. There was no difference between the overall survival and disease-free survival rates according to Chk2 status. In conclusion, Chk2 correlated with reduced expression of h-TERT and p27, but not with angiogenic factors. Chk2 expression also did not interfere in the outcome of the patients.

Adult↗
PubMed2004

Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells.

This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.

Adaptation, Physiological↗