Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by beta-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.
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PMID:Molecular basis for G2 arrest induced by 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine and consequences of checkpoint abrogation. 1606 71

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.
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PMID:Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe. 1615 83

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.
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PMID:Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation. 1695 7

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.
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PMID:SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage. 1879 17

Translesion synthesis by DNA polymerase eta (poleta) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking poleta are sensitive to these agents. To help define the consequences of poeta-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking poleta. We show that both cisplatin and carboplatin induce strong S-phase arrest in poleta-deficient XP30RO cells, associated with reduced expression of cyclin E and cyclin B. PIK kinase-mediated phosphorylation of Chk1, H2AX and RPA2 was strongly activated by both cisplatin and carboplatin, but phosphorylation of these proteins was induced earlier by cisplatin than by an equitoxic dose of carboplatin. Compared to Chk1 and H2AX phosphorylation, RPA2 hyperphosphorylation on serine4/serine8 is a late event in response to platinum-induced DNA damage. We directly demonstrate, using dual-labeling flow cytometry, that damage-induced phosphorylation of RPA2 on serine4/serine8 occurs primarily in the S and G(2) phases of the cell cycle, and show that the timing of RPA2 phosphorylation can be modulated by inhibition of the checkpoint kinase Chk1. Furthermore, Chk1 inhibition sensitizes poleta-deficient cells to the cytotoxic effects of carboplatin. Both hyperphosphorylated RPA2 and the homologous recombination protein Rad51 are present in nuclear foci after cisplatin treatment, but these are separable events in individual cells. These results provide insight into the relationship between cell cycle regulation and processing of platinum-induced DNA damage in human cells when poleta-mediated TLS is compromised.
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PMID:Characterization of the effects of cisplatin and carboplatin on cell cycle progression and DNA damage response activation in DNA polymerase eta-deficient human cells. 1971 47

Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.
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PMID:Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells. 1995 88

The tumor suppressive activities of the Kip-family of cyclin-dependent kinase (cdk) inhibitors often go beyond their role directly regulating the cell cycle. In this study, we show that p27 enhances Rad51 accumulation during repair of double-strand DNA breaks. Progression of platelet-derived growth factor (PDGF)-induced oligodendrogliomas was accelerated in mice lacking the cyclin-cdk binding activities of p27(kip1). To understand how p27 deficiency contributes, cell lines were developed from RCAS-PDGF infection of nestin-tv-a brain progenitor cells in culture. p27 deficiency did not affect cell proliferation in early passage cell lines; however, the absence of p27 affected chromosomal stability. In p27-deficient cells, the activation of Atm and Chk2 and the accumulation of gamma-H2AX was unaffected when compared with wild-type cells, and the number of phospho-histone H3 staining mitotic cells was decreased, consistent with G2/M checkpoint activation. However, the percentage of Rad51 foci-positive cells was decreased, and the kinase activity that targets the C-terminus of BRCA2, regulating BRCA2/Rad51 interactions, was increased in lysates derived from p27-deficient cells. Increased numbers of chromatid breaks in p27-deficient cells that adapted to the checkpoint were also observed. These findings suggest that Rad51-dependent repair of double-stranded breaks was hindered in p27-deficient cells, leading to chromosomal instability, a hallmark of cancers with poor prognosis.
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PMID:Defective DNA double-strand break repair underlies enhanced tumorigenesis and chromosomal instability in p27-deficient mice with growth factor-induced oligodendrogliomas. 2006 78

We developed a clonal WI-38hTERT/GFP-RAF1-ER immortal cell line to study RAF-induced senescence of human fibroblasts. Activation of the GFP-RAF1-ER kinase by addition of 4-hydroxy-tamoxifen led to a robust induction of senescence within one population doubling, accompanied by the assembly of heterochromatic foci. At least two pathways contribute in parallel to this senescence leading to the accumulation of p15, p16, p21 and p27 inhibitors of cyclin-dependent kinases (CKIs). Cells that traversed S phase after RAF1 kinase activation experienced a replicative stress manifested by phosphorylation of H2AX and Chk2 and synthesis of p21. However, about half the cells in the population were blocked without passing through S phase and did not show activation of DNA-damage checkpoints. When the cells were cultivated in 5% oxygen, RAF1 activation generated minimal reactive oxygen species, but RAF-induced senescence occurred efficiently in these conditions even in the presence of anti-oxidants or inhibitors of DNA checkpoint pathways. Despite the presence of heterochromatic foci, simultaneous knockdown of p16 and p21 with inactivation of the GFP-RAF1-ER kinase led to rapid reversion of the senescent state with the majority of cells becoming competent for long-term proliferation. These results demonstrate that replicative and oxidative stresses are not required for RAF-induced senescence, and this senescence is readily reversed upon loss of CKIs.
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PMID:Parallel pathways in RAF-induced senescence and conditions for its reversion. 2202 Mar 27

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress.
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PMID:SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba. 2292 76

Our results demonstrate that the addition of cisplatin after paclitaxel-induced mitotic arrest was more effective than individual treatment on gastric adenocarcinoma cells (MKN45). However, the treatment did not induce benefits in cells derived from lymph node metastasis (ST2957). Time-lapse microscopy revealed that cell death was caused by mitotic catastrophe and apoptosis induction, as the use of the caspase inhibitor z-VAD-fmk decreased cell death. We propose that the molecular mechanism mediating this cell fate is a slippage suffered by these cells, given that our Western blot (WB) analysis revealed premature cyclin B degradation. This resulted in the cell exiting from mitosis without undergoing DNA damage repair, as demonstrated by the strong phosphorylation of H2AX. A comet assay indicated that DNA repair was impaired, and Western blotting showed that the Chk2 protein was degraded after sequential treatment (paclitaxel-cisplatin). Based on these results, the modulation of cell death during mitosis may be an effective strategy for gastric cancer therapy.
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PMID:Targeting Chk2 improves gastric cancer chemotherapy by impairing DNA damage repair. 2327 Nov 72


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