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1.
Oncogene ; 35(6): 793-9, 2016 Feb 11.
Article in English | MEDLINE | ID: mdl-25961932

ABSTRACT

Regions of hypoxia occur in most solid tumors, and they are associated with a poor prognostic outcome. Despite the absence of detectable DNA damage, severe hypoxia (<0.1% O2) induces a DNA damage response, including the activation of p53 and subsequent induction of p53-dependent apoptosis. Factors affecting hypoxia-induced p53-dependent apoptosis are unclear. Here we asked whether H3K9me3, through mediating gene repression, could regulate hypoxia-induced p53-dependent apoptosis. Under hypoxic conditions, increases in H3K9me3 occur in an oxygen-dependent but HIF-1-independent manner. We demonstrate that under hypoxic conditions, which induce p53 activity, the negative regulator of p53, APAK, is repressed by increases in H3K9me3 along the APAK loci. APAK repression in hypoxia is mediated by the methyltransferase SETDB1 but not Suv39h1 or G9a. Interestingly, increasing hypoxia-induced H3K9me3 through pharmacological inhibition of JMJD2 family members leads to an increase in apoptosis and decreased clonogenic survival and again correlates with APAK expression. The relevance of understanding the mechanisms of APAK expression regulation to human disease was suggested by analysis of patients with colorectal cancer, which demonstrates that high APAK expression correlates with poor prognosis. Together, these data demonstrate the functional importance of H3K9me3 in hypoxia, and they provide a novel mechanistic link between H3K9me3, p53 and apoptosis in physiologically relevant conditions of hypoxia.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , DNA-Binding Proteins/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Histones/physiology , Tumor Suppressor Protein p53/physiology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Cell Hypoxia/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HCT116 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Oxygen/pharmacology , Tumor Cells, Cultured
2.
Br J Radiol ; 88(1047): 20140649, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25513745

ABSTRACT

Chromatin, the structure formed by the wrapping of approximately 146 base pairs of DNA around an octamer of histones, has a profound impact on numerous DNA-based processes. Chromatin modifications and chromatin remodellers have recently been implicated in important aspects of the DNA damage response including facilitating the initial sensing of the damage as well as subsequent recruitment of repair factors. Radiation is an effective cancer therapy for a large number of tumours, and there is considerable interest in finding approaches that might further increase the efficacy of radiotherapy. The use of radiation leads to the generation of DNA damage and, therefore, agents that can affect the sensing and repair of DNA damage may have an impact on overall radiation efficacy. The chromatin modifications as well as chromatin modifiers that have been associated with the DNA damage response will be summarized in this review. An emphasis will be placed on those processes that can be pharmacologically manipulated with currently available inhibitors. The rationale for the use of these inhibitors in combination with radiation will also be described.


Subject(s)
Chromatin/chemistry , DNA Damage/radiation effects , DNA Repair/radiation effects , DNA, Neoplasm/radiation effects , Neoplasms/radiotherapy , DNA, Neoplasm/genetics , Genomic Instability , Histones/metabolism , Humans , Molecular Structure , Neoplasms/genetics , Neoplasms/metabolism , Radiation, Ionizing
3.
Cell Death Dis ; 3: e441, 2012 Dec 06.
Article in English | MEDLINE | ID: mdl-23222511

ABSTRACT

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Isoxazoles/administration & dosage , Pancreatic Neoplasms/radiotherapy , Protein Kinase Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazines/administration & dosage , Radiation-Sensitizing Agents/administration & dosage , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Checkpoint Kinase 1 , DNA Damage/drug effects , DNA Damage/radiation effects , Female , Humans , Mice , Mice, Inbred BALB C , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phosphorylation/drug effects , Phosphorylation/radiation effects , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Radiation Tolerance
4.
Br J Cancer ; 107(2): 291-9, 2012 Jul 10.
Article in English | MEDLINE | ID: mdl-22713662

ABSTRACT

BACKGROUND: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy. METHODS: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser. RESULTS: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition. CONCLUSION: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Hypoxia/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Radiation Tolerance/drug effects , Ataxia Telangiectasia/genetics , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , DNA Damage , DNA Replication/drug effects , HCT116 Cells , HeLa Cells , Histones/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazines/pharmacology , Radiation Tolerance/genetics , Radiotherapy/methods , Repressor Proteins/genetics , Signal Transduction/drug effects , Sulfones/pharmacology , Tripartite Motif-Containing Protein 28
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