ve-821 and Ataxia-Telangiectasia

To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines.. Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis.. The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells.. Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

Topics: Antineoplastic Agents; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Bile Duct Neoplasms; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; DNA Damage; Humans; Morpholines; Protein Kinase Inhibitors; Pyrazines; Pyrones; Sulfones; Tumor Suppressor Protein p53

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.. 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.. 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.. 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.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Hypoxia; Cell Line, Tumor; Cell Survival; DNA Damage; DNA Replication; HCT116 Cells; HeLa Cells; Histones; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Phosphorylation; Protein Serine-Threonine Kinases; Pyrazines; Radiation Tolerance; Radiotherapy; Repressor Proteins; Signal Transduction; Sulfones; Tripartite Motif-Containing Protein 28