DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

• MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• Phosphorylation drug effects   radiation effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Proteome metabolism   MeSH
• Pyrazines pharmacology   MeSH
• Radiation-Sensitizing Agents pharmacology   MeSH
• Sulfones pharmacology   MeSH
• Gamma Rays * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH
• Proteome MeSH
• Pyrazines MeSH
• Radiation-Sensitizing Agents MeSH
• Sulfones MeSH

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.

• MeSH
• Apoptosis drug effects   radiation effects   MeSH
• Time Factors MeSH
• Chromones pharmacology   MeSH
• Phosphorylation drug effects   radiation effects   MeSH
• Histones metabolism   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Leukemia pathology   MeSH
• Humans MeSH
• Morpholines pharmacology   MeSH
• Cell Line, Tumor MeSH
• DNA Repair drug effects   radiation effects   MeSH
• DNA Damage MeSH
• Cell Proliferation drug effects   radiation effects   MeSH
• DNA-Activated Protein Kinase antagonists & inhibitors   MeSH
• Radiation-Sensitizing Agents pharmacology   MeSH
• Signal Transduction drug effects   radiation effects   MeSH
• Radiation Tolerance drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one MeSH Browser
• Chromones MeSH
• H2AX protein, human MeSH Browser
• Histones MeSH
• Protein Kinase Inhibitors MeSH
• Morpholines MeSH
• DNA-Activated Protein Kinase MeSH
• Radiation-Sensitizing Agents MeSH