Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors.
The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.
Discovery Sciences iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK. Catherine.Bardelle@astrazeneca.com
Catherine
C
Bardelle
Joanna
J
Boros
D064007:Ataxia Telangiectasia Mutated Proteins; D059447:Cell Cycle Checkpoints / Q000235:genetics; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D057809:HEK293 Cells; D019073:HT29 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011839:Radiation, Ionizing; D015398:Signal Transduction / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases