Inhibition of a DNA Repair Kinase ATM Leads to Cell Death in 3D Migration Independent of DNA Damage

Abstract

The DNA damage response (DDR) is a collection of processes by which a cell identifies and repairs a site of DNA damage. Many factors can cause DNA damage and initiate the repair machinery, such as UV, reactive oxygen species nucleases, or even drug treatments like etoposide. Recent studies of several cancer lines as well as immortalized epithelial cells (RPE-1) and primary dendritic cells have convincingly characterized nuclear envelope rupture and cell death in migration through narrow channel. ATM kinase phosphorylates H2AX (to γH2AX) as well as 53BP1 among other targets. A high dose of an ATM inhibitor (ATMi: KU-55933) increases migration-induced death of RPE-1 and HT1080 fibrosarcoma cells, particularly when combined with perturbations to the nuclear lamina or envelope repair. However, ATMi at high doses affects other pathways and ATM's roles beyond DNA repair now include a migration phenotype independent of DNA damage in MDA-MB-231 cells treated with siATM. Such results motivate dose-response assays here of ATMi in assessing relations between DNA damage and survival in pore migration of U2OS cells. Foci counts of phospho-ATM and of γH2AX (a target of ATM kinase) in 2D cultures were reduced by 50% at just 0.01 µM, consistent with pharmacological studies of ATM. Surprisingly, comet assays showed no DNA breaks even at 10-32 µM. Only at these very high ATMi concentrations did cells die: in 2D cultures, 50% of cells die at 66 µM ATMi, and this decreases to 37 µM and 14 µM in migration through 8 µm and 3 µm pores, respectively. However, ATM knockdown does not increase cell death in constricted migration, suggesting that high dose effects of ATMi involves pathways independent of DNA damage.