Histone H2AX becomes phosphorylated in response to high NaCl in apoptotic cells, but not in viable ones

Abstract

High NaCl induces DNA breaks. The breaks persist as long as NaCl remains elevated, but are rapidly repaired when NaCl is lowered. H2AX is phosphorylated in response to DNA damage from many causes, such as ionizing radiation, but whether this occurs in response to high NaCl has been controversial. In mIMCD3 cells H2AX does not become phosphorylated when osmolality is elevated to 500–600 mosmol/kg by adding NaCl (Am J Physiol Renal 285:F266,2003). In contrast, in HeLa cells elevation of osmolality to 500–600 mosmol/kg causes H2AX phosphorylation (Am J Physiol Renal 291:F1014,2006). The purpose of the present studies was to understand the difference. We find that 100% of mIMCD3 cells survive 24 hours after osmolality is increased to 500 mosmol/kg, but only 60% of Hela cells survive. Knowing that H2AX becomes phosphorylated during apoptosis, we identified apoptotic cells by fluorochrome labeling of activated caspases, and H2AX phosphorylation by immunostaining. Elevating NaCl to 500 mosmol/kg causes apoptosis in the HeLa cells and H2AX phosphorylation that is limited to the apoptotic cells. Exposure of HeLa cells to 400 mosmol/kg induces DNA breaks (Comet assay) without apoptosis or H2AX phosphorylation. We conclude that high NaCl‐induced DNA breaks do not cause H2AX phosphorylation in cells that survive, but that H2AX phosphorylation occurs in the cells that die by apoptosis. Supported by the Intramural Program of NHLBI