Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo

Abstract

Acute exposure of cells in culture to high NaCl damages DNA and impairs its repair. However, after several hours of cell cycle arrest, cells multiply in the hypertonic medium. Here, we show that, although adapted cells proliferate rapidly and do not become apoptotic, they nevertheless contain numerous DNA breaks, which do not elicit a DNA damage response. Thus, in adapted cells, Mre11 exonuclease is mainly present in the cytoplasm, rather than nucleus, and histone H2AX and chk1 are not phosphorylated, as they normally would be in response to DNA damage. Also, the adapted cells are deficient in repair of luciferase reporter plasmids damaged by UV irradiation. On the other hand, the DNA damage response activates rapidly when the level of NaCl is reduced. Then, Mre11 moves into the nucleus, and H2AX and chk1 become phosphorylated. Renal inner medullary cells in vivo are normally exposed to a variable, but always high, level of NaCl. As with adapted cells in culture, inner medullary cells in normal mice exhibit numerous DNA breaks. These DNA breaks are rapidly repaired when the NaCl level is decreased by injection of the diuretic furosemide. Moreover, repair of DNA breaks induced by ionizing radiation is inhibited in the inner medulla. Histone H2AX does not become phosphorylated, and repair synthesis is not detectable in response to total body irradiation unless NaCl is lowered by furosemide. Thus, both in cell culture and in vivo, although cells adapt to high NaCl, their DNA is damaged and its repair is inhibited.

Fig. 1.

Cells adapted to high NaCl have many DNA breaks, yet they survive and grow well. Cells were adapted to high NaCl, as described in Methods, then studied from passage 13 to 20. (A) Cells adapted to high NaCl (osmolality raised gradually to 500 mosmol/kg) appear similar to cells kept at 320 mosmol/kg. (B) Cells adapted by gradually raising NaCl to 500–550 mosmol/kg proliferate nearly as rapidly as cells kept at 320 mosmol/kg. (C) DNA breaks measured by single cell electrophoresis. Adapted cells have many DNA breaks. (D) Mitotic index, measured by immunostaining with antiphospho-histone H3 antibody. Mitotic index is not altered in adapted cells, indicating absence of G₂ arrest. (E) Caspase 3 activation, as a measure of apoptosis. Western blot detects cleavage to active form. There is no evidence of apoptosis (caspase 3 activation) whereas the adapted cells are proliferating, but caspase 3 is activated if they become overcrowded because they are allowed to grow beyond confluence (lane 3).

Fig. 2.

The classical DNA damage response is not activated in cells adapted to high NaCl and is activated when the level of NaCl is reduced. Adapted cells were returned to 320 mosmol/kg medium for 30 min. (A) Western blot analysis of Mre11 intracellular localization. In adapted cells, the proportion Mre11 in the cytoplasm increases, but the Mre11 translocates into nucleus after return to 320 mosmol/kg medium. (B) Western blot analysis of phosphorylation of chk1 and histone H2AX. Chk1 and H2AX are not phosphorylated in adapted cells, despite presence of DNA breaks, but become phosphorylated after return to 320 mosmol/kg. (C) Analysis of histone H2AX phosphorylation by immunocytochemistry. The percentage of P-H2AX-positive cells increases when adapted cells are returned to 320 mosmol/kg medium, indicating activation of DNA repair.

Fig. 3.

Repair of reporter plasmids damaged by UV is impaired in cells adapted to high NaCl. Cells were transfected with pRL-CMV-luciferase vector damaged by the doses of UV radiation that are shown. Luciferase expression, which depends on DNA repair within the cells, was measured 16 h after transfection.

Fig. 4.

DNA damage exists in mouse inner medullas in vivo under the normal condition of high NaCl and urea and is repaired rapidly when medullary osmolality is decreased by furosemide. (A) Time course of urine osmolality after furosemide injection (see Methods for details). (B) Single cell gel electrophoresis (comet) assay of DNA damage in cells from inner medulla and cortex. Damaged DNA appears in the “tails” of the “comets.” (Left) Representative nuclei stained with SYBR Green before furosemide treatment. (Right) The percentage of DNA in comet tails. (C) Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay of DNA breaks performed on mouse kidney sections. DNA damage is widespread in the inner medulla but not in cortex.

Fig. 5.

Histone H2AX is not phosphorylated in response to ionizing radiation at the normally elevated inner medullary osmolality in vivo but becomes phosphorylated when medullary osmolality is decreased by furosemide. Mice were given total body irradiation of 2.5 Gy, and some were injected with furosemide. (A) Immunocytochemical staining of phospho-H2AX on kidney sections. Red, DNA stained with propidium iodide; green, phospho-H2AX foci at locations of DNA damage. (B) Western blot analysis of H2AX phosphorylation in inner medulla and cortex.

Fig. 6.

DNA repair synthesis does not occur after induction of DNA damage by ionizing radiation at the normally high inner medullary osmolality in vivo but does when medullary osmolality is decreased by furosemide. Mice were given total body irradiation of 2.5 Gy, and some were injected with furosemide. All mice were injected with BrdUrd at the time of irradiation to label newly synthesized DNA. (Upper) Immunocytochemical staining of BrdUrd on kidney sections. (Lower) Staining of adjacent section with hematoxylin to show tissue structure.