p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication

We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mo smol) inhibits DNA replication and delays G(1)-S transition as concluded fr om analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorp oration rates. Lowering of p53 with p53 antisense oligonucleotide attenuate d such effects of hypertonicity, resulting in an increased number of apopto tic cells in S phase and cells with >4 N DNA. Results with synchronized cel ls are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphoryla ted on Ser(15) and translocates to the nucleus in S both in isotonic and hy pertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser( 15) phosphorylation, followed by a remarkable increase of DNA replication r ate, by failure of hypertonicity to inhibit it, and by reduction of cell nu mber during hypertonicity. Finally, inhibition of DNA replication by the DN A polymerase inhibitor aphidicolin significantly improves cell survival, co nfirming that keeping cells in G(1) and decreasing the rate of DNA replicat ion is protective and that these actions of p53 most likely are what normal ly help protect cells against hypertonicity.