This study demonstrates, by using neutral comet assay and pulsed field gel electrophoresis, that hyperosmotic stress causes DNA damage in the form of double strand breaks (dsb). Different solutes increase the rate of DNA dsb to different degrees at identical strengths of hyperosmolality. Hyperosmolality in the form of elevated NaCl (HNa) is most potent in this regard, whereas hyperosmolality in the form of elevated urea (HU) does not cause DNA dsb. The amount of DNA dsb increases significantly as early as 15 min after the onset of HNa. By using neutral comet and DNA ladder assays, we show that this rapid induction of DNA damage is not attributable to apoptosis. We demonstrate that renal inner medullary cells are able to efficiently repair hyperosmotic DNA damage within 48 h after exposure to hyperosmolality. DNA repair correlates with cell survival and is repressed by 25 M LY294002, an inhibitor of DNA-activated protein kinases. These results strongly suggest that the hyperosmotic stress resistance of renal inner medullary cells is based not only on adaptations that protect cellular proteins from osmotic damage but, in addition, on adaptations that compensate DNA damage and maintain genomic integrity. C ells adapt to osmotic stress by cell volume regulation and protection of protein structure, activity, and metabolism to maintain proper cell function. Such protective responses are necessary because osmotic stress alters cell volume, the concentration and stability of proteins, and the rate of biochemical reactions (1). Proteins are protected from osmotic stress by molecular chaperones and compatible organic osmolytes (2, 3). We recently showed that mouse renal inner medullary collecting duct (mIMCD3) cells respond to hyperosmotic stress by induction of cell cycle arrest, the tumor suppressor p53, and the growth arrest and DNA damage inducible proteins GADD45 and GADD153 (4,5). These responses are known hallmarks of signaling pathways that counteract DNA damage in mammalian cells (6, 7). DNA damage is defined as an alteration of DNA structure capable of causing cellular injury and reduction of viability or reproductive fitness of the organism (8). Thus, an induction of DNA repair pathways may be required to confer the high osmotic stress tolerance that is characteristic of renal inner medullary cells. However, in contrast to the wealth of knowledge about osmotic effects on cell volume and protein stability and function, little is known about the consequences of osmotic stress on DNA integrity in mammalian cells. Studies on several mammalian cell lines, including CHO cells (9), PAP-HT25 cells (10), human peripheral lymphocytes (11), and V79 cells (12), indicate that osmotic stress can lead to chromosomal aberrations. Such aberrations may be the consequence of an increased frequency of DNA double strand breaks (dsb) or result from an inhibition of constitutive DNA-repair mechanisms. Hyperosmotic stress inhibits inducible DNA repair pathways that are activated in response to ionizing radiation in some mammalian cell...

Because activation of p53 can trigger cell cycle arrest and apoptosis, it is necessary for a cell to suppress this activation until it is absolutely required for survival. The mechanisms underlying this important regulatory event are poorly understood. Here we show that nucleophosmin (NPM) acts as a natural repressor of p53 by setting a threshold for p53 activation in response to UV radiation. NPM binds to the p53 N terminus and inhibits p53 transcriptional activity by more than 70%. Our data indicate that the levels of NPM in a cell determine the UV dose at which the tumor suppressor p53 can be phosphorylated on Ser15. Moreover, we show that NPM is a substrate for the UV-activated protein kinase ATR and inhibits the UV-induced p53 phosphorylation at Ser15. In addition, NPM forms a complex with p53 and ATR in vivo. These data suggest that NPM is an early responder to DNA damage that prevents premature activation of p53. In normal cells, NPM could contribute to suppressing p53 activation until its functions are absolutely required while in cancer cells overexpression of NPM could contribute to p53 inactivation and tumor progression.Activation of the tumor suppressor p53 in response to DNA damage is an important event that prevents a normal cell from undergoing cellular aberrations that can lead to cancer progression. Because p53 can trigger cell cycle arrest or apoptosis, it is fundamental that the activation of p53 remains in check until it is absolutely required for cellular homeostasis. The restraint on p53 must be sufficient to allow normal growth and development while allowing it to retain the capacity for rapid induction in response to stress associated with tumor progression (28). In recent years, much emphasis has been put on the mechanisms that activate p53 in response to genotoxic stress, but little is known about how p53 functions are kept on hold under normal or low-stress conditions. Here we show data indicating that nucleophosmin (NPM) is a natural repressor of p53 that sets a threshold for p53 response to UV radiation.NPM, also known as B23, NO38, and numatrin (30), is a nucleolar protein that was initially identified as an important player in ribosome biogenesis (5). Since then a number of cellular activities associated with NPM indicate that the protein has multiple functions, especially in cell proliferation. For example, in anaplastic large-cell lymphoma NPM is fused to a receptor tyrosine kinase (anaplastic lymphoma kinase [ALK]) and works as an oncogene (10). NPM protein levels are 20 times higher in Novikoff hepatoma and 5 times higher in hypertrophic rat liver than in normal rat liver (5). NPM binds to pRb and synergistically stimulates DNA polymerase ␣ (25). NPM also binds to interferon regulatory factor 1 (IRF-1) and inhibits its tumor suppression function, probably by preventing expression of p21 (19). Another indication of NPM's role in cell proliferation is its association with the nucleolar organizer regions. The nucleolar organizer regions correlate with cell proliferation and tumor ...

Three GADD45 isoforms contribute to hypertonic stress phenotype of murine renal inner medullary cells. Am J Physiol Renal Physiol 283: F1020-F1029, 2002. First published July 2, 2002 10.1152/ajprenal.00118.2002.-Mammalian renal inner medullary (IM) cells routinely face and resist hypertonic stress. Such stress causes DNA damage to which IM cells respond with cell cycle arrest. We report that three growth arrest and DNA damage-inducible 45 (GADD45) isoforms (GADD45␣, GADDD45␤, and GADD45␥) are induced by acute hypertonicity in murine IM cells. Maximum induction occurs 16-18 h after the onset of hypertonicity. GADD45␥ is induced more strongly (7-fold) than GADD45␤ (3-fold) and GADD45␣ (2-fold). GADD45␣ and GADD45␤ protein induction is more pronounced and stable compared with the corresponding transcripts. Hypertonicity of various forms (NaCl, KCl, sorbitol, or mannitol) always induces GADD45 transcripts, whereas nonhypertonic hyperosmolality (urea) has no effect. Actinomycin D does not prevent hypertonic GADD45 induction, indicating that mRNA stabilization is the mechanism that mediates this induction. GADD45 induction patterns in IM cells exposed to 10 different stresses suggest isoform specificity, but similar functions, of individual isoforms during hypertonicity, heat shock, and heavy metal stress, when GADD45␥ induction is strongest (17-fold). These data associate all known GADD45 isoforms with the hypertonicity phenotype of renal IM cells. cell cycle; hypertonicity; nephrotoxins; kidney inner medulla CELLS OF THE MAMMALIAN RENAL inner medulla are routinely subjected to a wide range of osmolality as part of their function in renal urinary concentration. They express a phenotype that allows them to counteract the threat posed by hypertonicity and other stresses prevalent in the renal inner medulla. Because hypertonic stress represents such an immense threat to most human cells, it is critical to understand the molecular basis of the phenomenon and to study the cellular mechanisms by which cells minimize its consequences. Hypertonic stress damages proteins, leading to their unfolding and malfunction (35). This is compensated for by compatible and counteracting organic osmolytes, which are accumulated during hypertonic stress in many cell types, including renal inner medullary (IM) cells (reviewed in Refs. 4, 14, and 31). DNA is also threatened by hypertonic stress, which increases the amount of DNA double-strand breaks (19) and chromosomal aberrations (13).Our laboratory previously provided evidence that hypertonicity leads to activation of a complex network of intracellular signaling pathways, including MAPK pathways (20), the p53 pathway (8), and DNA-dependent protein kinases (19). Previous evidence also indicates that the growth arrest and DNA damage-inducible 45 (GADD45) family of genes is part of such networks (20, 33), but little is known about the osmotic regulation of the three mammalian GADD45 isoforms, prompting us to analyze this aspect of the hypertonic stress phenotype of IM cells.The first GADD4...