1Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, and 2Gene Response Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Submitted 12 February 2003 ; accepted in final form 1 April 2003
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ABSTRACT |
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renal cells; cell cycle arrest; H2AX; chk1
DNA damage detection is followed by DNA repair that is accompanied by cell cycle arrest (37). DNA repair involves recruitments of repair factors specific to different types of DNA damage. In the case of double-strand breaks, phosphorylation of histone H2AX is an important initial step in the recruitment of the repair factors and activation of the DNA repair (6, 26). The protein kinase Chk1 is activated in response to a variety of genotoxic agents and has an essential role in transducing the delay signal from damaged DNA to cell cycle machinery (5, 19, 21, 35, 36).
The recent discovery that high NaCl causes DNA double-strand breaks added it to the list of genotoxic stresses known to damage DNA (16). However, the mechanism by which high NaCl causes DNA damage is not known. The response to DNA damage caused by high NaCl includes induction of cell cycle checkpoints (11, 12, 17, 25), induction of the tumor suppressor p53 (10) and of the GADD45 growth arrest, and DNA damage-inducible proteins (7, 17).
Mre11 normally is a nuclear protein. However, in the present study, we show that high NaCl causes Mre11 to translocate out of the nucleus. In the presence of high NaCl, Mre11 remains in the cytoplasm even after UV or ionizing radiation (IR) that is known to induce DNA damage. Exclusion of Mre11 from the nucleus by high salt disrupts DNA damage signaling that is associated with failure of the DNA damage-repair network and leads to DNA damage accumulation. Also, Chk1 is not activated, but cell cycle delays still occur, evidently independent of chk1. When the level of NaCl is returned to normal, Mre11 returns to the nucleus and DNA repair is activated accompanied by chk1 phosporylation, indicating activation of DNA damage response. We propose that 1) by inhibiting DNA repair, high salt causes accumulation of the DNA breaks that normally occur during processes such as DNA replication and transcription or induced by genotoxic stresses and ordinarily are rapidly repaired and 2) by inducing chk1-independent cell cycle delay, high salt reduces accumulation of DNA damage until the DNA damage-response network can be reactivated.
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EXPERIMENTAL PROCEDURES |
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Mouse embryonic fibroblasts (MEF) were isolated from 13.5-day-old embryos. Each embryo with 1.0 ml trypsin was placed into syringe with an 18-gauge needle attached. The content was expelled, and this procedure was repeated twice. After incubation for 45 min at 37°C, trypsin was inactivated by complete medium (DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin). Isolated cells were harvested and cultured at 37°C in a humidified atmosphere of 95% air-5% CO2. All our primary culture preparations were conducted in conformity with the Guiding Principles in the Care and Use of Animals of the American Physiological Society according to protocol approved by the Institutional Animal Care and Use Committee (protocol 2-KE-32).
Primary dermal fibroblasts (DFs) were isolated from the skin of newborn mice. The skin was incubated in 0.25% trypsin at 4°C for 18 h. Dermis was separated from epidermis and incubated in 0.35% collagenase (Worthington Biomedical) for 30 min at 37% with gentle agitation. The suspension was pipetted several times to break the dermis apart. The cells were resuspended in the complete medium, plated, and cultured at 37°C in 5% CO2. Cells were grown in 45% DME low glucose and 45% Coon's Improved Medium mF-12 (Irvine Scientific) with 10% fetal bovine serum (HyClone) added.
Mouse second passage inner medullary (p2mIME) cells were prepared and grown as previously described (34). Osmolality of isotonic (control) medium was 320 mosmol/kgH2O. Hypertonic (high NaCl) medium was prepared by adding NaCl to the control medium.
IR. Cells were exposed to 58Gy from a 137Cs in a Shepherd Mark I irradiator. The medium was replaced with a fresh one before return to the incubator.
UV. Before treatment with UV, culture medium was removed and reserved. Cells were rinsed with cold PBS of the same osmolality as experimental medium and exposed to 15 J/m2 of UV light in a Stratalinker UV Crosslinker (Stratagene, no. 400071). The reserved medium was replaced before return to the incubator.
Protein sample preparation, Western blot analysis, and immunodetection. Total cell extract: cells were rinsed with PBS adjusted with NaCl to the same osmolality as the medium and then lysed and processed as previously described (12). Nuclear and cytoplasmic cell extracts were prepared with a Pierce N-Per kit (Pierce, no. 78833). Protein content was measured using the BCA Protein Assay (Pierce). Proteins were separated by SDS polyacrylamide gel electrophoresis. Immunodetection used specific antibodies against Mre11 (Oncogene, PC388), Chk1 (Santa Cruz, sc-7898), and phospho-Chk1 (Ser345) (Cell Signaling, no. 2341).
Immunostaining and analysis by laser-scanning cytometry. Cells were grown on eight-chamber slides and immunostained, as previously described (11, 12). Immunodetection used specific antibodies against Mre11 (Oncogene, PC388), phospho-H2AX (Ser139) (Upstate, 07164), and phospho-Histone H3 (Upstate, 06570). Bound primary antibodies were detected with Alexa 488 goat anti-rabbit IgG (green fluorescence) (Molecular Probes, no. A-11034). DNA was stained with propidium iodide (PI; red fluorescence). The slides were analyzed with a laser-scanning cytometer (CompuCyte, Cambridge, MA) as previously described (12). Briefly, integral green fluorescence from the nuclear area (defined by PI staining) was recorded as a measure of phospho-H2AX (Ser139), Mre11, or phospho-H3 content. Green maximal pixel brightness within nuclei was recorded to identify phospho-H2AX (Ser139)-positive cells. Integral red fluorescence was recorded as a measure of DNA content to identify cells in different phases of cell cycle. Regions of the cytograms that included (Mre11 cytograms) or excluded (phospho-H2AX cytograms) the majority of cells (90100%) in control samples were delineated by eye and percentage of cells in that region was determined for all experimental conditions.
Analysis of DNA damage by alkali comet assay. A comet assay kit (Trevigen, no. 4250050-K) was used according to the manufacturer's instructions. Cells were rinsed with PBS, scraped off the dish, resuspended in low melting-point aga-rose, and spread on microscopic slides. Slides were incubated for 1 h in lysis solution and then for 1 h in alkaline solution. Electrophoresis was performed at 4°C for 45 min in a horizontal apparatus at 1 V/cm and 300 mA in the alkaline solution. DNA was stained with SYBR Green. Distribution of DNA between the tail and the head of the comet was analyzed with Scion Image software (Scion, Frederick, MD).
Repair of Renilla luciferase reporter vector. pRL-CMV vector (Promega, no. E2261) was damaged by UV in a Stratalinker UV Crosslinker (Stratagene, no. 400071). mIMCD3 cells were placed in high NaCl (total osmolality 600 mosmol/kgH2O) for 1 h. Normal or UV-damaged pRL-CMV vectors were then transfected into the cells using TransFast Transfection Reagent (Promega, no. E2431). One hour later, the transfection reagent was washed out and cells were placed in either control or high-NaCl medium. Luciferase production was quantified after 16 h with a Renilla luciferase assay system (Promega, no. E2820). To estimate the repair efficiencies, data were normalized to values obtained from undamaged plasmids.
Number of viable cells. Cell number was estimated based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells. The cell proliferation reagent WST-1 was used according to the company's instructions (Roche, no. 1644807).
Analysis of DNA replication rate by BrdU labeling. Cells were pulse labeled for 45 min with 10 µM BrdU and stained for BrdU using BrdU labeling and a detection kit (Roche, no. 1296736). The amount of BrdU incorporated in newly synthesized DNA was measured and analyzed by laser-scanning cytometry as previously described (11).
Analysis of G2/M checkpoint by mitotic index measurements. Cells in mitosis were assessed by immunostaining with antiphospho-histone H3 antibody (mitotic marker) (Upstate Biotechnology, no. 06570). The percentage of mitotic cells was determined by laser-scanning cytometry as previously described (12).
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RESULTS |
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Mre11 is a nuclear protein that ordinarily accumulates at sites of the DNA damage (23). However, this does not occur following DNA damage induced by high NaCl. In fact, high NaCl causes Mre11 to translocate reversibly from the nucleus to the cytoplasm both in mIMCD3 and MEF cells (Fig. 1A). This causes a large and reversible reduction in nuclear Mre11 abundance (Fig. 1, A and B). p53 abundance and phosphorylation on Ser15 are increased by high NaCl in mIMCD3 cells (10). To test for a possible role of p53 in the high NaCl-induced translocation of Mre11, we examined p53-null cells. The translocation also occurs both in p53/MEFs and in p53/DF cells (Fig. 1A), excluding a role for p53 in this process. High NaCl has been reported to kill mIMCD3 cells by apoptosis (24, 25, 29). Consistent with this, elevated NaCl reduces mIMCD3 cell number (Fig. 1C). However, if the cells are returned to the control level of NaCl after 2 h, cell number decreases much less (Fig. 1C), associated with translocation of Mre11 back into the nucleus (Fig. 1, A and B).
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Histone H2AX is usually phosphorylated in response to DNA double-strand breaks (28) and recruits repair factors (6, 26). However, it is not phosphorylated in response to high NaCl (Fig. 2A). Genomic stress also usually results in phosphorylation of chk1 kinase, signaling cell cycle arrest (5, 19, 33, 35, 36). However, high NaCl reduces its phosphorylation in mIMCD3, which is independent of p53 as it also occurs in p53/DF cells (Fig. 2E).
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NaCl-induced translocation of Mre11 and inhibition of phosphorylation of histone H2AX and Chk1 suggest that high NaCl might inhibit repair of the DNA that it damages. When the high-NaCl concentration is reduced back to the control level, not only does Mre11 return to the nucleus (Fig. 1, A and B), but histone H2AX and chk1 become phosphorylated (Fig. 2, A and E), evidently in response to the as yet damaged DNA.
After NaCl concentration returns to normal, H2AX rapidly becomes phosphorylated, remains phosphorylated for several hours (Fig. 2C), and returns to basal level simultaneously with levels of DNA damage as measured by comet assay (Fig. 2D). The cells resume growth and survive much better (Fig. 1C). As seen from Fig. 2B, the DNA damage was accumulated with time and took place mostly in S-G2/M cells. The same time course and cell cycle position were obtained for chk1 phosphorylation in mIMCD3 cells (data not shown).
It is noteworthy that similar results were obtained with all the other types of cells that were tested in addition to mIMCD3 cells, including MEFs [wild type (wt) and p53/; Fig. 1A], DF (p53/; Figs. 1A and 2E), p2mIME cells (34) from wt mice (H2AX and chk1 phosphorylation were tested, data not shown), and p2mIME cells from p53/mice (H2AX phosphorylation was tested, data not shown).
These results provide an explanation for how high NaCl might cause DNA double-strand breaks. We previously showed that the lethality of high NaCl is greatest in the S phase of the cell cycle, while DNA is replicating (11). Mre11 complexes form not only at sites of exogenously induced DNA damage but also at sites of DNA replication (22). Removal of the Mre11 from frog extracts in which DNA is replicating induces double-strand breaks in the newly replicated DNA (8). Thus high NaCl might induce accumulation of DNA double-strand breaks in S phase cells under otherwise normal conditions, because breaks that normally occur during DNA replication and are rapidly repaired are not repaired in the absence of nuclear Mre11.
However, failure to repair transient breaks that occur during DNA replication does not explain the increased levels of H2AX phosphorylation in G2/M and G1 cells upon return to isotonic medium (Fig. 2B). Nevertheless, high NaCl does exclude Mre11 from nuclei in those phases of the cell cycle, which could cause DNA double-strand breaks by interrupting other processes involving Mre11. Also, additional processes, not dependent on Mre11, might contribute to the DNA damage. That the DNA damage depends on the tonicity of NaCl and is mediated by Mre11 is supported by the observation that high urea does not affect Mre11 localization (Fig. 1A, bottom) and does not cause DNA double-strand breaks (16).
High NaCl impairs repair of DNA damage caused by UV and IR. We next asked if high NaCl also impairs the response to DNA damage induced by UV and IR. After UV and IR, Mre11 normally remains in the nucleus (Figs. 3A and 4A), and histone H2AX (Figs. 2B and 4B) and Chk1 (Figs. 3C and 4C) become phosphorylated. However, when NaCl is high, Mre11 remains in the cytoplasm following UV or IR (Figs. 3A and 4A), and histone H2AX (Figs. 3B and 4B) and Chk1 (Figs. 3C and 4C) phosphorylation are much decreased. Then, when NaCl is returned to the control level, Mre11 moves into the nucleus (Figs. 3A and 4A), and histone H2AX (Figs. 3B and 4B) and Chk1 (Figs. 3C and 4C) phosphorylation increse greatly. We infer that high NaCl inhibits the DNA repair response that normally follows DNA damage induced by UV or IR but that the response ensues if the cells are returned to control medium. Accordingly, many cells are killed by UV if NaCl remains high, but not if NaCl is returned to the control level after 2 h (Fig. 3D).
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Histone H2AX phosphorylation normally occurs at sites of DNA double-strand breaks, identifying their presence and initiating repair. IR causes such breaks in all phases of cell cycle, resulting in H2AX phosphorylation throughout the cell cycle (Fig. 4B). After DNA damage by UV, on the other hand, the DNA lesions are transformed into double-strand breaks during DNA replication (18), resulting in phosphorylation of H2AX mostly in S phase cells (Fig. 3B). High NaCl prevents the H2AX phosphorylation that follows both UV (Fig. 3B) and IR (Fig. 4B), suggesting that it interferes with repair of the DNA damage that they cause.
The following experiments directly investigated the effect of high NaCl on repair of DNA damage. The test was cell-mediated repair of a UV-damaged CMV-renilla luciferase reporter vector. The vector does not produce luciferase until its DNA damage is repaired. We transfected cells with the damaged vector and measured the amount of luciferase produced after 16 h in control (320 mosmol/kgH2O) and high-NaCl (600 mosmol/kgH2O) media. High NaCl (600 mosmol/kgH2O) impairs DNA repair by both mIMCD3 cells and p53-null MEF (Fig. 3E). UV-induced DNA lesions generally are corrected by nucleotide excision repair (2). However, it is possible that proteins that repair double-strand DNA breaks were also involved.
In high NaCl, S and G2/M cell cycle delays induced by DNA damage are independent of chk1. Both Mre11 (9) and chk1 (5, 19, 21, 35, 36) deficiency leads to checkpoint activation defects. However, high NaCl activates checkpoints in all phases of the cell cycle (11, 25) despite suppression of Mre11 and Chk1. Activation and maintenance of G2/M arrest by high NaCl are not dependent on chk1 (12), consistent with the absence of chk1 phosphorylation (Fig. 2E). In the next experiment, we investigated how chk1 inactivation by high NaCl influences checkpoint activation in response to UV and IR.
To assess S phase checkpoint, we measured DNA replication rate by BrdU incorporation. In control medium (320 mosmol/kgH2O; Fig. 5A, left), either UV or IR reduces DNA replication rate. UCN-01, which inhibits chk1 activity, increases BrdU incorporation, whether or not the cells are radiated, consistent with a role for Chk1 in the suppression of DNA replication. In contrast, the inhibition of BrdU incorporation by high NaCl is not prevented by UCN-01 (Fig. 5A, right), suggesting that Chk1 is not involved in high NaCl-induced delay of the cell cycle in S. Furthermore, although UCN-01 inhibits the S phase delay caused by IR or UV in control medium (Fig. 5A, left), it does not when NaCl is high (Fig. 5A, right).
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To assess the G2/M checkpoint, we measured mitotic index, which depends on the G2/M transition rate. In control medium (Fig. 5B, left), either UV or IR reduces the percent of cells in mitosis, consistent with activation of the G2/M checkpoint. UCN-01 increases the percent of cells in mitosis in the control condition and after IR, but not after UV. This is consistent with previous reports that UV-induced G2/M delay depends on p38 but not on chk1 (4). In high NaCl, UCN-01 does not abrogate G2/M delay after IR or UV (Fig. 5B, right). Thus, in high NaCl, cells are still able to activate S and G/M checkpoints despite chk1 inactivation.
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DISCUSSION |
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Mre11 deficiency usually compromises the triggering of cell cycle checkpoints (9, 30). However, although high NaCl causes Mre11 to exit from the nucleus, cell cycle checkpoints are still activated and maintained for some time (Ref. 11 and Fig. 5, A and B). Stress-induced cell cycle arrest generally requires phosphorylation of Chk1, but in the presence of high NaCl, Chk1 is not phosphorylated in response to DNA damage and S and G2/M cell cycle arrests are not affected by inhibition of chk1 activity. Thus, when NaCl is high, cell cycle checkpoints are activated in response to DNA damage but by mechanisms independent of chk1.
There were previous indications that the mechanisms of cell cycle arrest following high NaCl might differ from those following other stresses. Compare, for example, the G2/M arrest that occurs after IR-induced DNA damage to that caused by high NaCl. In both cases, arrest occurs quickly and involves inhibition of cdc2 (3, 12). However, although IR acts through the ATM/ATR-chk1 kinase pathway (1, 19), high NaCl does not. Thus caffeine, an ATM/ATR inhibitor, and UCN-01, a chk1 inhibitor, impair the G2/M arrest following IR (1, 19) but not following high NaCl (12). Instead, p38 kinase is necessary for fast activation of G2/M arrest by high NaCl (12), similar to its role in response to UV radiation (4). High NaCl-induced activation of p38 is transient, but the arrest persists, becoming insensitive to p38 inhibition (12). Furthermore, ATM/ATR-chk1 inhibition abrogates IR- or UV-induced arrest (1, 3) but not high NaCl-induced arrest (12). When the NaCl level falls or when cells accumulate sufficient levels of compatible organic osmolytes in response to the hypertonicity (13), the cell cycle resumes.
Taken together, these findings indicate that the DNA damage response is salt sensitive and that disruption of DNA damage signaling by high NaCl impairs DNA repair and threatens genomic stability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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