High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:41 |只看该作者 |倒序浏览 |打印

作者：Natalia I. Dmitrieva, Dmitry V. Bulavin,  Maurice B. Burg作者单位：1 Laboratory of Kidney and Electrolyte Metabolism,National Heart, Lung, and Blood Institute, and GeneResponse Section, Center for Cancer Research, National Cancer Institute,National Institutes of Health, Bethesda, Maryland 20892
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      【摘要】
   High NaCl causes DNA double-strand breaks and cell cycle arrest, but themechanism of its genotoxicity has been unclear. In this study, we describe anovel mechanism that contributes to this genotoxicity. The Mre11 exonucleasecomplex is a central component of DNA damage response. This complex assemblesat sites of DNA damage, where it processes DNA ends for subsequent activationof repair and initiates cell cycle checkpoints. However, this does not occurwith DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exitsfrom the nucleus, DNA double-strand breaks accumulate in the S andG 2 phases of the cell cycle, and DNA repair is inhibited.Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response tothose stresses, as evidenced by absence of H2AX phosphorylation at places ofDNA damage and by impaired repair of damaged reporter plasmids. Activation ofchk1 by phosphorylation on Ser345 generally is required for DNA damage-inducedcell cycle arrest. However, chk1 does not become phosphorylated during highNaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UVradiation-induced phosphorylation of chk1, but cell cycle arrest still occurs,indicating the existence of alternative mechanisms for the S andG 2 /M delays. DNA breaks that occur normally during processes suchas DNA replication and transcription, as well as damages to DNA induced bygenotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage,accounting for the genotoxicity of high NaCl, and that cell cycle delayinduced by high NaCl slows accumulation of DNA damage until the DNAdamage-response network can be reactivated. ' Q( H0 y1 _4 e$ ?% s' y. T
      【关键词】 renal cells cell cycle arrest HAX chk. D0 v1 D  I$ R& l8 X9 ?
   DAMAGE TO CHROMOSOMAL DNA induces a complex cellular response designed to repair the DNA and delay cell cycle progression until it isrepaired ( 37 ). Damage to DNAactivates DNA repair systems and triggers checkpoints that prevent cell cycleprogression. The Mre11 complex heads the DNA damage response. It is amultisubunit nuclease that includes Mre11, Rad50, and Nbs1/Xrs2. This complex tethers the ends of DNA molecules and possesses a variety of DNA nuclease,helicase, ATPase, and annealing activities. It detects DNA damage andprocesses DNA ends for subsequent activation of DNA repair and cell cyclecheckpoints (reviewed in Ref. 9 ). All the members of Mre11complex are essential in vertebrates ( 20, 31, 38 ) and defects in thiscomplex lead to hypersensitivity to DNA damage and defects in triggering cellcycle checkpoints (reviewed in Ref. 9 ).

DNA damage detection is followed by DNA repair that is accompanied by cellcycle arrest ( 37 ). DNA repairinvolves recruitments of repair factors specific to different types of DNAdamage. In the case of double-strand breaks, phosphorylation of histone H2AXis an important initial step in the recruitment of the repair factors andactivation of the DNA repair( 6, 26 ). The protein kinase Chk1is activated in response to a variety of genotoxic agents and has an essentialrole in transducing the delay signal from damaged DNA to cell cycle machinery( 5, 19, 21, 35, 36 ).
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The recent discovery that high NaCl causes DNA double-strand breaks addedit to the list of genotoxic stresses known to damage DNA( 16 ). However, the mechanismby 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 tumorsuppressor p53 ( 10 ) and of theGADD45 growth arrest, and DNA damage-inducible proteins( 7, 17 ).
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Mre11 normally is a nuclear protein. However, in the present study, we showthat high NaCl causes Mre11 to translocate out of the nucleus. In the presenceof high NaCl, Mre11 remains in the cytoplasm even after UV or ionizingradiation (IR) that is known to induce DNA damage. Exclusion of Mre11 from the nucleus by high salt disrupts DNA damage signaling that is associated withfailure of the DNA damage-repair network and leads to DNA damage accumulation.Also, Chk1 is not activated, but cell cycle delays still occur, evidentlyindependent of chk1. When the level of NaCl is returned to normal, Mre11returns to the nucleus and DNA repair is activated accompanied by chk1phosporylation, indicating activation of DNA damage response. We propose that 1 ) by inhibiting DNA repair, high salt causes accumulation of the DNAbreaks that normally occur during processes such as DNA replication andtranscription or induced by genotoxic stresses and ordinarily are rapidlyrepaired and 2 ) by inducing chk1-independent cell cycle delay, highsalt reduces accumulation of DNA damage until the DNA damage-response networkcan be reactivated.

EXPERIMENTAL PROCEDURES

Cell cultures. Mouse inner medullary collecting duct cells (mIMCD3) were a gift from S. Gullans( 27 ).

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-gaugeneedle attached. The content was expelled, and this procedure was repeatedtwice. After incubation for 45 min at 37°C, trypsin was inactivated bycomplete medium (DMEM supplemented with 10% fetal bovine serum, 100 U/mlpenicillin, and 100 µg/ml streptomycin). Isolated cells were harvested andcultured at 37°C in a humidified atmosphere of 95% air-5% CO 2.All our primary culture preparations were conducted in conformity with theGuiding Principles in the Care and Use of Animals of the AmericanPhysiological Society according to protocol approved by the InstitutionalAnimal Care and Use Committee (protocol 2-KE-32).
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Primary dermal fibroblasts (DFs) were isolated from the skin of newbornmice. The skin was incubated in 0.25% trypsin at 4°C for 18 h. Dermis wasseparated from epidermis and incubated in 0.35% collagenase (WorthingtonBiomedical) for 30 min at 37% with gentle agitation. The suspension waspipetted several times to break the dermis apart. The cells were resuspended in the complete medium, plated, and cultured at 37°C in 5% CO 2.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.. R$ B( Y' y# H; w2 z$ x, a6 r
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Mouse second passage inner medullary (p2mIME) cells were prepared and grownas previously described ( 34 ).Osmolality of isotonic (control) medium was 320 mosmol/kgH 2 O.Hypertonic (high NaCl) medium was prepared by adding NaCl to the controlmedium.
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IR. Cells were exposed to 5-8Gy from a 137 Cs in aShepherd Mark I irradiator. The medium was replaced with a fresh one beforereturn to the incubator.4 B: |9 s3 J) d% c6 p8 d

UV. Before treatment with UV, culture medium was removed and reserved. Cells were rinsed with cold PBS of the same osmolality asexperimental medium and exposed to 15 J/m 2 of UV light in aStratalinker UV Crosslinker (Stratagene, no. 400071). The reserved medium wasreplaced before return to the incubator.  X8 B$ i% y, L
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Protein sample preparation, Western blot analysis, andimmunodetection. Total cell extract: cells were rinsed with PBS adjustedwith NaCl to the same osmolality as the medium and then lysed and processed aspreviously 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).
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Immunostaining and analysis by laser-scanning cytometry. Cells were grown on eight-chamber slides and immunostained, as previously described( 11, 12 ). Immunodetection usedspecific antibodies against Mre11 (Oncogene, PC388), phospho-H2AX (Ser139)(Upstate, 07-164), and phospho-Histone H3 (Upstate, 06-570). Boundprimary antibodies were detected with Alexa 488 goat anti-rabbit IgG (greenfluorescence) (Molecular Probes, no. A-11034). DNA was stained with propidiumiodide (PI; red fluorescence). The slides were analyzed with a laser-scanningcytometer (CompuCyte, Cambridge, MA) as previously described( 12 ). Briefly, integral greenfluorescence from the nuclear area (defined by PI staining) was recorded as ameasure of phospho-H2AX (Ser139), Mre11, or phospho-H3 content. Green maximalpixel 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 ofthe cytograms that included (Mre11 cytograms) or excluded (phospho-H2AXcytograms) the majority of cells (90-100%) in control samples weredelineated by eye and percentage of cells in that region was determined forall experimental conditions./ F5 K) f" y! M& j& K+ X: D
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Analysis of DNA damage by alkali comet assay. A comet assay kit(Trevigen, no. 4250-050-K) was used according to the manufacturer'sinstructions. Cells were rinsed with PBS, scraped off the dish, resuspended inlow melting-point aga-rose, and spread on microscopic slides. Slides wereincubated 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 apparatusat 1 V/cm and 300 mA in the alkaline solution. DNA was stained with SYBRGreen. Distribution of DNA between the tail and the head of the comet wasanalyzed with Scion Image software (Scion, Frederick, MD).4 f/ X3 [# Q3 Y3 @" G
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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 (totalosmolality 600 mosmol/kgH 2 O) for 1 h. Normal or UV-damaged pRL-CMVvectors were then transfected into the cells using TransFast TransfectionReagent (Promega, no. E2431). One hour later, the transfection reagent waswashed 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 repairefficiencies, data were normalized to values obtained from undamagedplasmids.0 M( C* N3 D$ i! v8 X* S: N/ w, U
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Number of viable cells. Cell number was estimated based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase inviable cells. The cell proliferation reagent WST-1 was used according to thecompany'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 BrdUlabeling and a detection kit (Roche, no. 1296736). The amount of BrdUincorporated in newly synthesized DNA was measured and analyzed bylaser-scanning cytometry as previously described( 11 ).+ d- E& j' O0 S4 n; a) o0 d

Analysis of G 2 /M checkpoint by mitoticindex measurements. Cells in mitosis were assessed by immunostaining withantiphospho-histone H3 antibody (mitotic marker) (Upstate Biotechnology, no.06-570). The percentage of mitotic cells was determined bylaser-scanning cytometry as previously described( 12 )., L: ]9 J9 y. \/ U% }( R+ M

RESULTS

High NaCl causes accumulation of DNA damage and inhibits the classicDNA damage response. Inspired by the report that osmotic stress in formof high NaCl induces DNA double-strand breaks ( 16 ), we examined the behaviorof some major components of the DNA damage network that are usually activatedin response to genotoxic stresses.  f, y2 Z0 Q0 H' [) h: b
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Mre11 is a nuclear protein that ordinarily accumulates at sites of the DNAdamage ( 23 ). However, thisdoes not occur following DNA damage induced by high NaCl. In fact, high NaClcauses Mre11 to translocate reversibly from the nucleus to the cytoplasm bothin mIMCD3 and MEF cells ( Fig.1 A ). This causes a large and reversible reduction innuclear Mre11 abundance ( Fig. 1, A and B ). p53 abundance and phosphorylation on Ser15 areincreased by high NaCl in mIMCD3 cells( 10 ). To test for a possible role of p53 in the high NaCl-induced translocation of Mre11, we examinedp53-null cells. The translocation also occurs both in p53-/-MEFsand in p53-/-DF cells ( Fig.1 A ), 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. 1 C ). However, ifthe cells are returned to the control level of NaCl after 2 h, cell numberdecreases much less ( Fig.1 C ), associated with translocation of Mre11 back into thenucleus ( Fig. 1, A and B ).! b' y6 C7 J# j  f
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Fig. 1. High NaCl causes reversible translocation of Mre11 from nucleus tocytoplasm. A and B : medium was changed from control (320mosmol/kgH 2 O) to high NaCl or urea (600 mosmol/kgH 2 O)and then 2 h later back to control. A : Western blot analysis of Mre11abundance in total cell extracts, cytoplasmic (cyto) and nuclear (nucl)fractions. wt, Wild type. B : nuclear Mre11 content in mouse innermedullary collecting duct cells (mIMCD3), immunocytochemistry, analyzed bylaser-scanning cytometry. C : viable mIMCD3 cells estimated fromcleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase. Cellnumber is relative to that (= 1) before NaCl was added at time 0.Survival is greater when cells are returned to 320 mosmol/kgH 2 Omedium after 2 h in high NaCl than when they remain in high-NaCl medium. wtMEFs, wild-type mouse embryonic fibroblasts.
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Histone H2AX is usually phosphorylated in response to DNA double-strand breaks ( 28 ) and recruitsrepair factors ( 6, 26 ). However, it is notphosphorylated in response to high NaCl( Fig. 2 A ). Genomicstress also usually results in phosphorylation of chk1 kinase, signaling cellcycle arrest ( 5, 19, 33, 35, 36 ). However, high NaClreduces its phosphorylation in mIMCD3, which is independent of p53 as it alsooccurs in p53-/-DF cells ( Fig.2 E ).- s8 ~, r& y% ?3 z' l' G8 q

Fig. 2. In high NaCl (600 mosmol/kgH 2 O), DNA damage accumulates mostlyin S and G 2 /M phases of the cell cycle, but DNA repair is notactivated nor is chk1 phosphorylated until cells return to 320mosmol/kgH 2 O. A - C : mIMCD3 cells wereimmunostained with anti-P-H2AX antibody (green), also stained with propidiumiodide (for DNA content) (red), and analyzed by laser-scanning cytometry. A : representative cytograms showing no increase in the number ofP-H2AX-positive cells (above the line) after change from 320 to 600mosmol/kgH 2 O by adding NaCl for 2 h and greatly increased number ofP-H2AX-positive cells on return to 320 mosmol/kgH 2 O medium for 0.5h. B : cells were exposed to high NaCl (600 mosmol/kgH 2 O; ). At indicated times, they were returned to 320 mosmol/kgH 2 Omedium for 0.5 h ( ). The number of cells that become P-H2AX positiveafter return for 0.5 h to 320 mosmol/kgH 2 O ( ) increases withmore previous hours in high NaCl (600 mosmol/kgH 2 O). The positivecells are mainly in S and G 2 /M phases of the cell cycle. C : cells were exposed to high NaCl (600 mosmol/kgH 2 O; ). After 2 h (indicated by arrows), some of them were returned to 320mosmol/kgH 2 O and fixed at indicated time points ( ). The levelof H2AX phosphorylation increases sharply after return to control medium butreturns to basal after several hours. D : single-cell gelelectrophoresis (comet) assay of DNA damage in mMCD3 cells. Damaged DNAappears in the "tails" of the "comets." Left :representative nuclei stained with SYBR Green. Right : ratio of DNA incomet tails/comet heads. High NaCl damages DNA, and the damage is repairedafter return to 320 mosmol/kgH 2 O medium. E : chk1phosphorylation on Ser345 decreases in high NaCl (600 mosmol/kgH 2 O)and then greatly increases after return to control (320mosmol/kgH 2 O). Western blot analysis of total chk1 and P-chk1(Ser345).

NaCl-induced translocation of Mre11 and inhibition of phosphorylation ofhistone H2AX and Chk1 suggest that high NaCl might inhibit repair of the DNAthat it damages. When the high-NaCl concentration is reduced back to thecontrol 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.3 F- L7 B" d; Q2 ^  `) U

After NaCl concentration returns to normal, H2AX rapidly becomes phosphorylated, remains phosphorylated for several hours( Fig. 2 C ), and returnsto basal level simultaneously with levels of DNA damage as measured by cometassay ( Fig. 2 D ). Thecells resume growth and survive much better( Fig. 1 C ). As seenfrom Fig. 2 B, the DNAdamage was accumulated with time and took place mostly in S-G 2 /Mcells. The same time course and cell cycle position were obtained for chk1phosphorylation in mIMCD3 cells (data not shown).1 L/ h1 D$ y* R3 L2 q
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It is noteworthy that similar results were obtained with all the othertypes of cells that were tested in addition to mIMCD3 cells, including MEFs[wild type (wt) and p53-/-; Fig. 1 A ], DF(p53-/-; Figs. 1 A and 2 E ), p2mIME cells( 34 ) from wt mice (H2AX andchk1 phosphorylation were tested, data not shown), and p2mIME cells fromp53-/-mice (H2AX phosphorylation was tested, data not shown).
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These results provide an explanation for how high NaCl might cause DNAdouble-strand breaks. We previously showed that the lethality of high NaCl isgreatest in the S phase of the cell cycle, while DNA is replicating( 11 ). Mre11 complexes form notonly at sites of exogenously induced DNA damage but also at sites of DNAreplication ( 22 ). Removal ofthe 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 underotherwise normal conditions, because breaks that normally occur during DNAreplication and are rapidly repaired are not repaired in the absence ofnuclear Mre11." v  G# P3 Y' j8 e$ X! B( ~$ R
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However, failure to repair transient breaks that occur during DNAreplication does not explain the increased levels of H2AX phosphorylation inG 2 /M and G 1 cells upon return to isotonic medium( Fig. 2 B ).Nevertheless, high NaCl does exclude Mre11 from nuclei in those phases of thecell cycle, which could cause DNA double-strand breaks by interrupting otherprocesses involving Mre11. Also, additional processes, not dependent on Mre11,might contribute to the DNA damage. That the DNA damage depends on thetonicity of NaCl and is mediated by Mre11 is supported by the observation thathigh urea does not affect Mre11 localization( Fig. 1 A, bottom ) and does not cause DNA double-strand breaks( 16 ).

High NaCl impairs repair of DNA damage caused by UV and IR. Wenext asked if high NaCl also impairs the response to DNA damage induced by UVand IR. After UV and IR, Mre11 normally remains in the nucleus (Figs. 3 A and 4 A ), and histone H2AX (Figs. 2 B and 4 B ) and Chk1 (Figs. 3 C and 4 C ) becomephosphorylated. However, when NaCl is high, Mre11 remains in the cytoplasm following UV or IR (Figs. 3 A and 4 A ), and histone H2AX(Figs. 3 B and 4 B ) and Chk1 (Figs. 3 C and 4 C ) phosphorylationare much decreased. Then, when NaCl is returned to the control level, Mre11moves into the nucleus (Figs. 3 A and 4 A ), and histone H2AX(Figs. 3 B and 4 B ) and Chk1 (Figs. 3 C and 4 C ) phosphorylation increse greatly. We infer that high NaCl inhibits the DNA repair response thatnormally follows DNA damage induced by UV or IR but that the response ensuesif the cells are returned to control medium. Accordingly, many cells arekilled by UV if NaCl remains high, but not if NaCl is returned to the control level after 2 h ( Fig.3 D ).
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Fig. 3. High NaCl inhibits repair of UV-induced DNA damage and prevents UV-inducedchk1 phosphorylation. A - C : after 1 h in control medium(320 mosmol/kgH 2 O) or high-NaCl medium (600mosmol/kgH 2 O), cells were exposed to UV. One hour later, some cellswere returned to control medium for 30 min. A : amount of Mre11analyzed by immunoblot. In the presence of high NaCl, most Mre11 iscytoplasmic, rather than nuclear, even after UV radiation. B : amountof Mre11 ( top ) and phosphorylated H2AX ( bottom ) in nuclei ofmIMCD3, analyzed by immunocytochemistry and laser-scanning cytometry. In thepresence of high NaCl, nuclear abundance of Mre11 and phosphorylated H2AX isreduced. C : mIMCD3 cell extracts were analyzed by immunoblot fortotal chk1 and P-chk1 (Ser345). High NaCl prevents UV-induced phosphorylationof Chk1. D : number of mIMCD3 cells, estimated from cleavage of thetetrazolium salt WST-1 by mitochondrial dehydrogenase 24 h after UV radiation.The cells were radiated in the presence of the levels of NaCl that are shown.Survival of cells that remain in high NaCl ( left ) falls progressivelywith increments in NaCl concentration. Many more cells originally in high NaClsurvive if they are returned to control osmolality 2 h after the UV radiation( right ). E : expression of luciferase by transfectedpRL-CMV-luciferase vector (means ± SE of 3 independent experiments,with triplicate measurements in each; * P t -test). Because the vector was damaged by the doses of UV radiationthat are shown, luciferase expression depends on DNA repair within the cells.DNA repair is impaired by high NaCl (600 mosmol/kgH 2 O) both inmIMCD3 ( left ) and p53-null MEFs ( right ).
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Fig. 4. High NaCl inhibits repair of ionizing radiation (IR)-induced DNA damage andprevents IR-induced chk1 phosphorylation. mIMCD3 in control medium (320mosmol/kgH 2 O) or after the first hour in high-NaCl (600mosmol/kgH 2 O) medium were exposed to 5Gy of IR. After 0.5 h, somecells in high-NaCl medium were returned to control medium for 0.5 h. A : amount of Mre11 analyzed by immunoblot. In the presence of highNaCl, most Mre11 is cytoplasmic, rather than nuclear, even after IR. B : amount of phosphorylated H2AX in nuclei of mIMCD3, analyzed byimmunocytochemistry and laser-scanning cytometry. In the presence of highNaCl, nuclear abundance of phosphorylated H2AX is reduced. C : mIMCD3cell extracts were analyzed by immunoblot for total chk1 and P-chk1 (Ser345).High NaCl prevents IR-induced phosphorylation of Chk1.
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Histone H2AX phosphorylation normally occurs at sites of DNA double-strandbreaks, identifying their presence and initiating repair. IR causes suchbreaks in all phases of cell cycle, resulting in H2AX phosphorylationthroughout the cell cycle ( Fig.4 B ). After DNA damage by UV, on the other hand, the DNA lesions are transformed into double-strand breaks during DNA replication( 18 ), resulting inphosphorylation of H2AX mostly in S phase cells( Fig. 3 B ). High NaClprevents the H2AX phosphorylation that follows both UV( Fig. 3 B ) and IR( Fig. 4 B ), suggesting that it interferes with repair of the DNA damage that they cause.

The following experiments directly investigated the effect of high NaCl onrepair of DNA damage. The test was cell-mediated repair of a UV-damagedCMV-renilla luciferase reporter vector. The vector does not produce luciferaseuntil its DNA damage is repaired. We transfected cells with the damaged vectorand measured the amount of luciferase produced after 16 h in control (320mosmol/kgH 2 O) and high-NaCl (600 mosmol/kgH 2 O) media. High NaCl (600 mosmol/kgH 2 O) impairs DNA repair by both mIMCD3 cells and p53-null MEF ( Fig.3 E ). UV-induced DNA lesions generally are corrected bynucleotide excision repair ( 2 ).However, it is possible that proteins that repair double-strand DNA breaks were also involved.

In high NaCl, S and G 2 /M cell cycle delaysinduced by DNA damage are independent of chk1. Both Mre11( 9 ) and chk1( 5, 19, 21, 35, 36 ) deficiency leads tocheckpoint activation defects. However, high NaCl activates checkpoints in allphases of the cell cycle ( 11, 25 ) despite suppression ofMre11 and Chk1. Activation and maintenance of G 2 /M arrest by highNaCl are not dependent on chk1( 12 ), consistent with theabsence of chk1 phosphorylation ( Fig.2 E ). In the next experiment, we investigated how chk1inactivation by high NaCl influences checkpoint activation in response to UVand IR.6 [1 p% Z1 Z4 I  x/ w% g
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To assess S phase checkpoint, we measured DNA replication rate by BrdUincorporation. In control medium (320 mosmol/kgH 2 O; Fig. 5 A, left ), either UV or IR reduces DNA replication rate. UCN-01, whichinhibits chk1 activity, increases BrdU incorporation, whether or not the cellsare radiated, consistent with a role for Chk1 in the suppression of DNAreplication. In contrast, the inhibition of BrdU incorporation by high NaCl isnot prevented by UCN-01 ( Fig.5 A, right ), suggesting that Chk1 is not involved in high NaCl-induced delay of the cell cycle in S. Furthermore, althoughUCN-01 inhibits the S phase delay caused by IR or UV in control medium( Fig. 5 A, left ), it does not when NaCl is high( Fig. 5 A, right ).

Fig. 5. Activation of S and G 2 /M checkpoints in high NaCl is independentof chk1. A and B : mIMCD3 in control medium (320mosmol/kgH 2 O) or following the first hour in high-NaCl medium (600mosmol/kgH 2 O) were exposed to UV or IR. One micromolar UCN-01, achk1 inhibitor, or vehicle (DMSO) was added 15 min before irradiation (resultsare representative of 2 independent experiments). A : DNA replicationrate was assessed 1 h after irradiation. In control medium ( left ),either UV or IR reduces DNA replication, and the rate of replication dependson chk1 activity. In high-NaCl medium ( right ), DNA replication rateis decreased independent of Chk1 activity because it is not affected byUCN-01. B : percentage of cells in mitosis, which depends on theG 2 /M transition rate, was assessed 1 h after irradiation. Incontrol medium ( left ), either UV or IR reduces the percentage ofcells in mitosis. UCN-01 increases the percentage of cells in mitosis in thecontrol condition and after IR but not after UV. In high NaCl( right ), the percentage of cells in mitosis is reduced either with orwithout IR or UV independent of chk1, because it is not affected by UCN-01. C : diagram summarizing disruption of the DNA damage-response networkby high NaCl (discussed in text).
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To assess the G 2 /M checkpoint, we measured mitotic index, which depends on the G 2 /M transition rate. In control medium( Fig. 5 B, left ), either UV or IR reduces the percent of cells in mitosis,consistent with activation of the G 2 /M checkpoint. UCN-01 increasesthe percent of cells in mitosis in the control condition and after IR, but notafter UV. This is consistent with previous reports that UV-inducedG 2 /M delay depends on p38 but not on chk1( 4 ). In high NaCl, UCN-01 doesnot abrogate G 2 /M delay after IR or UV( Fig. 5 B, right ). Thus, in high NaCl, cells are still able to activate S andG/M checkpoints despite chk1 inactivation.# D6 N0 e9 p; ]$ o" R3 W6 W4 h
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DISCUSSION

The findings presented here demonstrate that high NaCl dramatically modifies the response to DNA damage, as summarized in Fig. 5 C. Mre11normally resides in the nucleus, where it detects DNA damage and processes DNAends for subsequent activation of DNA repair and activation of cell cyclecheckpoints (reviewed in Ref. 9 ). High NaCl results inexclusion of Mre11 from the nucleus, disrupting DNA damage signaling, so thatDNA repair and chk1 kinase are not activated. In the present study, we assessed only two types of DNA repair, namely repair of double-strand breaks,monitored by H2AX phosphorylation, and nucleotide excision repair, monitoredwith a UV-damaged reporter vector. However, high NaCl presumably alsointerrupts additional processes involving Mre11, such as telomere maintenance,sister chromatid association, and homologous recombination (reviewed in Ref. 9 ), as well as other processesrequiring linked binding of two DNA substrates ( 15 ). The effect of high NaClon Mre11 resembles that of hyperthermia. Hyperthermia also induces Mre11translocation out of the nucleus and sensitizes cells to radiation( 32 ). It would be interesting to know whether the analogy carries further and hyperthermia disrupts repairof DNA damage, as does high NaCl./ o& \8 \$ y+ m' A: A7 N3 Q; z

Mre11 deficiency usually compromises the triggering of cell cyclecheckpoints ( 9, 30 ). However, although highNaCl 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 notphosphorylated in response to DNA damage and S and G 2 /M cell cyclearrests are not affected by inhibition of chk1 activity. Thus, when NaCl ishigh, cell cycle checkpoints are activated in response to DNA damage but bymechanisms independent of chk1./ X" t0 M; B4 \/ ?2 S2 ~$ b( q

There were previous indications that the mechanisms of cell cycle arrestfollowing high NaCl might differ from those following other stresses. Compare,for example, the G 2 /M arrest that occurs after IR-induced DNAdamage to that caused by high NaCl. In both cases, arrest occurs quickly andinvolves inhibition of cdc2 ( 3, 12 ). However, although IR actsthrough the ATM/ATR-chk1 kinase pathway( 1, 19 ), high NaCl does not. Thuscaffeine, an ATM/ATR inhibitor, and UCN-01, a chk1 inhibitor, impair the G 2 /M arrest following IR( 1, 19 ) but not following highNaCl ( 12 ). Instead, p38 kinaseis necessary for fast activation of G 2 /M arrest by high NaCl( 12 ), similar to its role inresponse 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 NaCllevel falls or when cells accumulate sufficient levels of compatible organicosmolytes in response to the hypertonicity( 13 ), the cell cycleresumes.  ^5 c4 H  {5 Y' P7 ?# q( L, E* Y

Taken together, these findings indicate that the DNA damage response issalt sensitive and that disruption of DNA damage signaling by high NaClimpairs DNA repair and threatens genomic stability.- H5 M8 k% D' L( B: P( I
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ACKNOWLEDGMENTS
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We are grateful to Q. Cai for preparing the p2mIME cells., G3 |3 b3 S+ C; G( N8 ^
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