英文文献
The Radiomimetic Enediyne C-1027Induces Unusual DNA Damage Responses to
Double-Strand Breaks?
Daniel R.Kennedy and Terry A.Beerman*
Department of Pharmacology and Therapeutics,Roswell Park Cancer Institute,Buffalo,New York14263
Recei V ed No V ember15,2005;Re V ised Manuscript Recei V ed January9,2006
ABSTRACT:Cells lacking the protein kinase ataxia telangiectasia mutated(ATM)have defective responses to DNA double-strand breaks(DSBs),including an inability to activate damage response proteins such as p53.However,we previously showed that cells lacking ATM robustly activate p53in response to DNA strand breaks induced by the radiomimetic enediyne C-1027.To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs,we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent.Like ionizing radiation (IR)and other radiomimetics,breaks induced by C-1027efficiently activate ATM by phosphorylation at Ser1981,yet unlike other radiomimetics and IR,DNA breaks induced by C-1027result in normal phosphorylation of p53and the cell cycle checkpoint kinases(Chk1and Chk2)in the absence of ATM.
In the presence of ATM,but under ATM and Rad3-related kinase(ATR)deficient conditions,C-1027 treatment resulted in a decrease in the level of Chk1phosphorylation but not in the level of p53and Chk2 phosphorylation.Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins.This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR.Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM,as long as ATR is present.
The successful detection of and response to DNA DSBs1 is crucial for cellular maintenance of genomic stability. Therefore,cells utilize a complex network of DNA strand break sensors,signal transmitters,and effectors to optimize DNA repair prior to mitosis,helping to prevent the conse-quences of genomic damage such as apoptosis,mitotic catastrophe,and irreversible growth arrest(1).Our under-standing of DNA DSB damage response mechanisms is primarily based upon IR studies.Following IR treatment, cells activate the phosphatidylinositol3-kinase-like protein kinase(PIKK)ATM through an autophosphorylation event (2).Activated ATM initiates a kinase cascade leading to cell cycle checkpoint responses and recruitment of repair proteins to sites of damaged DNA.For example,Chk2,a protein kinase directly phosphorylated by ATM at Thr68,mediates phosphorylation of Cdc25C and Cdk1,resulting in a G2/M checkpoint(3).Another important downstream target of ATM following IR treatment is p53-Ser15phosphorylation, which mediates the G1/S checkpoint(4-6).Cells without ATM function cannot efficiently activate checkpoint re-sponses to IR-induced DNA DSBs and are hypersensitive to IR treatment in regard to cell death and growth inhibition (7-9).
A complementary approach to studying PIKK responses to DNA DSBs utilizes radiomimetics such as enediynes.In comparison to IR,which induces multiple types of DNA damage[e.g.,thymidine dimers,purine deamination,single-strand breaks(SSBs),and DSBs],enediynes generally induce only DNA strand breaks(both SSBs and DSBs)(10,11). Despite differences in the nature of the DNA damage,cellular responses to treatment with enediynes such as neocarzinosta-tin(NCS)share hallmarks of the IR-induced damage response,including ATM-dependent activation of p53-Ser15, Nbs1-Ser343,and Chk2-Thr68(12-14),and cells lacking the ATM kinase are more readily killed by radiomimetic treatment than ATM-wild-type cells(11,15).Furthermore, enediynes have helped to define the nature of the ATM response to DNA breaks.For example,a study comparing damage induced by IR and calicheamicin,an enediyne known to induce almost exclusively DSBs,has shown that ATM activation strongly correlates with DSBs and not SSBs(16). Enediyne studies have also led to the discovery of DNA DS
B response pathways that do not strictly follow the IR model. We have shown that C-1027treatment induces activation of p53-Ser15in the absence of ATM,C-1027-induced cell growth inhibition does not increase in magnitude in cells lacking ATM,and radioresistant DNA synthesis is not observed(17).
Damage response mechanisms have also been identified that show while ATM is the primary PIKK to respond to
?This work was supported in part by National Cancer Institute Grants CA106312and CA16056(to T.A.B.).D.R.K.was supported by National Institutes of Health Training Grant CA09072-30.
*To whom correspondence should be addressed:Department of Pharmacology and Therapeutics,Roswell Park Cancer Institute,Elm and Carlton Streets,Buffalo,NY14263.Telephone:(716)845-3443. Fax:(716)845-1575.E-mail:terry.beerman@https://www.wendangku.net/doc/5d17753616.html,.
1Abbreviations:ATM,ataxia-telangiectasia mutated;ATR,ATM and Rad3-related;Chk,checkpoint kinase;DSBs,double-strand breaks; IR,ionizing radiation;kd,kinase dead;NCS,neocarzinostatin;PIKK, phosphatidylinositol3-kinase-like protein kinase;SSBs,single-strand breaks.3747
Biochemistry2006,45,3747-3754
10.1021/bi052334c CCC:$33.50?2006American Chemical Society
Published on Web02/28/2006
DNA DSBs,ATR can play a role,albeit the response is typically much more limited.The ATM-Chk2and ATR-Chk1pathways both can activate G2checkpoints by inducing inhibition of Cdc25C phosphatase(3,18).A recent study has now confirmed that either ATM or ATR can phospho-rylate p53-Ser15as well as the Chk kinases in response to IR,although generally the induced phosphorylation signals are significantly lower when cells are deficient in either of the kinases(8).In contrast,after C-1027treatment,cells lacking either ATM or ATR can still fully phosphorylate p53-Ser15(17).
This study elaborates on the nature of C-1027-induced DNA DSB damage responses in an attempt to identify additional cellular responses to DNA DSBs that are not solely dependent upon ATM.We have found that,like the cellular responses to IR or NCS treatment,ATM-Ser1981is rapidly phosphorylated after C-1027treatment,yet contrary to the IR-mimic NCS,DNA damage induced by C-1027does not require ATM to phosphorylate Chk2-Thr68and Chk1-Ser345.Furthermore,in response to C-1027-induced DNA DSBs,we found that ATR status does not affect Chk2 phosphorylation and only partially affects Chk1phospho-rylation.However,the C-1027-induced damage response requires either ATM or ATR,as simultaneous loss of both kinases results in a lower level of p53phosphorylation and a nearly complete loss of Chk1and Chk2phosphorylation. Additionally,an increased level of cell death in response to C-1027treatment is only observed when both PIKKs are compromised.
MATERIALS AND METHODS
Chemicals.C-1027,a gift from Taiho Pharmaceuticals Co. (Saitama,Japan),was stored at40μM and4°C in ddH2O, and neocarzinostatin,a gift from Bristol-Myers Squib Co. (Syracuse,NY),was stored at200μM and4°C in ddH2O. Cells.Isogenic ATM-null(AT169A)and ATM-restored (YZ510B)human fibroblast cell lines,a gift from P.Lu and Y.Shiloh,were grown at37°C and5%CO2in Dulbecco’s modified Eagle’s medium supplemented with10%fetal bovine serum and50μg/mL hygromycin to maintain selective pressure on the transfected constructs(19).Wild-type GM00536lymphoblast cells2were grown at37°C and 5%CO2in RPMI1640medium supplemented with10% fetal bovine serum.
siRNA.Oligofectamine(Invitrogen,Carlsbad,CA)was incubated at30°C for20min in the presence or absence of siRNA targeted against ATR(Dharmacon Inc.,Lafayette, CO)before being added to cells that had been incubated in serum-deficient medium.The siRNA was targeted to CCTC-CGTGATGTTGCTTGA,which corresponds to nucleotides 296-314of the ATR gene(20),and has previously been shown to be effective and selective against ATR in these cells(21).Control siRNA sequences were also examined to ensure that the knockdown was specific for ATR.After4h, an equal volume of medium supplemented with20%fetal bovine serum was added to return serum levels to normal.Forty-eight hours after siRNA treatment,cells were treated for1h with C-1027and used in subsequent assays. Immunoblotting.Cells with and without siRNA treatment were incubated with drugs at37°C for1h,harvested,and washed in phosphate-buffered saline.Total cellular extracts were prepared by directly adding SDS-PAGE buffer or by incubating cells on ice in lysis buffer[50mM Hepes(pH 7.6),150mM NaCl,5mM EDTA,5mM EGTA,0.5% Nonidet P-40,0.5%sodium deoxycholate,0.5%Triton X-100,50mM sodium fluoride,1mM sodium o-vanadate, 1mM -glycerophosphate,1mM phenylmethanesulfonyl fluoride,and protease inhibitors]for15min.The cell lysates were cleared by centrifugation,and the protein content was determined using the Bradford method(Bio-Rad).Equal amounts of protein were electrophoresed on SDS-polyacryl-amide gels and transferred to a PVDF membrane.The membranes were probed with primary antibodies,followed by secondary antibodies conjugated with horseradish per-oxidase.The following primary antibodies were used:anti-phospho-p53-Ser15,anti-phospho-Chk1-Ser345,and anti-phospho-Chk2-Thr68(Cell Signaling Technology,Beverly, MA),anti-phospho-ATM-Ser1981(Rockland Immunochem-icals,Gilbertsville,PA),anti-ATR(Santa Cruz Biotechnol-ogy,Santa Cruz,CA),and anti- -actin(Sigma,St.Louis, MO).Protein bands were visualized by enhanced chemilu-minescence,and phosphorylation levels were measured using a Personal Densitometer SI(Amersham Biosceinces,Pis-cataway,NJ).
Growth Inhibition Assay.Cells in six-well dishes were seeded at a density of2.5×105cells/well and were treated with drug24h later.Following a3day incubation,dishes were washed and cells were counted with a coulter counter (Beckman Coulter Inc.,Fullerton,CA).The degree of cell growth inhibition was calculated by comparing the number of treated to nontreated control cells.
Trypan Blue Exclusion.Cells growing in six-well dishes were seeded at a density of2.5×105cells/well.After24h, the cells were treated with drug.The cells were then incubated for24h,at which point the medium and cells were collected,spun,and incubated with a0.4%solution of trypan blue for10min at room temperature.Cell death was based on the percentage of trypan-stained cells in samples contain-ing g300cells.
Genomic DNA Damage.Following a30min drug treat-ment and harvest,cells were resuspended in PBS at a concentration of1×107cells/mL.Cells were then diluted into0.5%low-melting temperature agarose,placed on a frosted slide,and covered with a coverslip for2h at4°C. The slide was then immersed in alkaline lysis buffer[2.5M NaCl,0.1M EDTA,50mM Tris(pH10.0),10%DMSO, 1%Sarcosyl,and1%Triton X-100]in water overnight at4°C.The slides were then placed in electrophoresis buffer [30mM NaOH and10mM EDTA(pH13.0)]and equili-brated for20min before electrophoresis at27V for25min at4°C.The slides were then placed in0.4M Tris(pH7.5) three times for5min,followed by immersion in100% methanol,and then ethanol.The slides were dried and stained with15μg/mL ethidium bromide and covered with a coverslip.DNA damage was assessed as described elsewhere (22).
2In testing ATM phosphorylation,we used an intrinsically ATM-
wild-type cell line as opposed to the ATM-restored fibroblasts that were
used in other experiments,although both cell lines exhibited ATM-
Ser1981phosphorylation in response to C-1027treatment.
3748Biochemistry,Vol.45,No.11,2006Kennedy and Beerman
RESULTS
ATM Is Acti V ated by C-1027-Induced DNA Breaks.ATM-regulated DNA damage responses after IR and radiomimetic treatment are well-characterized.To test whether the unusual ATM-independent damage responses to C-1027are related to a weakened ability to activate ATM,we tested whether low levels of C-1027resulted in ATM-Ser1981autophos-phorylation (Figure 1).Like IR-induced responses,ATM was activated within minutes using exceedingly low levels (300fM)of C-1027treatment.NCS,another enediyne known to induce IR-like DNA damage responses,also activated ATM at concentrations that induce low levels of DNA breaks,consistent with the findings of others (14).
Verification that C-1027Can Induce p53-Ser15Phospho-rylation in a Manner Independent of ATM.In our previous study using IR or C-1027treatment,p53-Ser15phosphory-lation was significantly attenuated in an ATM-null line for the former but not the latter,when compared to nonisogenic ATM-wild-type cells (17).Also,unlike IR,C-1027treatment resulted in a similar cytotoxicity regardless of ATM status (17).However,since the cell lines were not isogenic,we could not rule out the possibility that the observed p53phosphorylation in the ATM-null cells was affected by the differing genotypes.To confirm that C-1027-induced DNA damage responses could be activated in the absence of ATM,p53-Ser15phosphorylation was assessed in isogenic ATM-wild-type and null cells after C-1027treatment.We found little difference in p53-Ser15phosphorylation between the two cell lines in response to C-1027treatment,while as expected,relative to the ATM-wild-type line,the level of p53-Ser15phosphorylation in the ATM-null cells was substantially reduced following NCS treatment (Figure 2).Similarly,we found that growth of the isogenic ATM-null cells when compared to that of ATM-wild-type cells was more inhibited by NCS but not by C-1027(17,19)(Figure 3).
C-1027Induces Robust p53Phosphorylation in Cells Deficient in either ATM or ATR but Not in Cells Deficient in Both Kinases .Previously,we showed that ATM-null cells respond to C-1027-induced damage like ATM-wild-type cells (Figures 2and 3)(17).Prior studies in our laboratory have also revealed that the amount of p53-Ser15phosphorylation observed after treatment with C-1027or with IR was unaffected by ATRkd expression (17).We have now extended these results by utilizing ATM-wild-type and -null cells in combination with siRNA targeted against ATR (Figure 4A,B).ATR levels averaged 15%of the control level after siRNA knockdown (Figure 4A),consistent with the findings of others using this siRNA sequence in this and in other mammalian cell lines (20,21).Reduction of ATR in ATM-wild-type cells had little effect on C-1027-induced phosphorylation of p53-Ser15(Figure 4A,B).In contrast,ATM-null cells with ATR depleted by siRNA exhibited an approximate 50%decrease in the level of p53-Ser15phos-phorylation in response to C-1027treatment (Figure 4A,B).Thus,unlike IR and other radiomimetics such as NCS,C-1027-induced phosphorylation of p53-Ser15is only de-creased in cells deficient in both ATM and ATR.
C-1027Induces Robust Chk2Phosphorylation in Cells
nM C-1027was measured by Western blotting.Similar to the p53results and unlike IR,NCS,and calicheamicin treatment,Chk2-Thr68was similarly phosphorylated in response to C-1027treatment regardless of ATM status (Figure 5A,B).Also,the diminishing ATR kinase activity caused by siRNA targeting of ATR in ATM-wild-type cells had little effect on Chk2phosphorylation in response to C-1027-induced DSB (Figure 5A,B),which is also in contrast to the IR model (8).Moreover,a diminished level of phosphorylation of Chk2was also observed following NCS treatment of ATR-reduced ATM-wild-type cells (data not shown).However,only ATM-null cells with siRNA-depleted ATR exhibited an almost complete loss of Chk2-Thr68phosphorylation in response to C-1027(Figure 5A,B).Thus,the ATM-independent DNA damage responses induced by C-1027extend to Chk2-Thr68phosphorylation.
F IGURE 1:C-1027treatment induces ATM activation.Wild-type human lymphoblast cells were treated with 0,0.3,or 3pM C-1027or 0.3and 3nM NCS for 15min at 37°C,and the cellular extracts were analyzed by Western blotting.Immunoblots were then probed with an antibody specific for phosphorylated ATM-Ser1981.
F IGURE 2:C-1027induces ATM-independent p53-Ser15phospho-rylation.ATM-null and restored human fibroblast cells were treated with 0or 1nM C-1027or 300nM NCS for 1h at 37°C,and the cellular extracts were analyzed by Western blotting.Immunoblots were then probed with an antibody specific for phosphorylated p53-Ser15.
F IGURE 3:ATM-null cells are not hypersensitive to C-1027treatment.ATM-null and restored human fibroblast cells were treated with 0or 30pM C-1027or 30nM NCS for 72h at 37°C,and the cells were harvested.The number of cells was counted and normalized as a percentage of the untreated control from three independent experiments.From these percentages,the ratio of growth inhibition between wild-type and ATM-null cells was determined.
C-1027Induces Unusual Damage Responses to Double-Strand Breaks Biochemistry,Vol.45,No.11,20063749
C-1027Induces Chk1Phosphorylation in Cells Deficient in either ATM or ATR but Not in Cells Deficient in Both Kinases .Loss of C-1027-induced activation of DNA damage response proteins seems to occur only when both ATM and ATR are depleted,in contrast to other radiomimetics and IR,where loss of ATM is sufficient to cause a substantial reduction in activation of these proteins.Phosphorylation of Chk1-Ser345could be a possible exception since it is activated directly by ATR following DNA damage (18).The level of Chk1phosphorylation is also known to be signifi-cantly decreased in the absence of ATM after IR treatment (8);thus,the level of Chk1phosphorylation would be expected to decrease with a loss of either ATM or ATR.In contrast,C-1027treatment of cells lacking ATM did not result in any decrease in the level of Chk1phosphorylation (Figure 6A,B).Moreover,in ATM-wild-type cells that are deficient in ATR,C-1027treatment resulted in an only partial loss of Chk1-Ser345phosphorylation (Figure 6A,B).Only cells deficient in both ATM and ATR exhibited a nearly complete loss of Chk1-Ser345phosphorylation after C-1027treatment (Figure 6A,B).
C-1027-Induced DNA Breaks Are Not Affected by ATM or ATR Status.To test if the amount of C-1027-induced breaks was similar under each kinase deficient condition,DNA breaks were measured by Comet analysis.3SiRNA-treated (either mock or ATR)ATM-wild-type or -null cells were incubated with C-1027under conditions used for Western analysis (1nM C-1027for 1h)and then analyzed
for induction of DNA breaks (Figure 7).PIKK status had little effect on the amount of DNA breaks observed after C-1027treatment,consistent with the idea that the difference in DNA damage response signaling is not related to the amount of DNA breaks induced.
The Le V el of C-1027-Induced Cell Death Increases in Cells Deficient in both ATM and ATR.Cells lacking ATM are unable to induce DNA damage checkpoint responses to DNA DSBs induced by IR and radiomimetics (except C-1027)and,as such,are more readily killed by these agents (6).Since reduction of C-1027-induced DNA damage response check-points for the most part requires loss of both ATM and ATR,we sought to determine whether hypersensitivity in regard to cell death would vary accordingly.We examined siRNA-treated (either mock or ATR)ATM-wild-type or -null cells for an increased level of cell death in response to C-1027treatment.Cells were treated with 0.3and 1nM C-1027for 24h and examined for their ability to exclude trypan blue.As in 3day growth inhibition (Figure 3)(17),there was no difference in C-1027-induced cell death in ATM-wild-type and ATM-null cells (Figure 8).Also consistent with our previous study was the fact that there was no change in drug-induced cell death between ATM-wild-type cells treated with mock siRNA or SiRNA targeted against ATR.In contrast,
3
Though Comet detects both SSBs and DSBs,C-1027induces almost exclusively DSBs (17),and our comet results are consistent with neutral pulse field gel electrophoresis estimates of DNA DSBs from C-1027-treated cells (Dr.M.McHugh,personal
communication).
F IGURE 4:Induction of p53-Ser15phosphorylation by C-1027is partially inhibited only in the absence of both ATM and ATR.(A)ATM-null and restored human fibroblast cells were pretreated for 48h with mock siRNA (ATR +)or siRNA directed toward ATR (ATR -)before being treated with 0or 1nM C-1027for 1h at 37°C.The cellular extracts were analyzed by Western blotting.Immunoblots were then probed with an antibody specific for phosphorylated p53-Ser15and quantitated using image quant software.(B)Each lane was normalized to a loading control ( -actin),and then the normalized value of each untreated signal was subtracted from the C-1027-treated signal under each kinase condition.Since the signal intensities can vary between experiments,the values of five independent experiments were converted to a percentage of the wild-type signal to estimate the loss of p53-Ser15
phosphorylation.
F IGURE 5:Induction of Chk2-Thr68phosphorylation by C-1027is greatly inhibited only in the absence of both ATM and ATR.(A)ATM-null and restored human fibroblast cells were pretreated for 48h with mock siRNA (ATR +)or siRNA directed against ATR (ATR -)before being treated with 0or 1nM C-1027for 1h at 37°C.The cellular extracts were analyzed by Western blotting.Immunoblots were then probed with an antibody specific for phosphorylated Chk2-Thr68and quantitated using image quant software.(B)Each lane was normalized to a loading control ( -actin),and then the normalized value of each untreated signal was subtracted from the C-1027-treated signal under each kinase condition.Since the signal intensities can vary between experiments,the values of five independent experiments were converted to a percentage of the wild-type signal to estimate the loss of Chk2-Thr68phosphorylation.
3750Biochemistry,Vol.45,No.11,2006Kennedy and Beerman
ATM-null cells that were treated for 48h with siRNA targeted against ATR exhibited a substantial increase in the level of cell death following C-1027treatment (Figure 8).Thus,unlike IR and other radiomimetics,where loss of ATM leads to an increased level of cell death,loss of both ATM and ATR is required for C-1027to recapitulate this response.DISCUSSION
Cells lacking ATM are hypersensitive with regard to cytotoxicity induced by IR or radiomimetic treatment,consistent with their inability to activate DNA damage
response proteins (6).This study further examined and expanded findings from this laboratory showing that after induction of DNA breaks by the enediyne C-1027,ATM-null cells robustly activated p53-Ser15and did not display hypersensitive growth inhibition in comparison to ATM-wild-type cells (17).Recent studies have demonstrated that phosphorylation of ATM-Ser1981occurs within minutes after low levels of IR treatment or treatment with radio-mimetics such as NCS and calicheamicin (2,14,16).This study revealed that the ATM-independent nature of C-1027-induced DNA damage responses is not due to an inability to activate ATM,as very low levels of C-1027treatment resulted in ATM-Ser1981phosphorylation (Figure 1).These results demonstrate a traditional role for ATM in response to C-1027-induced breaks.
In general,cells lacking ATM show a weakened response to DNA damage induced by IR and a wide variety of radiomimetics,including enediynes such as calicheamicin and NCS,as well as glycopeptides such as bleomycin (11).The notable exception to this scenario is C-1027,which induced similar p53phosphorylation in nonisogenic ATM-wild-type and ATM-null cell lines (17)and,now in this study,isogenic ATM-wild-type and -null cells,while the level of NCS-induced activation was greatly reduced in the cells lacking ATM (Figure 2).Similarly,the Chk kinase transducer proteins,located immediately downstream of ATM,are also activated differently after C-1027treatment when compared to treatment with IR or other radiomimetics.In response to C-1027treatment,Chk2and Chk1are robustly phosphorylated (Figures 5and 6),while after treatment with IR or other radiomimetics,cells lacking ATM exhibited markedly reduced levels of phosphorylation of Chk2and Chk1(8).
While all three damage response proteins were fully activated in the absence of ATM after C-1027-induced DNA DSBs,each protein was activated differently in cells deficient in ATR or both ATM and ATR.We found that after C-1027treatment as long as ATM is present when ATR is sup-pressed,both p53and Chk2were robustly phosphorylated while the level of Chk1phosphorylation was reduced by ～50%(Figures 4-6).In contrast,ATR activation of
DNA
F IGURE 6:Induction of Chk1-Ser345phosphorylation requires either ATM or ATR.(A)ATM-null and restored human fibroblast cells were pretreated for 48h with mock siRNA (ATR +)or siRNA directed against ATR (ATR -)before being treated with 0or 1nM C-1027for 1h at 37°C.The cellular extracts were analyzed by Western blotting.Immunoblots were then probed with an antibody specific for phosphorylated Chk1-Ser345and quantitated using image quant software.(B)Each lane was normalized to a loading control ( -actin),and then the normalized value of each untreated signal was subtracted from the C-1027-treated signal under each kinase condition.Since the signal intensities can vary between experiments,the values of five independent experiments were converted to a percentage of the wild-type signal to estimate the loss of Chk1-Ser345
phosphorylation.
F IGURE 7:Amount of C-1027-induced DNA strand breaks are not affected by PIKK status.Under the conditions used for Figures 4-6,the amount of DNA breaks induced by 1nM C-1027was examined under varying PIKK deficient conditions.Cells were harvested and placed on a microscope slide.Following cell lysis,electrophoresis,and the addition of ethidium bromide,the size of the DNA tail was viewed by fluorescence microscopy and DNA breaks were
quantitated.
F IGURE 8:Only cells deficient in both ATM and ATR exhibit increased levels of cell death in response to C-1027treatment.ATM-null and restored human fibroblast cells were pretreated for 48h with mock siRNA or siRNA directed against ATR before being treated with 0,0.3,or 1nM C-1027for 24h at 37°C.Cells were harvested from three independent experiments and assessed for the ability to exclude trypan blue.Three hundred cells from each sample were counted,and the percentage of cells excluding trypan blue was determined.
C-1027Induces Unusual Damage Responses to Double-Strand Breaks Biochemistry,Vol.45,No.11,20063751
damage response proteins in response to IR or other radiomimetics in ATM-null cells is not robust and occurs after longer periods of time and/or at high levels of DSBs when compared against ATM-wild-type cells(16,24).For example,ATR is thought to be responsible for Chk2 phosphorylation in response to high levels of IR or cali-cheamicin-induced DSBs in the absence of ATM(16).After IR treatment,the level of phosphorylation of both Chk kinases is diminished in cells deficient in ATR,although ATR status did not affect p53-Ser15phosphorylation(8,17). We obtained similar results after ATR deficient cells were treated with NCS(data not shown).While Chk1phospho-rylation is known to require ATR,the reason Chk2activation is lost under these conditions is less clear(6).Since ATM-mediated phosphorylation of Chk2in response to IR requires other damage response proteins such as Nbs1and DNA-PK,perhaps activation of these proteins also involves ATR (21,25).Taken together,these findings suggest that unlike the observed ATR-dependent activation of the Chk kinases in response to DSBs induced by IR and NCS,ATM can compensate for the loss of ATR when the breaks are induced by C-1027.
The notion that cells can readily use either ATM or ATR to respond to C-1027-induced DSBs is supported by the finding that the level of phosphorylation of p53is partially reduced,while the levels of Chk1and Chk2are drastically reduced,only when both PIKKs are lost(Figures4-6). While there was almost no phosphorylation of either of the Chk kinases,p53-Ser15phosphorylation levels were still significant,consistent with a previous observation that p53 can be activated by additional PIKK kinases such as DNA-PK and Smg1(26,27).Further study is needed to determine the reason for these differences in phosphorylation and whether other PIKKs are involved in the C-1027-induded damage response.
Our findings also revealed that PIKK status dictates not only DNA damage responses but also cell survival in response to DNA breaks.Thus,only cells lacking both ATM and ATR were hypersensitive(with regard to cell death)to C-1027treatment compared to wild-type cells and cells deficient in ATM or ATR(Figure8).In contrast,after IR or NCS treatment,deficiency in either ATM or ATR results in greater cell death(17,19).Thus,activation of cellular damage responses by C-1027is unique on the basis of PIKK status,and these responses correlate with cellular hypersen-sitivity with regard to cell death.
An important question is why C-1027-induced DNA damage responses differ from those induced by IR and other radiomimetics.Radiomimetics can be classified into two major groups,glycopeptides such as bleomycin,which interact with activated oxygen and a metal cation to damage DNA,and enediynes,which damage DNA by generation of free radicals that undergo a Bergman cycloaromatization reaction(11).Furthermore,enediynes can consist of a10-membered ring chromophore such as calicheamicin or a nine-membered ring chromophore surrounded by a hydrophobic protein such as that with NCS and C-1027(28).However, with the exception of C-1027,these various types of radiomimetics all produce DNA breaks that require ATM to robustly activate DNA damage responses and to prevent an increased level of cell death and growth inhibition.Thus, the particular characteristics of the C-1027protein chro-mophore structure are not likely to be the basis for induction of ATM-independent DNA damage responses.
While the major DNA lesions produced by enediynes are strand breaks,under anaerobic conditions in cell free systems, both interstrand cross-links and monoadducts have also been observed(29).In these studies,C-1027produced mainly covalent drug-DNA interstrand cross-links,NCS produced mostly monoadducts,and calicheamicin was less efficient at introducing either type of lesion(29).Possibly,C-1027 treatment of cells may also result in DNA interstrand cross-links that activate ATR in addition to DSBs,which activate ATM.Recent studies using mitomycin C,a known DNA interstrand cross-linker,have shown that cells utilize ATR to respond to DNA interstrand cross-links(30).There are also studies showing that enediynes can damage proteins or cleave RNA,which could also contribute to C-1027’s uniqueness as an enediyne(31,32).
It is also possible that the type of DNA strand break(either single or double)produced by IR or radiomimetics influences PIKK regulation of DNA damage responses.Enediyne treatments induce DNA strand breaks by abstracting hydro-gen from the C-1′,C-4′,and/or C-5′atoms of the DNA deoxyribose backbone(33,34).However,as the efficiency of hydrogen abstraction from each DNA strand differs with the agent,so does the frequency of single-to double-strand breaks.Even though IR and radiomimetics have ratios of SSBs to DSBs that vary from100SSBs for every one DSB for IR to almost exclusively DSBs for calicheamicin,all induce ATM-dependent damage responses(11,34-36). Thus,the SSB:DSB ratio of2:1induced by C-1027would likely not be a factor in the ATM dependence of the DNA damage response(37).
Could the types of DNA ends generated from the strand scission have an impact on the ATM dependence of the DNA damage response?C-1027-induced DNA DSBs have a2bp 3′overhang and produce a combination of3′-phosphogly-colate and base propenal ends on one strand,and either a nucleoside5′-aldehyde,2-deoxyribonolactone,or4′-hy-droxylated abasic site on the other(38).Similarly,NCS-induced damage also produces a2bp3′overhang and forms all of the DNA ends mentioned above(11).Therefore,it is unlikely that it is solely the products of C-1027-induced damage that can explain the observed responses to DNA DSBs in the absence of ATM.However,more subtle differences in DNA damage can exist between NCS and C-1027.For example,NCS reacts with bulges in the DNA structure associated with mismatch base pairing not seen with C-1027,which might influence how the damaged DNA triggers PIKK-dependent cell cycle checkpoint responses(39, 40).
The ability of radiomimetics to focus DNA damage to particular DNA sequences or regions in the genome might also influence the nature of the DNA damage response.In contrast to IR,radiomimetics such as NCS and bleomycin can preferentially cleave particular subsets of genomic DNA, such as actively transcribing regions(41).However,since these enediynes also induce ATM-dependent damage re-sponses,such regional DNA targeting is also unlikely to be a factor(41).Unlike IR,which induces undirected DNA damage,radiomimetics induce breaks by cleaving DNA at site-specific sequences.Although the highly preferred target sequences vary greatly,all of the agents besides C-1027
3752Biochemistry,Vol.45,No.11,2006Kennedy and Beerman
follow the IR model of ATM-dependent activation of damage responses(11,33,39,42).
Interestingly,one of C-1027’s preferred target sequences, GTTA,is contained in the telomere repeat GGGTTA(37). Telomeres are a repetitive DNA sequence8-15
found at the ends of chromosomes,and are responsible for maintaining cell viability and genomic stability(43).We recently showed that at equal levels of DSBs,C-1027 treatment caused telomere erosion accompanied by a degree of chromosomal aberrations and genomic instability signifi-cantly higher than that produced by IR treatment(44).Recent studies have also shown that telomere dysfunction activates repair responses in a partially ATM-independent manner (45).While it is unknown if ATR is involved in this telomere dysfunction response,the ATM and ATR yeast homologues are known to work together in the regulation of repair of damaged telomeres(46).Although more study is needed, preferential targeting of telomeres by C-1027could be contributing to the unique PIKK dependence of its induced damage response.
In conclusion,this study adds to the growing body of evidence which shows that DNA damage responses to DNA DSBs are not uniformly regulated by ATM.This study shows for the first time that loss of both ATM and ATR can be required to weaken activation of DNA damage response proteins and for an increase in the level of cell death.Future studies will determine if the ATM and ATR kinases have overlapping functions in response to C-1027-induced DNA damage and examine whether preferred targeting of telomere DNA and/or formation of DNA interstrand cross-links contributes to the unusual nature of the cellular responses to C-1027-induced breaks.
ACKNOWLEDGMENT
We thank Drs.Mary McHugh,Athena Lin,Adam Karpf, and Joel Huberman for critical reading of the manuscript. REFERENCES
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BI052334C
3754Biochemistry,Vol.45,No.11,2006Kennedy and Beerman
中英文参考文献格式
中文参考文献格式 参考文献（即引文出处）的类型以单字母方式标识： M——专著，C——论文集，N——报纸文章，J——期刊文章，D——学位论文，R——报告，S——标准，P——专利；对于不属于上述的文献类型，采用字母“Z”标识。 参考文献一律置于文末。其格式为： （一）专著 示例 [1] 张志建.严复思想研究[M]. 桂林：广西师范大学出版社，1989. [2] 马克思恩格斯全集：第1卷[M]. 北京：人民出版社，1956. [3] [英]蔼理士.性心理学[M]. 潘光旦译注.北京：商务印书馆，1997. （二）论文集 示例 [1] 伍蠡甫.西方文论选[C]. 上海：上海译文出版社，1979. [2] 别林斯基.论俄国中篇小说和果戈里君的中篇小说[A]. 伍蠡甫.西方文论选：下册[C]. 上海：上海译文出版社，1979. 凡引专著的页码，加圆括号置于文中序号之后。 （三）报纸文章 示例 [1] 李大伦.经济全球化的重要性[N]. 光明日报，1998-12-27，（3） （四）期刊文章 示例 [1] 郭英德.元明文学史观散论[J]. 北京师范大学学报（社会科学版），1995（3）. （五）学位论文 示例 [1] 刘伟.汉字不同视觉识别方式的理论和实证研究[D]. 北京：北京师范大学心理系，1998. （六）报告 示例 [1] 白秀水，刘敢，任保平. 西安金融、人才、技术三大要素市场培育与发展研究[R]. 西安：陕西师范大学西北经济发展研究中心，1998. （七）、对论文正文中某一特定内容的进一步解释或补充说明性的注释，置于本页地脚，前面用圈码标识。 参考文献的类型 根据GB3469-83《文献类型与文献载体代码》规定，以单字母标识： M——专著（含古籍中的史、志论著） C——论文集 N——报纸文章 J——期刊文章 D——学位论文 R——研究报告 S——标准 P——专利 A——专著、论文集中的析出文献 Z——其他未说明的文献类型 电子文献类型以双字母作为标识： DB——数据库 CP——计算机程序 EB——电子公告
化工英文文献翻译
Heavy Oil Development Technology of Liaohe Oilfield Han Yun (Scientific Research Information Department Exploration&Development Research Institute，Liaohe Oilfield Company) Liaohe Oilfield，the largest heavy oil production base in China，features in various reservoir types，deep burial，and wide range of crude oil viscosity．For many years，a series of technologies have been developed for different oil products and reservoir types of the oilfield，of which water flooding，foam slug drive，steam stimulation，steam drive,and SAGD are the main technologies. After continuous improvement，they have been further developed and played an important role in the development of heavy oil in the oilfield． Liaohe Oilfield is abundant in heavy oil resources，46％of the total proved reserves of Liaohe Oilfield Company. Horizontally the resources concentrates in the West Depression and the southern plunging belt of the Central Uplift in Liaohe Rift. Vertically,it is mainly distributed in Paleocene Shahejie Formation(ES). The distinctive geological feature of Liaohe 0ilfield is manifested in three aspects：first，the heavy oil reservoirs are deeply buried and 80％of them are buried more than 900m deep；second，the heavy oil viscosity ranges widely．For most of the reservoirs．the dead oil viscosity ranges in 100～100000mPa·s with the maximum 650000mPa·s.Third the reservoir types are various with complicated oil—water relationship，most of the reservoirs are edge water and bosom water reservoirs and there are also edge water reservoirs，top water reservoirs and bosom water reservoirs．For more than 20 years of development，Liaohe Oilfield has developed series of heavy oil development technologies for different oil products and different types of reservoirs，such as water flooding, foam slug drive,steam stimulation steam drive and SAGD．The most difficult issues have been overcome in the development of the super
100篇英文经典文献
share with 各位会计、财务专业的同学... （P.S.读英文期刊绝对是体力活...开读前一定要吃好睡好...） 这些是会计学的基础文献，是所有其他文献的参考文献~~~ 经典文献（The 100 articles with the highest citation index-until 1996） 参考：Lawrence D. Brown, 1996, “Influential Accounting Articles, Individuals, Ph. D Granting Institutions and Faculties; A Citational Analysis”, Accounting, Organizations and Society, Vol.21, NO.7/8, P726-728 1. Ball, R. and Brown, P., 1968, “An Empirical Evaluation of Accounting Income Numbers”, journal of Accounting Research, Autumn, pp. 159-178 1. 2.Watts R.L., Zimmerman J., 1978, “Towards a Positive Theory of the Determination of Accounting Standards”, The Ac counting Review, pp. 112-134 2. 3.Healy P.M, 1985, “The Effect of Bonus Schemes on Accounting Decisions”, Journal of Accounting and Economics, April, 85-107 3.Hopwood A. G., “Towards an Organizational Perspective for the Study of Accounting and Information S ystems”, Accounting, Organizations and Society (No. 1, 1978) pp. 3-14 4.Collins, D. W., Kothari, S. P., 1989, “An Analysis of Intertemporal and Cross-Sectional Determinants of Earnings Response Coefficients”, journal of Accounting & Economics, pp. 143-181 5.EastonP.D, Zmijewski M.E, 1989, “Cross-Sectional Variation in the Stock Market Response to Accounting Earnings Announcements”, Journal of Accounting and Economics, 117-141 6.Beaver, W. H., 1968, “The Information Content of Annual Earnings Announcements”, jo urnal of Accounting Research, pp. 67-92 7.Holthausen R.W., Leftwich R.W., 1983, “The Economic Consequences of Accounting Choice: Implications of Costly Contracting and Monitoring”, journal of Accounting & Economics, August, pp77-117 8.Patell J.M, 1976, “Corp orate Forecasts of Earnings Per Share and Stock Price Behavior: Empirical Tests. Journal of Accounting Research, Autumn, 246-276 9.Brown L.D., Griffin P.A., Hagerman R.L., Zmijewski M.E, 1987, “An Evaluation of Alternative Proxies for the Market’s Assessment of Unexpected Earnings”, Journal of Accounting and Economics, 61-87 10.Ou J.A., Penman S.H., 1989, “Financial Statement Analysis and the Prediction of Stock Returns”, Journal of Ac counting and Economics, Nov., 295-329 11.William H. Beaver, Roger Clarke, William F. Wright, 1979, “The Association between Unsystematic Security Returns and the Magnitude of Earnings Forecast Errors,” Journal of Accounting Research, 17, 316-340.
英文文献及中文翻译
毕业设计说明书 英文文献及中文翻译 学院：专 2011年6月 电子与计算机科学技术软件工程
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石油和天然气勘探中英文对照外文翻译文献
中英文对照外文翻译文献 (文档含英文原文和中文翻译) OIL UNDER ICE DETECTION: WHAT IS THE STATE-OF-THE-ART? Abstract. Since the exploration for oil and gas in the Canadian and US arctic commenced in the early 1970s, a need has been identified to develop technology to detect oil under ice. Both electromagnetic and acoustic sensors have been tried, but a practical field instrument has not been identified. Most proposed systems require that the equipment be operated from the ice surface in order to get adequate coupling and, for some systems, the snow must be removed from the ice. For many ice
situations, surface access is difficult and poses a severe safety issue. Two recent spills in Alberta used “high technology” ice augers to detect the presence of oil under the ice. Some potential new techniques are discussed and the basic principles of their operation described. Keywords: arctic, oil spill response, oil in ice, detection 1. Introduction The detection of oil under continuous ice cover has presented one of the most difficult challenges to the oil-spill technological community for the past two decades and there is still no operationally proven system available. Dickins (2000) under the sponsorship of the US Minerals Management Service conducted an excellent review of the status of oil-under-ice detection and this paper complements this review with a more detailed analysis of some systems. Dickins identified many false start concepts, which will not be discussed in this paper. In order to determine the design of a suitable oil-under-ice detector, the various situations under which oil may be found under a continuous ice sheet need to be considered. The oil must come from a sub-surface release since any surface release would either be on the ice surface or in a lead or other opening in the ice. Potential sources of sub-surface oil are a leak in a pipeline, the leakage from a submerged tank or vessel or a natural seep. Oil when trapped under ice does not spread rapidly or cover a large area due to natural
英文文献及中文翻译撰写格式
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每一插图和表格应有明确简短的图表名，图名置于图之下，表名置于表之上，图表号与图表名之间空一格。插图和表格应安排在正文中第一次提及该图表的文字的下方。当插图或表格不能安排在该页时，应安排在该页的下一页。 图表居中放置，表尽量采用三线表。每个表应尽量放在一页内，如有困难，要加“续表X.X”字样，并有标题栏。 图、表中若有附注时，附注各项的序号一律用阿拉伯数字加圆括号顺序排，如:注①。附注写在图、表的下方。 文中公式的编号用圆括号括起写在右边行末顶格，其间不加虚线。 8、文中所用的物理量和单位及符号一律采用国家标准，可参见国家标准《量和单位》(GB3100~3102-93)。 9、文中章节编号可参照《中华人民共和国国家标准文献著录总则》。
英文文献翻译
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经典会计英文文献目录100篇
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