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Coactivation of ATM ERK NF κB in the low dose radiation induced radioadaptive

Original Contribution

Coactivation of ATM/ERK/NF-κB in the low-dose radiation-induced radioadaptive response in human skin keratinocytes

Kazi Mokim Ahmed a ,1,Danupon Nantajit a ,Ming Fan a ,Jeffrey S.Murley b ,David J.Grdina b ,Jian Jian Li a ,c ,?

a Graduate Program of Radiation and Cancer Biology,Purdue University School of Health Sciences,West Lafayette,IN 47907,USA

b Department of Radiation and Cellular Oncology,University of Chicago,IL 60637,USA c

Cancer Research Center,Purdue University,West Lafayette,IN 47907,USA

a b s t r a c t

a r t i c l e i n f o Article history:

Received 9December 2008Revised 21February 2009Accepted 11March 2009

Available online 24March 2009Keywords:ATM ERK NF-κB

Low-dose radiation Keratinocytes Free radicals

Elucidating the molecular mechanism of the low-dose radiation (LDR)-mediated radioadaptive response is crucial for inventing potential therapeutic approaches to improving normal tissue protection in radiation therapy.ATM,a DNA-damage sensor,is known to activate the stress-sensitive transcription factor NF-κB upon exposure to ionizing radiation.This study provides evidence of the cooperative functions of ATM,ERK,and NF-κB in inducing a survival advantage through a radioadaptive response as a result of LDR treatment (10cGy X-rays).By using p53-inhibited human skin keratinocytes,we show that phosphorylation of ATM,MEK,and ERK (but not JNK or p38)is enhanced along with a twofold increase in NF-κB luciferase activity at 24h post-LDR.However,NF-κB reporter gene transactivation without a signi ?cant enhancement of p65or p50protein level suggests that NF-κB is activated as a rapid protein response via ATM without involving the transcriptional activation of NF-κB subunit genes.A direct interaction between ATM and NF-κB p65is detected in the resting cells and this interaction is signi ?cantly increased with LDR treatment.Inhibition of ATM with caffeine,KU-55933,or siRNA or inhibition of the MEK/ERK pathway can block the LDR-induced NF-κB activation and eliminate the LDR-induced survival advantage.Altogether,these results suggest a p53-independent prosurvival network involving the coactivation of the ATM,MEK/ERK,and NF-κB pathways in LDR-treated human skin keratinocytes,which is absent from mutant I κB cells (HK18/mI κB),which fail to express NF-κB activity.

?2009Published by Elsevier Inc.

Mammalian cells exposed to certain low levels of radiation show a temporary but signi ?cantly enhanced tolerance to a subsequent exposure to relatively higher doses of radiation [1–5].This radioadaptive phenotype is evidenced by the activation of an ef ?cient defense system to repair and eliminate radiation-mediated DNA injury [6,7]leading to enhanced cell survival [8,9].Several gene expression pro ?les have been described for the low-dose radiation (LDR)-mediated adaptive response in human cells [10,11].Furthermore,a prosurvival pathway initiated by transcription factor NF-κB is linked to an enhanced cell survival of mouse skin epithelial JB6cells if preexposed to a low dose of X-rays [12].However,the exact signaling pathways of the LDR-mediated adaptive response remain to be elucidated.

NF-κB is a well-characterized transcription factor that is involved in signaling during critical events of genotoxic stress [5,13,14].In resting cells,NF-κB is sequestered in the cytoplasm by binding to its speci ?c inhibitors I κBs [15].Under varied cytotoxic conditions,I κB kinase phosphorylates the NF-κB inhibitory protein I κB-α,resulting in I κB-α

degradation and NF-κB activation [16].The activated NF-κB (mainly the heterodimer of p65/p50)quickly translocates from the cytoplasm into the nucleus where it binds and up-regulates a wide variety of stress-responsive genes (e.g.,antiapoptotic and cell cycle regulators)[17].Although radiation-induced genes are involved in both cell death (i.e.,proapoptotic)and cell survival (i.e.,antiapoptotic),several NF-κB-controlled effector genes are shown to protect cells from cellular damage [12,18–21].Inhibition of I κB-αphosphorylation,which blocks NF-κB activation,increases the radiosensitivity of cells [21,22].However,the speci ?c upstream events required for NF-κB activation,especially in the context of network elements that are required for the LDR-induced adaptive radioresistance in human cells,remain unclear.

It has long been believed that the fate of an irradiated cell is in ?uenced by a complex and highly regulated signaling network [21,23,24],which contains the elements for sensing and repairing da-maged DNA [21,24,25].The ATM (ataxia telangiectasia mutated)protein,a serine –threonine kinase of the family of phosphatidylinositol kinase-related kinases,is proven to be an essential DNA-damage sensor that plays a critical role in the DNA-damage response [26].In resting cells,ATM forms an inactive dimer.In irradiated cells,ATM induces the rapid intermolecular autophosphorylation of Ser-1981that leads to dimer dissociation and ATM kinase activation [27].Cooperation between ATM and NF-κB is supported by the fact that A-T (ataxia telangiectasia)

Free Radical Biology &Medicine 46(2009)1543–1550

?Corresponding author.Graduate Program of Radiation and Cancer Biology,Purdue University School of Health Sciences,West Lafayette,IN 47907,USA.Fax:+17654961377.

E-mail address:jjli@https://www.sodocs.net/doc/5410755519.html, (J.J.Li).1

Present address:Department of Biological Chemistry,University of California at Irvine School of Medicine,Irvine,CA 92697,

USA.

0891-5849/$–see front matter ?2009Published by Elsevier Inc.doi:

10.1016/j.freeradbiomed.2009.03.012

Contents lists available at ScienceDirect

Free Radical Biology &Medicine

j o u r n a l h o me p a g e :w w w.e l sev i e r.c o m /l oc a t e /fr e e ra d b i o me d

patients exhibit a defect in NF-κB activation.Moreover,ectopic expres-sion of the ATM protein in A-T cells activates NF-κB in response to camptothecin,a DNA-damaging compound[28].In addition,ERK (extracellular signal-regulated kinase),a member of the mitogen-activated protein kinase(MAPK)family,is also an essential element in generating a stress response[29–31]and is also required for NF-κB induction[32,33].Although ATM,ERK,and NF-κB are suggested to participate in the stress response induced by various genotoxic insults [22,32,34],it is unknown whether a cooperative interaction between ATM,MEK/ERK,and NF-κB is responsible for the radioadaptive response induced by LDR in human cells.Following the observation of an adaptive radioprotective response in mouse skin epithelial cells,this study demonstrates a similar radioadaptive response in human skin kerati-nocytes that is associated with a coactivation ATM,MEK/ERK,and NF-κB.Because human keratinocytes are immortalized by the over-expression of human papillomavirus HPV18[35,36],which encodes E6 protein that in turn inactivates p53expression,this study is unique in that it demonstrates a p53-independent LDR-mediated radioadaptive protection process.The?nding that ATM is required for the activation of both MEK/ERK and NF-κB,and that it interacts with the NF-κB subunit p65,suggests that an ATM-initiated prosurvival network participates in the LDR-induced adaptive response that is observed in human skin cells.

Experimental procedures

Cells and radiation

The human keratinocyte cell line HK18was immortalized by transfection with the HVP18genome.HK18and the dominant negative mutant of IκB-transfected HK18(HK18/mIκB)cells were maintained in Dulbecco's minimum essential medium supplemented with10%fetal bovine serum(FBS;HyClone,Logan,UT,USA),penicillin(100units/ml), and streptomycin(100μg/ml)in a humidi?ed incubator(5%CO2).The culture conditions ensure the growth of HK18keratinocytes without inducing differentiation or growth arrest.ATM-de?cient?broblasts GM05849(purchased from Coriell Cell Repositories,Camden,NJ,USA) were maintained in Eagle's minimum essential medium supplemented with10%FBS and antibiotics.Exponentially growing cells that reached 70–80%con?uence were exposed to ionizing radiation(IR)at room temperature using a Cabinet X-ray System Faxitron Series(dose rate 0.028Gy/min,130kVp;Hewlett Packard,McMinnville,OR,USA)or GR-12irradiator equipped with a cobalt-60radiation source(dose rate 2.3Gy/min;U.S.Nuclear Corp.,Burbank,CA,USA).Cells shielded from the IR source were used as a sham-IR control.Radiation sensitivity was measured using both cell proliferation(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,or MTT)and clonogenic survival assays. Clonogenic survival assay

The plating ef?ciencyof HK18cells was determined before clonogenic assays were conducted.Based on the results of plating ef?ciency, identical numbers of HK18and HK18/mIκB cells were seeded for each of the treatment groups(10cGy and10cGy+2Gy)and the control group (sham-IR).Cells were plated into60-mm cell culture plates and exposed to sham or10cGy X-rays and then incubated for6h before exposure to 2Gyγ-rays(GR-12irradiator).Fourteen days postirradiation,plates were stained with Giemsa solution and colonies≥3mm in diameter were counted as surviving colonies and normalized to the clone numbers observed on nonirradiated cells[21,45].The plating ef?ciencies for the parental HK18and HK18/mIκB cells were18%and16,respectively.Each experiment was performed in triplicate and repeated at least three times. Cell viability assay

Exponentially growing HK18and HK18/mIκB cells were plated at cell densities of5000cells per well in96-well tissue culture plates for 18–24h.Cells were then exposed to sham or10cGy X-rays and incubated for6h before exposure to2Gyγ-rays.Twenty-four hours postirradiation, cell proliferation was measured by MTT assay according to the manu-facturer's instructions(Promega,Madison,WI,USA).The experiments were performed in triplicate and repeated a minimum of three times.

Western blotting

Cells were collected from the10-cm culture dishes and washed with PBS and lysed on ice in500μl of lysis buffer per dish(10mM Hepes,10mM KCl,1.5mM MgCl2,0.5mM DTT,1%NP-40,1mM PMSF, 25%glycerol,and0.2mM EDTA).Protein concentrations were determined using a BCA Protein Assay kit(Pierce,Rockford,IL,USA). Equal aliquots of protein(20μg/lane)were electrophoresed through5 or10%SDS–polyacrylamide gel and then transferred to nitrocellulose membrane(Bio-Rad,Hercules,CA,USA).The blot was?rst incubated with the appropriate primary antibody at a dilution of1:200–1000 and then with a horseradish peroxidase-conjugated secondary anti-body at a dilution of1:5000.The ECL system(Amersham Life Science, Arlington Heights,IL,USA)was used to visualize the speci?c protein. The antibodies to NF-κB p65and NF-κB p50and to MEK and phospho-MEK were bought from Upstate Biotechnology(Lake Placid,NY,USA) and Sigma(St.Louis,MO,USA),respectively.The antibodies against ERK1/2,p-ERK1/2,p38,p-p38,JNK1,p-JNK,actin,and cyclin D1were purchased from Santa Cruz Biotechnology(Santa Cruz,CA,USA).Anti-ATM and anti-p-ATM(Ser-1981)were obtained from Gene Tex(San Antonio,TX,USA)and Rockland(Gilbertsville,PA,USA),respectively. Chemical inhibitors with or without radiation

The MEK/ERK inhibitors PD98059and U0126(Sigma)were dissolved in DMSO.The ATM inhibitors caffeine(Calbiochem,San Diego,CA,USA)and KU-55933(a generous gift from Dr.David J.Chen at the University of Texas Southwestern Medical Center at Dallas,TX,USA) were dissolved in acidi?ed water(water:acetic acid98:2,v/v)and DMSO,respectively.HK18cells grown to70–80%con?uence in complete growth medium were incubated with vehicle(control)or50μM PD98059or5mM caffeine for2h and then10μM U0126or KU-55933 for1h.Treatments were terminated by replacing the medium with complete medium without inhibitor,and cell pellets were collected at 30min,1h,2h,4h,8h,and24h after exposure to a single dose of10cGy X-rays.Cell lysates were prepared from control and inhibitor/IR-treated cells for Western blotting.Inhibitor/IR-treated cells were assessed using a reporter luciferase assay and a clonogenic assay.

Reporter transfection and luciferase activity assay

HK18and HK18/mIκB cells plated in24-well plates were cotransfected with the NF-κB luciferase reporter containing an IL-6 promoter region(0.5μg)[36]along withβ-galactosidase reporters (0.05μg)for a6-h incubation followed by irradiation with a single dose of10cGy X-rays at room temperature.Control cells were sham-irradiated.Luciferase activity was measured at various time intervals postirradiation using20μl of total cell lysates and100μl of luciferase assay reagent(Promega)as previously described[12,36].An aliquot of the same cell lysates was used for the measurement ofβ-galactosidase activity to normalize luciferase activity.

Small interfering RNA(siRNA)design and transfection

siRNA designed to target ATM sequence had the sequence5′-AACATACTACTCAAAGACATT-3′.siRNA was synthesized with the Silen-cer siRNA construction kit(Ambion,Austin,TX,USA).Cells were seeded to achieve30–50%con?uence.Transfection of siRNA was performed using Lipofectamine RNAiMAX reagent(Invitrogen,Carlsbad,CA,USA) in a24-well dish with antibiotic-free medium for24h before

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transfection with 30nM siRNA.Scramble RNA duplex (Ambion)was included as control.Inhibition of ATM was determined by Western blotting at 48h posttransfection.All transfectants were maintained in antibiotic-free complete medium until collection for analysis.Immunoprecipitation

Whole-cell extracts treated with or without radiation were prepared in lysis buffer containing 10mM Hepes,10mM KCl,1.5mM MgCl 2,0.5mM DTT,1%NP-40,1mM PMSF,25%glycerol,and 0.2mM EDTA.Extracts were centrifuged at 4°C for 15min at 12,000rpm to remove insoluble materials.Before anti-ATM antibody was added to precipitate a speci ?c immunocomplex,extracts were precleared by a 1-h treatment with normal mouse IgG and 20μl of a 1:1slurry of protein G –Sepharose beads at 4°C on a Nutator (Pharmacia,Uppsala,Sweden).Immuno-precipitation (4h at 4°C)was preceded by 30min incubation with protein A or G beads.Beads were collected by brief centrifugation,washed four times with 1×PBS buffer containing 137mM NaCl,boiled in SDS gel loading buffer,fractionated on 5or 10%SDS –PAGE gel,and then Western blotted using p65or ATM antibody.Statistical analysis

Data are expressed as means±SE.Statistical signi ?cance among groups was determined using the Student t test and the differences were considered signi ?cant at P b 0.05.Results

NF-κB is required for the LDR-induced radioadaptive response in p53-de ?cient human skin keratinocytes

To determine whether NF-κB is involved in the radioadaptive response of human skin cells,we compared the radiosensitivity of an isogenic pair of human skin keratinocytes using HPV18-immortalized wild-type human skin keratinocytes (HK18)versus HK18stably

transfected with mutant I κB to inhibit NF-κB activation (HK18/mI κB)[37].Cells were exposed to LDR (10cGy X-rays)and then incubated for 6h before exposure to a challenge dose of 2Gy γ-rays.The 6-h gap after LDR treatment was required to induce the adaptive response in other cell systems [38,39],including our previously reported mouse skin epithelial JB6cells [12].Preexposure to LDR signi ?cantly enhanced cell survival in wild-type human skin kerati-nocytes HK18(Fig.1A),but failed to induce radioresistance in NF-κB-negative HK18/mI κB cells (Fig.1B).The potential role of NF-κB activation in this process was further illustrated through the use of a NF-κB-controlled luciferase reporter assay that demonstrated a signi ?cant enhancement in activity (Fig.2A).In contrast,LDR-mediated NF-κB activation was absent from HK18/mI κB cells (Fig.2B).Moreover,we observed that basal NF-κB activity in the sham-LDR control HK18cells was increased during the time course of the experiment (Fig.2A),an effect most likely due to cell proliferation.LDR-treated cells exhibited a signi ?cant elevation in NF-κB activity compared to corresponding controls at each time point (Fig.2A).Immunoblotting results showed a marginally increased p50

level,

Fig. 1.NF-κB is involved in LDR-induced radioadaptive response in human skin keratinocytes.(A)Human skin keratinocytes HK18and (B)HK18/mI κB,the stable trans-fectants of HK18cells containing mutant I κB,were irradiated with LDR (10cGy X-rays)and then incubated for 6h before exposure to a challenging dose of 2Gy γ-rays.Cell radiosen-sitivity was determined by cell proliferation (MTT,left)and clonogenic survival (right)assays after radiation.Cells treated with sham LDR were included as control (n

=3/group).

Fig.2.NF-κB activation without induction of p65and p50in LDR-treated HK18cells.(A)HK18and (B)HK18/mI κB cells were cotransfected with the NF-κB luciferase reporter and β-galactosidase reporter for 6h,and NF-κB luciferase activity was measured at the indicated time points after exposure to sham or a single low dose of 10cGy X-rays.Luciferase reporter activity was normalized to β-galactosidase (n =3/group).(C)Exponentially growing HK18cells were irradiated with a single dose of 10cGy X-rays and NF-κB p50and p65levels were measured by Western analysis (C,sham-LDR control;β-actin served as loading control).

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whereas the NF-κB subunit p65remained at a relatively constant level 24h after LDR treatment(Fig.2C).These results suggest that NF-κB plays an essential role in the radioadaptive response,and the LDR-mediated NF-κB activation in human skin cells is a rapid response that does not involve transcriptional activation of NF-κB subunit genes.

Inhibition of an ATM-blocked,LDR-activated,NF-κB-mediated radioadaptive response

It is known that ATM can play an important role in DNA-damage-induced activation of NF-κB[34].Western blots were performed to determine whether immediate NF-κB activation by LDR is ATM-dependent.The results showed that a10-cGy LDR exposure to X-rays induced the phosphorylation of ATM(p-ATM) after30min and extended to24h postirradiation(Fig.3A),without affecting total ATM protein levels.To examine whether this LDR-induced ATM phosphorylation was required for NF-κB activation,cells were treated with the ATM inhibitor caffeine(5mM;for2h)before LDR.A complete inhibition of LDR-induced NF-κB activity and adaptive resistance were observed(Fig.3B).Similar results were obtained with the more speci?c ATM inhibitor,KU-55933(10μM; Fig.3B,right).The basal activity of NF-κB was moderately reduced by both ATM inhibitors.Suppression of ATM expression by siRNA also eliminated the10-cGy X-ray-mediated survival advantage after exposure to2Gyγ-irradiation(Fig.3C).These results strongly indicate that ATM plays a critical role in the LDR-induced NF-κB activation and adaptive radioresistance.

LDR-induced NF-κB activation is mediated through the

MEK/ERK pathway

Although the MEK/ERK pathway is linked with NF-κB in a variety of stress-related responses[33],it is unclear whether the LDR-induced adaptive response requires MEK/ERK activation.Fig.4A shows a striking increase in phosphorylated MEK1/2detected at30min post-LDR(Fig.4A,top).Phosphorylated ERK1/2,a major substrate of MEK, was also increased.In contrast,phosphorylation of the other two subfamilies of MAPKs,e.g.,p38and JNK,was scarcely affected by LDR (Fig.4B).These results indicate that LDR-mediated MEK/ERK activation is involved in signaling LDR-mediated adaptive

resistance.

Fig.3.ATM is required for LDR-induced NF-κB activation and radioadaptive response.(A)LDR-induced ATM phosphorylation(pATM).HK18cells were irradiated with10cGy X-rays; total and phosphorylated ATM(Ser-1981)levels were detected by Western blotting(right shows densitometry data of the relative expression levels of pATM normalized to the expression levels ofβ-actin;C,sham-LDR control).(B)HK18cells were cotransfected with NF-κB luciferase andβ-galactosidase reporters for6h and then incubated with5mM caffeine(left)for2h or10μM KU-55933(right)for1h.Luciferase activity was measured24h after exposure to sham-IR or irradiation with a single dose of10cGy X-rays and normalized toβ-galactosidase(n=3).(C)HK18cells were treated with caffeine,KU-55933,or ATM siRNA and then exposed to2Gyγ-irradiation with or without preexposure to 10cGy X-rays.Radiosensitivity was determined by clonogenic survival(n=3;inset on the right shows a Western blot of HK18cells treated with ATM siRNA;Ab,antibody). 1546K.M.Ahmed et al./Free Radical Biology&Medicine46(2009)1543–1550

To determine whether MEK/ERK induction is linked to NF-κB activation,we treated HK18cells with the MEK/ERK inhibitor PD98059(Fig.4C,top)or U0126(Fig.4C,bottom)for 2h before LDR.Both inhibitors completely inhibited the LDR-induced increase in NF-κB activity,suggesting that MEK/ERK is required for NF-κB activation.

ATM is involved in LDR-induced MEK/ERK activation

The MEK/ERK pathway can be activated by ATM in response to DNA damage [32].To further understand ATM's function in LDR-induced activation of MEK/ERK,HK18cells were incubated with or without the ATM inhibitor caffeine (5mM)before exposure to a single LDR dose of 10cGy X-rays.The LDR-mediated MEK/ERK phospho-

rylation that is described in Fig.4A was totally eliminated by the addition of caffeine (see Fig.5A).A similar inhibition was described in HK18cells treated with the highly speci ?c ATM inhibitor KU-55933(10μM,Fig.5B)and in ATM-null human ?broblasts GM05849(Fig.5C).Given that LDR-induced NF-κB activity and MEK/ERK phosphorylation are eliminated by ATM inhibition (Figs.3B and Fig.5),we conclude that ATM activation is the initiating event required for a cooperative function between the MEK/ERK/NF-κB pathways that lead to the LDR-induced adaptive response.LDR-induced interaction between ATM and NF-κB p65

ATM is shown to directly interact with and phosphorylate speci ?c signaling elements including p53and https://www.sodocs.net/doc/5410755519.html,ing the

p53-

Fig.4.MEK/ERK activation is required for LDR-induced NF-κB activity.(A)HK18cells were irradiated with a single dose of 10cGy X-rays;total and phosphorylated MEK and ERK levels were detected by Western blot at the indicated times.Sham-LDR cells (0cGy)were collected at the same times as irradiated cells.Right shows densitometry results of p-MEK1/2and p-ERK1/2normalized to β-actin (C,sham-LDR control,β-actin as a loading control).(B)HK18cells were treated as in (A),and total and phosphorylated p38and JNK levels were detected by Western blot.(C)HK18cells were cotransfected with NF-κB luciferase and β-galactosidase reporters for 6h and then incubated with 50μM PD98059(top)or 10μM U0126(bottom)for 2h,followed by exposure to sham-LDR or a single dose of 10cGy X-rays.Luciferase reporter activity was measured at 24h postirradiation and normalized to β-galactosidase (?P =0.02compared to cells treated with control DMSO;n =3).

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inactivated HK18cells,we questioned whether ATM can directly interact with NF-κB subunits.We found that although a low level of p65/ATM interaction was detectable in sham-irradiated control HK18cells,a substantial amount of p65/ATM interaction was detected in 10-cGy-treated cells (Fig.6).No p65/ATM interaction was observed in the NF-κB-inactive HK18cells that were stably transfected with mI κB (Fig.6).The blot was also probed with an ATM antibody to ensure that ATM was pulled down in both cell lysates.The results indicate that a p53-independent interaction between ATM and NF-κB p65present in resting cells can be signi ?cantly elevated by the stress induced by low-dose radiation.Because p65lacks the Ser/Thr-Gln (SQ/TQ)motif that serves as a phosphoacceptor site for ATM kinase,ATM-mediated p65activation is more likely induced by a direct physical interaction with ATM,which may require an unknown kinase to activate NF-κB.Such a mechanism that accounts for this overriding of the interaction and activation of NF-κB by ATM requires further study.Discussion

Consistent with previously reported data regarding the adaptive response in mouse skin epithelial cells,we provide additional evidence that exposure to low-dose radiation (10cGy X-rays)can induce an adaptive radioresistance in human skin keratinocyte https://www.sodocs.net/doc/5410755519.html,ing p53-de ?cient HK18cells,the current study further suggests a cooperative function between the DNA-damage sensor protein ATM and stress kinases MEK/ERK along with the transcription factor NF-κB in the development of a low-dose radiation-mediated survival advantage.A novel complex of ATM and NF-κB p65detected in unirradiated control cells was strikingly increased after exposure to

LDR.Expression levels of p65and p50were not elevated by LDR and the ATM/p65complex was not observed in NF-κB-inhibited HK18/mI κB cells,indicating that the direct interaction between ATM and NF-κB is a predominant feature of LDR-induced NF-κB activation.

Skin is the largest organ in the human body.It is highly susceptible to oxidative stress present in the environment including that induced by exposure to low-dose radiation,which,along with other cytotoxic stress inducers,is a public concern due to its potential risk of gene mutation and carcinogenesis [40].Accumulating information suggests that exposure to a relatively low level of ionizing radiation can induce a radioadaptive response that leads to a signi ?cant reduction of radiation-induced injury.Preexposure of mammalian cells to LDR has been shown to enhance cell growth and survival and,importantly,increase the resistance to radiation-induced carcinogenesis [41,42].Other reports indicate that human lymphocytes become less suscep-tible to radiation after exposure to very low doses of X-rays [43].LDR-induced adaptive radioprotection has also been reported to protect against micronucleus formation and neoplastic transformation in C3H 10T1/2mouse embryo cells [44,45].Our present study identi ?es an adaptive radioprotection that involves the cooperative activation of ATM/ERK/NF-κB pathways induced by a 10-cGy X-ray exposure of human skin keratinocytes (Fig.1A).Key molecular elements in this adaptive pathway may serve as therapeutic targets with which to mitigate the extent of radiation injury in human skin.

NF-κB is actively involved in signaling responses to oxidative stress and ionizing radiation exposure [4,21].Several groups have tested the effects of NF-κB inhibitors on cellular radiosensitivity with results that support the concept that NF-κB activity is a necessary element for enhancing cell survival under high-dose radiation [37].Nontumori-genic cell lines obtained from mouse and human skin epithelial cells are very useful in vitro models for studying skin radioadaptive responses.NF-κB-mediated induction of the mitochondrial antiox-idant MnSOD has been previously observed by our group in mouse skin JB6P+epithelial cells [12].However,a similar NF-κB-mediated protective function has not been observed in human skin epithelial cells to date.We have reported that NF-κB is responsible for a major portion of the radioresistance observed in the cell population (HK18-IR)derived from human HK18keratinocytes exposed to fractionated doses of ionizing radiation (2Gy/fraction;total dose 60Gy)[37].Using microarray analysis,a group of stress-responsive genes was found to be activated in HK18-IR cells and about 25%of the up-regulated genes were identi ?ed as candidate genes responsive to NF-κB activation.In this study,we show that a single exposure to LDR (10cGy X-rays)can induce NF-κB-mediated luciferase reporter activity in HK18cells.However,in contrast to the LDR-induced signi ?cant expression of NF-κB subunits in JB6cells [12],the total protein levels of p65and p50were not increased (Fig.2).These

results

Fig.5.LDR-induced MEK/ERK phosphorylation was either eliminated by ATM inhibitors or absent from ATM-de ?cient GM05849cells.(A)HK18cells were treated with 5mM ATM inhibitor caffeine for 2h or (B)10μM KU-55933for 1h before exposure to 10cGy X-rays (C,sham-LDR control).The expression levels of the indicated proteins were detected by Western analysis.No detectable ATM phosphorylation was found in either ATM inhibitor-treated cells (A and B)or (C)the ATM-de ?cient GM05849cell

line.

Fig.6.LDR increased ATM/NF-κB p65complex formation.HK18and HK18/mI κB cells were irradiated with sham (0cGy)or 10cGy X-rays.Whole-cell lysates were immunoprecipitated (IP)with anti-ATM antibody followed by immunoblotting (IB)with p65or ATM antibody.Total lysates extracted from cells treated with 10cGy and preincubated with ATM antibody served as a negative (Neg.)control.

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provide the?rst evidence that the adaptive response is excluded in the HK18cells with inactive NF-κB owing to overexpression of mutant IκB (HK18/mIκB),suggesting that NF-κB activation is required for this response.Moreover,the present data demonstrate a potential mechanism that LDR-induced NF-κB activation in human skin cells is mediated via a rapid protein response and that the mode of radiation-induced NF-κB activation varies between human and mouse skin cells.

Another important?nding is the observed enhanced phosphoryla-tion of ATM in HK18cells exposed to LDR(10cGy X-rays).ATM,a key DNA-damage response protein,plays an essential role in many DNA-damage stress-responsive signaling pathways,including MEK/ERK [46]and NF-κB[47].Although both ATM and MEK/ERK pathways are shown to be actively involved in the radiation response,their exact function in the LDR-induced radioadaptive response is unknown.We previously reported that ERK phosphorylation down-regulates ATM/ NF-κB-mediated radioresistance in select human breast cancer MCF-7 cells after long-term exposure to therapeutic fractionated doses of radiation[48].In contrast to these data,this study identi?es an ATM-dependent activation of the MEK/ERK pathway after exposure to LDR (Fig.4).These results provide new evidence that MEK/ERK activity is differentially regulated in human cells(p53-competent breast cancer cells versus p53-inactive keratinocytes)under different stress condi-tions induced by high or low doses of radiation.The coordinated activation of the ATM/MEK/ERK/NF-κB network by LDR is further supported by the fact that blocking ATM with KU-55933or caffeine inhibits the phosphorylation of MEK and ERK(Figs.5A and B).In addition,blocking MEK/ERK activation with PD98059or U0126 eliminated LDR-induced NF-κB activation(Fig.4C).Interestingly,the sequence surrounding Ser-218and Ser-222within the activation loop domain of MEK fails to meet the criteria of an ATM consensus motif [49].Therefore,our data cannot con?rm a direct activation of the MEK/ ERK pathway by ATM,but rather suggest the involvement of an unknown intermediate kinase(s)that is responsible for MEK phos-phorylation in response to LDR.The MEK/ERK pathway is also implicated in NF-κB induction upon ectopic expression of p53in SAOS osteoblasts[33].A recent report indicates that activation of NF-κB by DNA damage is induced through the MEK/ERK pathway in p53-null cells in response to doxorubicin treatment[32].The p53-mediated NF-κB activation after DNA damage is believed to induce a proapoptotic response[33].Because HK18cells are p53inactive[37], this study strongly suggests that ATM-mediated NF-κB activation induces a prosurvival response via the MEK/ERK pathway.Our data also indicate that the DNA-damage sensor protein ATM can form a complex with NF-κB p65in resting cells and the complex formation can be enhanced by LDR.The possibility that the interaction could be via redox-sensitive modi?cations of cysteine or methionine residues on the proteins and not phosphorylation events needs to be clari?ed. Nonetheless,the interaction result suggests an active communication between ATM and NF-κB for enhancing cell survival.In addition, radiation-induced activation of MEK/ERK was ATM-dependent although there is no evidence for a direct interaction between ATM and MEK/ERK.Because the plating ef?ciency of HK18cells is low(18%), activation of the ATM/MEK/ERK/NF-κB pathway is expected to occur in a small number of cells capable of forming colonies.This is sup-ported by the fact that siRNA-mediated inhibition of ATM in HK18cells or NF-κB-inhibited HK18/mIκB cells failed to show a radioadaptive response.The detailed mechanism of the ATM/ERK/NF-κB pathway in radioadaptive cells derived from viable clones after10cGy+2Gy treatment will be determined in future studies.

In summary,this study suggests that an adaptive response can be induced by exposure to10cGy X-rays in HPV18-immortalized human skin keratinocytes.Coactivation of the ATM,MEK/ERK,and NF-κB pathways is required for the prosurvival effect exhibited against the cytotoxic effects of2Gyγ-irradiation.ATM was found to directly interact with the NF-κB subunit p65in unirradiated cells,and this interaction was signi?cantly enhanced in irradiated NF-κB wild-type cells but not in NF-κB-inhibited HK18/mIκB cells.Thus,the prosurvi-val ATM/ERK/NF-κB network in the low-dose radiation-induced radioadaptive response may offer an effective therapeutic approach to enhancing radiation tolerance in human skin cells.

Acknowledgments

We thank Dr.Shigeki Miyamoto(University of Wisconsin at Madison,WI,USA)for helpful comments and Dr.David Chen (University of Texas Southwestern Medical Center at Dallas,TX, USA)for providing the ATM inhibitor KU-55933.This work was supported by Department of Energy Grant DE-FG02-03ER63634and NIH NCI Grant RO1101990.

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The way常见用法

The way 的用法 Ⅰ常见用法: 1)the way+ that 2)the way + in which(最为正式的用法) 3)the way + 省略(最为自然的用法) 举例:I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法: 在当代美国英语中,the way用作为副词的对格,“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2)The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3)The way =how/ how much No one can imagine the way he missed her. 4)The way =because

免疫和炎症相关信号通路-精选.pdf

免疫与炎症相关信号通路 一、Jak/Stat Signaling:IL-6 Receptor Family Jak和Stat是许多调节细胞生长、分化、存活和病原体抵抗信号通路中的关 键部分。就有这样一个通路涉及到IL-6(gp130)受体家族,它帮助调节B 细胞的分化,浆细胞生成和急性期反应。细胞因子结合引起受体的二聚化同 时激活受体结合的Jak蛋白,活化的Jak蛋白对受体和自身进行磷酸化。这些磷酸化的位点成为带有SH2结构的Stat蛋白和接头蛋白的结合位臵,接头蛋白将受体和MAP激酶,PI3激酶/Akt还有其他的通路联系在一起。受体结合的Stat蛋白被Jak磷酸化后形成二聚体,转移进入细胞核调节目的基因的 表达。细胞因子信号传导抑制分子(SOCS)家族的成员通过同源或异源的 反馈减弱受体传递的信号。Jak或Stat参与其他受体蛋白的信号传导,在下 面Jak/Stat使用表格中有这方面的列举。研究人员已经发现Stat3和Stat5在一些实体肿瘤中被酪氨酸激酶而不是Jaks组成性激活。 JAK/STAT途径介导细胞因子的效应,如促红细胞生成素,血小板生成素, G-CSF,这些细胞因子分别是用于治疗贫血,血小板减少症和中性粒细胞减 少症的蛋白质类药物。该途径也通过干扰素介导信号通路,干扰素可以用来 作为抗病毒和抗增殖剂。研究人员发现,失调的细胞因子信号有助于癌症的 发生。异常的IL-6的信号或导致自身免疫性疾病,炎症,癌症,如前列腺癌 和多发性骨髓瘤的发生。Jak抑制剂目前正在多发性骨髓瘤模型中进行测试。Stat3具有潜在促癌性(原癌基因),在许多癌症中持续的表达。在一些癌细 胞中,细胞因子信号传导和表皮生长因子受体(EGFR)家族成员之间存在交流。 Jak激活突变是恶性血液病中主要的分子机制。研究人员已经在Jak2假激酶域中发现一个特有的体细胞突变(V617F),这个突变常常发生于真性红细 胞增多症,原发性血小板增多症和骨髓纤维化症患者。这个突变导致Jak2的病理激活,同时激活控制红细胞,巨核细胞和粒细胞增殖分化的促红细胞 生成素(EPO),血小板生成素(TPO)和G-CSF等的受体。而Jak1的功能获得性体细胞突变已发现存在于成人急性淋巴细胞性白血病当中。体细胞激活突变已经证明存在于小儿急性淋巴细胞白血病(ALL)患者中。此外,

The way的用法及其含义(二)

The way的用法及其含义(二) 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语: 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势,忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中

(完整版)the的用法

定冠词the的用法: 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...

“the way+从句”结构的意义及用法

“theway+从句”结构的意义及用法 首先让我们来看下面这个句子: Read the followingpassageand talkabout it wi th your classmates.Try totell whatyou think of Tom and ofthe way the childrentreated him. 在这个句子中,the way是先行词,后面是省略了关系副词that或in which的定语从句。 下面我们将叙述“the way+从句”结构的用法。 1.the way之后,引导定语从句的关系词是that而不是how,因此,<<现代英语惯用法词典>>中所给出的下面两个句子是错误的:This is thewayhowithappened. This is the way how he always treats me. 2.在正式语体中,that可被in which所代替;在非正式语体中,that则往往省略。由此我们得到theway后接定语从句时的三种模式:1) the way+that-从句2)the way +in which-从句3) the way +从句 例如:The way(in which ,that) thesecomrade slookatproblems is wrong.这些同志看问题的方法

不对。 Theway(that ,in which)you’re doingit is comple tely crazy.你这么个干法,简直发疯。 Weadmired him for theway inwhich he facesdifficulties. Wallace and Darwingreed on the way inwhi ch different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way(that) hedid it. I likedthe way(that) sheorganized the meeting. 3.theway(that)有时可以与how(作“如何”解)通用。例如: That’s the way(that) shespoke. = That’s how shespoke.

way 用法

表示“方式”、“方法”,注意以下用法: 1.表示用某种方法或按某种方式,通常用介词in(此介词有时可省略)。如: Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法,其后可接不定式或of doing sth。 如: It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词),也可跟由that 或in which 引导的定语从句,但是其后的从句不能由how 来引导。如: 我不喜欢他说话的态度。 正:I don’t like the way he spoke. 正:I don’t like the way that he spoke. 正:I don’t like the way in which he spoke. 误:I don’t like the way how he spoke. 4.注意以下各句the way 的用法: That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看,你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题: ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A,而不选其他几项,则要弄清选项中含way的四个短语的不同意义和用法,下面我们就对此作一归纳和小结。 一、in a way的用法 表示:在一定程度上,从某方面说。如: In a way he was right.在某种程度上他是对的。注:in a way也可说成in one way。 二、on the way的用法 1、表示:即将来(去),就要来(去)。如: Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示:在路上,在行进中。如: He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示:(婴儿)尚未出生。如: She has two children with another one on the way.她有两个孩子,现在还怀着一个。 She's got five children,and another one is on the way.她已经有5个孩子了,另一个又快生了。 三、by the way的用法

The way的用法及其含义(一)

The way的用法及其含义(一) 有这样一个句子:In 1770 the room was completed the way she wanted. 1770年,这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么?其意义如何?在阅读时,学生经常会碰到一些含有the way 的句子,如:No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中,the way之后的部分都是定语从句。第一句的意思是,“没人知道他是怎样发明这台机器的。”the way的意思相当于how;第二句的意思是,“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中,the way也是as的含义。随着现代英语的发展,the way的用法已越来越普遍了。下面,我们从the way的语法作用和意义等方面做一考查和分析: 一、the way作先行词,后接定语从句 以下3种表达都是正确的。例如:“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如:“火灾如何发生的,有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.

肿瘤常见信号通路

1 JAK-STAT信号通路 1) JAK与STAT蛋白 JAK-STAT信号通路是近年来发现的一条由细胞因子刺激的信号转导通路,参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。与其它信号通路相比,这条信号通路的传递过程相对简单,它主要由三个成分组成,即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。 (1) 酪氨酸激酶相关受体(tyrosine kinase associated receptor) 许多细胞因子和生长因子通过JAK-STAT信号通路来传导信号,这包括白介素2?7(IL-2?7)、GM-CSF(粒细胞/巨噬细胞集落刺激因子)、GH(生长激素)、EGF (表皮生长因子)、PDGF (血小板衍生因子)以及IFN(干扰素)等等。这些细胞因子和生长因子在细胞膜上有相应的受体。这些受体的共同特点是受体本身不具有激酶活性,但胞内段具有酪氨酸激酶JAK的结合位点。受体与配体结合后,通过与之相结合的JAK的活化,来磷酸化各种靶蛋白的酪氨酸残基以实现信号从胞外到胞内的转递。 (2) 酪氨酸激酶JAK(Janus kinase) 很多酪氨酸激酶都是细胞膜受体,它们统称为酪氨酸激酶受体(receptor tyrosine kinase, RTK),而JAK却是一类非跨膜型的酪氨酸激酶。JAK是英文Janus kinase的缩写,Janus在罗马神话中是掌管开始和终结的两面神。之所以称为两面神激酶,是因为JAK既能磷酸化与其相结合的细胞因子受体,又能磷酸化多个含特定SH2结构域的信号分子。JAK蛋白家族共包括4个成员:JAK1、JAK2、JAK3以及Tyk2,它们在结构上有7个JAK同源结构域(JAK homology domain, JH),其中JH1结构域为激酶区、JH2结构域是“假”激酶区、JH6和JH7是受体结合区域。 (3) 转录因子STAT(signal transducer and activator of transcription)STAT被称为“信号转导子和转录激活子”。顾名思义,STAT在信号转导和转录激活上发挥了关键性的作用。目前已发现STAT家族的六个成员,即STAT1-STAT6。STAT蛋白在结构上可分为以下几个功能区段:N-端保守序列、DNA结合区、SH3结构域、SH2结构域及C-端的转录激活区。其中,序列上最保守和功能上最重要的区段是SH2结构域,它具有与酪氨酸激酶Src的SH2结构域完全相同的核心序列“GTFLLRFSS”。 2) JAK-STAT信号通路 与其它信号通路相比,JAK-STAT信号通路的传递过程相对简单。信号传递过程如下:细胞因子与相应的受体结合后引起受体分子的二聚化,这使得与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化作用而活化。JAK激活后催化受体上的酪氨酸残基发生磷酸化修饰,继而这些磷酸化的酪氨酸位点与周围的氨基酸序列形成“停泊位

way 的用法

way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗? 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法,方式”讲时,如表示“以……方式”,前面常加介词in。如例1; ●way作“方法,方式”讲时,其后可接不定式to do sth.,也可接of doing sth. 作定语,表示做某事的方法。如例2,例3;

PD-1和PDL-1信号通路阻断剂的研究

肿瘤逃逸是目前肿瘤免疫治疗公认的障碍。患者肿瘤细胞自身的缺陷和免疫系统的功能障碍一起作用促进了肿瘤的疯狂生长。T细胞在免疫治疗肿瘤的过程中处于中心地位,如果能调动体内的杀伤武器,那将是最有效的也是最安全的治疗肿瘤的途径。 肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。肿瘤免疫治疗的过程中早期肿瘤特异性的CD8+T细胞是激活的,随着肿瘤生长到后期失去了杀伤的功能;IFN-γ的分泌功能持续增强,对肿瘤产生抵抗力。 T细胞的活化除了需要通过APC递呈MHC-抗原肽给抗原特异性T细胞提供第一信号外,还需要一系列协同刺激分子提供第二信号,进而才能使T细胞达到生理活化阈值产生正常的免疫应答,这在理论上更好地解释了免疫系统对自身和非自身抗原产生免疫学应答时精确而微妙的调节机制。如果缺少共刺激分子提供的第二信号,将会导致T细胞的无反应性或特异性免疫耐受甚至进入凋亡,因此,正性和负性协同刺激信号的调节及两者之间的平衡在极体免疫应答的整个过程中起着重要的调节作用。 PD-1(programmed death-1)最初是在凋亡的T细胞杂交瘤中得到的,由于其和细胞凋亡相关而被命名为程序性死亡-1受体。 PDL-1(B7-H1)属于B7家族,具有IgV和IgC样区、跨膜区及胞浆区尾部,PDL-1与其T 细胞上的受体PD1相互作用,在免疫应答的负性调控方面发挥着重要作用;该分子具有广泛的组织表达谱,在一些肿瘤细胞系上有较高的表达,许多研究均表明其与肿瘤的免疫逃逸机制相关。肿瘤部位的微环境可诱导肿瘤细胞上的PDL-1的表达,且表达广泛,表达的PDL-1有利于肿瘤的发生和生长,诱导抗肿瘤T细胞的凋亡。转染PDL-1基因的P815肿瘤细胞系在体外可抵制特异性CTL的裂解,将其接种小鼠体内后具有更强的致瘤性和侵袭性。这些生物学特性均可通过阻断PDL-1而逆转。敲除PD1基因的小鼠,阻断PDL-1/PD-1通路,则接种肿瘤细胞不能形成肿瘤。 目前,PD-1的配体被证实有两个,分别是PDL-1(B7-H1)和PDL-2(B7-DC).PDL-1蛋白广泛表达于抗原提呈细胞(APCs)、活化T、B细胞、巨噬细胞、胎盘滋养层、心肌内皮和胸腺皮质上皮细胞。在许多人类肿瘤组织中均可检测到PDL-1蛋白的表达,且许多癌组织较正常组织中的PDL-1表达水平明显上调。应用免疫组织化学方法,已先后在乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤等人类肿瘤组织中检测到PDL-1蛋白的表达,且PDL-1的表达水平和患者的临床及预后紧密相关。 国内外研究进展-PD1/PDL1在肿瘤免疫治疗中的作用 在抗肿瘤免疫中,通常遵循肿瘤特异性T细胞活化、T细胞增值、肿瘤浸润和T细胞记忆应答加强的步骤。研究表明,肿瘤细胞以及肿瘤微环境中的APCs表达的PDL-1均可经PD-1/PDL-1信号通路抑制肿瘤抗原特异性T细胞的活化,下调T细胞介导的肿瘤免疫应答。 PDL-1单抗能有效抑制局部肿瘤生长; 阻断PD-1/PDL-1信号可以促进肿瘤抗原特异性T细胞的增值,发挥杀伤肿瘤细胞的作用;阻断肿瘤细胞上相关PDL-1信号可上调浸润CD8+T细胞IFN-γ的分泌,表明PD-1/PDL-1信号通路的阻断在以诱导免疫应答为目的的肿瘤免疫应答中发挥作用; 选择抗PDL-1单抗配合肿瘤疫苗进行肿瘤免疫治疗可有效加强肿瘤疫苗的免疫激活

the-way-的用法讲解学习

t h e-w a y-的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

免疫疗法—IDO信号通路 (Qwbio)

免疫疗法—IDO信号通路(Qwbio) 免疫逃避是识别恶性肿瘤的标志之一,研究者一直在努力地研究以明确能使癌细胞回避宿主免疫系统的复杂机制。癌症细胞以便于生存、增长、入侵及恶性肿瘤细胞的转移使用吲哚胺2,3-双氧酶(IDO)途径来抑制宿主的免疫应答。IDO通路在许多的癌症中是非常活跃的,对于T细胞袭击提供直接防御。这个IDO路径在许多的抗原提呈细胞中也非常活跃,导致对肿瘤相关抗原的外周耐受性。 IDO有两种同工酶,IDO1和IDO2,通常涉及到氨基酸色氨酸的分解代谢。启维益成代理IDO具有免疫阻尼效应,通过抑制巨噬细胞和效应T细胞应答。这个免疫抑制的确切机制目前并不清楚,但是他可能涉及了敏感T细胞色氨酸饥饿或色氨酸代谢过程中有毒代谢物(犬尿氨酸)的积累,导致细胞周期停滞或在有癌症细胞的环境下导致效应T细胞死亡。IDO的表达经常能直接激活关闭免疫应答反应的调节性T细胞,这放大了抑制效。 IDO的抑制剂能够预防这种免疫抑制并容许识别和对抗癌细胞的免疫细胞的激活。因此作为研发新一类的免疫治疗抗癌剂,IDO将会是继PD-1和CTLA通路之后一个新的研究热点。重要的是,这是一个广泛可适用的信号通路,而不是特定于任何特定形式的癌症。IDO 抑制能配合化疗和免疫治疗药物及临床疗法一起结合使用,包括使用单克隆抗体策略来免疫检查靶标如CTLA-4、PD-1和PD-L1。 许多大型药物公司已经意识到了开发IDO信号通路的价值(金融和治疗价值)。几个IDO 抑制剂在早期的临床试验已经展现出很好的抗肿瘤活性,且有许多的公司正积极地投资于这些领域。近期Genentech (Roche)公司与NewLink Genetics公司达成一致意见,将NewLink’s 的两个IDO 抑制剂(NLG919 和indoximod)的研发估价为十亿美元。上周Bristol Meyers Squibb公司购买了Flexus Biosciences的F001287(Flexus公司的小分子IDO1抑制剂)花费了12.4亿元,这个小分子抑制剂能作用于相关的酶,TDO(色氨酸-2,3双加氧酶)。Incyte公司正积极地调查他们自己的IDO1抑制剂(INCB24360)和Merck公司的anti-PD-1免疫疗法((MK-3475))、AstraZeneca/ MedImmune公司的anti-PDL1抑制剂(MEDI4736)和Genentech 公司的PD-L1免疫检查抑制剂((MPDL3280A))的使用情况。 启维益成代理的Bps目前提供的IDO相关的Products: 产品名称中文名称Item number IDO1 protein IDO1 蛋白71182 IDO2 protein IDO2 蛋白71194 TDO protein TDO 蛋白71195 INCB024360 inhibitor INCB024360 抑制剂27338 NLG919 inhibitor NLG919 抑制剂27337 即将推出 IDO1 Assay Kit IDO1 分析试剂盒 IDO2 Assay Kit IDO2 分析试剂盒 TDO Assay Kit TDO 分析试剂盒

免疫和炎症相关信号通路

免疫与炎症相关信号通路 1、 Jak/Stat Signaling:IL-6 Receptor Family Jak和Stat是许多调节细胞生长、分化、存活和病原体抵抗信号通路中的关键部分。就有这样一个通路涉及到IL-6(gp130)受体家族,它帮助调节B细胞的分化,浆细胞生成和急性期反应。细胞因子结合引起受体的二聚化同时激活受体结合的Jak蛋白,活化的Jak 蛋白对受体和自身进行磷酸化。这些磷酸化的位点成为带有SH2结构的Stat蛋白和接头蛋白的结合位置,接头蛋白将受体和MAP激 酶,PI3激酶/Akt还有其他的通路联系在一起。受体结合的Stat蛋白被Jak磷酸化后形成二聚体,转移进入细胞核调节目的基因的表 达。细胞因子信号传导抑制分子(SOCS)家族的成员通过同源或异源的反馈减弱受体传递的信号。Jak或Stat参与其他受体蛋白的信号传导,在下面Jak/Stat使用表格中有这方面的列举。研究人员已经发现Stat3和Stat5在一些实体肿瘤中被酪氨酸激酶而不是Jaks组成性激活。 JAK/STAT途径介导细胞因子的效应,如促红细胞生成素,血小板生成素,G-CSF,这些细胞因子分别是用于治疗贫血,血小板减少症和中性粒细胞减少症的蛋白质类药物。该途径也通过干扰素介导信号通路,干扰素可以用来作为抗病毒和抗增殖剂。研究人员发现,失调的细胞因子信号有助于癌症的发生。异常的IL-6的信号或导致自身免疫性疾病,炎症,癌症,如前列腺癌和多发性骨髓瘤的发生。Jak抑制剂目前正在多发性骨髓瘤模型中进行测试。Stat3具有潜在促癌性(原癌基因),在许多癌症中持续的表达。在一些癌细胞中,细胞因子信号传导和表皮生长因子受体(EGFR)家族成员之间存在交流。 Jak激活突变是恶性血液病中主要的分子机制。研究人员已经在Jak2假激酶域中发现一个特有的体细胞突变(V617F),这个突变常常发生于真性红细胞增多症,原发性血小板增多症和骨髓纤维化

way的用法总结大全

way的用法总结大全 way的用法你知道多少,今天给大家带来way的用法,希望能够帮助到大家,下面就和大家分享,来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地,大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。

way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句

the_way的用法大全教案资料

t h e_w a y的用法大全

The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆,下面的可以不看了 另外,在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的

the way 的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.

The way的用法及其含义(三)

The way的用法及其含义(三) 三、the way的语义 1. the way=as(像) Please do it the way I’ve told you.请按照我告诉你的那样做。 I'm talking to you just the way I'd talk to a boy of my own.我和你说话就像和自己孩子说话一样。 Plant need water the way they need sun light. 植物需要水就像它们需要阳光一样。 2. the way=how(怎样,多么) No one can imagine the way he misses her.没人能够想象出他是多么想念她! I want to find out the way a volcano has formed.我想弄清楚火山是怎样形成的。 He was filled with anger at the way he had been treated.他因遭受如此待遇而怒火满腔。That’s the way she speaks.她就是那样讲话的。 3. the way=according as (根据) The way you answer the questions, you must be an excellent student.从你回答问题来看,你一定是名优秀的学生。 The way most people look at you, you'd think a trash man was a monster.从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物。 The way I look at it, it’s not what you do that matters so much.依我看,重要的并不是你做什么。 I might have been his son the way he talked.根据他说话的样子,好像我是他的儿子一样。One would think these men owned the earth the way they behave.他们这样行动,人家竟会以为他们是地球的主人。

way的用法

一.Way:“方式”、“方法” 1.表示用某种方法或按某种方式 Do it (in) your own way. Please do not talk (in) that way. 2.表示做某事的方式或方法 It’s the best way of studying [to study] English.。 There are different ways to do [of doing] it. 3.其后通常可直接跟一个定语从句(不用任何引导词),也可跟由that 或in which 引导的定语从句 正:I don’t like the way he spoke. I don’t like the way that he spoke. I don’t like the way in which he spoke.误:I don’t like the way how he spoke. 4. the way 的从句 That’s the way (=how) he spoke. I know where you are from by the way you pronounce my name. That was the way minority nationalities were treated in old China. Nobody else loves you the way(=as) I do. He did not do it the way his friend did. 二.固定搭配 1. In a/one way:In a way he was right. 2. In the way /get in one’s way I'm afraid your car is in the way, If you are not going to help,at least don't get in the way. You'll have to move-you're in my way. 3. in no way Theory can in no way be separated from practice. 4. On the way (to……) Let’s wait a few moments. He is on the way Spring is on the way. Radio forecasts said a sixth-grade wind was on the way. She has two children with another one on the way. 5. By the way By the way,do you know where Mary lives? 6. By way of Learn English by way of watching US TV series. 8. under way 1. Elbow one’s way He elbowed his way to the front of the queue. 2. shoulder one’s way 3. feel one‘s way 摸索着向前走;We couldn’t see anything in the cave, so we had to feel our way out 4. fight/force one’s way 突破。。。而前进The surrounded soldiers fought their way out. 5.. push/thrust one‘s way(在人群中)挤出一条路He pushed his way through the crowd. 6. wind one’s way 蜿蜒前进 7. lead the way 带路,领路;示范 8. lose one‘s way 迷失方向 9. clear the way 排除障碍,开路迷路 10. make one’s way 前进,行进The team slowly made their way through the jungle.

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