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High cytotoxic sensitivity of the oligodendrocyte precursor cells to HSP90 inhibitors

Short Communication

High cytotoxic sensitivity of the oligodendrocyte precursor cells to HSP90inhibitors in cell cultures

Alberto Alcazar a ,?,Cristina Cid b

a Department of Investigation,Hospital Ramón y Cajal,Ctra.Colmenar km 9.1,E-28034,Madrid,Spain b

Center for Astrobiology,CSIC-INTA,Torrejon de Ardoz,Spain

a b s t r a c t

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

Received 15October 2008Revised 18December 2008Accepted 22December 2008Available online 7January 2009

Keywords:

Heat shock protein 90HSP90inhibitors Geldanamycin

17-allylamino-17-demethoxygeldanamycin Radicicol

Oligodendrocyte precursor cells O-2A cells Cell death

Oligodendrocyte precursor cells (OPCs)are able to proliferate,and most differentiate into post-mitotic oligodendrocytes that contribute to remyelination.We have previously studied the expression of heat shock protein90(HSP90)in OPCs.The ansamycins,such as geldanamycin and 17-allylamino-17-demethoxygeldana-mycin (17-AAG)act as speci ?c HSP90inhibitors,are potent anti-tumor agents and are currently undergoing clinical trials.This work investigated the effect of HSP90inhibitors on cultured OPCs.Geldanamycin,17-AAG and another chemically unrelated HSP90inhibitor,radicicol,were extremely cytotoxic for OPCs.The IC50values of geldanamycin,17-AAG and radicicol for OPCs were 7.1,10.7and 137nM,respectively,compared to 1000–2000nM for preoligodendrocytes,astrocytes and neurons.Adult OPCs were found to be susceptible to HSP90inhibitors in a similar fashion to perinatal cells.OPC death induced by these HSP90inhibitors led to a signi ?cant decrease in the oligodendrocyte population.The present results highlight that OPCs are uniquely sensitive to HSP90inhibitors.Geldanamycin and 17-AAG,which penetrate the blood-brain barrier,are novel cancer chemotherapeutic agents and we noted that,in anti-cancer therapy with these drugs,the OPCs may be compromised.

Oligodendrocyte precursor cells (OPCs),also known as O-2A progeni-tor cells,have been identi ?ed in cultured rodent cells and in the adult rodent and human central nervous system (CNS)(Raff et al.,1983a;Roy et al.,1999;Scolding et al.,1999;Shi et al.,1998).These cells retain characteristics of multipotent CNS stem cells (Kondo and Raff,2000),are able to proliferate,and most differentiate into post-mitotic oligodendro-cytes (Temple and Raff,1986).The human cells are immunophenotypi-cally similar to their rodent counterparts and those of the adult rodent CNS (Armstrong et al.,1992;Roy et al.,1999;Scolding et al.,1999).OPCs are extremely ef ?cient at myelin repair and contribute to remyelination (Blakemore and Keirstead,1999;Scolding et al.,1999).We have previously studied the expression of heat shock protein 90(HSP90)in OPCs (Cid et al.,2004,2005).HSP90is a conserved molecular chaperone (e.g.,rat HSP90βonly differs in 10amino acids out of 723from the human form of the protein)and is largely expressed in the cytosol,which is essential for the posttranslational control.The activity of HSP90is regulated by ATP.The ansamycin antibiotics,such as geldanamycin and their derivates,and the macrolactone radicicol interact with the ATP-binding site and act as speci ?c HSP90inhibitors (Chaudhury et al.,2006).Through speci ?c interaction with a single molecular target,these HSP90inhibitors cause

the destabilization and eventual degradation of HSP90client proteins.Among the HSP90inhibitors,the geldanamycin-derived compounds have shown potent anti-tumor activity in experimental models,and are promising in preclinical model systems,including in vitro and in vivo glioma models (Newcomb et al.,2007;Jane and Pollack,2008),and are currently in phase I and II clinical trials (Zhang et al.,2007).The present work investigated the potential toxicity of these inhibitors for oligoden-drocyte lineage cells:OPCs and pre-oligodendrocytes.HSP90inhibitors geldanamycin,its derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG),and the chemically unrelated radicicol were used,and their effects were studied on cultured OPCs,pre-oligodendrocytes,astrocytes and neurons.

Primary cell cultures were prepared from cerebral hemispheres,as previously described (Cid et al.,2004).All animal procedures were in accordance with The Declaration of Helsinki and Spanish legislation for the use of animals in biomedical experimentation.Cell suspensions from cerebral hemispheres of 17–19-day-old Sprague –Dawley rat embryos were seeded and kept at 37°C in a 7.5%CO 2atmosphere in high-glucose Dulbecco's medium supplemented with 15%heat-inactivated fetal calf serum.After 24h,cell cultures were placed and maintained in a serum-free medium as described (Cid et al.,2004)and named as culture medium.Cells were plated at a ?nal density of 33–36×103cells/cm 2.OPCs were phenotypically identi ?ed by their characteristic bipolar morphology and A2B5-positive (A2B5+)immunoreactivity (Raff et al.,1983a )labeling with mouse monoclonal A2B5IgM antibody (Eisenbarth et al.,1979)(Chemicon).The oligodendrocyte population was studied by

Experimental Neurology 216(2009)511–514

Abbreviations:17-AAG,17-allylamino-17-demethoxygeldanamycin;GFAP,glial ?brillary acidic protein;HSP90,heat shock protein 90;OPCs,oligodendrocyte precursor cells.

?Corresponding author.Fax:+34913369016.

E-mail address:alberto.alcazar@hrc.es (A.

10.1016/j.expneurol.2008.12.022

Contents lists available at ScienceDirect

Experimental Neurology

j o u r n a l h o me p a g e :w w w.e l s e v i e r.c om /l o c a t e /y e x n r

labeling the cells with mouse monoclonal O4IgM antibody(Sommer and Schachner,1981)(Chemicon),a marker of pre-oligodendrocytes,cells that have differentiated into the oligodendrocyte lineage(Armstrong et al.,1992).These cells were phenotypically identi?ed by their multipolar morphology and O4+immunoreactivity(Sommer and Schachner,1981). Rabbit polyclonal anti-glial?brillary acidic protein(GFAP)antibody (Dako)and mouse monoclonal anti-β-tubulin isotype III(βIII-tubulin) IgG antibody(Sigma)were used as markers for astrocytes and neurons, respectively.As previously described(Cid et al.,2005),in6-day-old cell cultures,the content of A2B5+OPCs was12±1%of the cells;very few O4+ pre-oligodendrocyte cells were present(≤1±0.2%);and GFAP+astrocytes andβIII-tubulin+neurons represented23±3%and63±9%,respectively, of all cells.From day6to12of culture,a gradual reduction in OPCs was observed,while the number of pre-oligodendrocytes correspondingly increased to10–12%of all cells,and the number of astrocytes and neurons remained constant over time(Cid et al.,2004).GFAP+andβIII-tubulin+cells were negative for A2B5(Supplementary material).

Cells cultured for6days were untreated(control cells)or treated with the HSP90inhibitors geldanamycin(Alexis),17-AAG and radicicol (both from Sigma)at concentrations of0.01nM to50μM for2h,to establish their cytotoxic effect.The cells were then washed and placed in culture medium for24h,and cell viability was assessed by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)-based viability assay(Roche Applied Science).HSP90inhibitors signi?cantly reduced cell viability in a dose-dependent manner(Supplementary material).Both17-AAG and geldanamycin induced signi?cant cell death at a concentration of100nM or higher,with an IC50of617and623nM, respectively.Radicicol at1μM was necessary to induce a signi?cant reduction in cell viability,with an IC50of1523nM.However,10nM17-AAG or geldanamycin was toxic for6–8%of cells in culture.We analyzed the type of dead cells in these cultures by immuno?uorescence with A2B5,O4,anti-GFAP and anti-βIII-tubulin antibodies(Fig.1).Interest-ingly,17-AAG(and geldanamycin;data not shown)was found to induce signi?cant cell death only in A2B5+OPCs,without affecting the other types of cells studied(O4+pre-oligodendrocytes,GFAP+astrocytes and βIII-tubulin+neurons)(Fig.1).Similar results were showed when 100nM radicicol was used(Fig.1A).The selective high toxicity of HSP90 inhibitors for OPCs was demonstrated by dose–response curves(Fig.1

B).

Fig.1.HSP90inhibitors are selectively cytotoxic for OPCs.Cultured cells were treated with or without HSP90inhibitors for1.5h,washed,and placed in culture medium for24h.Cells were?xed with3.7%paraformaldehyde for10min,and incubated for an immuno?uorescence assay for1h at room temperature,or overnight at4°C with the speci?c primary antibody A2B5,O4,anti-GFAP or anti-βIII-tubulin,for identi?cation of OPCs,pre-oligodendrocytes,astrocytes or neurons,respectively.Cells were washed and then exposed to the corresponding?uorochrome-conjugated secondary antibodies for1h at room temperature,mounted with anti-fade solution,and visualized by?uorescence microscopy.When anti-GFAP or anti-βIII-tubulin antibodies were used,the?xed cells were permeabilized in ethanol/acetic acid(19:1,v/v)for15min at?20°C.(A)Comparative susceptibility of OPCs,pre-oligodendrocytes,astrocytes and neurons to HSP90inhibitors.Cells cultured for6or12days(12-d)were untreated(control,white bars)or treated with10nM17-AAG(gray bars)or 100nM radicicol(black bars)for1.5h.Cell death was evaluated by immuno?uorescence assay by comparing the number of immunolabeled cells after treatment with the number in control experiments.The cells counted were all intact,stained and showed a full cell body.At least100–200cells from nine(3×3)or more?elds per coverslip were counted.Two independent observers counted the labeled cells in each experiment.Data are the mean±SEM of three or four independent experiments run in duplicate.Each experiment was performed with a different batch of cultured cells.??p b0.01compared to untreated cells(Student's t test).(B)Dose–response curves for HSP90inhibitors.Cells cultured for6or 12days(12-d)were treated with17-AAG(top),geldanamycin(middle)or radicicol(bottom),?xed and immunolabeled as described above.The labeled OPCs(circles),pre-oligodendrocytes-from12-d cultures-(triangles),astrocytes(inverted triangles)and neurons(squares)were counted as in A,and the number of each type of cell is represented as a percentage with reference to their number in untreated control cultures(de?ned as100%).Data are the mean of three or four independent experiments run in duplicate.Each experiment was performed with a different batch of cultured cells.The curves have a sigmoidal?t;±SEMs are omitted to simplify the?gure.(C,D)6-day cell cultures were untreated (control)(C)or treated with10nM17-AAG(D),?xed,and OPCs were labeled with A2B5antibody and?uorescein-conjugated anti-mouse IgM secondary antibody(Chemicon) (green).(E,F)HSP90inhibitors decreased the oligodendrocyte population.Cells cultured for6days were untreated(control)(E)or treated with10nM17-AAG(F)and placed in serum-free culture medium for another6days.Cells were?xed and labeled with O4antibody and rhodamine-red-X-conjugated anti-mouse IgM secondary antibody(Jackson)(red). The?gure shows intact bipolar A2B5+(C)and multipolar O4+(E)cells,and A2B5+(D)and O4+(F)cells as“footprints”in representative immuno?uorescence experiments.Scale bar inμm.

512Short Communication

Concentrations of≥100nM17-AAG or geldanamycin,or≥1μM radicicol, induced the extinction of the OPC population and became toxic for the other cells studied(Fig.1B).The IC50values of17-AAG,geldanamycin and radicicol for OPCs were10.7,7.1and137nM,respectively,compared to985,936and1,844nM,respectively,for neurons(Table1). Concentrations of≤1nM17-AAG or geldanamycin,or≤10nM radicicol were not toxic for any of the cells studied.Geldanamycin shares biological activities with17-AAG(Schulte and Neckers,1998)and was cytotoxic for OPCs at similar doses than17-AAG(see above);therefore, for subsequent experiments,only17-AAG was used.

O4+pre-oligodendrocytes were not susceptible to HSP90inhibitors in the range of concentrations toxic for OPCs,i.e.,10–100nM17-AAG or 100–500nM radicicol(Fig.1B).However,since most OPCs differentiate into pre-oligodendrocytes,it is important to demonstrate that OPC death induced by HSP90inhibitors leads to a signi?cant decrease in the oligodendrocyte population.As described above,6-day-old cell cultures contain OPCs and a low number of O4+pre-oligodendrocyte cells, whereas,in12-day-old cultures,the number of O4+pre-oligodendro-cytes increases in a manner that corresponds to the reduction in OPCs. Accordingly,6-day cell cultures were treated with10nM17-AAG or 100nM radicicol for1.5h,and were washed and placed in fresh serum-free culture medium for another6days.These12-day-cultured cells were?xed and labeled with the O4antibody in an immuno?uorescence assay for the identi?cation of pre-oligodendrocytes(Figs.1E and F).The number of O4+pre-oligodendrocytes in culture was signi?cantly reduced after treatment of OPCs with10nM17-AAG or100nM radicicol (61.3±4.4and72.6±5.5%,respectively,compared to100%for untreated controls;p b0.02,one-sample t test),and the labeled cells remained as O4+“footprints”(Fig.1F).As in the OPC population,this effect was dose-dependent,and augmented when HSP90inhibitor concentration was increased,but it was not observed when cells were treated with0.01–1.0nM17-AAG or0.01–10nM radicicol(data not shown).

It was of interest to con?rm the results described above in adult OPCs,to establish the potential biological effect of HSP90inhibitors in clinical treatment.OPCs from the optic nerve of N60-day-old rats were prepared according to the procedure of Raff et al.(Raff et al.,1983a,b),to obtain adult oligodendrocyte lineage cells.Two-day-old optic nerve cultures consist largely of OPCs,and maintaining the cells in low serum medium yields cultures enriched in oligodendrocytes.In7-day-old optic nerve cultures,multipolar oligodendrocytes were evident under phase contrast microscopy and represented N95%of the living cells.Two-day-old OPC cultures from the adult optic nerve were treated with HSP90 inhibitors for1.5h,and then washed and placed in fresh culture medium for another5days.These7-day cultures were?xed and labeled with O4 antibody to identify pre-oligodendrocytes in immuno?uorescence assays.The number of O4+pre-oligodendrocytes in culture was signi?cantly reduced after treatment of adult OPCs with10nM17-AAG or0.5μM radicicol(55.9±1.2and8±2.3%,respectively,compared to 100%for the untreated control cells;p b0.001,one-sample t test).As expected,0.01–1.0nM17-AAG or0.01–10nM radicicol did not induce any effect in treated adult OPCs.

The present work investigated the potential toxicity of HSP90 inhibitors in OPCs and its effect on oligodendrocyte production.The ansamycins geldanamycin and17-AAG were tested and compared to radicicol,another chemically unrelated HSP90inhibitor that also interacts with the ATP-binding site of HSP90(Chaudhury et al.,2006), to establish whether the observed effects were mediated by binding of the inhibitor to the ATP-binding domain of HSP90and to exclude any unknown and HSP90-unrelated effect.Geldanamycin,17-AAG and radicicol were highly cytotoxic for OPCs.The calculated IC50of geldanamycin and17-AAG for OPCs was～10nM,100-fold lower than that for pre-oligodendrocytes,astrocytes or neurons.This IC50value was in the range described for mouse and human tumor cells(Kamal et al.,2003),and compared to811–1193nM for oligodendrocytes, astrocytes and neurons,which is in the range described for normal mouse and human tissues(Kamal et al.,2003).The IC50of radicicol for OPCs was137nM,which was also signi?cantly lower than the1821–1959nM for other cells studied.

OPCs are myelin-forming after their differentiation into post-mitotic oligodendrocytes(Levine et al.,2001).Investigating whether HSP90 inhibitors are toxic for OPCs that develop into oligodendrocytes is important for considering the possible prevention of remyelination during therapy with these drugs.We demonstrated that HSP90 inhibitors induced OPC death and led to a signi?cant reduction in the oligodendrocyte population.Furthermore,because HSP90inhibitors are currently undergoing clinical trials in adult patients,we considered it of interest to con?rm the described biological effects of these drugs in adult cells.Adult OPCs express many of the same phenotypic marker antigens as their perinatal counterparts,although they differ in terms of their cell cycle duration and time course of differentiation,both of which are slower(Levine et al.,2001).Experiments were performed using adult OPCs,which were found to be susceptible to HSP90inhibitors in a similar fashion to perinatal OPCs,which led to a notable reduction in the adult oligodendrocyte population.

This study demonstrates that OPCs are uniquely sensitive to HSP90 inhibitors,compared to other cells from the CNS,including differ-entiated pre-oligodendrocytes.Nanomolar concentrations of these inhibitors induced signi?cant OPC death,without affecting the other types of cells studied.We propose that this?nding may be related with the fact that OPCs particularly express HSP90on their surface (Cid et al.,2004,2005).Recently,it has been described that surface HSP90interacts speci?cally with the extracellular domain of HER2 (ErbB2),a key client protein that mediates cell motility,differentiation and proliferation(Sidera et al.,2008).Cell-surface HSP90would be more accessible to interact with HSP90inhibitors and low dose of them would be suf?cient to inhibit this particular HSP90,leading to the degradation of client proteins,i.e.HER2,and the induction of cy-totoxicity.The nanomolar cytotoxicity of geldanamycin and17-AAG—which penetrate the blood-brain barrier(Egorin et al.,2001)—for OPCs,is coincident with the nanomolar anti-tumor activity of this family of drugs.These HSP90inhibitors exhibit potent anti-tumor activity and are novel cancer chemotherapeutic agents currently in phase I and II clinical trials(Neckers and Neckers,2002;Zhang et al., 2007).Among them,several clinical trials(Solit et al.,2007;Weigel et al.,2007;Bagatell et al.,2007)have reported peak plasma concentra-tions of17-AAG in the range～5–15μM.These17-AAG concentrations, according to pharmacokinetic studies of Egorin et al.(2001),would give a brain concentration of17-AAG from5to10-fold lower. Assuming this calculation,OPCs with a17-AAG IC50of10.7nM may be damaged.We noted in the present study that,during therapy with these drugs,the OPC population may be compromised and remyelina-tion limited.

Acknowledgments

We are indebted to Ms.M.Gómez-Calcerrada for her technical assistance.This work was supported by ISCIII grants08/0761and

Table1

IC50of HSP90inhibitors for OPCs,pre-oligodendrocytes,astrocytes and neurons

IC50(nM)

17-AAG Geldanamycin Radicicol

OPCs10.7±17.1±1137±14 Pre-oligodendrocytes921±104916±981821±196 Astrocytes1,193±160811±831959±312 Neurons985±156936±1201844±228

Cultured cells treated with17-AAG,geldanamycin or radicicol were?xed and immunolabeled with A2B5,O4,anti-GFAP and anti-βIII-tubulin antibodies as described in Fig.1to detect and count OPCs,pre-oligodendrocytes,astrocytes and neurons,respectively.IC50(±SEM)for HSP90inhibitors was calculated from dose–response curves(Fig.1B).513

Short Communication

RETICS-RD06/0026/0008,and grant CTM2008-00304,from Spanish Ministry of Science and Innovation.

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.expneurol.2008.12.022. References

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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.

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；

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.

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.

the way的用法大全

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 =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.

“the-way+从句”结构的意义及用法知识讲解

“the way+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 following passage and talk about it with your classmates. Try to tell what you think of Tom and of the way the children treated 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 the way how it happened. 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the way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) these comrades look at problems is wrong.这些同志看问题的方法不对。

The way(that ,in which)you’re doing it is completely crazy.你这么个干法，简直发疯。 We admired him for the way in which he faces difficulties. Wallace and Darwin greed on the way in which 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) he did it. I liked the way (that) she organized the meeting. 3.the way(that)有时可以与how(作“如何”解)通用。例如： That’s the way (that) she spoke. = That’s how she spoke. I should like to know the way/how you learned to master the fundamental technique within so short a time. 4．the way的其它用法：以上我们讲的都是用作先行词的the way，下面我们将叙述它的一些用法。

定冠词the的12种用法

定冠词the的12种用法定冠词the 的12 种用法，全知道?快来一起学习吧。下面就和大家分享，来欣赏一下吧。定冠词the 的12 种用法，全知道? 定冠词the用在各种名词前面，目的是对这个名词做个记号，表示它的特指属性。所以在词汇表中，定冠词the 的词义是“这个，那个，这些，那些”，可见，the 即可以放在可数名词前，也可以修饰不可数名词，the 后面的名词可以是单数，也可以是复数。定冠词的基本用法： (1) 表示对某人、某物进行特指，所谓的特指就是“不是别的，就是那个!”如： The girl with a red cap is Susan. 戴了个红帽子的女孩是苏珊。 (2) 一旦用到the，表示谈话的俩人都知道说的谁、说的啥。如：

The dog is sick. 狗狗病了。(双方都知道是哪一只狗) (3) 前面提到过的，后文又提到。如： There is a cat in the tree.Thecat is black. 树上有一只猫，猫是黑色的。 (4) 表示世界上唯一的事物。如： The Great Wall is a wonder.万里长城是个奇迹。(5) 方位名词前。如： thenorth of the Yangtze River 长江以北地区 (6) 在序数词和形容词最高级的前面。如： Who is the first?谁第一个? Sam is the tallest.山姆最高。但是不能认为，最高级前必须加the，如： My best friend. 我最好的朋友。 (7) 在乐器前。如： play the flute 吹笛子

Way的用法

Way用法 A:I think you should phone Jenny and say sorry to her. B:_______. It was her fault. A. No way B. Not possible C. No chance D. Not at all 说明：正确答案是A. No way，意思是“别想！没门！决不！” 我认为你应该打电话给珍妮并向她道歉。没门！这是她的错。再看两个关于no way的例句：（1）Give up our tea break? NO way! 让我们放弃喝茶的休息时间？没门儿！（2）No way will I go on working for that boss. 我决不再给那个老板干了。 way一词含义丰富，由它构成的短语用法也很灵活。为了便于同学们掌握和用好它，现结合实例将其用法归纳如下：一、way的含义 1. 路线

He asked me the way to London. 他问我去伦敦的路。 We had to pick our way along the muddy track. 我们不得不在泥泞的小道上择路而行。 2. （沿某）方向 Look this way, please. 请往这边看。 Kindly step this way, ladies and gentlemen. 女士们、先生们，请这边走。 Look both ways before crossing the road. 过马路前向两边看一看。 Make sure that the sign is right way up. 一定要把符号的上下弄对。 3. 道、路、街，常用以构成复合词 a highway（公路），a waterway（水路），a railway（铁路），wayside（路边）

way与time的特殊用法

way/time的特殊用法 1、当先行词是way意思为”方式.方法”的时候,引导定语从句的关系词有下列3种形式： Way在从句中做宾语 The way that / which he explained to us is quite simple. Way在从句中做状语 The way t hat /in which he explained the sentence to us is quite simple. 2、当先行词是time时,若time表示次数时,应用关系代词that引导定语从句,that可以省略; 若time表示”一段时间”讲时,应用关系副词when或介词at/during + which引导定语从句 1.Is this factory _______ we visited last year? 2.Is this the factory-------we visited last year? A. where B in which C the one D which 3. This is the last time _________ I shall give you a lesson. A. when B that C which D in which 4.I don’t like the way ________ you laugh at her. A . that B on which C which D as 5.He didn’t understand the wa y ________ I worked out the problem. A which B in which C where D what 6.I could hardly remember how many times----I’ve failed. A that B which C in which D when 7.This is the second time--------the president has visited the country. A which B where C that D in which 8.This was at a time------there were no televisions, no computers or radios. A what B when C which D that