王　鋒　胡啟立　李　健　趙　嚴(yán)　劉　華　湯茂春　夏小俊　孟宏波　宋振順　徐曉蓉△

(1同濟(jì)大學(xué)附屬上海市第十人民醫(yī)院消化內(nèi)科, 2普外科　上?！?00072)

Akt通過磷酸化p27誘導(dǎo)膽管癌細(xì)胞生長

王鋒1胡啟立2李健2趙嚴(yán)1劉華1湯茂春1夏小俊1孟宏波2宋振順2徐曉蓉1△

(1同濟(jì)大學(xué)附屬上海市第十人民醫(yī)院消化內(nèi)科,2普外科上海200072)

【摘要】目的探討Akt和p27蛋白控制膽管癌細(xì)胞生長的分子機(jī)制。方法利用突變載體、突變失活載體以及位點突變基因的轉(zhuǎn)染技術(shù),流式細(xì)胞儀檢測PI3K、轉(zhuǎn)染各類載體對膽管癌細(xì)胞周期的影響,免疫印跡、免疫熒光檢測PI3K抑制劑、轉(zhuǎn)染各類載體對P27蛋白表達(dá)及分布的影響。結(jié)果PI3K抑制劑LY294002引起膽管癌細(xì)胞株G1期細(xì)胞周期阻滯,并引起p27蛋白入核;Akt能通過誘導(dǎo)p27蛋白出細(xì)胞核而解除LY294002的G1期細(xì)胞周期阻滯;Akt通過磷酸化野生型p27蛋白第157位蘇氨酸逆轉(zhuǎn)細(xì)胞周期阻滯。 結(jié)論Akt通過磷酸化p27第157位蘇氨酸來誘導(dǎo)p27細(xì)胞內(nèi)重定位于細(xì)胞質(zhì),促進(jìn)膽管癌細(xì)胞的生長。

【關(guān)鍵詞】膽管癌;p27;Akt;磷酸化;細(xì)胞周期

膽管癌是源自膽管上皮細(xì)胞的惡性腫瘤,流行病學(xué)研究顯示,膽管癌的發(fā)病率和死亡率在過去30 年間明顯呈進(jìn)行性增加趨勢[1]。針對膽管癌發(fā)病機(jī)制,尋找靶基因進(jìn)行靶向治療是目前研究的熱點。磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)/蛋白激酶B(protein kinase B,PKB)信號通路在肺癌、結(jié)腸癌、胰腺癌等惡性腫瘤細(xì)胞增殖、細(xì)胞周期、分化、凋亡過程中發(fā)揮重要作用,提示腫瘤的不良預(yù)后[2-3]。

p27作為細(xì)胞周期依賴性蛋白激酶抑制劑,能與cyclinE/CDK2和cyclinD/CDK4復(fù)合物緊密結(jié)合,同時其也是一種腫瘤抑制基因。研究發(fā)現(xiàn)包括膽管癌在內(nèi)的幾乎所有人類惡性腫瘤中,均存在p27蛋白的表達(dá)下調(diào)或缺失,是公認(rèn)的抑癌蛋白。p27抑制cyclinE/CDK2從而引起細(xì)胞周期阻滯在G1期,且p27只有在細(xì)胞核中才能抑制細(xì)胞的生長。PKB又稱Akt,能抑制p27的活性,PI3K信號通路可激活A(yù)kt發(fā)生磷酸化,Akt的激活也是膽管癌增長所需要的,然而,p27和Akt控制膽管癌細(xì)胞生長的分子機(jī)制目前尚不清楚。我們認(rèn)為膽管癌細(xì)胞中Akt誘導(dǎo)p27功能的喪失,是依賴于p27磷酸化以及p27重新定位于細(xì)胞核之外所引起。

因此,本研究擬通過觀察PI3K/Akt通路抑制劑對膽管癌細(xì)胞周期的影響,以及p27蛋白質(zhì)水平及定位的變化,利用突變載體、突變失活載體以及位點突變基因的轉(zhuǎn)染技術(shù),以初步闡明Akt通過調(diào)控p27進(jìn)而對膽管癌細(xì)胞生長產(chǎn)生影響。

材 料 和 方 法

材料和試劑人肝膽管癌細(xì)胞HCCC-9810細(xì)胞株來自中國科學(xué)院上海生物所細(xì)胞庫,RPMI-1640 培養(yǎng)基、胎牛血清購自美國Gibco公司，胰蛋白酶購自美國Invitrogen公司。PI3K抑制劑LY294002、MEK信號通路抑制劑U0126、p27 Kip1抗體、α-tubulin抗體、RCC1抗體購自美國Cell Signaling公司,二抗購自美國Santa Cruz公司。第157位蘇氨酸被替換為丙氨酸的p27蛋白(p27-T157A-GFP)、標(biāo)記GFP的p27蛋白由美國羅切斯特大學(xué)Hong W.Yao教授惠贈。突變激活A(yù)kt載體(mutationally activated Akt,Myr-Akt)、顯性失活突變Akt(dominate negative Akt,dn-Akt)購自美國Addgene公司。細(xì)胞核蛋白與細(xì)胞質(zhì)蛋白抽提試劑盒購自上海碧云天生物技術(shù)有限公司。

Western blot法檢測常規(guī)利用試劑盒分別提取細(xì)胞核蛋白和細(xì)胞質(zhì)蛋白或提取細(xì)胞總蛋白。利用BCA法測定蛋白濃度。取30 μg蛋白樣品,以10%SDS-PAGE凝膠電泳后,轉(zhuǎn)移至PVDF膜,脫脂牛奶室溫封閉2 h,加入一抗(1∶1 000) 4 ℃孵育過夜,加入二抗(1∶3 000)孵育1 h,洗膜后,以化學(xué)發(fā)光液顯像,暗室中進(jìn)行X光片壓片、顯影、定影后得到目的蛋白表達(dá)變化條帶。取重復(fù)3次實驗后的平均值作為實驗結(jié)果。

免疫熒光染色不同處理的細(xì)胞爬片后,經(jīng)4%甲醛固定10 min,0.5%Triton X-100破膜20 min,PBS清洗,一抗4 ℃ 孵育過夜,PBS清洗,37 ℃孵育熒光二抗1 h,DAPI染核,以含抗熒光淬滅劑的封片液封片。免疫熒光經(jīng)共聚焦顯微鏡觀察。

細(xì)胞周期采用流式細(xì)胞術(shù)檢測細(xì)胞周期。對于各組不同處理的細(xì)胞,用胰蛋白酶消化并制備細(xì)胞懸液,加入75%的冰乙醇4 ℃固定過夜,再用PBS洗2次,加入終濃度為50 mg/L的核糖核酸酶A(RNase A),37 ℃孵育30 min,加入終濃度為50 mg/L的PI,4 ℃ 閉光染色10 min后用流式細(xì)胞儀檢測。用Cell Quest獲取、Mod Fit軟件分析各組細(xì)胞的周期分布。

數(shù)據(jù)統(tǒng)計分析組間數(shù)據(jù)比較使用Student′st檢驗進(jìn)行分析,所有數(shù)據(jù)均使用SPSS 11.0軟件進(jìn)行處理。P<0.05為差異有統(tǒng)計學(xué)意義。

結(jié)果

LY294002對細(xì)胞周期及p27的影響與DMSO處理的對照組相比較,HCCC-9810細(xì)胞經(jīng)PI3K抑制劑LY294002(50 μmol/L)處理后,導(dǎo)致G1期細(xì)胞數(shù)目增加(P=0.008 9),而MEK抑制劑U0126(20 μmol/L)并不能誘導(dǎo)G1期的細(xì)胞數(shù)目發(fā)生變化(圖1A)。同時,PI3K抑制劑LY294002能增加HCCC-9810細(xì)胞中p27蛋白的總含量,而MEK抑制劑U0126對其并不產(chǎn)生影響(圖1B)。

LY294002可誘導(dǎo)p27蛋白進(jìn)入細(xì)胞核為評估p27蛋白在細(xì)胞內(nèi)的具體分布情況及明確其發(fā)揮作用的機(jī)制,我們進(jìn)一步檢測了PI3K抑制劑和MEK信號通路抑制劑對p27在細(xì)胞質(zhì)和細(xì)胞核分布情況的影響,結(jié)果顯示,PI3K抑制劑LY294002能增加p27在HCCC-9810細(xì)胞核中的表達(dá),并且降低其在細(xì)胞質(zhì)中的表達(dá)(圖1C)。而MEK信號通路抑制劑U0126對p27蛋白在細(xì)胞質(zhì)和細(xì)胞核的分布情況并無影響。RCC1和α-tubulin的Western blot實驗結(jié)果代表細(xì)胞核和細(xì)胞質(zhì)成分的純度。

A:Inhibitor of PI3K induces G1 arrest in HCCC-9810 cells.The percentage of cells in G1 phase was increased by the PI3K inhibitor,LY294002,compared with the DMSO control.MEK inhibitor,U0126,did not alter the number of cells in G1 phase.B:LY294002,increased p27 protein level compared to the DMSO control whereas U0126 had no effect.C:LY294002 decreased p27 content in the cytoplasm and increased p27 in the nuclear fractions.However,the MEK inhibitor did not alter p27 distribution in HCCC-8910 cells.The immunblots for RCC1 and alpha tubulin show appropriate distributions in the nuclear and cytoplasmic fractions,indicating purity of the fractions.(1)P<0.01,vs.DMSO group.

圖1PI3K抑制劑通過增加p27入核引起膽管癌

HCCC-9810細(xì)胞株發(fā)生G1期細(xì)胞阻滯

Fig 1Inhibitor of PI3K induces G1 phase arrest and increases

nuclear p27 accumulation in HCCC-9810 cell lines

Akt依賴性的p27蛋白再定位參與G1期細(xì)胞阻滯與對照組相比,突變激活A(yù)kt載體可誘導(dǎo)細(xì)胞中Akt蛋白含量增加(圖2B),顯示Myr-Akt工作良好。相對于轉(zhuǎn)染空質(zhì)粒組,LY294002對轉(zhuǎn)染GFP細(xì)胞的G1期細(xì)胞數(shù)目并沒有影響;而在共轉(zhuǎn)染Myr-Akt和GFP的細(xì)胞中,Myr-Akt增加了Akt總量,LY294002降低了G1期的細(xì)胞數(shù)目(圖2A,P=0.007 8)。這些數(shù)據(jù)表明,Akt的過度表達(dá)解除了LY294002誘導(dǎo)的G1期阻滯。

進(jìn)而,我們研究Akt對p27在細(xì)胞內(nèi)的定位情況。Myr-Akt載體誘導(dǎo)細(xì)胞高表達(dá)Akt,dn-Akt降低Akt表達(dá),收集各組細(xì)胞的細(xì)胞質(zhì)、細(xì)胞核蛋白進(jìn)行免疫印跡試驗。結(jié)果顯示高表達(dá)Akt的膽管癌細(xì)胞中,p27在細(xì)胞質(zhì)中含量增加,而在細(xì)胞核中含量減少;相反,在低表達(dá)Akt的膽管癌細(xì)胞中p27在細(xì)胞質(zhì)中含量減少，在細(xì)胞核中含量增加(圖2C)。上述數(shù)據(jù)表明,Akt可導(dǎo)致p27再定位到細(xì)胞質(zhì)中,從而參與到G1期細(xì)胞阻滯的過程。

A:The representative of immunoblotting of Akt in cells transfected with Myc-Akt or dn-Akt.B:The percentage number of cells in G1 arrest due to LY294002 in cells transfected with empty vector,GFP or co-transfected with Myr-Akt.C:The representative of immunoblotting of p27 in the cytoplasm and nucleus in cells transfected with Myc-Akt or dn-Akt,treated with LY294002 or not.(1)P<0.01,vs. Control group.

圖2Akt依賴性的p27在HCCC-9810細(xì)胞內(nèi)再

定位緩解LY294002誘導(dǎo)的G1期細(xì)胞阻滯

Fig 2Akt relieves G1 arrest induced by LY294002

through Akt-dependent p27 subcellular

localization in HCCC-9810 cells

Akt磷酸化p27蛋白第157位蘇氨酸引起p27出核為了解Akt引起p27細(xì)胞內(nèi)再定位的機(jī)制,我們采用免疫熒光染色觀察p27的細(xì)胞內(nèi)定位(圖3A)。結(jié)果顯示,細(xì)胞轉(zhuǎn)染野生型p27時,p27蛋白幾乎完全定位在細(xì)胞核;細(xì)胞轉(zhuǎn)染野生型p27和Myr-Akt載體,p27蛋白在細(xì)胞質(zhì)和細(xì)胞核中均有分布;而細(xì)胞同時轉(zhuǎn)染第157位蘇氨酸磷酸化位點突變的p27(p27-T157A)和Myr-Akt載體時,p27蛋白仍主要分布在細(xì)胞核內(nèi)。

進(jìn)而，我們采用定量方法檢測p27在細(xì)胞質(zhì)和細(xì)胞核中的表達(dá)量(圖3B)。在轉(zhuǎn)染野生型p27的細(xì)胞中，增加Akt的表達(dá)可增加p27在細(xì)胞質(zhì)中的含量并降低其在細(xì)胞核中的含量。相反,在細(xì)胞中轉(zhuǎn)染第157位蘇氨酸磷酸化位點突變的p27,Akt的表達(dá)對p27的分布影響不大。數(shù)據(jù)表明Akt依賴性的p27重定位需要p27在第157位點磷酸化后才能出核,從而影響其對細(xì)胞周期的阻滯作用。

A:The representative image of immunofluorescence staining of p27,Akt in HCCC-8910 cell lines.B:The Quantitative content of p27 distributed in nucleus and cytoplasm in cells transfected with p27 or p27-T157A,with Akt or not.Scale bar=10 μm.

圖3p27細(xì)胞內(nèi)再定位依賴于Akt磷酸化p27

第157位蘇氨酸

Fig 3Akt-dependent phosphorylation of p27 at T157

causes p27 accumulation in the cytoplasm

Akt通過磷酸化p27第157位蘇氨酸解除LY294002的G1期阻滯突變激活表達(dá)的Akt可以逆轉(zhuǎn)HCCC-9810的細(xì)胞周期阻滯,是由于野生型p27蛋白所誘導(dǎo),而不是磷酸化缺失的p27突變體。

相比轉(zhuǎn)染空載體,轉(zhuǎn)染野生型p27蛋白能增加細(xì)胞在G1期的數(shù)目。野生型p27蛋白與Akt的共表達(dá)能降低G1期的細(xì)胞數(shù)目(P=0.006 7)。相比轉(zhuǎn)染空載體,轉(zhuǎn)染第157位蘇氨酸突變的p27蛋白不僅能誘導(dǎo)G1期細(xì)胞增多,還阻斷了Akt降低G1期細(xì)胞數(shù)目的功能。結(jié)果提示,Akt引起p27蛋白在第157位點的磷酸化導(dǎo)致p27出核,并抑制了其G1期細(xì)胞阻滯的特性,從而促進(jìn)了細(xì)胞生長(圖4)。

The percentage number of cells in G1 arrest in cells transfected with p27,p27-T157A or Myr-Akt.(1)P<0.01.

圖4Akt通過磷酸化野生型p27第157位蘇氨酸解除LY294002

對膽管癌細(xì)胞HCCC-9810的G1期阻滯

Fig 4Akt rescues HCCC-9810 cells from the cell-cycle

delay induced by LY294002 through wild-type p27

phosphorylation at Thr157

討論

本研究在膽管癌中探對了Akt和p27調(diào)控腫瘤細(xì)胞生長的機(jī)制。結(jié)果顯示抑制PI3K/Akt信號通路可誘導(dǎo)細(xì)胞發(fā)生G1期阻滯,同時p27蛋白在細(xì)胞核內(nèi)表達(dá)增加,這種細(xì)胞周期阻滯可被上調(diào)的Akt所解除,p27在細(xì)胞核內(nèi)的表達(dá)也相應(yīng)減少,后續(xù)實驗進(jìn)一步證實Akt是通過p27蛋白Thr157位點磷酸化誘導(dǎo)出核,出核的p27和細(xì)胞周期蛋白結(jié)合后轉(zhuǎn)換到促細(xì)胞周期進(jìn)展的模式。我們認(rèn)為膽管癌細(xì)胞生長失調(diào)是由于PI3K/Akt信號通路的失調(diào),導(dǎo)致p27磷酸化水平降低,p27細(xì)胞質(zhì)移位導(dǎo)致喪失其生長抑制的特性,從而促進(jìn)腫瘤細(xì)胞生長。

膽管癌是多基因、多因素共同作用的結(jié)果,PI3K/Akt信號通路的失調(diào)對細(xì)胞增殖、凋亡具有重要的調(diào)控作用[4],在維持細(xì)胞惡性生物學(xué)行為中起重要作用。Akt作為PI3K的下游靶蛋白,是PI3K/Akt信號通路的核心,目前認(rèn)為Akt的活性調(diào)節(jié)主要依賴于PI3K,Ser473和Thr308位點的磷酸化是Akt激活的必要條件,磷酸化Akt(pAkt)是PI3K/Akt信號通路的效應(yīng)核心。磷酸化的pAkt可通過誘導(dǎo)抗凋亡蛋白的表達(dá)以及磷酸化凋亡調(diào)節(jié)蛋白從而抑制腫瘤細(xì)胞凋亡[5]。PI3K/Akt信號通路主要激活途徑是帶有磷酸化位點的結(jié)構(gòu)域與磷脂分子PIP3(磷脂酰肌醇3磷酸)結(jié)合后將PI3K亞單位p85募集到近細(xì)胞膜附近,p85磷酸化是PI3K激活的啟動及激活因子,從而進(jìn)一步激活增殖及凋亡相關(guān)基因,改變細(xì)胞周期,調(diào)控腫瘤細(xì)胞的生物學(xué)行為[6-7]。pAkt處于細(xì)胞內(nèi)多種信號途徑的交匯點,通過激活或抑制下游30余種靶蛋白,參與多種細(xì)胞活動及物質(zhì)代謝調(diào)節(jié),尤其與促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡、調(diào)控細(xì)胞周期等密切相關(guān)[8]。PI3K/Akt可通過mTOR來磷酸化eIF4E和p70S6K,影響細(xì)胞增殖,PI3K抑制劑可抑制mTOR,發(fā)生G1期阻滯,影響腫瘤細(xì)胞生長[8-9]。Akt升高還可激活p21激酶Pak1,繼而調(diào)節(jié)p21活性,導(dǎo)致細(xì)胞增殖[10];也可直接磷酸化p27,使其在細(xì)胞內(nèi)堆積,防止其對細(xì)胞周期的阻滯作用[11]。LY294002通透細(xì)胞后特異性抑制PI3K/Akt信號途徑,包括Akt的磷酸化,我們的研究顯示LY294002可引起膽管癌細(xì)胞HCCC-9810發(fā)生G1期阻滯。這種G1期阻滯還伴有p27蛋白在核內(nèi)分布的增多,這種細(xì)胞阻滯也可被外源增加的Akt所解除,同時,p27蛋白在細(xì)胞核內(nèi)的分布也相應(yīng)減少。這提示p27蛋白在PI3K/Akt通路調(diào)控細(xì)胞周期、促進(jìn)細(xì)胞增殖中發(fā)揮一定的作用。

p27是一種相對分子質(zhì)量為27 000的細(xì)胞周期抑制性蛋白[12],它能與cyclinE/CDK2、cyclinD/CDK4復(fù)合物緊密結(jié)合,其基因定位于12號染色體的12p12.0～12p13.1之間,含有3個外顯子和1個內(nèi)含子[13]。p27蛋白分子的N端含有2個絲氨酸磷酸化位點,介導(dǎo)CDK激酶活性的抑制;C端含有2個蘇氨酸磷酸化位點,與抑制H1組蛋白磷酸化有關(guān)。p27是CDK1重要的家族成員,可以結(jié)合Cyclin D/CDK復(fù)合物,是細(xì)胞周期的負(fù)調(diào)控因子[14]。p27蛋白在膽管癌中通常也是低表達(dá)狀態(tài),且p27水平與預(yù)后相關(guān),高表達(dá)組生存期更高[15-16]。Shiraso等[17]針對p27設(shè)計的缺失Jab1結(jié)合區(qū)域的p27-jab-d有抑制膽管癌細(xì)胞生長的作用。受到惡性增殖信號的刺激,細(xì)胞中p27的蛋白表達(dá)水平下降,這是由于其合成下降,同時蛋白翻譯后泛素蛋白酶體降解速度也提高[18]。p27蛋白水平功能的下降削弱了其對CDK或cyclin/CDK復(fù)合物活性的抑制效應(yīng),導(dǎo)致CDK的活化效應(yīng)遞增,使細(xì)胞能夠完成細(xì)胞周期G1到S期的重要轉(zhuǎn)換,驅(qū)動細(xì)胞周期的運行。Akt也可以使p27的Ser10位點、Thr187位點以及Thr157位點發(fā)生磷酸化,并且Thr157位點位于p27的細(xì)胞核內(nèi)定位信號區(qū)域中。當(dāng)PKB磷酸化p27的Thr157位點后,p27不能進(jìn)入細(xì)胞核內(nèi)發(fā)揮G1期阻滯的作用[11],而使細(xì)胞增殖、分裂過快、過多并導(dǎo)致腫瘤形成。我們的實驗結(jié)果也顯示,在膽管癌細(xì)胞中,Akt激活后通過磷酸化p27的Thr157位點從細(xì)胞核進(jìn)入細(xì)胞質(zhì),從而喪失抑制細(xì)胞周期的功能,促進(jìn)細(xì)胞生長。

正常膽管上皮細(xì)胞中,細(xì)胞周期蛋白依賴性的細(xì)胞生長是通過p27蛋白與cyclin/CDK在細(xì)胞核中的相互作用而抑制的。在膽管癌細(xì)胞中,各類生長因子和細(xì)胞因子通過細(xì)胞膜受體,激活PI3K/Akt信號通路,磷酸化的Akt可導(dǎo)致核內(nèi)的p27蛋白在T157位點磷酸化,觸發(fā)p27蛋白的核移出,使得p27與cyclinE/CDK2、cyclinD/CDK4復(fù)合物解離,細(xì)胞轉(zhuǎn)換到促細(xì)胞周期進(jìn)展的模式,導(dǎo)致分裂加快、腫瘤形成。我們認(rèn)為,下調(diào)Akt或突變p27第157磷酸化位點,均可調(diào)控p27的亞細(xì)胞定位,達(dá)到G1期阻滯、抑制增殖的目的,可作為膽管癌治療的靶點之一。

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Akt induces cell growth through phosphorylation p27 in cholangiocarcinoma cell

WANG Feng1, HU Qi-li2, LI Jian2, ZHAO Yan1, LIU Hua1, TANG Mao-chun1, XIA Xiao-jun1,MENG Hong-bo2, SONG Zhen-shun2, XU Xiao-rong1△

(1DepartmentofGastroenterology,2DepartmentofGeneralSurgery,ShanghaiTenthPeople′sHospital,TongjiUniversity,Shanghai200072,China)

【Abstract】ObjectiveTo investigate the molecular mechanisms for Akt and p27 in control of cholangiocarcinoma cell growth.MethodsMutationally activated Akt,dominate negative Akt,empty vector and phosphorylation deficient p27 were used in our study.Cholangiocarcinoma HCCC-9810 cells were treated with the above vectors,proteins or PI3K inhibitor.The cell cycle was measured by flow cytometry.The protein level and distribution of p27 were detected by Western blot and immunofluorescence.ResultsLY294002,an inhibitor of PI3K,induced G1 phase arrest in cholangiocarcinoma cell lines and increased p27 into the nucleus.Akt released LY294002-induced G1 phase arrest through increasing cytoplasmic p27 protein level.Akt reversed cells from the cell cycle arrest through wild type p27 phosphorylation at Thr157.ConclusionsAkt induced p27 phosphorylation at Thr157 and p27 relocalization to an extranuclear subcellular distribution in cholangiocarcinoma cells.

【Key words】cholangiocarcinoma;p27;Akt;phosphorylation;cell cycle

【中圖分類號】R753.8

【文獻(xiàn)標(biāo)識碼】A

doi:10.3969/j.issn.1672-8467.2016.03.003

(收稿日期:2016-01-29;編輯:王蔚)

國家自然科學(xué)基金(81472578,81570578); 上海市自然科學(xué)基金(15ZR1432800)

△Corresponding authorE-mail:xuxr@#edu.cn

*This work was supported by the National Natural Science Foundation of China(81472578,81570578) and the Natural Science Foundation of Shanghai,China (15ZR1432800).