摘要
目的
建立人急性早幼粒白血病细胞株HL60分别向粒系和单核系分化模型,运用比较蛋白质组学分析鉴定HL60细胞在甾醇类新药(NSC67657)和全反式维甲酸(all-trans retinoic acid ,ATRA)诱导下表达差异的蛋白质分子,并对其中筛选出来的差异蛋白β-catenin相关蛋白1(β-catenin-interacting protein 1,ICAT)进行功能研究。
策略与方法
1.验证NSC67657对CEBPα的促进作用并构建细胞分化模型:在基因和蛋白水平分析CEBPα在NSC67657作用下的表达变化情况,然后分别用NSC67657和ATRA作用HL60细胞,通过光镜、MTT试验、电镜、细胞表面分化抗原检测,细胞化学染色等方面,分析细胞分化方向,分化程度,以及诱导细胞分化最适药物浓度。通过流式细胞技术和电镜的联合使用,分析HL60细胞在诱导分化过程中凋亡的发生情况,保证后续蛋白筛查所得差异蛋白大部分和分化相关,而非和凋亡相关。
2. HL60细胞全蛋白双向电泳分离条件的优化:分别采用标准蛋白提取法、三氯乙酸/丙酮沉淀法、超声破碎法等蛋白提取方法,在传统电泳和改良电泳条件下进行细胞全蛋白分离,并做蛋白点计数数和凝胶重复性分析,寻找新的针对HL60细胞省时高效的双向电泳条件。
3.运用比较蛋白质组在最适电泳分离条件下,分析HL60细胞在分化前,和向单核、粒系分化后的差异表达蛋白谱:依照前面优化双向电泳分离条件,对分化前和向单核系、粒系分化后HL60细胞总蛋白进行分离,用PDquest(7.0)软件对扫描的二维凝胶电泳图像进行分析,筛选出HL60细胞分化前后差异表达蛋白斑点。候选差异表达蛋白斑点经胶内胰酶酶解,MALDI-TOF-MS分析,得到肽指纹图谱(peptide mass fingerprinting, PMF),使用Mascot软件在Swissprot数据库中检索鉴定差异表达蛋白质,并对差异蛋白质进行生物信息学分析。
4.验证仅出现在NSC67657处理组的差异蛋白点ICAT的表达差异情况,并对ICAT蛋白进行细胞内定位:使用NSC67657和ATRA分别作用HL60细胞,诱导其向两系分化,分别用RT-PCR、Western blot方法检测细胞分化前后ICAT在mRNA与蛋白水平的变化情况;然后双染ICAT蛋白和HL60细胞核,采用激光共聚焦的方法观察ICAT蛋白在细胞内的表达情况,并对其进行细胞内定位。
5. ICAT真核表达载体的构建、转染,并对转染细胞ICAT基因、蛋白的过表达进行验证:采用RT-PCR方法扩增加入了EcoR I和BamH I两个酶切位点的ICAT基因CDS全长序列,并克隆入带有红色荧光标签的真核表达载体pDsRed-C1,通过改良电转染技术,导入白血病HL60细胞和THP-1细胞,然后采用RT-PCR、Western blot方法对转染后细胞中ICAT基因和蛋白的高表达进行验证。
6.成功转染ICAT基因的HL60细胞生物学特征分析:采用MTT方法观察转染ICAT基因前后的HL60细胞在NSC67657作用下的增殖情况;应用流式细胞技术,细胞化学染色,电镜等方法检测转染前后及加有药物NSC67657作用前后细胞的分化情况。
7. ICAT基因在其他白血病细胞中的功能初探:采用流式细胞技术、和细胞化学染色方法观察NSC67657处理未转染和转染了pDsRed-ICAT真核表达载体的THP-1细胞分化情况。
结果
1.药物作用HL60细胞后,CEBPα基因和蛋白的表达呈现先升高后下降的趋势;NSC67657和ATRA对急性早幼粒细胞株HL60的增殖有明显抑制作用,半数抑制浓度(50% inhibitory concentration, IC50)分别是10μM和2μM。当用10μM NSC67657和2μM ATRA作用HL60细胞5天后,CD14和CD11b的表达分别达到90%以上,并出现明显肾形、畸形核的单核系分化细胞和呈杆状、分叶核的粒系分化细胞;胞浆可见较多嗜天青颗粒。未见细胞膜的磷脂酰丝氨酸外翻,细胞核染色质固缩及凋亡小体出现等凋亡现象;
2.在传统的高压聚焦的条件下,三种蛋白抽提方法所得蛋白分离条件不佳,可见大量横条纹和片状阴影;改良低压聚焦条件下,三种蛋白抽提方法所得蛋白分离均有所改善,三氯乙酸/丙酮沉淀法所得蛋白损失严重;超声破碎法所得蛋白点重复性差;标准蛋白抽提方法所得蛋白点数多,重复性好,结果可靠。蛋白分布于4～7居多,采用非线性pH3～10胶条最佳。
3.通过对分化前后的二维凝胶电泳图像进行对比分析,发现约有70个蛋白斑点表达有差异,对其中63个差异蛋白斑点进行MALDI-TOF-MS分析后,鉴定出了46种可能的差异表达蛋白。其中有24个差异蛋白点的变化趋势在两系分化中是相同的,有9个蛋白点仅在单核系分化后表达有差异,有13个蛋白点仅在粒系分化后表达有差异。
4. ICAT蛋白是仅在HL60细胞单核系分化前后表达差异的蛋白分子之一。通过验证,NSC67657处理后,ICAT在mRNA与蛋白水平都明显高于ATRA处理组和非处理对照组;激光共聚焦可见ICAT蛋白大多表达于细胞核和细胞浆中。
5.通过酶切、胶回收、测序,真核表达载体pDsRed-ICAT构建成功。随着改良电穿孔技术的支持,在成功转染了pDsRed-ICAT表达载体的HL60细胞中,验证了ICAT基因和蛋白的表达均明显增加。
6. pDsRed-ICAT真核载体转染HL60细胞前后,表面分化抗原CD14的表达无明显变化;NSC67657处理后,pDsRed-ICAT载体转染HL60细胞的增殖相对于对照组减慢更为明显,分化速度明显加快。
7. NSC67657可以诱导THP-1细胞向单核系分化,药物处理转染了pDsRed-ICAT真核载体的THP-1细胞相对于对照组,细胞增殖和分化无明显差异。
结论
1. NSC67657可以促进HL60细胞CEBPα的表达,并且10μM的NSC67657和2μM的ATRA可以在不伴随凋亡发生的情况下,成功诱导HL60细胞向单核系和粒系分化。
2.低压聚焦为真核细胞蛋白质组分析提供了又一高效,省时,可靠的蛋白质分离方法。
3.鉴定出的50种差异表达蛋白可能在NSC67657和ATRA诱导细胞分化的途径中起到重要作用。
4. ICAT蛋白仅在NSC67657诱导HL60细胞向单核系分化过程中表达升高。
5.外源性ICAT基因在HL60细胞和THP-1细胞中成功表达。
6. ICAT基因的过表达可以促进NSC67657诱导HL60细胞的分化进程,但不影响THP-1细胞的分化,说明ICAT蛋白可能是粒单系祖细胞定向分化的新的药物作用靶点。
Objective
To construct the models of promyelocytic leukemia HL60 cells’granulocytic and monocytic differentiation, and use comparative proteome method to separate and identify differentially expressed proteins in HL60 cells which are induced either by NSC67657 or ATRA. In addition, to further the functional research of ICAT which is screened out from these differentially expressed proteins.
Method
1. The verification of the activity of NSC67657 on the expression of CEBPα,and the construction of differentiating models: HL60 cells were treated by NSC67657 and ATRA respectively. Then the differentiating level, direction could be observed, and the most suitable drug concentration for introduction could be screened out by light microscope observation, MTT assay, ultramicrostucture analysis and cellular surface antigen detection. What’s more, the analysis of apoptosis was also performed as the cooperated detection by flow cytometry analysis and ultracmicrostructure observation. Only in this way, the proteins we screened out from differentiating HL60 cells were associated with cellular differentiation, but not apoptosis.
2. The modification of two dimensional electrophoretic separation of total proteins in HL60 cells: Three methods for protein extraction were employed: standard protein extraction, TCA/Ac precipitation and transonic quassation to extract total protein of HL60 cells with traditional and modified procedure. Then the protein spots could be counted and repeatation could be analyzed, which were the prerequisites for screening out the most suitable way for two dimensional electrophoresis.
3. Analysis differentially expressed proteins in HL60 cells when they were induced into granulocyte and monocyte in the most suitable way for separation: the total protein in differenting HL60 cells was separated using former mentioned suitable method, and then the 2DE gel image could be outlined and quantified using PDQuest 7.0 software. Matrix assisted laser desorption/isonization time of flying mass spectrometry (MALDI-TOF-MS) was adopted to identify differentia protein spots and then mascot software was used to search for proteins in Swissprot database.
4. Verify the expressing level of ICAT which was only differentially expressed in HL60 cells when they were treated by NSC67657, then locate the protein ICAT in HL60 cells: HL60 cells differentiation were induced by NSC67657 and ATRA respectively. Then RT-PCR and Western blot method were employed to verify the expressing level of ICAT mRNA and protein when cells were treated by the drugs. Furthermore, ICAT protein could be located using fluorescent microscope by fluorescent staining method.
5. Constructing eukaryotic expressing vector and verifying mRNA and protein overexpression after the transfection: The CDS fragment of ICAT gene which was inserted two incision enzyme recognization sites was cloned using RT-PCR method in pDsRed-C1 that was labeled by a red fluorescence tag. Then the transfection of recombinent ICAT into HL60 and THP-1 cells was performed using a modified electroporating procedure. RT-PCR and Western blot methods were employed to verificate the overexpression of ICAT mRAN and protein.
6. Analysis of biological character of sucessfully transfected HL60 cells: The proliferation of NSC67657 treated HL60 cells could be detected by MTT assay when recombinant ICAT was successfully transfected. Then the differentiation of drug treated or untreated, transfectd or untransfected HL60 cells was detected by flow cytometry analysis, chemical staining and ultramicrostructure observation.
7. An initial functional research about ICAT protein in other leukemic cells: THP-1 cell line was selected and subjected to NSC67657 treatment before or after the transfection of ICAT eukaryotic expressing vector.
Results
1. The expression of CEBPαmRNA and protein were elevated firstly and then down-regulated when HL60 cells were treated by NSC67657. NSC67657 and ATRA could noticeably inhibit the proliferation of HL60 cells and the IC50(50% inhibitory concentration)dose was about 10μM and 2μM respectively; The percentage of CD14 and CD11b positive cells could up to over 90% when HL60 cells were treated by these two drugs. At the same time, the kidney like and anisotrophy like caryomorphism which were frequently appeared in monocyte, and rhabditiform like and sublobe like caryomorphism which were frequently appeared in granulocyte, could be easily found with many azurophil granules in endochylema . The apoptosis could not be found in drug treated HL60 cells.
2. Under traditional focusing process with high voltage, chiastic stripe and lamellar shadow could be found everywhere on 2DE gels, by which protein spots were not that easy to investigate. However, the separation of proteins which were extracted by all the three methods was significantly up graded. But, the quantity of protein lost much using TCA/Ac precipitation, and the repeatation of 2DE gel images was low using transonic quassation. Inversely, not only protein quantity, but also 2DE gel images repeatation could be siginificantly improved using standard protein extraction under modified focusing procedure. Most of protein spots arrange from pH 4 to 7, so pH3~10 NL IPG was the best choice for further experiment.
3. Through the analysis of 2DE gel images, more than 70 protin spots showed differentially expressed, among which 63 protein spots were identified by MALDI-TOF-MS, and then 50 protein spots were found to be potential candidates as differentially expression during cellular differentiation. 25 of these 50 protein spots were either elevated or down regulated both in NSC67657 and ATRA treated HL60 cells, and 10 protein spots were only differentially expressed in NSC67657 treated HL60 cells, 15 protein spots were only differentially expressed in ATRA treated HL60 cells.
4. ICAT protein was one of 10 protein spots which were only differentially expressed in NSC67657 treated HL60 cells. The expressing levels of ICAT mRNA and protein were significantly up-regulated in NSC67657 treated group compared to untreated and ATRA treated group. The subcellular location of ICAT protein is cellular nucleus and endochylema.
5. The pDsRed-ICAT eucaryotic expressing vector was successfully constructed. And the expressing level of ICAT mRNA and protein was promoted after transfection.
6. The expression of CD14 did not change significantly between pDsRed-ICAT transfected and untransfected HL60 cells. However, the expression of CD14 in pDsRed-ICAT was greatly enhanced by the treatment of NSC67657, and the cellular proliferation was significantly inhibited.
7. THP-1 cell line could be induced into monocytic differentiation after the treatment of NSC67657. However, cellular differentiation and proliferation of pDsRed-ICAT transfected THP-1 cells did not show marked differences compared to untransfected THP-1 cells when they were treated by NSC67657.
Conclusions
1. NSC67657 was a potential activator of CEBPα. HL60 cells could be induced into monocytic and granulocytic differentiation without the heppening of apoptosis under 10μM NSC67657 and 2μM ATRA.
2. Focusing with low voltage was a new effective, reliable, time saved method for the eukaryocyte proteome analysis.
3. 50 differentially expressed proteins which had been identified might play critical roles in the differentiation of HL60 cells induced by NSC67657 and ATRA.
4. The expressing level of ICAT protein was elevated only in NSC67657 treated HL60 cells.
5. The overexpression of ICAT gene in HL60 cells and THP-1 cells had been successfully constructed.
6. The overexpression of ICAT protein could enhance the monocytic differentiation of NSC67657 treated HL60 cells, however, ICAT over-expression could not sensitize THP-1 cells under NSC67657 treatment. We concluded that ICAT might exert its effect only on granulo-monocytic progenitor cell stage, but not in monocytic progenitor cell stage.
引文
[1] Huang ME, Ye YC, Chen SR., Chai JR, Lu JX, Zhoa L, Gu LJ , Wang ZY.1988. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia[J]. Blood 72: 567-572.
[2] Erik D, Harris CJG, Joseph W, et al. A sterol mesylate activator of CEBPαsignaling induces monocytic differentiation in human leukemia cells in vitro and in vivo[J]. Proc Amer Assoc Cancer Res, 2006, 47,Abstract #4619.
[3] Behre G, Singh SM, Liu H,et al.Ras Signaling Enhances the Activity of C/EBPalpha to Induce Granulocytic Differentiation by Phosphorylation of Serine 248[J].J Biol Chem,2002, 277(29): 26293– 26299.
[4] Whiteside TL,Vujanovic NL, Herberman RB. Natural killer cells and tumor therapy[J]. Current Topics in Microbiology and Immunology, 1998, 230:221-224.
[5] Peter KJ. Stopping replication, at the begainning[J]. Nature Chemical Biology, 2008,4:331-332.
[6] Frank L, David YL, David AZ, et al. Effective Tumor Therapy with Plasmid-encoded Cytokines Combined with in Vivo Electroporation[J]. Cancer Research,2001, 61:3281-3284.
[7] Westendorf JJ, Yamamoto CM, Lenny N, et al. The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation[J]. Mol. Cell.Biol, 1998,18(1):322-333.
[8] Yan J, George PS. Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ? Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells[J]. Cancer Research, 2004,64:370-377.
[1] Suh N,Luyengi L,Fong HH,et al.Discovery of natural product chemopreventive agents utilizing HL60 cell differentiation as a model[J].Anticancer Res. 1995;15(2):233-239
[2] Saravanan RS,Rose JKC.A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues[J].Proteomics.2004;4:2522-2532
[3] Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Ana1 Biochem.1976;72:248-254
[4]牟芝蓉,吴玉章,李晋涛。双向电泳分析比较维甲酸诱导HL60细胞分化的蛋白表达差异[J]。第三军医大学学报。2002;24(10):1153-1156
[5] Voss T,Ahom H,Haberl P,et a1.Correlation of clinical data withpmteomics profiles in 24 patients with B-cell chronic lymphocytic leukemia[J] . Int J Cancer.2001;91:180-186
[6] Behre G,Reddy VA,Tenen DG,et a1.Proteomic analysis of transcription factor interactions in myeloid stem cell development and leukemia[J].Expert Opinion on Therapeutic Targets.2002;6:491-495
[7] Zhang J,Kalkum M,Yamamura S,et al.E protein silencing by the leukemogenic AML1一ETO fusion protein[J].Science.2004;305:1286-1289
[8] Cochran DA,Evans CA,Blinco D,et a1.Proteomic Analysis of Chronic Lymphocytic Leukemia Subtypes with Mutated or Unmutated Ig VH Genes[J].Mol Cell Proteomics.2003;2:1331-1341
[9] Kaiser T, Kamal H, Rank A, et al. Proteomics applied to clinical follow up of patients after allogeneic hematopoietic stem cell transplantation[J]. Blood. 2004;104: 340-349
[1] Wittmann LB, Graack HR, Pohl T. Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry[J]. Proteomics, 2006,6:4688-4703.
[2] Hellman U, Wemstedt C, Gonez J, et al. Non-decoloured In-gel Digestion of Coomassie Blue-stained Proteins [J]. Anal Biochem, 1995,224(1):451-455.
[3] Jungmook L, Frank C, Eek-hoon J, et al. Ectopic Expression of Axin Blocks Neuronal Differentiation of Embryonic Carcinoma P19 Cells[J]. Biol. Chem,2003:278:13487-13495.
[4] Fleck RA, Romero-Steiner S, Nahm MH. Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies[J]. Clin Diagn Lab Immunol, 2005,12(1):19-27.
[5] Uckun FM, Evans WE, Forsyth CJ, et al. Biotherapy of B-cell precursor leukemia by targeting genistein to CD19-associated tyrosine kinases[J]. Science, 1995;267:889-891
[6] Myers DE., Jun X, Waddick KG, et al. Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells[J]. Proc Natl Acad Sci, 1995;92:9575-9579
[7] Guo DH, Li MY, Zhang Y, et al. A functional variant of SUMO4, a new IKBαmodifier, is associated with type 1 diatetes[J]. Nature Genetics 2004;36:837-841
[8] Gill AW, Neil DP. Transcriptional Cross Talk between NF-κB and p53[J].Molecular and Cellular Biology,1999;19:3485-3495
[9] Maiguel D, Pei Z, Masashi M, et al. Medium chain fatty acid acyl-CoA synthetase ACSF2 may play a role in neuronal differentiation[J]. The FASEB Journal ,2006;20:A948
[10] Reed JC. Mechanisms of Bcl-2 family protein function and dysfunction in health and disease[J]. Behring Inst Mitt, 1996;97:72-100
[11] Scott CK, Diane EB, David BS, et al. BCL-2 Cooperates with PromyelocyticLeukemia Retinoic Acid ReceptorαChimeric Protein (PMLRARα) to Block Neutrophil Differentiation and Initiate Acute Leukemia[J]. The Journal of Experimental Medicine, 2001;193:531-544
[12] Paul P. Wnt signaling and cancer[J]. Genes&Development 2000;14:1837-1851.
[13] Tago K, Nakamura T, Nishita M, et al. Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein[J]. Genes Dev, 2000;14: 1741–1749.
[14] Von KJ, Winbeck G, Asbrand C, et al. Hot spots in beta-catenin for interactions with LEF-1, conductin and APC[J]. Nat Struct Biol, 2000;7: 800–807.
[1] Miller JR, Moon RT. Signal transduction throughβ-catenin and specification of cell fate during embryogenesis[J].Genes & Dev,1996,10: 2527-2539.
[2] Cadigan, KM, Nusse R. Wnt signaling: A common theme in animal development[J]. Genes & Dev,1997,11: 3286-3305.
[3] Clevers H Wetering M. TCF/LEF factor earn their wings[J]. Trends Genet,1997,13: 485-489.
[4] Eastman Q, Grosschedl R. Regulation of LEF-1/TCF transcription factors by Wnt and other signals[J]. Curr. Opin. Cell Biol,1999,11: 233-240.
[5] Tiffany MP, William HM, Frank P.The Response of CD24–/low/CD44+ Breast Cancer–Initiating Cells to Radiation[J]. JNCI,2006,24:1777-1785.
[6] Tamai K, Semenov M, Kato Y, et al. LDL-receptor-related proteins in Wnt signal transduction[J]. Nature,2000,407:530-535.
[7] Paul P. Wnt signaling and cancer[J].Genes&Development,2000,14:1837-1851.
[8] Hue HL, Ruiwen Z, Rex CH, et al. Wnt / -Catenin Signaling Pathway as Novel Cancer Drug Targets[J]. Current Cancer Drug Targets,2004,4: 653-671.
[9] W. James N, Roel N. Convergence of Wnt, beta-Catenin, and Cadherin Pathways[J].Science,2004,303:1483-1487.
[10] Yusuke H, Yasuhiro I, Hidenori T, et al. The Wnt/β-catenin pathway directs neuronal differentiation of cortical neural precursor cells[J]. Development ,2004,131:2791-2801.
[11] van den Brink GR, Bleuming SA, Hardwick JCH, et al. Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation[J].NatureGenetics,2004,36: 277-282.
[12] Tago K, Nakamura T, Nishita M, et al. Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein[J]. Genes Dev,2000,14: 1741–1749.
[13] Von KJ, Winbeck G, Asbrand C, et al.Hot spots in beta-catenin for interactions with LEF-1, conductin and APC[J]. Nat Struct Biol,2000,7: 800–807.
[14] Shtutman M, Zhurinsky J, Simcha I, et al.The cyclin D1 gene is a target of theβ-catenin/LEF-1 pathway[J]. Proc Natl Acad Sci,1999,96:5522–5527.
[15] Brabletz T, Herrmann K, Jung A, et al. Expression of nuclearβ-catenin and c-myc is correlated with tumor size but not with proliferative activity of colorectal adenomas[J]. Am J Pathol ,2000,156:865–870.
[16] He TC, Chan TA, Vogelstein B, et al. PPAR is an APC-regulated target of nonsteroidal anti-inflammatory drugs[J]. Cell,1999, 99:335–345.
[17] Oosterwegel MA, van de Wetering ML, Holstege FC, et al. TCF-1, a T cell-specific transcription factor of the HMG box family, interacts with sequence motifs in the TCRβand TCR enhancers[J]. Int Immunol,1991,3:1189–1192.
[18] Crawford HC, Fingleton BM, Rudolph-Owen LA, et al. The metalloproteinase matrilysin is a target of ?-catenin transactivation in intestinal tumors[J]. Oncogene,1999,18:2883–2891.
[19] Mikami T, Saegusa M, Mitomi H, et al. Significant correlations of E-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas[J]. Am J Clin Pathol,2001,116:369–376.
[20] Behrens J. Control of ?-catenin signaling in tumor development[J]. Ann NY Acad Sci ,2000,910:21–33.
[21] Peifer M, Polakis P. Wnt signaling in oncogenesis and embryogenesis–a look outside the nucleus[J]. Science,2000,287:1606–1609.
[22] Van Es JH, Barker N, Clevers H. You Wnt some, you lose some: oncogenes in the Wnt signaling pathway[J]. Curr Opin Genet Dev,2003,13:28–33.
[23] Barker N, Morin PJ, Clevers H. The yin-yang of TCF/?-catenin signaling[J]. Adv Cancer Res,2000, 77:1–24.
[24] Barker N, Clevers H. Catenins, Wnt signaling and cancer[J]. Bioessays,2000,22:961–965.
[25] Chaplinski TJ, Niedel JE. Cyclic Nucleotide-induced Maturation of Human Promyelocytic Leukemia Cells[J]. J. Clin. Invest,1982,70: 953-964.
[26] Breitman TR., Selonick SE,Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid Proc[J]. Natl. Acad. Sci. ,1980,77:2936-2940.
[27] Julie LH, Kleanthis GX. Biological Role of the CCAAT/Enhancer-binding Protein Family of Transcription Factors[J]. THE JOURNAL OF BIOLOGICAL CHEMISTRY,1998,273:28545-28548.
[28] Darlington, GJ, Ross SE, MacDougald OA. The role of C/EBP genes in adipocyte differentiation[J]. J Biol Chem,1998,273:30057–30060
[1] Jiang Y,Xue ZH,Shen WZ,et al. Desferrioxmine induces leukemic cell differentiation potentially by hypoxia-inducible factor-1 that augments transcriptional activity of CCAAT/enhancer binding protein alpha[J].Leukemia, 2005,19:1239-1247
[2] Behre G., Singh SM., Liu H. Bortolin, M. Christopeit, H. S. Radomska, J. Rangatia, W. Hiddemann, A. D. Friedman, and D. G. Tenen.Ras Signaling Enhances the Activity of C/EBPalpha to Induce Granulocytic Differentiation by Phosphorylation of Serine 248[J].J Biol Chem., 2002,277(29): 26293- 26299
[3] Zhang DE, Zhang P, Wang ND,et al.Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAAT enhancer binding protein alpha-deficient mice[J].Proc Natl Acad Sci USA,1997,94:569-574
[4] Godambe SA, Chaplin DD, Takova T, et al. A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene[J]. Mol Cell Biol,1995,15(1):112-119.
[1] Evans MJ,Kaufman M.Establishment in culture of pluripotent cells from mouse embryos[J].Nature.1981;292:154
[2] Martin G.Isolation of a pluripotent cell line from early embryos cultured in medium conditioned by teratocarcinoma cells[J].Proc Natl Acad Sci USA.1981;78:7634
[3] Andrews PW.From teratocarcinomas to embryonic stem cells[J].Biol Sci.2002;357: 405
[4] Kanatsu-Shinohara M,Inoue K,Lee J,et al.Generation of pluripotent stem cells from neonatal mouse testis[J]. Cell.2004;119:1001~1012
[5] Mclaren A,Durcova-Hills G.Germ cells and pluripotent stem cells in the moiuse[J].Reprod Fertil Dev.2001;13:661~664
[6] Szakmary A,Cox D,Wang Z,et al.Regulatory relationship among piwi,pumilio,and bag-of-marbles in drosophila germline stem cell self-renewal and differentiation[J].Current Biology.2005;15(2):171~178
[7] Ramalho-Santos M, Yoon S., Matsuzaki Y.,et al.‘‘Stemness’’: transcriptional profiling of embryonic and adult stem cells[J], Science, 2002; 298: 597~600
[8] Sperger JM, Chen X, Draper JS,et al.Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors[J]. Proc Natl Acad Sci USA . 2003;100:13350~13355
[9] Kurisaki A, Hamazaki TS., Okabayashi K,et al.Chromatin-related proteins in pluripotent mouse embryonic stem cells are downregulated after removal of leukemia inhibitory factor[J]. Biochem Biophys Res Commun. 2005;335: 667~675
[10] Nagano K, Taoka M, Yamauchi Y,et al. Largescale identification of proteins expressed in mouse embryonic stem cells[J]. Proteomics,2005; 5:1346~1361
[11] Fujino RS,Ishikawa Y,Tanaka,K,et al.Capillary morphogenesis gene(CMG)-1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells[J].Mol Reprod Dev,2006;73:955~966
[12] Hajime K,Yasuhiro K,Masaharu H,et al.A comparison study in the proteomic signatures of multipotent germline stem cells,embryonic stem cells,and germlinestem cells[J].Biochemical and Biophysical Research Communications.2007: 53:259~ 267
[13] Nishigaki R, Shinohara T, Toda T,et al. An extra human chromosome 21 reduces mlc-2a expression in chimeric mice and Down syndrome, Biochem. Biophys[J]. Res. Commun. 2002; 295:112~118
[14] Kadota M, Nishigaki R, Wang CC, et al.Proteomic signatures and aberrations of mouse emb- ryonic stemcells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome[J]. Neuroscience. 2004;129:325–335
[15] Wendy P,George QD,Peter Z,et al.Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling[J].PNAS. 2004;101 (9):2900~2905
[16] Reubinoff, BE, Pera MF, Fong CY,et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro[J].Nat. Biotechnol. 2000; 18: 559~559
[17] Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation[J].J Cell Biochem.1997;64:278~294
[18] Bruder SP,Kurth AA,Shea M,et al.Bone regeneration by implantation of purified, culture- expanded human mesenchymal stem cells[J].J Orthop Res.1998;16:155
[19] Barry FP,Murphy JM.Mesenchymal stem cells:clinical applications and biological charac- terization[J].Int J Biochem Cell Biol.2004;36(4):568~584
[20] Fang B,Shi M,Zhao RC,et al.Multiorgan engraftment and multilineage differentiation by human fetal bone marrow Flk1+/CD31-/CD34- progenitors. J Hematother Stem Cell Res[J]. 2003;12(6):603~613
[21] Fang B,Liao L,Zhao RC,et al.Multipotency of FLK1CD34 progenitors derived from human fetal bone marrow[J].J Lab Clin Med.2004;143(4):230~240
[22] Guo H,Fang B,Zhao RC,et al.Hemangioblastic characteristics of fetal bone marrow-derived FLK1(+)CD31(-)CD34(-)cells[J].Exp Hematol.2003, 31(7):650~658
[23] Hu Y,Liao L,Zhao RC,et al.Isolation and identification of mesenchymal stem cells from human fetal pancreas[J].J Lab Clin Med.2003;141(5):342~349
[24] Deng W,Han Q,Zhao RC,et al.Allogeneic bone marrow-derived flk-1+Sca-1- mesenchymal stem cells leads to stable mixed chimerism and donor-specific tolerance[J].Exp Hematol .2004;32(9):861~867
[25] Robert E,Feldmann JR,Karen B,et al.Stem cell proteomes:A profile of human mesenchymal stem cells derived from umbilical cord blood[J].Electrophoresis. 2005;26: 2749~2758
[26] Rosada C, Justesen J, Melsvik D, et al. The Human Umbilical Cord Blood: A Potential Source for Osteoblast Progenitor Cells[J].Calcif. Tissue Int. 2003; 72: 135~142.
[27] Panepucci R A, Siufi,J L., Silva A R, et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells[J]. Stem Cells, 2004, 22(7): 1263~1278
[28] Li J, Sensebe L, Herve P, Charbord P,et al. Nontransformed colony-derived stromal cell lines from normal human marrows. II. Phenotypic characterization and differentiation pathway[J]. Exp Hematol 1995;23:133~141
[29] Ogawara M, Inagaki N, Tsujimura K, et al. Differential targeting of protein kinase C and CaM kinase II signalings to vimentin[J]. J.Cell Biol. 1995, 131(4):1055~1066
[30] Eriksson J , He T, Trejo-Skalli A V, et al. Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments[J] .J. Cell Sci. 2004, 117, 919~932
[31] Maki M, Matsukawa N, Yuasa H, et al. Decreased expression of hippocampal cholinergic neurostimu- lating peptide precursor protein mRNA in the hippocampus in Alzheimer disease .J.Neuropathol[J]. Exp. Neurol. 2002, 61, 176–185
[32] Lorenz K, Lohse M J, Quitterer U,et al. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2[J]. Nature. 2003; 426:574~579
[33] Yeung K, Janosch P, McFerran B,et al. Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein[J].Mol. Cell. Biol. 2000; 20: 3079~3085
[34] Leonard JF, Patricia AZ,Chen L,et al. Differential Expression Profiling of Membrane Proteins by Quanti- tive Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteoblast Differentiation[J]. Stem Cell.2005;23:1367~1377
[35] Bianco P, Robey PG. Stem cells in tissue engineering[J]. Nature 2001;414:118–121
[36] Bruder SP, Horowitz MC, Mosca JD et al. Monoclonal antibodies reactive with human osteogenic cell surface antigens[J]. Bone 1997;21:225~235
[37] Simonsen JL, Rosada C, Serakinci N et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells[J]. Nat Biotechnol 2002;20:592~596
[38] Abdallah BM, Haack-Sorensen M, Burns JS et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite of extensive proliferation[J]. Biochem Biophys Res Commun 2005;326:527~538
[39] Gronthos S, Zannettino AC, Hay SJ et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[J]. J Cell Sci 2003;116:1827~1835
[40] Kim KH, Song MJ, Yoo EJ et al. Regulatory role of GSK3 for transcriptional activity of ADD1/SREBP1c[J]. J Biol Chem 2004;279:1999~2006
[41] Kusakabe T, Maeda M, Hoshi N et al. Fatty acid synthase is expressed mainly in adult hormone-sensitive cells or cells with high lipid metabolism and in proliferating fetal cells[J]. J Histochem Cytochem 2000;48:613~622
[42] Irina K,Blagoy B,Mandana HS,et al.Mechanism of Divergent Growth Factor Effect in Mesenchymal Stem Cell Differentiation[J]. Science.2005; 308:1472~1477
[43] Pawson T,P Nash. Assembly of Cell Regulatory Systems Through Protein Interaction[J].Domains. Science. 2003; 300:445
[44] P Blume-Jensen, T Hunter. Oncogenic kinase singaling[J].Nature .2001;411:355
[45] Schlessiger J. Cell Signaling by Receptor Tyrosine Kinases[J].Cell.2000;103:211
[46] Szymkiewicz I, Shupliakov O, Dikic I,et al. Larvicidal Cry proteins from Bacillus thurin- giensis are released in root exudates of transgenic B. thuringiensis corn,potato,and rice but not of B. thuringiensis canola,cotton, and tobacco[J].Biochem.2004;383~387
[47] Amit M, Shariki C, Margulets V, et al. Feeder Layer-and Serum-Free Culture of Human Embryonic Stem Cells[J] .Biol. Reprod,2003; 68:2150~2156
[48] Xu C,Inokuma MS,Denham J,et al. Feeder-free growth of undifferentiated human embryonic stem cells[J].Nat. Biotechnol.2000; 19:971~974.
[49] Czyz J, Wobus AM. Embryonic stem cell differentiation: The role of extracellular factors[J]. Differentiation. 2001; 68:167~174.
[50] Chambers I, Smith,A. Self-renewal of teratocarcinoma and embryonic stem cells[J]. Oncogene 2004;23: 7150~7160
[51] Ernst M, Oates A, Dunn, A R. gp130-mediated Signal Transduction in Embryonic Stem Cells Involves Activation of Jak and Ras/Mitogen-activated Protein Kinase Pathways[J] . J. Biol. Chem. 1996; 271:30136~30143
[52] Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science.1998; 282: 1145~1147
[53] Doetschman TC, Eistetter H, Katz M, et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.J. Embryol[J]. Exp. Morphol. 1985; 87:27~45
[54] Guo X, Ying W, Wan J, et al. Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro[J]. Electrophoresis 2001;22:3067–3075
[55] Prudhomme W, Daley GQ, Zandstra P,et al. Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling[J]. Proc. Natl. Acad. Sci. U. S. A..2004; 101:2900~2905
[56] Lawrence GP,Douglas JB,Jean-Francois C,et al.Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling[J]. Molecular & Cellular Proteomics. 2006; 5:57~67.
[57] Nagano K, Taoka M, Yamauchi Y, et al. Largescale identification of proteins expressed in mouse embryonic stem cells[J]. Proteomics. 2005; 5:1346~1361
[58] Christonph Z,Bernd G,Nian G,et al.Protemic profiling of human stem cells derived from umbilical cord blood[J].Biochemical and Biophysical Research Communications. 2005;328:968 ~972
[59] Wen T,Mu W,Emily D,et al.Comparative proteomic analysis of human CD34+ stem/ progenitor cells and mature CD15 myeloid cells[J].Stem Cells.2004;22:1003~ 1014
[60] Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells[J]. Science 2002;298:597~600
[61] Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells[J]. Nature 2001;414:105~111
[62] Richard DU,Duncan LS,David B,et al.Quantitative proteomics reveals posttranslational control as a regulator factor in primary hematopoietic stem cells[J].Blood.2006;17(12):4687~4694
[63] Osawa M,Nakamura K,Nishi N,et al.In vivo self-renewal of c-Kit(+)Sca-1(+)Lin (low/-) hemo -poietic stem cells[J].J Immunol.1996;156:3207~3214
[64] Zhong JF,Zhao Y,Sutton S,et al.Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells[J].Proc Natl Acad Sci USA.2005;102:2448 ~2453
[65] Wenger RH.Cellular adaptation to hypoxia:O-2-sensing protein hydroxylases, hypoxia- inducible transcript- tion factors,and O-2-regulated gene expression[J]. FASEB J.2002;16:1151~ 1162
[66] Hammerer-Lercher A,Mair J,Bonatti J,et al.Hypoxia induces heat shock protein expression in human coron- ary artery bypass grafts[J].Cardiovasc Res.2001;50:115~ 124
[67] Lin J,Zhang XL,Sejas DP,et al.Hypoxia-induced nucleophosmin protects cell death through inhibition of p53[J].J Biol Chem.2004;279:41275~41279
[68] Krishnamachary B,Berg-Dixon S,Kelly B,et al.Regulation of colon carcinoma cell invasion by hypoxia- inducible factor 1[J].Cancer Res.2003;63:1138~1143
[69] Ito K,Hirao A,Arai F,et al.Regulation of oxidative stress by ATM is required for self-renewal of haemato- poietic stem cells[J].Nature.2004:434:997~1002
[70] Reya T,Morrison SJ,Clarke MF,et a1.Stem cells,cancer,and cancer stem cells[J]. Nature.2001; 414:105~111
[71] Tracy W. Work on breast cancer stem cells raises questions about treatment strategies[J].Journal of the National Cancer Institute.2003.95(11):774~775
[72] Hayman MW,Przyborski SA.Proteomic identification of biomarkers expressed by human pluripotent stem cells[J].Biochemical and Biophysical Research Communications.2004; 316 :918~923
[73] Andrews PW,Przyborski SA,Thomson JA.Embryonal carcinoma cells as embryonic stem cells,in:D.R.Marshak,R.Cardner,D.Gottlieb(Eds.) [J].Stem cell Biology,Cold Spring Harbor Press,Monograph 40,2001
[74] Stewart R,Christie V,Przyborski SA,et al.Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes[J].Stem Cells.2003;21:248~256
[75] Przyborski SA.Isolation of human embryonal carcinoma stem cells by immuno- magnetic sorting[J].Stem Cells. 2001;19:500~504
[76] Przyborski SA,Smith S,Wood A.Transcriptional profiling of neuronal differentiation by human embryonal carcinoma stem cells in vitro.Stem Cells[J] .2003; 21:459~471
[77] Ota J,Yamashita Y,Okawa K,et al.Proteomic analysis of hematopoitic stem cell-like fractions in leukemic disorders[J].Oncogene.2003;22:5720~5728
[78] Lian Z, Kluger Y, Greenbaum DS, et al. Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line[J]. Blood.2002; 100:3209~3220
[79] Ursula MG, Süleyman E,Udo S, et al.In vitro differentiation of endothelial cells from AC133-positive progenitor cells[J].Blood.2000;95(10):3106-3112
[1] Lozzio CB,Lozzio BB.Human chronic myelogenous leukemia cell line with positive philadelphia chromosome[J]. Blood,1975;45:321-333.
[2] Chung TW,Choi HJ,Lee YC ,et al. Molecular mechanism for transcriptional activation of ganglioside GM3 synthase and its function in differentiation of HL-60 cell[J]. Glycobiology. 2005; 15(3):233-244.
[3] Passioura T, Dolnikov A ,Shen S ,et al. N-ras-induced growth suppression of myeloid cells is mediated by IRF-1[J]. Cancer Res. 2005;65(3): 797-804.
[4] Athanasion M, Mavrothalassitis G, Sun L, et al.Transcription factors in erythroid differentiation-FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells[J]. Leukemia . 2000; 14(3): 439-463
[5] Munoz-Alonso MJ, Acosta JC, Richard C, et al. P21 Cip1 and P27 Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells[J]. J Biol Chem. 2005; 280(18): 18120-18129.
[6] Xu Y, Voelter MS, Mahlknecht U, et al. The histone clearetylase inhabitorsuberoylanilide hyoroxamic acid down-regulates expression levels of Bcr-abl, c-myc and HDAC3 in chronic myelid leukemia cell line[J].Int J Mol Med. 2005; 15(1): 169-172.
[7] Michele C, Timo S, Karen H .et al. P53-mediated downregulation of chk1 abrogates the DNA damage-induced G2M checkpoint in K562 cell, resulting in increased apotosis[J] . British Journal of Haematology .2002;116:421-428.
[8] Ferrreira R, Ohneda K, Yamamoto M, et al. GATA-1 function, a program for transcription factors in hematopoiesis[J]. Mol Cell Biol. 2005; 25(4): 1215-1227.
[9] Orian S, Todd G, Channing Y.et al. ABC-me: a novel mitochondrial transportorer induced by GATA-1 during erythroid differentiation[J]. The EMBO Journal. 2000; 19(11): 2492-2502.
[10] Yi ZC, Wang Z, Li HX, et al. Effect of chebulinic acid on differentiation of human leukemia K562 cells[J]. Acta Pharmacol Sin. 2004;25(2):231-238.
[11] Yi Z ,Wang Z, Li H, et al.Inhabiting effect of tellimagrandin I on chemically induced differentiation of human lLeukemia K562 cell[J].Toxicol Lett. 2004;147(2): 109-119.
[12] Kamaleldin E, Elagib MX, Isa M, et al. Jun blockade of erythropoiesis role for repression of GATA-1 by HERP2[J]. Molecular and Cellular Biology .2004:7779-7794.
[13] Kohmura K, Miyakawa Y, Kawai Y, et al. Different roles of p38 MAPK and ERK in STI571-induced multi-lineage differentiation of K562 cells[J]. J Cell Physiol. 2004; 198(3):370-376.
[14] Horiguchi TJ, Kawaro T, Saito S, et al. Ectopic cyclin D1 expression blocks STI571-induced erythroid differentiation of K562 cells[J]. Leu Res .2004; 28(6): 623-629.
[15] Vladan PC, Reginald D, Smith B, et al. Hydroxyurea induces fetal hemoglobin by the metric oxide-dependent activation of soluble guanylyl cyclase[J]. J Clin Invest. 2003;111(2): 231-239.
[16] Ariel S, Michel V, Maria I, et al. The exosome pathway in K562 cells is regulatedby Rab11[J]. Journal of Cell Science.2002; 15: 2505-2515.
[17] Bianchi N,Chiarabell C, Borgatti M, et al. Accumulation of [gamma]-globin and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine[J]. Blackwell Science Ltd. 2001:113(4-Ⅱ): 951-961.
[18] Motoi M, Xiao FC,Tatsuhiko F,et al.Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells[J]. Cancer Science,2005,94(6):557-563
[19] Yoshitsugn K, Terukazu T, Akira K, et al. Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase[J]. The EMBO Journal.2001;20(20): 5666-5677
[20] Davies J, Evans B, Jenney M, et al. Effect of chemotherapeutic agents on the function of primary human osteoblast-like cells derived from children[J]. J Clin Endocrinol Metab. 2003; 88(12): 6088-6097.
[21] Bohmer RM. IL-3-dependent early erythropoiesis is stimulated by autocrine transforming growth factor beta[J]. Stem Cells . 2004; 22 (2): 216-24.
[22] Patricia E, Burger, Pauline T. Basic fibroblast growth factor modulates the expression of glycophorin A and c-kit and inhibits erythroid differentiation in K562 cells[J]. J Cell Physiol. 2002; Jan; 190(1): 83-91.
[23] Huang HM, Chang TW, Liu JC. Basic fibroblast growth factor antagonizes activin A-mediated growth inhibition and hemoglobin synthesis K562 cells by activating ERK1/2 and deactivating p38 kinase[J]. Biochem Biophys Res Commun.2004;320(4):1247-1252