摘要
研究背景
前列腺癌是西方国家最常见的恶性肿瘤,其发病率居美国和西欧国家男性恶性肿瘤的第一,二位,死亡率位于男性恶性肿瘤患者的首位。我国前列腺癌的发病率目前虽然远低于西方国家,但近年来前列腺癌的检出率和发病率呈明显上升趋势,已位居泌尿系肿瘤的第三位,并且一半以上前列腺癌患者确诊时已经是局部进展型和转移型前列腺癌,失去了手术根治性治疗的机会,只能应用内分泌激素治疗。70%-80%前列腺癌细胞会逐渐转变成对激素治疗耐受或没有反应的状态,即所谓的雄激素非依赖性前腺癌。目前,任何对于雄激素非依赖性前列腺癌的治疗,疗效均有限,寻找新的治疗方法迫在眉睫。
基因治疗是将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用,从而达到治疗疾病的目的。自从1990年对一个因为腺苷脱氨酶(ADA)缺乏而造成重症联合性免疫缺陷症(SCID)的4岁小女孩进行临床基因治疗获得成功以来,基因治疗在这20年有了飞速的发展。腺病毒载体是目前基因治疗中最常用的载体。FEZ1基因是Ishii H等(1999)对原发性食管癌进行研究时发现的。FEZ1基因全名为F37／食管癌相关基因编码亮氨酸拉链基因序列,简称FEZ1/LZTS1。在肺癌,胃癌,食管癌,膀胱癌,乳腺癌细胞相关研究中均发现FEZ1基因mRNA和FEZ1蛋白表达缺失或显著下降。目前FEZ1基因被认为是候选的抑癌基因,目前考虑FEZ1基因可能通过调节微管和有丝分裂等机制抑制肿瘤细胞生长。目前应用FEZ1基因进行基因治疗已经在MCF7(乳腺癌细胞株)AT 6.2(鼠前列腺癌细胞株)和LNCaP(雄激素依赖型前列腺癌细胞株)SW780细胞(膀胱癌系)细胞中取得了相应的进展。
本课题试图将FEZ1基因引入雄激素非依赖型前列腺癌细胞DU145的基因治疗。用RT-PCR和Western blot技术检测FEZ1基因在不同的前列腺细胞株的差异,PCR技术扩增FEZ1基因片段,并用CloneEZ技术和LR体外重组技术将FEZ1基因片段克隆至腺病毒载体,酶切和测序方法对重组腺病毒载体pAd-FEZ1的鉴定。用MTT法、Hoechst染色法、流式细胞分析法等方法分析感染重组腺病毒载体pAd-FEZ1的DU145细胞增殖凋亡。
目的：了解不同前列腺细胞株中FEZ1基因表达的差异。方法：利用RT-PCR和Western blot技术检测正常前列腺上皮细胞(RWPE-1),雄激素非依赖型前列腺癌细胞株DU-145,前列腺癌细胞株ALAV中FEZ1基因的mRNA量和FEZ1蛋白量,比较三组细胞FEZ1基因的表达差异结果：在三株细胞中均有FEZ1基因的表达,前列腺正常上皮细胞RWPE-1中的表达最强,而前列腺癌细胞DU145、ALAV中的表达量相对较低。结论：FEZ1基因在前列腺正常上皮组织高表达,而在前列腺肿瘤细胞株中低表达。两种肿瘤细胞株表达无明显差异,为FEZ1基因在前列腺癌细胞生长影响研究提供了实验基础。
目的：扩增人FEZl基因,构建和鉴定重组腺病毒表达载体,稳定表达FEZ1基因的前列腺癌细胞株DU145。方法：(1)采用PCR技术扩增FEZ1基因片段,并用CloneEZ技术和LR体外重组技术将FEZ1基因片段克隆至腺病毒载体,构建好重组腺病毒载体pAd-FEZ1后,酶切和测序方法对重组腺病毒载体pAd-FEZ1的鉴定,扩增重组腺病毒rAd-FEZ1并测定滴度。(2)选用最佳的感染复数(multiplicity ofinfection, MOI)的rAd-FEZ1感染DU145,建立重组腺病毒rAd-FEZ1 DU145组腺病毒空载体DU145组,和空白对照DU145组,用RT-PCR Western Blot技术免疫荧光技术,测量FEZl基因的表达,确认构建重组腺病毒rAd-FEZ1 DU145细胞成功。结果：成功扩增FEZ1基因cDNA序列并构建和鉴定重组腺病毒表达载体rAd-FEZ1,成功获得稳定表达FEZ1基因的前列腺癌细胞株DU145。结论：FEZ1基因重组腺病毒载体的成功构建和鉴定,稳定表达FEZ1基因的前列腺癌细胞株DU145的成功构建为前列腺癌细胞生长影响研究提供了实验基础。
目的：观察重组FEZ1基因腺病毒对前列腺癌细胞株DU145生物学特性的影响。方法：测定细胞生长曲线(MTT),Hoechst染色法观察细胞生长形态,流式细胞分析法分析细胞增殖凋亡。结果：(1)MTT法结果,重组腺病毒rAd-FEZ1感染组DU145细胞生长速度慢于腺病毒空载体对照DU145组和空白对照DU145组(p<0.05).。腺病毒对照组和正常DU145细胞对照组生长速度无明显差别(p>0.05)(2) Hoechst染色法显示FEZ1基因过表达后,48小时的感染,细胞核会呈致密浓染,或呈碎块状致密浓染,异样细胞核明显增多,明显出现凋亡。(3) AnnexinⅤ/PI流式细胞分析法重组腺病毒组凋亡率较腺病毒对照组和正常DU145细胞对照组高(p<0.05).,腺病毒对照组和正常DU145细胞对照组凋亡率无明显差别(p>0.05)。结论：FEZ1基因的高表达诱导雄激素非依赖型前列腺癌DU145细胞凋亡和抑制细胞生长增殖。FEZ1基因显示了基因治疗雄激素非依赖型前列腺癌的巨大潜力。
Background Prostate cancer is the most frequent malignant tumor in western counties.Its incidence rate situated the first or second place in maleness tumors in the United States and Western Europe.The mortality rate situate the first place in maleness malignant tumors in the United States and Western Europe. At present, the incidence of prostate cancer in our country is much lower than western countries, but in recent years, it's incidence ascended significantly and situated the third place in urinary tract tumors. More than half patients with prostate cancer were already partly progressive and metastatic prostate cancer and missed opportunity of radical treatment; they can only be applied endocrine hormone treatment. Moreover 70%-80% of prostate cancer patients were to gradually transformed into a state of tolerance or lack of response to hormone, namely, the so-called androgen-independent prostate carcinoma (AIPC). At present, the efficacy of any therapy to AIPC efficacy is very limited. It is extremely urgent to find new treatments.
Gene therapy is a new method of treatment that created by combining modern medicine and molecular biology. The gene therapy has been growing rapidly in this 20 year since the first success of clinical gene therapy for a four-year-old girl with immunodeficiency disorder (SCID) causing by adenosine deaminize (ADA) deficiency in 1990. Adenovirus vector for gene therapy is the most commonly used carrier. Ishii H(1999), found a candidate fragment F37 which was found in normal cell lines and not found in cancer cell lines by cloning, sequencing methods to analysis of tumor and cell line cDNA in the study of primary esophageal cancer. Ishii H named F37/Esophageal cancer-related gene-coding leucine-zipper motif (F37/esophageal cancer related gene encoding leucine-zipper gene sequences, known FEZ1/LZTS1). At present FEZ1 gene is considered a candidate tumor suppressor gene, FEZ1 gene may inhibit the tumor cell growth by regulating the microtubule and mitotic.The FEZ1 gene mRNA and protein expression of the FEZ1 were found missing or decreased significantly in the lung, stomach, esophagus, bladder cancer, breast cancer cells lines.Application of gene therapy to breast cancer cell line MCF7, AT6.2 (mouse prostate cancer cell line) and LNCaP (androgen-dependent prostate cancer cell line) SW780 cells (bladder cancer line), all the cell growth and proliferation had a noticeable inhibition.
First We Use RT-PCR and Western blot to detect FEZ1 gene expression in prostate cell lines. The next we use PCR amplify FEZ1 gene fragments, use CloneEZ technology and LR in vitro recombinant technology clone FEZ1 gene fragment into adenovirus vector. Then we use PCR and sequencing Methods identify recombinant adenovirus vector pAd-FEZ1. At last we use MTT method, Hoechst staining, flow cytometer analyze, to analyze the proliferation, apoptosis of the DU145 cell lines.We wish that we can give a new idea for androgen-independent prostate carcinoma.
Objective:To study the FEZ1 gene expression in different prostate cell lines. Methods:RT-PCR and Western blot were used to examine the expression of FEZ1 gene in normal prostate epithelial cells (RWPE-1), androgen-independent prostate cancer cell line DU-145, prostate cancer cell line ALAV. Results:All the cells have the FEZ1 gene expression, and the RWPE-1 FEZ1 gene expression was the maximum, two kinds of tumor cell lines expression have no significant difference. Conclusion: All the cells have the FEZ1 gene expression; the RWPE-1 expression was the highest. This work provides the base for the following study.
Objective:To amplify human FEZ1 gene, construct and identify the FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145.Methods:(1) We used PCR to amplify FEZ1 gene fragment., used cloneEZ technology and LR in vitro recombination technology clone FEZ1 gene fragment into adenovirus vector. Constructed recombinant adenoviral vector pAd-FEZ1 well, we identified FEZ1 gene recombinant adenovirus vector by the enzyme digestion and sequencing. (2) Choosed the best multiplicity of infection (multiplicity ofinfection, MOI) of rAd-FEZ1 infected DU145, constructed the recombinant adenovirus rAd-FEZ1 DU145 group, adenovirus vector DU145 group, and blank control DU145 group. To study FEZ1 gene expression with RT-PCR, Western-Blot technique and immunofluorescence technique to confirm construct recombinant adenovirus rAd-FEZ1 DU145 group successfully. Results: The FEZ1 gene was amplified successfully and the FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145 were constructed and identified successfully. Conclusion:The FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145 were constructed and identified successfully. This work provided the base for the following study.
Objective:To observe the biology effect of the FEZ1 gene recombinant adenovirus to prostate cancer cell line DU145. Methods: Measured the cell growth curves by MTT method. Hoechst staining cells in morphology, flow cytometry analysis method explored cell proliferation and apoptosis. Results:(1) MTT method results:the growth rate of the recombinant adenovirus rAd-FEZ1 infection DU145 cell line group was lower than the adenovirus vector control DU145 group and blank control DU145 group. The growth rate of the adenovirus control group and blank control group, had no significant difference (p> 0.05), There was a significant difference (p<0.05). between the growth rate of the two groups of cells and recombinant adenovirus rAd-FEZ1 group, (2) Hoechst staining showed dense nucleus wound stain or a dense stain Chunky, abnormal nuclei increased significantly, a marked apoptosis after being infected for 48 hours (3) AnnexinⅤ/PI flow cytometry analysis showed the apoptosis rate of recombinant adenovirus rAd-FEZ1 infection DU145 group was higher than the blank control group and the control adenovirus group. The apoptosis rate of the adenovirus control group and blank control group, had no significant difference (p> 0.05) There was a significant difference (p<0.05). between the apoptosis rate of the two groups of cells and recombinant adenovirus rAd-FEZ1 group Conclusion:FEZ1 gene can FEZ1 protein induce apoptosis and inhaibit proliferation androgen indepent prostate cancer DU145 cell lines. FEZ1 gene shows great potential for the gene therapy of androgen independent carcinoma of prostate.
引文
[1.]Ruchlin HS, Pellissier JM. An economical overview of prostate carcinoma. Cancer,2001,92:2796-810.
[2.]Jemal A, Tiwari RC, Murray T, et al. Cancer statistics.2004, CA cancer J clin.2004,154(10):8-29.
[3.]顾方六,刘玉立.50年泌尿男生殖系肿瘤发病和构成情况的变迁.中华泌尿外科杂志,2002,23(2)：88-90.
[4.]刘耀庭,李秀霞,程伟.泌尿外科肿瘤发病情况的变化.临床泌尿外科杂志,1997,12(1)：47-48.
[5.]徐志兵,王国民,孙立安等.上海市市区前列腺癌发病情况调查分析.中国临床医学,2003,10(3)：344-346.
[6.]Goodin S, Rao KV, Dipaola RS. State-of-the-Art Treatment of Metastatic Hormone-Refractory Prostate Cancer. Oncologist,2002,7:360-70.
[7.]Pronzato P. Rondini M. Hormonotherapy of advanced prostate cancer. Ann Onc01.2005; 16(suppl):iv80-iv84.
[8.]Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med.2004; 351:1513-1520.
[9.]Tannock IF, de Wit R, Berry WR, et al; for the TAX 327 Investigators. Docetaxel plus prednisoneor mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med.2004;351:1502-1512.
[10.]Anderson W F, Blaese R M, Culver K, et al. The ADA human gene therapy clinical protocol:points to consider response with clinical protocol. Hum Gene Ther,1990,1:331-362
[11.]Office of Biotechnology Web site. Available at:http://www4.od.nih.gov/oba. Accessed November 2006
[12.]Ishii H,Baffa R, Numata SI,et al,The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors [J].Proc natl acad sci U S A,1999,96(7);3928-3933
[13.]Toyooka S, Fukuyama Y, Wistuba 11, et al, Differential expression of FEZ1/LZTS1 gene in lung cancers and their cell cultures [J].Clin Cancer Res,2002,8(7):2292-2297、
[14.]Vecchione A, Ishii H, Shiao YH, et al. Fezl/lzsl alterations in gastric carcinoma [J].Clin cancer res,2001,7(6):1546-1552
[15.]闫尧,陈香宇,陈军,等, 食管癌组织中FEZ基因mRNA的表达改变,胃肠病学和肝病学杂志,2007,16(3)：284
[16.]Vecchione A, Ishii H, Baldssarre G, et al.FEZl/LZTSl is down-regulated in high-grade bladder cancer, and its restoration suppresser tumorigenicity in transitional cell carcinoma cells [J]. Am J Pathol,2002,160 (4):1345-1352
[17.]Chen L, Zhu Z, Sun X et al:Down-regulation of tumor suppressor gene FEZ1/LZTS1 in breast carcinoma involves promoter methylation and associates with metastasis. [J] Breast Cancer Res Treat.2009 116(3):471-478
[18.]Ishii H, Vecchione A, Murakumo Y,et al. FEZ 1/LZTS 1gene at 8p22 suppresses cancer cell growth and regulates mitosis [J]. Proc Natl Acad Sci USA,200198(18):10374-1037
[19.]Nonaka D Nonaka D, Fabbri A, Roz L et al, Reduced FEZ1/LZTS1 expression and outcome prediction in lung cancer [J]. Cancer Res.2005 Feb 15; 65(4): 1207-1212
[20.]Hosseini HA, Ahani A, Galehdari H et al.Frequent loss of heterozygosity at 8p22 chromosomal region in diffuse type of gastric cancer. [J] World J Gastroenterol.2007,13(24):3354-3358
[21.]KnowlesMA, Aveyard JS, Taylor CF, et al. Mutation analysis of the 8p candidate tumour suppressor genes DBC2 (RHOBTB2) and LZTS1 in bladder cancer[J]. Cancer Lett,2005,225 (1):121-130.
[22.]Cabeza-Arvelaiz Y, SepulvedaJL, Lebovitz RM, et al.Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22[J]. Oncogene,2001,20(31):4169-4179.
[23.]Califano D, Pignata S, Pisano C et al:FEZ1/LZTS1 protein expression in ovarian cancer. [J] J Cell Physiol.2010 Feb;222(2):382-386
[24.]ZanesiNAO,Vecchione A,Ishii H, etal.Targeted disruption of the FEZ1/LZTS1 gene predisposes mice to cancer[J]. Proc AmAssoc Cancer Res,2005(46):590.
[25.]Cai D, Hoppe AD',Swanson JA. Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells [J].J Cell Biol,2004:176(1):51-63
[26.]Blasius TL,CaiD, Jih GT. Two binding partners cooperate to activate the molecular motor kinesin-1 [J].J Cell Biol,2007,176 (1):11-17.
[27.]Yew PR. Ubiquitin-mediated proteolysis of vertebrate GI-and S-phase regulators. [J] J Cell Physiot,2001,187(1):1-10.
[28.]Rudolph J:Cdc25 Phosphatases:Structure, Specificity, and Mechanism. Biochemestry 2007,46:3595-3604.
[29.]Pines J:Mitosis:a matter of getting rid of the right protein at the right time. Trends Cell Biol 2006,16:55-63.
[30.]Nigg EA:Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2001,2:21-32.
[31.]Smits VA, Medema RH:Checking out the G(2)/M transition. Bio-chim Biophys Acta 2001,1519:1-12.
[32.]Vecchione A, Baldassarre G, Ishii H et al, Fezl/Lztsl absence impairs Cdkl/Cdc25C interaction during mitosis and predisposes to cancer development [J]. Cancer Cell 2007,11:275-289.
[33.]Chen F, Zhang Z, Bower J et al,:Arsenite-induced Cdc25C degradation is through the KEN-box and ubiquitin-proteasome pathway [J].Proc Natl Acad Sci USA 2002,99:1990-1995.
[34.]Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science,2000, 288(5466):669-672
[35.]Han YSaito H, Thummala NR, et al, Adenovirus-mediated gene therapy of liver diseases. Seminars in liverdisease.1999.19:49-59.
[36.]WolffJA, Wilfianms, PW,Acsadi G, et al. Conditions affecting direct gene transfer into rodent tousleinvivo, Bio techniques.1991; 11:474-85.
[37.]Michael MG. Cancer gene therapy:all awkward adolescence. Cancer gene therapy.2003; 10:501-508.
[38.]Guillot C, Mathieu RCoathalem H, et al, Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4-Ig expression. J Immmol,2000,164:5258-5268
[39.]S-A Im, J-S Kim, C Gomez-Manzano, et al. Inhibition ofbreast cancer growth in vivo by anti-angiogenesis gene therapy with adenovirus mediated antisense-VEOF. British Journal cancer.2001; 84(9):1252-1257
[40.]Vorburger S A.Hunt K K.Adenoviral gene therapy.Oncologist.2002, 7(1):46-59
[41.]Rowe W P.Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. [J] Proc soc Exp Biol Med.1953,84:570-573.
[42.]Han YSaito H, Thummala NR, et al, Adenovirus-mediated gene therapy of liver diseases. Seminars in liverdisease.1999.19:49-59
[43.]Morgan RA,Anderson WF. Human gene therapy. Annu Rev Biochem 1993; 62:191-217.
[44.]Mill R, Curicl DT. Towards the use ofreplicative adenovirus vectors for cancer gene therapy. Gene Therapy.1996; 3:557.
[45.]Yibin Wang, Shuang Huang. Adenovirus technology for the gene manipulation and functional studies, Drug Discovery Today.2000; 5:10-16.
[46.]Wurn F, Bernard A. Large-scale transient expression in mammalian cells for recombinant protein production.Curr Opin Biotechnol,1999,10:156
[47.]Bushman W, Thompson J F, Vargas L et al. Control of directionality in lambda site specific recombination. Science,1985,230(4278):906-911
[48.]Landy A, Dunamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem,1989,58:913-949
[49.]Hartley J L, Temple G F, Brasch M A. DNA cloning using in vitro site-specfic recombination. Genome Research,2000,10(11):1788-1795.
[50.]Walbout A J, Sordella R, LuX, et al. Protein ininteraction mapping in C.elegans using proteins involved in vulval development Science,2000,287(5450):116-122
[51.]Rudolph C, Schreier P H, Uhrig J F. Peptide-mediated broad-specific.plant resistance to tospoviruses. Proc Natl Acad Sci.2003.100 (8) 4429-4434.
[52.]Grand-PetterT. Bouillot A, Perrot A, et al,SCAP ligands are potent new lipid-lowering drugs.Nat Med,2001,7(12):1332-1338
[53.]陈其军,安瑞同海梦,等使用与Gateway技术兼容的T载体获得入门克隆生物化学与生物物理进展,2004.31(10)：951-954.
[54.]司梅文倩,宋文强,朱玉贤,等利用Gateway克隆技术大规模克隆拟南莽转录园子分子植物育种,2004,2(3)：358-364
[55.]Wurn F, Bernard A. Large-scale transient expression in mammalian cells for recombinant protein production.Curr Opin Biotechnol,1999,10:156
[56.]]Nadeau I, Kamen A.Production of adenvirus vector for gene therapy.Biotechnol Adv,2003,20:475
[57.]Graham FL,Prevec L. Method in molecular biology. Gene Transfer and Expression Protocol.1991:7.
[58.]Frrira CG, Epping M, Kruyt FE, et al. Apoptosis:target of cancer therapy. Clinical Cancer Research,2002,8:2024-2034.
[59.]陈辉熔.前列腺癌凋亡通路及分子调控.国外医学泌尿外科分册,2004,24(2)：157-60.
[60.]王瑞年,主编。分子肿瘤学概论。上海：上海科技教育出版社。2001；25.78
[61.]马文丽,郑文岭,主编。分子肿瘤学。北京：科学出版社。2003：98.236.
[62.]纪春祥,于金明,主编。肿瘤学。北京：科学出版社。2003：15.145.
[63.]Chen C, Hyytinen ER, Sun X, et al. Deletion, mutation, and loss of expression of KLF6in human prostate Cancer. Am JPathol.2003Apt; 162(4):1349-1354.
[64.]王瑞年,主编。分子肿瘤学概论。上海：上海科技教育出版社。2001；25.78
[65.]Turashvili G Bouchal J, Burkadze G. Wnt signaling pathway in mammary gland development and carcinogenesis. Pathobiology.2006; 73(5):213-223.
[66.]Soofiakumaran P, Langley SE, Laing RW. COX-2 inhibition:a possible role in the management ofprostate cancer J Chemother.2007; 19(1):21-32.
[67.]Schmid HP,Engeler DS, Pummer K, et al. Prevention ofprostate cancer:more questions than data, Recent Results Cancer Res.2007; 174:101-107.
[68.]Dobosy JR,Roberts. Fu VX,et al, The expanding role of epigenetics in the development, diagnosis and treatment of prostate cancer and beIligll prostatic hyperplasia. J Urol.2007; 177(3):822-831.
[69.]殷序梅,张建业。Bcl-2基因与肿瘤相关性及在细胞凋亡中的作用。临床肿瘤学杂志。2002：4：25-26.
[70.]Watson RW,Fitzpatrick JM. Targeting apoptosis in prostate cancer:focus on caspases and inhibitors ofapoptosis proteins. BJU Int.2005; 96(2):30-34.
[1]Ishii H,Baffa R,Numata SI,et al,The Fezl gene at chromosome 8p22 encodes a leucine-zipper protein,and its expression is altered in multiple human tumors [J].Proc natl acad sci U S A,1999,96(7);3928-3933
[2]Buchanan SG, Gay NJ. Structural and functional diversity in the leucine-rich repeat family of proteins [J]. Prog BiophysMolBiol,1996,65(1-2):1-44
[3]Ishii H, Vecchione A, Murakumo Y,et al. FEZ 1/LZTS lgene at 8p22 suppresses cancer cell growth and regulates mitosis[J]. Proc Natl Acad Sci USA,200198(18):10374-10379
[4]Toyooka S, Fukuyama Y, Wistuba 11, et al, Differential expression of FEZ1/LZTS 1 gene in lung cancers and their cell cultures [J].Clin Cancer Res, 2002,8(7):2292-2297
[5]Nonaka D Nonaka D, Fabbri A, Roz L et al, Reduced FEZ1/LZTS1 expression and outcome prediction in lung cancer [J]. Cancer Res.2005 Feb 15;65(4):1207-1212
[6]Vecchione A, Ishii H, Baldssarre G, et al.FEZ1/LZTS1 is down-regulated in high-grade bladder cancer, and its restoration suppresser tumorigenicity in transitional cell carcinoma cells [J]. Am J Pathol,2002,160 (4):1345-1352
[7]KnowlesMA, Aveyard JS, Taylor CF, et al. Mutation analysis of the 8p candidate tumour suppressor genes DBC2 (RHOBTB2) and LZTS1 in bladder cancer[J]. Cancer Lett,2005,225 (1):121-130.
[8]柳建军,叶木石,曹军,等,FEZ1、P34在膀胱尿路上皮癌中的表达意义,宁夏医学杂志2008,30(7)：582
[9]Hawkins GA,Mychaleckyj JC, Zheng SL, et al. Germline sequence variants of the LZTS1 gene are associated with prostate cancer risk[J]. Cancer Genet Cytogenet,2002,137(1):1-7.
[10]Vecchione A, Ishii H, Shiao YH, et al. FEZ1/LZTS1 alterations in gastric carcinoma[J]. Clin Cancer Res,2001,7 (6):1546-1552.
[11]Hosseini HA, Ahani A, Galehdari H et al.Frequent loss of heterozygosity at 8p22 chromosomal region in diffuse type of gastric cancer. [J] World J Gastroenterol.2007,13(24):3354-3358
[12]闫尧,陈香宇,陈军,等, 食管癌组织中FEZ1基因mRNA的表达改变,胃肠病学和肝病学杂志,2007,16(3)：284
[13]Chen L, Zhu Z, Sun X et al:Down-regulation of tumor suppressor gene FEZ1/LZTS 1 in breast carcinoma involves promoter methylation and associates with metastasis. [J] Breast Cancer Res Treat.2009 116(3):471-478
[14]Califano D, Pignata S, Pisano C et al:FEZ1/LZTS1 protein expression in ovarian cancer. [J] J Cell Physiol.2010 Feb;222(2):382-386
[15]Vecchione A, Galetti TP, Gardiman M et al:Collecting duct carcinoma of the kidney:an immunohistochemical study of 11 cases. [J] BMC Urol 2004, 9(4):11.
[16]Ono K, et al. Down-regulation of FEZ1/LZTS1 gene with frequent loss of heterozygosity in oral squamous cell carcinomas. [J] Oncol. (2003) 23:297
[17]Cabeza-Arvelaiz Y, SepulvedaJL, Lebovitz RM, et al.Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22[J]. Oncogene,2001,20(31):4169-4179.
[18]ZanesiNAO,Vecchione A,Ishii H, etal. Targeted disruption of the FEZ1/LZTS1 gene predisposes mice to cancer [J]. Proc AmAssoc Cancer Res,2005(46):590.
[19]Hadfield JA,Duck S, Hirst N et al. Tubulin and microtubules as targets for anticancer drugs. [J] Prog Cell Cycle Res.2003,5:309-325
[20]Paul G Mckean,Sue Vaughan et al. The extend tublin superfamily. [J] J CeU Sci,2001,114:2723-2733.
[21]Vale, R. D, Reese, T. S Sheetz M. P et al, Identification of a Novel Force-generating Protein, Kinesin, in Microtubule-based Motility[J]. Cell, 1985,42,39-50.
[22]Blasius TL,CaiD, Jih GT. Two binding partners cooperate to activate the molecular motor kinesin-1[J].J Cell Biol,2007,176 (1):11-17.
[23]CaiD, Hoppe AD,Swanson JA. Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells [J].J Cell Biol,2004:176(1):51-63.
[24]Hartwell LH. Genetic control of the cell division cycle in yeast [J]. J Mol Biol,1971,51:627-637.
[25]Lohka MJ, Hayes MK, Mailer JL. Purification of muturation-promoting factor, an intracellular regulator of early mitotic events[J]. Pric Natl Acad Sic, 1988,85:3009-3013.
[26]Sofia J C, Jang SJ, Khuri FR, et al. Overexpression of cyclln Blin early-stage non-small cell lung cancer and its clinical implication[J]. Cancer Res,2000, 60(15):4000-4004.
[27]Takeno S, Nognchi T, Kikuchi R, et al. Pmgnosfic value of cyclin B1 in patients witll esophageal squamous cell carcinoma[J]. Caneer,2002,94(11): 2874-2881.
[28]Nozoe T, Korenaga D, Kabashima A, et al. Significance of cyclin B1 expression as an independent pmgnostic indicator of patients with squamous cell carcinoma of the esophagus [J]. Clin Cancer Res,2002,8(3):817-822.
[29]Yew PR. Ubiquitin-mediated proteolysis of vertebrate GI-and S-phase regulators. [J] J Cell Physiot,2001,187(1):1-10.
[30]Rudolph J:Cdc25 Phosphatases:Structure, Specificity, and Mechanism. Biochemestry 2007,46:3595-3604.
[31]Pines J:Mitosis:a matter of getting rid of the right protein at the right time. Trends Cell Biol 2006,16:55-63.
[32]Nigg EA:Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2001,2:21-32.
[33]Smits VA, Medema RH:Checking out the G(2)/M transition. Bio-chim Biophys Acta 2001,1519:1-12.
[34]Vecchione A, Baldassarre G, Ishii H et al, Fez1/Lzts1 absence impairs Cdkl/Cdc25C interaction during mitosis and predisposes to cancer development [J]. Cancer Cell 2007,11:275-289.
[35]Chen F, Zhang Z, Bower J et al,:Arsenite-induced Cdc25C degradation is through the KEN-box and ubiquitin-proteasome pathway [J]. Proc Natl Acad Sci USA 2002,99:1990-1995.
[36]Honda A, Miyoshi K, Baba K, et al. Expression of fasciculation and elongation protein zeta-1(FEZ1) in the developing rat brain[J].Mol Brain Res,2004,122(1):89-92.
[37]Fujita T, Ikuta J, Hamada J, et al.Identification of a tissue nonspecific homologue of axonal fasciculation and elongation protein zeta-1 [J].Biochem Biophys Res Commun,2004,313(3):738-774.
[38]Miyoshi K, Honda A, Baba K et al. Disrupted-in-schizophrenia 1, a candidate gene for schizophrenia, participates in neurite out growth [J]. Mol Psychiatry, 2003;8:685-694
[39]Okumura F, Hatakeyama S, Matsumo to M, et al.Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis[J].J Biol Chem,2004,279(51):53533-53543.
[40]Ikuta J,Maturana A,Fujita T, et al. Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility[J].Biochem Biophys Res Commun,2007,353(1): 127-132.
[41]SuzukiT,Okada Y, Semba S, et al. Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules:role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation [J]. J Biol Chem,2005,280 (26):24948-24956.
[42]Jeong JY,Einhorn Z, Mercurio S, et al. Determinant of zebrafish basal forebrain dopaminergic neurons and is regulated by the conserved zinc finger protein Tof/Fezl [J].Proc Natl Acad SciU SA,2006,13(103):5143-5148.
[43]Jeong JY, Einhorn Z, Mathur P, et al.Patterning the zebrafish diencephalon by the conserved zinc finger protein FEZ1[J]. Development,2007,134(1): 127-136.
[44]Haedicke J, Brown C, Naghavi MH. et al.The brain-specific factor FEZ1 is a determinant of neuronal susceptibility to HIV-1 infection. [J].Proc Natl Acad Sci U S A.2009 106(33):14040-14045.
[45]Miyoshi K,Honda A,Baba K, et al. Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite out-growth[J]. Mol Psychiatry, 2003,8(7):685-694.
[46]Lipska BK, Mitkus SN, Mathew SV, et al. Functional genomics in postmortem human brain:abnormalities in a DISCI molecular pathway in schizophrenia [J]. Dialogues Clin Neurosci,2006,8 (3):353-357.
[47]Matsuzaki S, Tohyama M. Molecular mechanism of schizophrenia with reference to disrupted-in-schizophrenia 1 (DISC1)[J]. Neurochem Int,2007, 51(2-4):165-172.
[48]Nicodemus KK, Callicott JH, Higier RGHum Genet.et al,Evidence of statistical epistasis between DISCI, CIT and NDEL1 impacting risk for schizophrenia:biological validation with functional neuroimaging [J]. Hum Genet 2010,12 (4) 123-126
[49]Rastogi A, Zai C, Likhodi O et al, Genetic association and post-mortem brain mRNA analysis of DISCI and related genes in schizophrenia[J].Schizophr Res. 2009;114(1-3):39-49.
[50]Matsuzaki S, Tohyama M et al. Molecular mechanism of schizophrenia with reference to disrupted-in-schizophrenia 1 (DISC1)[J]. Neurochem Int.2007 51(2-4):165-172
[51]Sakae N, Yamasaki N, Kitaichi K et al,Mice lacking the schizophrenia-associated protein FEZ1 manifest hyperactivity and enhanced responsiveness to psychostimulants [J]. Hum Mol Genet.2008; 17(20): 3191-3203.
[52]Yamada K, Nakamura K, Minabe Y et al, Association analysis of FEZ1 variants with schizophrenia in Japanese cohorts[J]. Biol Psychiatry. 2004;56(9):683-690.
[53]Koga M, Ishiguro H, Horiuchi Y et al, Failure to confirm the association between the FEZ1 gene and schizophrenia in a Japanese population[J]. Neurosci Lett.2007;417(3):326-329.
[1]RuchlinH S, PellissierJ M. An economical overview of prostate carcinoma.Cancer,2001,92:2796-2810
[2]Jemal A, Tiwari RC, Murray T, et al.Cancer statistics.2004,CA Cancer J Clin. 2004,154(10):8-29
[3]顾方六,刘玉立,50年泌尿男生殖系肿瘤发病和构成情况的变迁.中华泌尿外科杂志,2002,,23(2)：.88-90
[4]刘耀庭,李秀霞,程伟.泌尿外科肿瘤发病情况的变化.临床泌尿外科杂志,1997,12(1)：47-48
[5]Goodin S, Rao KV, Dipaola RS. State-of-the-art Treatment of Metastatic Hormone-Refractory Prostate Cancer. Oncologist,2002,7:360-370
[6]Pronzato P, Rondini M. Hormonotherapy of advanced prostate cancer. Ann Oncol.2005;16(suppl):iv80-iv84
[7]Kantoff PW, Halabi S, Conaway M, Picus J, Kirshner J, Hars V, et al. Hydrocortisone with or without mitoxantrone in men with hormone-refractory prostate cancer:result of the cancer and leukemia group B 9182 study. J Clin Oncol 1999; 17:2506-2513
[8]Anderson W F, Blaese R M, Culver K, et al. The ADA human gene therapy clinical protocol:points to consider response with clinical protocol. Hum Gene Ther,1990,1:331-362
[9]Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science,2000, 288(5466):669-672
[10]Patel P, Ashdown D, James N, et al. Is gene therapy the answer for prostate cancer. Protate Cancer Prostatic Dis,2004,7 suppl:s14-9.
[11]Nagpal ML, Chen Y, Lin T. Effects of overexpression of Cxcl10 (cytokine-responsiver gene-2) on MA-10 mouse Leydig tumor cell steroidogenesis and proliferation. Endocrinol,2004,183(3):585-594.
[12]Kaliberov SA, Kaliberova LN, Stockard CR, et al. Adenovirus-mediated FLT1-tageted proapoptotic gene therapy of human prostate cancer.Ol Ther, 2004,10(6):1059-70
[13]Satoh T, The BS, Timme TL, et al.Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients. Int J Radiat Oncol Biol Phys,2004,59(2):562-571
[14]Kraaij R, van Rijswijk AL, Oomen MH, et al. Prostate specific membrane antigen (PSMA) is a tissue-specific target for adenoviral transduction of prostate cancer in vitri. Prostate,2004,62(3):253-259
[15]Liu M, Acres B, Balloul JM, BIzouame N, et al.Gene-based vaccines and immunotherapeutics. Proc Natl Acad Sci USA,2004,101 suppl 2:14567-71
[16]Deweese TL, van der Poel H, Li S, et al. A phase I trial of CV706, a replication competenet, PSA selective oncolytic adenovirus,for the treatment of locally recurrent prostate cancer following radiation therapy.Cancer Res, 2001,61(20):7464-72
[17]Pisters IL, Pettaway CA, Troncoso P. Evidence that transfer of functional p53 protein results in increased apoptosis in prostate cancer.Clin Cancer Res,2004, 10(8):2587-93
[18]Eastham JA, Hall SJ, Sehgal I, et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res.1995,55(22);5151-5155
[19]Chakravarti A, Heydon K, Wu CL, et al. loss of p16 expression is of prognostic significance in locally advanced prostate cancer:an analysis from the Radiatiaon Therapy Oncology Group protocol 86-10. J Clin Obcol,2003, 21(17):3328-34
[20]Hernandez I, Maddison LA,Wei Y, et al.Prostate-specific expression of p53(R172L) differentially regulates p21, Bax, and mdm2 to inhibit prostate cancer progression and prolong survival. Mol Cancer Res,2003,1 (14):1036-47
[21]Enhanced systemic T-cell activation after in situ gene therapy with radiotherapy in prostate cancer patients. Int J Radiat Oncol Biol Phys, 2004,59(2):562-71
[22]Tsui KH, Wu L. Chang PL, et al. Identifying the combination of the transcriptional regulatory sequences on prostate specific antigen and human glanular kallikrein genes. J Urol,2004,172(5 Pt 1):2029-34
[23]Teh BS, Ayala G, Aguilar L, et al. Phase Ⅰ-Ⅱ trial evaluating combined intensity-modulated radiotherapy and in situ gene therapy with or without hormonal therapy in treatment of prostate cancer-interim report on PSA response and biopsy data. Int J Radiat Oncol Biol Phys,2004,58(5):1520-9
[24]Pantuck AJ, Vanophoven A,Gitlitz BJ, et al. Phase I trail of antigen-specific gene therapy using a recombinant vaccinia virus encoding MUC-1 and IL-2 in MUC-1-positive patients with advanced prostate cancer. J Immunoyher,2004,27(3):240-253
[25]Kubo H, Cardner TA, Wada Y, et al. Phase Ⅰ dose escalation clinical trial of adenovirus vector carrying osteoealcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormonerefractory prostate cancer. Hum Gene Ther,2003,14(3):227-41.
[26]Saito Y, Miyahara R, Gopalan B,et al. Selective induction of cell cycle arrest and apoptosis in human prostate cancer cells through adenoviral transfer of the melanoma differentiation-associated-7(mda-7)/interleukin-24(IL-24) gene. Cancer Gene Ther,2004.
[27]Steiner MS, Gingrich JR, Chauhan RD. Prostate cancer gene therapy. Stag Oncol Clin N Am,2002,11:607-620.
[28]Hall SJ, Mutchnik SE, Yang G, et al. Cooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer. Cancer Gene Ther,1999,6(1):54-63.
[29]Wildner O, Morris JC The role of the E1B 55 kDa gene product in oncolytic
[30]adenoviral vectors expressing herpes simplex virus-tk:assessment of antitumor efficacy and toxicity. Cancer Res,2000,60(15):4167-74
[31]Kirm D. oncolytic virotherapy for cancer with the adenovirus dl1520 (Onyx-015):results of Phase Ⅰ and Ⅱ trails. Exp Opin Ther,2001,1(3): 525-538.
[32]Lebedeva IV, Sarkar D, Su ZZ, et al. Bcl-2 and Bel-x(L) differentially
[33]protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7. Mda-7/IL-24. Oncogene,2003,22(54): 8758-73.
[34]Boueher PD, Im MM, Freytag SO, Shewach DS. A novel mechanism of synergistic cytotoxicity with 5-fluorocytosine and ganeiclovir in double suicide gene therapy [J]. Cancer Res,2006,66(6):3230-3237.
[35]Lipinski KS, Pelech S, Mountain A et al. Nitroreductase-based therapy of prostate cancer, enhanced by raising expression of heat shock protein 70, actsthrongh increased anti-tumour immunity [J]. Cancer Immunol Immunother,2006,55(3):347-354.
[36]FujitaT, Teh BS, Timme TL, et al Sustained long-term immune responses after in situ gene therapy combined with radiotherapy and hormonal therapy in prostate cancer patients [J]. Int J Radiat Oncol Biol Phys,2006,65(1):84-90.
[37]Raja Gahaglia c, Diaz de Durana Y, Graham FL, et al. Attenuation of the glucocorticoid response during Ad5IL-12 adenovirus vector treatment enhances natural killer cell--mediated killing of MHC class 1-negative LNCaP prostate tumors[J]. Cancer Res,2007,67(5):2290-2297.
[38]Zhao P, Zhu YH, Wu JX, et al. Adenovirus-mediated delivery of human IFNgamma gene inhibits prostate cancer growth [J]. Life Sci,2007,81 (9): 695-701.
[39]Fujita T, Timme TL, Tabata K et al. Cooperative effects of adenoviral vecter-mediated interleukin 12 gene therapy with radiotherapy in a preclinical model of metastatic prostate cancer [J]. Gene Ther,2007,14(3):227-236
[40]Tsai CH, Hong JH, Hsieh KF, et al. Tetracycline-regulated intratumoral expression of interleukin-3 enhances the efficacy of radiation therapy for murine prostate cancer [J]. Cancer Gene Ther,2006,13(12):1082-1092.
[41]Butch PA, Croghan GA, Gastineau DA, et al. Immunothempy(APC8015, Pmvenge) targeting prostatic acid phosphatase can induce durable remission of metastatic androgen-independent prostate cancer:a Phase 2 trial[J]. Prostate,2004,60(3):197-04.
[42]Young JG, Green NK, Mautner V, et al. Combining gene and immunotherapy for prostate cancer[J]. Prostate Cancer Prostatic Dis,2008,11(2):187-193.
[43]Lee J, Wang A, Hu Q, et al. Adenovirus-mediated intefferon-beta gene transfer inhibits angiogenesis and progression of orthotopic tumors of human prostate cancer cells in nude mice [J]. Im J Oncol,2006,29(6):1405-1412.
[44]Guan M, Jiang H, Xu C, et al.Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2[J]. Cancer Biol Ther,2007,6(3):419-425.
[45]Raikwar SP, Temm CJ, Raikwar NS, et al. Adenoviral vectors expressing human endostafinangiostatin and soluble Tie2:enhanced suppression of tumor growth and antiangiogenic effects in a prostate tumor model [J]. Mol Ther, 2005,12(6):1091-1100.
[46]Li x, Liu YH, Lee SJ, et al. Prostate restricted replicative adenovirus expressing human endostatin-angiostatin fusion gene exhibiting dramatic antitumor efficacy [J]. Clin Cancer Res,2008,14(1):291-299.
[47]Sarkar D, Lebedeva IV, Su ZZ, et al. Eradication of therapy-resistant human prostate tumors using a cancer tenninator virus [J]. Cancer Res,2007,67(11): 5434-5442.
[48]Naruishi K, Timme TL, Kusaka N, et al. Adanoviral vector-mediated PRVP-I gene-modified tumor cell-based vaccine suppresses the development of experimental prostate cancer [J]. Cancer Gene Ther,2006,13(7):658-663.
[49]Zhang Y, Yu J, Until E, et al. Monogene and polygene therapy for the treatment of experimental prostate cancer by use of apoptotic genes bax and bad driven by
[50]the prostate-specific promoter ARR(2)PB[J]. Hum Gene/her,2002,13(17) 2051-2064.
[51]Li X, Liu YH, Zhang YP, et al. Fas ligand delivery by a prostate-restricted replicative adenovirus enhances safety and antitumor efficacy [J].Clin Cancer Res,2007,13(18 Pt 1) 5463-5473
[52]Dzojic H, Loskog A, Totterman TH, et al. Adenovirus-mediated CD40 ligand therapy induces tumor cell apoplosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model [J]. Prostate,2006,66(8):831-838.
[53]Scattoni V, Montorsi F, Picchio M, et al. Diagnosis of local recurrence after radical prostatectomy[J]. BJU Int,2004,93(5):680-688.
[54]Mitrofanova E, Unfer R, Vahanian N, et al. Rat sodium iodide symporter allows using lower dose of 131I for cancer therapy [J]. Gene Ther, 2006,13(13): 1052-1056.
[55]DeWeese TL, van der Poel H, Li S, et al. A phase I trial of CV706。 a replication-competent, PSA selective oncolytie adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy [J]. Cancer Res, 2001,61(20):7464-7472.
[56]Small EJ, Carducci MA, Burke JM, et al. A phase (?) trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate Cancer[J]. Mol Ther,2006,14(1):107-117.
[57]Nasu Y, Saika T, Ebara S, et al. Suicide gene therapy with adenoviral delivery of HSV-TK gene for patients with local recurrence of prostate cancer after hormonal therapy [J]. Mol Ther,2007,15(4):834-840.
[58]Shirakawa T, Terao S, Hinata N, et al. Long-term Outcome of phase I/11 clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer[J]. Hum Gene Ther,2007,18(12):1225-1232.
[59]Freytag SO, Movsas B, Aref I, et al. Phase I trial of replication-competent adenovirus-mediated suicide gene therapy combined with IMBT for prostate cancer[J]. Mol Ther,2007,15(5):1016-1023.