跳到主要內容

臺灣博碩士論文加值系統

(3.236.225.157) 您好!臺灣時間:2022/08/16 00:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:楊錄
研究生(外文):Percy Luk Yeung
論文名稱:氯化鎘與抗癌藥物對大鼠熱休克蛋白八十六基因之調控
論文名稱(外文):Transactivation and Regulation of Rat hsp86 induced by Cadmium Chloride and Antitumor Drugs
指導教授:張大慈
指導教授(外文):Margaret Dah-Tsyr Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:67
中文關鍵詞:熱休克蛋白八十六氯化鎘膠達那黴素瑞迪士可黴素起動子轉錄因子
外文關鍵詞:Heat shock protein 86cadmium chlorideGeldanamycinradicicolpromotertranscription elements
相關次數:
  • 被引用被引用:0
  • 點閱點閱:524
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
目前哺乳動物的熱休克蛋白九十 (HSP90) 已知有兩種類蛋白,分別為緊迫誘發的熱休克蛋白八十六 (HSP86) 與持續的熱休克蛋白八十四 (HSP84)。已知重金屬與抗癌藥物會誘使HSP86的表現,然而hsp86基因之調控機制還不清楚,我們以對大鼠hsp86起動子的研究來探討重金屬與抗癌藥物緊迫對hsp86基因調控的機制。我們以全長大鼠hsp86的起動子找出可能存在的轉錄因子,以及對幾個哺乳類動物的hsp86起動子作DNA序列比對,我們發現在-270到+1的DNA序列在很高的相似性,這說明了哺乳類動物之間hsp86基因是以類似的起動子來調控的。為了探討大鼠hsp86起動子與特定轉錄因子的活性,全長的以及其一系列刪除突變的起動子分別建構到含螢火蟲螢光酵素的載體內,以螢光酵素的活性來換算起動子在正常的情況,氯化鎘刺激以及抗癌藥物(膠達那黴素,瑞迪士可黴素,17-AAG)處理下的活性。我們發現-478至+1這區域的起動子是為最小的必要起動子,而-270至+1區域的活性比-478至+1區域與-148至+1區域高兩倍,這說明了HSE(-84)是hsp86基因最重要的促進因子,而GC(-154)有正調控的作用。另外,我們發現在氯化鎘刺激下NF-kappaB(-395)有負調控的作用,而CRE(-1177)對氯化鎘有促進的作用。為了要尋找第二主要的轉錄因子,我們建構了一系列HSE突變的起動子,結果發現了NF-kappaB(-395)在正常情況以及氯化鎘緊迫下扮演了一個很重要的角色。

HSP86 and HSP84 are mammalian heat shock proteins identified to be the inducible and constitutive isoform of HSP90, respectively. However, the regulatory mechanism of hsp86 upon heavy metal and antitumor drugs stress are still unclear. In this study, rat hsp86 promoter region was used to investigate the mechanism of hsp86 expression induced under stress. The full length promoter region of rat hsp86 was used to search the putative transcription elements in hsp86 promoter from transcription element database. We also conducted sequence analysis between promoters of several hsp86 in mammals. The sequence comparison results showed that the -270 to +1 region of mammalian hsp86 promoters is almost identical, suggesting the importance of functional conservation. To test whether the -270 to +1 region is sufficient and to characterize the individual cis-acting elements, full length and serial deletions of the predicted hsp86 promoter isolated from genomic library were constructed into firefly luciferase reporter plasmid. The functional analyses of these promoter constructs were first verified under normal condition and then tested under the stimulation of cadmium chloride, geldanamycin, 17-allylamino, 17-demethoxygeldanamycin, and radicicol in 9L rat brain tumor (RBT) cells. The same minimal essential promoter region of rat hsp86 located within the —471 to +1 region is identified under normal condition, and stimulation of the heavy metal cadmium, and three anticancer drugs. Our experimental results identified two more regulatory regions than the hypothetic result from conserved sequence analysis data of the -270 to +1 region. The functional analysis indicated, consistently, that within the hypothetic -270 to +1 region, the heat shock element (HSE) containing region from —137 to +1 plays the main transactivation role, and the GC containing region (from —265 to —137) exhibits a positive regulatory activity. However, the nuclear factor kappa chain transcription B cells (NF-kappaB) containing region (from -471 to —265) has a newly found negative regulatory activity and a distal region containing cAMP responsive element (CRE) from —1418 to —1177 is identified as a possible cadmium-specific, responsive region. To detect the secondary important elements, HSE was specifically mutated from the deletion constructs and the results identified NF-kappaB as the secondary effective element during cadmium treatment. No obvious secondary element was found under normal condition indicating the NF-kappaB element is required but not sufficient for the basal level expression of hsp86.

Abstract (Chinese) i
Abstract (English) ii
Acknowledgements iv
Contents v
List of Tables vi
List of Figures vii
List of Appendixes ix
Abbreviations x
Introduction 1
Materials and Methods 4
Results 11
Discussion 18
References 22
Figure Legends 27

Bannai, S., H. Sato, and S. Taketani. 1991. Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase. Biochim. Biophys. Acta. 1092:175-179.
Beyersmann, D., and S. Hechtenberg. 1997. Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol. Appl. Pharmacol. 144:247-265.
Brugge, J.S., E. Erikson, and R.L. Erikson. 1981. The specific interaction of the Rous Sarcoma virus transforming protein pp60src, with two cellular proteins. Cell. 25:363-372.
Cotto, J.J., M. Kline, and R.I. Morimoto. 1995. Activation of Heat Shock Factor 1 DNA Binding Precedes Stress-induced Serine Phosphorylation EVIDENCE FOR A MULTISTEP PATHWAY OF REGULATION. J Biol Chem. 271:3355-8.
Csermely, P., T. Schnaider, C. Soti, and Z. Prohaszka. 1998. The 90-kDa molecular chaperone family : structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79:129-168.
Dale, E.C., X. Yang, S.K. Moore, and G. Shyamala. 1997. Murine 86-kDa heat shock protein gene and promoter. Cell Stress Chaperones. 2:87-93.
Felts, S.J., B.A. Owen, P. Nguyen, J. Trepel, D.B. Donner, and D.O. Toft. 2000. The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties. J Biol Chem. 275:3305-12.
Galan, A., M.L. Garcia-Bermejo, A. Troyano, N.E. Vilaboa, E. de Blas, M.G. Kazanietz, and P. Aller. 2000. Stimulation of p38 Mitogen-activated Protein Kinase Is an Early Regulatory Event for the Cadmium-induced Apoptosis in Human Promonocytic Cells. J Biol Chem. 275:11418-24.
Gaubin, Y., F. Vaissade, F. Croute, B. Beau, J.P. Soleilhavoup, and J.C. Murat. 2000. Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung cell-line. Biochim. Biophys. Acta. 1495:4-13.
Hartl, F.U. 1996. Molecular chaperones in cellular protein folding. Nature. 381:571-580.
Hickey, E., S.E. Brandon, G. Smale, D. Lloyd, and L.A. Weber. 1989. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein. Mol. Cell. Biol. 9:2615-2626.
Hsia, C.Y., S. Cheng, A.M. Owyang, S.F. Dowdy, and H.C. Liou. 2002. c-Rel regulation of the cell cycle in primary mouse B lymphocytes. Int. Immunol. 14:905-916.
Hung, J.J., T.J. Cheng, M.D. Chang, K.D. Chen, H.L. Huang, and Y.K. Lai. 1998a. Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells. J Cell Biochem. 71:21-35.
Hung, J.J., T.J. Cheng, Y.K. Lai, and M.D. Chang. 1998b. Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells. J. Biol. Chem. 273:31924-31931.
Imaizumi, K., K. Miyoshi, T. Katayama, T. Yoneda, M. Taniguchi, T. Kudo, and M. Tohyama. 2001. The unfolded protein response and Alzheimer's disease. Biochim. Biophys. Acta. 1536:85-96.
Jaiswal, R.K., E. Weissinger, W. Kolch, and G.E. Landreth. 1996. Nerve growth factor-mediated activation of the mitogen-activated protein (MAP) kinase cascade involves a signaling complex containing B-Raf and HSP90. J. Biol. Chem. 271:23626-23629.
Jakob, U., and J. Buchner. 1994. Assisting spontaneity : the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem. Sci. 19:205-211.
Johnson, B.D., R.J. Schumacher, E.D. Ross, and D.O. Toft. 1998. Hop modulates Hsp70/Hsp90 interactions in protein folding. J. Biol. Chem. 273:3679-3686.
Kwon, H.J., M. Yoshida, R. Nagaoka, T. Obinata, T. Beppu, and S. Horinouchi. 1997. Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression. Oncogene. 15:2625-31.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.
Lee, M.J., H. Nishio, H. Ayaki, M. Yamamoto, and K. Sumino. 2002. Upregulation of stress response mRNAs in COS-7 cells exposed to cadmium. Toxicology. 174:109-17.
Lee, W.C., K.Y. Lin, C.M. Chen, Z.T. Chen, H.J. Liu, and Y.K. Lai. 1991. Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells. J. Cell. Physiol. 149:66-76.
Lindquist, S., and E. Craig. 1988. The heat-shock proteins. Annu. Rev. Genets. 22:631-677.
Metz, K., J. Ezernieks, W. Sebald, and A. Duschl. 1996. Interleukin-4 upregulates the heat shock protein Hsp90alpha and enhances transcription of a reporter gene coupled to a single heat shock element. FEBS Lett. 385:25-8.
Minami, Y., H. Kawasaki, Y. Miyata, K. Suzuki, and I. Yahara. 1991. Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90. J. Biol. Chem. 266:10099-10103.
Moore, S.K., C. Kozak, E.A. Robinson, S.J. Ullrich, and E. Appella. 1989. Murine 86- and 84-kDa heat shock proteins, cDNA sequences, chromosome assignments, and evolutionary origin. J. Biol. Chem. 264:5343-5351.
Nathan, D.F., and S. Lindquist. 1995. Mutational analysis of Hsp90 function : interactions with a steroid receptor and a protein kinase. Mol. Cell. Biol. 15:3917-3925.
Nishizawa, J., A. Nakai, K. Matsuda, M. Komeda, T. Ban, and K. Nagata. 1999. Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart. Circulation. 99:934-41.
Oikawa, T., H. Ito, H. Ashino, M. Toi, T. Tominaga, I. Morita, and S. Murota. 1993. Radicicol, a microbial cell differentiation modulator, inhibits in vivo angiogenesis. Eur J Pharmacol. 241:221-7.
Pailhoux, E., B. Vigier, D. Vaiman, L. Schibler, A. Vaiman, E. Cribiu, C. Nezer, M. Georges, J. Sundstrom, L.J. Pelliniemi, M. Fellous, and C. Cotinot. 2001. Contribution of domestic animals to the identification of new genes involved in sex determination. J. Exp. Zool. 209:700-708.
Pan, J., B. Hinzmann, W. Yan, F. Wu, J. Morser, and Q. Wu. 2002. Human and murine corin genes: Genomic structures and functional GATA elements in their promoters. J. Biol. Chem.
Pirkkala, L., P. Nykanen, and L. Sistonen. 2001. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J. 15:1118-1131.
Prodromou, C., S.M. Roe, R. O'Brien, J.E. Ladbury, P.W. Piper, and L.H. Pearl. 1997. Identification and structural characterization of the ATP/ADP-binding site in the hsp90 molecular chaperone. Cell. 90:65-75.
Schlatter, H., T. Langer, S. Rosmus, M.L. Onneken, and H. Fasold. 2002. A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits. Biochem J. 362:675-84.
Schulte, T.W., and L.M. Neckers. 1998. The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to Hsp90 and shares important biologic activities with geldanamycin. Cancer Chemother. Pharmacol. 42:273-9.
Sorger, P.K., and H.R. Pelham. 1987. The glucose-regulated protein grp94 is related to heat shock protein hsp90. J. Mol. Biol. 194:341-344.
Stebbins, C.E., A.A. Russo, C. Schneider, N. Rosen, F.U. Hartl, and N.P. Pavletich. 1997. Crystal structure of an Hps90-geldanamycin complex : targeting of a protein chaperone by an antitumor agent. Cell. 89.
Stephanou, A., V. Amin, D. A. Isenberg, S. Akira, T. Kishimoto, and D. S. Latchman. 1997. Interleukin 6 activates heat-shock protein 90beta gene expression. Biochem. J. 321:103-106
Stephanou, A., D. A. Isenberg, K. Nakajima, and D. S. Latchman. 1999. Signal transducer and activator of transcription-1 and heat shock factor-1 interact and activate the transcription of the Hsp-70 and Hsp-90beta gene promoters. J. Biol. Chem. 274:1723-1728.
Stohs, S.J., and D. Bagchi. 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18:321-336.
Supko, J.G., Hickman R. L., Grever M. R., and M. L. 1995. Preclinical pharmacological evaluation of geldanamycin as an antitumor agent. Cancer Chemother. Pharmacol. 36:305-15.
Thirumalai, D., and G.H. Lorimer. 2001. Chaperonin-mediated protein folding. Annu Rev Biophys Biomol Struct. 30:245-269.
Vasconcelos, M.H., S.C. Tam, J.E. Hesketh, M. Reid, and J.H. Beattie. 2002. Metal- and tissue-dependent relationship between metallothionein mRNA and protein. Toxicol. Appl. Pharmacol. 182:91-97.
Whitesell, L., E.G. Mimnaugh, B. De Costa, C.E. Myers, and L.M. Neckers. 1994. Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci U S A. 91:8324-8.
Xia, W., and R. Voellmy. 1997. Hyperphosphorylation of Heat Shock Transcription Factor 1 Is Correlated with Transcriptional Competence and Slow Dissociation of Active Factor Trimers. J. Biol. Chem. 272:4094-4102.
Yoshida, H., K. Haze, H. Yanagi, T. Yura, and K. Mori. 1998. Identification of the cis-Acting Endoplasmic Reticulum Stress Response Element Responsible for Transcriptional Induction of Mammalian Glucose-regulated Proteins. J. Biol. Chem. 273:33741-33749.
Yoshida, H., T. Okada, K. Haze, H. Yanagi, T. Yura, M. Negishi, and K. Mori. 2001. Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6alpha and 6beta that activates the mammalian unfolded protein response. mol Cell Biol. 21:1239-48.
Zhang, S.L., J. Yu, X.K. Cheng, L. Ding, F.Y. Heng, N.H. Wu, and Y.F. Shen. 1999. Regulation of human hsp90alpha gene expression. FEBS Lett. 444:130-135.
Zuo, J., D. Rungger, and R. Voellmy. 1995. Multiple Layers of Regulation of Human Heat Shock Transcription Factor 1. Mol Cell Biol. 15:4319-4330.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top