進階搜尋


 
系統識別號 U0026-0812200910212625
論文名稱(中文) p53於Saos-2細胞中對於rat Mrp3基因轉錄調控之研究
論文名稱(英文) Transcriptional regulation of multidrug resistance-associated protein 3 (Mrp3) gene expression by p53 in Saos-2 cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 90
學期 2
出版年 91
研究生(中文) 吳筱珈
研究生(英文) Hsiao-Chia Wu
學號 s2689402
學位類別 碩士
語文別 中文
論文頁數 90頁
口試委員 指導教授-黃金鼎
口試委員-賴明德
口試委員-呂增宏
中文關鍵字 多重抗藥性相關性蛋白  轉錄調控 
英文關鍵字 Sp1  p53  Mrp3  transcriptional regulation  multidrug resistance-associated protein 
學科別分類
中文摘要 中文摘要
多重抗藥性相關性蛋白(Multidrug resistance-associated protein, MRP)為ATP binding cassette轉送器超級家族中,重要的轉送器之一。而目前發現的多重抗藥性相關性蛋白家族,是由MRP1~MRP7所組成,其分別也都在體內扮演著不同的生理角色。MRP3在體內為一有機陰離子的轉送器,目前MRP3也被認為是體內膽鹽的腸肝循環中扮演重要角色的轉送器之一。在1992年的文獻指出,MDR基因啟動區的活性表現會受到p53的調控。當轉染野生型p53時會抑制MDR啟動區的活性,而轉染突變型p53時,反而會抑制野生型p53對MDR啟動區的調控。1998年的文獻亦指出,MRP1基因的表現會受到轉錄因子 p53及Sp1的調控,Sp1會增強MRP1 基因啟動區的活性,而野生型p53會抑制Sp1所刺激增加的MRP1基因啟動區的活性。在我們實驗室已發表的文獻中指出,rat Mrp3基因啟動區上有五個Sp1 binding site (-436~440,-213~-219,-107~-118,-69~-74)。我們將rat Mrp3基因啟動區做 5’端不同長度deletion,並在其後接上luciferase reporter gene,同時將其與利用tetracycline off系統調控之野生型p53於Saos2細胞進行轉染,找出p53最主要影響在rat Mrp3 基因啟動區-157~-19的位置。而先前我們實驗室發表的文獻已指出,在-157~-19 啟動區上的第三與第四個Sp1 binding site對於rat Mrp3 基因啟動區的活性扮演著重要角色,但在我們的實驗結果中發現野生型p53並非藉由第四與第五個Sp1 binding site來抑制rat Mrp3基因啟動區的活性,若將第三個Sp1 binding site前的半個p53 binding site 去除也不影響野生型p53抑制rat Mrp3 基因啟動區活性。而當野生型p53表現會抑制rat Mrp3基因啟動區活性表現時,一些癌症中常見的突變型p53 (V143A, R248Q, R249S, R283P )卻無法抑制rat Mrp3基因啟動區的活性表現。因此,依據我們的實驗結果目前已知p53確實可調控Mrp3 基因啟動區的活性,但其機轉並非藉由結合於第四或第五個Sp1 binding site或第三個Sp1 binding site前的半個p53 binding site來進行其抑制作用。
英文摘要 Abstrate
Multidrug resistance-associated protein (MRP) is an important subfamily of the ATP-binding cassate superfamily. Seven MRP isoforms (MRP1~MRP7) have been identified in humans and have been shown to exhibit a tissue-specific distribution and play different physiological functions. MRP3 is an organic anion transporter in the normal physiological condition and it may play an important role in enterohepatic circulation of the bile salt. It was reported that MDR gene promoter is regulated by p53. The MDR promoter activity is down regulated by co-transfected with wild type p53, but when co-transfected with mutant type p53, the MDR promoter activity which is down regulated by wild type p53 can be reversed. In another report, it has been found that Sp1 can upregulate the MRP1 promoter activity, but wild type p53 can repress the activity which is stimulated by Sp1. Our previous studies indicated that there are five Sp1 binding sites (-436~440,-213~-219,-107~-118,-69~-74) in the rat Mrp3 promoter region. We used the 5’ deletion method to get different length of the rat Mrp3 promoter, and then constructed the different rat Mrp3 promoter fragment to the pGL3-basic plasmid adjacent to the luciferase reporter gene. Then, the tetracycline off system was used to express p53 or not. When co-transfect rat Mrp3 promoter reporter gene and p53 in Saos-2 TetOff cells, we found that wild-type p53 inhibited the rat Mrp3 promoter activity at the major region of –157~-19. In our previous studies indicated that the third and fourth Sp1 binding site (-107~-118) play important role in rat Mrp3 promoter activity, but in this study we find the wild type p53 does not inhibit rat Mrp3 promoter activity through the fourth or the fifth Sp1 binding site, or through the half p53 binding in front of the third Sp1 binding site. And four mutant type p53 (V143A, R248Q, R249S, R283P) were found to lose the ability to inhibit the rat Mrp3 promoter activity. In our study, we consider that rat Mrp3 gene expression is regulated by p53, and tumor-derived mutant p53 mutated in the p53 DNA binding domain abolish the p53 repression activity of Mrp3.
論文目次 目錄
中文摘要……………………………………………………………….……..1
英文摘要………………………………………………………………….…..3
縮寫檢索表……………………………………………………………..….…5
第一章 緒論…………………………………………...……………………..6
第二章 實驗材料……………………………………………..…………….15
第三章 實驗方法……………………………………………………..…….24
第四章 實驗結果
第一節 探討p53是否調控Mrp3基因啟動區的表現………...…….………49
第二節 探討野生型p53抑制Mrp3基因啟動區之機轉……………....…….53
第三節 探討突變型p53是否調控Mrp3基因啟動區的表現………………..56
第四節 探討野生型p53對於老鼠迴腸細胞IEC-18中內生性Mrp3 mRNA表現之影響58
第五章 總結與討論60
第六章 參考文獻66
附表74
附圖78
參考文獻 第六章 參考文獻
Allikmets, R., Gerrard, B., Hutchinson, A., and Dean, M. (1996) Characterirization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database. Hum. Mol. Genet. 5: 1649-1655
Aloin-Grinstein, R., Schwartz, D., and Rotter, V. (1995) Accumulation of wild type p53 protein upon g-irradiation induces a G2-dependent immunoglobobulin k light chain gene expression. EMBO J. 14: 1392-1401, 1995
Barak, Y., Juven, T., Haffner, R., and Oren, M. (1993) mdm2 expression is induced by wild type p53 activity. EMBO J. 12: 461-468
Borst, P., Evers, R., Kool, M., and Wijnholds, J. (1999) The multidrug resistance protein family. Biochim. Biophys. Acta 1461: 347-357
Borst, P., Evers, R., Kool, M., and Wijnholds, J. (2000) A family of drug transporters: the multidrug resistance-associated proteins. J. Natl. Cancer Inst. 92: 1295-1302
Buschmann, T., Minamoto, T., Wagle, N., Fuchs, S.Y., Adler, V., Mai, M., and Ronai, Z. (2000) Analysis of JNK, Mdm2, and p14(ARF) contribution to the regulation of mutant p53 stability. J. Mol. Biol. 295:10009-1021
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavietich, N.P. (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265: 346-355
Cole, S.P.C., Bhardwaj, G., Gerlach, J.H., Mackie, J.E., Grant, C.E., Almquist, K.C., Stewart, A.J., Kurz, E.U., Duncan, A.M.V., and Deeley, R.G. (1992) Overexpession of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 258: 1650-1654
Cole, S.P. (1999) Re: Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter protein [letter]. J. Natl. Cancer Insat. 91: 888-889
Donner, M.G., and Keppler, D. (2001) Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 34: 351-359
el-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., and Vogelstein, B. (1992) Definition of a consensus binding site for p53. Nat. Genet. 1: 45-49
el-Deiry,W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D., Mercer, W.E., Kinzler, K.W., and Vogelstein, B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75:817-825
Farmer, G., Friedlander, P., Colgan, J., Manley, J.L., and Prives, C. (1996) Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein. Nucleic acids Res. 24: 4281-4288
Giaccia, A.J., and Kastan, M.B. (1998) The complexity of p53 modulation: emerging patterns from divergent signalsm. Genes. Dev. 12: 2973-2983
Gottlieb, E., Haffner, R., von Ruden T., Wangner, E.F., Oren, M. (1994) Downregulation of wild type p53activity interferes with apoptosis of IL-3-dependent hematopoietic cells following Il-3 withdrawal. EMBO J. 13:1368-1374
Guengerich , F.P. (1995) Cytochrome P450: structure, metabolism, and biochemistry(second edition), Chapter 14, edited by Paul R. Oritz de Montellano. Plenum Press, New Yourk
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., and Elledge, S.J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75: 805-816
Higgins, C.F. (1992) ABC transporters: from microorganisms to man. Ann. Rev. Cell Biol. 8: 67-113
Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T. and Sugiyama, Y. (1998) Hepatic expression of multidrug resistance-associated protein like proteins maintained in Eisai hyperbilirubinemic rats. Mol. Pharm. 53: 1068-1075
Hirohashi, T., Suzuki, H. and Sugiyama, Y. (1999) Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J. Biol. Chem. 274: 15181-15185, 1999
Hirohashi, T., Suzuki, H., Takikawa, H. and Sugiyama, Y. (2000) ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3). J. Biol. Chem. 275: 2905-2910
Hopper, E., Belinsky, M.G., Zeng, H., Tosolini, A., Testa, J.R., and Kruh, G.D. (2001) Analysis of the structure and expression pattern of MRP7 (ABCC10), a new member of the MRP subfamily. Cancer Lett. 162: 181-191
Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.L.,and Kim, S.-H. (1998) Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396: 703-707
Inokuchi, A., Hinoshita, E., Iwamoto, Y., Kohno, K., Kuwano, M., and Uchiumi, T. (2001) Enhanced expression of the human multidrug resistance protein 3 by bile salt in human enterocytes. A transcriptional control of a plausible bile acid transporter. J. Biol. Chem. 276: 46822-46829
Kastan, M.B., Zhan, Q., El-Deiry, W.S., Carrier, F., Jacks, T., Walsh, W.V., Plunkett, B.S., Vogelstein, B., and Fornace, A.J., Jr. (1992) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71:587-597
Keppler, D. and König J. (1997) Expression and localization of the conjugate export pump encoded by MRP2 (cMRP/cMOAT) gene in liver. FASEB J. 11: 509-516
Keppler, D., Kamisako, T., Leier, I., Cui, Y., Nies, A.T., Tsujii, H., and König J. (2000) Localization, substrate specificity, and drug resistance conferred by conjugate export pimps of the MRP family. Adv. Enzyme. Regul. 40:339-349
Kern, S.E., Kinzler, K.W., Bruskin, S., Jarosz, D., Friedman, P., Prives, C., and Vogelstein, B. (1991) Identification of p53 as a sequence-specific DNA-binding protein. Science 252: 1708-1711
Khew-Voon,C., Kazumitsu, U., Ira, P., and Michael, M.G. (1992) Modulation of activity of the promoter of the human MDR1 gene by ras and p53. Science 255: 459-461
Koutsodontis G, Tentes I, Papakosta P, Moustakas A, and Kardassis D. (2001) Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein. J. Biol. Chem. 276: 29116-25
Kao,H.H., Huang, J.D., and Chang, M.S. (2002) cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter. Gene 286: 299-306
Lane, D.P., and Crawford, L.V. (1979) T antigen is bound to a host protein in SV40- transformed cells. Nature 278: 261-263
Leslie, E.M., Deeley, R.G. and Cole, S.P. (2001) Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters. Toxicology 167: 3-23
Li, B. and Lee, M.,Y. (2001) Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1. J. Biol. Chem. 276: 29729-39
Liu, X., and Berk, A., J. (1995) Reversal of in vitro p53 squelching by both TFIIB and TFIID. Mol. Cell Biol. 15: 6474-6478
Liu, X., Miller, C.W., Koeffler, P.H., and Berk, A.J. (1993) The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription. Mol. Cell. Biol. 13: 3291-3300,
Lotem, J., and Sachs, L. (1993) Regulation by bcl-2, c-mymc, and p53 of susceptibility toinduvtion of by heat shock and cancer chemotherapy copounds indifferentiation-competent and-defective myeloid leukemic cell. Cell Growth Differ. 4: 41-47
Lu, X., and Lane, D.P. (1993) Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell 75: 765-778
Mack, D.H., Vartikar, J., Pipas,J.M., and Laimins, L.A. (1993) Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53. Nature 363: 281-283
Maki, C.G.., and Howley, P.M. (1997) Ubiquitination of p53 and p21 is differentially affected y ionizing radiation and UV radiation. Mol Cell Biol. 17: 355-363
Maltzman, W., and Czyzyk, L. (1694) UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell Biol. 4:1689-1694
Marin, M.C., Jost, C.A., Brooks, L.A., Irwin, M.S., O’Nions, J., Tidy, J.A., James, N., McGregor, J.M., Harwood, C.A., Yulug, I.G., Vousden, K.H., Allday, M.J., Gusterson, B, Ikawa, S., Hinds, P.W., Crook, T., and Kaelin, W.G.Jr. (2000) A common polymorphism acts as an intragenic modifier of mutant p53 behavior. Nat. Genet. 25: 47-54
McAleer, M.A., Breen, M.A., White, N.L. and Mathews, N. (1999) pABC11(also known as MOAT-C and MRP5), a member of the ABC family of proteins, has anion transporter activity but does not confer multidrug resistance when overexpressed in human embryonic kidney 293 cells. J. Biol. Chem. 274: 23541-23548
Miyashita, T., and Reed, J.C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80: 293-299
Momand, J., Wu, H.H., and Dasgupta, G. (2000) MDM2-master regulator of the p53 tumor suppressor protein. Gene 25: 15-29
Nelson, W. G., and Kastan, M. (1994) DNA strand breaks: the DNA template alterations that trigger p53 dependent DNA damage response pathways. Mol. cell Biol. 14:1815-1823
Okamoto, K., and Beach, D. (1994) Cyclin G us a transcriptional target of the p53 tumor suppressor protein. EMBO J. 13: 4816-4822
Oude, Elferink R.P., Meijer, D.K., Kuipers, F., Jasen, P.L., Groen, A.K. and Griithuis, G.M. (1995) Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport. Biochim. Biophys. Acta 1241: 215-268
Perry, M.E., Piette, J., Zawadzki, J.A., Harvey, D., and Levine, A.J. (1993) The mdm-2 gene is induced in response to UV light in a p53-dependent manner. Proc. Natl. Acad. Sci. U.S.A. 90: 11623-11627
Ptashne M. (1988) How eukaryotic transcriptional activators work. Nature 335: 683-689
Rost, D., Mahner, S., Sugiyama, Y., and Stremmel, W. (2002) Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 282: G720-6
Seto, E., Usheva, A., Zambetti, G.P., Momand, J., Horiloshi, N., Weinmann, R., Levine, A.J., and Shenk, T. (1992) Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc. Natl. Sci. U.S.A. 89: 12028-12032
Sharom, F.J., Liu, R., Romsicki, Y., Lu, P. (1999) Insights into the structure and substrate interaction of the P-glycoprotein multidrug transporter from spectroscopic studies. Biochim. Biophys. Acta 1461: 327-345
Sigal, A., and Rotte, V. (2000) Oncogenic mutaions of the p53 tumor suppressor the demons of the guardian of the genome. Cancer Res. 60: 6788-6793
Takada, T., Suzuki, H., and Sugiyama, Y. (2000) Characterization of 5’-flanking region of human MRP3. Biochem. Biophys. Res. Commun. 270: 728-732
Takenaka,O., Horie, T., Kobayashi, K., Suzuki, H., and Suguyama Y. (1995) Kinetic analysis of hepatobiliary transport for conjugated metabolites in the perfused liver of mutant rats (EHBR) with hereditary conjugated hyperbilirubinemia. Pharm. Res. (NY) 12: 1746-1755
Truant, R., Xiao, H., Ingles, C.J., and Greenblatt, J. (1993) Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. J. Biol. Chem. 268: 2284-2287
Tzeng, S.J. and Huang, J.D. (2002) Transcriptional regulation of the rat Mrp3 promoter in intestine cells. Biochem. Biophys. Res. Commun. 291: 270-7
Vogelstein, B., Lane, D., and Levine A.J. (2000) Surfing the p53 net work. Nature 408:307-310
Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J. (1982) Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. : 945-951
Wang, Q., and Beck W.T. (1998) Transcriptional suppression of multidrug resistance-associated protein (MRP) gene expression by wild-type p53. Cancer Res. 58: 5762-9
Wu, X., Bayle, J.H., Olson, D., and Levine, A.J. (1993) The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 7: 1126-1132
Xiong, Y., Hannon, G..J., T., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993) p21 is a universal inhibitor of cyclin kinases. Nature 366: 701-704
Yamazaki, M., Suzuki, H. and Sugiyama, Y. (1996) Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics. Pharm. Res. (NY) 13: 497-513
Yamazaki, M., Akiyama, S., Niinuma, K., Nishigaki, R. and Sugiyama, Y. (1997) Biliary excretion of pravastatin in rats: contribution of the excretion pathway mediated by canalicular multispecific organic anion transporter. Drug Metab. Dispos. 25: 1123-1129, 1997
Zeng, H., Liu, G., Rea, P.A. and Kruh, G.D. (2000) Transport of amphipathic anions by human multidrug reisitance protein 3. Cancer Res. 60: 4779-4784
Zhan,Q., Fan, S., Bae, I., Guillouf, C., Liebermann, D.A., O’Connor, P.M., and Fornace, A.J. (1994) Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis. Oncogene 9: 3743-3751
Zhang, D., Cao, D., Russell, R. and Pizzorno, G. (2001) p53-dependent suppression of uridine phosphorylase gene expression through direct promoter interaction. Cancer Res. 61: 6899-905
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2003-07-22起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2003-07-22起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw