砷化物抑制Galectin-1基因之表現及機制之研究

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：11

、訪客IP：3.144.17.45

姓名

潘雅芳(Ya-Fang Pan) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

砷化物抑制Galectin-1基因之表現及機制之研究
(The mechanism of arsenic inhibit galectin-1 gene expression.)

相關論文

★ 三氯乙烯與四氯乙烯對人類肺癌細胞之毒性研究	★ Galectin-1 與 Thioredoxin peroxidase II 基因之選殖及表現
★ 亞砷酸鈉誘引分裂中期停滯細胞之蛋白質體研究	★ Galectin-1蛋白與亞砷酸鈉毒性與結合作用之研究
★ 氧化壓迫與p53參與三氯乙烯及四氯乙烯誘導人類肺癌細胞凋亡之研究	★ Thioredoxin Peroxidase II蛋白與亞砷酸鈉毒性與結合作用之研究
★ 環型類	★ 亞砷酸鈉與Galectin-1蛋白交互作用之研究
★ PAG蛋白質對三氧化二砷誘發急性前骨髓性白血癌細胞毒性之角色研究	★ Galectin-1蛋白對細胞生長於幾丁聚醣膜上的影響及調控機制之研究
★ 低劑量亞砷酸鈉誘引第一型血紅素氧化酶調控路徑之研究	★ 砷化物誘導Thioredoxin Peroxidase II蛋白之表現及機制之研究
★ Galectin-1蛋白促進老鼠軟骨細胞於幾丁聚醣修飾之聚乳酸-聚乙酸醇共聚物支架生長之研究	★ Galectin-1對亞砷酸鈉毒性影響之研究
★ Galectin-1誘導前脂肪細胞分化機制之研究	★ 氧化性壓迫誘導Thioredoxin peroxidase II蛋白之表現機制研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

長期暴露在含有砷化物的環境之下，已知會造成肝臟的損傷與周邊血管疾病，如烏腳病及周邊神經病變等。此外長期暴露於砷化物中會提高皮膚癌，肺癌及膀胱癌發生的機會。而根據本實驗室之前的研究指出，三價砷在中國倉鼠卵巢細胞中會抑制一種特定蛋白-Galectin-1 (Gal-1) 的表現。而Gal-1蛋白是一種β-半乳糖苷蛋白，它能參與細胞貼附，增生與計劃性的細胞死亡。本論文進一步研究砷化物抑制Gal-1之表現及調控機制。
根據目前之研究結果指出三價砷對HFW，H1299，H460，KB，3T3等細胞內之Gal-1的量並無抑制現象。而在HaCaT與IRC細胞中Gal-1的表現量則會隨著砷化物處理的劑量而降低。砷化物在生物體中之代謝主要是經由甲基化之過程，而基因表現也與驅動子 (promoter) 之甲基化息息相關。因此我們進一步去探討在哺乳動物細胞中，砷化物是否會改變Gal-1基因之promoter區域的DNA methylation，而去抑制Gal-1基因之表現。我們將DNA利用二種對甲基化之敏感性不同的限制酶裁剪後，再以依照Gal-1基因之promoter序列所設計之primer，經PCR放大後，結果顯示在亞砷酸鈉所處理過的IRC細胞中，其Gal-1的promoter區域會有高度甲基化的情形。此外，將細胞前處理5-azadeoxycytidine (5-aza，一種甲基轉移酶的抑制劑)，則可回復受亞砷酸鈉抑制的Gal-1基因mRNA及蛋白質表現。此一結果顯示，砷化物抑制Gal-1基因的表現是在transcription的層面，且與Gal-1基因promoter區段的過度甲基化有關。

摘要(英)

Chronic exposure to arsenic has been associated with liver damage, peripheral vascular disease and peripheral neuropathy. Furthermore, exposure to excessive levels of arsenic has been correlated with an increased incidence of skin, lung and bladder cancers. The preliminary study in our laboratory showed that arsenite inhibits galectin-1 (gal-1) expression in CHOA cells. Gal-1 is a member of a growing family of animal ?-galactoside-binding proteins that are involved in the regulation of cell adhesion and immune function as well as proliferation and apoptosis. In this study, we further investigate the mechanism of arsenite-inhibited Gal-1 expression.
The present results showed that arsenite has no effect on the expression of Gal-1 in HFW, H1299, H460, KB, 3T3 cells, however arsenite treatment results in dose-dependent inhibition of Gal-1 expression in HaCaT and IRC cells. Arsenite is metabolized in vivo by methylation and DNA methylation in promoter region involves in regulating gene expression. We propose that alteration of gal-1 DNA methylation of the promoter region by arsenic might contribute to the inhibition of gal-1 gene expression in mammalian cells. The DNA was first digested with methylation-sensitive/insensitive restriction enzymes, and PCR-amplified with primers derived from the promoter region of gal-1. The results showed that the promoter region of Gal-1 gene is hypermethylated in arsenite-treated IRC cells. Moreover, the arsenite-inhibited expression of Gal-1 mRNA and protein can be effectively restored by pre-treatment of cells with 5-azadeoxycytidine (5-aza, a methyltransferase inhibitor). The results suggested that arsenite inhibits Gal-1 expression in transcription level, and is mediated through DNA hypermethylation of the Gal-1 promoter region.

關鍵字(中)

★ β-半乳糖苷蛋白
★ 砷化物

關鍵字(英)

★ galectin-1
★ arsenic

論文目次

圖目錄……………………………………………………………IV
縮寫表…………………………………………………………….V
第一章、緒論
I. 砷化物的簡介………………………………………………1
II. Galectin-1的簡介…………………………………………..4
III. DNA甲基化對基因轉錄之影響………………………….7
IV.實驗室初步結果……………………………………………7
V. 研究動機……………………………………………………7
VI. 研究目的…………………………………………………..8
第二章、材料與方法
實驗材料
1.細胞株……………………………………………………9
2.抗體………………………………………………………10
實驗方法
培養與保存
1.1細胞的培養及處理………………………………….10
蛋白質之定量及測定及純化製備相關
2.1細胞的收取………………………………………….11
2.2蛋白質定量………………………………………….12
2.3 SDS-PAGE分析（Sodiumdodecyl sulfate
polyacrylamide gel electrophoresis）……………….12
2.4西方點墨法（Western Blot）………………………13
細胞中Gal-1 mRNA表現量的測定
3.1 全RNA的萃取……………………………………..13
3.2 反轉錄反應………………………………………….14
3.3 聚合酶連鎖反應…………………………………….15
3.4 PCR產物之分析…………………………………...15
DNA甲基化的測定
4.1 genomic DNA的抽取與純化………………………16
4.2 核酸限制酶剪切……………………………………17
4.3 聚合酶連鎖反應……………………………………17
DNA 甲基化序列分析
5.1 Bisulfite modification………………………………..18
5.2 聚合酶連鎖反應……………………………………19
第三章、結果……………………………………………21
第四章、討論……………………………………………25
第五章、結論與展望……………………………………28
圖表………………………………………………………29
第六章參考文獻…………………………………………40
附錄………………………………………………………47

參考文獻

張光寧. (1999) 4-aminophenylarsine oxide親和性管柱鑑定中國倉鼠卵巢細胞中三種受亞砷酸鈉調控之蛋白. 國立中央大學碩士論文.
李麗雯. (2001) Galectin-1蛋白與亞砷酸鈉毒性與結合作用之研究. 國立中央大學碩士論文.
陳怡均. (2001) Galectin-1與Thioredoxin peroxidase II基因之選殖及表現. 國立中央大學碩士論文.
黃琦斐. (2002) 亞砷酸鈉與Galectin-1蛋白交互作用之研究. 國立中央大學碩士論文.
Allen, H.J., D. Sucato, B. Woynarowska, S. Gottstine, A. Sharma, and R.J. Bernacki. (1990) Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro. J. Cell Biolchem. 43: 43-57.
Attwood, J. T., R. L. Yung, B. C. Richardson. (2002) DNA methylation and the regulation of gene transcritpion. Cell. Mol. Life Sci. 59: 241-257.
Barondes, S.H., V. Castronovo, D.N. Cooper, R.D. Cummings, K. Drickamer, T. Feizi, M.A. Gitt, J. Hirabayashi, C. Hughes, K. Kasai, and et al. (1994a) Galectins: a family of animal beta-galactoside-binding lectins. Cell. 76: 597-598.
Barondes, S.H., D.N. Cooper, M.A. Gitt, and H. Leffler. (1994b) Galectins. Structure and function of a large family of animal lectins. J. Biol. Chem. 269:20807-20810.
Bates, M.N., A.H. Smith, and C. Hopenhayn-Rich. (1992) Arsenic ingestion and internal cancers: A review. Am. J. Epidemiol. 135: 462-476.
Benvenuto, G., M. L. Carpentieri, P. Salvatore, L. Cindolo, C. B. Bruni,
L. Chiariotti, (1996). Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region. Mol. Cell. Biol. 16: 2736-2743.
Berolero, F., G. Pozzi, E. Sabbioni, and U. Saffiotti. (1987) Cellular
uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation. Carcinogenesis. 8: 803-808.
Bianchet, M.A., H. Ahmed, G.R. Vasta, and L.M. Amzel. (2000) Soluble beta-galactosyl-binding lectin (galectin) from toad ovary: crystallographic studies of two protein-sugar complexes. Proteins. 40: 378-388.
Buchet, J.P., and R. Lauwerys. (1985) Study of inorganic arsenic methylation by rat liver in vitro: relevance for the interpretation of observation in man. Arch. Toxicol. 57: 125-129.
Chen, C.J., C.W. Chen, M.M. Wu, and T.L. Kuo. (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer. 66: 888-892.
Chen, G.Q., J. Zhu, X.G. Shi, J.H. Ni, H.J. Zhong, G.Y. Si, X.L. Jin, W. Tang, X.S. Li, S.M. xong, Z.X. Shen, G.L. Sun, J. Ma, P. Zhang, T.D. Zhang, C. Gazin, T. Naoe, S.J. Chen, Z.Y. Wang, and Z. Chen. (1996) In virto studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 88: 1052-1061.
Chen, M.L., R.S. King, and H.J. Armbrecht. (1990) Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats. J. Cell Physiol.143: 488-493.
Chiariotti, L., G. Benvenuto, R. Zarrilli, E. Rossi, P. Salvatore, V. Colantuoni, C. B. Bruni, (1994) Activation of the galectin-1 (L-14-I) gene from nonexpressing differentiated cells by fusion with undifferentiated and tumorigenic cells. Cell Growth Differ. 5: 769-775.
Chiou, H.Y., Y.M. Hsueh, K.F. Liaw, S.F. Horng, M.H. Chiang, Y.S. Pu, J.S.N. Lin, C.H. Huang, and C.J. Chen. (1995) Incidence of internal cancers and ingested inorganic arsenic: A seven-year follow-up study in Taiwan. Cancer Res. 55: 1296-1300.
Cho, M., and R.D. Cummings. (1995a) Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization. J. Biol. Chem. 270: 5198-5206.
Cho, M., and R.D. Cummings. (1995b) Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis. J. Biol. Chem. 270: 5207-5212.
Choi, J.Y., A.J. Van Wijnen, F. Asiam, J.D. Leszyk, J.L. Stein, G.S. Stein, J.B. Lian, and S. Penman. (1998) Developmental association of the beta-galcetoside binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts. J. Cell Sci. 111: 3035-3043.
Cindolo, L., G. Benvenuto, P. Salvatore, R. Pero, G. Salvatore, V. Mirone, D. Prezioso, V. Altieri, C.B. Bruni, and L. Chiariotti. (1999) Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas. Int. J. Cancer. 84: 39-43.
Cooper, D.N.W., and S.H. Barondes. (1990) Evidence for export of a muscle lectin from cytosol to extracellular matrix and for anovel secretory. J. Cell Biol.110: 1681-1691.
Cooper, D.N.W., S.M. Massa, and S.H. Barondes. (1991) Endogenous muscle lectin inhibits myoblast adhesion to laminin. J. Cell Biol. 115: 1437-1448.
Delnomdedieu, M., M.M. Basti, J.D. Otvos, and D.J. Thomas. (1994) Reduction and binding of arsenate and dimethylarsinate by glutathione: a magnetic resonance study. Chem. Biol. Interact. 90: 139-55.
Dodd, J., and T.M. Jesell. (1986) Cell surface glycoconjugates and carbohydrate-binding proteins: possible recognition signals in sensory neuron development. J. Exp. Biol. 124: 225-238.
Fan, S.R., I.C. Ho, F.L. Yeoh, C.J. Lin, and T.C. Lee. (1996) Squalene inhibits sodium arsenite-induced sister chromatid exchanges and micronuclei in Chinese hamster ovary-K1 cells. Mutat. Res. 368: 165-169.
Fischer, A.B., J.P. Buchet, and R. Lauwerys. (1985) Arsenic uptake, cytotoxicity and detoxification studied in mammalian cells in culture. Arch. Toxicol. 57: 168-172.
Gonzalgo, M. L., G. Liang, C. H. Spruck, J. M. Zingg, W. M. Rideout, P. A. Jones. (1997) Identification and characterization of differentially medthylated regions of genomic DNA by methylation-sensitive arbitrarily primed PCR. Cancer Res. 57: 594-599.
Gorbsky, G.J. (1994) Arsenic in human medicine, arsenic in the environment. Part2: Human health and ecosystem effects, New York.
Herman, J., J. R. Graff, S. Myohanen, B. D. Nelkin, S. B. Baylin, (1996) Methylation-specific PCR: A noel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA. 93: 9821-9826.
Horie, H., and T. Kadoya. (2000) Identification of oxidized galectin-1 as an initial repair regulatory factor after axotomy in peripheral nerves. Neurosci. Res. 38: 131-137.
Horie, H., T. Kadoya, Y. Inagaki, and Y. Sohma. (2000) [Oxidized galectin-1 is a new type factor to promote nerve regeneration]. Seikagaku. 72: 1245-1249.
Huang, R.N., and T.C. Lee. (1996) Cellular uptake of trivalent arsenite and pentavalent arsenate in KB cells cultured in phosphate-free medium. Toxicol. Appl. Pharmacol.136: 243-9.
Hughes, R. C. (2001) Galectins as modulators of cell adhesion. Biochimie. 83: 667-676.
Hughes, M. F. (2002) Arsenic toxicity and potential mechanisms of action. Toxicol Lett. 133: 1-16.
Hughtes, R.C. (1999) Secretion of the galectin family of mammalian carbonhydrate-binding protein. Biochim. Biophys. Acta. 1473: 172-185.
Hynes, M.A., L.B. Buck, M. Gitt, S. Barondes, J. Dodd, and T.M. Jesell. (1989) Carbonhydrate recognition in neuronal development: structure, expression of surface oligosaccarides and beta-galactoside-binding lectins. Clin. Symp.145: 189-210.
Iglesias, M.M., G.A. Rabinovich, V. Ivanovic, C. Sotomayor, and C. Wolfenstein-Todel. (1998) Galectin-1 ovine placenta amino-acid sequence, physicochemical properties and implications in T-cell death. Eur. J. Biochem. 252: 400-7.
Inagaki, Y., Y. Sohma, H. Horie, R. Nozawa, and T. Kadoya. (2000) Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties. Eur. J. Biochem. 267: 2955-64.
Joubert, R., M. Caron, and D. Bladier. (1987) Brain lectin-mediated agglutinability of dissociated cells from embryonic and postnatal mouse brain. Brain Res. 433: 146-150.
Kapahi, P., T. Takahashi, G. Natoli, S.R. Adams, Y. Chen, R.Y. Tsien, and M. Karin. (2000) Inhibition of NF-kB activation by arsenite through reaction with a critical cysteine in the activation loop of IkB kinase. J. Biol. Chem. 275: 36062-36066.
Lee, T.C., and I.C. Ho. (1995) Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts. Arch. Toxicol. 69: 498-504.
Li, W., and I.N. Chou. (1992) Effects of sodium arsenite on the cytoskeleton and cellular glutathione levels in cultured cells. Toxicol. Appl. Pharmacol.114: 132-9.
Liang, G., C. E. Salem, M. C. Yu, H. D. Nguyen, F. A. Gonzales, T. T. Nguyen, P. W. Nichols, P. A. Jones. (1998) DNA methlation differences associated with tumor tissues identified by genome scanning analysis. Genomics 53: 260-268.
Liu, Y.C., and H. Huang. (1997) Involvement of calcium-dependent protein kinase C in arsenite-induced genotoxicity in Chinese hamster ovary cells. J. Cell Biochem. 64: 423-33.
Liu, F. T., R. J. Patterson, J. L. Wang. (2002). Intracellular functions of galectins. Biochimica et Biophysica Acta. 1572:263-273.
Mass, M. J. and L. Wang (1997) Arsenic alters cytosine methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells: a model for a mechanism of carcinogenesis. Mutat. Res. 386: 263-277.
Mathews, C.K., and K.E. van Holde. (1996) Biochemistry.The Benjamin/Cummings Publishing company, Inc. Menlo Park, CA.
Mazurek, N., J. Conklin, J.C. Byrd, A. Raz, and R.S. Bresalier. (2000) Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands. J. Biol. Chem. 275: 36311-15.
Menzel, D.B., H.K. Hamadeh, E. Lee, D.M. Meacher, V. Said, R.E. Rasmussen, H. Greene, and R.N. Roth. (1999) Arsenic binding proteins from human lymophoblastoid cells. Toxicol. Lett. 105: 89-101.
Moiseeva, E.P., Q. Javed, E.L. Spring, and D.P. de Bono. (2000) Galectin 1 is involved in vascular smooth muscle cell proliferation. Cardiovasc. Res. 45: 493-502.
Perillo, N.L., M.E. Marcus, and L.G. Baum. (1998) Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death. J. Mol. Med. 76: 402-412.
Perillo, N.L., K.E. Pace, J.J. Seihamer, and L.G. Baum. (1995) Apoptosis of T cells mediated by galectin-1. Nature. 78: 736-739.
Robertson, K. D. and A. P. Wolffe (2000) DNA methylation in health and disease. natrue reviews: genetics 1: 11-19.
Salvatore, P., C. Contursi, G. Benvenuto, C. B. Bruni, L. Chiariotti, (1995) Characterization and functional dissection of the galectin-1 gene promoter. FEBS Lett. 373: 159-163.
Salvatore, P., G. Benvenuto, M. Caporaso, B. C. Bruni, L. Chiariotti, (1998) High resolution methylation analysis of the galectin-1 gene promoter region in ezpressing and nonexpressing tissues. FEBS Lett. 421: 152-158.
Tseng, W.P. (1977) Effects and dose-response relationships of skin
cancer and blackfoot disease eith arsenic. Environ. Health Perspect. 19: 109-119.
Vahter, M., and J. Envall. (1983) In vivo reduction of arsenate in mice and rabbits. Environ. Res. 32: 14-24.
Vogt, B.L., and T.G. Rossman. (2001) Effects of arsenite on p53, p21 and cyclin D expression in normal human fibroblasts --- a possible mechanism for arsenite's comutagenicity. Mutat. Res. 478: 159-168.
Wang, H.F., and T.C. Lee. (1993) Glutathione s-transferase facilitates the excretion of arsenic from arsenic-resistant chinese hamster ovary cells. Biochem. Biophys. Res. Commun.192: 1093-1099.
Wang, T.S., C.F. Kuo, K.Y. Jan, and H. Huang. (1996) Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species. J. Cell. Physiol. 169: 256-268.
Wen, W.N., T.L. Lieu, H.J. Chang, S.W. Wuu, M.L. Yau, and K.Y. Jan. (1981) Baseline and sodium arsenite-induced sister chromatid exchange in cultured lympocytes from patients with Blackfoot disease and healthy persons. Hum. Genet. 59: 201-203.
Yamanaka, K., K. Mishima, Y. Nagashima, A. Asai, Y. Sanai, and T. Kirino. (2000) Expression of galectin-1 mRNA correlates with the malignant potential of Human Gliomas and expression of antisense galectin-1 inhibits the growth of 9 Glioma cells. J. Neurosci. Res. 59: 722-730.
Yamaoka, K., A. Ingendoh, S. Tsubuki, Y. Nagai, and Y. Sanai. (1996) Structural and functional characterization of a novel tumor-derived rat galectin-1 having transforming growth factor (TGF) activity: the relationship between intramolecular disulfide bridges and TGF activity. J. Biochem. Tokyo 119: 878-886.
Yamauchi, H., K. Takahashi, M. Mashiko, and Y. Yamamura. (1989) Biological monitoring of arsenic exposure of gallium arsenide- and inorganic arsenic-exposed workers by determination of inorganic arsenic and its metabolites in urine and hair. Am. Ind. Hyg. Assoc. J. 50: 606-12.
Yih, L.H., and T.C. Lee. (1999) Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts. Mutat. Res. 440: 75-82.
Zhao, C. Q., M. R. Young, B. A. Diwan, T. P.Coogan, M. P. Waalkes, (1997) Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc. Natl. Acad Sci. USA 94: 10907-10912.
Zhong, C. X. and M. J. Mass (2001) Both hypomethylation and hypermethylation of DNA associated with arsenite exposure in culture of human cells identified by methylation-sensitive arbitrarily-primed PCR. Toxicology Lett. 122: 223-234.

指導教授

黃榮南(Rong-Nan Huang)

審核日期

2003-7-14

推文