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研究生:徐心儀
研究生(外文):Hsin-Yi Shyu
論文名稱:鈣離子調控的含氧自由基參與9L大白鼠腦瘤細胞中膠達納黴素所誘發的葡萄糖調控蛋白78表現
論文名稱(外文):INVOLVEMENT OF CALCIUM MEDIATED REACTIVE OXYGEN SPECIES IN INDUCTIVE GRP78 EXPRESSION BY GELDANAMYCIN IN 9L RAT BRAIN TUMOR CELLS
指導教授:黎耀基黎耀基引用關係
指導教授(外文):Prof. Yiu-Kay Lai
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
中文關鍵詞:膠達納黴素葡萄糖調控蛋白78鈣離子蛋白質激脢C beta II含氧自由基
外文關鍵詞:geldanamycingrp78calciumPKCbeta IIreactive oxygen species
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膠達納黴素 (geldanamycin, GA) 是熱休克蛋白90 (90 kDa heat shock protein, HSP90) 的抑制物且可能成為有效的抗癌藥物。在先前的研究中,記錄了膠達納黴素的訊號傳遞路徑造成內質網逆境 (endoplasmic reticulum stress, ER stress) 的產生﹔且在我們的實驗室中證實含氧自由基 (reactive oxygen species, ROS) 在其中扮演重要的訊號傳遞角色。葡萄糖調控蛋白78 (glucose regulated protein 78, GRP78) 位於內質網中,其擁有保護子的功能,可以幫助新合成的蛋白質摺疊成正確的構型;並且在內質網逆境中會被大量表現出來以調節細胞生理。
在此篇研究中,我們以9L大白鼠腦瘤細胞 (9L Rat Brain Tumor cells) 為材料;探討鈣離子、含氧自由基與其二者相互關係在膠達納黴素所誘使的葡萄糖調控蛋白78中所扮演的角色。我們發現膠達納黴素誘使的葡萄糖調控蛋白78表現主要操作在轉錄層次且需要完善的蛋白質合成過程。藉由螢光顯微鏡監測細胞質內鈣離子含量的變化,我們發現耗盡細胞內胞器所儲存的鈣離子或防止細胞外的鈣離子流入細胞內會影響膠達納黴素所產生的鈣離子訊號。當我們利用BAPTA/AM或EGTA將細胞內或外的鈣離子予以螯合會減少膠達納黴素對葡萄糖調控蛋白78的誘使現象,暗示鈣離子的變化對葡萄糖調控蛋白78的誘使現象是必需的。此外,U73122減少膠達納黴素對葡萄糖調控蛋白78的誘使現象也暗示所監測到的鈣離子變化可能是仰賴磷脂脢C (phospholipase C) 的作用。
為了分析可能參與訊號傳遞的蛋白質激脢,我們藉由蛋白質激脢抑制物的前處理發現膠達納黴素的訊號傳遞可能是透過蛋白質激脢C (protein kinase C, PKC),且可能是蛋白質激脢CβII (PKCβII) 這個專一的亞型。前處理 genistein也有顯著的葡萄糖調控蛋白78減少現象,暗示著酪胺酸激脢也參與在膠達納黴素訊號傳遞路徑中。
另一方面,抗氧化物N-acetyl-L-cysteine (NAC) 防止膠達納黴素對葡萄糖調控蛋白78的誘使現象暗示膠達納黴素處理下細胞內有含氧自由基的產生。此外,我們更進一步地發現在不同化學組成物的含氧自由基中,氫氧根自由基 (hydroxyl radical) 的角色重於其他者。而且,我們是第一個發現在膠達納黴素處理下鈣離子訊號產生活化蛋白質激脢CβII,且進而產生含氧自由基訊號。簡言之,我們發現在9L大白鼠腦瘤細胞中膠達納黴素對葡萄糖調控蛋白78的誘使是透過鈣離子、磷脂脢C、蛋白質激脢CβII、酪胺酸激脢和含氧自由基的產生。

Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of HSP90 and has been implicated as a potent anti-cancer drug. Previous studies reported that endoplasmic reticulum stress (ER stress) was evoked with GA, and in our laboratory reactive oxygen species (ROS) was found to play important signal transduction roles. Glucose regulated protein 78 (GRP78) is mainly located in lumen of ER and has chaperone function to assist new synthesized proteins folding to correct conformations, and it was induced in response to ER stress.
In this study, we investigated the roles of calcium, ROS and their interrelationship in GA-induced GRP78 expression in 9L rat brain tumor (RBT) cells. The induction of GRP78 by GA requires intact process of protein synthesis and is regulated mainly in transcription level. By monitoring cytoplasmic calcium contents with fluorescence microscopy, the calcium signaling evoked by GA was influenced by depletion of organelle-stored calcium or by preventing the influx of calcium across the plasma membrane. Chelation of intracellular calcium with 1,2-bis(2-amino- phenoxy)ethane-N,N,N’,N’,-tetraacetic acid (BAPTA/AM) or extracellular calcium with ethylene glyco-bis(β-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) reduces GRP78 induction in GA-treated 9L RBT cells, implying that calcium signaling is required for GRP78 induction. The compound 1-96-[17beta- 3-methoxyestra-1,3,5(10)-trien-17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122) decreases the GA-induced GRP78 expression, suggesting that this calcium mobilization might be dependent on phospholipase C.
To assess the possible kinases involved, inhibitors were used and coupled with calcium monitoring. We found that the increase of cytoplasmic calcium concentration in GA signal transduction pathway may act through protein kinase C (PKC) and the specific subtype involved may be PKCβII. Significant reduction of GRP78 induction was also evident when genistein was applied, indicating the involvement of protein tyrosine kinase.
On the other hand, antioxidants prevent GRP78 induction by GA, implying ROS generation in vivo under GA treatment. To distinguish the contribution of diverse chemical species of ROS, our results indicate that GA-induced GRP78 expression was prominently blocked by antioxidants specific for hydroxyl radical. To delineate the causal effects of these intermediates, we show that increase in cytoplasmic calcium concentration activating PKCbII precedes the generation of ROS. In conclusion, we investigated the regulation of inductive expression of GRP78 in 9L RBT cells by GA treatment and identified that this signal transduction pathway is acting through calcium, PLC, PKCβII, and protein tyrosine kinase and then driving the ROS generation to concert this induction.

中文摘要……………………………………………………………………………1
ABSTRACT……………………………………………………………………2
INTRODUCTION………………………………………………………………4
EXPERIMENTAL PROCEDURES………………………………………………6
RESULTS……………………………………………………………………10
DISCUSSION…………………………………………………………………18
REFERENCES…………………………………………………………………22
FIGURE LEGENDS……………………………………………………………26
FIGURES……………………………………………………………………30

1. Asano, N., Kizu, H., Oseki, K., Tomioka, E., Matsui, K., Okamoto, M., and Baba, M. (1995) J. Med. Chem. 38, 2349-2356
2. Arora, P. D., Silvestri, L., Ganss, B., Sodek, J., and McCulloch, C. A. (2001) J. Biol. Chem. 276, 14100-14109
3. Barone, M. V., Crozat, A., Tabaee, A., Philipson, L., and Ron, D. (1994) Genes Dev. 8, 453-464
4. Bhat, V. B., and Madyastha, K. M. (2001) Biochem. Biophys. Res. Commun. 288, 1212-1217
5. Billecke, S. S., Bender, A. T., Kanelakis, K. C. Murphy, P. J., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 277, 20504-20509
6. Blobe, G. C., Khan, W. A., Halpern, A. E., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 10627-10635
7. Blobe, G. C., Stribling, D. S., Fabbro, D., Stabel, S., and Hannun, Y. A. (1996) J. Biol. Chem. 271, 15823-15830
8. Bolanos, J. P. and Medina, J. M. (1996) J. Neurochem. 66, 2091-2099
9. Browman, K. E., Kantor, L., Richardson, S., Badiani, A., Robinson, T. E. and Gnegy, M. E. (1998) Brain Res. 814, 112-119
10. Buchner, J. (1999) Trends Biochem. Sci. 24, 136-141. Review
11. Buckley, B. J., and Whorton, A. R. (1997) Am. J. Physiol. 273, C1298-C1305
12. Cao, X., Zhou, Y., and Lee, A. S. (1995) J. Biol. Chem. 270, 494-502
13. Carrasco-Marin, E., Paz-Miguel, J. E., Lopez-Mato, P., Alvarez-Dominguez, C., and Leyva-Cobian, F. (1998) Immunology 95, 314-321
14. Caspersen, C., Pedersen, P. S., and Treiman, M. (2000) J. Biol. Chem. 275, 22363-22372.
15. Chan., T. M., Chen, E., Tatoyan, A., Shargill, N. S., Pleta, M., and Hochstein, P. (1986) Biochem. Biophys. Res. Commun. 139, 439-445
16. Chen, L. Y., Chiang, A. S., Hung, J. J., Hung, H. I., and Lai. Y. K. (2000) J. Cell. Biochem. 78, 404-416
17. Di Monte, D. A., Royland, J. E., Irwin, I., Langston, J. W. (1996) Neurotoxicology 17, 697-703. Review
18. Duchen, M. R. (2000) J. Physiol. 529, 57-68. Review
19. Finkel, T., and Holbrook, N. J. (2000) Nature 408, 239-247. Review
20. Foley, P., and Riederer, P. (2000) J. Neurol. 247, II82-II94. Review
21. Freiden, P. J., Gaut, J. R., and Hendershot, L. M. (1992) EMBO J. 11, 63-70
22. Gau, R. J., Yang, H. L., Suen, J. L., and Lu, F. J. (2001) Biochem. Biophys. Res. Commun. 283, 743-749
23. Goodnight, J. A., Mischak, H., Kolch, W., and Mushinski, J. F. (1995) J. Biol. Chem. 270, 9991-10001
24. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450
25. Gschwendt, M., Kittstein, W., and Johannes, F. J. (1998) FEBS Lett. 421, 165-168.
26. Hampton, R. Y. (2000) Curr. Biol. 10, R518-R521. Review
27. Haze, K., Yoshida, H., Yanagi, H., Yura, T., and Mori, K. (1999) Mol. Biol. Cell 10, 3787-3799
28. Ichimiya, M., Chang, S. H., Liu, H., Berezesky, I. K., Trump, B. F., and Amstad, P. A. (1998) Am. J. Physiol. 275, C832-C839
29. Imaizumi, K., Miyoshi, K., Katayama, T., Yoneda, T., Taniguchi, M., Kudo, T., and Tohyama, M. (2001) Biochim. Biophys. Acta. 1536, 85-96. Review
30. Kass, G. E., Duddy, S. K., and Orrenius, S. (1989) Biochem. J. 260, 499-507
31. Kassenbrock, C. K., and Kelly, R. B. (1989) EMBO J. 8, 1461-1467
32. Kaufman, R. J. (1999) Genes. Dev. 13, 1211-1233. Review
33. Kumagai, Y., Nakajima, H., Midorikawa, K., Homma-Takeda, S., and Shimojo, N. (1998) Chem. Res. Toxicol. 11, 608-613
34. Kumar, S., Bharti, A., Mishra, N. C., Raina, D., Kharbanda, S., Saxena, S., and Kufe, D. (2001) J. Biol. Chem. 276, 17281-17285
35. Laemmli, U. K. (1970) Nature 227, 680-685
36. Laitusis, A. L., Brostrom, M. A., and Brostrom, C. O. (1999) J. Biol. Chem. 274, 486-493
37. Lawson, B., Brewer, J. W., and Hendershot, L. M. (1998) J. Cell. Physiol. 174, 170-178
38. Lee, A. S. (2001) Trends Biochem. Sci. 26, 504-510. Review
39. Lee, W. C., Lin, K. Y., Chen, C. M., Chen, Z. T., Liu, H. J., and Lai, Y. K. (1991) J. Cell. Physiol. 149, 66-76
40. Leno, G. H., and Ledford, B. E. (1990) FEBS Lett. 276, 29-33
41. Li, M., Baumeister, P., Roy, B., Phan, T., Foti, D., Luo, S., and Lee, A. S. (2000) Mol. Cell. Biol. 20, 5096-5106
42. Lievremont, J. P., Rizzuto, R., Hendershot, L., and Meldolesi, J. (1997) J. Biol. Chem. 272, 30873-30879
43. Liu, H., Bowes, R. C., van de Water, B., Sillence, C., Nagelkerke, J. F., and Stevens, J. L. (1997) J. Biol. Chem. 272, 21751-21759
44. Liu. T., Liu, X., Wang, H., Moon, R. T., and Malbon, C. C. (1999) J. Biol. Chem. 274, 33539-33544
45. Ludowyke, R. I., Holst, J., Mudge, L. M., and Sim, A. T. (2000) J. Biol. Chem. 275, 6144-6152
46. Machida, K., Tanaka, T., Fujita, K., and Taniguchi, M. (1998) J. Bacteriol. 180, 4460-4465
47. Mattson, M. P., LaFerla, F. M., Chan. S. L., Leissring, M. A., Shepel, P. N., and Geiger, J. D. (2000) Trends Neurosci. 23, 222-229. Review.
48. Meldolesi, J., and Pozzan, T. (1998) Trends Biochem. Sci. 23, 10-14. Review
49. Michell, B. J., Chen, Z. P., Tiganis, T., Stapleton, D., Katsis, F., Power, D. A., Sim, A. T., and Kemp, B. E. (2001) J. Biol. Chem. 276, 17625-17628
50. Miyake, H., Hara, I., Arakawa, S., and Kamidono, S. (2000) J. Cell. Biochem. 77, 396-408
51. Muid, R. E., Dale, M. M., Davis, P. D., Elliott, L. H., Hill, C. H., Kumar, H., Lawton, G., Twomey, B. M., Wadsworth, J., and Wilkinson, S. E. (1991) FEBS Lett. 293, 169-172
52. Munster, P. N., Srethapakdi, M., Moasser, M. M., and Rosen, N. (2001) Cancer Res. 61, 2945 — 2952
53. Murakami, Y., Fukazawa, H., Mizuno, S., and Uehara, Y. (1994) Biochem. J. 301, 57-62
54. Murakami, Y., Mizuno, S., Hori, M., and Uehara, Y. (1988) Cancer Res. 48, 1587-1590
55. Murphy, C. T., Poll, C. T., and Westwick, J. (1995) Cell Calcium 18: 245-251
56. Myhre, O., and Fonnum, F. (2001) Biochem. Pharmacol. 62, 119-128
57. Neckers, L., Mimnaugh, E., and Schulte, T. W. (1999) Drug Resist. Updat. 2, 165-172
58. Ohguchi, K., Banno, Y., Nakashima, S., and Nozawa, Y. (1995) Biochem. Biophys. Res. Commun. 211, 306-311
59. Okatani, Y., Wakatsuki, A., and Reiter, R. J. (2000) Biochem. Biophys. Res. Commun. 277, 470-475
60. Pai, K. S., Mahajan, V. B., Lau, A., and Cunningham, D. D. (2001) J. Biol. Chem. 276, 32642-32647
61. Parker, R., Phan, T., Baumeister, P., Roy, B., Cheriyath, V., Roy, A. L., and Lee, A. S. (2001) Mol. Cell. Biol. 21, 3220-3233
62. Paschen, W. (1996) Med. Hypotheses 47, 283-288. Review.
63. Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624
64. Resendez, E., Ting, J., Kim, K. S., Wooden, S. K., and Lee, A. S. (1986) J. Cell Biol. 103, 2145-2152
65. Reynaud, S., Duchiron, C., and Deschaux, P. (2001) Toxicol. Appl. Pharmacol. 175, 1-9
66. Roe, S. M., Prodromou, C., O’Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1999) J. Med. Chem. 42, 260-266
67. Rose, C. R., and Konnerth, A. (2001) Nature 4, 773-774.
68. Satoh, M., Nakai, A., Sokawa, Y., Hirayosih, K., and Nagata, K. (1993) Exp. Cell Res. 205, 76-83
69. Scheibel, T., and Buchner, J. (1998) Biochem. Pharmacol. 56, 675-682. Review
70. Schulte, T. W., Akinaga, S., Murakata, T., Agatsuma, T., Sugimoto, S., Nakano, H., Lee, Y. S., Simen, B. B., Argon, Y., Felts, S., Toft, D. O., Neckers, L. M., and Sharma, S. V. (1999) Mol Endocrinol. 13, 1435-1448
71. Schulte, T. W., Akinaga, S., Soga, S., Sullivan, W., Stensgard, B., Toft, D., and Neckers, L. M. (1998) Cell Stress Chaperones 3, 100-108
72. Singh, S., Farhan, A. S., and Hadi, S. M. (1998) Neurosci. Lett. 258, 69-72
73. Soderling, T. R. (1990) J. Biol. Chem. 265, 1823-1826
74. Spear, E., and Ng, D. T. (2001) Traffic 2, 515-523. Review
75. Staddon, J. M., Bouzyk, M. M., and Rozengurt, E. (1992) J. Biol. Chem. 267, 25239-25245
76. Stevens, J. L., Liu, H., Halleck, M., Bowes, R. C., Chen, Q. M., and van de Water, B. (2000) Toxicol. Lett. 112-113, 479-486. Review
77. Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S., and Walter, P. (2000) Cell 101, 249-258
78. Uehara, Y., Murakami, Y., Mizuno, S., and Kawai, S. (1988) Virology 164, 294-298
79. Weizsaecker, M., Deen, D. F., Rosenblum, M. L., Hoshino, T., Gutin, P. H., and Baker, M. (1981) J. Neurol. 224, 183-192
80. Whitesell, L., Mimnaugh, E. G., De Costa, B., and Myers, C. E. (1994) Proc. Natl. Acad. Sci. 91, 8324-8328
81. Wu, K-D., Bungard, D., and Lytton, J. (2001) Am. J. Physiol. Cell Physiol. 280, C843-C851
82. Zhang, C., Gong, Y., Ma, H., An, C., Chen, D., and Chen, Z. L. (2001) Biochem. J. 355, 653-661
83. Zou, J., Guo, Y., Guettouche, T., Smith D. F., and Voellmy, R. (1998) Cell 94, 471-480

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