臺灣博碩士論文加值系統

倉鼠頰囊袋鱗狀上皮細胞癌GST-P mRNA表現之研究
本實驗的目的在於以倉鼠頰囊袋鱗狀上皮細胞癌的動物實驗模式（animal model），對癌化的組織及正常的組織中GST-P mRNA的表現量做比較。實驗方法為將實驗組的動物以致癌物DMBA塗抹於頰囊袋共12週使其致癌，對照組則以礦物油代替。十二週後取下倉鼠之頰囊袋萃取RNA後，再以RT-PCR放大後來比較GST-P mRNA的表現量。實驗結果可發現到實驗組之倉鼠頰囊袋於12週的DMBA塗抹後，於外觀及組織學上已有鱗狀上皮細胞癌的情形發生，對照組則無。於RT-PCR之結果方面，實驗組有明顯之GST-P mRNA表現的現象，但對照組則無。此結果和以往之化學免疫染色方法檢測GST-P的結論對應。然而影響GST-P mRNA表現的機制甚多，仍待進一步的探討。

Placental glutathione S-transferase (GST-P) has been regarded as a tumor marker in many types of cancers including oral squamous cell carcinoma. The objective of this study is to investigate the expression of GST-P mRNA in DMBA-induced squamous cell carcinoma of hamster buccal pouch mucosa. Ten Syrian golden hamsters in the experimental group were painted with 0.5% DMBA in the buccal pouches for 12 weeks (three times a week). Other ten hamsters in the control group were similarly treated with mineral oil. After 12-week painting, all the animals were sacrificed and their pouches were removed. Half of these pouches were histologically examined. Other half of the pouches were used for total RNA extraction and RT-PCR was then performed to analyze the GST-P mRNA expression. Grossly and histologically, all the pouches of the DMBA-treated group showed well- differentiated squamous cell carcinoma, however, those of the control group remained unchanged. The results of RT-PCR revealed overexpression of GST-P mRNA in the buccal pouches of the experimental group, while no expression in control group was noted. These results were consistent with our previous works using immunohistochemical technique. However, factors involving overexpression of mRNA are vast and the mechanism underlying the overexpression of GST-P mRNA needs further elucidation.

中文摘要 2
英文摘要 3
緒論 5
癌化（carcinogenesis）概觀 5
Glutathione S-transferase (GST)之性質 7
GST與癌症 9
研究目的 11
材料與方法 14
Carcinomas induced by DMBA 倉鼠頰囊袋癌之誘發 14
Extraction of RNA 萃取RNA 14
Analysis of RNA Expression 分析RNA的表現 17
結果 21
一、 大體及組織學之觀察（Gross and histological examination） 21
二、 GST-P mRNA表現 22
討論 24
參考文獻 28

1. Kumar,V., et al. Basic Pathology, 5th ed. W.B. Saunders.1992
2. Weinberg RA. Sci Ame 1996; Sept:32-33
3. Fearon ER, et al. Cell 1990; 61:759
4. Boyer TD. The glutathione S-transferase: An update. Hepatology 1989; 9: 486-496
5. Morgenstern R, DePierre JW, Jomvall H. NE. Microsomal glutathione transferases: primary structure. J Biol Chem 1985; 260:13976-13983
6. Mannervik B, Danielson UH. Glutathione S-transferases -Structure and catalytic activity. Crit Rev Biochem 1988; 23: 283-337
7. Meister A, Anderson ME. Glutathione. Ann Rev Biochem 1983; 52:771-760
8. Meister A. Selective modification of glutathione metabolism. Science 1983; 220: 472477
9. Mannervik B. The isoenzymes of glutathione transferase. Adv Enzymol Relat Areas Mol Biol 1985; 57:357-417
10. Van Bladeren PJ, Van Ommmen B. The inhibition of glutathione S-tranferase: mechanism, toxic consequenceses and therapeutic benefits. Pharmacol Ther 1991;37:35-46
11. Boyland E, Chasseaud LF. The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis. Adv Enzymol Relat Areas Mol Biol 1969; 32: 173
12. Jacoby WB. The glutathione transferases: a group of multifunctional detoxification proteins. Adv Enzymol 1978; 46: 383-415
13. Bass NM, Kirsch RE, Tuft SA, et al. Ligandin heterogeneity: evidence that the two nonidentical subunits are the monoers of two distinct proteins. Biochim Biophy Acta 1977; 492: 163-175
14. Mannervik B, Alin P, Guthenberg C, et al. Identification of three classes of cytosofic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc Natl Acad Sci USA 1985; 82: 7202-7206
15. Tsushida S, Sato K. Glutathione transferases and cancer. Crit Rev Biochem Mol Biol 1992; 27:337-384
16. Sato K. Glutathione transferases as markers of preneoplasia and neoplasia. Adv Cancer Res 1989;52:205-55
17. Sato K, Kitahara A, Satoh K, et al. The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis. Gann 1984; 75: 199-202
18. Satoh K, Kitahara A, Soma Y, et al. Purification, induction and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc Natl Acad Sci USA 1985; 82:3964-3968
19. Suzuki T, Johnstan PN. Structure and organization of the human alfa class glutathione s-transferase genes and related pseudogenes. Genomics 1993;18:680-6
20. Board PG, Webb GC. Isolation of a cDNA clone and localization of the human glutathione S-transferase 3 genes to chromosome bands 11q3 and 12q13-14. Ann Hum Genet 1989;53:205-13
21. Ross VL, Board PG, Webb GC. Chromosomal mapping of the human mu class glutathione S-transferases to 1p13. Genomics 1993;18:87-91.
22. Tan KL, Webb GC. Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase with overall survival in patients with gastric carcinoma. Proc Am Assoc Cancer Res 1996;37:192
23. Kano T, Sakai M. Structure and expression of a human class pi glutathione S-transferase messenger RNA. Cancer res 1987;47:5626-30
24. Cowell IG, Dixon KH. The structure of the human glutathione S-transferase pi gene. Biochem J 1988;255:
25. Tsuchida S, Sekine Y, Shineha R, et al. Elevation of the placental glutathione Stransferase form (GST-π) in tumor tissues and the levels in sera of patients with cancer. Cancer Res 1989; 49: 5225-5229
26. Di Ilio C, Del Boccio G, Aceto A, et al. Elevation of glutathione transferase activity in human lung tumor. Carcinogenesis (London) 1988; 9:335-340
27. Mannervik B, Castro VM, Danielson UH, et al. Expression of class Pi glutathione transferase in human malignant melanoma cells. Carcinogenesis (London) 1987; 8:1929-1932
28. Shiratori Y, Soma Y, Maruyama H, et al. Immunohistochemical detection of glutathione S-transferase placental form (GST-π) in dysplastic and neoplastic human cervix lesions. Cancer Res 1987- 47: 6806-6809
29. Gutherberg C, ManneMk B. Glutathione transferase n from human placenta is identical or close related to glutathione S-transferase P from erythrocytes. Biochim Biophys Acta 1981; 661:255-260
30. Niitsu Y, Takahashi Y, Saiot T, et al. Serum glutathione S-transferase π as a tumor marker for gastrointestinal malignancies. Cancer 1989- 63:317
31. Hirata S, Tetsuyo 0, Kohama G, et al. Significance of glutathione S-transferase-π as a tumor marker in patients with oral cancer. Cancer 1992; 70:2381-2387
32. Zhang L. The value of placental glutathione S-transferase and r -glutamyl traspeptidase as markers of altered foci during hamster pouch carciongenesis. Carcinogenesis 1994- 15:105-109
33. Zhang L, Yjao Y, Priddy R. Increase in placental glutathione S-transferase in human oral epithelial dysplastic lesions and squamous cell carcinoma. J Oral Pathol Med 1994; 23:75-79
34. Chen YK, Lin LM. Immunohistochemical demonstration of epithelial glutathione S-transferase isoenzymes in normal, benign, malignant human oral mucosa. J Oral Pathol Med 1995; 24:316-321
35. Chen YK and Lin LM. Sequential expression of placental glutathione S-transferase (GST-P) during DMBA-induced hamster buccal pouch squamous cell carcinogenesis. J Oral Pathol Med 1996; 25:388-394
36. Mulder TP. Glutathion S-transferases and glutathione in human head and neck cancer. Carcinogenesis 1995; 16:619-624
37. Henderson CJ, Smith AG et. al. Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases. Proc Natl Acad Sci USA 1998;95(9):5275-80
38. Bongers V, Snow GB et. al. Second primary head and neck squamous cell carcninoma predicted by the glutathione S-transferase expression in healthy tussue in the direct vicinity of the first tumor. Lab Invest 1995;73:503-510
39. Palvelic ZP, Wang X, Gleich L, et al. Overexpression of glutathione S-transferase pi messenger RNA and its relationship to gene amplification in head and neck squamous cell carcinoma. Eur Arch Otorhinolargyngo 1997; 254(suppl. 1): S114-S146
40. Iersel M, Vergagen H, Bladeren PJ. The role of biotransformation in dietary (anti)carcinogenesis. Mutation Research 1999; 259-270
41. Harries LW, Stubbins MJ, Forman D, et al. Identification of genetic polymorphisms at glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis, 1997; 18:641-644
42. Ali-Osman R, Akande O, Antoun G, et al. Molecular cloning, characterization and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. J Biol Chem, 1997; 272:10004-10012
43. Moffat GJ, McLaren AW, Wolf CR. Transcriptional and post-transcriptional mechanisms can regulate cell-specific expression of the human Pi-class glutathione S-transferase gene. Biochem J, 1997; 324:91-95
44. Moffat GJ, McLaren AW, Wolf CR. Involvement of Jun and Fos proteins in regulating transcriptional activation of the human Pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells. J Biol Chem, 1994; 269: 16397-16402
45. Lee WH, Morton RA, Epstein J, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione s-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA, 1994; 91:11733-11737
46. Henderson CJ, Smith AG, Brown JU, et al. Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases. Proc Natl Acad Sci USA, 1998; 95:5275-5280
47. Kuzmich S, Vanderveer LA, Ealsh ES, et al. Increased levels of glutathione S-transferase π transcript as a mechanism of resistance to ethacrynic acid. Biochem J, 1992;281:219-224
48. Palvelic ZP, Wang X, Gleich L, et al. Overexpression of glutathione S-transferase pi messenger RNA and its relationship to gene amplification in head and neck squamous cell carcinoma. Eur Arch Otorhinolargyngo 1997; 254(suppl. 1): S114-S146.
49. Iersel M, Vergagen H, Bladeren PJ. The role of biotransformation in dietary (anti)carcinogenesis. Mutation Research 1999; 259-270
50. Alberts B, Bray Dennis, Lewis J, et al. Molecular biology of the cell, 3rd ed. Garland Publishing, 1994
51. Ali-Osman R, Akande O, Antoun G, et al. Molecular cloning, characterization and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. J Biol Chem, 1997; 272:10004-10012
52. Moffat GJ, McLaren AW, Wolf CR. Transcriptional and post-transcriptional mechanisms can regulate cell-specific expression of the human Pi-class glutathione S-transferase gene. Biochem J, 1997; 324:91-95
53. Moffat GJ, McLaren AW, Wolf CR. Involvement of Jun and Fos proteins in regulating transcriptional activation of the human Pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells. J Biol Chem, 1994; 269: 16397-16402
54. Lee WH, Morton RA, Epstein J, et al. Cytidine methylation of regulatory sequences near the pi-class glutathione s-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci USA, 1994; 91:11733-11737
55. Henderson CJ, Smith AG, Brown JU, et al. Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases. Proc Natl Acad Sci USA, 1998; 95:5275-5280
56. Kuzmich S, Vanderveer LA, Ealsh ES, et al. Increased levels of glutathione S-transferase π transcript as a mechanism of resistance to ethacrynic acid. Biochem J, 1992;281:219-224
57. Ookawa K, Nakamo H, Kakizaki I et al. Identification of Glutathione S-transferase p-1 as the class Pi form dominantly expressed in mouse hepatic adenomas. Jpn J Cancer Res 1998; 89:641-648
58. Morrow CS, Chiu Jason, Cowan KH. Posttranscriptional control of glutathione S-transferase πgene expression in human breast cancer cells. J Biol Chem, 1992; 267: 10544-10550
59. Boyle O, Macfarlane GJ, Maisonneuve P, et al. Epidemiology of mouth cancer in 1989: a review. J R Soc Med, 1990; 83:724-730
60. 衛生署,1999. http://www.doh.gov.tw/new/sp/88kill/TMCAN88.htm
61. Salley. Experimental carcinogenesis in the cheek pouch of the syrian hamster. J Dental Res 1954; 33:253-262
62. Morris. Factors influencing experimental carcinogenesis in the hamster cheek pouch. J Dental Res 1961; 40:3-15
63. Macaughan FM, Brown AL, Harrison DJ. The effect of inhibition of glutathione S-transferase P in the growth of the Jukat human T cell line. J Pathol 1994; 172:357-62.
64. Chen YK, Lin LM. glutathione S-transferase isoenzyme expression in polycyclic aromatic hydrocarbon-induced hamster buccal pouch mucsa. Oral Oncol 1998; 34:180-5.
65. Lin CC, Chen YK, Lin LM. Placental glutathione S-transferase isoenzyme expression during promotion of two-stage hamster cheek-pouch carcinogenesis. Arch Oral Biol 1999; 44:525-9.
66. Shimizu K, Toriyama F, Yoshida H. The expression of placental-type glutathione S-transferase (GST-p) in human cutaneous squamous cell carcinoma and normal human skin. Virchows Archiv 1995; 425: 589-92.
67. Hatayama, I, Satoh K, Sato K. A cDNA sequence coding a class pi glutathione S-transferase of mouse. Nucleic Acid Res 1990, 18: 4606