摘要
目的：检测多肽：N-乙酰氨基半乳糖转移酶家族的mRNA表达，并初步了解该酶在肿瘤的发生、发展等过程中的作用及辐射与中药的影响。方法和内容：采用RT-PCR方法，检测(1)多肽：N-乙酰氨基半乳糖转移酶1、2、9(ppGalNAcT1、T2、T9)在不同肿瘤细胞中的mRNA表达；(2)肿瘤细胞经γ射线照射后多肽：N-乙酰氨基半乳糖转移酶-2(ppGalNAcT-2)的mRNA表达变化及几种药物对肿瘤细胞受照射后多肽：N-乙酰氨基半乳糖转移酶-2mRNA表达的影响。结果：通过不同肿瘤细胞ppGalNAcT-2mRNA表达差异比较，脑神经胶质瘤细胞株SHG44中的表达甚低，而胃癌细胞株SGC-7901、白血病细胞株K562很高，其次为卵巢癌细胞(CD40+)，而多发性骨髓瘤细胞株表达较少。其大小次序为白血病细胞株＞胃癌细胞株＞卵巢癌细胞株＞多发性骨髓瘤细胞株＞NIH3T3成纤维细胞株和脑神经胶质瘤细胞株，这结果可能与ppGalNAcT-2为合成O-糖链的关键酶且O-糖链主要存在于粘蛋白相符。另外ppGalNAcT-1在各种细胞株中呈低表达，ppGalNAcT-9则在卵巢癌细胞株和白血病细胞株中几乎没有表达。肿瘤细胞经γ射线照射后ppGalNAcT-2的表达有所降低，且与照射剂量呈负相关性；加入不同的药物后，受照的肿瘤细胞中ppGalNAcT-2的表达增加，提示这些药物对辐照具有生物防护作用。结论：以上实验结果说明ppGalNAcT-1、9在一些肿瘤细胞中的表达水平普遍较低，而ppGalNAcT-2在不同肿瘤细胞中的表达均较高且有差异，由内胚层来源的细胞如胃癌细胞株中ppGalNAcT-2的表达较高，外胚层来源的细胞如神经胶质瘤细胞株中ppGalNAcT-2表达量较低；K562细胞经照射后ppGalNAcT-2的表达量减
多肚:N一乙酞氨基半乳糖转移酶家族(PPG洲AcTs)在不同肿瘤细胞中mRNA表达谱研究及辐射与中药影响的初步研究
中文摘要
少，说明辐照可以使PPGalNAcT一2的表达受到抑制，加入某些药物后PPGalNAcT-2
的表达量又有所回升，而我们同时进行的基质金属蛋白酶(MMPS)的表达谱研究
结果正好相反，至少证明PPGalNAcT一2与肿瘤的一些生物学特性有关，如肿瘤的浸
润与转移等。通过这一开创性的工作，初步为寻找GalNAcT一2及MMPS的特异性
抑制剂或激活剂打下基础，也为临床上肿瘤的诊断、治疗等提供了实验依据。
In order to know the function of the polypeptide: N-acetylgalactosaminyltransferase-
Ts(ppGalNAC-Ts) in the course of tumour's generating and developing , we examined the
enzyme's expression at mRNA level with RT-PCR method. Methods: The first part is to
examine the expression of ppGalNACT-1, 2, 9 in different rumour cells. The second
part is to find out the variation of ppGalNAcT2's expression in tumour cells exposed to
Y -rays and the effect of several remedies added to the culture medium. Results: 1) In
SHG44 neuroglia cancer cells, the expression of ppGalNAcT-2 is extremely low, while in
SGC-7901 gastric carcinoma cells and K562 Leukaemia cells, it's very high, the lesser
being the ovary cancer cells(CD+); but in multiple medulla carcinoma cells ,the
expression of ppGalNAcT-2 is rare. The order is: K562 Leukaemia cells > SGC-7901
gastric carcinoma cells > ovary cancer cells(CD+) > multiple medulla carcinoma cells >
NIH3T3 fibroblast cells and SHG44 neuroglia cancer cells, this result probably corresponds with the viewpoint that ppGalNAc-T2 is the key-enzyme in biosynthesis of O-glycan and O-glycan mostly consists in mucin. Aside this, the expression of ppGalNAc Tl appears low, and ppGalNAc T9 is barely expressed in ovary cancer cells and in Leukaemia cells . 2) After irradiated by -rays, the expression of ppGalNAcT-2 in tumour cells was somewhat reduced, and it was negatively reletive to the dosage; The
expression of ppGalNAcT-2 was somewhat enhanced in the irradiated tumour cells after
several remedies were added to the culture medium, this phenomenon hinted us these remedies had the biologic protection at cell levels. Conclusion: These results hereinbefore evinced that the standards of expression of ppGalNAcTl and T9 were lower, but that of ppGalNAcT-2 was higher and differed in different tumour cells; the expression of ppGalNAcT-2 was all high in cells that originated from endoblast, such as gastric carcinoma cells; but it was low in cells which originated from ectoderm, such as neuroglia cancer cells. After irradiation, the expression of ppGalNAcT2 was decreased in K562 cells, from which we concluded that irradiation could repress the expression of ppGalNAcT2, but some remedies could make the expression of ppGalNAcT2 somewhat promoted again., and this situation was reverse to that of MMPs. Through the experiment, we primarily confirmed that ppGalNAcT2 is relative to some biological specificity of cancer cells, such as tumour's invasiveness and migration. This initial work
is the foundation in seeking particular depressor or activator of ppGalNAcTs and MMPs, and it will provide the experimental evidence to cancer diagnosis and clinical therapy.
引文
[1] Kobata A, Eur. J.Biochem. 1992, 709,483-501
[2] MASANORI BABA. et al. ANTIMICROBIAL. AGENTS AND CHEMOTHERAPY.1988,32:1742-1745
[3] 张遒哲．糖基转移酶与肿瘤标志．生命的化学．1996，16(1)：35
[4] Beyer T A,Sadler J E, et al. Advance Enzymol.1981, 52-53
[5] 杨清香，曹军卫等．糖基转移酶和去糖基转移酶。氨基酸和生物资源．1997，19(2) ：38-43
[6] Hagan F K,Ten Hagen K G,Tabak L A. Polypeptide N-Acetylgalactosaminyltransferases. In Taniguchi N, Honde K,Fukuda M. Handbooks of glycosyltransferase and related genes.Tokyo, Springer Chapter 22, p167-173
[7] Hideki Ohta et al. Clinica Acta,1993:214:83
[8] 王克夷．糖基转移酶的研究进展．生物化学与生物物理进展．1994，21(1)：9
[9] Shimodaira K, Nakayama J, Nakamura N, Hasebe O, Katsuyama T, Fukuda M.Carcinoma-associated expression of core 2 beta-1,6-N-acetylglucosaminyltransferase gene in human colorectal cancer:role of O-glycans in tumor progression. Cancer Res,1997, 57:5201-5206
[10] 陈惠黎．糖蛋白糖链与疾病．糖复合物的结构和功能．上海医科大学出版社，1997，104-34
[11] Blithe D L. Biological function of oligosaccharide on glycoproteins. Trends Glycosci Glycotechnol,1993,5:81-98
[12] Rudd P M, Dwek R A. Glycosylation:Heterogeneity and 3D structure of proteins in Farman G D, edi. Critical review in biochemistry and molecular biology.1997(1),1-100
[13] Rosen J D, Bertozzi C R. The selectins and their ligands. Curr Opin Cell Biol.1994,6:663-673
[14] Brockhausen I. Pathway of O-glycan biosynthesis in cancer cells. Biochim Biophys Acta.1999,1473-67-95
[15] Schachter H,Brockhausen I. The biosynthesis of serine(threonine)-N-acetylgalactosamine-linked carbohydrate moieties. In: Allen H J, Kisailus E C(eds).Glycoconyugate, Composition, Struture and Function. New York,Basel, Hongkong:Marcel Dekker Inc,1992, pp263-331
[16] White T, Bennett EP, Takio K, Sorensen T, Bonding N, Clausen H. Purification and cDNA cloning of a human UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem 1995,270:24156-24165
[17] Yan Zhang,Hiroko Iwasaki, Han Wang, Takashi Kudo, Timothy B.Cloning and Characterization of a New Human UDP-N-Acetyl-α-D-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase,Designated pp-GalNAc-T13,That Is Specifically Expressed in Neurons and Synthesizes GalNAc α-Seriine/Threonine Antigen.J Biol Chem 2003, 278:573-584
[18] Wandalls HH, Hassan H, Mirgorodskaya E, Kristensen AK, Roepstorff P, Bennett EP,Nielsen PA, Hollingsworth MA, Burchell J,Taylor-Papadimitriou J, Clausen H.Substrate Specificities of three members of the human UDP-N-Acetyl-α-D-
galactosamine:Polypeptide N-acetyl-galactosaminyltransferase family, GalNAc-T1,-T2, and T3. J Biol Chem 1997, 272:23510-23514
[19] Brockhausen I, Yang J, Lehotay M, Ogata S, Itzkowitz S. Pathway of mucin O-glycosylation in normal and malignant rat colonic epithelial cells reveal a mechanism for cancer-associated Sialyl-Tn antigen expression. Biol Chem.2001,382:219-232
[20] 顾晓松，谭湘林，丁斐．分子生物学理论与技术．第一版，北京：北京科学技术出版社，2002，118-120
[21] J.萨姆布鲁克，E.F弗里特，T.曼尼阿蒂斯．分子克隆实验指南．第二版，北京 2001，
[22] Kudo T, Ikehara Y, Togayachi A, Morozumi K, Watanabe M, Nakamura M, Nishihara S, Narimatsu H. Up-regulation of a set glycosyltransferase genes in human colorectal cancer. Lab Invest,1998,78(7):797-811
[23] Petretti T, Schulze B, Schlag PM, Kemmner W. Altered mRNA expression of glycosyitransferases in human gassstric carcinomas. Biochim Biophys Acta,1999,1428:209-218
[24] Ten Hagen KG, Bedi GS,Tetaer D, Kingsley PD, Hagen FK, Balys MM, Beres TM,Degand P, Tabak LA. Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9. J Biol Chem 2001,276:17395-17404
[25] Fred K. Hagen, Keith Nehrke. cDNA Cloning and Expression of a Family of UDP-N-acetyl-D-galactosamine:polypeptide N-Acetylgalactosaminyltransferase sequence Homologs from Caenorhabditis elegans.J Biol Chem 1998, 273:8268-8277
[26] Qian LW, Mizumoto K, Urashima T, Nagai N, Nakajima M.Radiation-induced
increase in invasive potential of human pancreatic cancer cells and its blockade by a matrix metalloproteinase inhibitor. Clin Cancer Res, 2002, 8(4):1223-1227
[27] Wild-Bode C, Weller M, Rimner A, Dichgans J, Wick W. Subletal irradiation promotes migration and invasiveness of glioma cells;implications for radiotherapy of human giloblatoma. Cancer Res, 2001,61(6):2744-2750
[28] Hanisch F-G, Muller S, Hassan H, Clausen H,Zachara N, Gooley AA, Paulsen H,Alving K, Peter-Katalinic J.Dynamic epigenetic regulation of O-glycosylation by UDP-N-acetyl-galactosamine:polypeptide N-acetylgalactosaminyltransferase.Site specific glycosylation of MUC1 repeat peptide influences the substrate qualitys at adjacent or distant Ser/Thr positions. J Biol Chem 1999,274:9946-9954