染色体不稳定与口腔黏膜癌变
|
摘要
基因不稳定是人类肿瘤发生发展的中心环节,染色体不稳定(chromosome instability, CIN)是见于人类大多数恶性肿瘤中的一种基因不稳定形式,包括口腔鳞状细胞癌(Oral squamous cell carcinomas, OSCC)。非整倍体作为CIN的特征性表现形式可以出现在相当比例的OSCC及癌前病损,并与细胞的恶性转化进程有关,已经被成功地应用于预测口腔癌前病损的癌变进程,但其形成机制仍不清楚。近年来研究显示:细胞有丝分裂检查点(mitotic checkpoint)功能缺陷和中心体异常可能是肿瘤细胞CIN(非整倍体)形成的重要原因,但这一推测还没有在包括OSCC在内的人类大量实体瘤中得到全面证实。
因此,本研究旨在探讨口腔鳞状细胞癌细胞染色体不稳定的潜在机制及其相关调控,了解其与口腔黏膜癌变的关系及其在口腔癌前损害预后判断中的作用和意义。
研究材料与方法如下:(1)以7株体外培养的非整倍体口腔鳞状细胞癌(OSCC)细胞为研究对象,采用免疫荧光染色(IF)、激光共聚焦显微镜观察及荧光活化细胞分类(FACS)等方法,观察非整倍体OSCC细
Genetic instability is currently considered to be central to the development of human cancer. Chromosome instability (CIN) exemplified by aneuploidy was seen in the early stage of many tumors, including oral squamous cell carcinomas (OSCC). It is currently considered that the functional defects of mitotic checkpoint and centrosome abnormalities may play important roles in the development of aneuploidy in tumor cells. This study is an attempt to elucidate the possible underlying mechanisms of CIN in oral squamous cell carcinoma and their possible roles in oral carcinogenesis.In this study, (1) Seven aneuploidy OSCC cell lines were investigated for the functional status of mitotic checkpoint by using FACA to investigate the percentage of cells arrested at G2/M after incubated in nocodazole, and the status of centrosomes in these cell lines were investigated by using double Immunofluorescence staining with antibodies against gamma-tubulin and pericentrin. (2) Formalin-fixed, paraffin-embedded tissues of 12 cases of normal oral epithelium, 22 case of dysplasia with different degree epithelium dysplasia and 32 cases of OSCC with different differentiation were investigated for centrosome status by using double immunofluorescence staining with antibodies against gamma-tubulin and cytokeratin. The differences and the change trend of centrosome status in these groups were statistically analyzed by
SPSS 10.0. (3) The same OSCC cell lines were investigated for the expression of Cycling Cdk2, p27/KipK p21/Wafl by western blot and the correlation between the expression of the proteins and centrosome amplification were analyzed.The results showed: (1) All aneuploidy OSCC cell lines exhibited high percentages of cells arrested at G2/M phase of the cell cycle after 18 hours incubation in nocodazole indicating an intact mitotic checkpoint, while centrosome abnormalities, numerical amplification such as more than two centrosomes per cell as well as morphological defects such as clusters and/or huge in size of centrosomes, were observed in a fraction of OSCC cells (0.4%-18.8%) in all OSCC cell lines. (2) Normal oral epithelium showed normal centrosomes in epithelium cells, while 16 out of 22 cases (72.73%) of dysplasia (DYS) and 27 out of 32 cases (84.38%) of OSCC showed the evidence of centrosome amplification and morphological abnormalities characterized by huge size, clump or supernumerary centrosomes in a small fraction of epithelium or tumor cells. The percentage of cells with abnormal centrosomes increased gradually from mild-dysplasia epithelium to poorly differentiated OSCC, which positively correlated with the histological\cytologic grade of oral precancerous lesions and OSCC (r=0.955, P<0.01) . The percentage of cells with abnormal centrosomes in OSCC is higher than that in dysplasia (t= 5.26, PO.01), while there were no significantly differences between the dysplasia with OSCC outcome and that without OSCC outcomes. (3) The degree of centrosome amplification positively correlated with the ratios of protein expression of CyclinE/p21wafl (r =0.710, p<0.05) and CyclinE/p27kipI (r=0.848, pO.Ol )in all OSCC cell lines
investigated.In all, it is suggested that (1) The CIN phenotype (aneuploidy) in OSCC cells may not attribute to the defects of mitotic checkpoint, while the abnormalities of centrosome is likely a contributing factor towards CIN in OSCC cells. (2) Centrosome amplification was an early event and that might play a role in the establishment and perhaps the progression of OSCC. There might be some direct mechanistic relationship between centrosome defects and the cellular morphological phenotype characteristic of dysplasia and OSCC, Centrosome amplification could be served as an alternative diagnostic indicator of dysplasia but not a effective predictive marker for the progression of oral precancerous lesions. (3) Unbalance of the expression of CyclinEN Cdl^ p27/Kipl, p21/Wafl might be responsible for centrosome amplification in OSCC cells and the increase of the ratios of CyclinE/p27kipl and/or CyclinE/p21wafl was likely to be one of the reasons for centrosome amplification in OSCC cells.
因此,本研究旨在探讨口腔鳞状细胞癌细胞染色体不稳定的潜在机制及其相关调控,了解其与口腔黏膜癌变的关系及其在口腔癌前损害预后判断中的作用和意义。
研究材料与方法如下:(1)以7株体外培养的非整倍体口腔鳞状细胞癌(OSCC)细胞为研究对象,采用免疫荧光染色(IF)、激光共聚焦显微镜观察及荧光活化细胞分类(FACS)等方法,观察非整倍体OSCC细
Genetic instability is currently considered to be central to the development of human cancer. Chromosome instability (CIN) exemplified by aneuploidy was seen in the early stage of many tumors, including oral squamous cell carcinomas (OSCC). It is currently considered that the functional defects of mitotic checkpoint and centrosome abnormalities may play important roles in the development of aneuploidy in tumor cells. This study is an attempt to elucidate the possible underlying mechanisms of CIN in oral squamous cell carcinoma and their possible roles in oral carcinogenesis.In this study, (1) Seven aneuploidy OSCC cell lines were investigated for the functional status of mitotic checkpoint by using FACA to investigate the percentage of cells arrested at G2/M after incubated in nocodazole, and the status of centrosomes in these cell lines were investigated by using double Immunofluorescence staining with antibodies against gamma-tubulin and pericentrin. (2) Formalin-fixed, paraffin-embedded tissues of 12 cases of normal oral epithelium, 22 case of dysplasia with different degree epithelium dysplasia and 32 cases of OSCC with different differentiation were investigated for centrosome status by using double immunofluorescence staining with antibodies against gamma-tubulin and cytokeratin. The differences and the change trend of centrosome status in these groups were statistically analyzed by
SPSS 10.0. (3) The same OSCC cell lines were investigated for the expression of Cycling Cdk2, p27/KipK p21/Wafl by western blot and the correlation between the expression of the proteins and centrosome amplification were analyzed.The results showed: (1) All aneuploidy OSCC cell lines exhibited high percentages of cells arrested at G2/M phase of the cell cycle after 18 hours incubation in nocodazole indicating an intact mitotic checkpoint, while centrosome abnormalities, numerical amplification such as more than two centrosomes per cell as well as morphological defects such as clusters and/or huge in size of centrosomes, were observed in a fraction of OSCC cells (0.4%-18.8%) in all OSCC cell lines. (2) Normal oral epithelium showed normal centrosomes in epithelium cells, while 16 out of 22 cases (72.73%) of dysplasia (DYS) and 27 out of 32 cases (84.38%) of OSCC showed the evidence of centrosome amplification and morphological abnormalities characterized by huge size, clump or supernumerary centrosomes in a small fraction of epithelium or tumor cells. The percentage of cells with abnormal centrosomes increased gradually from mild-dysplasia epithelium to poorly differentiated OSCC, which positively correlated with the histological\cytologic grade of oral precancerous lesions and OSCC (r=0.955, P<0.01) . The percentage of cells with abnormal centrosomes in OSCC is higher than that in dysplasia (t= 5.26, PO.01), while there were no significantly differences between the dysplasia with OSCC outcome and that without OSCC outcomes. (3) The degree of centrosome amplification positively correlated with the ratios of protein expression of CyclinE/p21wafl (r =0.710, p<0.05) and CyclinE/p27kipI (r=0.848, pO.Ol )in all OSCC cell lines
investigated.In all, it is suggested that (1) The CIN phenotype (aneuploidy) in OSCC cells may not attribute to the defects of mitotic checkpoint, while the abnormalities of centrosome is likely a contributing factor towards CIN in OSCC cells. (2) Centrosome amplification was an early event and that might play a role in the establishment and perhaps the progression of OSCC. There might be some direct mechanistic relationship between centrosome defects and the cellular morphological phenotype characteristic of dysplasia and OSCC, Centrosome amplification could be served as an alternative diagnostic indicator of dysplasia but not a effective predictive marker for the progression of oral precancerous lesions. (3) Unbalance of the expression of CyclinEN Cdl^ p27/Kipl, p21/Wafl might be responsible for centrosome amplification in OSCC cells and the increase of the ratios of CyclinE/p27kipl and/or CyclinE/p21wafl was likely to be one of the reasons for centrosome amplification in OSCC cells.
引文
1. Silverman S Jr, Gorsky M. Epidemiologic and demographic update in oral cancer: California and national data--1973 to 1985. J Am Dent Assoc. 1990 May;120(5):495-9.
2. Li M. leukoplakia: molecular umderstanding of pre-malignant lesions and implications for clinical management. Molecular Medicine Today. 1997; 3(6): 442-448
3. Warnakulasuriya S. Lack of molecular markers to predict malignant potential of oral precancer. J Pathol. 2000 Mar;190(4):407-9.
4. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998 Dec 17;396(6712):643-9
5. Reith A, Sudbo J. Impact ofgenomic instability in risk assessment and chemoprevention of oral premalignancies. Int J Cancer 2002 Sep 20; 101(3):205-9
6. Sen S. Aneuploidy and cancer. Curt Opin Oncol 2000;12:82-8
7. Scully C, Field JK, Tanzawa H., Genetic aberrations in oral or head and neck squamous cell carcinoma 2: chromosomal aberrations[J]. Oral Oncology 2000,Feb(36):311-327
8. Jon Sudbo, Wanja K, Bjorn R, et al. DNA content as a prognostic marker in patients with oral leukoplakia. N Engl J Med, 2001 Apr; 344(17):1270-1278
9. Hong Ai, Jose EB, Zhaoxing P, et al. Identification of individuals at high risk for head and neck carcinogenesis using chromosome aneuploidy detected by fluorescence in situ hybridization. Mutation Research 1999, 439:223-232
10. Bharadwaj R, Yu H. The spindle checkpoint, aneuploidy, and cancer. Oncogene. 2004 Mar 15;23(11):2016-27.
11. Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, Doxsey SJ. Centrosome defects and genetic instability in malignant tumors. Cancer Res 1998 Sep 1 ;58(17):3974-85
12. Yoon DS, Wersto RP, Zhou W, Chrest FJ, Garrett ES, Kwon TK, Gabrielson E. Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer. Am J Pathol 2002 Aug;161(2):391-7
13. Lingle WL, Barrett SL, Negron VC, D'Assoro AB, Boeneman K, Liu W, Whitehead CM, Reynolds C, Salisbury JL. Centrosome amplification drives chromosomal instability in breast tumor development. Proc NatlAcad Sci U S A 2002 Feb 19;99(4):1978-83
14. Reider CL, Cole RW, Khodjakov A, Sluder G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. Journal of Cellular Biology 1995(130):94I-948
15. Reider CL, Schultz A, Cole R, Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. Journal of Cellular Biology 1994(127): 1301-1310
16. Paulovich,A.G., Toczyski,D.P., & Hartwell,L.H. When checkpoint fail. Cell 88, 315-321(1997).
17. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature. 1998 Mar 19;392(6673):300-3.
18. Takahashi T, Haruki N, Nomoto S, Masuda A, Saji S, Osada H, Takahashi T. Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers. Oncogene. 1999 Jul 29;18(30):4295-300.
19. Doxsey S. The centrosome—a tiny organelle with big potential. Nat Genet. 1998 Oct;20(2):104-6.
20. Lingle W L, Lute W H, Ingle J N, Maihle N J and Salisbury J L. Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity[J]. Proc. Natl. Acad. Sci. USA, 1998, 95(6):2950-2955
21. Gustafson LM, Gleich LL, Fukasawa K, Chadwell J, Miller MA, Stambrook PJ, Gluckman JL. Centrosome hyperamplification in head and neck squamous cell carcinoma: a potential phenotypic marker of tumor aggressiveness. Laryngoscope 2000 Nov; 110(11): 1798-801
22. Fry AM, Mayor T, Nigg EA. Regulating centrosomes by protein phosphorylation. Curr Top Dev Biol 2000;49:291-312(Review)
23. Tighe A, Johnson VL, Albertella M, Taylor SS. Aneuploid colon cancer cells have a robust spindle checkpoint. EMBO Rep 2001 Jul;2(7):609-14
24. Stephen J. Elledge.Cell Cycle Checkpoints: Preventing an Identity Crisis. Science 1996(274),1664-1672 ).
25. Nakagawa H, Yokozaki H, Oue N, Sugiyama M, Ishikawa T, Tahara E, Yasui W. No mutations of the Bubl gene in human gastric and oral cancer cell lines. Oncol Rep. 2002 Nov-Dec;9(6):1229-32.
26. Hinchcliffe EH, Chuan Li, Thompson EA, Mailer JL, Sluder G. Requirement of Cdk2-CyclinE activity for repeated centrosome reproduction in xenopus egg extracts. SCIENCE 1999 Feb, 283(5): 851-854
27. Hinchcliffe EH, Sluder G. "It Takes Two to Tango": understanding how centrosome duplication is regulated throughout the cell cycle. Genes & Dev. 2001, 15:1167-1181
28. Schuyler SC, Pellman D. Search, capture and signal: games microtubules and centrosomes play. Journal of Cell Science 114: 247-255
29. Ghadimi BM, Sackett DL, Difilippantonio MJ, Schrock E, Neumann T, Jauho A, Auer G, Ried T. Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. Genes Chromosomes Cancer 2000 Feb; 27(2): 183-90
30. Gisselsson D, Jonson T, Yu C, Martins C, Mandahl N, Wiegant J, Jin Y, Mertens F, Jin C. Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres. Br J Cancer 2002 Jul 15;87(2):202-7
31. Carroll PE, Okuda M, Horn HF, Biddinger P, Stambrook PJ, Gleich LL, Li YQ, Tarapore P, Fukasawa K. Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene 1999 Mar 18; 18(11): 1935-44
32. Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Centrosome Defects Can Account for Cellular and Genetic Changes That Characterize Prostate Cancer Progression. Cancer Res. 2001;61::2212-2219
33. Haruki N, Harano T, Masuda A, Kiyono T, Takahashi T, Tatematsu Y, Shimizu S, Mitsudomi T, Konishi H, Osada H, Fujii Y, Takahashi T. Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation. Am J Pathol 2001 Oct;159(4): 1345-52
34. Pinborg JJ, Reichartt PA, Smith CJ. Histological typing of cancer and precancer of the oral mucsa 2nd[M]. Edication Springer. World Health Organization, 1997:35-38
35. Boveri T.(1914). Zur Frage der Entstehung maligmer tumoren . Jena: Fischer Verlag (English translation by Boveri M. (1929). The Origin of Malignant Tumors. Baltimore: Waverly Press
36. Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Abnormal centrosome amplification in the absence of p53. Science 1996 Mar 22;271 (5256): 1744-7
37. Wang XJ, Greenhalgh DA, Jiang A, He D, Zhong L, Medina D, Brinkley BR, Roop DR. Expression of a p53 mutant in the epidermis of transgenic mice accelerates chemical carcinogenesis. Oncogene 1998 Jul 9; 17(1):35-457
38. Sato N, Mizumoto K, Nakamura M, Maehara N, Minamishima YA, Nishio S, Nagai E, Tanaka M. Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells. Cancer Genet Cytogenet 2001 Apt 1 ; 126(1): 13-9
39.蔡扬,李秉琦.中心体异常、染色体不稳定和癌发生的相关性.临床口腔医学杂志,2003,19(9):571—573.
40. Tarapore P, Horn HF, Tokuyama Y, Fukasawa K. Direct regulation of the centrosome duplication cycle by the p53-p21Waf1/Cip 1 pathway. Oncogene 2001 May 31 ;20(25):3173-84
41. Morris VB, Brammall J, Noble J, Reddel R. p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study. Exp Cell Res 2000 Apt 10;256(1):122-30
42. Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Nail Acad Sci U S A 1998 Oct 27;95(22):12983-8
43. Mussman JG, Horn HF, Carroll PE, Okuda M, Tarapore P, Donehower LA, Fukasawa K. Synergistic induction of centrosome hyperamplification by loss of p53 and cyclin E overexpression. Oncogene 2000 Mar 23; 19(13): 1635-46
44. Andrew W. Murray. CELL CYCLE: Centrioles at the Checkpoint. Science 2001 Feb 23 ;291 (5508): 1499-502
45. Jonathon Pines. Four-dimensional control of the cell cycle. Nature cell biology. july 1999 volume 1 issue 3 pp E73 - E79 (Review)
46. Lacey KR, Jackson PK, Sterans T. Cyclin-dependant kinase control of centrosome duplication. Proc Natl Acad Sci U S A 1999 March27;96(22):2817-2822
47. Tokuyama Y, Horn HF, Kawamura K, Tarapore P, Fukasawa K. Specific phosphorylation of nucleophosomin on Thrl99 by Cyclin-dependent kinase-2-CyclinE and its role in centrosome deplication. The Journal of Biological Chemistry, 2001, 276(24):21529-21537
48. Matsumoto Y, Hayashi K, Nishida E. Cyclin-dependent kinase2(Cdk2) is required for centrosome duplication in mammalian cells. Curr. Biol. 1999; 9:429-432
49. Fukasawa K. Introduction[J]. Oncogene, (2002) 21,6140-6145
50. Khodjakov A, Rieder CL. Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression. J Cell Biol 2001 Apr 2; 153(1): 237-42
51. Balczon R, Bao L, Zimmer WE, Brown K, Zinkowski RP, Brinkley BR. Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells. J Cell Biol 1995 Jul; 130(1): 105-15
52. Robert JM. Evolving ideas about Cyclins.Cell 1999 July; 98(1): 129-132
2. Li M. leukoplakia: molecular umderstanding of pre-malignant lesions and implications for clinical management. Molecular Medicine Today. 1997; 3(6): 442-448
3. Warnakulasuriya S. Lack of molecular markers to predict malignant potential of oral precancer. J Pathol. 2000 Mar;190(4):407-9.
4. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998 Dec 17;396(6712):643-9
5. Reith A, Sudbo J. Impact ofgenomic instability in risk assessment and chemoprevention of oral premalignancies. Int J Cancer 2002 Sep 20; 101(3):205-9
6. Sen S. Aneuploidy and cancer. Curt Opin Oncol 2000;12:82-8
7. Scully C, Field JK, Tanzawa H., Genetic aberrations in oral or head and neck squamous cell carcinoma 2: chromosomal aberrations[J]. Oral Oncology 2000,Feb(36):311-327
8. Jon Sudbo, Wanja K, Bjorn R, et al. DNA content as a prognostic marker in patients with oral leukoplakia. N Engl J Med, 2001 Apr; 344(17):1270-1278
9. Hong Ai, Jose EB, Zhaoxing P, et al. Identification of individuals at high risk for head and neck carcinogenesis using chromosome aneuploidy detected by fluorescence in situ hybridization. Mutation Research 1999, 439:223-232
10. Bharadwaj R, Yu H. The spindle checkpoint, aneuploidy, and cancer. Oncogene. 2004 Mar 15;23(11):2016-27.
11. Pihan GA, Purohit A, Wallace J, Knecht H, Woda B, Quesenberry P, Doxsey SJ. Centrosome defects and genetic instability in malignant tumors. Cancer Res 1998 Sep 1 ;58(17):3974-85
12. Yoon DS, Wersto RP, Zhou W, Chrest FJ, Garrett ES, Kwon TK, Gabrielson E. Variable levels of chromosomal instability and mitotic spindle checkpoint defects in breast cancer. Am J Pathol 2002 Aug;161(2):391-7
13. Lingle WL, Barrett SL, Negron VC, D'Assoro AB, Boeneman K, Liu W, Whitehead CM, Reynolds C, Salisbury JL. Centrosome amplification drives chromosomal instability in breast tumor development. Proc NatlAcad Sci U S A 2002 Feb 19;99(4):1978-83
14. Reider CL, Cole RW, Khodjakov A, Sluder G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. Journal of Cellular Biology 1995(130):94I-948
15. Reider CL, Schultz A, Cole R, Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. Journal of Cellular Biology 1994(127): 1301-1310
16. Paulovich,A.G., Toczyski,D.P., & Hartwell,L.H. When checkpoint fail. Cell 88, 315-321(1997).
17. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature. 1998 Mar 19;392(6673):300-3.
18. Takahashi T, Haruki N, Nomoto S, Masuda A, Saji S, Osada H, Takahashi T. Identification of frequent impairment of the mitotic checkpoint and molecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in human lung cancers. Oncogene. 1999 Jul 29;18(30):4295-300.
19. Doxsey S. The centrosome—a tiny organelle with big potential. Nat Genet. 1998 Oct;20(2):104-6.
20. Lingle W L, Lute W H, Ingle J N, Maihle N J and Salisbury J L. Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity[J]. Proc. Natl. Acad. Sci. USA, 1998, 95(6):2950-2955
21. Gustafson LM, Gleich LL, Fukasawa K, Chadwell J, Miller MA, Stambrook PJ, Gluckman JL. Centrosome hyperamplification in head and neck squamous cell carcinoma: a potential phenotypic marker of tumor aggressiveness. Laryngoscope 2000 Nov; 110(11): 1798-801
22. Fry AM, Mayor T, Nigg EA. Regulating centrosomes by protein phosphorylation. Curr Top Dev Biol 2000;49:291-312(Review)
23. Tighe A, Johnson VL, Albertella M, Taylor SS. Aneuploid colon cancer cells have a robust spindle checkpoint. EMBO Rep 2001 Jul;2(7):609-14
24. Stephen J. Elledge.Cell Cycle Checkpoints: Preventing an Identity Crisis. Science 1996(274),1664-1672 ).
25. Nakagawa H, Yokozaki H, Oue N, Sugiyama M, Ishikawa T, Tahara E, Yasui W. No mutations of the Bubl gene in human gastric and oral cancer cell lines. Oncol Rep. 2002 Nov-Dec;9(6):1229-32.
26. Hinchcliffe EH, Chuan Li, Thompson EA, Mailer JL, Sluder G. Requirement of Cdk2-CyclinE activity for repeated centrosome reproduction in xenopus egg extracts. SCIENCE 1999 Feb, 283(5): 851-854
27. Hinchcliffe EH, Sluder G. "It Takes Two to Tango": understanding how centrosome duplication is regulated throughout the cell cycle. Genes & Dev. 2001, 15:1167-1181
28. Schuyler SC, Pellman D. Search, capture and signal: games microtubules and centrosomes play. Journal of Cell Science 114: 247-255
29. Ghadimi BM, Sackett DL, Difilippantonio MJ, Schrock E, Neumann T, Jauho A, Auer G, Ried T. Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. Genes Chromosomes Cancer 2000 Feb; 27(2): 183-90
30. Gisselsson D, Jonson T, Yu C, Martins C, Mandahl N, Wiegant J, Jin Y, Mertens F, Jin C. Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and neck tumours with dysfunctional telomeres. Br J Cancer 2002 Jul 15;87(2):202-7
31. Carroll PE, Okuda M, Horn HF, Biddinger P, Stambrook PJ, Gleich LL, Li YQ, Tarapore P, Fukasawa K. Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression. Oncogene 1999 Mar 18; 18(11): 1935-44
32. Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Centrosome Defects Can Account for Cellular and Genetic Changes That Characterize Prostate Cancer Progression. Cancer Res. 2001;61::2212-2219
33. Haruki N, Harano T, Masuda A, Kiyono T, Takahashi T, Tatematsu Y, Shimizu S, Mitsudomi T, Konishi H, Osada H, Fujii Y, Takahashi T. Persistent increase in chromosome instability in lung cancer: possible indirect involvement of p53 inactivation. Am J Pathol 2001 Oct;159(4): 1345-52
34. Pinborg JJ, Reichartt PA, Smith CJ. Histological typing of cancer and precancer of the oral mucsa 2nd[M]. Edication Springer. World Health Organization, 1997:35-38
35. Boveri T.(1914). Zur Frage der Entstehung maligmer tumoren . Jena: Fischer Verlag (English translation by Boveri M. (1929). The Origin of Malignant Tumors. Baltimore: Waverly Press
36. Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Abnormal centrosome amplification in the absence of p53. Science 1996 Mar 22;271 (5256): 1744-7
37. Wang XJ, Greenhalgh DA, Jiang A, He D, Zhong L, Medina D, Brinkley BR, Roop DR. Expression of a p53 mutant in the epidermis of transgenic mice accelerates chemical carcinogenesis. Oncogene 1998 Jul 9; 17(1):35-457
38. Sato N, Mizumoto K, Nakamura M, Maehara N, Minamishima YA, Nishio S, Nagai E, Tanaka M. Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells. Cancer Genet Cytogenet 2001 Apt 1 ; 126(1): 13-9
39.蔡扬,李秉琦.中心体异常、染色体不稳定和癌发生的相关性.临床口腔医学杂志,2003,19(9):571—573.
40. Tarapore P, Horn HF, Tokuyama Y, Fukasawa K. Direct regulation of the centrosome duplication cycle by the p53-p21Waf1/Cip 1 pathway. Oncogene 2001 May 31 ;20(25):3173-84
41. Morris VB, Brammall J, Noble J, Reddel R. p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study. Exp Cell Res 2000 Apt 10;256(1):122-30
42. Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Nail Acad Sci U S A 1998 Oct 27;95(22):12983-8
43. Mussman JG, Horn HF, Carroll PE, Okuda M, Tarapore P, Donehower LA, Fukasawa K. Synergistic induction of centrosome hyperamplification by loss of p53 and cyclin E overexpression. Oncogene 2000 Mar 23; 19(13): 1635-46
44. Andrew W. Murray. CELL CYCLE: Centrioles at the Checkpoint. Science 2001 Feb 23 ;291 (5508): 1499-502
45. Jonathon Pines. Four-dimensional control of the cell cycle. Nature cell biology. july 1999 volume 1 issue 3 pp E73 - E79 (Review)
46. Lacey KR, Jackson PK, Sterans T. Cyclin-dependant kinase control of centrosome duplication. Proc Natl Acad Sci U S A 1999 March27;96(22):2817-2822
47. Tokuyama Y, Horn HF, Kawamura K, Tarapore P, Fukasawa K. Specific phosphorylation of nucleophosomin on Thrl99 by Cyclin-dependent kinase-2-CyclinE and its role in centrosome deplication. The Journal of Biological Chemistry, 2001, 276(24):21529-21537
48. Matsumoto Y, Hayashi K, Nishida E. Cyclin-dependent kinase2(Cdk2) is required for centrosome duplication in mammalian cells. Curr. Biol. 1999; 9:429-432
49. Fukasawa K. Introduction[J]. Oncogene, (2002) 21,6140-6145
50. Khodjakov A, Rieder CL. Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression. J Cell Biol 2001 Apr 2; 153(1): 237-42
51. Balczon R, Bao L, Zimmer WE, Brown K, Zinkowski RP, Brinkley BR. Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells. J Cell Biol 1995 Jul; 130(1): 105-15
52. Robert JM. Evolving ideas about Cyclins.Cell 1999 July; 98(1): 129-132