口腔癌為台灣男性十大癌症死因的第四位。在台灣，嚼食檳榔是造成口腔癌的主要原因之一。CDK2AP1為一生長抑制基因，負向調節Cyclin-dependent kinase 2 (CDK2)的活性。人類p12CDK2AP1蛋白質表現在口腔癌上有減少或消失的情形。此外，研究顯示人類配對錯誤修補基因MLH1，在其蛋白質表現低的情況下易與微衛星不穩定的區域的出現具有正相關性。微衛星不穩定的大腸直腸癌細胞株其p12CDK2AP1蛋白質的表現量有減少或降低的趨勢。因此，本研究探討CDK2AP1基因在口腔癌的mRNA與蛋白質表現量，並探討其與臨床特質與疾病存活的關係。首先我們經由即時定量PCR (Quantitative reverse transcription polymerase chain reaction)分析44位口腔癌病人的細胞中CDK2AP1 mRNA的表現量，結果顯示在正常口腔組織的表現量較口腔癌組織高，但未達統計差異(P>0.05)。同樣的藉由西方點墨法分析p12CDK2AP1蛋白質表現量，發現口腔癌組織的p12CDK2AP1與CDK2，兩者蛋白質都有表現較高的情形(P<0.05)。而在四株口腔癌細胞Ca9-22、CAL27、SAS、TW2.6與一株正常角質細胞株HaCaT中，與HaCaT相較之下p12CDK2AP1及CDK2蛋白質分別在Ca9-22和TW2.6表現量最高。藉由免疫細胞染色（Immunocytochemistry）的技術，顯示p12CDK2AP1蛋白質在Ca9-22、CAL27、SAS及HaCaT細胞表現在細胞核與細胞質的位置，而在嗜食檳榔患者所建立之TW2.6細胞株主要表現在細胞核。另一方面我們分析了p12CDK2AP1與臨床病理因子、存活及嚼食檳榔的關係。在所收集的167個口腔頰黏膜鱗狀上皮細胞癌檢體中，利用免疫組織染色（Immunohistochemistry）結果顯示，雖然口腔癌患者的核內p12CDK2AP1表現較低，但無論在細胞核(P=0.157)或質(P=0.350)的p12CDK2AP1蛋白質，其表現量與臨床病理因子、存活及嚼食檳榔均未達顯著相關。此外，定序方式檢測CDK2AP1 3’-UTR在四株口腔癌組織及六個低表現人類MLH1的口腔癌組織的基因體DNA，並未發現微衛星不穩定的區域。由本文結果可知: (1) p12CDK2AP1在大部分口腔癌細胞株及一株正常角質細胞株HaCaT表現在細胞核與細胞質，而在嗜食檳榔口腔癌細胞株TW2.6則是主要表現在細胞核內；(2)雖然口腔癌患者的核內p12CDK2AP1表現較低，但在細胞質或核之p12CDK2AP1蛋白質，其表現量與臨床病理因子、存活及嚼食檳榔均未達顯著相關；(3)六個低表現人類MLH1的口腔癌組織的基因體DNA，並未發現CDK2AP1 3’-UTR微衛星不穩定的區域。

Oral cancer is now the fourth leading cause of male cancer mortality in Taiwan. Betel quid chewing is one of the main causes of oral cancer in Taiwan. CDK2AP1 is a growth suppressor gene that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12CDK2AP1 protein is reduced and/or lost in oral cancers. Mutations in microsatellite-like sequence of CDK2AP1 gene in microsatellite instability colorectal cancer are associated with down-regulated CDK2AP1 transcription. This mutation was due to down-regulation of one DNA repair protein, MLH1. In order to understand whether CDK2AP1 mRNA and protein expression levels associate with betel-chewing oral cancers, we firstly analyzed 44 oral cancer specimens (normal and tumor, in pairs) by quantitative reverse transcription polymerase chain reaction and Western blotting. Immunohistochemistry was used to examine p12CDK2AP1 protein expression in another 167 buccal mucosa squamous cell carcinoma tissues. We have demonstrated that the expression levels of CDK2AP1 mRNAs were slightly higher in normal oral tissues than those in tumor tissues (P>0.05). Similarly, p12CDK2AP1 and CDK2 protein expression levels were up-regulated in oral cancer tissues than in normal tissues by Western blot analysis (P<0.05). Among Ca9-22, CAL27, SAS and in betel-chewing oral cancer cells TW2.6 and human normal skin keratinolial cells (HaCaT) that we examined, p12CDK2AP1 and CDK2 proteins were detected to be highest expressed in Ca9-22 and TW2.6 cells, respectively, when compared to HaCaT cells. Immunocytochemistry indicated p12CDK2AP1 expressed in nucleus and cytoplasm in Ca9-22, CAL27, SAS and HaCaT cells, however predominant present in nucleus in TW2.6 cells. On the other hand, immunohistochemistry demonstrated that nuclear (P=0.157) and cytoplasmic p12CDK2AP1 (P=0.350) in 167 patients with buccal mucosa squamous cell carcinoma were slightly down-regulated. Reduction of nuclear p12CDK2AP1 was not significantly correlated with any clinicopathologic characteristics or prognosis. Direct sequencing indicated that lack of microsatellite-like instability of CDK2AP1 3’-UTR in four oral cancer cell lines, HaCaT and six patients with down-regulated MLH1 protein. In conclusion, we demonstrated that: (1) p12CDK2AP1 was located in both the nucleus and cytoplasm in most oral cancer cell lines and HaCaT cells but predominate present in the nucleus in betel-chewing oral cancer cells, TW2.6; (2) Reduction of nuclear p12CDK2AP1 in buccal mucosa squamous cell carcinoma tissues were identified, however, not significantly correlated with any clinicopathologic characteristics, prognosis or betel chewing; (3) In six patients with down-regulated MLH1, lack of micorsatellite-like instability in the CDK2AP1 3’-UTR region has been found.

Abstract
Chinese iii
English v
Abbreviation vii
Introduction 1
Materials and Methods 6
Results 14
Discussions 19
References 23
Tables 28
Figures 36
Appendix 53

台灣地區主要死因分析 (2004)，行政院衛生署統計資料。http://www.doh.gov.tw/statistic/index.htm
Boland, C. R. (1997). Genetic pathways to colorectal cancer. Hosp Pract (Minneap) 32, 79-84, 87-96.
Burns, J. E., Baird, M. C., Clark, L. J., Burns, P. A., Edington, K., Chapman, C., Mitchell, R., Robertson, G., Soutar, D., and Parkinson, E. K. (1993). Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines. Br J Cancer 67, 1274-1284.
Carbonelli, D. L., Duran, H. A., Schwint, A., and Molinari de Rey, B. (1994). Nucleolar organizer regions as a marker of incipient transformation in a model of experimental carcinogenesis. Virchows Arch 425, 165-170.
Choy, A. T., van Hasselt, C. A., Chisholm, E. M., Williams, S. R., King, W. W., and Li, A. K. (1992). Multiple primary cancers in Hong Kong Chinese patients with squamous cell cancer of the head or neck. Cancer 70, 815-820.
Daigo, Y., Suzuki, K., Maruyama, O., Miyoshi, Y., Yasuda, T., Kabuto, T., Imaoka, S., Fujiwara, T., Takahashi, E., Fujino, M. A., and Nakamura, Y. (1997). Isolation, mapping and mutation analysis of a human cDNA homologous to the doc-1 gene of the Chinese hamster, a candidate tumor suppressor for oral cancer. Genes Chromosomes Cancer 20, 204-207.
Dash, B. C., and El-Deiry, W. S. (2005). Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity. Mol Cell Biol 25, 3364-3387.
Dhooge, I. J., De Vos, M., and Van Cauwenberge, P. B. (1998). Multiple primary malignant tumors in patients with head and neck cancer: results of a prospective study and future perspectives. Laryngoscope 108, 250-256.
Figueiredo, M. L., Kim, Y., St John, M. A., and Wong, D. T. (2005). p12CDK2-AP1 gene therapy strategy inhibits tumor growth in an in vivo mouse model of head and neck cancer. Clin Cancer Res 11, 3939-3948.
Gump, J., Stokoe, D., and McCormick, F. (2003). Phosphorylation of p16INK4A correlates with Cdk4 association. J Biol Chem 278, 6619-6622.
Hu, M. G., Hu, G. F., Kim, Y., Tsuji, T., McBride, J., Hinds, P., and Wong, D. T. (2004). Role of p12(CDK2-AP1) in transforming growth factor-beta1-mediated growth suppression. Cancer Res 64, 490-499.
Huang, M. J., Yeh, K. T., Shih, H. C., Wang, Y. F., Lin, T. H., Chang, J. Y., Shih, M. C., and Chang, J. G. (2002). The correlation between CpG methylation and protein expression of P16 in oral squamous cell carcinomas. Int J Mol Med 10, 551-554.
Kim, Y., McBride, J., Zhang, R., Zhou, X., and Wong, D. T. (2005a). p12(CDK2-AP1) mediates DNA damage responses induced by cisplatin. Oncogene 24, 407-418.
Kim, Y., Ohyama, H., Patel, V., Figueiredo, M., and Wong, D. T. (2005b). Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect. J Biol Chem 280, 23273-23279.
Kinzler, K. W., and Vogelstein, B. (1996). Lessons from hereditary colorectal cancer. Cell 87, 159-170.
Ko, Y. C., Huang, Y. L., Lee, C. H., Chen, M. J., Lin, L. M., and Tsai, C. C. (1995). Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 24, 450-453.
Koff, A., Giordano, A., Desai, D., Yamashita, K., Harper, J. W., Elledge, S., Nishimoto, T., Morgan, D. O., Franza, B. R., and Roberts, J. M. (1992). Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 257, 1689-1694.
Kohno, Y., Patel, V., Kim, Y., Tsuji, T., Chin, B. R., Sun, M., Bruce Donoff, R., Kent, R., Wong, D., and Todd, R. (2002). Apoptosis, proliferation and p12(doc-1) profiles in normal, dysplastic and malignant squamous epithelium of the Syrian hamster cheek pouch model. Oral Oncol 38, 274-280.
Kwan, H. W. (1976). A statistical study on oral carcinomas in Taiwan with emphasis on the relationship with betel nut chewing: a preliminary report. Taiwan Yi Xue Hui Za Zhi 75, 497-505.
Lippman, S. M., Sudbo, J., and Hong, W. K. (2005). Oral cancer prevention and the evolution of molecular-targeted drug development. J Clin Oncol 23, 346-356.
Neville, B. W., and Day, T. A. (2002). Oral cancer and precancerous lesions. CA Cancer J Clin 52, 195-215.
Pande, P., Mathur, M., Shukla, N. K., and Ralhan, R. (1998). pRb and p16 protein alterations in human oral tumorigenesis. Oral Oncol 34, 396-403.
Peltomaki, P., and Vasen, H. F. (1997). Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology 113, 1146-1158.
Pines, J. (1995). Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J 308 ( Pt 3), 697-711.
Reed, A. L., Califano, J., Cairns, P., Westra, W. H., Jones, R. M., Koch, W., Ahrendt, S., Eby, Y., Sewell, D., Nawroz, H., et al. (1996). High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res 56, 3630-3633.
Saito, T., Nakajima, T., and Mogi, K. (1999). Immunohistochemical analysis of cell cycle-associated proteins p16, pRb, p53, p27 and Ki-67 in oral cancer and precancer with special reference to verrucous carcinomas. J Oral Pathol Med 28, 226-232.
Sartor, M., Steingrimsdottir, H., Elamin, F., Gaken, J., Warnakulasuriya, S., Partridge, M., Thakker, N., Johnson, N. W., and Tavassoli, M. (1999). Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines. Br J Cancer 80, 79-86.
Schneeberger, C., Eder, S., Swoboda, H., Ullrich, R., and Zeillinger, R. (1998). A differential PCR system for the determination of CCND1 (cyclin D1) gene amplification in head and neck squamous cell carcinomas. Oral Oncol 34, 257-260.
Sharma, D. C. (2003). Betel quid and areca nut are carcinogenic without tobacco. Lancet Oncol 4, 587.
Sherr, C. J. (1996). Cancer cell cycles. Science 274, 1672-1677.
Sherr, C. J., and Roberts, J. M. (1995). Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9, 1149-1163.
Shintani, S., Mihara, M., Terakado, N., Nakahara, Y., Matsumura, T., Kohno, Y., Ohyama, H., McBride, J., Kent, R., Todd, R., et al. (2001). Reduction of p12DOC-1 expression is a negative prognostic indicator in patients with surgically resected oral squamous cell carcinoma. Clin Cancer Res 7, 2776-2782.
Shintani, S., Ohyama, H., Zhang, X., McBride, J., Matsuo, K., Tsuji, T., Hu, M. G., Hu, G., Kohno, Y., Lerman, M., et al. (2000). p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein. Mol Cell Biol 20, 6300-6307.
Silverman, S., Jr., and Griffith, M. (1972). Smoking characteristics of patients with oral carcinoma and the risk for second oral primary carcinoma. J Am Dent Assoc 85, 637-640.
Slaughter, D. P., Southwick, H. W., and Smejkal, W. (1953). Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6, 963-968.
Somers, K. D., Merrick, M. A., Lopez, M. E., Incognito, L. S., Schechter, G. L., and Casey, G. (1992). Frequent p53 mutations in head and neck cancer. Cancer Res 52, 5997-6000.
Todd, R., Donoff, R. B., and Wong, D. T. (1997). The molecular biology of oral carcinogenesis: toward a tumor progression model. J Oral Maxillofac Surg 55, 613-623; discussion 623-615.
Todd, R., McBride, J., Tsuji, T., Donoff, R. B., Nagai, M., Chou, M. Y., Chiang, T., and Wong, D. T. (1995). Deleted in oral cancer-1 (doc-1), a novel oral tumor suppressor gene. Faseb J 9, 1362-1370.
Tsuji, T., Duh, F. M., Latif, F., Popescu, N. C., Zimonjic, D. B., McBride, J., Matsuo, K., Ohyama, H., Todd, R., Nagata, E., et al. (1998). Cloning, mapping, expression, function, and mutation analyses of the human ortholog of the hamster putative tumor suppressor gene Doc-1. J Biol Chem 273, 6704-6709.
van Oijen, M. G., Tilanus, M. G., Medema, R. H., and Slootweg, P. J. (1998). Expression of p21 (Waf1/Cip1) in head and neck cancer in relation to proliferation, differentiation, p53 status and cyclin D1 expression. J Oral Pathol Med 27, 367-375.
Warnakulasuriya, K. A., Tavassoli, M., and Johnson, N. W. (1998). Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma. J Oral Pathol Med 27, 376-381.
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323-330.
Wong, D. T., Todd, R., Tsuji, T., and Donoff, R. B. (1996). Molecular biology of human oral cancer. Crit Rev Oral Biol Med 7, 319-328.
Yuan, Z., Sotsky Kent, T., and Weber, T. K. (2003). Differential expression of DOC-1 in microsatellite-unstable human colorectal cancer. Oncogene 22, 6304-6310.