在烏腳病地區，砷的暴露是造成膀胱癌的其中一種環境因子。而不同的砷濃度可能促使細胞生長亦可能造成細胞凋亡。由於缺乏適當的動物模式研究砷所引起的癌症，砷暴露的危險指標濃度一直無法確立。此外，砷導致癌症發生的相關機制也尚未了解。Aurora-A 是一種與細胞分裂相關的蛋白，當它過度表現時會造成中心體不正常增加、染色體不穩定以及細胞走向癌化等現象。在我們過去的研究中，發現在烏腳病地區的臨床檢體中，Aurora-A的過量表現與砷暴露所導致的膀胱癌有高度相關性。而在我的細胞實驗當中發現到，0.5 M砷暴露24小時即可活化Aurora-A的啟動子活性 (promoter activity) 以及增加其RNA的表現量。此外，砷處理E7細胞所造成的Aurora-A 過量表現會持續一到四週。而在機制方面，我們發現Aurora-A 基因增幅 (gene amplification) 並未參與基因的活化。進一步發現砷活化Aurora-A 的啟動子活性可能透過E2F1這個轉錄因子活化所造成。動物實驗方面，我們發現細胞增生的指標性蛋白PCNA 及BrdU標示以及 Aurora-A在子宮及膀胱當中表現量有增加。簡言之，低濃度長時間的砷暴露會促使Aurora-A藉由轉錄因子的調控過度表現，而非藉由基因增幅。而此一現象除了在細胞實驗中，也可以在動物實驗當中得到應證。本研究證實在低濃度1 M之砷暴露下短時間內(兩星期)即可活化Aurora-A並可能與膀胱癌之形成有關，本研究之發現可做為最低砷暴露量及日後檢測膀胱癌等砷暴露相關癌症的參考。

In blackfoot-disease endemic areas, arsenic exposure is an environmental factor which correlates with bladder cancer formation. Depending on the dosage, arsenic may trigger the cells undergoing either proliferation or apoptosis-related cell death. Because of lack of the proper animal model to study arsenic induced tumorigenesis, the accurate risk level of arsenic exposure has not been determined. In addition, the mechanism of arsenic-related tumorigenesis remains unclear. Aurora-A is a mitotic kinase, over-expression of Aurora-A leads to centrosome amplification, chromosomal instability and cell transformation. Our previous study revealed that Aurora-A is over-expressed in the bladder cancer patients from blackfoot-disease endemic areas. Our cell line study reveals that arsenic exposure at 0.5 M for 24 hr is able to induce Aurora-A activation based on promoter activity and RNA analysis. Furthermore, Aurora-A over-expression was sustained for 1 to 4 weeks by chronic treatment of immortalized bladder cell line E7 with NaAsO2. Aurora-A gene amplification was not detected in the long time arsenic treated E7 cells. The expression level of E2F1 transcription factor is increased in the presence of arsenic. It is plausible that arsenic-related Aurora-A over-expression is regulated by E2F transcription factor 1 (E2F1). Proliferating cell nuclear antigen (PCNA), Bromodeoxyuridine (BrdU) (cell proliferation markers) and Aurora-A expression were increased in mice uterus after arsenic (over 0.5 M) exposure for 1 month. Altogether, arsenic exposure at low concentration induces Aurora-A over-expression through transcriptional regulation, but not through gene amplification both in vitro and in vivo.

[1] I.C. Ch'i, and R.Q. Blackwell, A controlled retrospective study of Blackfoot disease, an endemic peripheral gangrene disease in Taiwan. Am J Epidemiol 88 (1968) 7-24.
[2] K.P. Chen, and H.Y. Wu, Epidemiologic studies on blackfoot disease in Taiwan, China. 6. Effect of the piped water supply on occurrence and disease progress of blackfoot disease. Taiwan Yi Xue Hui Za Zhi 68 (1969) 291-5.
[3] W.P. Tseng, Prognosis of blackfoot disease. A 10-year follow-up study. Taiwan Yi Xue Hui Za Zhi 69 (1970) 1-21.
[4] F.J. Lu, C.K. Yang, and K.H. Ling, [Physico-chemical characteristics of drinking water in blackfoot endemic areas in Chia-I and Tainan Hsiens]. Taiwan Yi Xue Hui Za Zhi 74 (1975) 596-605.
[5] C.J. Chen, Y.C. Chuang, T.M. Lin, and H.Y. Wu, Malignant neoplasms among residents of a blackfoot disease-endemic area in Taiwan: high-arsenic artesian well water and cancers. Cancer Res 45 (1985) 5895-9.
[6] C.J. Chen, Y.C. Chuang, S.L. You, T.M. Lin, and H.Y. Wu, A retrospective study on malignant neoplasms of bladder, lung and liver in blackfoot disease endemic area in Taiwan. Br J Cancer 53 (1986) 399-405.
[7] P.H. Chiang, M.S. Huang, C.J. Tsai, E.M. Tsai, C.H. Huang, and C.P. Chiang, Transitional cell carcinoma of the renal pelvis and ureter in Taiwan. DNA analysis by flow cytometry. Cancer 71 (1993) 3988-92.
[8] W.P. Tseng, Effects and dose--response relationships of skin cancer and blackfoot disease with arsenic. Environ Health Perspect 19 (1977) 109-19.
[9] M.M. Wu, T.L. Kuo, Y.H. Hwang, and C.J. Chen, Dose-response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol 130 (1989) 1123-32.
[10] H.Y. Chiou, Y.M. Hsueh, K.F. Liaw, S.F. Horng, M.H. Chiang, Y.S. Pu, J.S. Lin, C.H. Huang, and C.J. Chen, Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res 55 (1995) 1296-300.
[11] H. Shi, X. Shi, and K.J. Liu, Oxidative mechanism of arsenic toxicity and carcinogenesis. Mol Cell Biochem 255 (2004) 67-78.
[12] C.Y. Yang, H.F. Chiu, C.C. Chang, S.C. Ho, and T.N. Wu, Bladder cancer mortality reduction after installation of a tap-water supply system in an arsenious-endemic area in southwestern Taiwan. Environ Res 98 (2005) 127-32.
[13] R. Ruiz-Ramos, L. Lopez-Carrillo, A.D. Rios-Perez, A. De Vizcaya-Ruiz, and M.E. Cebrian, Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins, NF-kappaB activation and cell proliferation in human breast cancer MCF-7 cells. Mutat Res (2008).
[14] T.G. Rossman, Mechanism of arsenic carcinogenesis: an integrated approach. Mutat Res 533 (2003) 37-65.
[15] M. Carmena, and W.C. Earnshaw, The cellular geography of aurora kinases. Nat Rev Mol Cell Biol 4 (2003) 842-54.
[16] Y.S. Lin, L.J. Su, C.T. Yu, F.H. Wong, H.H. Yeh, S.L. Chen, J.C. Wu, W.J. Lin, Y.L. Shiue, H.S. Liu, S.L. Hsu, J.M. Lai, and C.Y. Huang, Gene expression profiles of the aurora family kinases. Gene Expr 13 (2006) 15-26.
[17] A.R. Barr, and F. Gergely, Aurora-A: the maker and breaker of spindle poles. J Cell Sci 120 (2007) 2987-96.
[18] D.T. Ross, U. Scherf, M.B. Eisen, C.M. Perou, C. Rees, P. Spellman, V. Iyer, S.S. Jeffrey, M. Van de Rijn, M. Waltham, A. Pergamenschikov, J.C. Lee, D. Lashkari, D. Shalon, T.G. Myers, J.N. Weinstein, D. Botstein, and P.O. Brown, Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 24 (2000) 227-35.
[19] M.M. Tanner, M. Tirkkonen, A. Kallioniemi, C. Collins, T. Stokke, R. Karhu, D. Kowbel, F. Shadravan, M. Hintz, W.L. Kuo, and et al., Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes. Cancer Res 54 (1994) 4257-60.
[20] R. Giet, and C. Prigent, Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J Cell Sci 112 ( Pt 21) (1999) 3591-601.
[21] G. Vader, and S.M. Lens, The Aurora kinase family in cell division and cancer. Biochim Biophys Acta 1786 (2008) 60-72.
[22] M. Kimura, S. Kotani, T. Hattori, N. Sumi, T. Yoshioka, K. Todokoro, and Y. Okano, Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 272 (1997) 13766-71.
[23] D.L. Stenoien, S. Sen, M.A. Mancini, and B.R. Brinkley, Dynamic association of a tumor amplified kinase, Aurora-A, with the centrosome and mitotic spindle. Cell Motil Cytoskeleton 55 (2003) 134-46.
[24] R. Giet, C. Petretti, and C. Prigent, Aurora kinases, aneuploidy and cancer, a coincidence or a real link? Trends Cell Biol 15 (2005) 241-50.
[25] S. Anand, S. Penrhyn-Lowe, and A.R. Venkitaraman, AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol. Cancer Cell 3 (2003) 51-62.
[26] D. Zhang, T. Hirota, T. Marumoto, M. Shimizu, N. Kunitoku, T. Sasayama, Y. Arima, L. Feng, M. Suzuki, M. Takeya, and H. Saya, Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models. Oncogene 23 (2004) 8720-30.
[27] J.R. Bischoff, L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, C.S. Chan, M. Novotny, D.J. Slamon, and G.D. Plowman, A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J 17 (1998) 3052-65.
[28] H. Zhou, J. Kuang, L. Zhong, W.L. Kuo, J.W. Gray, A. Sahin, B.R. Brinkley, and S. Sen, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 20 (1998) 189-93.
[29] S. Sen, H. Zhou, R.D. Zhang, D.S. Yoon, F. Vakar-Lopez, S. Ito, F. Jiang, D. Johnston, H.B. Grossman, A.C. Ruifrok, R.L. Katz, W. Brinkley, and B. Czerniak, Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst 94 (2002) 1320-9.
[30] Y.S. Tseng, C.C. Tzeng, C.Y. Huang, P.H. Chen, A.W. Chiu, P.Y. Hsu, G.C. Huang, Y.C. Wang, and H.S. Liu, Aurora-A overexpression associates with Ha-ras codon-12 mutation and blackfoot disease endemic area in bladder cancer. Cancer Lett 241 (2006) 93-101.
[31] Y.T. Chang, The effect of arsenic treatment on Aurora-A promoter methylation and screening for novel Ras-related proteins. Master Thesis of Department of Microbiology and Immunology in National Cheng Kung University. (2008) 1-80.
[32] M. Esteller, CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21 (2002) 5427-40.
[33] A.P. Feinberg, and B. Vogelstein, Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301 (1983) 89-92.
[34] S. Nambu, K. Inoue, and H. Saski, Site-specific hypomethylation of the c-myc oncogene in human hepatocellular carcinoma. Jpn J Cancer Res 78 (1987) 695-704.
[35] P.M. Rao, A. Antony, S. Rajalakshmi, and D.S. Sarma, Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver. Carcinogenesis 10 (1989) 933-7.
[36] J.S. Ray, M.L. Harbison, R.M. McClain, and J.I. Goodman, Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse live tumors. Mol Carcinog 9 (1994) 155-66.
[37] R.S. Okoji, R.C. Yu, R.R. Maronpot, and J.R. Froines, Sodium arsenite administration via drinking water increases genome-wide and Ha-ras DNA hypomethylation in methyl-deficient C57BL/6J mice. Carcinogenesis 23 (2002) 777-85.
[38] K.E. Eblin, T.G. Bredfeldt, and A.J. Gandolfi, Immortalized human urothelial cells as a model of arsenic-induced bladder cancer. Toxicology 248 (2008) 67-76.
[39] J. Li, M. Gorospe, J. Barnes, and Y. Liu, Tumor promoter arsenite stimulates histone H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human diploid fibroblasts. J Biol Chem 278 (2003) 13183-91.
[40] T. Yoshida, H. Yamauchi, and G. Fan Sun, Chronic health effects in people exposed to arsenic via the drinking water: dose-response relationships in review. Toxicol Appl Pharmacol 198 (2004) 243-52.
[41] S. Suzuki, L.L. Arnold, T. Ohnishi, and S.M. Cohen, Effects of inorganic arsenic on the rat and mouse urinary bladder. Toxicol Sci 106 (2008) 350-63.
[42] T.G. Rossman, A.N. Uddin, F.J. Burns, and M.C. Bosland, Arsenite is a cocarcinogen with solar ultraviolet radiation for mouse skin: an animal model for arsenic carcinogenesis. Toxicol Appl Pharmacol 176 (2001) 64-71.
[43] F.J. Burns, A.N. Uddin, F. Wu, A. Nadas, and T.G. Rossman, Arsenic-induced enhancement of ultraviolet radiation carcinogenesis in mouse skin: a dose-response study. Environ Health Perspect 112 (2004) 599-603.
[44] S.M. Cohen, L.L. Arnold, M. Eldan, A.S. Lewis, and B.D. Beck, Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit Rev Toxicol 36 (2006) 99-133.
[45] S.M. Cohen, T. Ohnishi, L.L. Arnold, and X.C. Le, Arsenic-induced bladder cancer in an animal model. Toxicol Appl Pharmacol 222 (2007) 258-63.
[46] M.P. Waalkes, J.M. Ward, and B.A. Diwan, Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers. Carcinogenesis 25 (2004) 133-41.
[47] M.P. Waalkes, J. Liu, and B.A. Diwan, Transplacental arsenic carcinogenesis in mice. Toxicol Appl Pharmacol 222 (2007) 271-80.
[48] M.P. Waalkes, J.M. Ward, J. Liu, and B.A. Diwan, Transplacental carcinogenicity of inorganic arsenic in the drinking water: induction of hepatic, ovarian, pulmonary, and adrenal tumors in mice. Toxicol Appl Pharmacol 186 (2003) 7-17.
[49] S.H. Liou, J.C. Lung, Y.H. Chen, T. Yang, L.L. Hsieh, C.J. Chen, and T.N. Wu, Increased chromosome-type chromosome aberration frequencies as biomarkers of cancer risk in a blackfoot endemic area. Cancer Res 59 (1999) 1481-4.
[50] A. Hernandez-Zavala, E. Cordova, L.M. Del Razo, M.E. Cebrian, and E. Garrido, Effects of arsenite on cell cycle progression in a human bladder cancer cell line. Toxicology 207 (2005) 49-57.
[51] P.P. Simeonova, S. Wang, W. Toriuma, V. Kommineni, J. Matheson, N. Unimye, F. Kayama, D. Harki, M. Ding, V. Vallyathan, and M.I. Luster, Arsenic mediates cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation. Cancer Res 60 (2000) 3445-53.
[52] L. Vega, M. Styblo, R. Patterson, W. Cullen, C. Wang, and D. Germolec, Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicol Appl Pharmacol 172 (2001) 225-32.
[53] J.J. Bower, S.S. Leonard, and X. Shi, Conference overview: molecular mechanisms of metal toxicity and carcinogenesis. Mol Cell Biochem 279 (2005) 3-15.
[54] T.C. Lee, N. Tanaka, P.W. Lamb, T.M. Gilmer, and J.C. Barrett, Induction of gene amplification by arsenic. Science 241 (1988) 79-81.
[55] K. Salnikow, and A. Zhitkovich, Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium. Chem Res Toxicol 21 (2008) 28-44.
[56] M. Yamamoto, S. Hirano, C.F. Vogel, X. Cui, and F. Matsumura, Selective activation of NF-kappaB and E2F by low concentration of arsenite in U937 human monocytic leukemia cells. J Biochem Mol Toxicol 22 (2008) 136-46.
[57] K.J. Trouba, E.M. Wauson, and R.L. Vorce, Sodium arsenite-induced dysregulation of proteins involved in proliferative signaling. Toxicol Appl Pharmacol 164 (2000) 161-70.
[58] L. He, H. Yang, Y. Ma, W.J. Pledger, W.D. Cress, and J.Q. Cheng, Identification of Aurora-A as a direct target of E2F3 during G2/M cell cycle progression. J Biol Chem 283 (2008) 31012-20.
[59] M. Kimura, C. Uchida, Y. Takano, M. Kitagawa, and Y. Okano, Cell cycle-dependent regulation of the human aurora B promoter. Biochem Biophys Res Commun 316 (2004) 930-6.
[60] A.Y. Olsson, A. Feber, S. Edwards, R. Te Poele, I. Giddings, S. Merson, and C.S. Cooper, Role of E2F3 expression in modulating cellular proliferation rate in human bladder and prostate cancer cells. Oncogene 26 (2007) 1028-37.
[61] W. Ouyang, J. Li, Q. Ma, and C. Huang, Essential roles of PI-3K/Akt/IKKbeta/NFkappaB pathway in cyclin D1 induction by arsenite in JB6 Cl41 cells. Carcinogenesis 27 (2006) 864-73.
[62] S. Prajapati, Z. Tu, Y. Yamamoto, and R.B. Gaynor, IKKalpha regulates the mitotic phase of the cell cycle by modulating Aurora A phosphorylation. Cell Cycle 5 (2006) 2371-80.
[63] S. Balakrishnan, P.T. Uppala, D.S. Rupa, L. Hasegawa, and D.A. Eastmond, Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o-phenylphenol. Mutagenesis 17 (2002) 89-93.
[64] G.J. Ahlborn, G.M. Nelson, R.D. Grindstaff, M.P. Waalkes, B.A. Diwan, J.W. Allen, K.T. Kitchin, R.J. Preston, A. Hernandez-Zavala, B. Adair, D.J. Thomas, and D.A. Delker, Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice. Toxicology (2009).