口腔癌盛行於世界，尤其是東南亞國家。在台灣，口腔癌更位居男性癌症死亡原因的第四名，其主要是與嚼食檳榔的習慣有關。近來的研究報告指出，嚼檳榔時，會產生活性氧分子，因此可能導致口腔中的組織的氧化傷害，而產生口腔癌化現象。我們實驗室曾在結直腸癌與血癌細胞中發現氧化壓力可使DNA 錯誤配對修復機制失去校對能力，因DNA錯誤配對修復機制的缺失所引起的。此外已知氧化壓力抑制DNA錯誤配對修復活性，以致失去校正及修復錯誤序列之能力，進而造成細胞發生微衛星DNA不穩定性，最終，此突變現象累積過多後使得正常細胞轉化為癌症細胞。因此，我們假設，利用檳榔子內容物引發口腔癌細胞增加微衛星DNA不穩定性。為了證明此假設，我使用了美國國家癌症機構所建議的聚合酵素鏈鎖反應方法以及西方點墨法來測定口腔癌細胞株與口腔癌病人組織檢體中是否有表現出微衛星DNA的不穩定性。並且建立一個螢光微衛星DNA不穩定性的偵測系統，以利於快速檢測口腔癌細胞中的微衛星DNA不穩定性，而未來將可進而用於篩選預防口腔癌的食品或藥物。

Oral cancer prevails over the world, particularly in Southeast Asian countries. In Taiwan oral cancer ranks
the sixth cause of cancer mortality, mainly due to a custom of betel quid (BQ) chewing. Recent studies have demonstrated that reactive oxygen species (ROS) are generated in the oral cavity during BQ chewing. ROS is known to inactivate the function of DNA mismatch repair system and leads to increased microsatellite
instability (MSI) in colon cancer and erythro-leukemia cells. Therefore, we hypothesize that ROS generated by
the betel nut ingredients can increase MSI in human
oral cancer cells. To test this hypothesis, I have examined whether oral cancer cell lines and patient samples display the MSI phenotype using a multiplex fluorescent PCR method and the Western bolt analysis.
In addition, I have designed and established an in-vivo fluorescent MSI reporter system for future screening of cancer-preventive compounds.

1. Daftary D. The situation in high risk areas of the
world: Risk Markers for Oral Diseases. Cambridge
University Press, 1990;2.
2. SR Moore NJ, AM Pierce and DF Wilson. The
epidemiology of mouth cancer: a review of global
incidence. Oral Dis 2000;6:65-74.
3. Ko YC HY, Lee CH, Chen MJ, Lin LM and Tsai CC.
Betel quid chewing, cigarette smoking and alchol
consumption related to oral cancer in Taiwan.
J Oral Pathol Med. 1995;24:450-9.
4. Thomas S. Slaked lime and betel nut cancer in
Papua New Guinea. lancet 1992;340:577-8.
5. IARC. Monographs on the Evaluation of Carcinogenic
Risk of Chemicals to Humans. IARC 1985;37.
6. Sharan RN. Association of betel nut with
carcinogenesis. Cancer J 1996;9:13-9.
7. Sen S TG, Sharma A. Betel cytotoxicity. J
Ethnopharmacol 1989;26:217-47.
8. Pillai R, Balaram,P and Reddiar,K.S. Pathogenesis
of oral submucous fibrosis: relationship to risk
factors associated with oral cancer. Cancer J
1992;69:2011-20.
9. Jeng JH, Chang,M.C and Hahn,L.J. Role of areca nut
in betel quid-associated chemical carcinogenesis:
current awareness and future perspectives.
Oral Oncol 2001;37:477-92.
10.Thomas S KJ. Betel quid and oral cancer: a review.
Oral Oncol 1993;29(B):251-5.
11.Rao A. Evaluation of the carcinogenicity of
different preparations of areca nut in mice. Int J
Cancer 1989;43:728-32.
12.Ranadive K. Betel quid chewing and oral cancer:
experimental studies on hamsters. Int J Cancer
1979;24:835-43.
13.Chang M. Areca nut extract and arecoline induced
the cell cycle arrest but not apoptosis of
cultured oral KB epithelial cell: association
of glutathione, reactive oxygen species and
mitochondrial membrane potential. Carcinogenesis
2001;22:1527-35.
14.Lee PH, Chang, M.C., Chang,W.H.,Wang, T.M.,Wang,
Y.J., Hahn, L.J., Ho, Y.S., Lin, C.Y., Jeng, J.H.,
Chang,M.C and Hahn,L.J. Prolonged exposure to
arecoline arrested human KB epithelial cell growth:
regulatorymechanisms of cell cycle and apoptosis.
Toxicology 2006;220:81-90.
15.Tsai Y. Arecoline, a major alkaloid of areca nut,
inhibits p53, repress DNA repair and triggers DNA
response in human epithelial cells. Toxicology
2008;249:230-7.
16.Hwang L.S. WCK, Sheu M. J. and Kao L. S. Phenolic
compounds of piper betle flower as flavoring and
uenronal activity modulating agents. ACS Symp
1993;506:200-13.
17.Egawa S. Genomic instability of microsatellite
repeats in prostate cancer: relationship to
clinicopathologic variables. 1995 1995;55:2418-21.
18.Suzuki Y. Eugenol-mediated superoxide generation and
cytotoxity in guinea pig neutrophils. Jpn J Pharmacol
1985;39:381-6.
19.Azuma Y ON, Ueda N and Takagi N. Pharmacological
studies on the anti-inflammatory action of phenolic
compounds. J Dent Res 1986;65:53-6.
20.Modrich P. Mechanisms and biological effect of
mismatch repair. Annu Rev Genet 1991;25:229-53.
21.Jiricny J. Replication errors: cha(lle)nging the
genome. EMBO J 1998;17:6427-36.
22.Modrich P. Strand specific mismatch repair in
mammalian cells. J Biol Chem 1997;272:24727-30.
23.Jiricny J. The multifaceted mismatch-repair
system. Mol Cell Biol 2006;7:335-46.
24.Amiram R. Human DNA Repair Genes. Envir Mol Mut
2001;37:241-83.
25.Li GM MP. Restoration of mismatch repair to nuclear
extracts of H6 colorectal tumor cells by a
heterodimer of human MutL homologs. Proc Natl Acad
Science USA 1995;92:1950-4.
26.Drummond JT LG, Longley J, Modrich P. Isolation of
an hMSH2-p160 heterodimer that restore DNA mismatch
repair to tumor cells. Science 1995;268:1909-12.
27.Palombo F GP, Iaccarino I, Lettieri T, Hughes M, D
Arrigo A, Truong O, Hsuan JJ and Jiricny J. GTBP,
a 160-kilodalton protein essential for
mismatch-binding activity in human cells. Science
1995;268:1912-4.
28.Alani E. The Saccharomyces cerevisiae Msh2 and Msh6
proteins form a complex that specifically binds to
duplex oligonucleotides containing mismatched DNA
base pairs. Mol Cell Biol 1996;16:5604-15.
29.Habraken Y SP, Prahash L, Praksh S. Binding of
insertion/deletion DNA mismatches by the heterodimer
of yeast mismatch repair proteins MSH2 and MSH3.
Curr Biol 1996;6:1185-7.
30.Genschel J LS, Drummond JT and Modrich P. Isolation
of MutS from human cells and comparison of the
mismatch repair specificities of MutS and MutS. J
Biol Chem 1998;273:19895-901.
31.Palombo F II, Nakajima E, Ikejima M, Shimada T and
Jiricny J. hMutS, a heterodimer of hMSH2 and hMSH3,
binds to insertion/deletion loops in DNA. Curr Biol
1996;6:1181-4.
32.Schmutte C MR, Sadoff MM, Guerette S, Overhauser J
and Fishel R. Human exonuclease I interact with the
mismatch repair protein hMSH2. Cancer Res 1998;58:
4537-42.
33.Tishkoff DX FN, Gaida GM and Kolodner RD. A novel
mutation avoidance mechanism dependent on
S. cerevisiae RAD27 is distinct from DNA mismatch
repair. Cell 1997;88:253-63.
34.Longley MJ PA, Modrich P. DNA polymerase alpha is
required for human mismatch repair in vitro. J Biol
Chem 1997;272:10917-21.
35.Umar A BA, Simon JA, Thomas DC, Clark AB, Liskay RM
and Kunkel TA. Requirement for PCNA in DNA mismatch
repair at a step preceding DNA resynthesis. Cell
1996;87:65-73.
36.Lin YL SM, Chen C, Kolodner R, Wood RD and Dutta A.
The evolutionary conserved zinc finger motif in the
largest protein of human replication protein A is
required for DNA replication and mismatch repair but
not for nucleotide excision repair. J Biol Chem
1998;273:1453-61.
37.Kolodner R MG. Eukariotic DNA mismatch repair. Curr
Opin Cancer Dev 1999;9:89-96.
38.Mukund V. Katti PKRaVSG. Differential Distribution
of Simple Sequence Repeats in Eukaryotic Genome
Sequences. Mol Biol Evol 2001;18:1161-7.
39.IHGC. Initial sequencing and analysis of the human
genome. Nature 2001;409:860-921.
40.Li YC, A. B. Korol, T. Fahima, A. Beiles and E.
Nevo. Microsatellites: genomic distribution,
putative functions and mutational mechanisms: a
reivew. Mol Ecol 2002;11:2453-65.
41.Fishel R LM, Rao MRS, Copeland NG, Jenkins NA,
Garber J, Kane M and Kolodner R. The human mutator
gene homolog MSH2 and its association with
hereditary nonpolyposis colon cancer. Cell
1993;75:1027-38.
42.Leach F NN, Papadopoulos N, Liu B, Jen J, Parsons
R, Peltomaki P, Sistonen P, Aaltonen L,
Nystrom-Lahti M, Guan XY,, Zhang J MP, Yu J,
Kao F, Chen D, Cerosaletti K, Fournier R, Todd S,
Lewis T, Leach R, Naylor S, Weissenbach J,
Mecklin J,, Jarvinen H PG, Hamilton S, Green J,
Jass J, Watson P, Lynch H, Trent J,
de la Chapelle A, Kinzler K and Vogelstein B.
Mutations of a MutS homolog in hereditary
non-polyposis colorectal cancer. Cell 1993;
75:1215-25.
43.Parsons R LG, Longley M, Fang W, Papadopoulos N,
Jen J, de la, Chapelle A, Kinzler K, Vogelstein B
and Modrich P. Hypermutability and mismatch repair
deficiency in RER+ tumor cells. Cell 1993;
75:1227-36.
44.Bronner C BS, Morrison P, Warren G, Smith L,
Lescoe M, Kane M, Earabino C, Lipford J,
Lindblom A, Tannergard P, Bollag R, Godwin A,
Ward D, Nordenskjold M, Fishel R, Kolodner R and
Liskay R. Mutation in the DNA mismatch repair gene
homologue hMLH1 is associated with hereditary non-
polyposis colon cancer. Nature 1994;368:258-61.
45.Nicolaides N PN, Liu B, Wel Y, Carter K, Ruben S,
Rosen C, Haseltine W, Fleischmann R, Fraser C,
Adams M, Venter J, Dunlopo M, Hamilton S,
Petersen G, de la Chapelle A, Vogelstein B and
Kinzler K. Mutations of two PMS homologues in
hereditary nonpolyposis colon cancer. Nature
1994;371:75-80.
46.Papadopoulos N NN, Wei Y, Ruben S, Carter K,
Rosen C, Haseltine W, Fleischmann R, Fraser C,
Adams M, Venter J, Hamilton S, Petersen G,
Watson P, Lynch H, Peltomaki P, Mecklin J, de la
Chapelle A, Kinzler K and Vogelstein B. Mutations
of a mutL homolog in hereditary colon cancer.
Science 1994;263:1625-9.
47.Eshleman JR LE, Bowerfind GK, Parsons R,
Vogelstein B, Willson JKV, Veigl ML, Sedwick WD
and Markowitz SD. Increased mutation rate at the
hprt locus accompanies microsatellite instability
in colon cancer. Oncogene 1995;10:33-7.
48.Bhattacharyya NP SA, Ganesh A, Groden J and
Meuth M. Mutator phenotypes in human colorectal
carcinoma cell lines. Proc Natl Acad Sci USA
1994;91:6319-23.
49.Shibata D PM, Ionov Y, Malkhosyan S, Perucho M.
Genomic instability in repeated sequences is an
early somatic event in colorectal tumorigenesis
that persists after transformation. Nat Genet
1994;6:273-81.
50.Kane MF LM, Gaida GM, Lipman J, Mishra R,
Goldman H, Jessup JM, Kolodner R. Methylation of
the hMLH1 promoter correlates with lack of
expression of hMLH1 in sporadic colon tumors and
mismatch repair-defective human tumor cell lines.
Cancer Res 1997;57:808-11.
51.Herman JG UA, Polyak K, Graff JR, Ahuja N, Issa JP,
Markowitz S, Willson JK, Hamilton SR, Kinzler KW
et al. Incidence and functional consequences of
hMLH1 promoter hypermethylation in colorectal
carcinoma. Proc Natl Acad Sci USA 1998;95:6670-5.
52.Veigl ML KL, Olechnowicz J, Ma AH, Lutterbaugh JD,
Periyasamy S, Li GM, Drummond J, Modrich PL,
Sedwick WD and Markowitz SD. Biallelic inactivation
of hMLH1 by epigenetic gene silencing, a novel
mechanism causing human MSI cancers. Proc Natl Acad
Sci USA 1998;95:8698-702.
53.Esteller M CL, Matias-Guiu X, Mutter GL, Prat J,
Baylin SB and Herman JG. hMLH1 promoter
hypermethylation is an early event in human
endometrial tumorigenesis. Am J Pathol 1999;1
55:1767-72.
54.Wheeler JM BN, Kim HC, Tomlinson IP, Mortensen NJ,
Bodmer WF. Mechanisms of inactivation of mismatch
repair genes in human colorectal cancer cell lines:
The predominant role of hMLH1. Proc Natl Acad Sci
USA 1999;96:10296-301.
55.Cunningham JM CE, Tester DJ, Kim CY, Roche PC,
Burgart LJ and Thibodeau SN. Hypermethylation of
the hMLH1 promoter in colon cancer with
microsatellite instability. Cancer Res 1998;
59:3455-60.
56.Kuismanen SA HM, Salovaara R, Schweizer P,
Aaltonen L, de la Chapelle A, Nyström-Lahti M,
Peltomäki P. Epigenetic phenotypes distinguish
microsatellite-stable and -unstable colorectal
cancers. Proc Natl Acad Sci USA 1999;96:12661-6.
57.Moslein G TD, Lindor NM, Honchel R, Cunningham JM,
French AJ, Halling KC, Schwab M, Goretzki P and
Thibodeau SN. Microsatellite instability and
mutation analysis of hMSH2 and hMLH1 in patients
with sporadic, familial and hereditary colorectal
cancer. Hum Mol Genet 1996;5:1245-52.
58.Christoph Lengauer KWKaBV. Genetic instabilities
in human cancers. Nature 1998;396:643-9.
59.Li Mao DJL, Melvyn S. Tockman, Yener S. Erozan,
Frederic Askin and David Sidransky. Microsatellite
alterations as clonal markers for the detection of
human cancer. Proc Nati Acad Sci USA 1994;91:9871-5.
60.Gonzalez-Zulueta M ea. Microsatellite instability
in bladder cancer. Cancer Res 1993;53:5620-3.
61.Yee C RN, Verrier CS and Parl FF. Microsatellite
instability and loss of heterozygosity in breast
cancer. Cancer Res 1994;54:1641-4.
62.Risinger JI BA, Kohler MF, Watson P, Lynch HT
and Boyd J. Genetic instability of microsatellites
in endometrial carcinoma. Cancer Res 1993;53:5100-3.
63.Caduff R. Clinical and pathological significance of
microsatellite instability in sporadic endometrial
carcinoma. Am J Pathol 1996;148:1671-8.
64.Lin J. Microsatellite instability in gastric
carcinoma with special references to
histopathology and cancer stages. 11 1995:1879-82.
65.Pieretti M. Genetic alterations distinguish
different types of ovarian tumors. Int J Cancer
1995;64:434-40.
66.El-Naggar AK HK, Huff V, Clayman GL, Luna MA and
Bat-sakis JG. Microsatellite instability in
preinvasive and invasive head and neck squamous
carcinoma. Am J Pathol 1996;148:2067-72.
67.Ishwad CS PR, Rossie KM, Appel BN, Johnson JT and
Myers EN, et al. Microsatellite instability in oral
cancer. Int J Cancer 1995;64:332-5.
68.Tsung-Yun L. Oxidative damage to DNA induced by
areca nut extract. Mut Res 1996;367:25-31.
69.Chang CL, G. Marra, D. P. Chauhan, H. T. Ha, D. K.
Chang, L. Ricciardiello, A. Randolph, J. M.
Carethers and C. R. Boland. Oxidative stress
inactivates the human DNA mismatch repair system.
Am J Physiol Cell Physiol 2002;283:C148-C54.
70.Aimee L ea. Induction of microsatellite instability
by oxidative DNA damage. Proc Natl Acad Sci USA
1998;95:12486-73.
71.Christoph Gasche CLC, Jennifer Rhees, Ajay Goel
and C. Richard Boland. Oxidative Stress Increases
Frameshift Mutations in Human Colorectal Cancer
Cells. Cancer Res 2001:7444-8.
72.Papadopoulos N, Nicolaides, N. C., Wei, Y. F.,
Ruben, S. M., Carter, K. C., Rosen, C. A.,
Haseltine, W. A., Fleischman, R. D., Fraser, C. M.,
Adams, M. D., Venter, J. C., Hamilton, S. R.,
Peterson, G. M. Watson, P., Lynch, H. 1., Peltomaki,
P., Mecklin J. P., de la Chapelle, A., Kinzler, K. W
and Vogelstein, B. Mutation of a mutL homolog in
hereditary colon cancer. Science 1994;263:1625-9.
73.Denizot F LR. Rapid colorimetric assay for cell
growth and survival. Modifications to the
tetrazolium dye procedure giving improved
sensitivity and reliability. J Immunol Methods
1986;89:271-7.
74.Karin D. Berg CLG, Richard E. Thompson, Stanley R.
Hamilton,, Constance A. Griffin aJRE. Detection of
Microsatellite Instability by Fluorescence Multiplex
Polymerase Chain Reaction. J Mol Diag 2000;2:20-8.
75.Richard BC SNT, Stanley R. Hamilton, David Sidransky,
James R. Eshleman, Randall W. Burt, Stephen J.
Meltzer, Miguel A. Rodriguez-Bigas, Riccardo Fodde,
G. Nadia Ranzani and Sudhir Srivastava. A National
Cancer Institute Workshop on Microsatellite
Instability for Cancer Detection and Familial
Predisposition: Development of International
Criteria for the Determination of Microsatellite
Instability in Colorectal Cancer. Cancer Res
1997;58:5248-57.
76.Karin D. Detection of Microsatellite Instability
by Fluorescence Multiplex Polymerase Chain
Reaction. J Mol Diag 2000;2:20-8.
77.Dietmaier W, Bocker T, Kullman F, Fishel R and
Ruschoff J. Diagnostic microsatellite instability:
definition and correlation with mismatch repair
expression. Cancer Res 1997;57:4749-56.
78.Bocker T, Diermann, J., Friedl, W., Gebert, J..
Holinski-Feder, E., Karner-, Hanusch JvK-DMKKMGS,
H. K. WHC, Fishel, R and Ruschoff. J.
Microsatellite instability analysis: a multicenter
study for reliability and quality control. Cancer
Res 1997;57:4739-43.
79.Ionov Y PA, Malkhosyan S, Shibata D and Perucho M.
Ubiquitous somatic mutations in simple repeated
sequences reveal a new mechanism for colonic
carcinogenesis. Nature 1993;363:558-61.
80.Shibata D, Peinado, M. A., Ionov, Y., Malkhosyan,
S and Perucho, M. Genomic instability in repeated
sequences is an early somatic event on colorectal
tumongenesis that persists after transformation.
Nat Genet 1995;6:273-81.
81.Baylin SB HJ, Graff JR, Vertino PM and Issa JP.
Alterations in DNA methylation: a fundamental
aspect of neoplasia. Adv Cancer Res 1998;72:141-96.
82.Jeng JH, et al. Arecoline cytotoxicity on human
oral mucosal fibroblasts was related to cellular
thiol and esterase activities. Food Chem Toxicol
1999;37:891-7.
83.Jeng JH, et al. Effects of areca nut,
inflorescence piper betle extracts and arecoline
on cytotoxicity, total and unscheduled DNA synthesis
in cultured gingival keratinocytes. J Oral Pathol
Med. 1999;28:64-71.
84.Alex D. Mutations at coding repeat sequences in
mismatch repair-deficient human cancers: Toward a
new concept of target genes for instability. Cancer
Res 2002;62:2447-54.