統計上，慢性的砷暴露和一些疾病的發生有密切關係，如：皮膚
癌、肝癌、膀胱癌、肺癌以及烏腳病等等。在本實驗室前人的研
究中顯示HeLa S3 細胞經過5 μM 亞砷酸鈉處理，會有大約35 %
的細胞停置在有絲分裂中期，而處理staurosporine （蛋白質激酉每
抑制劑）之後，會促使中期停滯細胞離開有絲分裂期。在本研究
中，我們試著觀察亞砷酸鈉所誘導下來的中期停滯細胞經過
staurosporine 作用後，其顯微結構的變化以及蛋白質的修飾作
用。結果顯示亞砷酸鈉所誘引下來的細胞經過staurosporine 處理
之後，染色體以及拉長與濃染的紡綞體會解構、核膜會開始再形
成且會產生多核的現象。除此之外，藉由二維電泳的分析，我們
也觀察到亞砷酸鈉誘引下來的細胞，經過staurosporine 處理後，
其蛋白質表現的改變，然而，這些蛋白質表現的變化仍需要經過
MALDI-TOF 或是二維電泳-西方點墨法做進一步確認。

and air. Epidemiological evidences have revealed that arsenic
exposure is associated with increased incidences of a variety of
diseases, such as Blackfoot Disease, and skin, liver, bladder and lung
cancers. Previous studies in our laboratory have shown that
treatment of HeLa S3 cells with 5 μM arsenite leads to about 35% of
the cells arrested in the mitotic metaphase. Treatment of
arsenite-arrested mitotic metaphase cells with staurosporine (protein
kinase inhibitor) enforces them exiting from mitosis. In this study,
we try to observe changes of ultrastructure and investigate protein
modifications by staurosporine on arsenite-arrested mitotic cells.
Our results show that treatment of arsenite-arrested mitotic cells
with staurosporine leads to dissolution of chromosome and
elongated and condensed spindle, reformation of nuclear envelope
and generation of multiple nuclei. In addition, proteins modified by
staurosporine treatment on arsenite-arrested mitotic cells are also
observed by two-dimentional gel electrophoretic analysis. However,
these proteins should be further identified and characterized by
MALDI-TOF or 2D-Western techniques.

參考文獻
Abe, K., M. Yoshida, T. Usui, S. Horinouchi and T. Beppu (1991)
Highly synchronous culture of fibroblasts from G2 block caused
by staurosporine , a potent inhibitor of protein kinases, Exp Cell
Res, 192, 122-127.
Alberts, A.S., A.M. Thorburn, S. Shenolikar, M.C. Mumby and J.R.
Ferimisco (1993) Regulation of cell cycle progression and
nuclear affinity of the retinblastoma protein by protein
phosphatases, Proc Natl Acad Sci USA, 90, 388-392.
Aposhian, H.V. (1989) Biochemical toxicology of arsenic. Rev
Biochem Toxicol, 10, 265-299.
Barrett, J.C., P.W. Lamb, T.C. Wang and T.C. Lee (1989)
Mechanisms of arsenic-induced cell transformation, Biol Trace
Elem Res, 21, 421-429.
Becker, W.M., J.B. Reece and M.F. Poenic (1996) The World of the
Cell, New York.
Blasina, A., I.V. de Weyer, M.C. Laus, W.H. Luyten, A.E. Parker
and C.H. McGowan (1999) A human homologue of the
checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase,
Curr Biol, 9, 1- 10.
Brady, H.J., M.G. Gil-Gomez, J. Kirberg and A.J. Berns (1996) Bax
alpha perturbs T cell development and affects cell cycle entry of
T cells, EMBO J, 15, 6991-7001.
39
Bruno, S., B. Ardelt, J.S. Skierski, F. Traganos and Z.
Darzynkiewicz (1992) Different effects of staurosporine, an
inhibitor of protein kinases, on the cell cycle and chromatin
structure of normal and leukemic lymphocytes, Cancer Res, 51,
470-473.
Buchet, J.P. and R. Lauwerys (1985) Study of inorganic arsenic
methylation by rat liver in vitro: relevance for the interpretation
of observations in man, Arch Toxicol, 57, 125-129.
Chen, C.J., Y.C. Chuang, T.M. Lin and H.Y. Wu (1985) Malignant
neoplasms among residents of a blackfoot disease-endemic area
in Taiwan: high-arsenic artesian well water and cancers, Cancer
Res, 45, 5895-5899.
Chen, C.J., Y.M. Hsueh, M.S. Lai, M.P. Shyu, S.Y. Chen, M.M. Wu,
T.L. Kuo and T.Y. Tai (1995) Increased prevalence of
hypertension and long-term arsenic exposure, Hypertension, 25,
53-60.
Chen, G.Q., J. Zhu, X.G. Shi, J.H. Ni, H.J. Zhong, G.Y. Si, X.L. Jin,
W. Tang, X.S. Li, S.M. Xong, Z.X. Shen, G.L. Sun, J. Ma, P.
Zhang, T.D. Zhang, C. Gazin, T. Naoe, S.J. Chen, Z.Y. Wang
and Z. Chen (1996) In vitro studies on cellular and molecular
mechanisms of arsenic trioxide (As2O3) in the treatment of
acute promyelocytic leukemia: As2O3 induces NB4 cell
apoptosis with downregulation of Bcl-2 expression and
modulation of PML-RAR alpha/PML proteins, Blood, 88,
1052-1061.
Chen, P.L., P. Scully, J.-Y. Shew, J. W. Wang and W.-H. Lee (1989)
40
Phophorylation of the retinoblastoma gene product is modulated
during the cell cycle and cellular differentiation, Cell, 56,
1193-1198.
Crissman, H.A., D. M. Gadbois, R. A. Tobey and E.M. Bradbury
(1991) Transformed mammalian cells are deficient in
kinase-mediated control of progression through the G1 phase of
the cell cycle, Proc Natl Acad Sci USA, 88, 7580-7584.
Crumpton, N.E., A.J. Hain, R. Jaussi and W. Burkart (1993)
Staurosporine- and radiation-mediated G2-phase cell cycle
blocks are equally released by caffeine, Rediation Res, 135,
372-378.
Davis, P.D., C. Hill, H.E. Keech, G. Lawton, J.S. Nixon and A.D.
Sedgwick (1989) Potent selective inhibitors of protein kinase C,
FEBS Lett, 259, 61-63.
Desai, A. and T.J. Mitchison (1997) Microtubule polymerization
dynamics, Annu Rev Cell Dev Biol, 13, 83-117.
Dopp, E., J. Saedler, H. Stopper, D.G. Weiss and D. Schiffmann
(1995) Mitotic disturbances and micronucleus induction in
Syrian hamster embryo fibroblast cells caused by asbestos fibers,
Environ Health Perspect, 103, 268-271.
Eichenlaub, R.U. and I. Betzandahl (1995) Chloral hydrate induced
spindle aberrations, metaphase I arrest and aneuploidy in mouse
oocytes, Mutagenesis, 10, 477-486.
Erickson, H.P. and E.T. O’Brien (1992) Microtubule dynamic
instability and GTP hydrolysis, Annu Rev Biophys Biomol
Struct, 21, 145-166.
41
Epe, B., U. Harttig, H. Stopper and M. Metzler (1990) Covalent
binding of reactive estrogen metabolites to microtubular protein
as a possible mechanism of aneuploidy induction and neoplastic
cell transformation, Environ Health Perspect, 88, 123-127.
Fan, S.R., I.C. Ho, F.L. Yeoh, C.J. Lin and T.C. Lee (1996) Squalene
inhibits sodium arsenite-induced sister chromatid exchanges and
micronuclei in Chinese hamster ovary-K1 cells, Mutat Res, 368,
165-169.
Fujita-Yamaguchi, Y., and S. Kathuria (1988) Characterization of
receptor tyrosine-specific protein kinases by the use of
inhibitors. Staurosporine is a 100-times more potent inhibitor of
insulin receptor than IGF-Ⅰreceptor, Biochem Biophys Res
Commun, 157, 955-962.
Furukawa, Y., J.A. DeCaprio, A. Freedman, Y. Kanakura, M.
Nakamura, T.J. Ernst, D.M. Livingston and J.D. Griffin (1990)
Expression and state of phosphorylation of the retinoblastoma
susceptibility gene product in cycling and non-cycling human
hematopoietic cells, Proc Natl Acad Sci USA, 87, 2770-2774.
Gorbsky, G.J. (1994) Arsenic in human medicine, Arsenic in the
environment. Part 2:Human health and ecosystem effects, New
York.
Gurley, L.R., R.A. Walters, J.H. Jett and R.A. Tobey (1980)
Response of CHO cell proliferation and histone phosphorylation
to sodium arsenite, J Toxicol Environ Health, 6, 87-105.
Gurr, J.R., Y.C. Lin, I.C. Ho, K.Y. Jan and T.C. Lee (1993) Induction
42
of chromatid breaks and tetraploidy in Chinese hamster ovary
cells by treatment with sodium arsenite during the G2 phase,
Mutat Res, 319, 135-42.
Haldar, S., A. Basu and C.M. Croce (1997) Bcl2 is the guardian of
microtubule integrity, Cancer Res, 57, 229-233.
Hall, L.L., J.P. Th’ng, X. W. Guo, R. L. Teplitz and E.M. Bradbury
(1996) A brief staurosporine treatment of mitotic cells triggers
premature exit from mitosis and polyploidy cell formation,
Cancer Res, 56, 3551-3559.
Hei, T.K., S.X. Liu and C. Waldren (1998) Mutagenicity of arsenic
in mammalian cells: role of reactive oxygen species, Proc Natl
Acad Sci USA, 95, 8103-8107.
Horio, T. and H. Hotani (1986) Visualization of the dynamic
instability of individual microtubules by dark-field microscopy,
Nature, 321, 605-607.
Huang, R.N., I.C. Ho, L.H. Yih and T.C. Lee (1995) Sodium arsenite
induces chromosome endoreduplication and inhibits protein
phosphatase activity in human fibroblasts, Environ Mol
Mutagen, 25, 188-196.
Huang, R.N. and T.C. Lee (1996) Cellular uptake of trivalent
arsenite and pentavalent arsenate in KB cells cultured in
phosphate-free medium, Toxicol Appl Pharmacol, 136,
243-249.
Huang, S.C. and T.C. Lee (1998) Arsenite inhibits mitotic division
and perturbs spindle dynamics in HeLa S3 cells, Carcinogenesis,
19, 889-896.
43
IARC (1980) Carcinogenesis of arsenic of arsenic compounds,
IARC, 23, 37-141.
Karsenti, E., J. Newport, R. Hubble and M. Kirschner (1984)
Interconversion of metaphase and interphase microtubule arrays,
as studied by the injection of centrosomes and nuclei into
Xenopus eggs, J Cell Biol, 98, 1730-1745.
Kawakami, K., H. Futami, J. Takahara and K. Yamaguchi (1996)
UCN-1, 7-hydroxyl-staurosporine, inhibits kinase activity of
cyclin-depentent kinases and reduces the phosphorylation of the
retinoblastoma susceptibility gene product in A549 human lung
cancer cell line, Biochem Biophys Res Commun, 219, 778-783.
Knowles, F.C. (1985) Reactions of lipoamide dehydrogenase and
glutathione reductase with arsonic acids and arsonous acids,
Arch Biochem Biophys, 242, 1-10.
Lee, T.C. and I.C. Ho (1995) Modulation of cellular antioxidant
defense activities by sodium arsenite in human fibroblasts, Arch
Toxicol, 69, 498-504.
Lee, T.C., J.L. Ko and K.Y. Jan (1989) Differential cytotoxicity of
sodium arsenite in human fibroblasts and Chinese hamster
ovary cells, Toxicology, 56, 289-299.
Li, W. and I.N. Chou (1992) Effects of sodium arsenite on the
cytoskeleton and cellular glutathione levels in cultured cells,
Toxicol Appl Pharmacol, 114, 132-139.
Linette, G., P.Y. Li, K. Roth and S.J. Korsmeyer (1996) Cross talk
between cell death and cell cycle progression: BCL-2 regulates
NFAT-mediated activation, Proc Natl Acad Sci USA, 93,
44
9545-9552.
Ling, Y.H., T. Tornos and R. Perez-Soler (1998) Phosphorylation of
Bcl-2 is a marker of M phase events and not a determinant of
apoptosis, J Biol Chem, 273, 18984-18991.
Liu, Y.C. and H. Huang (1997) Involvement of calcium-dependent
protein kinase C in arsenite-induced genotoxicity in Chinese
hamster ovary cells, J Cell Biochem, 64, 423-433.
Ludlow, J.W., J. Shen, J.M. Pipas, D.M. Livingston and J.A.
DeCaprio (1993) Specific enzymatic dephosphoyrylation of the
retinoblastoma protein, Mol Cell Biol, 13, 367-372.
Lynn, S., J.N. Shiung, J.R. Gurr and K.Y. Jan (1998) Arsenite
stimulates poly(ADP-ribosylation) by generation of nitric oxide,
Free Radic Biol Med, 24, 442-449.
Marafante, E., M. Vahter and J. Envall (1985) The role of the
methylation in the detoxication of arsenate in the rabbit, Chem
Biol Interact, 56, 225-238.
Matsuoka, S., M. Huang and S.J. Elledge (1998) Linkage of ATM to
cell cycle regulation by the Chk2 protein kinase, Science, 282,
1893- 1897.
Mazel, S., D. Burtrum and H.T. Petrie (1996) Regulation of cell
division cycle progression by bcl-2 expression: a potential
mechanism for inhibition of programmed cell death, J Exp Med,
183, 2219-2226.
McNally, F., J. (1999) Microtubule dynamics: controlling split ends,
Curr Biol, 9, R274-R276.
45
Michael, A. D. and C.L., Cepko (2000) p57 Kip2 regulates progeni-
tor cell proliferation and amacrine interneuron development in
the mouse retina, Development, 127, 3593-3605.
Mitchison, T., J. and M.W. Kirschner (1984) Dynamic instability of
microtubules growth, Nature, 312, 237-242.
Mitchison, T., J., (1992) Self-organization of polymer-motor systems
in the cytoscheleton, Phil Trans R Soc Lond B, 336, 99-106.
Morton, W.E. and D.A. Dunnette (1994) Health Effect of
Environmental Arsenic, In Arsenic in the environment, New
York.
Murray, A. (1994) Cell cycle checkpoints, Curr Opin Cell Biol, 6,
872-876.
Nelson, K.W. (1977) Industrial contributions of arsenic to the
environment, Environ Health Perspect, 19, 31-34.
Nevins, J.R. (1992) E2F: a link between the RB tumor suppressor
protein and viral oncoproteins, Science, 258, 424-429.
Newton, K. and A. Strasser (1998) The Bcl-2 family and cell death
regulation, Curr Opin Genet Dev, 8, 68-75.
Oberly, T.J., C.E. Piper and D.S. McDonald (1982) Mutagenicity of
metal salts in the L5178Y mouse lymphoma assay, J Toxicol
Environ Health, 9, 367-376.
O’Reilly, L.D., C.S. Huang and A. Strasser (1996) The cell death
inhibitor Bcl-2 and its homologues influence control of cell
cycle entry, EMBO J, 15, 6979-6990.
Peng, C.Y., P.R. Graves, R.S. Thoma, Z. Wu, A.S. Shaw and H.
Piwnica-Worms (1997) Mitotic and G2 checkpoint control:
46
regulation of 14-3-3 protein binding by phosphorylation of
Cdc25C on serine-216, Science, 277, 1501- 1505.
Ramirez, P., D.A. Eastmond, J.P. Laclette and P. Ostrosky Wegman
(1997) Disruption of microtubule assembly and spindle
formation as a mechanism for the induction of aneuploid cells
by sodium arsenite and vanadium pentoxide, Mutat Res, 386,
291-298.
Rein, K.A., B. Borrebaek and J. Bremer (1979) Arsenite inhibits
beta-oxidation in isolated rat liver mitochondria, Biochim
Biophys Acta, 574, 487-494.
Riley, D.J., E.Y.-H.P. Lee and W.-H. Lee (1994) The retinoblastoma
protein: more then a tumor suppressor, Annu Rev Cell Biol, 10,
1-29.
Schnier, J.B., D.M. Gadbois, K. B. Nishi and E. Morton (1994) The
kinase inhibitor staurosporine induces G1 arrest at two points:
Effect on retinoblastoma protein phosphorylation and
cyclin-dependent kinase 2 in normal and trasformed cells,
Cancer Res, 54, 5959-5964.
Seymour, C.B. and C. Mothersill (1988) The effect of glycolysis
inhibitors on the radiation response of CHO-K1 cells, Radiat
Environ Biophys, 27, 49-57.
Shan, B. and W. -H. Lee (1994) Deregulated expression of E2F-1
induces S-phase entry and leads to apoptosis, Mol Cell Biol, 14,
8166-8173.
Sherr, C.J., and J.M. Roberts (1995). Inhibitors of mammalian G1
cyclin-dependent kinases, Genes Dev, 9, 1149-1163.
47
Stokke, T., L. Smedshammer, T.S. Jonassen, H.K. Skarstad, K.
Skarstad and H.B. Steen (1997) Uncoupling of the order of the
S and M phases: effects of staurosporine on human cell cycle
kinases, Cell Prolif, 30, 197-218.
Sweeney, C.M., M. Murphy, S.E. Kubelka, S.E., Ravnik, C.F.
Hawkins, D.J. Wolgemuth and M. Carrington (1996) A distinct
cyclin A is expressed in germ cells in the mouse, Development,
122, 53-64.
Tamaoki, T., H. Nomoto, I. Takahashi, Y. Kato, M. Morimoto and F.
Tomita (1986) Staurosporine, a potent inhibitor of
phospholipid/Ca
++
depentent protein kinase, Biochem Biophys
Res Commun, 135, 397-402.
Terui Y., Y. Furukawa, J. Kikuchi, S. Iwase, K. Hatake and Y. Miura
(1998) Bcl-x is a regulatory factor of apoptosis and
differentiation in megakaryocytic lineage cells, Exp Hematol,
26, 236-244.
Terui, Y., Y. Furukawa, K. Sakoe, M. Ohta and M. Saito (1995)
Expression of differentiation-related phenotypes and apoptosis
are independently regulated during myeloid cell differentiation,
J Biochem, 117, 77-84.
Therman, E. and M. Susman (1993) Human Chromosome: Structure,
Behavior, Effects., New York.
Thompson, D.J. (1993) A chemical hypothesis for arsenic
methylation in mammals, Chem Biol Interact, 88, 89-114.
Tseng, C.H., C.K. Chong, C.J. Chen and T.Y. Tai (1996)
Dose-response relationship between peripheral vascular disease
48
and ingested inorganic arsenic among residents in blackfoot
disease endemic villages in Taiwan, Atherosclerosis, 120,
125-133.
Tseng, W.P. (1977) Effects and dose--response relationships of skin
cancer and blackfoot disease with arsenic, Environ Health
Perspect, 19, 109-119.
Tseng, W.P., H.M. Chu, S.W. How, J.M. Fong, C.S. Lin and S. Yeh
(1968) Prevalence of skin cancer in an endemic area of chronic
arsenicism in Taiwan, J Natl Cancer Inst, 40, 453-463.
Vairo, G.K., M. Innes and J.M. Adams (1996) Bcl-2 has a cell cycle
inhibitory function separable from its enhancement of cell
survival, Oncogen, 13, 1511-1519.
Vig, B.K., M.L. Figueroa, M.N. Cornforth and S.H. Jenkins (1984)
Chromosome studies in human subjects chronically exposed to
arsenic in drinking water, Am J Ind Med, 6, 325-338.
Wen, W.N., T.L. Lieu, H.J. Chang, S.W. Wuu, M.L. Yau and K.Y.
Jan (1981) Baseline and sodium arsenite-induced sister
chromatid exchanges in cultured lymphocytes from patients
with Blackfoot disease and healthy persons, Hum Genet, 59,
201-203.
Yamauchi, H., K. Takahashi, M. Mashiko and Y. Yamamura (1989)
Biological monitoring of arsenic exposure of gallium arsenide-
and inorganic arsenic-exposed workers by determination of
inorganic arsenic and its metabolites in urine and hair, Am Ind
Hyg Assoc J, 50, 606-612.
Yang, R., C. Muller, V. Huynh, Y.K. Fung, A.S. Yee and H.P.
Koeffler (1999) Functions of cyclin A1 in the cell cycle and its
interactions with transcription factor E2F1 and RB family of
proteins, Mol Cell Biol, 19, 2400-2407.
Yee, C., H.I. Krishnan, C.C. Baker, R. Schlegel and P.M. Howley
(1985) Presence and expression of human papillomavirus
sequences in human cervical carcinoma cell lines, Am J Pathol,
119, 361-366.
Yen, A., S. Varvayanis and J.D. Platko (1993)
12-O-tetradecanoyl-phorbol-13-acetate and staurosporine
induce increased retinoblastoma tumor suppressor gene
expression with megakaryocytic differentiation of leukemic
cells, Cancer Res, 53, 3085-3091.
Yih, L.H., H.M. Huang, K.Y. Jan and T.C. Lee (1991) Sodium
arsenite induces ATP depletion and mitochondrial damage in
HeLa cells, Cell Biol Int Rep, 15, 253-264.
Yih, L.H., I.C. Ho and T.C. Lee (1997) Sodium arsenite disturbs
mitosis and induces chromosome loss in human fibroblasts,
Cancer Res, 57, 5051-5059.
Yih, L.H. and T.C. Lee (1999) Effects of exposure protocols on
induction of kinetochore-plus and -minus micronuclei by
arsenite in diploid human fibroblasts, Mutat Res, 440, 75-82.
Zhai, Y., P.J. Kronebusch, P.M. Simon and G.G. Borisy (1996)
Microtubule dynamics at the G2/M transition: abrupt breakdown of
cytoplasmic microtubules at nuclear envelope breakdown and
implications for spindle morphofenesis, J Cell Biol, 135, 201-214.