臺灣博碩士論文加值系統

NG108-15 細胞經thapsigargin (TG)處理後，會耗空鈣庫引起胞內鈣離子上升與
caspase 3 活化且細胞也會死亡(Chin et al.,2002)。本論文沿用NG108-15 細胞
為模式，探討TG 引起之死亡中，胞內鈣離子與caspase 3 活化以及細胞死亡三者的
關係。NG108-15 細胞，經鈣離子螯合劑BAPTA 處理，TG 仍會引起細胞死亡；給予
caspase 3 抑制劑Ac-DEVD-CHO 作用，細胞死亡則會被抑制；若同時給予Ac-DEVD-CHO
與BAPTA，相同地細胞死亡也受到抑制，因此，在NG108-15 中，TG 是透過caspase
3 活化造成細胞死亡，且與胞內鈣濃度上升無關。此現象是否普遍存在於其他細胞？
以豬大動脈平滑細胞進行類似的實驗。給予廣效型capase 抑制劑ZVADfmk 作用，TG
所引起的細胞死亡會被減弱。而單獨給予BAPTA，細胞死亡卻會增加。若同時給予
ZVADfmk 與BAPTA，細胞死亡的情形也會增加。因此，只要有BAPTA，不論有無處理
抑制劑ZVADfmk，細胞死亡都會增加，單獨的給予ZVADfmk 卻能夠降低TG 引起的死
亡程度。因此，豬大動脈平滑肌細胞中，TG 引起的細胞死亡除依賴caspase 活化之
外，也與胞內鈣有密切關係。豬大動脈平滑肌細胞經TG 處理1 小時以後，會造成
cytochrome C 由粒線體位移至cytosol。同時給予BAPTA-AM 及抑制caspase，會促
使更多cytochrome C 位移。此結果與前述細胞死亡之結果一致，所以cytochrome C
位移是造成細胞死亡的原因之一。cytochrome C 為粒線體內膜之週邊蛋白，其與粒
線體內膜結合可能很需要鈣離子參與。一旦給予BAPTA，則將鈣移走，其結合力量
瓦解，致使更多cytochrome C 位移。單獨的抑制caspase，雖然可以使細胞死亡減
弱，但對於TG 引起的cytochrome C 位移，卻未有等比例相對的減弱。TG 也能促使
粒線體穩定的吸收鈣，及使其膜電位瓦解。前者是以Rhod2，後者則是使用TMRM 測
量。TG 促使的粒線體吸收鈣先於膜電位的瓦解，且兩者均可被permeability
transition pore（PTP）之抑制劑，cyclosporin A 抑制。cyclosporin A 也可抑制
TG 誘發的cytochrome C 位移及細胞死亡。所以，豬大動脈平滑肌細胞TG 造成鈣庫
耗空後，會活化caspase，也會活化經由開啟PTP，造成cytochrome c 位移，caspase
與cytochrome C 位移，兩者加成性的造成細胞死亡。

In neuroblastoma x glioma NG108-15 cells, depletion of Ca2+ stores by
thapsigargin caused an increase of the cytosolic Ca2+ concentration ([Ca2+]i),
activation of caspase-3 and cell death. To characterize the relationship among [Ca2+]i
increase, caspase-3 activation and cell death, we determine the effect of caspase-3
inhibitor (DEVD-CHO) and Ca2+ chelator (BAPTA) on thapsigargin-induced cell
death in NG108-15 cells. Cell death was quantified by counting the percent of cells
with DNA fragmentation. The dose of BAPTA and DEVD-CHO used were efficient
to inhibited thapsigargin-induced [Ca2+]i increase and caspase-3 activation,
respectively, while having no effect on caspase-3 activity and [Ca2+]i increase,
respectively. In the presence of DEVD-CHO, cells were protected from
thapsigargin-induced cell death regardless of the presence of BAPTA or not, while
thapsigargin-induced cell death remained the same when [Ca2+]i increase was chelated.
Our data indicate that depletion of Ca2+ stores is required for cell death, but not
sufficient. It further depends on the activation of caspase-3, while being insensitive to
the increase of [Ca2+]i in NG108-15 cells. Similar experiments were undertaken in
cultured porcine aortic smooth muscle cells. Addition of ZVAD-fmk alone (broad
caspases inhibitor) partially attenuated thapsigargin-induced cell death determined by
trypan blue exclusion, while BAPTA enhanced cell death. The effect of BAPTA on the
enhancement of thapsigargin-induced cell death is not relieved by the addition of
ZVAD-fmk. Thus, in porcine aortic smooth muscle cells, thapsigargin-induced cell
death partly attributed to the activation of caspases. Translocation of cytochrome C
from mitochondria fraction to cytosol was observed after treatment of porcine aortic
smooth muscle cells with thapsigargin for one hr. The translocation of cytochrome C
was further enhanced when BAPTA and ZVAD-fmk were present. This is consistent
with thapsigargin-induced cell death indicating that cell death is partly attributed to
the translocation of cytochrome C. Thapsigargin-induced cytochrome C translocation
was not correlated reduced as observed in cell death when BAPTA was applied
simultaneously. BAPTA may remove the Ca2+ that holds cytochrome C with
mitochondrial inner membrane as a peripheral membrane protein. Ca2+ uptake within
mitochondria and the collapse of mitochondrial membrane potential were evoked by
thapsigargin and the former preceded the latter. Both could be inhibited by
cyclosporin A. Cyclosporin A also blocked the translocation of cytochrome C and cell
death induced by thapsigargin. In conclusion, in porcine aortic smooth muscle cells,
after Ca2+ stores depletion induced by thapsigargin, activation of caspases and
translocation of cytochrome C via the opening of permeability transition pores occur.
Activation of caspases and translocation of cytochrome C additively cause cell death.

I
正文目錄
頁次
正文目錄••••••••••••••••••••••I
圖表目錄••••••••••••••••••••••II
縮寫表•••••••••••••••••••••••Ⅲ
中文摘要••••••••••••••••••••••Ⅴ
英文摘要••••••••••••••••••••••VI
緒論••••••••••••••••••••••••1
實驗材料與方法•••••••••••••••••••10
結果••••••••••••••••••••••••17
討論••••••••••••••••••••••••22
參考文獻••••••••••••••••••••••40

40
參考文獻
Baimbridge, K.G., Celio, M.R. and Rogers, J.H. Calcium-binding proteins in the
nervous system. Trends Neurosc, 15: 303-308., 1992.
Barnard, E. A. The transmitter-gated channels: a range of receptor types and
structures. Trends Pharmacol Sci, 17: 305-309., 1996.
Bernardi, P. and Petronilli, V. The permeability transition pore as a mitochondrial
calcium release channel: a critical appraisal. J Bioenerg Biomembr, 28: 131-138.,
1996.
Berridge, M. J. Elementary and global aspects of calcium signalling. J Physiol, 499:
291-306., 1997.
Berridge, M. J. Neuronal calcium signaling. Neuron, 21: 13-26., 1998.
Berridge, M. J., Lipp, P., and Bootman, M. D. The versatility and universality of
calcium signalling. Nat Rev Mol Cell Biol, 1: 11-21., 2000.
Broekemeier, K. M. and Pfeiffer, D. R. Inhibition of the mitochondrial permeability
transition by cyclosporin A during long time frame experiments: relationship between
pore opening and the activity of mitochondrial phospholipases. Biochemistry, 34:
16440-16449., 1995.
Carafoli, E. Biogenesis: plasma membrane calcium ATPase: 15 years of work on the
purified enzyme. Faseb J, 8: 993-1002., 1994.
Carafoli, E. Intracellular calcium homeostasis. Annu Rev Biochem, 56: 395-433,
1987.
Chin, T. Y., Hwang, H. M., and Chueh, S. H. Distinct effects of different
calcium-mobilizing agents on cell death in NG108-15 neuroblastoma X glioma cells.
Mol Pharmacol, 61: 486-494., 2002.
Cryns, V. and Yuan, J. Proteases to die for. Genes Dev, 12: 1551-1570., 1998.
Denton, R. M. and McCormack, J. G. Ca2+ as a second messenger within
mitochondria of the heart and other tissues. Annu Rev Physiol, 52: 451-466, 1990.
Di Lisa, F. and Bernardi, P. Mitochondrial function as a determinant of recovery or
death in cell response to injury. Mol Cell Biochem, 184: 379-391., 1998.
41
Dousa, T. P., Chini, E. N., and Beers, K. W. Adenine nucleotide diphosphates:
emerging second messengers acting via intracellular Ca2+ release. Am J Physiol, 271:
C1007-1024., 1996.
Du, C., Fang, M., Li, Y., Li, L., and Wang, X. Smac, a mitochondrial protein that
promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.
Cell, 102: 33-42., 2000.
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. A
caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature, 391: 43-50., 1998.
Friel, D. D. TRP: its role in phototransduction and store-operated Ca2+ entry. Cell, 85:
617-619., 1996.
Galione, A. Ca(2+)-induced Ca2+ release and its modulation by cyclic ADP-ribose.
Trends Pharmacol Sci, 13: 304-306., 1992.
Ghosh, A. and Greenberg, M. E. Calcium signaling in neurons: molecular
mechanisms and cellular consequences. Science, 268: 239-247., 1995.
Ghosh, T. K., Bian, J., and Gill, D. L. Intracellular calcium release mediated by
sphingosine derivatives generated in cells. Science, 248: 1653-1656., 1990.
Grynkiewicz, G., Poenie, M. and Tsien, R.Y. A new generation of Ca2+ indicators with
greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-50., 1985.
Gunter, T. E., Gunter, K. K., Sheu, S. S., and Gavin, C. E. Mitochondrial calcium
transport: physiological and pathological relevance. Am J Physiol, 267: C313-339.,
1994.
Hamprecht, B. Structural, electrophysiological, biochemical, and pharmacological
properties of neuroblastoma x glioma cell hybrids in cell culture. Int. Rev. Cytol., 49:
99-170., 1977.
Hoth, M. and Penner, R. Depletion of intracellular calcium stores activates a calcium
current in mast cells. Nature, 355: 353-356., 1992.
Khodorov, B., Pinelis, V., Storozhevykh, T., Yuravichus, A., and Khaspekhov, L.
Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the
glutamate-induced calcium response in cultured cerebellar granule cells. FEBS Lett,
458: 162-166., 1999.
42
Korge P. Weiss JN. Thapsigargin directly induces the mitochondrial permeability
transition. European Journal of Biochemistry. 265(1):273-80, 1999
Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T. J.,
Kirschner, M. W., Koths, K., Kwiatkowski, D. J., and Williams, L. T.
Caspase-3-generated fragment of gelsolin: effector of morphological change in
apoptosis. Science, 278: 294-298., 1997.
Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. Structural
determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing
activity. J Biol Chem, 264: 1608-1615., 1989.
Lehr HA. Mankoff DA. Corwin D. Santeusanio G. Gown AM. Application of
photoshop-based image analysis to quantification of hormone receptor expression in
breast cancer. Journal of Histochemistry & Cytochemistry. 45(11):1559-65, 1997
Li, L. Y., Luo, X., and Wang, X. Endonuclease G is an apoptotic DNase when
released from mitochondria. Nature, 412: 95-99., 2001.
Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. Induction of apoptotic
program in cell-free extracts: requirement for dATP and cytochrome c. Cell, 86:
147-157., 1996.
Liu, X., Zou, H., Slaughter, C., and Wang, X. DFF, a heterodimeric protein that
functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.
Cell, 89: 175-184., 1997.
Marks, A. R. Intracellular calcium-release channels: regulators of cell life and death.
Am J Physiol, 272: H597-605., 1997.
Martinou, J. C. and Green, D. R. Breaking the mitochondrial barrier. Nat Rev Mol
Cell Biol, 2: 63-67., 2001.
McPherson, P. S. and Campbell, K. P. The ryanodine receptor/Ca2+ release channel. J
Biol Chem, 268: 13765-13768., 1993.
Medema, J. P., Toes, R. E., Scaffidi, C., Zheng, T. S., Flavell, R. A., Melief, C. J.,
Peter, M. E., Offringa, R., and Krammer, P. H. Cleavage of FLICE (caspase-8) by
granzyme B during cytotoxic T lymphocyte-induced apoptosis. Eur J Immunol, 27:
3492-3498., 1997.
Meir, A., Ginsburg, S., Butkevich, A., Kachalsky, S. G., Kaiserman, I., Ahdut, R.,
Demirgoren, S., and Rahamimoff, R. Ion channels in presynaptic nerve terminals and
control of transmitter release. Physiol Rev, 79: 1019-1088., 1999.
43
Miyamoto, T., Restrepo, D., Cragoe, E. J., Jr., and Teeter, J. H. IP3- and
cAMP-induced responses in isolated olfactory receptor neurons from the channel
catfish. J Membr Biol, 127: 173-183., 1992.
Neher, E. and Augustine, G.J. Calcium gradients and buffers in bovine chromaffin
cells. J. Physiol. 450, 273-301., 1992.
Nelson, P., Christian, C. and Nirenberg, M. Synapse formation between clonal
neuroblastoma X glioma hybrid cells and striated muscle cells. Proc. Nat. Acad.
Sci..73: 123-127., 1976.
Nireberg, M., Wlison, S., Higashida, W., Rotter, A., Krueger, K., Busis, N., Ray, R.,
Kenimer, J.G. and Adler, M. Modulation of synapse formation by cyclic adenosine
monophosphate. Science, 222: 794-799., 1983.
Orth, K., Chinnaiyan, A. M., Garg, M., Froelich, C. J., and Dixit, V. M. The
CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death
substrate lamin A. J Biol Chem, 271: 16443-16446., 1996.
Penner, R., Fasolato, C., and Hoth, M. Calcium influx and its control by calcium
release. Curr Opin Neurobiol, 3: 368-374., 1993.
Philipson, K. D. and Nicoll, D. A. Sodium-calcium exchange. Curr Opin Cell Biol, 4:
678-683., 1992.
Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J. Molecular and cellular physiology
of intracellular calcium stores. Physiol Rev, 74: 595-636., 1994.
Reeves, J. P. Molecular aspects of sodium-calcium exchange. Arch Biochem Biophys,
292: 329-334., 1992.
Rheaume, E., Cohen, L. Y., Uhlmann, F., Lazure, C., Alam, A., Hurwitz, J., Sekaly, R.
P., and Denis, F. The large subunit of replication factor C is a substrate for caspase-3
in vitro and is cleaved by a caspase-3-like protease during Fas- mediated apoptosis.
Embo J, 16: 6346-6354., 1997.
Rosenberg, R. N., Vance, C.K., Morrion, M., Prashad, Neyne, I. and Baskin, F.
Differentiation of neuroblatoma, glioma and hybrid cella as measured by synthesis of
specific protein species: evidence for neuroblast X glioblast reciprocal genetic
regulation. J. Neurochem., 30: 1343-1355., 1978
Ross, R. The smooth muscle cell. II. Growth of smooth muscle in culture and
formation of elastic fibers. J. Cell Biol, 50: 172-86., 1971.
44
Rudel, T. and Bokoch, G. M. Membrane and morphological changes in apoptotic cells
regulated by caspase-mediated activation of PAK2. Science, 276: 1571-1574., 1997.
Saade, G. R., Gray, G., Belfort, M. A., Carpenter, R. J., Jr., and Moise, K. J., Jr.
Ultrasonographic measurement of crown-rump length in high-order multifetal
pregnancies. Ultrasound Obstet Gynecol, 11: 438-444., 1998.
Santella, L. and Carafoli, E. Calcium signaling in the cell nucleus. Faseb J, 11:
1091-1109., 1997.
Stennicke, H. R. and Salvesen, G. S. Properties of the caspases. Biochim Biophys
Acta, 1387: 17-31., 1998.
Stout, A. K., Raphael, H. M., Kanterewicz, B. I., Klann, E., and Reynolds, I. J.
Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat
Neurosci, 1: 366-373., 1998.
Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri, T., Litwack, G.,
Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C.
Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1
beta-converting enzyme-related proteases with distinct substrate recognition
properties are active in apoptosis. Proc Natl Acad Sci U S A, 93: 8395-8400., 1996.
Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri, T., Litwack, G.,
Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C.
Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1
beta-converting enzyme-related proteases with distinct substrate recognition
properties are active in apoptosis. Proc Natl Acad Sci U S A, 93: 8395-8400., 1996.
Tsien, R. W. and Tsien, R. Y. Calcium channels, stores, and oscillations. Annu Rev
Cell Biol, 6: 715-760, 1990.
Wen, L. P., Fahrni, J. A., Troie, S., Guan, J. L., Orth, K., and Rosen, G. D. Cleavage of
focal adhesion kinase by caspases during apoptosis. J Biol Chem, 272: 26056-26061.,
1997.
Wiegand, U. K., Corbach, S., Prescott, A. R., Savill, J., and Spruce, B. A. The trigger
to cell death determines the efficiency with which dying cells are cleared by
neighbours. Cell Death Differ, 8: 734-746., 2001.
Wu, L., Katz, S., and Brown, G. R. Inositol 1,4,5-trisphosphate-, GTP-, arachidonic
acid- and thapsigargin- mediated intracellular calcium movement in PANC-1
microsomes. Cell Calcium, 15: 228-240., 1994.
45
Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., and Birnbaumer, L.
trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+
entry. Cell, 85: 661-671., 1996.
Zoratti, M. and Szabo, I. Electrophysiology of the inner mitochondrial membrane. J
Bioenerg Biomembr, 26: 543-553., 1994.