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Activation of caspases and mitochondria in FTY720-mediated apoptosis in human T cell line Jurkat
International Immunopharmacology 1 Ž2001. 2011–2021 www.elsevier.comrlocaterintimp
Activation of caspases and mitochondria in FTY720-mediated apoptosis in human T cell line Jurkat Masayuki Fujino a,b,1, Xiao-Kang Li a,1, Lei Guo a , Takashi Amano b, Seiichi Suzuki a,) a
Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8509, Japan b Department of Zootechnical Science, Tokyo UniÕersity of Agriculture, Tokyo, Japan Received 10 April 2001; received in revised form 4 June 2001; accepted 13 June 2001
Abstract FTY720, a novel immunosuppressive drug originally derived from a metabolite from Isaria sinclairii, is known to induce apoptosis in lymphocytes. In this study, we investigated the involvement of caspases and mitochondria in FTY720-mediated apoptosis using Jurkat cells, a human T cell line. Our results indicated that FTY720-induced activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not activated. We also observed in the FTY720-treated cells a loss of mitochondrial membrane potential, a release of cytochrome c into cytosol and an exposed phosphatidylserine ŽPS. at the outer surface of the cell membrane. Pretreatment with a peptide inhibitor, benzyloxycarbonyl-Asp-CH 2 COC-2, 6-dichlorobenzene ŽZ-Asp-CH 2-DCB., prevented apoptosis and externalization of phosphatidylserine, whereas the inhibitor did not prevent the mitochondrial events. This suggests that caspases may play a role downstream of the mitochondrial pathway. Therefore, caspase cascade in FTY720-treated cells may be initiated by activation of mitochondria. q 2001 Elsevier Science B.V. All rights reserved. Keywords: FTY720; Apoptosis; Caspase; Mitochondria; Jurkat cells
1. Introduction
AbbreÕiations: Z-Asp-CH 2 -DCB, benzyloxycarbonyl-AspCH 2 COC-2, 6-dichlorobenzene; Ac-YVAD-CHO, acetyl-Try-ValAla-Asp-aldehyde; Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-IETD-CHO, acetyl-Ile-Glu-Thr-Asp-aldehyde; Ac-LEHD-CHO, acetyl-Leu-Glu-His-Asp-aldehyde; PS, phosphatidylserine; PI, propidium iodide; DCm , mitochondrial memX brane potential; DiOC 6 Ž3., 3, 3 -dihexyloxacarbocynine iodide; Ac, acetyl; CHO, aldehyde; pNA, p-nittoanilide. ) Corresponding author. Tel.: q81-3-3414-8121; fax: q81-33411-7309. E-mail address: [email protected] ŽS. Suzuki.. 1 Masayuki Fujino and Xiao-Kang Li contributed equally to this work.
Apoptosis is an active energy-dependent phenomenon that is characterized by morphological changes, including loss of cell volume, plasma membrane blebbing and cell shrinkage, expression of phosphatidylserine ŽPS. on the outer leaflet of the cell membrane, chromatin condensation, and nuclear fragmentation w1x. Caspases, a family of cystein proteases, are responsible for the biochemical and morphological changes associated with apoptosis and play a critical role in its execution phase w2,3x. It has been proposed that AinitiatorB caspases with long prodomains, such as caspases 2 ŽNedd-2rICH1., 8
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 3 0 - 8
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ŽMACHrFLICErMch5., 9 ŽMch6, ICE-LAP6., and 10 ŽMch4., activate AeffectorB caspases directly or indirectly, including caspases 3 ŽYamarCPP32r apopain., 6 ŽMch2., and 7 ŽICE-LAP3rMch3r CMH-1. w2,4,5x. These effector caspases then cleave intracellular substrates, such as ICADrDFF45 w6x and PARP w2x, during the execution phase. Mitochondria have been proposed to act as amplifiers in Fas- and granzyme B-mediated apoptosis; i.e., when activated, caspase 8 cleaves a cytosolic substrate, and cytochrome c is released from the mitochondrial membrane w7–9x. Cytochrome c, which is usually present in the mitochondrial intermembrane space, is released into the cytosol following the induction phase of apoptosis by many stimuli, including receptor-mediated signals ŽFas, tumor necrosis factor receptor ŽTNFR.. and chemotherapeutic agents w10–12x. The release of mitochondrial cytochrome c and activation of caspase 3 in receptor-mediated apoptosis can be blocked by a broadspectrum caspase inhibitor. However, the release of cytochrome c appears to be caspase-independent in apoptosis induced by a chemotherapeutic factor or irradiation, since it is not inhibited by the broadspectrum caspase inhibitor w11,13–15x. FTY720, a synthetic derivative of ISP-I isolated from Isaria sinclairii w16x, is a potent immunosuppressant. It prolonged the survival of skin w17x, heart w18x, and liver w19x allografts in rats, and renal allografts in dogs w20x. The drug also prevented the development of graft-versus-host disease in rats w21x. Furthermore, accelerated lymphocyte apoptosis has been demonstrated to contribute to the potent immunosuppressive effects of FTY720 w22x. Recent reports showed that FTY720 induces cell death selectively in mature T-lymphocyte, especially CD4positive cells, in peripheral blood without a depression of bone marrow w23x. The drug-induced apoptosis was not related to Fas-antigen w24x. It was also demonstrated that FTY720-mediated apoptosis was involved in activation of caspases and mitochondria w25–29x. In the present study, we investigated the mechanism of FTY720-mediated apoptosis in detail using a human T-lymphocyte cell line ŽJurkat cells.. We concluded that FTY720-mediated apoptosis was dependent on the activation of caspases through the activation of mitochondria. The activation of cas-
pases 8 and 10 may not be associated with the pathway of the death receptor but are activated in the amplification loop of apoptosis mediated by FTY720.
2. Materials and methods 2.1. Reagents and antibodies RPMI1640 medium and bovine serum were purchased from Sigma ŽSt. Louis, MO. and Life Technologies ŽRockville, MD., respectively. FTY720 was provided by Yoshitomi Pharmaceutical Industries ŽOsaka, Japan.. We purchased benzyloxycarbonyl-Asp-CH 2 COC-2, 6-dichlorobenzene ŽZ-Asp-CH 2-DCB., acetyl-Try-Val-Ala-Asp-aldehyde ŽAc-YVAD-CHO., acetyl-Asp-Glu-Val-Asp-aldehyde Ž Ac-DEVDCHO., acetyl-Ile-Glu-Thr-Asp-aldehyde ŽAc-IETDCHO., and acetyl-Leu-Glu-His-Asp-aldehyde ŽAcLEHD-CHO. from Peptide Institute ŽOsaka, Japan.. These compounds were dissolved in dimethylsulfoxide ŽDMSO. and then added to the cell culture at a final concentration of 100 m M Ž1% DMSO in the culture medium.. The cells incubated without these compounds were also cultured with 1% DMSO-containing RPMI1640 alone. We also purchased DiOC 6 Ž3. from Molecular Probes ŽEugene, OR., anti-cytochrome c antibody Ž7H8.2 C12. and Annexin-V-FITC from Pharmingen ŽSan Diego, CA., Alexa Fluore 488-conjugated rabbit anti-mouse IgG from Molecular Probes, and Hoechst No. 33342 from Sigma. 2.2. Cell culture Jurkat cells, a human T-lymphocyte cell line, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and kanamycin 75 mgrl. 2.3. Caspase actiÕity assay The activities of caspases 1, 2, 3, 5, 6, 8, 9 and 10 were determined using a colorimetric protease assay kit ŽMBL, Aichi, Japan.. Briefly, a cell pellet was suspended in 50 m l of chilled cell lysis buffer and
M. Fujino et al.r International Immunopharmacology 1 (2001) 2011–2021
incubated on ice for 10 min. After centrifugation at 10,000 = g for 1 min, the supernatant was stored at y7 8C. A proteolytic reaction was carried out in reaction buffers containing 50 m g of cytosolic protein extracts and acetyl-Try-Val-Ala-Asp-pNA ŽAcYVAD-pNA., acetyl-Val-Asp-Val-Ala-Asp-pNA ŽAc-VDVAD-pNA., acetyl-Asp-Glu-Val-Asp-pNA ŽAc-DEVD-pNA ., acetyl-Trp-Glu-His-Asp-pNA ŽAc-WEHD-pNA ., acetyl-Val-Glu-Ile-Asp-pNA ŽAc-VEID-pNA., acetyl-Ile-Glu-Thr-Asp-pNA ŽAcIETD-pNA., acetyl-Leu-Glu-His-Asp-pNA ŽAcLEHD-pNA., and acetyl-Ala-Glu-Val-Asp-pNA ŽAc-AEVD-pNA. at 20 nM each. The reaction mixtures were incubated at 37 8C for 2 h, and the formation of p-nitroanilide was measured at 405 nm using a Wallac1420 ALBOsx microtiter plate reader ŽAmersham Pharmacia Biotech, Tokyo.. 2.4. Determination of protein The protein concentration was determined with a DC protein assay kit ŽBio-Rad, Hercules, CA. using bovine serum albumin as a standard. 2.5. Chromatin condensation and determination of DNA fragmentation We washed the cells with PBS and suspended them in 10 m M of Hoechst 33342. The nuclear morphology was then monitored under a fluorescence microscope ŽOlympus Optical, Tokyo.. DNA extraction and gel electrophoresis were conducted with an ApoLadder EX DNA extraction kit ŽTakara, Shiga, Japan.. TdT-mediated dUTP nick-end labeling ŽTUNEL. was performed using an Apoptosis Screening Kit wako ŽWako, Osaka, Japan. according to the manufacturer’s protocol.
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tion for 15 min in a CO 2 incubator at 37 8C. The stained cells Ž10,000 cells. were analyzed by a fluorescence-activated cell sorter ŽFACSort, Becton Dickinson, San Jose, CA.. Green fluorescence was monitored through a 530r20-nm band-pass filter. 2.7. Immunocytochemistry After cytospin centrifugation Ž1200 rpm, 5 min., the cells were fixed for 2 min with methanolracetone Ž1:1. at room temperature. Thereafter, non-specific binding was blocked by incubation in 4% Block Ace ŽDainippon Pharmaceutical, Tokyo. for 10 min at room temperature. The cells were then washed once with PBS and incubated with anti-cytochrome c antibodies diluted 1:150 in PBS containing 0.4% Block Ace solution for 3 h at 37 8C in a humidified chamber. We removed excess antibody binding by washing the cells with PBS Žthree times, 5 min. and added Alexa Fluore 488-conjugated anti-mouse IgG antibody diluted 1:10 in PBS containing 0.4% Block Ace. One hour after incubation at room temperature with protection against light, the cells were rinsed with PBS for 3 min, incubated for 1 min with PBS containing 500 ngrml of propidium iodide ŽPI., and then washed three times with PBS for 5 min. Finally, we smeared the cells on a glass slide and mounted a cover slip using PERMA FLUOR ŽShandonr Lipshaw, Pittsburgh, PA.. 2.8. Statistical analysis The Student’s t-test was used for comparison of paired and unpaired analysis. p value of - 0.05 was considered statistically significant.
3. Results 2.6. Flow cytometry 3.1. FTY720 induced apoptosis in Jurkat cells An increase in the externalization of PS on the cell surface was detected by incubating with Annexin-V-FITC ŽPharmingen. in a binding buffer Ž10 mM HEPES pH7.4, 140 mM NaCl, 2.5 mM CaCl 2 .. DiOC 6 Ž3. dissolved in DMSO was added to the cells at a final concentration of 40 nM to detect a mitochondrial membrane potential Ž DCm ., The cells were centrifuged and resuspended in PBS after incuba-
We have demonstrated that FTY720 induced apoptosis in vitro using several cell lines and primary cells w22–27,30,31x. In addition, we have vigorously investigated the time course and dose–response of the drug with various cell lines, including Jurkat cells, HL60 cells and DU145 cells w25,26,31x. In the study using Jurkat cells w26x, it has clearly
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demonstrated that FTY720 caused DNA fragmentation in a dose-dependent manner Ž1–10 m M.. Exposure to 10 m M FTY720 resulted in DNA fragmentation beginning at 2 h and reaching maximum at 5 h. Based on this study, we treated Jurkat cells with 10 m M FTY720 for 4 h in the present investigation. The treatment resulted in the appearance of a large number of cells exhibiting typical signs of apoptosis, as similar as previous paper w26x, with May-GrunwaldGiemsa staining Ždata not shown.. The morphology of the apoptotic cells included membrane blebbing, cytoplasmic shrinking, and chromatin condensation. This feature was highlighted by staining with the fluorogenic substrate Hoechst No. 33342 ŽFig. 1A.. Agarose gel electrophoresis of DNA isolated from the FTY720-treated cells showed the typical ladder formation based on internucleosomal fragmentation ŽFig. 1B.. The externalization of PS is known as one of typical findings of apoptosis, which may be related to the clearance of apoptotic cells by phagocytes w32x. Thus, we investigated whether FTY720 increased its externalization. As shown in Fig. 1C, the externalization was markedly increased in the cells by treatment with FTY720. 3.2. ActiÕation of caspases 2, 3, 6, 8, 9 and 10, but not caspases 1 and 5, in FTY720-treated cells Previous studies demonstrated that caspase 3 w25,27x andror caspase 1 w27x were activated in response to FTY720, although the involvement of other caspases was unclear. In this study, we explored the involvement with several caspases in an FTY720-initiated proteolytic cascade. We analyzed the activity of the caspases using caspase-specific substrates Ac-YVAD-pNA Žcaspase 1 ., AcVDVAD-pNA Žcaspase 2., Ac-DEVD-pNA Žcaspase 3., Ac-WEHD-pNA Žcaspase 5., Ac-VEID-pNA Žcaspase 6., Ac-IETD-pNA Žcaspase 8., Ac-LEHDpNA Žcaspase 9., and Ac-AEVD-pNA Žcaspase 10.. The cells were treated with FTY720 and harvested sequentially at different time intervals. DNA fragmentation was observed in a time-dependent manner, as shown in Fig. 2A. In accordance with the induction of apoptosis, FTY720 triggered the activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not processed ŽFig. 2B.. Although Matsuda et al. w27x previously demonstrated that FTY720 acti-
Fig. 1. Characteristics of FTY720 mediated apoptosis in Jurkat cells. The cells were cultured for 4 h with 10 m M FTY720 or without any drug ŽControl.. ŽA. Chromatin condensation of the FTY720-treated cells stained with Hoechst 33342. ŽB. DNA fragmentation on agarose gel electrophoresis. Nuclear DNA was extracted from Jurkat cells incubated with FTY720. ŽC. Externalization of PS. Cells were stained with Annexin-V-FITC. FITCpositive cells were detected by FACS analysis.
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vated caspase 1 to induce apoptosis in WR19L cells, the present study could not observe the activation of caspase 1 in Jurkat cells. Caspases with long prodomains, including caspases 2, 8, 9 and 10, are generally considered to activate upstream in the cell death pathway and are associated with death receptor molecules on the cell surface, such as Fas or TNFR w33,34x. Therefore, this result indicated that FTY720 activates the upstream caspases in the apoptotic pathway in Jurkat cells, whereas caspases 1 and 5 were not activated. 3.3. Inhibition of FTY720-mediated apoptosis by caspase inhibitors We studied the effect of specific caspase inhibitors Ž100 m M. on DNA fragmentation to assess the role of caspases in FTY720-mediated apoptosis. As shown in Fig. 3A, the inhibitors of caspase 3 ŽAc-DEVD-CHO., caspase 8 ŽAc-IETD-CHO., and caspase 9 ŽAc-LEHD-CHO., and a broad spectrum caspase inhibitor ŽZ-Asp-CH 2-DCB. prevented DNA fragmentation, while the caspase 1 inhibitor ŽAcYVAD-CHO. did not. We also observed that Z-AspCH 2-DCB inhibited the externalization of PS ŽFig. 3B.. These observations suggest that FTY720-mediated apoptosis was associated with caspases 3, 8 and 9, but not caspase 1. 3.4. Inhibition of caspases 3 and 8 by inhibitors We and other investigators w26x previously demonstrated the preventive effect of caspase 3 inhibitor Ac-DEVD-CHO on FTY720-mediated apop-
Fig. 2. FTY720-mediated apoptosis and activation of caspases in Jurkat cells. The cells were cultured for 4 h with 10 m M FTY720. ŽA. Kinetics of FTY720-induced apoptosis. After incubation for the indicated times, cells were harvested and DNA fragmentation was determined by gel electrophoresis. ŽB. Caspase activity increased with elapsed time after treatment with FTY720. Specific substrates were as follows: Ac-YVAD-pNA Žcaspase 1., Ac-VDVAD-pNA Žcaspase 2., Ac-DEVD-pNA Žcaspase 3., AcWEHD-pNA Žcaspase 5., Ac-VEID-pNA Žcaspase 6., Ac-IETDpNA Žcaspase 8., Ac-LEHD-pNA Žcaspase 9., and Ac-AEVDpNA Žcaspase 10.. Data are expressed as mean"SE from three different samples. The asterisks indicated p- 0.05 compared with control sample Ž0 h..
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tosis. Caspase 3 belongs to the cluster of effector caspases and plays an important role in the execution phase in receptor-mediated and chemotherapeutic agent-mediated apoptosis. Therefore, we also assessed the preventive effect of various caspase in-
hibitors Ž100 m M. on the activation of caspase 3. The results demonstrated that Ac-DEVD-CHO, AcIETD-CHO, Ac-LEHD-CHO, and Z-Asp-CH 2-DCB decreased caspase 3 activation, whereas Ac-YVADCHO did not ŽFig. 4A.. Caspase 8, an initial and significant caspase activated by death receptors w26x, was also activated in association with FTY720-mediated apoptosis ŽFig. 2B.. We observed that the activation of caspase 8 was prevented by Ac-DEVD-CHO, Ac-IETD-CHO, Ac-LEHD-CHO, and Z-Asp-CH 2-DCB but not by Ac-YVAD-CHO ŽFig. 4B.. In receptor-mediated apoptosis, Ac-IETD-CHO prevented caspase 3 activation and Ac-DEVD-CHO did not prevent caspase 8 activation w35x. Ac-IETD-CHO did not completely prevent the activation of caspase 3 in the FTY720treated cells. In contrast, Ac-DEVD-CHO completely prevented caspase 8 activation. These results indicate that caspase 8 may not be an essential caspase in the induction of apoptosis by FTY720. 3.5. ActiÕation of mitochondria-controlled apoptotic pathway by FTY720 Recent evidence has demonstrated that mitochondria play a key role in the events leading to caspase activation. Alteration of mitochondrial functions, including a loss of DCm , was found to play a major role in the apoptotic process induced by chemotherapeutic agents in the activation of death-associated receptors w36–42x. Mitochondria with a loss of DCm release apoptogenic proteins, such as cytochrome c and an apoptosis-inducing factor ŽAIF., from the intermembrane space into the cytosol, where the proteins can activate caspases and endonucleases
Fig. 3. Effects of caspase inhibitors on apoptosis in Jurkat cells cultured with FTY720. Jurkat cells were preincubated for 1 h with DMSO, Z-Asp-CH 2 -DCB, Ac-YVAD-CHO, Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO and then treated with 10 m M FTY720 for 4 h. ŽA. Apoptosis was evaluated by TUNEL method using Apoptosis Screening Kit wa ko . Data are expressed as mean" SD from three different samples. The asterisks indicated p- 0.05 compared with the DMSO group. ŽB. Apoptosis was also evaluated by the externalization of PS. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution medium for caspase inhibitors, and then cultured with FTY720. Control: Cells incubated without any drug.
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in Fig. 5A, a loss of DCm was observed 0.5 h after the treatment. Cytochrome c forms a complex with the Apaf-1 in cytosol, resulting in a cleavage of procaspase 9 and subsequent activation of other cas-
Fig. 4. Effects of caspase inhibitors on activation of caspases 3 and 8 in Jurkat cells after treatment with FTY720. The cells were preincubated for 1 h with DMSO, Z-Asp-CH 2 -DCB, Ac-YVADCHO, Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO and then cultured with 10 m M FTY720 for 4 h. ŽA. Evaluation of caspase 3 activation. Samples taken at intervals after treatment with FTY720 were examined for caspase 3 activation with its specific substrate. Its activation was assessed 4 h after treatment with FTY720. ŽB. Evaluation of caspase 8 activation. Samples taken at intervals after treatment with FTY720 were examined for caspase 8 activation with its specific substrate. Its activation was assessed 4 h after treatment with FTY720. Data are expressed as mean"SD from three different samples. The asterisks indicated p- 0.05 and the double asterisks indicated p- 0.001 compared with the DMSO group. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution buffer for caspase inhibitors, and then cultured with FTY720. Control: Cells incubated without any drug.
w10,11,13,36,37,40,42–44x. We treated the Jurkat cells with FTY720 at a concentration of 10 m M and harvested them at different time intervals. As shown
Fig. 5. Activation of mitochondria in Jurkat cells after treatment with FTY720. ŽA. Analysis of mitochondria DCm after FTY720treatment. The loss of DCm was determined by staining the FTY720-treated cells with the potential-sensitive fluorochrome DiOC 6 Ž3.. After incubation with FTY720 for the indicated times, the cells were analyzed FACS. Data are expressed as mean"SD. The asterisks indicated p- 0.05 compared with the control group Ž0 h.. ŽB. Release of cytochrome c from mitochondria to cytosol. Jurkat cells were treated with 10 m M FTY720 for 4 h. The cells were stained with monoclonal antibody to cytochrome c Žgreen. and PI for nuclear morphology Žred.. Control: Cells cultured for 4 h in the absence of FTY720.
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pases w10,11,13,36,37,40,42–44x. We demonstrate the translocation of cytochrome c into the cytosol in Jurkat cells at 4 h after FTY720-treatment ŽFig. 5B..
3.6. Caspase inhibition dose not affect mitochondrial eÕents in FTY720-treated cells Since a caspase inhibitor blocks mitochondrial events in receptor-mediated apoptosis, the activation of caspases may induce a loss of DCm , probably via a direct effect on the DCm pore complex w37,45x. This suggests that caspases act both upstream and downstream of the mitochondrial pathway. However, mitochondrial events mediated by non-receptor signals were not blocked by caspase inhibitors. In the present study, a loss of DCm ŽFig. 6A. and a release of cytochrome c ŽFig. 6B. were not blocked by Z-Asp-CH 2-DCB Ž100 m M.. This indicates that FTY720 activates the mitochondrial pathway in a caspase-independent manner.
4. Discussion The potent immunosuppressive effect of FTY720 has been demonstrated in cell culture systems and animal transplantation models w16–21,46–49x. Along with other investigators, we have shown that FTY720 induced apoptosis specifically in lymphocytes both in vitro w26,27,30x and in vivo w22–24x. However, the precise molecular event in FTY720-mediated apoptosis is not yet completely clear. Cystein proteases of the caspase family are important regulators of the apoptotic process w2x. Jurkat cells treated with FTY720 exhibited various features of apoptosis, including chromatin condensation, DNA fragmentation, and externalization of PS ŽFig. 1.. Since apoptosis did not occur when the cells were
Fig. 6. Caspase-independent activation of mitochondria. Jurkat cells preincubated with broad-caspase inhibitor ŽZ-ASP-CH 2 DCB. were cultured with FTY720 Ž10 m M. for 4 h. Data are expressed as mean"SD. ŽA. Effect of the caspase inhibitor on the loss of DCm . We evaluated the loss of DCm after treatment with FTY720 in Jurkat cells preincubated with Z-Asp-CH 2-DCB. ŽB. Effect of the caspase inhibitor on release of cytochrome c from mitochondria to cytosol. After treatment with FTY720, the cells preincubated with Z-Asp-CH 2 -DCB were stained with monoclonal antibody to cytochrome c Žgreen. and PI for nuclear morphology Žred.. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution medium for the caspase inhibitor, and then cultured with FTY720. Control: Cells incubated without any drug.
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pretreated with Z-Asp-CH 2-DCB, a broad-spectrum inhibitor of caspases, it was evidently dependent on caspase activity ŽFig. 3.. In the presence of AcDEVD-CHO Ža caspase 3 inhibitor., Ac-IETD-CHO Ža caspase 8 inhibitor., or Ac-LEHD-CHO Ža caspase 9 inhibitor., FTY720 greatly reduced the ability to induce apoptotic cell death. However, Ac-YVADCHO Ža caspase 1 inhibitor. did not have a preventive effect on FTY720-mediated apoptosis ŽFig. 3., indicating that caspase 1 is not involved. Caspases 2, 3, 6, 8, 9 and 10 were activated in a time-dependent manner when the cells were treated with FTY720 ŽFig. 2B.. It appeared that the caspase cascade occurred first by the activation of the initiator caspases, followed by activation of the effector caspases. The initiators were caspases 2, 8, 9 and 10, which have long prodomains, and the effectors were caspases 3, 6 and 7, with short prodomains. Caspase 8 is activated in the beginning of receptor-mediated apoptosis by binding the adapter molecule to its long prodomain. In contrast, the apoptosis induced by chemotherapeutic agents was initiated via an activation of caspase 9 by binding cytochrome c, ATP, and Apaf-1 to its long prodomain w34x. Our study demonstrated that caspases 8 and 9 were activated almost simultaneously in the treated cells ŽFig. 2.. Thus, it is still unclear which caspases are activated initially in FTY720-associated apoptosis. Ac-IETDCHO and Ac-LEHD-CHO inhibited activation of caspase 3 to prevent apoptosis ŽFig. 3. in Jurkat cells treated with FTY720 ŽFig. 4.. Although caspases 8 and 9 were involved in the FTY720-induced apoptosis, we did not observe an activation of caspases 1 and 5. Furthermore, a specific inhibitor for caspase 1, Ac-YVAD-CHO, did not inhibit the apoptosis or block the activation of the effector caspases, whereas other inhibitors for initiator caspases ŽAc-IETD-CHO and Ac-LEHD-CHO. did. Slee et al. w50x recently showed that cytochrome c initiated activation of caspases, including caspases 2, 3, 6, 7, 8, 9 and 10, and that a complex of ATP, Apaf-1, and cytochrome c activated caspase 9, which then activated caspases 3 and 7. Activated caspase 3 cleaved caspases 2 and 6, followed by cleavage of the initiator caspases, such as caspases 8 and 10, by activated caspase 6. Activated caspase 8 reversely cleaved caspase 3 and Bid, resulting in upregulation of the downstream cascade. These observations sug-
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gest that the caspase cascade constitutes a loop of amplification. Sun et al. demonstrated that Fas-induced release of cytochrome c is caspase-dependent, while chemically induced caspase activation is initiated by the release of cytochrome c from the mitochondria to cytosol. Since a broad caspase inhibitor ŽZ-VAD-FMK. did not prevent a chemically induced release of cytochrome c, caspase activation was not involved in its release w34x. In this study, we demonstrated that FTY720 activated mitochondria, resulting in a loss of mitochondrial membrane potential, and a release of cytochrome c to cytosol ŽFig. 5.. However, caspase activation was not involved in mitochondrial activation ŽFig. 6.. It was also suggested that FTY720mediated apoptosis is not associated with apoptogenic receptors, including Fas and TNFR. This is in agreement with a previous study using an lpr mouse w22,24x. In addition, the drug-treated cells induced a loss of mitochondrial membrane potential and a release of cytochrome c into cytosol. These observations indicate that the direct targets of FTY720 may not be caspases, but mitochondria. This is in agreement with a other report. w28x. Several reports demonstrated the caspase activation and mitochondrial involvement during FTY720-mediated apoptosis w25–28x. Nagahara et al. w28x indicated a loss of mitochondrial membrane potential and a release of cytochrome c were upstream events of caspase activation. In this paper, they studied the activation of caspases 1 and 3, but not other caspases. Therefore, downstream events of caspase activation were still unclear. In the present study, we clearly demonstrated that caspases 2, 3, 6, 8, 9 and 10 are activated downstream of mitochondrial involvement in FTY720-mediated apoptosis. In conclusion, our data demonstrated that the activation of caspases in FTY720-treated Jurkat cells was initiated by cytochrome c released from mitochondria. The activation of initiator caspases may be associated with an amplification loop through a mitochondria-controlled pathway.
Acknowledgements We thank Drs. Hiromitsu Kimura, Shin Enosawa and Torayuki Okuyama for their comments and use-
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ful suggestions. We also thank Naoko Funeshima, Yusuke Kitazawa and Shuhei Fukuda for their technical assistance. This study was supported by grants from Minister of Health and Welfare Ž12KO-2, H12-saisei-016., Minister of Education ŽGrant No. 10307030., and a grant for Organized Research Combination System from the Science and Technology Agency in Japan.
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w14x Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 1998; 17Ž1.:37–49. w15x Zhuang J, Dinsdale D, Cohen GM. Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondria prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential. Cell Death Differ 1998;5Ž11.:953–62. w16x Adachi K, Kohara T, Nakano N, Arita M, Chiba K, et al. Design, synthesis, and structure–activity relationship of 2substituted-2-amino-1,2-propanediols: discovery of a novel immunosuppressant, FTY720. Bioorg Med Chem Lett 1995; 5:853–6. w17x Chiba K, Hoshino Y, Suzuki C, Masubuchi Y, Yanagawa Y, et al. FTY720, a novel immunosuppressant possessing unique mechanisms: I. Prolongation of skin allograft survival and synergistic effect in combination with cyclosporine in rats. Transplant Proc 1996;28Ž2.:1056–9. w18x Hoshino Y, Suzuki C, Ohtsuki M, Masubuchi Y, Amano Y, et al. FTY720, a novel immunosuppressant possessing unique mechanisms: II. Long-term graft survival induction in rat heterotopic cardiac allografts and synergistic effect in combination with cyclosporine A. Transplant Proc 1996;28Ž2.: 1060–1. w19x Suzuki S, Enosawa S, Kakefuda T, Shinomiya T, Amari M, et al. A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Transplantation 1996;61Ž2.: 200–5. w20x Kawaguchi T, Hoshino Y, Rahman F, Amano Y, Higashi H, et al. FTY720, a novel immunosuppressant possessing unique mechanisms: III. Synergistic prolongation of canine renal allograft survival in combination with cyclosporine A. Transplant Proc 1996;28Ž2.:1062–3. w21x Masubuchi Y, Kawaguchi T, Ohtsuki M, Suzuki C, Amano Y, et al. FTY720, a novel immunosuppressant, possessing unique mechanisms: IV. Prevention of graft versus host reactions in rats. Transplant Proc 1996;28Ž2.:1064–5. w22x Li XK, Shinomiya T, Enosawa S, Kakefuda T, Amemiya H, et al. Induction of lymphocyte apoptosis by a novel immunosuppressant FTY720: relation with Fas, Bcl-2 and Bax expression. Transplant Proc 1997;29Ž1–2.:1267–8. w23x Enosawa S, Suzuki S, Kakefuda T, Li XK, Amemiya H. Induction of selective cell death targeting on mature Tlymphocytes in rats by a novel immunosuppressant, FTY720. Immunopharmacology 1996;34Ž2–3.:171–9. w24x Suzuki S, Li XK, Shinomiya T, Enosawa S, Amemiya H, et al. The in vivo induction of lymphocyte apoptosis in MRLlprrlpr mice treated with FTY720. Clin Exp Immunol 1997; 107Ž1.:103–11. w25x Wang JD, Takahara S, Nonomura N, Ichimaru N, Toki K, et al. Early induction of apoptosis in androgen-independent prostate cancer cell line by FTY720 requires caspase-3 activation. Prostate 1999;40Ž1.:50–5. w26x Matsuda S, Minowa A, Suzuki S, Koyasu S. Differential activation of c-Jun NH2-terminal kinase and p38 pathways
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w39x Zamzami N, Susin SA, Marchetti P, Hirsch T, GomezMonterrey I, et al. Mitochondrial control of nuclear apoptosis wsee commentsx. J Exp Med 1996;183Ž4.:1533–44. w40x Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996;184Ž4.:1331–41. w41x Decaudin D, Geley S, Hirsch T, Castedo M, Marchetti P, et al. Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 1997;57Ž1.:62–7. w42x Fulda S, Scaffidi C, Susin SA, Krammer PH, Kroemer G, et al. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem 1998; 273Ž51.:33942–8. w43x Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, et al. Cytochrome c and dATP-dependent formation of Apaf1rcaspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91Ž4.:479–89. w44x Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1998;1366Ž1–2.:151–65. w45x Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med 1998;187Ž8.:1261–71. w46x Suzuki S, Li XK, Shinomiya T, Enosawa S, Kakefuda T, et al. Induction of lymphocyte apoptosis and prolongation of allograft survival by FTY720. Transplant Proc 1996;28Ž4.: 2049–50. w47x Suzuki S, Enosawa S, Kakefuda T, Amemiya H, Hoshino Y, et al. Long-term graft acceptance in allografted rats and dogs by treatment with a novel immunosuppressant, FTY720. Transplant Proc 1996;28Ž3.:1375–6. w48x Suzuki K, Kazui T, Kawabe A, Li XK, Funeshima N, et al. Immunosuppressive effect of FTY 720 on rat pancreas allograft. Transplant Proc 1998;30Ž7.:3417–8. w49x Suzuki T, Shimamura T, Jin MB, Yokota R, Fukai M, et al. Dose-dependent study of a novel immunosuppressant, FTY720, with the canine renal allograft transplantation model. Transplant Proc 1999;31Ž1–2.:1208–9. w50x Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 1999;144Ž2.:281– 92.
Activation of caspases and mitochondria in FTY720-mediated apoptosis in human T cell line Jurkat Masayuki Fujino a,b,1, Xiao-Kang Li a,1, Lei Guo a , Takashi Amano b, Seiichi Suzuki a,) a
Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8509, Japan b Department of Zootechnical Science, Tokyo UniÕersity of Agriculture, Tokyo, Japan Received 10 April 2001; received in revised form 4 June 2001; accepted 13 June 2001
Abstract FTY720, a novel immunosuppressive drug originally derived from a metabolite from Isaria sinclairii, is known to induce apoptosis in lymphocytes. In this study, we investigated the involvement of caspases and mitochondria in FTY720-mediated apoptosis using Jurkat cells, a human T cell line. Our results indicated that FTY720-induced activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not activated. We also observed in the FTY720-treated cells a loss of mitochondrial membrane potential, a release of cytochrome c into cytosol and an exposed phosphatidylserine ŽPS. at the outer surface of the cell membrane. Pretreatment with a peptide inhibitor, benzyloxycarbonyl-Asp-CH 2 COC-2, 6-dichlorobenzene ŽZ-Asp-CH 2-DCB., prevented apoptosis and externalization of phosphatidylserine, whereas the inhibitor did not prevent the mitochondrial events. This suggests that caspases may play a role downstream of the mitochondrial pathway. Therefore, caspase cascade in FTY720-treated cells may be initiated by activation of mitochondria. q 2001 Elsevier Science B.V. All rights reserved. Keywords: FTY720; Apoptosis; Caspase; Mitochondria; Jurkat cells
1. Introduction
AbbreÕiations: Z-Asp-CH 2 -DCB, benzyloxycarbonyl-AspCH 2 COC-2, 6-dichlorobenzene; Ac-YVAD-CHO, acetyl-Try-ValAla-Asp-aldehyde; Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-IETD-CHO, acetyl-Ile-Glu-Thr-Asp-aldehyde; Ac-LEHD-CHO, acetyl-Leu-Glu-His-Asp-aldehyde; PS, phosphatidylserine; PI, propidium iodide; DCm , mitochondrial memX brane potential; DiOC 6 Ž3., 3, 3 -dihexyloxacarbocynine iodide; Ac, acetyl; CHO, aldehyde; pNA, p-nittoanilide. ) Corresponding author. Tel.: q81-3-3414-8121; fax: q81-33411-7309. E-mail address: [email protected] ŽS. Suzuki.. 1 Masayuki Fujino and Xiao-Kang Li contributed equally to this work.
Apoptosis is an active energy-dependent phenomenon that is characterized by morphological changes, including loss of cell volume, plasma membrane blebbing and cell shrinkage, expression of phosphatidylserine ŽPS. on the outer leaflet of the cell membrane, chromatin condensation, and nuclear fragmentation w1x. Caspases, a family of cystein proteases, are responsible for the biochemical and morphological changes associated with apoptosis and play a critical role in its execution phase w2,3x. It has been proposed that AinitiatorB caspases with long prodomains, such as caspases 2 ŽNedd-2rICH1., 8
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 3 0 - 8
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ŽMACHrFLICErMch5., 9 ŽMch6, ICE-LAP6., and 10 ŽMch4., activate AeffectorB caspases directly or indirectly, including caspases 3 ŽYamarCPP32r apopain., 6 ŽMch2., and 7 ŽICE-LAP3rMch3r CMH-1. w2,4,5x. These effector caspases then cleave intracellular substrates, such as ICADrDFF45 w6x and PARP w2x, during the execution phase. Mitochondria have been proposed to act as amplifiers in Fas- and granzyme B-mediated apoptosis; i.e., when activated, caspase 8 cleaves a cytosolic substrate, and cytochrome c is released from the mitochondrial membrane w7–9x. Cytochrome c, which is usually present in the mitochondrial intermembrane space, is released into the cytosol following the induction phase of apoptosis by many stimuli, including receptor-mediated signals ŽFas, tumor necrosis factor receptor ŽTNFR.. and chemotherapeutic agents w10–12x. The release of mitochondrial cytochrome c and activation of caspase 3 in receptor-mediated apoptosis can be blocked by a broadspectrum caspase inhibitor. However, the release of cytochrome c appears to be caspase-independent in apoptosis induced by a chemotherapeutic factor or irradiation, since it is not inhibited by the broadspectrum caspase inhibitor w11,13–15x. FTY720, a synthetic derivative of ISP-I isolated from Isaria sinclairii w16x, is a potent immunosuppressant. It prolonged the survival of skin w17x, heart w18x, and liver w19x allografts in rats, and renal allografts in dogs w20x. The drug also prevented the development of graft-versus-host disease in rats w21x. Furthermore, accelerated lymphocyte apoptosis has been demonstrated to contribute to the potent immunosuppressive effects of FTY720 w22x. Recent reports showed that FTY720 induces cell death selectively in mature T-lymphocyte, especially CD4positive cells, in peripheral blood without a depression of bone marrow w23x. The drug-induced apoptosis was not related to Fas-antigen w24x. It was also demonstrated that FTY720-mediated apoptosis was involved in activation of caspases and mitochondria w25–29x. In the present study, we investigated the mechanism of FTY720-mediated apoptosis in detail using a human T-lymphocyte cell line ŽJurkat cells.. We concluded that FTY720-mediated apoptosis was dependent on the activation of caspases through the activation of mitochondria. The activation of cas-
pases 8 and 10 may not be associated with the pathway of the death receptor but are activated in the amplification loop of apoptosis mediated by FTY720.
2. Materials and methods 2.1. Reagents and antibodies RPMI1640 medium and bovine serum were purchased from Sigma ŽSt. Louis, MO. and Life Technologies ŽRockville, MD., respectively. FTY720 was provided by Yoshitomi Pharmaceutical Industries ŽOsaka, Japan.. We purchased benzyloxycarbonyl-Asp-CH 2 COC-2, 6-dichlorobenzene ŽZ-Asp-CH 2-DCB., acetyl-Try-Val-Ala-Asp-aldehyde ŽAc-YVAD-CHO., acetyl-Asp-Glu-Val-Asp-aldehyde Ž Ac-DEVDCHO., acetyl-Ile-Glu-Thr-Asp-aldehyde ŽAc-IETDCHO., and acetyl-Leu-Glu-His-Asp-aldehyde ŽAcLEHD-CHO. from Peptide Institute ŽOsaka, Japan.. These compounds were dissolved in dimethylsulfoxide ŽDMSO. and then added to the cell culture at a final concentration of 100 m M Ž1% DMSO in the culture medium.. The cells incubated without these compounds were also cultured with 1% DMSO-containing RPMI1640 alone. We also purchased DiOC 6 Ž3. from Molecular Probes ŽEugene, OR., anti-cytochrome c antibody Ž7H8.2 C12. and Annexin-V-FITC from Pharmingen ŽSan Diego, CA., Alexa Fluore 488-conjugated rabbit anti-mouse IgG from Molecular Probes, and Hoechst No. 33342 from Sigma. 2.2. Cell culture Jurkat cells, a human T-lymphocyte cell line, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and kanamycin 75 mgrl. 2.3. Caspase actiÕity assay The activities of caspases 1, 2, 3, 5, 6, 8, 9 and 10 were determined using a colorimetric protease assay kit ŽMBL, Aichi, Japan.. Briefly, a cell pellet was suspended in 50 m l of chilled cell lysis buffer and
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incubated on ice for 10 min. After centrifugation at 10,000 = g for 1 min, the supernatant was stored at y7 8C. A proteolytic reaction was carried out in reaction buffers containing 50 m g of cytosolic protein extracts and acetyl-Try-Val-Ala-Asp-pNA ŽAcYVAD-pNA., acetyl-Val-Asp-Val-Ala-Asp-pNA ŽAc-VDVAD-pNA., acetyl-Asp-Glu-Val-Asp-pNA ŽAc-DEVD-pNA ., acetyl-Trp-Glu-His-Asp-pNA ŽAc-WEHD-pNA ., acetyl-Val-Glu-Ile-Asp-pNA ŽAc-VEID-pNA., acetyl-Ile-Glu-Thr-Asp-pNA ŽAcIETD-pNA., acetyl-Leu-Glu-His-Asp-pNA ŽAcLEHD-pNA., and acetyl-Ala-Glu-Val-Asp-pNA ŽAc-AEVD-pNA. at 20 nM each. The reaction mixtures were incubated at 37 8C for 2 h, and the formation of p-nitroanilide was measured at 405 nm using a Wallac1420 ALBOsx microtiter plate reader ŽAmersham Pharmacia Biotech, Tokyo.. 2.4. Determination of protein The protein concentration was determined with a DC protein assay kit ŽBio-Rad, Hercules, CA. using bovine serum albumin as a standard. 2.5. Chromatin condensation and determination of DNA fragmentation We washed the cells with PBS and suspended them in 10 m M of Hoechst 33342. The nuclear morphology was then monitored under a fluorescence microscope ŽOlympus Optical, Tokyo.. DNA extraction and gel electrophoresis were conducted with an ApoLadder EX DNA extraction kit ŽTakara, Shiga, Japan.. TdT-mediated dUTP nick-end labeling ŽTUNEL. was performed using an Apoptosis Screening Kit wako ŽWako, Osaka, Japan. according to the manufacturer’s protocol.
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tion for 15 min in a CO 2 incubator at 37 8C. The stained cells Ž10,000 cells. were analyzed by a fluorescence-activated cell sorter ŽFACSort, Becton Dickinson, San Jose, CA.. Green fluorescence was monitored through a 530r20-nm band-pass filter. 2.7. Immunocytochemistry After cytospin centrifugation Ž1200 rpm, 5 min., the cells were fixed for 2 min with methanolracetone Ž1:1. at room temperature. Thereafter, non-specific binding was blocked by incubation in 4% Block Ace ŽDainippon Pharmaceutical, Tokyo. for 10 min at room temperature. The cells were then washed once with PBS and incubated with anti-cytochrome c antibodies diluted 1:150 in PBS containing 0.4% Block Ace solution for 3 h at 37 8C in a humidified chamber. We removed excess antibody binding by washing the cells with PBS Žthree times, 5 min. and added Alexa Fluore 488-conjugated anti-mouse IgG antibody diluted 1:10 in PBS containing 0.4% Block Ace. One hour after incubation at room temperature with protection against light, the cells were rinsed with PBS for 3 min, incubated for 1 min with PBS containing 500 ngrml of propidium iodide ŽPI., and then washed three times with PBS for 5 min. Finally, we smeared the cells on a glass slide and mounted a cover slip using PERMA FLUOR ŽShandonr Lipshaw, Pittsburgh, PA.. 2.8. Statistical analysis The Student’s t-test was used for comparison of paired and unpaired analysis. p value of - 0.05 was considered statistically significant.
3. Results 2.6. Flow cytometry 3.1. FTY720 induced apoptosis in Jurkat cells An increase in the externalization of PS on the cell surface was detected by incubating with Annexin-V-FITC ŽPharmingen. in a binding buffer Ž10 mM HEPES pH7.4, 140 mM NaCl, 2.5 mM CaCl 2 .. DiOC 6 Ž3. dissolved in DMSO was added to the cells at a final concentration of 40 nM to detect a mitochondrial membrane potential Ž DCm ., The cells were centrifuged and resuspended in PBS after incuba-
We have demonstrated that FTY720 induced apoptosis in vitro using several cell lines and primary cells w22–27,30,31x. In addition, we have vigorously investigated the time course and dose–response of the drug with various cell lines, including Jurkat cells, HL60 cells and DU145 cells w25,26,31x. In the study using Jurkat cells w26x, it has clearly
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demonstrated that FTY720 caused DNA fragmentation in a dose-dependent manner Ž1–10 m M.. Exposure to 10 m M FTY720 resulted in DNA fragmentation beginning at 2 h and reaching maximum at 5 h. Based on this study, we treated Jurkat cells with 10 m M FTY720 for 4 h in the present investigation. The treatment resulted in the appearance of a large number of cells exhibiting typical signs of apoptosis, as similar as previous paper w26x, with May-GrunwaldGiemsa staining Ždata not shown.. The morphology of the apoptotic cells included membrane blebbing, cytoplasmic shrinking, and chromatin condensation. This feature was highlighted by staining with the fluorogenic substrate Hoechst No. 33342 ŽFig. 1A.. Agarose gel electrophoresis of DNA isolated from the FTY720-treated cells showed the typical ladder formation based on internucleosomal fragmentation ŽFig. 1B.. The externalization of PS is known as one of typical findings of apoptosis, which may be related to the clearance of apoptotic cells by phagocytes w32x. Thus, we investigated whether FTY720 increased its externalization. As shown in Fig. 1C, the externalization was markedly increased in the cells by treatment with FTY720. 3.2. ActiÕation of caspases 2, 3, 6, 8, 9 and 10, but not caspases 1 and 5, in FTY720-treated cells Previous studies demonstrated that caspase 3 w25,27x andror caspase 1 w27x were activated in response to FTY720, although the involvement of other caspases was unclear. In this study, we explored the involvement with several caspases in an FTY720-initiated proteolytic cascade. We analyzed the activity of the caspases using caspase-specific substrates Ac-YVAD-pNA Žcaspase 1 ., AcVDVAD-pNA Žcaspase 2., Ac-DEVD-pNA Žcaspase 3., Ac-WEHD-pNA Žcaspase 5., Ac-VEID-pNA Žcaspase 6., Ac-IETD-pNA Žcaspase 8., Ac-LEHDpNA Žcaspase 9., and Ac-AEVD-pNA Žcaspase 10.. The cells were treated with FTY720 and harvested sequentially at different time intervals. DNA fragmentation was observed in a time-dependent manner, as shown in Fig. 2A. In accordance with the induction of apoptosis, FTY720 triggered the activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not processed ŽFig. 2B.. Although Matsuda et al. w27x previously demonstrated that FTY720 acti-
Fig. 1. Characteristics of FTY720 mediated apoptosis in Jurkat cells. The cells were cultured for 4 h with 10 m M FTY720 or without any drug ŽControl.. ŽA. Chromatin condensation of the FTY720-treated cells stained with Hoechst 33342. ŽB. DNA fragmentation on agarose gel electrophoresis. Nuclear DNA was extracted from Jurkat cells incubated with FTY720. ŽC. Externalization of PS. Cells were stained with Annexin-V-FITC. FITCpositive cells were detected by FACS analysis.
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vated caspase 1 to induce apoptosis in WR19L cells, the present study could not observe the activation of caspase 1 in Jurkat cells. Caspases with long prodomains, including caspases 2, 8, 9 and 10, are generally considered to activate upstream in the cell death pathway and are associated with death receptor molecules on the cell surface, such as Fas or TNFR w33,34x. Therefore, this result indicated that FTY720 activates the upstream caspases in the apoptotic pathway in Jurkat cells, whereas caspases 1 and 5 were not activated. 3.3. Inhibition of FTY720-mediated apoptosis by caspase inhibitors We studied the effect of specific caspase inhibitors Ž100 m M. on DNA fragmentation to assess the role of caspases in FTY720-mediated apoptosis. As shown in Fig. 3A, the inhibitors of caspase 3 ŽAc-DEVD-CHO., caspase 8 ŽAc-IETD-CHO., and caspase 9 ŽAc-LEHD-CHO., and a broad spectrum caspase inhibitor ŽZ-Asp-CH 2-DCB. prevented DNA fragmentation, while the caspase 1 inhibitor ŽAcYVAD-CHO. did not. We also observed that Z-AspCH 2-DCB inhibited the externalization of PS ŽFig. 3B.. These observations suggest that FTY720-mediated apoptosis was associated with caspases 3, 8 and 9, but not caspase 1. 3.4. Inhibition of caspases 3 and 8 by inhibitors We and other investigators w26x previously demonstrated the preventive effect of caspase 3 inhibitor Ac-DEVD-CHO on FTY720-mediated apop-
Fig. 2. FTY720-mediated apoptosis and activation of caspases in Jurkat cells. The cells were cultured for 4 h with 10 m M FTY720. ŽA. Kinetics of FTY720-induced apoptosis. After incubation for the indicated times, cells were harvested and DNA fragmentation was determined by gel electrophoresis. ŽB. Caspase activity increased with elapsed time after treatment with FTY720. Specific substrates were as follows: Ac-YVAD-pNA Žcaspase 1., Ac-VDVAD-pNA Žcaspase 2., Ac-DEVD-pNA Žcaspase 3., AcWEHD-pNA Žcaspase 5., Ac-VEID-pNA Žcaspase 6., Ac-IETDpNA Žcaspase 8., Ac-LEHD-pNA Žcaspase 9., and Ac-AEVDpNA Žcaspase 10.. Data are expressed as mean"SE from three different samples. The asterisks indicated p- 0.05 compared with control sample Ž0 h..
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tosis. Caspase 3 belongs to the cluster of effector caspases and plays an important role in the execution phase in receptor-mediated and chemotherapeutic agent-mediated apoptosis. Therefore, we also assessed the preventive effect of various caspase in-
hibitors Ž100 m M. on the activation of caspase 3. The results demonstrated that Ac-DEVD-CHO, AcIETD-CHO, Ac-LEHD-CHO, and Z-Asp-CH 2-DCB decreased caspase 3 activation, whereas Ac-YVADCHO did not ŽFig. 4A.. Caspase 8, an initial and significant caspase activated by death receptors w26x, was also activated in association with FTY720-mediated apoptosis ŽFig. 2B.. We observed that the activation of caspase 8 was prevented by Ac-DEVD-CHO, Ac-IETD-CHO, Ac-LEHD-CHO, and Z-Asp-CH 2-DCB but not by Ac-YVAD-CHO ŽFig. 4B.. In receptor-mediated apoptosis, Ac-IETD-CHO prevented caspase 3 activation and Ac-DEVD-CHO did not prevent caspase 8 activation w35x. Ac-IETD-CHO did not completely prevent the activation of caspase 3 in the FTY720treated cells. In contrast, Ac-DEVD-CHO completely prevented caspase 8 activation. These results indicate that caspase 8 may not be an essential caspase in the induction of apoptosis by FTY720. 3.5. ActiÕation of mitochondria-controlled apoptotic pathway by FTY720 Recent evidence has demonstrated that mitochondria play a key role in the events leading to caspase activation. Alteration of mitochondrial functions, including a loss of DCm , was found to play a major role in the apoptotic process induced by chemotherapeutic agents in the activation of death-associated receptors w36–42x. Mitochondria with a loss of DCm release apoptogenic proteins, such as cytochrome c and an apoptosis-inducing factor ŽAIF., from the intermembrane space into the cytosol, where the proteins can activate caspases and endonucleases
Fig. 3. Effects of caspase inhibitors on apoptosis in Jurkat cells cultured with FTY720. Jurkat cells were preincubated for 1 h with DMSO, Z-Asp-CH 2 -DCB, Ac-YVAD-CHO, Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO and then treated with 10 m M FTY720 for 4 h. ŽA. Apoptosis was evaluated by TUNEL method using Apoptosis Screening Kit wa ko . Data are expressed as mean" SD from three different samples. The asterisks indicated p- 0.05 compared with the DMSO group. ŽB. Apoptosis was also evaluated by the externalization of PS. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution medium for caspase inhibitors, and then cultured with FTY720. Control: Cells incubated without any drug.
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in Fig. 5A, a loss of DCm was observed 0.5 h after the treatment. Cytochrome c forms a complex with the Apaf-1 in cytosol, resulting in a cleavage of procaspase 9 and subsequent activation of other cas-
Fig. 4. Effects of caspase inhibitors on activation of caspases 3 and 8 in Jurkat cells after treatment with FTY720. The cells were preincubated for 1 h with DMSO, Z-Asp-CH 2 -DCB, Ac-YVADCHO, Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO and then cultured with 10 m M FTY720 for 4 h. ŽA. Evaluation of caspase 3 activation. Samples taken at intervals after treatment with FTY720 were examined for caspase 3 activation with its specific substrate. Its activation was assessed 4 h after treatment with FTY720. ŽB. Evaluation of caspase 8 activation. Samples taken at intervals after treatment with FTY720 were examined for caspase 8 activation with its specific substrate. Its activation was assessed 4 h after treatment with FTY720. Data are expressed as mean"SD from three different samples. The asterisks indicated p- 0.05 and the double asterisks indicated p- 0.001 compared with the DMSO group. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution buffer for caspase inhibitors, and then cultured with FTY720. Control: Cells incubated without any drug.
w10,11,13,36,37,40,42–44x. We treated the Jurkat cells with FTY720 at a concentration of 10 m M and harvested them at different time intervals. As shown
Fig. 5. Activation of mitochondria in Jurkat cells after treatment with FTY720. ŽA. Analysis of mitochondria DCm after FTY720treatment. The loss of DCm was determined by staining the FTY720-treated cells with the potential-sensitive fluorochrome DiOC 6 Ž3.. After incubation with FTY720 for the indicated times, the cells were analyzed FACS. Data are expressed as mean"SD. The asterisks indicated p- 0.05 compared with the control group Ž0 h.. ŽB. Release of cytochrome c from mitochondria to cytosol. Jurkat cells were treated with 10 m M FTY720 for 4 h. The cells were stained with monoclonal antibody to cytochrome c Žgreen. and PI for nuclear morphology Žred.. Control: Cells cultured for 4 h in the absence of FTY720.
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pases w10,11,13,36,37,40,42–44x. We demonstrate the translocation of cytochrome c into the cytosol in Jurkat cells at 4 h after FTY720-treatment ŽFig. 5B..
3.6. Caspase inhibition dose not affect mitochondrial eÕents in FTY720-treated cells Since a caspase inhibitor blocks mitochondrial events in receptor-mediated apoptosis, the activation of caspases may induce a loss of DCm , probably via a direct effect on the DCm pore complex w37,45x. This suggests that caspases act both upstream and downstream of the mitochondrial pathway. However, mitochondrial events mediated by non-receptor signals were not blocked by caspase inhibitors. In the present study, a loss of DCm ŽFig. 6A. and a release of cytochrome c ŽFig. 6B. were not blocked by Z-Asp-CH 2-DCB Ž100 m M.. This indicates that FTY720 activates the mitochondrial pathway in a caspase-independent manner.
4. Discussion The potent immunosuppressive effect of FTY720 has been demonstrated in cell culture systems and animal transplantation models w16–21,46–49x. Along with other investigators, we have shown that FTY720 induced apoptosis specifically in lymphocytes both in vitro w26,27,30x and in vivo w22–24x. However, the precise molecular event in FTY720-mediated apoptosis is not yet completely clear. Cystein proteases of the caspase family are important regulators of the apoptotic process w2x. Jurkat cells treated with FTY720 exhibited various features of apoptosis, including chromatin condensation, DNA fragmentation, and externalization of PS ŽFig. 1.. Since apoptosis did not occur when the cells were
Fig. 6. Caspase-independent activation of mitochondria. Jurkat cells preincubated with broad-caspase inhibitor ŽZ-ASP-CH 2 DCB. were cultured with FTY720 Ž10 m M. for 4 h. Data are expressed as mean"SD. ŽA. Effect of the caspase inhibitor on the loss of DCm . We evaluated the loss of DCm after treatment with FTY720 in Jurkat cells preincubated with Z-Asp-CH 2-DCB. ŽB. Effect of the caspase inhibitor on release of cytochrome c from mitochondria to cytosol. After treatment with FTY720, the cells preincubated with Z-Asp-CH 2 -DCB were stained with monoclonal antibody to cytochrome c Žgreen. and PI for nuclear morphology Žred.. DMSO: Cells that were preincubated only with 1% DMSO-containing RPMI1640, a solution medium for the caspase inhibitor, and then cultured with FTY720. Control: Cells incubated without any drug.
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pretreated with Z-Asp-CH 2-DCB, a broad-spectrum inhibitor of caspases, it was evidently dependent on caspase activity ŽFig. 3.. In the presence of AcDEVD-CHO Ža caspase 3 inhibitor., Ac-IETD-CHO Ža caspase 8 inhibitor., or Ac-LEHD-CHO Ža caspase 9 inhibitor., FTY720 greatly reduced the ability to induce apoptotic cell death. However, Ac-YVADCHO Ža caspase 1 inhibitor. did not have a preventive effect on FTY720-mediated apoptosis ŽFig. 3., indicating that caspase 1 is not involved. Caspases 2, 3, 6, 8, 9 and 10 were activated in a time-dependent manner when the cells were treated with FTY720 ŽFig. 2B.. It appeared that the caspase cascade occurred first by the activation of the initiator caspases, followed by activation of the effector caspases. The initiators were caspases 2, 8, 9 and 10, which have long prodomains, and the effectors were caspases 3, 6 and 7, with short prodomains. Caspase 8 is activated in the beginning of receptor-mediated apoptosis by binding the adapter molecule to its long prodomain. In contrast, the apoptosis induced by chemotherapeutic agents was initiated via an activation of caspase 9 by binding cytochrome c, ATP, and Apaf-1 to its long prodomain w34x. Our study demonstrated that caspases 8 and 9 were activated almost simultaneously in the treated cells ŽFig. 2.. Thus, it is still unclear which caspases are activated initially in FTY720-associated apoptosis. Ac-IETDCHO and Ac-LEHD-CHO inhibited activation of caspase 3 to prevent apoptosis ŽFig. 3. in Jurkat cells treated with FTY720 ŽFig. 4.. Although caspases 8 and 9 were involved in the FTY720-induced apoptosis, we did not observe an activation of caspases 1 and 5. Furthermore, a specific inhibitor for caspase 1, Ac-YVAD-CHO, did not inhibit the apoptosis or block the activation of the effector caspases, whereas other inhibitors for initiator caspases ŽAc-IETD-CHO and Ac-LEHD-CHO. did. Slee et al. w50x recently showed that cytochrome c initiated activation of caspases, including caspases 2, 3, 6, 7, 8, 9 and 10, and that a complex of ATP, Apaf-1, and cytochrome c activated caspase 9, which then activated caspases 3 and 7. Activated caspase 3 cleaved caspases 2 and 6, followed by cleavage of the initiator caspases, such as caspases 8 and 10, by activated caspase 6. Activated caspase 8 reversely cleaved caspase 3 and Bid, resulting in upregulation of the downstream cascade. These observations sug-
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gest that the caspase cascade constitutes a loop of amplification. Sun et al. demonstrated that Fas-induced release of cytochrome c is caspase-dependent, while chemically induced caspase activation is initiated by the release of cytochrome c from the mitochondria to cytosol. Since a broad caspase inhibitor ŽZ-VAD-FMK. did not prevent a chemically induced release of cytochrome c, caspase activation was not involved in its release w34x. In this study, we demonstrated that FTY720 activated mitochondria, resulting in a loss of mitochondrial membrane potential, and a release of cytochrome c to cytosol ŽFig. 5.. However, caspase activation was not involved in mitochondrial activation ŽFig. 6.. It was also suggested that FTY720mediated apoptosis is not associated with apoptogenic receptors, including Fas and TNFR. This is in agreement with a previous study using an lpr mouse w22,24x. In addition, the drug-treated cells induced a loss of mitochondrial membrane potential and a release of cytochrome c into cytosol. These observations indicate that the direct targets of FTY720 may not be caspases, but mitochondria. This is in agreement with a other report. w28x. Several reports demonstrated the caspase activation and mitochondrial involvement during FTY720-mediated apoptosis w25–28x. Nagahara et al. w28x indicated a loss of mitochondrial membrane potential and a release of cytochrome c were upstream events of caspase activation. In this paper, they studied the activation of caspases 1 and 3, but not other caspases. Therefore, downstream events of caspase activation were still unclear. In the present study, we clearly demonstrated that caspases 2, 3, 6, 8, 9 and 10 are activated downstream of mitochondrial involvement in FTY720-mediated apoptosis. In conclusion, our data demonstrated that the activation of caspases in FTY720-treated Jurkat cells was initiated by cytochrome c released from mitochondria. The activation of initiator caspases may be associated with an amplification loop through a mitochondria-controlled pathway.
Acknowledgements We thank Drs. Hiromitsu Kimura, Shin Enosawa and Torayuki Okuyama for their comments and use-
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ful suggestions. We also thank Naoko Funeshima, Yusuke Kitazawa and Shuhei Fukuda for their technical assistance. This study was supported by grants from Minister of Health and Welfare Ž12KO-2, H12-saisei-016., Minister of Education ŽGrant No. 10307030., and a grant for Organized Research Combination System from the Science and Technology Agency in Japan.
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