In TCR-Stimulated Thymocytes Induction of Chromosomal DNA Degradation Caspase-Activated Deoxyribonuclease in the Possible Involv

Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes This information is current as of September 28, 2021. Takuya Nagata, Hiroyuki Kishi, Qing Li Liu, Tomoyasu Yoshino, Tadashi Matsuda, Zhe Xiong Jin, Kimie Murayama, Kazuhiro Tsukada and Atsushi Muraguchi J Immunol 2000; 165:4281-4289; ; doi: 10.4049/jimmunol.165.8.4281 Downloaded from http://www.jimmunol.org/content/165/8/4281

References This article cites 42 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/8/4281.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes1

Takuya Nagata,* Hiroyuki Kishi,* Qing Li Liu,* Tomoyasu Yoshino,* Tadashi Matsuda,* Zhe Xiong Jin,* Kimie Murayama,‡ Kazuhiro Tsukada,† and Atsushi Muraguchi2*

TCR engagement of immature CD4؉CD8؉ thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identiﬁed two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated

DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. Downloaded from We puriﬁed the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B speciﬁcally inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and http://www.jimmunol.org/ Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes. The Journal of Immunology, 2000, 165: 4281–4289.

n the thymus, CD4ϩCD8ϩ thymocytes expressing low levels DNA fragmentation, occur (10). Caspases play an inevitable role of ␣␤-TCR are subjected to both positive and negative se- in an initiation phase as well as an effector phase of apoptosis. I lection events (1). Positive selection ensures the survival and Initiator caspases (caspases 8, 9, and 10) cleave and activate ef- differentiation of cells capable of recognizing foreign Ag in the fector caspases (caspases 3, 6, and 7). Mitochondria play an im- context of self-MHC, whereas negative selection eliminates im- portant role in the activation of caspases. Some types of apoptotic mature thymocytes expressing self-reactive TCRs by the induction stimuli induce dysregulation of the mitochondrial transmembrane by guest on September 28, 2021 ⌬⌿ of apoptosis. It is generally believed that the avidity of the inter- potential ( m) and the release of cytochrome c from the inter- action between their TCR and the MHC/peptide complex deter- membrane space (11). Free cytochrome c, making complexes with mines the fates of thymocytes for positive or negative selection (2, caspase 9 and Apaf-1, activates caspase 3 (12). The resulted acti- 3). Concerning molecules or signal transduction pathways leading vated caspases, in turn, cleave multiple cytoplasmic and nuclear to positive or negative selection of thymocytes, it was reported that substrates (13). DNA fragmentation factor 40 (DFF40)3/caspase- the ZAP-70 and Vav are essential for both positive and negative activated DNase (CAD) exists as a complex with DFF45/inhibitor selection (4, 5). Furthermore, it was reported that the Ras/Raf/ of CAD (ICAD) in the normal cell, and when activated caspase 3 mitogen-activated kinase kinase 1/extracellular regulated kinase cleaves DFF45/ICAD, DFF40/CAD is released as an active form pathway and the calcineurin pathway are necessary for positive and induces nuclear condensation and DNA fragmentation (14, selection (6, 7), whereas the mitogen-activated kinase kinase 6/p38 15). However, mice deﬁcient in the genes encoding the above- pathway and c-Jun N-terminal kinase may be involved in the neg- mentioned apoptosis-inducing molecules showed no defect of negative selection of thymocytes (8, 9). How these pathways lead to ative selection of thymocytes (16), suggesting that an alternative the distinct fates of thymocytes is still unclear. signaling pathway(s) for negative selection of self-reactive-thymo- In apoptotic cells, multiple structural changes, such as plasma cytes may exist. and nuclear membrane blebbing, chromatin condensation, and It has been reported that stimulation of the CD3/TCR complex of immature thymocytes with anti-CD3 mAb induces DNA degradation and cell death through the endogenous pathway of apo- † *Department of Immunology and Second Department of Surgery, Faculty of Med- ptosis (17). In this study we show that in vivo stimulation of thy- icine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan; and ‡Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Jyun- mocytes with anti-CD3 mAb or a natural ligand such as OVA in tendo University School of Medicine, Tokyo, Japan DO11.10 TCR-transgenic mice generates activities that cause ap- Received for publication May 1, 2000. Accepted for publication July 24, 2000. optotic changes and chromosomal DNA fragmentation of naked The costs of publication of this article were defrayed in part by the payment of page nuclei as well as the activity to degrade plasmid DNA. We puriﬁed charges. This article must therefore be hereby marked advertisement in accordance the molecule that is responsible for plasmid DNA degradation with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. 3 Abbreviations used in this paper: DFF40, DNA fragmentation factor 40; CAD, 2 Address correspondence and reprint requests to Dr. Atsushi Muraguchi, Department caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; Cyp B, cy- Ј of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, clophilin B; CEB, cell extract buffer; DiOC6(3), 3,3 -dihexyloxacarbocyanine iodide; 2630 Sugitani, Toyama 930-0194, Japan. E-mail address: [email protected] LS100-s, LS100 from anti-CD3-stimulated thymocytes.

from the cytosol of in vivo anti-CD3-stimulated thymocytes. De- mM EDTA, 2 M sucrose, and 10% (v/v) glycerol), and homogenized using termination of the N-terminal amino acid sequence revealed that a motor-driven 30 ml Teflon-glass homogenizer until Ͼ90% of the cells its sequence is identical with that of cyclophilin B (Cyp B), a were enucleated. The homogenate was diluted to 85 ml with homogenization buffer, layered in three 27-ml aliquots over three 10-ml cushions of the member of cyclophilins that normally localizes in microsome frac- same buffer, and centrifuged at 24,000 rpm for 30 min at 4°C in an SW28 tion. Our data indicate that stimulation of thymocytes with anti- rotor (Beckman Instruments, Palo Alto, CA). The combined nuclear pellets CD3 mAb induces activation of CAD, which is responsible for were resuspended in 50 ml of a mixture of homogenization buffer and generation of internucleosomal DNA fragmentation, as well as the glycerol (9/1, v/v), using a Teflon-glass homogenizer. This homogenate was layered over two 10-ml cushions as described above and centrifuged release of Cyp B from the microsome to the cytosolic/nuclear frac- under the same conditions. Pelleted nuclei were resuspended in 0.5 ml of tion, which directly or indirectly causes chromosomal DNA deg- nuclei storage buffer (10 mM PIPES (pH 7.40, 80 mM KCl, 20 mM NaCl, radation. Thus, our results pose the possibility that active CAD and 250 mM sucrose, 5 mM EGTA, 1 mM DTT, 0.5 mM spermidine, 0.2 mM Cyp B function in harmony on the cell death of TCR-stimulated spermine, and 50% (v/v) glycerol) at a concentration of 1 ϫ 106 nuclei/␮l Ϫ thymocytes. and stored at 80°C. Assay of apoptosis-inducing activity Materials and Methods To investigate the nuclear DNA fragmentation-inducing activity, reaction Mice and Ab buffer (1 mM HEPES (pH 7.0), 4 mM ␤-glycerophosphate, 5 mM NaCl, 2 ␮ ICR mice (4 wk old), C57BL/6 mice (8 wk old), and DO11.10 mice (4 wk mM ATP, 1 mM creatine phosphate, and 5 g/ml creatine kinase), various amounts of the cell fractions, CEB, and 2 ϫ 106 nuclei in a final volume old; a gift from Dr. D. Y. Loh, Nippon Roche, Kamakura, Japan) were bred ␮ in our animal facility. Anti-mouse CD3⑀ Ab (clone 145-2C11) was purified of 200 l were incubated at 37°C for various time periods. After incubation, nuclei were collected by centrifugation for 10 min at 10,000 ϫ g, then from the culture supernatant of hybridoma cells by protein A-Sepharose column. ␮ Downloaded from Anti-mouse Cyp B Ab was a gift from Dr. J. G. Sutcliffe (Research Institute of resuspended in 20 l of resuspension buffer (50 mM Tris-HCl (pH 8.0), 10 Scripps Clinic, La Jolla, CA), and anti-mouse CD28 Ab (clone PV-1) was pro- mM EDTA, 0.5% (w/v) sodium lauroyl sarkosinate, and 0.5 mg/ml provided by Dr. R. Abe (Science University of Tokyo, Noda, Japan). teinase K), and incubated at 50°C for 2 h. Ten microliters (0.5 mg/ml) of RNase A was added to each sample and incubated at 50°C for an additional Analysis of apoptosis by flow cytometry 2 h. Samples were heated to 70°C, and 10 ␮l of 1% (w/v) low gelling temperature agarose containing 10 mM EDTA (pH 8.0), 0.25% (w/v) bro- Anti-CD3-stimulated or nonstimulated thymocytes (4 ϫ 105/ml) were in- mophenol blue, and 40% (w/v) sucrose was mixed with each sample before

cubated with FITC-conjugated annexin V (1 ␮g/ml; Sigma, St. Louis, MO) loading into the dry wells of a 2% (w/v) agarose gel containing 0.1 ␮g/ml http://www.jimmunol.org/ for 15 min at room temperature or with 3,3Ј-dihexyloxacarbocyanine io- ethidium bromide. To investigate plasmid DNA degradation activity, var- dide (DiOC6(3); 40 nM; Aldrich, Milwaukee, WI) for 15 min at 37°C. ious amounts of the cell fractions and 1 ␮g plasmid DNA in a final volume Stained cells were analyzed by FACSCalibur (Becton Dickinson, San Jose, of 20 ␮l were incubated at 37°C for 30 min and assayed with 1.5% (w/v) CA). Acquisition of data was performed without gating on forward light agarose gel electrophoresis. DNA fragmentation- and DNA degradation- scatter. A minimum of 104 events were acquired for each sample. The inducing activities were assessed by the analysis of densitograph (Atto, staining pattern of the majority of nonstimulated thymocytes was regarded Tokyo, Japan). For examining the morphology of apoptotic nuclei, 2 ϫ 106 high as annexin V negative and DiOC6(3) (live cell), and the percentage of nuclei were incubated with the cell lysates, and an aliquot (6 ␮l) of the low the annexin V-positive, DiOC6(3) cell population (apoptotic cells) was nuclei was stained with 10 ␮g/ml 4,6-diamino-2-phenylindole (Sigma) in calculated. 200 mM sucrose, 5 mM MgCl2, 15 mM PIPES (pH 7.4), 80 mM KCl, 15 mM NaCl, 5 mM EDTA, and 3.7% (v/v) formaldehyde. The nuclei were Preparation of thymocyte cell extracts observed under a fluorescence microscope (Olympus, Tokyo, Japan). by guest on September 28, 2021 Fifty micrograms of anti-CD3 mAb or control IgG was i.p. injected into Construction of expression vectors ICR mice, or 2.3 ␮g of OVA or 3.4 ␮g of BSA was injected into DO11.10 mice. After the indicated periods, the cell extracts of thymocytes were The open reading frame of mouse Cyp B cDNA with the coding region of prepared according to the method described by Enari et al. (18) with some Flag tag was amplified by PCR from cDNA synthesized from total RNA of modifications. In brief, thymocytes were washed with PBS, pH 7.4, fol- mouse thymocytes and cloned into the EcoRI and XhoI sites of pME18S lowed by a single wash with 5 ml of cell extract buffer (CEB; 50 mM (gift from Dr. Maruyama, University of Tokyo, Tokyo, Japan) to produce PIPES (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and Flag-tagged Cyp B expression vector (Cyp B-Flag). N-terminal signal se- 10 mM cytochalasin B) containing a mixture of protease inhibitors (1 mM quence-deleted Cyp B was also amplified and cloned into the EcoRI site of PMSF, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 5 ␮g/ml antipain, and 1 the pGEX-4T-1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) ␮g/ml chymopain). Cells were spun down and transferred to a Dounce to produce the GST fusion protein of Cyp B (GST-Cyp B). Mouse ICAD homogenizer (Wheaton, Millville, NJ), allowed to swell by the addition of cDNA was provided by Dr. S. Nagata (Osaka University, Osaka, Japan). an adequate volume of CEB, and disrupted by freezing and thawing once. ICAD cDNA was inserted into the EcoRI site of pGEX-4T-1 vector to After grinding with the pestle, cell lysis was monitored by staining an produce the GST-ICAD protein. We also obtained pcDNA3-HA-CAD and aliquot of the cell suspension with methyl green and observation under a pcDNA-3-Flag-DFF45 vectors from Dr. Nuń˜ez (University of Michigan, microscope. The cell lysate was then transferred to a 1.5-ml microcentri- Ann Arbor, MI). fuge tube and centrifuged at 4°C for 15 min at 700 ϫ g. The supernatant was carefully collected without disturbing the nuclear pellets and was used Preparation of recombinant active CAD as cell extracts. The protein concentration of the cell extracts or the purified ␮ preparation was measured by protein assay (Bio-Rad, Hercules, CA). Two hundred micrograms of pcDNA3-HA-CAD vector and 50 gof pcDNA-3-Flag-DFF45 vector were cotransfected into 2 ϫ 107 293T cells Subcellular fractionation of cell extracts by a calcium phosphate method. Twenty-four hours after transfection, 293T cells were harvested and lysed in 1 ml of TBS (25 mM Tris-HCl (pH Cell extract was centrifuged at 7,000 ϫ g at 4°C for 30 min and separated 7.5) and 150 mM NaCl) containing 1% Nonidet P-40. The cell lysate was into the pellet of the mitochondria-rich fraction (P7) and the supernatant. incubated with 1 ␮g of anti-Flag Ab (Upstate Biotechnology, Lake Placid, Then, the supernatant was ultracentrifuged at 100,000 ϫ g at 4°C for 90 NY) bound to protein A-Sepharose FF beads (Amersham Pharmacia Bio- min and separated to the pellet containing the microsomal fraction (P100) tech). After washing the beads with CEB, 10 ␮l of them was incubated and the supernatant. Finally, the supernatant was further ultracentrifuged at with appropriate amount of recombinant human caspase 3 (Chemicon, Te- 100,000 ϫ g at 4°C for 13 h. After centrifugation, the supernatant was mecula, CA), and the supernatant was used as an active CAD preparation. separated into two phases: the lower fraction was orange (LS100), and the upper fraction was colorless (US100). Each fraction was suspended to the Purification of the LS100 fraction same volume with CEB. All purification steps were conducted at 4°C, using a Vision automatic fast Preparation of liver nuclei protein liquid chromatography station (PE Biosystems Japan, Chiba, Ja- pan). Four hundred fifty milligrams of LS100 from thymocytes stimulated All procedures were performed on ice. Livers removed from C57BL/6 with anti-CD3 mAb for 20 h was applied to 40 ml of Q-Sepharose beads mice were minced, put in a 30 ml of homogenization buffer (10 mM (Amersham Pharmacia Biotech) that was equilibrated with CEB containing HEPES (pH 7.6), 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 1% (w/v) 3-(1-pyridinio)-1-propanesulfonate (Fluka Chemika, Buchs, The Journal of Immunology 4283

Switzerland). After 15-min rotation, the tube was centrifuged at 3000 rpm for 1 min, and the supernatant was applied to 40 ml of hydroxyapatite beads (Bio-Rad). After 15-min rotation, the tube was centrifuged at 3000 rpm for 1 min, and the supernatants were removed. CEB containing 200 mM KCl was added to the beads and rotated for 15 min. Then the tube was centrifuged at 3000 rpm for 1 min, and the supernatants were dialyzed against CEB at 4°C for 3 h. After dialysis, the sample was applied to a Mono S column (Amersham Pharmacia Biotech) equilibrated with CEB and eluted with a 0–1 M linear KCl gradient. Active fraction (eluted at 0.5 M KCl) was loaded onto a Superdex 200 gel ﬁltration column equilibrated and eluted with CEB. After the Superdex 200 column fractionation, each fraction was assayed for DNA degradation activity. Peptide sequence analysis The sample was electrophoresed on a 12% SDS-polyacrylamide gel and transferred onto an Immobilon-PSQ membrane (Millipore, Bedford, MA). After staining with Coomassie Brilliant Blue (PhastGel Blue R, Amersham Pharmacia Biotech), a band of 20 kDa was cut out from the membrane and sequenced using a protein sequencer (G1005A, Hewlett Packard, Palo Alto, CA). The amino acid sequence was searched against the databases Swiss Prot and TrEMBL.

Purification of GST-ICAD and GST-Cyp B protein Downloaded from Escherichia coli, strain DH5, containing GST-ICAD or GST-Cyp B expression plasmid, was grown (37°C) to an OD at 600 nm of 0.5 in 800 ml of Luria-Bertoni medium containing 100 ␮g/ml of ampicillin. Isopropyl- ␤-D-thiogalactopyranoside (1 mM) was added to the culture medium, and the cells were grown for an additional 3 h. Cells were harvested, resus- FIGURE 1. Cell death execution of thymocytes after in vivo anti-CD3 pended in 15 ml of TBS containing 1% Triton X-100 and 10% glycerol, stimulation. A, Apoptotic changes in cell membrane and mitochondrial ϫ http://www.jimmunol.org/ and rotated for1hat4°C. After centrifugation at 14,000 g for 30 min, membrane properties in anti-CD3-stimulated thymocytes. Mice were i.p. the supernatant was mixed with 1 ml of glutathione-Sepharose 4B beads injected with anti-CD3 mAb or control Ab (50 ␮g/mouse), and thymocytes (Amersham Pharmacia Biotech) and rotated at 4°C overnight. The beads were prepared at various times after injection and stained with annexin V were washed with TBS. Bound GST-ICAD or GST-Cyp B protein was or DiOC6(3). Stained cells were analyzed by flow cytometry. The percent- eluted with elution buffer (50 mM Tris-HCl (pH 8.0) and 10 mM gluta- low thione) and dialyzed against CEB. ages of annexin V positively stained cells and DiOC6(3) cells of anti- CD3-stimulated thymocytes compared with those of control Ab-stimulated Transfection of Cyp B into EL4 cells and their TCR stimulation thymocytes is shown. B, Degradation of chromosomal DNA in thymocytes Cyp B-Flag expression vector (5 ␮g) was transfected into 5 ϫ 106 of the after anti-CD3 stimulation. Mice were i.p. injected with anti-CD3 mAb or T lymphoma cell line EL4 by the DEAE-dextran method. Cells were in- control Ab (50 ␮g/mouse). Chromosomal DNA was extracted from 2 ϫ 6 cubated for 12 h in the RPMI medium containing 10% FCS. Transfected 10 thymocytes at various times after injection and analyzed by agarose gel by guest on September 28, 2021 cells were stimulated by immobilized anti-CD3 and anti-CD28 mAbs (50 electrophoresis. DNA fragmentation- and degradation-inducing activities ␮g/ml) and incubated for an additional 12 h. After incubation, cells were assessed by densitograph are shown at the bottom. harvested, lysed, and fractionated into nuclear, microsomal (P100), and cytosolic (LS100 ϩ US100) fractions as described. Western blot analysis 1B). These data show that in vivo TCR stimulation of thymocytes Each subcellular fraction was separated by SDS-PAGE and transferred to induces apoptotic changes in thymocytes, such as a change in cell a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The surface membrane property, a decrease in membrane potential of membrane was blocked with 5% nonfat milk in 0.1% Tween-20, 25 mM Tris-Cl (pH 7.5), and 150 mM NaCl for 4 h followed by incubation with mitochondria, and chromosomal DNA fragmentation followed by anti-Flag Ab, M2 (Upstate Biotechnology), for another2hatroom tem- chromosomal DNA degradation. perature. The membrane was washed three times with 0.1% Tween-20, 25 mM Tris-Cl (pH 7.5), and 150 mM NaCl and incubated with goat anti- Cell death-inducing molecules in cell lysate from TCR- mouse IgG conjugated with HRP (EY Laboratories, San Mateo, CA), de- stimulated thymocytes veloped using chemiluminescence (ECL Plus Western blotting detection reagents, Amersham Pharmacia Biotech), and then exposed on RX-U film To analyze the cell death-inducing molecules in TCR-stimulated (Fuji Film, Tokyo, Japan). thymocytes, we injected anti-CD3 mAb into ICR mice and investigated the abilities of cell extracts of thymocytes to induce Results changes in morphology and chromosomal DNA in the isolated Cell death of thymocytes induced by in vivo TCR stimulation liver nuclei using a cell-free system. We also investigated the ac- To elucidate the mechanism of cell death of in vivo TCR-stimu- tivity of cell extracts to induce the degradation of naked DNA. As lated thymocytes, we i.p. injected anti-CD3 mAb into ICR mice shown in Fig. 2A, the cell extract of anti-CD3-stimulated thymo- and examined changes in cell surface membrane property, mito- cytes induced shrinking of nuclei as well as its chromatin conden- chondrial membrane potential, and chromosomal DNA of thymo- sation. The cell extract of thymocytes from control Ab-injected mice cytes at various times after injection. As shown in Fig. 1A, the did not induce these morphological changes. The cell extract from low percentages of annexin V-positive cells and DiOC6(3) cells anti-CD3-stimulated thymocytes also caused chromosomal DNA gradually increased after anti-CD3 injection. As previously re- fragmentation of the liver nuclei and plasmid DNA degradation ported (19), internucleosomal DNA fragmentation of thymocytes (Fig. 2B). These activities were gradually increased at 12–24 h was detected at 12 h after anti-CD3 stimulation (Fig. 1B). It was after anti-CD3 injection. Cell extracts prepared from the thymo- noteworthy that chromosomal DNA degradation of thymocytes, cytes of control Ab-injected mice did not induce either nuclear shown as the smear of DNA electrophoresis pattern, started 24 h chromosomal DNA fragmentation or plasmid DNA degradation. after anti-CD3 injection. Thymocytes of control Ab-injected mice To test whether the stimulation of thymocytes with more physio- did not cause either DNA fragmentation or DNA degradation (Fig. logical ligand for TCR rather than anti-CD3 mAb generates similar 4284 INVOLVEMENT OF CYP B IN DNA DEGRADATION IN APOPTOTIC THYMOCYTES

methods, into four fractions: mitochondria (P7), microsome (P100), and two cytosolic fractions, LS100 and US100, as described in Materials and Methods (Fig. 3A). Electron microscopy revealed that P7 dominantly contained mitochondria, and P100 mainly contained membrane vesicles, endoplasmic reticulum la- mellae, and ribosomes (data not shown). Each fraction was incubated with normal liver nuclei or plasmid DNA, and activities that induce nuclear DNA fragmentation or plasmid DNA degradation were investigated. As shown in Fig. 3B, both nuclear DNA fragmentation-inducing activity and DNA degradation activity were detected in P100 as well as LS100 of anti-CD3-stimulated thymocytes. Neither nuclear DNA fragmentation-inducing activity nor plasmid DNA degradation activity was found in any fraction of the cell extract of thymocytes from control Ab-injected mice. The corresponding fractions (P100 and LS100) of the cell extracts of thymocytes from OVA-stimulated DO11.10 mice also showed nuclear DNA fragmentation-inducing activity and plasmid DNA degradation activity (Fig. 3C), as found in the anti-CD3-stimulated

cell extract. The fractions of the cell extracts from thymocytes of Downloaded from BSA-injected DO11.10 mice did not show these activities. Biochemical characterization of LS100 of cell extract from anti- CD3-stimulated thymocytes Further biological and biochemical characterization of the mole-

cule(s) that are responsible for nuclear DNA fragmentation and/or http://www.jimmunol.org/ plasmid DNA degradation were performed using LS100 from anti- CD3-stimulated thymocytes (LS100-s). As shown in Fig. 4A, FIGURE 2. Nuclear morphological changes, nuclear DNA fragmenta- LS100-s induced nuclear DNA fragmentation and plasmid DNA tion, and DNA degradation induced by cell extracts of in vivo anti-TCR- degradation in a dose-dependent fashion. Kinetics studies showed stimulated thymocytes. A, Nuclear morphological changes induced by cell that nuclear DNA fragmentation by LS100-s was observed at 15 extract of anti-CD3-stimulated thymocytes. Mice were injected i.p. with min and became clearer at 30 or 60 min after incubation with the ␮ anti-CD3 or control Ab (50 g/mouse), and cell extracts of thymocytes nuclei. DNA degradation by LS100-s was detected at 5 min and were prepared at 20 h after injection. Cell extracts (5 mg/ml) were then became clearer at 15–30 min (Fig. 4B). When LS100-s was pre-

incubated with liver nuclei for 60 min and stained with 4,6-diamino-2- by guest on September 28, 2021 treated with proteinase K or preincubated at 68°C for 10 min, both phenylindole, and the morphology of nuclei was observed under a fluorescence microscope. B, Chromosomal DNA fragmentation of nuclei and DNA fragmentation-inducing activity and DNA degradation ac- plasmid DNA degradation induced by cell extracts of anti-CD3-stimulated tivity were completely abrogated (Fig. 4C), indicating that the thymocytes. Cell extracts (5 mg/ml) were prepared from thymocytes at molecule(s) responsible for DNA fragmentation and DNA degra- various times after anti-CD3 or control Ab injection into ICR mice and dation in LS100-s is a heat-labile protein(s). incubated with liver nuclei for 60 min or with plasmid DNA for 30 min. The cation requirements for a molecule(s) in LS100-s to induce Nuclear DNA (upper panel) and plasmid DNA (lower panel) were ana- nuclear DNA fragmentation and plasmid-DNA degradation were lyzed by agarose gel electrophoresis. C, Chromosomal DNA fragmentation also investigated. As shown in Fig. 4D, both DNA fragmentation of nuclei and plasmid DNA degradation induced by cell extracts of OVA- and DNA degradation occurred in the presence of Mg2ϩ with or stimulated DO.11.10 thymocytes. Cell extracts (5 mg/ml) were prepared without Ca2ϩ. They were not induced in the presence of Ca2ϩ from thymocytes at various times after OVA or BSA injection into ϩ ϩ alone. These data show that Mg2 ions, but not Ca2 ions, are DO.11.10 transgenic mice and were incubated with liver nuclei or plasmid DNA as described above. Nuclear DNA (upper panel) and plasmid DNA required for the protein(s) to induce DNA fragmentation and DNA (lower panel) were analyzed by agarose gel electrophoresis. DNA frag- degradation. mentation- and degradation-inducing activities are shown at the bottom. Enari et al. recently identified CAD that induces nuclear DNA fragmentation using the cell-free system (15). They also identified activities, we used DO11.10 TCR-transgenic mice, in which trans- ICAD. To examine whether CAD plays a role in the nuclear DNA gene encodes TCR recognizing an OVA peptide in the context of fragmentation activity of LS100-s, the recombinant GST fusion I-Ad (20). We i.p. injected OVA or BSA into DO11.10 mice, pre- protein of ICAD (GST-ICAD) was prepared and added to pared the cell extract from the thymocytes at various periods after LS100-s, and nuclear DNA fragmentation-inducing activity was injection, and examined their abilities to induce changes in nuclear analyzed. As shown in Fig. 5A, nuclear DNA fragmentation-in- chromosomal DNA or plasmid DNA. As shown in Fig. 2C, the cell ducing activity in LS100-s was inhibited in a dose-dependent man- extract of OVA-stimulated thymocytes at 24 or 48 h after injection ner by GST-ICAD, but not by GST. Western blot analysis of induced nuclear DNA fragmentation and plasmid DNA degrada- LS100-s with anti-CAD Ab revealed a band of CAD protein in tion, but the cell extract of thymocytes of BSA-injected mice did LS100-s (data not shown). These results indicate that the molecule not induce these activities. These results indicate that anti-CD3- responsible for DNA fragmentation of nuclei in LS100-s is CAD. stimulated cell extracts may reconstitute the death-signaling path- It was worthwhile to note that the plasmid DNA degradation ac- way of the negative selection of immature thymocytes in vitro. tivity in LS100-s was not completely inhibited by GST-ICAD (Fig. 5A). Enari et al. previously demonstrated that the DNA degrada- Subcellular localization of cell death-inducing activities tion activity of CAD was completely inhibited by ICAD (15). By To investigate which subcellular organelles contain these activi- using our cell-free system, CAD also digested plasmid DNA, and ties, we separated cell extracts, using differential centrifugation its DNA degradation activity was completely inhibited by ICAD at The Journal of Immunology 4285 Downloaded from

FIGURE 3. Subcellular localization of cell death-inducing activity. A, Subcellular fractionation procedure. Cell extracts of thymocytes were fractionated into the mitochondria-rich fraction (P7), the microsome fraction (P100), and two different cytosolic fractions (LS100 and US100) as described in Materials and Methods. B, Nuclear DNA fragmentation or plasmid DNA degradation induced by subcellular fractions from thymocytes of anti-CD3 or control Ab-injected ICR mice (left), or OVA- or BSA-injected DO11.10 mice (right). Cell lysates from thymocytes of ICR mice injected with anti-CD3 or control Ab (left) or those from thymocytes of DO11.10 mice injected with OVA or BSA (right) were prepared after 20 h of injection, and their subcellular fractions were prepared as described above. Two hundred microliters of cell lysate or each subcellular fraction (P7, P100, LS100, and US100) was incubated with http://www.jimmunol.org/ liver nuclei for 60 min or plasmid DNA for 30 min. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom. the dose that completely inhibited DNA fragmentation activity of activity of LS100 was inhibited by anti-Cyp B Ab in a dose-de- CAD (Fig. 5B). These results suggest that a molecule other than pendent manner but not by control Ab, indicating that Cyp B par- CAD is also involved in DNA degradation activity in LS100-s. ticipates in DNA degradation activity in LS100-s. To delineate the Thus, we tried to purify the protein that is responsible for DNA activity of Cyp B in more detail, the GST fusion protein of Cyp B degradation in LS100-s. (GST-Cyp B) was prepared, and its activity to induce DNA deg- by guest on September 28, 2021 radation was investigated. GST-Cyp B induced degradation of Puriﬁcation and characterization of DNA degradation- chromosomal DNA in nuclei as well as plasmid DNA in a dose- inducing protein dependent manner, but did not induce internucleosomal DNA frag- To purify DNA degradation-inducing protein in LS100-s, LS-100-s mentation in nuclei (Fig. 7B). (total of 450 mg protein collected from 2000 anti-CD3-injected mice) To examine the possibility that Cyp B and CAD synergistically was fractionated by Q-Sepharose, hydroxyapatite, Mono S, and Su- execute apoptotic nuclear changes, we characterized the nuclear perdex 200 columns. The DNA degradation-inducing protein did not DNA degradation activity and plasmid DNA degradation activity bind to the Mono Q column, but bound to hydroxyapatite and Mono of LS100-s, CAD, Cyp B, or CAD plus Cyp B. As shown in Fig. S columns. The activity was eluted from hydroxyapatite column with 7C, LS100-s caused internucleosomal DNA fragmentation in nu-

0.2 M KPO4 and from Mono S column with 0.5–0.6 M KCl. Overall clei as well as plasmid DNA degradation. A low concentration of puriﬁcation of this protein was 631.5-fold, with a yield of 0.1% (Table active CAD induced internucleosomal DNA fragmentation stron- I). After further fractionation by Superdex 200 column chromatogra- ger than LS100, but hardly degraded plasmid DNA. In the pres- phy, each fraction was examined for the ability to induce DNA deg- ence of both CAD and Cyp B, they caused not only internucleo- radation. As shown in Fig. 6, DNA degradation activity was detected somal DNA fragmentation but also degradation of chromosomal only in the fractions 39 and 40. When each fraction was electropho- DNA and plasmid DNA (Fig. 7, C and D). These data showed that resed on a SDS-polyacrylamide gel, a band around 20 kDa was de- not only CAD but also Cyp B is required to reconstitute DNA tected only in the fractions 39 and 40 (Fig. 6). However, this band was fragmentation and DNA degradation activity in LS100-s. not detected in the corresponding fractions of LS100 from PBS- injected mouse thymocytes (data not shown). The protein of this band was extracted, and the amino acid se- Cyc B is released from microsomal fraction to cytosolic/nuclear quence was determined (NDKKKGPKVT). By using Swiss Prot fractions of a T cell line by TCR stimulation and TrEMBL databases, it was revealed that this sequence is iden- It was reported that premature Cyp B contained a signal sequence tical with the sequence of N-terminal of Cyp B. targeting the protein to the endoplasmic reticulum and existed in microsome fraction in rat hepatocytes (21). To examine whether Cyp B together with CAD induces degradation of chromosomal Cyp B is released from microsome fraction to cytosol and moves DNA in apoptotic nuclei into nuclei by stimulation of TCR-CD3 complexes, we constructed To examine whether Cyp B is involved in the DNA degradation- an expression vector for Flag-tagged Cyp B (Cyp B-Flag) and inducing activity in LS100-s, LS100-s was incubated with anti- transfected it into a T lymphoma cell line, EL4. EL4 cells were Cyp B polyclonal Ab or control Ab and then incubated with plas- then stimulated with immobilized anti-CD3 and anti-CD28 mAbs, mid DNA. As shown in Fig. 7A, DNA degradation-inducing and subcellular distributions of Cyp B were examined by Western 4286 INVOLVEMENT OF CYP B IN DNA DEGRADATION IN APOPTOTIC THYMOCYTES Downloaded from

FIGURE 4. Biochemical characterization of apoptosis-inducing activity in LS100 from anti-CD3-stimulated thymocytes. A, Dose response of DNA fragmentation- or DNA degradation-inducing activity. Various concentrations of LS100 were incubated with liver nuclei or plasmid DNA, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. B, Kinetics of DNA fragmentation or DNA http://www.jimmunol.org/ degradation activity. LS100 (50 ␮g/ml) was incubated with liver nuclei or plasmid DNA for various times at 37°C, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. C, Sensitivity of DNA fragmentation- or DNA degradation-inducing activity to proteinase K or heat treatment. LS100 (50 ␮g/ml) was pretreated with proteinase K or RNase A beads for 5 min at 4°C (left)or preincubated at 37°C (control) or 68°C for 10 min (right) and then incubated with liver nuclei or plasmid DNA. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis.

D, Cation requirements for LS100-induced DNA fragmentation or DNA by guest on September 28, 2021 degradation. LS100 (50 ␮g/ml) was incubated with liver nuclei or plasmid DNA in the presence of 5 mM EGTA and 5 mM EDTA (shown as Ϫ/Ϫ), 2mMMg2ϩ and 5 mM EGTA (shown as Mgϩϩ),2mMCa2ϩ and5mM EDTA (shown as Caϩϩ),or2mMMg2ϩ and2mMCa2ϩ (shown as ϩϩ ϩϩ Mg /Ca ). Nuclear DNA (upper panel) and plasmid DNA (lower FIGURE 5. Effect of ICAD on nuclear DNA fragmentation or DNA panel) were analyzed by agarose gel electrophoresis. DNA fragmentation- degradation induced by LS100 or CAD. A, Effect of ICAD on LS100- and degradation-inducing activities are shown at the bottom. induced nuclear DNA fragmentation or plasmid DNA degradation. Various concentrations of LS100 (lane 1,0␮g/ml; lane 2,10␮g/ml; lane 3,25 ␮g/ml; lane 4,50␮g/ml) or 50 ␮g/ml of LS100 together with various blot analysis using anti-Flag Ab (Fig. 8). In the fractions of non- concentrations of GST-ICAD or GST (lanes 5 and 8,1␮g/ml; lanes 6 and stimulated EL4 cells, a high level of Cyp B was detected in the 9,3␮g/ml; lanes 7 and 10,6␮g/ml) were incubated with liver nuclei or microsome fraction, but lower levels of Cyp B were found in the plasmid DNA. Chromosomal DNA fragmentation (upper panel) and plas- cytosolic and nuclear fractions. On the contrary, when EL4 cells mid DNA degradation (lower panel) were assessed by agarose gel elec- were stimulated with anti-CD3/CD28 mAb, higher levels of Cyp B trophoresis. B, Effect of ICAD on nuclear DNA fragmentation and plasmid were detected in the nuclear and cytosolic fractions compared with DNA degradation induced by active CAD. Ten microliters of recombinant CAD/ICAD complex was treated with various concentrations of active those in the microsome fraction. These data show that Cyp B is caspase 3 (lane 1, 0 U/ml; lane 2, 1 U/ml; lane 3, 2 U/ml; lanes 4–6,3 released from the microsome fraction to the cytosolic/nuclear frac- U/ml), and incubated with liver nuclei (upper panel) or plasmid DNA tion by the signaling from TCR-CD3 complexes on EL4 cells. (lower panel) in the absence (lanes 1–4) or the presence of GST-ICAD (lane 5,6␮g/ml; lane 6,12␮g/ml). Chromosomal DNA fragmentation and plasmid Discussion DNA degradation were analyzed by agarose gel electrophoresis. DNA frag- In this study, using a cell-free system, we showed that the cell mentation- and degradation-inducing activities are shown at the bottom. extracts from anti-CD3 mAb and natural Ag (OVA)-stimulated thymocytes contain activities to induce morphological changes in naked nuclei and two distinct endonuclease activities (Figs. 2–5), cytes and may be responsible for DNA fragmentation activity in one that induced internucleosomal nuclear DNA fragmentation TCR-simulated thymocytes. In agreement with our results, Clay- and the other that induced DNA degradation. With regard to in- ton et al. demonstrated that an inhibitor of caspases inhibited ap- ternucleosomal DNA fragmentation activity, Enari et al. recently optosis of thymocytes using a TUNEL assay (22). As the TUNEL reported that CAD caused internucleosomal DNA fragmentation in assay should reﬂect DNA fragmentation in apoptotic cells, their nuclei in vitro (15). DNA fragmentation activity in TCR-activated results indicated the involvement of caspases in the activation of thymocyte extracts was completely inhibited by ICAD (Fig. 5), nucleases. Concerning the DNA degradation activity, Enari et al. indicating that CAD was activated by TCR stimulation in thymo- (15) demonstrated that activated CAD showed DNase activity to The Journal of Immunology 4287

Table I. Puriﬁcation of DNA degradation-inducing molecule from LS100

Stepa Protein (mg) Total Activityb (U) Speciﬁc Activity (U/mg) Yield (%)

S100 1,062 10,620 10 100 LS100 450 10,350 23 97 Q-Sepharose 189 8,316 44 78 Hydroxyapatite 99 7,476 84 78 Mono S 0.44 926.2 2,105 8.7 Superdex 200 0.0015 9.47 6,315 0.1

a S100 fraction from anti-CD3-stimulated thymocytes (1062 mg) was fractionated to LS100 and US100 by ultracentrifugation, and LS100 was puriﬁed by each fast protein liquid chromatography column chromatography. b One unit of activity was arbitrarily deﬁned as the density of the degradation pattern of the plasmid DNA (1 ␮g) generated by incubation with 10 ␮g/ml anti-CD3-activated LS100.

degrade plasmid DNA. However, Toh et al. showed that a human duced by the anti-CD3-stimulation (Fig. 1B), indicating that Cyp B homologue of CAD, DFF40, alone could not cause degradation of is involved in this process. plasmid DNA (23). In the present study we demonstrated that a Cyp B contains N-terminal signal sequence (21), is produced in low dose of CAD induced internucleosomal DNA fragmentation, endoplasmic reticulum, and is secreted into biological ﬂuids such Downloaded from but hardly degrades the chromosomal DNA into smaller pieces as milk and plasma (34, 35). Spik and his colleagues demonstrated when incubated with naked nuclei (Fig. 7C). that peripheral blood T lymphocytes possess binding sites of Cyp Interestingly enough, ICAD did not completely inhibit the plas- B that internalize extracellular Cyp B into cells (36). To date, the mid DNA degradation activity in the cell extracts of TCR-stimu- fate and the function of internalized Cyp B are not clear. In Fig. 8 lated thymocytes, suggesting that a DNase(s) other than CAD is it is shown that Cyp B is released from microsome fraction into

also activated by TCR stimulation in the thymocytes (Fig. 5A). We cytosol as well as the nuclear fraction of a T cell line by signals http://www.jimmunol.org/ purified this protein and revealed that its N-terminal amino acid from TCR. This result coincides with an assumption that Cyp B sequence is identical with that of Cyp B (Fig. 6). Cyclophilin, may be involved in chromosomal DNA degradation in apoptotic cyclosporin A-binding protein, has five family members: Cyp A thymocytes stimulated by TCR. How is Cyp B in the microsome (18 kDa) (24), Cyp B (21 kDa) (21), Cyp C (23 kDa) (25), Cyp D fraction translocated into cytoplasm and nuclei? Concerning this (19 kDa) (26), and Cyp 40 (40 kDa) (27). With respect to the question, Peitsch et al. demonstrated that DNase I, produced in nuclease activity, Montague et al. demonstrated that the nuclease endoplasmic reticulum, is involved in nuclear DNA degradation purified from glucocorticoid-treated rat thymocytes was highly ho- during apoptosis and proposed that the mechanism by which mologous to Cyp A (28) and that recombinant Cyp A, Cyp B, and DNase I gains access to nuclei is through the breakdown of the Cyp C degrade plasmid DNA (29) in a Ca2ϩ-orMg2ϩ-dependent endoplasmic reticulum and nuclear membrane during apoptosis by guest on September 28, 2021 manner. They showed that Cyp C could generate 50-kb DNA frag- (32). In the case of cytochrome c, several models were proposed ments when incubated with naked nuclei; however, they did not for its release of cytochrome c from mitochondria during the pro- show whether Cyp A, B, or C is involved in DNA degradation in cess of apoptosis (37): 1) cytochrome c is released as a result of the apoptotic cells and especially they did not demonstrate whether rupture of outer mitochondrial membrane; and 2) cytochrome c is Cyp B could degrade chromosomal DNA in nuclei. Thus, the role of Cyp B in thymocyte apoptosis has been obscure. In this study we showed that Abs to Cyp B dose dependently inhibited DNA degradation activity in cytosolic fraction prepared from anti-CD3- activated thymocytes, and that the recombinant Cyp B degrades chromosomal DNA in nuclei as well as plasmid DNA (Fig. 7). Our results demonstrated, for the first time, the possible involvement of Cyp B in the DNA degradation activity in TCR-stimulated thymocytes. Regarding nucleases observed in apoptotic cells, at least three types of nucleases were reported. The first is a nuclease that degrades chromosomal DNA into an approximately 50-kb fragment. This includes apoptosis-inducing factor (30) and Cyp C (29). The second is a nuclease that induces internucleosomal DNA fragmentation, a hallmark of an apoptotic nuclear change, including DNase II and CAD (15, 31). The last is a nuclease that breaks chromosomal DNA into smaller pieces. This involves DNase I (32) and Cyp B in this study. Recently, Wu et al. demonstrated that NUC-1, a Caenorhabditis elegans DNase II homologue, is involved in DNA degradation in apoptotic cells and that activation of NUC-1 FIGURE 6. Purification of DNA degradation-inducing protein in LS100 by Superdex 200 gel filtration chromatography. A, DNA degradation ac- may degrade internucleosomally fragmented DNA in apoptotic tivity in fractions separated by Superdex 200. An aliquot (20 ␮l) of each cells (33). They observed more TUNEL-reactive nuclei in NUC- fraction was incubated with plasmid DNA, and DNA degradation was an- 1-deficient embryos than in the wild-type embryos. Thus, DNA alyzed by agarose gel electrophoresis. Nuclease activity was defined as in degradation is one of the major aspects of the apoptotic process. In Table I. B, SDS-PAGE of fractions separated by Superdex 200. An aliquot (20 this respect we observed the chromosomal DNA degradation after ␮l) of fractions was electrophoresed on an SDS-polyacrylamide gel, and pro- internucleosomal DNA fragmentation in apoptotic thymocytes in- teins were visualized by silver staining. Each fraction corresponds to A. 4288 INVOLVEMENT OF CYP B IN DNA DEGRADATION IN APOPTOTIC THYMOCYTES Downloaded from

FIGURE 7. Involvement of Cyp B in DNA degradation activity in LS100. A, Inhibition of plasmid DNA degradation activity in LS100 by anti-Cyp B Ab. Various amounts of LS100 (lane 1,0␮g/ml; lane 2,10␮g/ml; lane 3,25␮g/ml; lanes 4–9,50␮g/ml) were incubated with anti-Cyp B Ab or rabbit IgG (lanes 5 and 8,1␮g/ml; lanes 6 and 9,3␮g/ml; lanes 7 and 10,9␮g/ml) for 30 min at 4°C. After incubation, plasmid DNA (1 ␮g) was incubated with each mixture for 60 min at 37°C and analyzed by agarose gel electrophoresis. B, Induction of chromosomal and plasmid DNA degradation by recombinant Cyp B. Various concentrations of GST-Cyp B or GST (lanes 1 and 6,0␮g/ml; lanes 2 and 7,5␮g/ml; lanes 3 and 8,10␮g/ml; lanes 4 and http://www.jimmunol.org/ 9,15␮g/ml; lanes 5 and 10,30␮g/ml) were incubated with liver nuclei or plasmid DNA, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. C and D, Degradation of CAD-generated internucleosomal DNA fragments by recombinant Cyp B. Liver nuclei (upper panel) or plasmid DNA (lower panel) was incubated with buffer alone (control), LS100 (50 ␮g/ml), active CAD (pretreated with1Uof caspase 3), GST-Cyp B (10 ␮g/ml), or active CAD and GST-Cyp B (C) or with various concentrations of GST-Cyp B or GST (lanes 1 and 6,0␮g/ml; lanes 2 and 7,5␮g/ml; lanes 3 and 8,10␮g/ml; lanes 4 and 9,15␮g/ml; lanes 5 and 10,30␮g/ml) with active CAD (pretreated with 0.5 U of caspase 3; D) for 60 min at 37°C and analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom. released by the formation of a pore in the outer membrane. With tivity was inhibited by ICAD (data not shown). CAD was puriﬁed by guest on September 28, 2021 regard to the latter case, it was demonstrated that the Bcl-2 family, from the cytoplasm of apoptotic cells (18), and at the present time including Bax and Bcl-2, possesses a pore-forming ability (38, 39) it is not clear whether CAD is localized in the microsomal fraction. and that Bax could release cytochrome c from isolated mitochon- In this respect, it has been shown that procaspase 12 is localized in dria (40). Bcl-2 is shown to be localized not only on mitochondrial the microsomal fraction and released to the cytoplasm by endo- membrane, but also on endoplasmic reticulum (41). Thus, it may plasmic reticulum stress (42). We are now investigating the rela- be possible to assume that a proapoptotic Bcl-2 family forms a tionship between CAD in the cytoplasm and CAD-like molecule in pore on the membrane of endoplasmic reticulum to release Cyp B the microsomal fraction as well as its physiological role in apo- during apoptosis, although this is merely a speculation. ptotic cells. In the beginning of this study we unexpectedly observed DNA fragmentation activity in the microsomal fraction of TCR-stimulated thymocytes in addition to the cytosolic fraction. This activity may also be attributed to a CAD-like molecule(s), since this ac-

FIGURE 8. Release of Cyp B from cytoplasmic microsome fraction to FIGURE 9. A model for the DNA degradation mechanism in TCR- cytosol/nuclear fractions of EL4 T cells by TCR stimulation. EL4 cells stimulated thymocytes. Signals from TCR activate caspase 3. Active (5 ϫ 106) transfected with Cyp B-Flag expression vector were incubated in caspase 3 cleaves the ICAD/CAD complex, and free CAD is localized in the presence or the absence of immobilized-anti-CD3 and anti-CD28 mAbs nuclei to induce chromosomal DNA fragmentation. Simultaneously, Cyp B for 12 h. After incubation, cells were fractionated into nuclear, microsomal, localized in endoplasmic reticulum (ER) is released into cytosol by the and cytosolic fractions. Total cell lysate and each fraction (50 ␮g of protein) signals from TCR, translocated into nuclei, and ﬁnally degrades CAD- were separated on SDS-PAGE, and the blot was probed with anti-Flag Ab. fragmented chromosomal DNA into small pieces. The Journal of Immunology 4289

Based on these results, we propose a model for chromosomal 18. Enari, M., A. Hase, and S. Nagata. 1995. Apoptosis by a cytosolic extract from DNA degradation that was seen in TCR-stimulated thymocytes as Fas-activated cells. EMBO J. 14:5201. 19. Smith, C. A., G. T. Williams, R. Kingstone, E. J. Jenkinson, and J. T. Owen. follows. TCR engagement of immature thymocytes induces acti- 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in vation of CAD. The engagement also induces the release of Cyp B immature T cells in thymic cultures. Nature 337:181. 20. Iwabuchi, K., K. Nakayama, R. L. McCoy, F. Wang, T. Nishimura, S. Habu, from microsome fraction to cytosol fraction. Activated CAD and K. M. Murphy, and D. Y. Loh. 1992. Cellular and peptide requirements for in vitro Cyp B are translocated into nuclei and exert their activities in clonal deletion of immature thymocytes. Proc. Natl. Acad. Sci. USA 89:9000. harmony on degrading chromosomal DNA after they are translo- 21. Hasel, K. W., J. R. Glass, M. Godbout, and J. G. Sutcliffe. 1991. An endoplasmic reticulum-specific cyclophilin. Mol. Cell. Biol. 11:3484. cated into the nucleus (Fig. 9). Further biochemical analysis is 22. Clayton, L. K., Y. Ghendler, E. Mizoguchi, R. J. Patch, T. D. Ocain, K. Orth, necessary to elucidate the signal transduction from TCR causing A. K. Bhan, V. M. Dixit, and E. L. Reinherz. 1997. T-cell receptor ligation by the release of Cyp B from endoplasmic reticulum as well as the peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection. EMBO J. 16:2282. release of cytochrome c from mitochondria. 23. Toh, S. Y., X. Wang, and P. Li. 1998. Identification of the nuclear factor HMG2 as an activator for DFF nuclease activity. Biochem. Biophys. Res. Commun. 250: Acknowledgments 598. 24. Handschumacher, R. E., M. W. Harding, J. Rice, R. J. Drugge, and We thank H. Hirano for helpful suggestions about purifying Cyp B protein, D. W. Speicher. 1984. Cyclophilin: a specific cytosolic binding protein for cy- R. Mineki and N. Shindo for determining the protein sequence, D. Y. Loh closporin A. Science 226:544. for providing the DO11.10 mice, Dr. J. G. Sutcliffe for Ab to Cyp B, Dr. R. 25. Schneider, H., N. Charara, R. Schmitz, S. Wehrli, V. Mikol, M. G. Zurini, Abe for anti-mouse CD28, Dr. S. Nagata for mouse ICAD cDNA, Dr. K. V. F. Quesniaux, and N. R. Movva. 1994. Human cyclophilin C: primary struc- ture, tissue distribution, and determination of binding specificity for cyclosporins. Maruyama for pME18S vector, and Dr. G. Nuń˜ez for HA-CAD and Flag- Biochemistry 33:8218. DFF45 expression vectors. 26. Bergsma, D. J., C. Eder, M. Gross, H. Kersten, D. Sylvester, E. Appelbaum, Downloaded from D. Cusimano, G. P. Livi, M. M. McLaughlin, and K. Kasyan. 1991. The cyclophilin multigene family of peptidyl-prolyl isomerases: characterization of three References separate human isoforms. J. Biol. Chem. 266:23204. 1. Von Boehmer, H., and P. Kisielow. 1990. Self-nonself discrimination by T cells. 27. Kieffer, L. J., T. Thalhammer, and R. E. Handschumacher. 1992. Isolation and char- Science 248:1369. acterization of a 40-kDa cyclophilin-related protein. J. Biol. Chem. 267:5503. 2. Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, H. P. Pircher, 28. Montague, J. W., M. L. Gaido, C. Frye, and J. A. Cidlowski. 1994. A calcium- R. M. Zinkernagel, and S. Tonegawa. 1994. Evidence for a differential avidity dependent nuclease from apoptotic rat thymocytes is homologous with cyclophi- model of T cell selection in the thymus. Cell 76:651. lin. J. Biol. Chem. 269:18877. 3. Sebzda, E., V. A. Wallace, J. Mayer, R. S. Yeung, T. W. Mak, and P. S. Ohashi. 29. Montague, J. W., F. M. J. Hughes, and J. A. Cidlowski. 1997. Native recombinant http://www.jimmunol.org/ 1994. Positive and negative thymocyte selection induced by different concentra- cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis-trans- tions of a single peptide. Science 263:1615. isomerase activity: potential roles of cyclophilins in apoptosis. J. Biol. Chem. 4. Negishi, I., N. Motoyama, K. Nakayama, K. Nakayama, S. Senju, 272:6677. S. Hatakeyama, Q. Zhang, C. A. Chang, and D. Loh. 1995. Essential role for 30. Susin, S. A., H. K. Lorenzo, N. Zamzami, I. Marzo, B. E. Snow, G. M. Brothers, ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435. J. Mangion, E. Jacotot, P. Costantini, M. Loeffler, et al. 1999. Molecular char- 5. Kong, Y. Y., K. D. Fischer, M. F. Bachmann, S. Mariathasan, I. Kozieradzki, acterization of mitochondrial apoptosis-inducing factor. Nature 397:441. M. P. Nghiem, D. Bouchard, A. Bernstein, P. S. Ohashi, and J. M. Penninger. 1998. 31. Barry, M. A., and A. Eastman. 1993. Identification of deoxyribonuclease II as an Vav regulates peptide-specific apoptosis in thymocytes. J. Exp. Med. 188:2099. endonuclease involved in apoptosis. Arch. Biochem. Biophys. 300:440. 6. Alberola-Ila, J., K. A. Forbush, R. Seger, E. G. Krebs, and R. M. Perlmutter. 32. Peitsch, M. C., B. Polzar, H. Stephan, T. Crompton, H. R. MacDonald, 1995. Selective requirement for MAP kinase activation in thymocyte differenti- H. G. Mannherz, and J. Tschopp. 1993. Characterization of the endogenous de- ation. Nature 373:620.

oxyribonuclease involved in nuclear DNA degradation during apoptosis (pro- by guest on September 28, 2021 7. Wang, C. R., K. Hashimoto, S. Kubo, T. Yokochi, M. Kubo, M. Suzuki, grammed cell death). EMBO J. 12:371. K. Suzuki, T. Tada, and T. Nakayama. 1995. T cell receptor-mediated signaling ϩ ϩ 33. Wu, Y. C., G. M. Stanfield, and H. R. Horvitz. 2000. NUC-1, a Caenorhabditis events in CD4 CD8 thymocytes undergoing thymic selection: requirement of elegans DNase II homolog, functions in an intermediate step of DNA degradation calcineurin activation for thymic positive selection but not negative selection. during apoptosis. Genes Dev. 14:536. J. Exp. Med. 181:927. 34. Price, E. R., M. Jin, D. Lim, S. Pati, C. T. Walsh, and F. D. McKeon. 1994. 8. Sugawara, T., T. Moriguchi, E. Nishida, and Y. Takahama. 1998. Differential Cyclophilin B trafficking through the secretory pathway is altered by binding of roles of ERK and p38 MAP kinase pathways in positive and negative selection cyclosporin A. Proc. Natl. Acad. Sci. USA 91:3931. of T lymphocytes. Immunity 9:565. 9. Rincon, M., A. Whitmarsh, D. D. Yang, L. Weiss, B. Derijard, P. Jayaraj, 35. Allain, F., C. Boutillon, C. Mariller, and G. Spik. 1995. Selective assay for CyPA R. J. Davis, and R. A. Flavell. 1998. The JNK pathway regulates the in vivo and CyPB in human blood using highly specific anti-peptide antibodies. J. Im- deletion of immature CD4ϩCD8ϩ thymocytes. J. Exp. Med. 188:1817. munol. Methods 178:113. 10. Wyllie, A. H., J. F. R. Kerr, and A. R. Currie. 1980. Cell death: the significance 36. Carpentier, M., F. Allain, B. Haendler, A. Denys, C. Mariller, M. Benaissa, and of apoptosis. Int. Rev. Cytol. 68:251. G. Spik. 1999. Two distinct regions of cyclophilin B are involved in the recog- 11. Kroemer, G., N. Zamzami, and S. A. Susin. 1997. Mitochondrial control of ap- nition of a functional receptor and of glycosaminoglycans on T lymphocytes. optosis. Immunol. Today 18:44. J. Biol. Chem. 274:10990. 12. Li, P., D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, 37. Martinou, J. C., S. Desagher, and B. Antonsson. 2000. Cytochrome c release from and X. Wang. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/ mitochondria: all or nothing. Nat. Cell. Biol. 2:41. caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479. 38. Antonsson, B., F. Conti, A. Ciavatta, S. Montessuit, S. Lewis, I. Martinou, 13. Thornberry, N. A., and Y. Lazebnik. 1998. Caspases: enemies within. Science L. Bernasconi, A. Bernard, J. J. Mermod, G. Mazzei, et al. 1997. Inhibition of 281:1312. Bax channel-forming activity by Bcl-2. Science 277:370. 14. Liu, X., P. Li, P. Widlak, H. Zou, X. Luo, W. T. Garrard, and X. Wang. 1998. The 39. Schlesinger, P. H., A. Gross, X. M. Yin, K. Yamamoto, M. Saito, G. Waksman, 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and and S. J. Korsmeyer. 1997. Comparison of the ion channel characteristics of chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. USA 95:8461. proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. USA 94: 15. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, and S. Nagata. 11357. 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its 40. Eskes, R., B. Antonsson, A. Osen-Sand, S. Montessuit, C. Richter, R. Sadoul, inhibitor ICAD. Nature 391:43. G. Mazzei, A. Nichols, and J. C. Martinou. 1998. Bax-induced cytochrome c 16. Los, M., S. Wesselborg, and K. S. Osthoff. 1999. The role of caspases in devel- release from mitochondria is independent of the permeability transition pore but ϩ opment, immunity, and apoptotic signal transduction: lessons from knockout highly dependent on Mg2 ions. J. Cell Biol. 143:217. mice. Immunity 10:629. 41. Reed, J. C. 1994. Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 17. Shi, Y. F., R. P. Bissonnette, N. Parfrey, M. Szalay, R. T. Kubo, and D. R. Green. 124:1. 1991. In vivo administration of monoclonal antibodies to the CD3 T cell receptor 42. Nakagawa, T., H. Zhu, N. Morishima, E. Li, J. Xu, B. A. Yankner, and J. Yuan. complex induces cell death (apoptosis) in immature thymocytes. J. Immunol. 2000. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cyto- 146:3340. toxicity by amyloid-␤. Nature 403:98.