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. 2002 Apr;105(4):399–406. doi: 10.1046/j.1365-2567.2002.01347.x

The regulation of DNAse activities in subcellular compartments of activated thymocytes

Takuya Nagata *, Hiroyuki Kishi *, Qin Li Liu *, Tadashi Matsuda *, Tsuneo Imanaka †, Kazuhiro Tsukada ‡, Dongchon Kang §, Atsushi Muraguchi *
PMCID: PMC1782686  PMID: 11985660

Abstract

Thymocytes expressing self-reactive T-cell receptors (TCR) are eliminated in the thymus through a TCR-mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase-activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co-localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase-3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase-3-dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony.

Introduction

In the thymus, CD4+8+ (double positive) thymocytes expressing low levels of T-cell receptor (TCR)-αβ are subjected to both positive and negative selection events.1 Positive selection ensures the survival and differentiation of cells capable of recognizing foreign antigen in the context of self-major histocompatibility complex (MHC), whereas negative selection events eliminate immature thymocytes expressing self-reactive TCRs by the induction of apoptosis. It is currently thought that the avidity of the interaction between TCR and the MHC–peptide complex determines the fate of positive or negative selection,2,3 and many investigators have been engaged in delineating the signal pathways leading to positive or negative selection. It has been reported that ZAP-70 and Vav are essential for both positive and negative selection4,5 and that the Ras/Raf/MKK/Erk pathway and the calcineurin pathway are necessary for positive selection.6,7 On the contrary, it has been shown that the MKK6/p38 pathway and c-Jun N-terminal protein kinase (JNK) are involved in negative selection.8,9 However, how these pathways lead to the distinct fates of thymocytes is still unclear.

Apoptosis is a form of programmed cell death which occurs under various developmental and physiological conditions that require the selective elimination of cells from tissues and organs. During apoptosis, multiple structural changes occur, such as plasma and nuclear membrane blebbing, chromatin condensation and DNA fragmentation.10 Regarding the signal transduction leading to DNA fragmentation, caspases play an inevitable role in both an initiation phase (such as caspases 8, 9 and 10, the main function of which is to activate downstream caspases) and an effector phase (such as caspases 3, 6 and 7, which are dismantling cellular proteins). In the mitochondria-mediated apoptotic pathway, cytochrome c is released from mitochondria and makes complexes with caspase-9 and Apaf-1 to activate caspase-9, followed by caspase-3 activation. The active form of caspase-3 in turn activates DNA fragmentation factor 40 (DFF40)/caspase-activated DNAse (CAD) by degrading DFF45/inhibitor of CAD (ICAD), which causes DNA fragmentation in the nuclei of apoptotic cells.11,12 In a previous study, we have shown that in vivo TCR engagement of thymocytes induced activation of CAD and the release of cyclophilin B from endoplasmic reticulum (ER) and both exert their activities in harmony on degrading chromosomal DNA.13 However, the pathways from TCR engagement to DNA fragmentation in thymocytes through activated DFF40/CAD remains obscure.

In this study, we examined (by TCR stimulation) how and where CAD is activated in thymocytes. Our data show that the CAD/ICAD complex exists not only in nucleus and cytosol, but also in microsome of thymocytes, and TCR engagement of thymocytes induces degradation of ICAD localized in both cytosol and microsome, which finally causes chromosomal DNA fragmentation.

Materials and methods

Cells and antibodies

Cos-7 cells and 293 T cells were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 10% fetal calf serum (FCS). Anti-mouse CD3ε monoclonal antibody (mAb) was purified from the culture supernatant of a hybridoma (clone 145-2C11) by using a protein A–sepharose column (Pharmacia, Uppsala, Sweden). Anti-mouse CAD/DFF40/CPAN and anti-mouse ICAD/DFF45 polyclonal antibodies were purchased from Santa Cruz (Santa Cruz, CA) and Imgenex (San Diego, CA), respectively. Anti-HA and anti-Flag (clone M2) mAbs were purchased from Santa Cruz and Covance (Richmond, CA), respectively. Antibody against endonuclease G (endoG) was obtained from Dr D. Kang, Kyushu University (Fukuoka, Japan). Recombinant caspase-3 was purchased from Chemicon (Temecula, CA).

Preparation of cell extract and nuclei, and subcellular fractionation

Fifty micrograms of anti-CD3 antibody or control antibody was injected intraperitoneally into 4-week-old ICR mice. After 20 hr, thymocytes were obtained from the mice and cell extract was prepared as described previously.13,14 Protein concentrations were determined using a protein assay (Bio-Rad, Hercules, CA). Cell extract was further fractionated into mitochondria-rich, microsomal and cytosolic fractions, according to the method described by Ferren et al.15 Nuclear protein was extracted from the nuclear pellet as described by Schreiber et al.16 In brief, the nuclear pellet was suspended in buffer C (20 mm HEPES, pH 7·9; 0·4 m NaCl; 1 mm EDTA; 1 mm EGTA; 1 mm dithiothreitol [DTT]; and 0·25 m sucrose), and shaken vigorously at 4° for 15 min. After centrifugation (15 000 g, 4°) for 15 min, supernatants (nuclear fraction) were harvested and the protein concentration was measured.

Sucrose density-gradient centrifugation

To separate organelles of thymocytes, 0·6 ml of cell extract was layered over a linear gradient of sucrose (from 1·10 to 1·27 in density) and centrifuged (100 000 g, 4°) for 90 min in a VTi 65 rotor (Beckman, Palo Alto, CA). Successive gradient fractions (10 fractions, each 1 ml) were collected from the bottom, dialysed against cell extract buffer (CEB; 50 mM PIPES (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 10 mM cytochalasin B),13 and assayed for enzyme distribution and apoptosis-inducing activity. Density distribution of lactate dehydrogenase (LDH), cytochrome c oxidase, NADH cytochrome c reductase, and esterase were analysed as described previously.17,18

Assay for apoptosis-inducing activity

DNA fragmentation-inducing activity was determined as described previously.13 Briefly, different amounts of the cell extract or subcellular fractions, and 2 × 106 murine liver nuclei in a reaction buffer, were incubated in a final volume of 200 µl at 37° for 60 min. After incubation, chromosomal DNA was prepared from nuclei and analysed on 2% agarose containing 0·1 µg/ml ethidium bromide. Where indicated, the cell extract or subcellular fractions were incubated with 6 µg/ml of glutathione S-transferase (GST)-ICAD (prepared as described previously; see ref. 13) for 30 min at 4° prior to incubation with liver nuclei.

Transfection

Two-hundred micrograms of pcDNA3-HA-CAD vector and 50 µg of pcDNA-3-Flag-DFF45 vector13 were cotransfected into 2 × 107 Cos-7 cells or 293T cells by using a calcium phosphate method. Where indicated, pEYFP-ER (Clontech, Palo Alto, CA), encoding a fusion protein of enhanced fluorescent protein (EYFP) and ER-targeting sequence at the 5′ and 3′ end, was cotransfected. After 48 hr of incubation, transfected cells were harvested and used for each experiment.

Immunoblotting

Each fractionated protein was solubilized in sodium dodecyl sulphate (SDS) sample buffer, separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (12·5% gel) and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad) at 100 V for 60 min. After treatment with 5% non-fat milk, the membranes were incubated with primary antibodies against mouse CAD, ICAD, calnexin, HA-, or Flag-epitope. Horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG was used as the secondary antibody and proteins were visualized by using the enhanced chemiluminescence system (Renaissance; NEN Life Science Products Inc., Boston, MA).

Immunocytochemistry

For immunocytochemistry analysis, thymocytes and EYFP/HA-CAD/Flag-ICAD-transfected Cos-7 cells were fixed for 30 min in phosphate-buffered saline (PBS) containing 3·7% formaldehyde and then permeabilized with 0·5% Triton-X-100 for 5 min. After blocking in 3% non-fat milk for 30 min at room temperature, cells were incubated with polyclonal rabbit anti-CAD (1 : 100), monoclonal mouse anti-HA (1 : 100), or anti-Flag (1 : 100), at 37° for 30 min. Bound antibody was detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (EY Laboratories, San Mateo, CA) or Tetramethylrhodamine B isothiocyanate (TRITC)-conjugated rabbit anti-mouse IgG (EY Laboratories) and analysed using a confocal laser-scanning microscope (Carl Zeiss, Jena, Germany).

Results

Subcellular localization of DNA fragmentation-inducing activity and CAD/ICAD in in vivo anti-TCR-stimulated or unstimulated thymocytes

In order to determine the subcellular localization of molecules(s) responsible for DNA fragmentation in in vivo anti-TCR-stimulated thymocytes, ICR mice were injected intraperitoneally with anti-CD3 Ab and subcellular fractions of thymocytes were prepared. We then investigated the ability of each fraction to induce chromosomal DNA fragmentation in a cell-free system as previously described.13 As shown in Fig. 1(a), the cytosol fraction and the microsome fraction from anti-CD3-stimulated thymocytes induced DNA fragmentation, while none of the fractions from unstimulated thymocytes caused DNA fragmentation. When the fractions from anti-CD3-stimulated thymocytes were incubated with the recombinant ICAD, the DNA fragmentation-inducing activities in the microsome and cytosol fraction were completely abolished, indicating that the molecule responsible for the DNA fragmentation-inducing activity is CAD (Fig. 1b).

Figure 1.

Figure 1

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Subcellular localization of DNA fragmentation-inducing activity, caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) in in vivo anti-T-cell receptor (TCR)-stimulated or unstimulated thymocytes. (a) DNA fragmentation-inducing activities of subcellular fractions of in vivo TCR-stimulated or unstimulated thymocytes in a cell-free system. Fifty micrograms of anti-CD3 monoclonal antibody (mAb) or control Ab was injected intraperitoneally into ICR mice. Twenty hours later, cell lysates of thymocytes were separated into mitochondria (Mit), microsome (Mic) and cytosol (Cyt) fractions, as described in the Materials and methods. Normal liver cell nuclei separated from mice were incubated with 200 µl of each subcellular fraction at 37° for 2 hr, and DNA fragmentation was examined by agarose-gel electrophoresis. Lys, cell lysate; Nuc, nuclei fraction. (b) Inhibition of DNA fragmentation-inducing activity by ICAD. The cell lysate or the subcellular fractions prepared from anti-CD3-stimulated thymocytes were incubated with GST-ICAD for 30 min, and then DNA fragmentation-inducing activity was analysed as described above. (c) Immunoblot analysis of CAD and ICAD in unstimulated or TCR-stimulated thymocytes. Fifteen microlitres of total lysate or of each subcellular fraction, prepared as described above, were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and the blot was probed with anti-CAD Ab, or anti-ICAD Ab, respectively. Molecular weight (MW) markers are indicated on the left. (c) Immunoblot analysis of endonuclease G (endoG) in unstimulated or TCR-activated thymocytes. Nuclear fractions prepared from unstimulated or TCR-activated thymocytes were separated by SDS–PAGE, as described above, and the blot was probed with anti-endoG Ab. MW markers are indicated on the left.

We then investigated the subcellular localization of CAD and ICAD, in unstimulated or anti-CD3-stimulated thymocytes, using immunoblot analysis. As shown in Fig. 1(c), both CAD and ICAD were localized in nuclear, microsome and cytosol fractions of unstimulated thymocytes. When thymocytes were stimulated with anti-CD3, the concentration of ICAD in the microsome fraction was decreased and ICAD in the cytosol fraction was hardly detectable, while CAD in these fractions was clearly detectable. These results demonstrated that the CAD/ICAD complex resides in nuclear, microsome and cytosol fractions, and that engagement of the TCR on thymocytes induces cleavage of ICAD in microsome and cytosol fractions, and the resulting activated CAD induces DNA fragmentation in the isolated nuclei.

In the nuclear fraction of anti-CD3-stimulated thymocytes, degradation-inducing activity was observed. This activity was not inhibited by ICAD (Fig. 1b), suggesting that a DNAse other than CAD was activated by TCR stimulation. It has recently been reported that a mitochondrion-specific nuclease, endoG, is translocated to the nucleus and cleaves DNA.19 We examined whether the nuclear fraction of anti-CD3-stimulated thymocytes contains endoG, by Western blot analysis using anti-endoG antibody. As shown in Fig. 1(d), a 30 000-molecular weight (MW) band (corresponding to endoG) was detected in the nuclear fraction of anti-CD3-stimulated thymocytes, but not in unstimulated thymocytes. This result strongly suggests that endoG is one of the nucleases responsible for cleaving DNA independently of CAD in the nuclear fraction of activated thymocytes.

CAD was originally identified as a DNA fragmentation-inducing molecule activated by caspase-3.12 To examine whether caspase-3 induces the activation of CAD in microsome and/or cytosol in thymocytes, lysate or subcellular fractions separated from unstimulated thymocytes were incubated with recombinant active caspase-3 and the DNA fragmentation-inducing activity was analysed. As shown in Fig. 2(a), although none of the subfractions from unstimulated thymocytes showed DNA fragmentation-inducing activity, DNA fragmentation-inducing activity was induced in microsome and cytosol fractions by treatment with active caspase-3. These activities were inhibited by ICAD (Fig. 2b). These data suggest that CAD localized in microsome and cytosol fractions were activated by caspase-3.

Figure 2.

Figure 2

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Induction of DNA fragmentation-inducing activity in microsome/cytosol fractions from unstimulated thymocytes by recombinant caspase-3 (casp3). (a) Activation of DNA fragmentation-inducing activity by casp3. Microsome (Mic) or cytosol (Cyt) fractions were separated from unstimulated thymocytes and 200 µl of each fraction was incubated with 3 U of casp3 for 2 hr. After incubation, each sample was incubated with isolated nuclei and DNA fragmentation-inducing activity was examined as described in Fig. 1. One unit of casp3 is equivalent to the enzyme activity that cleaves 1 nmol of the caspase substrate DEVD-pNA. Lys, cell lysate. (b) Inhibition of DNA fragmentation-inducing activity by the inhibitor of caspase-activated deoxyribonuclease (ICAD). Mic or Cyt fractions from unstimulated thymocytes were incubated with casp3, as described above, incubated with GST-ICAD (see Fig. 1) and then DNA fragmentation activity was analysed.

Localization of CAD and ICAD in organelles separated by sucrose density-gradient ultracentrifugation

To more precisely determine the localization of CAD/ICAD in thymocytes, cell lysate from unstimulated thymocytes, from which nuclei had been removed, was separated into 10 fractions using sucrose density-gradient ultracentrifugation. After separation, the organelle-specific enzyme activities (LDH in cytosol, cytochrome c oxidase in mitochondria and cytochrome c reductase in ER) in each fraction were determined. Simultaneously, immunoblot analysis of calnexin (ER membrane-bound protein), CAD and ICAD in each fraction was performed. As shown in Fig. 3, LDH activity was detected in fractions 9 and 10. Cytochrome c oxidase activity was found mainly in fractions 3 and 4, suggesting that these fractions contain most of the mitochondria. Cytochrome c reductase activity and calnexin were detected broadly in fractions 2–8, indicating that proteins localized in the ER are distributed in fractions 2–8. A strong cytochrome c reductase activity was noted in fractions 9 and 10, which may be a result of enzyme release from the ER to cytosol during the fractionation procedure. As shown in Fig. 3(b), both CAD and ICAD were detected in fractions 2–8 (the ER fraction) and fractions 9–10 (the cytosol fraction), confirming that CAD and ICAD are localized in both the ER and cytosol.

Figure 3.

Figure 3

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Localization of caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) in organelles separated by sucrose density-gradient ultracentrifugation. (a) Localization of organelle-specific enzyme activity. Cell lysate from unstimulated thymocytes was layered on the linear gradient of sucrose and separated into 10 fractions after centrifugation, and enzyme activities of lactate dehydrogenase (LDH) (localized in the cytosol), cytochrome c oxidase (localized in the mitochondria) and cytochrome c reductase (localized in the endoplasmic reticulum) in each fraction were determined. (b) Western blot of fractionated cell lysate of unstimulated thymocytes probed by anti-calnexin, anti-CAD and anti-ICAD.

CAD and ICAD are localized in the microsome and cytosol fractions in CAD/ICAD-transfected Cos-7 cells

To confirm the localization of CAD/ICAD protein in microsome and cytosol in other cells, we transfected HA-CAD/Flag-ICAD cDNA into Cos-7 cells and DNA fragmentation-inducing activity, as well as localization of HA-CAD/Flag-ICAD, in each subcellular fraction were determined prior to or after treatment with active caspase-3. As shown in Fig. 4, no DNA fragmentation-inducing activity was detected in subcellular fractions of the transfectants, but HA-CAD/Flag-ICAD was detected in microsome and cytosol fractions, as determined by immunoblot with anti-CAD and anti-Flag Abs. When these subcellular fractions were treated with recombinant active caspase-3, potent DNA fragmentation-inducing activity was induced in the microsome and cytosol fractions. This treatment of the microsome and cytosol fractions with active caspase-3 induced degradation of Flag-ICAD, while CAD remained in these fractions, indicating that active caspase-3 degraded ICAD to convert inactive CAD to active CAD in the microsome and cytosol fractions.

Figure 4.

Figure 4

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Localization of DNA fragmentation-inducing activity, caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) in subcellular fractions of CAD/ICAD cDNA-transfected 293T cells. (a) DNA fragmentation induced by active caspase-3-treated or untreated subcellular fractions from CAD/ICAD-transfected 293T cells. HA-CAD cDNA and Flag-ICAD cDNA were cotransfected into 293T cells (5 × 107) and the lysate of transfectants was fractionated as described in Fig. 2. Two-hundred microlitres of each fraction was treated with or without 3 U of active caspase-3 (casp3), and DNA fragmentation-inducing activity was examined as described in Fig. 1. (b) Immunoblot analysis of CAD and ICAD in untreated or active casp3-treated lysate and its subcellular fractions. Fifteen microlitres of total lysate or of each subcellular fraction was separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted with anti-CAD antibody (Ab) or anti-Flag Ab. Anti-CAD Ab, instead of anti-HA, was used because HA-Tag of HA-CAD could be degraded by casp3. Indeed, we detected two bands of CAD by immunoblot with CAD Ab of which the upper band was thought to be intact HA-CAD and the lower band HA-deficient-HA-CAD. Molecular weight (MW) markers are indicated on the left. Cyt, cytosol; Lys, cell lysate; Mic, microsome; Mit, mitochondria.

Analysis of CAD localization in thymocytes and Cos-7 transfectants by immunofluorescence staining

To further investigate the localization of CAD in thymocytes, immunocytochemistry using confocal microscopy was performed. When thymocytes were stained with anti-CAD Ab, CAD was found to be localized mainly in the cytoplasm (Fig. 5a). Although dotted stains were observed in the cytoplasm, and this suggested the localization of CAD in ER, it was hard to discriminate cytosol and ER as a result of the fact that thymocytes have a very narrow cytoplasmic space. To determine the location of CAD in a different cell, we transfected Cos-7 cells with cDNA for HA-CAD and Flag-ICAD together with pEYFP-ER encoding EYFP fused with the ER-targeting sequence. Transfectants were then immunostained with anti-HA and analysed using confocal microscopy. As shown in Fig. 5(b), the cytoplasm was positively stained with only anti-HA Ab and the ER was stained with not only EYFP but also anti-HA Ab, showing that CAD is localized in both cytosol and ER. The results show that CAD localizes to ER in the Cos-7 transfectants.

Figure 5.

Figure 5

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Immunocytochemistry of caspase-activated deoxyribonuclease (CAD) in thymocytes and CAD-cDNA transfected Cos-7 cells. (a) Immunocytochemistry of normal thymocytes. Thymocytes from normal ICR mice were stained with anti-CAD antibody (Ab) (green) and analysed by confocal microscopy. (b) Immunocytochemical staining of HA-CAD/Flag-ICAD transfected Cos-7 cells. Cos-7 cells were transfected with HA-CAD/Flag-ICAD and the EYFP-ER vector encoding the endoplasmic reticulum (ER)-localizing EYFP fusion protein (green). After transfection, cells were stained with anti-HA Ab (red) and analysed by confocal microscopy. CAD localized in ER is shown in yellow and CAD localized in cytosol in red. cDNA of both CAD and its inhibitor (ICAD) were transfected because CAD is not expressed by CAD cDNA alone.12

Active CAD is released from microsome by treating microsome with active caspase-3

Our results suggested that CAD is localized as an inactive form (probably in a type of CAD–ICAD complex) in both cytosol and microsome, and that the inactive CAD localized on microsome is converted to active CAD by caspase-3-mediated ICAD degradation. In order to clarify whether active CAD is released from microsome by treatment with caspase-3, the microsome fractions from thymocytes or 293T cells, which had been transfected with cDNAs encoding CAD and ICAD, were treated with active caspase-3. The sample was then centrifuged into a precipitate fraction (that contained mainly ER) and a supernatant fraction (that contained soluble molecules), and the DNA fragmentation-inducing activity of each fraction was examined. As shown in Fig. 6, the supernatant fraction of caspase-3-treated microsome of thymocytes and transfectants exhibited DNA fragmentation-inducing activity, which was inhibited by ICAD. The supernatant of untreated microsome did not show DNA fragmentation-inducing activity. Addition of active caspase-3 to the supernatant of untreated microsome did not induce DNA fragmentation (data not shown). These results suggest that caspase-3 exerted its activity to the CAD–ICAD complex in microsome to degrade ICAD, and the activated CAD was released from microsome to cytosol, and after translocation into nuclei, it caused chromosomal DNA fragmentation.

Figure 6.

Figure 6

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Release of activated caspase-activated deoxyribonuclease (CAD) from the endoplasmic reticulum (ER) by treatment with caspase-3 (casp3). Microsome fractions prepared from thymocytes or 293T cells transfected with cDNA from CAD or its inhibitor (ICAD) were untreated (lanes 1 and 2) or treated (lanes 3 and 4) with active casp3 for 2 hr at 37°. The fractions were ultracentrifuged at 100 000 g for 90 min, and separated into precipitates (ppt; lanes 1 and 3) and supernatant (sup; lanes 2 and 4). Each sample was incubated with purified liver nuclei for 2 hr at 37° in the absence (lanes 1–4) or presence (lane 5) of ICAD, and DNA fragmentation-inducing activity of each fraction was examined by agarose-gel electrophoresis.

Discussion

This study describes the localization and function of CAD/ICAD in in vivo TCR-stimulated thymocytes. First, it was shown that CAD/ICAD are localized in cytosol, microsomal and nuclear fractions in unstimulated thymocytes, and that following in vivo TCR stimulation, CAD was activated with degradation of ICAD, and DNA fragmentation in nucleus was induced (Fig. 1,Fig. 3). Second, it was demonstrated that CAD, located in both cytosol and microsome, was activated by recombinant caspase-3. Finally, we demonstrated that CAD localized in the microsomal fraction of unstimulated thymocytes is activated by caspase-3, and is released from microsome into soluble fraction by cleavage of ICAD (Fig. 6).

There are many conflicting reports regarding the localization of CAD and/or ICAD. First, it was shown that DFF/CAD and ICAD are localized in the cytosol (S100 fraction) of WR19L, Jurkat, or HeLa cells.12,20 Second, the nuclear localization of the CAD/ICAD complex was reported in HeLa or DFF40/DFF45 cDNA-transfected CV-1 cells.11,21,22 Third, it was reported that ICAD was localized in the nuclei of thymic cortical epithelial cells and medullar lymphocytes and in both nuclei and cytosol of interfollicular lymphocytes of spleen and Hodgkin's lymphoma cells.23 Taken together, it is probable that the localization of CAD and ICAD may vary in different cell types. With this in mind, we transfected HeLa cells or Cos-7 cells with CAD/ICAD cDNA and (using confocal microscopy) we found that CAD was localized mainly in the nuclei in HeLa cells (data not shown) and in the ER in Cos-7 cells (Fig. 5), confirming that the localization of CAD/ICAD differs from cell to cell.

In this study, CAD localized in microsome was activated by caspase-3 and released from microsome, finally causing DNA fragmentation in TCR-stimulated thymocytes (Fig. 1,Fig. 6). Regarding activation of caspase-3 in thymocytes in response to TCR stimulation, we and others have shown that in vitro TCR engagement of thymocytes induces disruption of mitochondrial membrane potential,5 cytochrome c release from mitochondria24 and caspase-3 activation.25,26 Although how in vivo anti-CD3 stimulation induces activation of caspase-3 in thymocytes has not been determined, it is conceivable that a similar cell death pathway through mitochondria/cytochrome c is activated in an in vivo situation.

Regarding the activation of CAD in cytosol and ER by caspase-3 and generation of DNA fragmentation, it was shown that CAD remains inactive while it binds to ICAD. However, once caspase-3 cleaves the ICAD at two sites (DETD and DAVD at amino acid positions 117 and 224, respectively), CAD is converted to an active form which in turn generates DNA fragmentation.27 These sites could be cleaved not only by caspase-3, but also by several other proteases of caspase family members. It has been reported previously that active caspase-7 could directly cleave ICAD, although the efficiency was much less compared with caspase-3.28 It was also demonstrated that granzyme B, caspase-6 and caspase-8 prompted cleavage of ICAD indirectly by activating caspase-3 and/or caspase-7.28 Furthermore, it was found that active caspase-7 was translocated to liver mitochondrial as well as microsomal fractions following Fas activation by agonistic anti-Fas antibody.29 Thus, it is conceivable that activated caspase-7, together with caspase-3, is involved in the cleavage of ICAD and activation of CAD in cytosol and microsome, which finally induces DNA fragmentation in TCR-stimulated thymocytes.

In summary, we have demonstrated that CAD/ICAD are located in nuclear, cytosol and microsome in thymocytes, and CAD localized in cytosol and microsome is activated with degradation of ICAD in response to TCR stimulation. The physiological role of location of CAD/ICAD in microsome has still to be determined.

Acknowledgments

We thank Sanae Hirota, Yusuke Hayashi and Masafumi Toriyabe for technical assistance, and Kaoru Hata for secretarial work. This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.

Abbreviations

DFF40

DNA fragmentation factor 40

CAD

caspase-activated DNAse

DFF45

DNA fragmentation factor 45

ICAD

inhibitor of caspase-activated DNAse

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