The FASEB Journal express article 10.1096/fj.02-0020fje. Published online June 7, 2002. Stressful death of T-ALL tumor cells following treatment with the antitumor agent Tetrocarcin-A Inge Tinhofer*,�, Gabriele Anether*, Monika Senfter*, Kristian Pfaller�, David Bernhard�, Mitsunobu Hara§, and Richard Greil*,�

*Laboratory of Molecular Cytology, Department of Hematology and Oncology, �Tyrolean Cancer Research Institute, and �Institute of Anatomy and Histology, University of Innsbruck, Innsbruck, Austria; and §Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co. Ltd., Shimotogari 1188, Nagaizumi-cho, Suntougun, Shizuoka-ken 411, Japan Corresponding author: Inge Tinhofer, Laboratory of Molecular Cytology, Department of Hematology and Oncology, University of Innsbruck, A-6020 Innsbruck, Austria. E-mail: Inge.Tinhofer@uibk.ac.at ABSTRACT The T-ALL cell lines CCRF-CEM and Jurkat were studied for their sensitivity toward apoptosis induced by tetrocarcin-A (TC-A), an antibacterial and antitumor agent isolated from the actinomycete Micromonospora. This substance promoted cell death via a mitochondrial signaling pathway, that is, by activation of Bid and Bax, loss of the mitochondrial transmembrane potential, release of cytochrome c, and activation of effector caspases, even under conditions of Bcl-2 overexpression. Furthermore, sensitivity to TC-A was not dependent on expression of wild-type caspase-8. In contrast, this apoptotic pathway was inhibited markedly by pretreatment of cells with cycloheximide, an inhibitor of de novo protein synthesis. cDNA microarray chip analysis revealed that TC-A induced a significant up-regulation of members of the heat shock protein family known to be involved in the endoplasmic reticulum (ER)-stress-induced apoptotic program. The activation of caspase-12, the central inducer caspase involved in ER-stress by TC-A treatment, is in concordance with this result. These results show that, in T-ALL cells, TC-A induces an apoptotic machinery via mitochondrial and ER signaling, which is not inhibited by aberrant expression/function of important regulators of death receptor- and drug-induced apoptosis. Key words: ER-stress • heat shock protein • apoptosis

A

poptotic signaling pathways have received much attention because the induction of apoptosis seems to be the predominant mode of action of chemotherapeutic drugs (1, 2). However, attempts to eliminate tumor cells by the induction of apoptosis frequently fail

because of the selective advantage of tumor cells, which either express elevated levels of anti-apoptotic proteins, have down-regulated important mediators of the main apoptotic signaling pathways, and/or express mutated forms or even lack the expression of tumor-suppressor genes involved in the initiation of apoptosis. Thus, the identification of agents that might negate these tumor-specific survival advantages by inducing signals that bypass these regulators are of particular clinical interest.

Mitochondrial components and functions are important regulators of apoptotic pathways. Early events in cells triggered to undergo apoptosis often include a decrease in the mitochondrial transmembrane potential (∆Ψm) and the release of pro-apoptotic proteins stored in the intermembrane space of mitochondria, such as caspase-2 and -9 (3), cytochrome c (4), Smac/Diablo (5), and apoptosis-inducing factor (AIF) (6). Among the genes known to regulate apoptotic signals induced by cytotoxic drugs and proceeding via mitochondria, those belonging to the Bcl-2 family play an important role. The pro-apoptotic members of this family have been shown to act on mitochondrial membranes, either directly by forming channels in mitochondrial membranes (i.e., Bax, Bak) or indirectly by oligomerization with Bax (7). The function of these pro-apoptotic proteins is inhibited by direct interaction with anti-apoptotic members of the family, that is, Bcl-2, Bcl-XL, and Bcl-w. High levels of Bcl-2 expression have been detected in a variety of tumor types as a result of chromosomal translocation (8) or loss of negative transcriptional control by wild-type p53 (9). Based on the well-established role of Bcl-2 as a critical regulator of cell death processes, Bcl-2 antisense strategies have been developed for reversal of resistance to apoptosis of leukemia and lymphoma cells that overexpress Bcl-2 (10). Beside this potential therapeutic strategy, new drugs, which either inactivate the anti-apoptotic function of Bcl-2 or bypass the Bcl-2-mediated control of apoptosis, could be clinically exploited. In addition to the apoptotic pathway mediated via mitochondrial signals and regulated by Bcl-2 family members, a different signaling pathway has been described, which starts with the activation of caspase-8, a component of the death receptor signaling complex (DISC), and is insensitive to overexpression of Bcl-2 (11). Unlike its proximal role in death receptor signaling, in drug-induced apoptosis caspase-8 functions rather as an amplifying executioner caspase downstream of caspase-3 activation (12, 13). Inactivation of caspase-8 gene by DNA hypermethylation in neuroblastoma (14, 15), malignant brain tumors, and melanoma (15) mediates resistance to both death-receptor as well as drug-induced apoptosis (15), which underscores its importance in both of the apoptotic pathways described above. TC-A was isolated from a Micromonospora culture and has been characterized to have antibiotic as well as antitumor functions (16). In a recent study, it was reported that HeLa cells with ectopic high expression of Bcl-2 or Bcl-XL were resistant to Fas and tumor necrosis factor (TNF) as well as to staurosporine treatment, but could be resensitized to these pro-apoptotic stimuli by pretreatment with TC-A (17). In detailed biochemical analyses, it was demonstrated that it was the protective function of Bcl-2 and of Bcl-XL on mitochondria that was abolished by TC-A pretreatment, while the substance alone showed no cytotoxic activity. However, the exact molecular action of TC-A on Fas-, TNF- and staurosporine-induced apoptosis was not completely clarified by this study. In the present study, we raised the question whether TC-A might be applicable as a single pro-apoptotic agent in tumor therapy by using T-ALL as a model. We focused on the elucidation of the apoptotic signaling pathway and its dependence on Bcl-2 and caspase-8, the known important regulators of drug-induced apoptosis. MATERIALS AND METHODS Reagents and Antibodies The following reagents and antibodies were used in this study: tetrocarcin-A (Kyowa Hakko Kogyo Co, Shizuoka, Japan); fludarabine phosphate (Schering, Berlin, Germany), vincristine (Pharmacia and Upjohn, Vienna, Austria); cycloheximide, tunicamycin, brefeldin A, and pan-

caspase inhibitor zVAD-fmk (all from Calbiochem, San Diego, CA); A23187 (Sigma, Vienna, Austria); anti-human Bcl-2 (Dako, Vienna, Austria); anti-human Bax (Santa Cruz, Santa Cruz, CA); anti-human Bid, anti-human caspase-3, -7, and -9 and anti-human cytochrome c (all from Pharmingen, San Diego, CA); anti-cytochrome c oxidase (Molecular Probes, Leiden, The Netherlands); anti-human caspase-8 (Upstate Biotechnologies, Lake Placid, NY); anti-human Fas (clone CH11, Immunotech, Marseille, France); anti-human HSP70 (for immunoblotting: Stressgen Biotechnologies, Scabo Scandic, Vienna, Austria; for flow cytometric analysis: NeoMarkers, Fremont, CA); anti-human HSP110 and anti-human grp78 (Stressgen). Monoclonal mouse antibody against human caspase-12 (18) was kindly provided by Junying Yuan (Harvard Medical School, Boston, MA). Cell lines and culture conditions We used the T-ALL cell line C7H2-2A10 (C7H2-VC), which is a subclone of the CEM-C7H2, a glucocorticoid-sensitive subline of CCRF-CEM. CEM-C7H2-9C3 and -9F3 (C7H2-Bcl-2) are derivatives of the C7H2-2A10 cell line that are stably transfected with human bcl-2 cDNA (19). These subclones were kindly provided by Reinhard Kofler (Department of Experimental Pathology, University of Innsbruck, Austria). The Jurkat cell line was kindly provided by Gottfried Baier (Department of Medical Biology and Human Genetics, University of Innsbruck, Austria). A subclone of the Jurkat cell line with a spontaneous mutation in the caspase-8 gene was kindly provided by Peter Juo (Harvard Medical School) (20). All cell lines were cultured in RPMI-1640 media (PAA Laboratories, Linz, Austria) supplemented with 10 % heat-inactivated fetal calf serum (PAA Laboratories), 2 mmol/l L-glutamine (GIBCO, Grand Islands, NY), and 100 µg/ml gentamycin (GIBCO) at 37°C in a humidified atmosphere containing 5 % CO2. Detection of phosphatidylserine exposure on apoptotic cells Staining of cells with the combination of Annexin V/FITC and propidium iodide was performed according to the manufacturer's instructions for detection of early (Annexin V/FITC+/PI�); late (Annexin V/FITC+/PI+) apoptotic cells and both subpopulations together were considered to represent the total fraction of apoptotic cells (21). Briefly, 2.5 × 105 cells were incubated with saturating concentrations of Annexin V/FITC (Alexis, Läufelfingen, Switzerland) and propidium iodide (Sigma) for 15�30 min at room temperature (RT) and were analyzed immediately by flow cytometry. MTT assay For determination of the metabolic activity of cells, the CellTiter96 Assay (Promega, Madison, WI) was used according to the manufacturer�s protocol. Briefly, the metabolic activity of cells could be recorded by the cells� ability to convert the tetrazolium component of a dye solution into formazan. The product was quantified photometrically by absorbance reading at 570 nm. Detection of DNA strand breaks For detection of double-strand breaks yielding low-molecular-weight DNA fragments, we stained the cellular DNA content following cell permeabilization with propidium iodide solution (Triton X-100 0.1%, propidium iodide 50 µg/ml, sodium citrate 0.1%) according to the protocol of Nicoletti et al. (22). Cells with cleaved DNA and hence lower stainability ('sub-G1' peak) were quantified by flow cytometry.

Assessment of the mitochondrial transmembrane potential (∆Ψm) The breakdown of the mitochondrial transmembrane potential (∆Ψm) was followed by staining cells with the dual-emission potential-sensitive probe 5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazolylcarbocyanine iodide (10 µmol/l) (JC-1, Molecular Probes) for 20 min at RT. This fluorochrome has been demonstrated to be a reliable probe for assessing ∆Ψm changes during apoptosis (23). Immediately after being washed with phosphate-buffered saline (PBS), the fluorescence signal intensity of FL-2 (representing cells with high mitochondrial transmembrane potential) and of FL-1 (cells with low mitochondrial transmembrane potential) was analyzed by flow cytometry. Immunoblotting Cells (5 × 106) were resuspended in 100 µl lysis buffer (50 mmol/l Tris/HCL, pH 7.5; 150 mmol/l NaCl; 2 mmol/l EDTA; 1 mmol/l EGTA supplemented with 25 µg/ml leupeptin; 25 µg/ml aprotinin; and 1% Triton X-100) and three times were frozen and thawed in liquid nitrogen. The samples were cleared by centrifugation (14,000 × g, 30 min, 4°C) and were corrected for protein concentrations. SDS/PAGE was performed under reducing conditions on Tris/glycine-buffered gels (Novex, San Diego, CA). Proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) by semidry blotting (220 mA, 70 min, 4ûC). The membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk, followed by incubation with the primary antibody (Ab) at a dilution of 1:1000. Horseradish peroxidase (HRP)-conjugated Ab (Dako, Copenhagen, Denmark) were used as secondary Ab (dilution 1:1000). HRP-labeled antibodies were detected in a luminol-based chemiluminescent reaction (Amersham Life Sciences, Buckinghamshire, UK). As an internal control for equal protein amounts, tubulin-α was detected. cDNA microarray analysis Cells (6 × 5 × 106/well) were left untreated or stimulated with TC-A (1µmol/l) for 8 h and washed with PBS, and total RNA was isolated by using TRIzol reagent (GIBCO Life Technologies, Lofer, Austria) according to the manufacturer�s protocol. Briefly, cells were transferred to reaction tubes, pelleted, resuspended in 1 ml TRIzol reagent, and incubated for 5 min at RT. Subsequently, we added chloroform, and the suspension gently mixed, incubated for 3 min at RT, and centrifuged for 15 min at 4°C and 12,000 × g. The upper phase was collected, mixed with 500 µl isopropanol, incubated at RT for 10 min, and centrifuged for 10 min at 4°C at 12,000 × g. The pellet was resuspended in 1 ml ice-cold ethanol (75%) and centrifuged for 5 min at 4°C at 7,500 × g. The supernatant was discarded and the pellet was air-dried for 10 min at RT, then resuspended in 15 µl Tris/HCl buffer (10 mmol/l, pH 7.5) and heated to 60°C for 10 min. We determined RNA content and purity photometrically. Finally, parallel samples (sixfold) were pooled to a total of at least 260 µg RNA, freezed, and out-sourced for hybridization and analysis (PIQORTM Cell death chip; Human, Memorec, Cologne, Germany). For data analysis, we used GenePix Pro3.0 software (Axon Instruments, Union City, CA). For further detailed information on PIQORTM cDNA array systems, see Tomiuk and Hofmann (24) or visit www.memorec.com/technologien.htm. Scanning electron microscopy (SEM)

C7H2-VC or C7H2-bcl2 cells (1 × 106 cells/ml) were left untreated or stimulated with 1 µmol/l TC-A for the indicated time. Subsequently, they were washed, pelleted, and resuspended to a final concentration of 4 × 106 cells/ml. Of this cell suspension, 100 µl was spinned onto a cover slip (6 mm) by using a cytospin (Shandon, Pittsburgh, PA). Coverslips with cells were immediately fixed for 5 h in 2.5% glutaraldehyde in 0.2 mol/l cacodylate buffer (pH 7.3). Specimens were then washed in cacodylate buffer, osmificated for 1 h in 1% OsO4 in distilled water, and gradually dehydrated with ethanol following critical point drying in CO2 (BAL-TEC CPD 030 Balzers, Vaduz, Liechtenstein). They were mounted on aluminum stubs with colloidal silver, sputtered with 10 nm Au/Pd (BAL-TEC MED 020 Balzers), and examined with a Zeiss scanning electron microscope. RESULTS TC-A-induced apoptosis in CCRF-CEM cells is delayed but not inhibited by Bcl-2 overexpression To test whether TC-A treatment triggers apoptosis in T-ALL cells and whether apoptosis can be blocked by overexpression of Bcl-2, we treated the T-ALL CEM-C7H2 vector control cell line (C7H2-VC) with increasing doses of TC-A and compared the extent of apoptosis induction with that occurring in subclones stably overexpressing Bcl-2. As seen in Figure 1 A�D, the C7H2-VC cell line underwent cell death in a dose-dependent manner characterized by phosphatidylserine exposure (Fig. 1A), changes in cell volume and granularity (Fig. 1B), and DNA fragmentation (Fig. 1C). These changes were accompanied by the reduction of metabolic activity as measured by the MTT assay (Fig. 1D). Significant ultrastructural changes characteristic for apoptotic cells, that is, reduction of cell size, smoothing of cell surface, and formation of apoptotic bodies, were observed by scanning electron microscopy (Fig. 1E). With none of the different methods applied, a significantly different sensitivity could be detected in the Bcl-2-overexpressing subclone (Fig. 1A�E). However, because we observed a slightly reduced percentage of apoptotic cells in the Bcl-2-overexpressing subclone, we also determined the time dependence of TC-A-induced apoptosis in both cell line models (Fig. 2A, B). Bcl-2 overexpression delayed the onset of apoptosis (Fig. 2A), but when these cells were incubated for longer than 24 h (up to 7 days, Fig. 2B), the partial protection of cells by Bcl-2 was abolished by TC-A. The effector phase of TC-A induced apoptosis comprises mitochondrial signals To characterize the signal cascade triggered by TC-A, we focused on the mitochondrial pathway to apoptosis. The analysis of the mitochondrial transmembrane potential (∆Ψm) revealed that TC-A acted on mitochondria and reduced ∆Ψm in the C7H2-VC cell lines in a dose- and time-dependent manner (Fig. 3A, B). Again, Bcl-2 overexpression delayed but did not block TC-A-mediated loss of ∆Ψm (Fig. 3A, B). As expected, TC-A also induced the efflux of cytochrome c into the cytosol as detected by immunofluorescence analysis by using a specific anti-cytochrome c antibody (Fig. 3C). Bid and Bax, both pro-apoptotic members of the Bcl-2 family, have been demonstrated to contribute to the loss of ∆Ψm. Bid cleavage induced by death receptor signaling is mediated by caspase-8 (25), whereas Bax processing has been shown in a calpain- (26) or caspase-3-dependent manner (27) following cytotoxic treatment. Our analyses revealed that TC-A treatment led to the cleavage of Bid and Bax in a time-dependent manner (Fig. 4A). In parallel, Bax staining of TC-A-treated cells yielded a particulate pattern, which reflected the mitochondrial distribution (Fig. 4B, middle image). The mitochondrial association of Bax was underlined further by its co-localization with the respiratory chain enzyme cytochrome c oxidase

(Fig. 4B, right image). Bax cleavage was accompanied by an increase in binding of a specific anti-Bax antibody, raised against the native form of Bax, and used in this study for flow cytometric (Fig. 4C) and fluorescence microscopic studies (Fig. 4B). This increase in binding suggests that conformational changes of the protein had occurred. Cleavage of Bid and Bax could be inhibited by preincubation of cells with the pan-caspase inhibitor zVAD-fmk, which suggests that their processing occurred downstream of caspase activation (Fig. 4D). TC-A induced apoptosis proceeds via caspase activation Because we observed TC-A-induced cleavage of Bid and Bax, we wanted to determine which set of caspases would be activated by TC-A treatment. Applying immunoblot analyses, we analyzed caspase-3, -7, -8, and -9 and found that each of these caspases was proteolytically cleaved at least 12 h after TC-A addition (Fig. 5). Using these biochemical approaches, it is impossible to elucidate the exact sequence of caspase activation because of the different sensitivity of the antibodies used. Activation of caspase-3, -7, and -8, however, seemed to precede the processing of caspase-9. Although activation of caspase-8 was found in C7H2 (Fig. 5) and Jurkat cells (data not shown), in a Jurkat cell line lacking caspase-8 (20) we observed similar sensitivity toward apoptosis induction by TC-A compared with the wild-type caspase-8-expressing cell line (Fig. 6). In contrast, loss of caspase-8 completely abrogates apoptosis induced by the agonistic anti-Fas mab CH11 (data not shown). These data argue against an important role of caspase-8 in TC-A-induced apoptosis. The central role of caspases in this cell death program, however, is underlined by the almost complete inhibition of TC-A-induced cell death by the pan-caspase inhibitor zVAD (Fig. 7). De novo protein synthesis is required for TC-A-induced apoptosis Preincubation of C7H2 or Jurkat cells with cycloheximide, an inhibitor of protein synthesis, added either up to 16 h before or simultaneously with TC-A, proved to efficiently reduce the percentage of cells undergoing apoptosis (Fig. 8), whereas Fas-induced apoptosis used as negative control remained unchanged (data not shown). Thus, to screen for potential inducer(s) of TC-A apoptosis, we used a cDNA microarray system, which enabled us to analyze in parallel 200 genes known to be involved in apoptosis. C7H2-VC cells were stimulated with 1 µmol/l TC-A for 8 h (a time interval in which degradation of cellular targets were not yet detectable), and total RNA was prepared for microarray analysis as described (see Materials and Methods). As presented in Table 1, in cells stimulated with TC-A, 10% of the genes analyzed were regulated (genes with >twofold differential mRNA expression levels were considered as regulated). Among these 20 regulated genes identified, there were four transcription factors and six members of the heat shock protein (HSP) family. The strong increase of HSP, especially of HSP70 (>35-fold), suggested the activation of a program known as endoplasmic reticulum (ER)-induced stress by TC-A. HSP70 up-regulation is specific for TC-A treatment Expression of HSP70 observed to be up-regulated in the cDNA microarray analysis was analyzed at the protein level. For this purpose, cells were stimulated with TC-A (1 µmol) for 4, 8, and 24 h; stained for intracellular HSP70, and analyzed by flow cytometry (Fig. 9A). Stimulation with an anti-Fas antibody (250 ng/ml, 8 h) and heat treatment (42°C for 1 h, followed by 37°C for 24 h) served as negative and positive controls, respectively. Alternatively, cells lysates samples were prepared and subjected to immunoblotting (Fig. 9B). With both techniques, we could confirm the results of the microarray analysis that a strong up-regulation of

HSP70 is induced by TC-A, which resembles a cellular response to heat treatment but is absent in the Fas-stimulated cell sample despite comparable extent of apoptosis induction (data not shown). The signal for intracellular HSP70 decreased if cells were treated with TC-A for 24 h (Fig. 9A, B), which might result from an increase in membrane permeability and the diffusion of HSP70 into the supernatant (I. Tinhofer, unpublished observations). Analyzing the protein expression levels of HSP110 and grp78, two other proteins involved in the ER stress response, we found that HSP110 was significantly up-regulated in TC-A treated cells (Fig. 9C), whereas grp78 protein expression remains almost unchanged (data not shown), confirming the data from the microarray analysis (Table 1). We then analyzed two other pro-apoptotic agents with distinct mechanisms of action for their effect on HSP70 expression levels, that is, vincristine, a vinca alkaloid leading to cytoskeletal changes and fludarabine phosphate, a purine analog involved in the inhibition of DNA synthesis. Neither by flow cytometry (Fig. 9D) nor by immunoblot analyses (data not shown) could we detect changes in the expression levels of HSP70 following treatment with these drugs. Characterization of the ER-stress response triggered by TC-A The ER-stress response, also called the unfolded protein response (UPR), is a surveillance system in the ER, which regulates essentially all aspects of ER function and continuously coordinates the processing and degradation pathways for unfolded proteins. This system is activated in many situations that increase the levels of unfolded proteins in the ER lumen. Recent evidence shows that procaspase-12, a novel protease associated to ER-membranes, is cleaved in a calpain-dependent manner generating active caspase-12 and initiating ER-stress-induced apoptosis (28). Because the up-regulation of six members of the heat shock protein family suggested the induction of a stress response by TC-A, we questioned whether the TC-A-induced apoptosis might be driven by caspase-12 activation. To answer this question, we stimulated C7H2-VC or Jurkat cells with 1 µmol/l TC-A for 0, 4, 12, 16, or 24 h and performed immunoblot analysis of whole cell lysates to detect procaspase-12. As shown in Figure 10 for C7H2-VC cells, we observed a decrease in the specific signal for procaspase-12, which started 12 h after the addition of TC-A. Essentially identical results were obtained in Jurkat cells (data not shown). Because it has been reported that the activation of caspase-12 can also be mediated by caspase-7 (29) and because we observed active caspase-7 in lysates of TC-A treated cells, we asked whether caspase-12 processing following TC-A treatment occurs upstream or downstream of other caspases. C7H2-VC cells untreated or pretreated for 1 h with zVAD-fmk (20 µmol/l) were left unstimulated or stimulated with 1 µmol/l TC-A for 24 h. The processing of caspase-12 was then detected by immunoblot analysis. As seen in Figure 10B, the processing of caspase-12 could be inhibited by zVAD-fmk, which suggests that caspases other than caspase-12 are activated upstream, and these are involved in the processing of caspase-12. DISCUSSION Our present study elucidates the apoptotic pathway induced by treatment of T-ALL tumor cells with the antibacterial and anticancer agent TC-A. In this tumor model, TC-A alone was an efficient inducer of apoptosis in a dose- and time-dependent manner. Our analyses revealed further that the apoptotic features observed in cells treated with TC-A comprised phosphatidylserine exposure and DNA fragmentation, loss of mitochondrial transmembrane potential ∆Ψm, cleavage of Bid and Bax described to activate their pro-apoptotic function (26,

30), and activation of a broad spectrum of caspases. The induction of apoptosis was also reflected by the formation of apoptotic bodies. The signal cascade triggered by TC-A was independent of Bcl-2 expression levels and also occurred in tumor cells lacking caspase-8. The role of Bcl-2 expression in the modulation of chemosensitivity of tumor cells is controversial. Evidence has indicated that, although apoptosis induction correlated well with chemosensitivity of tumor cell lines, Bcl-2 expression levels did not, at least in the majority of cell lines tested (31). However, retrospective analyses of samples from ovarian cancer patients revealed an inverse relation of Bcl-2 levels and the initial responses to chemotherapy and identified Bcl-2 as independent prognostic factor (32). In tumors of the lymphatic system, such as B-chronic lymphocytic leukemia, no correlation of Bcl-2 expression alone but of Bcl-2/Bax ratios with drug-induced apoptosis has been found (33�35). Based on these data, antisense strategies were developed and demonstrated successful in increasing chemosensitivity of tumor cells lines in vitro (36�38) or in murine xenograft models (39, 40). Beside modification of bcl-2 gene expression at the translational step, drugs that inhibit the molecular interaction of Bcl-2 and its pro-apoptotic binding partners leading to the release of the latter, and thus to a more direct induction of cell death, should represent possible candidates for clinical antitumor therapy. Our data presented here imply that TC-A might fulfill such criteria: 1) we found that following TC-A treatment, Bax was cleaved into a 18k-Da fragment (Fig. 4A) in a caspase-dependent manner (Fig. 4D). An early N-terminal cleavage of Bax in etoposide-treated Jurkat cells has been described recently (26). In that study, it was found that the processing of the full-protein Bax to an 18-kDa fragment occurred in a caspase-independent manner, but was mediated by calpain. Most importantly, Bax/p18 colocalized to mitochondria had lost its ability to interact with Bcl-2 and, when expressed ectopically, had direct cytochrome-releasing ability in the transfected clone. 2) In our study, TC-A treatment of cells resulted in increased binding of a specific anti-Bax antibody, which might be caused by a conformational change of Bax (Fig. 4C). Such conformational changes in the C-terminus of Bax have been reported to be triggered by Bid (41), leading to the insertion of Bax into mitochondrial membranes (42). In fact, our immunofluorescence studies of Bax and, as a control for mitochondrial localization, of cytochrome c oxidase revealed the association of Bax to mitochondria in TC-A-treated cells (Fig. 4B). Beside the activation of Bax by Bid or other effector molecules involved in the apoptotic program, all these molecular changes could also have resulted from a direct interaction of TC-A with Bax. Such a direct interaction with the Bcl-2 homology domain 3 (BH3) of Bcl-XL has been demonstrated recently for antimycin A, also an antibiotic agent (43). This molecular interaction led to a paradoxical correlation of Bcl-XL expression levels (normally with anti-apoptotic function) with sensitivity toward apoptosis induced by antimycin-A. This paradox might be explained by antimycin-induced release of pro-apoptotic binding partners or by the conversion of an anti-apoptotic into a pro-apoptotic protein by post-translational alterations, for example, by proteolytic cleavage as described for Bcl-2 (44) and Bcl-XL (45). We observed that TC-A treatment led to the activation of caspases (Fig. 5), which was indispensable for apoptosis induction (Fig. 7). However, sensitivity to apoptosis by TC-A was independent of the caspase-8 expression status of the T-ALL tumor cell lines used (Fig. 6). In contrast to divergent results obtained from experiments designed to elucidate the role of death receptors in drug-induced apoptosis (46�48), the activation of caspase-8 has been identified as a common principle in both death receptor- (30) and drug-mediated cell death (48, 49). The

clinical relevance of caspase-8 defects was demonstrated by a recent study showing that, in cell lines or primary samples derived from neuroblastoma patients, loss of caspase-8 mRNA expression was found to result from promotor hypermethylation and was correlated with N-MYC gene amplification (14). The latter is associated with a more aggressive form of this tumor characterized by the frequent occurrence of drug resistance. In addition, reintroduction of caspase-8 into neuroblastoma cell lines resensitized these cells to drug-induced apoptosis (50). The importance of caspase-8 silencing by gene hypermethylation for the development of drug-resistance in a broader spectrum of tumor models was demonstrated recently (15). For evaluation of the significance of caspase-8 deficiences in primary tumor cells of T-ALL patients, further analyses in a similar detailed manner are required. The apoptotic features observed following TC-A treatment and described above are all part of a signaling cascade triggered by a variety of different pro-apoptotic stimuli, such as activation of death receptors or treatment with cytotoxics. In particular, TC-A-induced apoptosis could be blocked by coincubation of cells with cycloheximide (CHX), an inhibitor of protein synthesis (Fig. 7), which supports the assumption that CHX prevents the synthesis of molecules necessary for induction of apoptosis by TC-A. To identify possible inducers, we used the cDNA microarray technique, by which we observed up-regulation of six members of the HSP family following TC-A treatment, pointing to the induction of ER-stress by TC-A (Table 1). ER-stress can be induced by a variety of signals. These include diminished protein glycosylation caused by starvation or treatment with drugs, such as tunicamycin, increased production of misfolded mutant proteins or unassembled protein subunits and altered lumenal ion content caused by ionophores or heavy metals (51). The common result of these treatments is a state of ER-stress in which the burden of unfolded proteins exceeds the capacity of the ER machinery to deal with them. As a result, a stress signal, the so-called unfolded protein response (UPR), is transmitted from the lumen to the cytosol via transmembrane kinases resulting in translational attenuation (52). In addition, transcription of proteins is activated; these proteins reside in the ER, have chaperone function, and can buffer a dangerous accumulation of unfolded proteins (53). Similar to the cellular response to DNA damage, in case of irreversible damage, an intrinsic apoptotic pathway is triggered. This pathway is initiated by the activation of caspase-12, a caspase shown to be associated with the ER and mediating ER-stress in a Ca2+- and calpain-dependent manner (18). Consistent with the induction of an irreversible ER-stress response by TC-A are our observations of the cleavage of procaspase-12 (Fig. 10A), which was inhibited by pretreatment of cells with zVAD-fmk. Both calpain and caspases have been described recently to link ER-stress and caspase-12 activation: 1) In the study of Nakagawa et al., deprivation of oxygen and glucose induced caspase-12 activation in glial cells, which was mediated by calpain but not by caspases (28). 2) An alternative activation signal was found to involve caspase-7 which, following ER-stress, translocates from the cytosol to the surface of the ER and associates with procaspase-12 leading to its activation (29). From the results of our immunoblot analyses and our inhibitory experiments with zVAD-fmk, we hypothesize that in the TC-A mediated signaling pathway caspase-12 is activated downstream of caspase-3 and -7 (Fig. 5, 10). However, the exact mechanism by which TC-A induces caspase-12 activation in T-ALL cells and its sequence in the signal cascade, remains to be determined. The up-regulation of HSP70 and of HSP110 observed in TC-A-treated cells (Fig. 9, Table 1) suggests that ER functions are heavily disturbed, and, as a result, HSP are induced to limit cellular damage by their ability to bind to unfolded proteins, to inhibit protein aggregation, and to restore the function of denatured proteins (54). Besides its role in protein folding, HSP70 has also been demonstrated to actively suppress cell death by inhibiting caspase processing at the

apoptosome (55). HSP70 was found to be highly expressed in human tumor tissue and in tumor cell lines. This finding, together with the ability of HSP70 to protect cells from a wide range of apoptotic and necrotic stimuli, underscores the relevance of this HSP in tumorgenesis (56). In our present study, HSP70 failed to exert its survival function in TC-A-induced apoptosis. One explanation is that HSP70 has different functions depending on the tumor model, and/or the functions change when very high amounts of this protein accumulate in cells. An intriguing alternative hypothesis is that HSP70 is generally unable to block TC-A-induced apoptosis, which would further support the application of TC-A in tumor therapy. Beside the protective intracellular function of HSPs, they have been identified as highly important regulators of immunogenicity of cancer, representing a danger signal when cells are lysed and activating professional antigen-presenting cells to elicit a specific anti-tumor response (57). The impact of HSP induction in apoptotic tumor cells by TC-A for activating APC and eliciting anti-tumor T-cell responses will be the focus of future studies. CONCLUSION TC-A might represent an attractive anti-tumor agent because it induces stressful death via a mitochondrial and ER signaling, which is inhibited neither by aberrant expression/function of important regulators of death receptor- and drug-induced apoptosis nor by survival proteins involved in tumorigenesis. ACKNOWLEDGMENTS This work was supported by a grant of the Austrian Science Foundation (to I.T., T95-PAT; to D.B., P14482-MOB), the Austrian National Bank (to R.G., ÖNB-8222), the Academy of Sciences (to G.A.), the Tyrolean Cancer Aid, and the Austrian Cancer Aid and the Province of Tyrol (to R.G.). G.A. was supported by the Verein für Klinische Malignom und Zytokinforschung. We are indebted to Rajam Csordas for critical reading and editorial assistance. REFERENCES 1. Houghton, J. A. (1999) Apoptosis and drug response. Curr Opin Oncol 11, 475-481. 2. Makin, G. and Hickman, J. A. (2000) Apoptosis and cancer chemotherapy. Cell Tissue Res

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Received January 23, 2002; revised April 17, 2002.

Table 1 cDNA microarray analysis of CEM-C7H2 cells. Candidate list >twofold differential expression Ratio mRNA expression levels of TC-A treated

: untreated cells

Transcription factors

AP-1 3.3

FOS-B 2.1

STAT2 2.6

STAT4 2.0

Bcl-2 family members

A1 2.2

BIK –3.7

Cell cycle regulators

P21 (CIP1/WAF1) 3.2

Heat shock proteins

HSP105: (HSP105 OR HSP110) 7.2

HSP70 34.7

HSPCA: (HSPCA OR HSPC1 OR HSP90A) 6.7

BAG-3: SODD-RELATED PROTEIN BAG3 7.2

OSP94: (OSP94) OSMOTIC STRESS PROTEIN 94

3.7

BIP: (HSPA5 OR GRP78) 2.5

Fig. 1

Figure1. Sensitivity to TC-A-induced apoptosis in CCRF-CEM cells is independent of Bcl-2 overexpression. C7H2 vector control (black bars) or Bcl-2 overexpressing cells (white bars) were stimulated with TC-A at the indicated concentrations for 24 h. Subsequently, increase in phosphatidylserine exposure (A), changes in cell volume and granularity (B), and extent of DNA fragmentation (C) were determined by flow cytometry. The mean values of at least four independent experiments ± SEM are presented. D) Metabolic activity by TC-A was quantified in two independent experiments by using the MTT assay. The result from one representative analysis is presented. E) Ultrastructural characteristics of C7H2-VC (upper panel) and C7H2-Bcl2 cells (lower panel) left untreated (left side) or treated for 8 h with 1µmol/l TC-A (right side) were analyzed by SEM. Arrows indicate apoptotic bodies. Bars = 5 µm.

Fig. 2

Figure 2. Time dependence of TC-A-induced apoptosis. C7H2-VC (black bars) and Bcl-2 overexpressing cells (white bars) were stimulated for the indicated times (A, 0–24 h; B, 1–7 d), and the percentage of apoptotic cells was determined by using the Annexin V/FITC/PI assay. The mean values of at least five independent experiments ± SEM (A) and of two independent analyses (B) are presented.

Fig. 3

Figure 3. TC-A induces apoptosis via mitochondrial signaling. C7H2-VC (black bars) or Bcl-2-overexpressing cells (white bars) were left untreated or stimulated with increasing concentrations of TC-A (A) or with 1µmol/l TC-A for the indicated times (B). Each bar represents mean ± SEM of five determinations. C) The redistribution of cytochrome c from mitochondria in untreated cells to the cytosol in C7H2-VC cells treated with 1µmol/l TC-A for 12 h was detected by immunofluorescence staining followed by microscopic analysis.

Fig. 4

Figure 4. TC-A induces Bid/Bax cleavage, translocation and conformational changes of Bax. A) Immunoblot analyses against Bid and Bax from lysates of C7H2-VC cells left untreated or stimulated with 1µmol/l TC-A for the indicated times are presented. The specific signals for the uncleaved and cleaved form of Bid and Bax, respectively, are indicated by arrows. B) The redistribution of Bax from cytosol to mitochondria in untreated C7H2-VC cells (left image) or cells treated with 1µmol/l TC-A for 12 h (middle image) was detected by immunofluorescence staining and microscopic analysis. As a control for mitochondria-associated staining patterns, C7H2-VC cells were stained for cytochrome c oxidase (right image). C) Flow cytometric detection of Bax in permeabilized C7H2-VC cells untreated (thin solid line) or treated with 1µmol/l TC-A for 12 h (bold solid line) revealed an increase in staining intensity, while the signal from cells stained with the negative isotype control antibody (dotted and dashed lines) remained unchanged. D) Immunoblot analysis of Bid (upper panel), Bax (middle panel), and, as internal loading control, of tubulin-α (lower panel) in lysates from C7H2-VC cells left untreated or treated with TC-A (1 µmol/l, 12 h) or ZVAD-fmk (20 µmol/l, 16 h) alone or in the indicated combinations.

Fig. 5

Figure 5. TC-A induces caspase activation. C7H2-VC cells were left untreated or stimulated for the indicated times with 1 µmol/l TC-A. Cell lysates were analyzed for caspase-3, -7, -8 and -9 by immunoblotting. As internal loading control for equal amounts of protein, tubulin-α was detected.

Fig. 6

Figure 6. Lack of caspase-8 does not influence TC-A sensitivity. Jurkat cells expressing either wt caspase-8 (black bars) or lacking caspase-8 due to a spontaneous mutation (white bars) were stimulated with the indicated concentrations of TC-A for 24 h. Bars represent mean ± SEM of five independent experiments.

Fig. 7

Figure 7. Caspase activation is indispensable for TC-A-induced cell death. C7H2-VC cells were left untreated or stimulated with TC-A (1 µmol/l), zVAD-fmk (20 µmol/l) or the combination of both for 24 h, and the percentage of apoptosis by using the Annexin V/FITC/PI assay was determined by flow cytometry. Mean values of five independent experiments ± SEM are given.

Fig. 8

Figure 8. TC-A-induced cell death requires de novo protein synthesis. C7H2-VC cells or Jurkat cells were incubated with TC-A (1 µmol/l, 24 h), cycloheximide (1 mmol/l, 28 h) or the combination of both and the percentage of apoptosis by using the Annexin V/FITC/PI assay was determined by flow cytometry. Bars represent mean ± SEM of five independent experiments.

Fig. 9

Figure 9. HSP70 induction by TC-A treatment. C7H2-VC cells were left untreated or stimulated with 1 µmol/l TC-A for the indicated times. Stimulation of cells with anti-Fas antibody CH11 (250 ng/ml for 8 h) and heat treatment of cells (42°C for 30 min) was used for negative and positive control, respectively. A) The expression of HSP70 was determined in permeabilized cells by immunofluorescence staining followed by flow cytometric analysis and (B) in cell lysates by immunoblot analysis. C) The up-regulation of HSP-110 was detected by immunoblot analysis in C7H2-VC cells treated for 24 h with 1 µmol/l TC-A. Heat treatment served as positive control. D) Stimulation of C7H2-VC with vincristine (10 µmol/l) or fludarabine phosphate (100 µg/ml) for 24 h failed to induce HSP70 expression in C7H2-VC cells as determined by flow cytometry.

Fig. 10

Figure 10. ER-stress response in TC-A treated cells includes procaspase-12 cleavage. C7H2-VC cells were left untreated or treated with 1 µmol/l TC-A for the indicated times (A). Alternatively, cells were left untreated or stimulated with TC-A (1 µmol/l), zVAD-fmk (20 µmol/l), or the combination of both for 24 h (B). Subsequently, the specific signal for procaspase-12 in cell lysates was detected by immunoblot analysis. Tubulin-α served as internal loading control.