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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.02-0020fje. |
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2
* Laboratory of Molecular Cytology, Department of Internal Medicine, University of Innsbruck, A-6020 Innsbruck, Austria;
Tyrolean Cancer Research Institute at the University of Innsbruck, A-6020 Innsbruck, Austria;
Institute of Anatomy and Histology, University of Innsbruck, A-6020 Innsbruck, Austria; and
Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co. Ltd., Shimotogari 1188, Nagaizumi-cho, Suntougun, Shizuoka-ken 411, Japan
2Correspondence: Laboratory of Molecular Cytology, Department of Hematology and Oncology, University of Innsbruck, A-6020 Innsbruck, Austria. E-mail: Inge.Tinhofer{at}uibk.ac.at
SPECIFIC AIMS
Cytotoxic agents frequently fail to induce apoptosis in neoplasias because tumor cells express increased levels of anti-apoptotic proteins, have down-regulated important executor molecules of the main apoptotic signaling pathways, and/or express mutated forms or even lack the expression of genes involved in the initiation of apoptosis. Thus, identification of agents that might antagonize these tumor-specific survival advantages by inducing signals that bypass these regulators are of clinical interest. The present study was designed to characterize the molecular mechanisms of the Bcl-2-antagonizing agent Tetrocarcin-A (TC-A) in the T-ALL tumor model.
PRINCIPAL FINDINGS
1. TC-A directly induced apoptosis in CCRF-CEM cells in a Bcl-2-independent manner
To characterize the proapoptotic effect of TC-A in T-ALL cells and its dependence on Bcl-2, we treated the CEM-C7H2 vector control cell line (C7H2-VC) and subclones stably overexpressing Bcl-2 with increasing doses of TC-A. Independent of Bcl-2 expression levels, induction of apoptosis was observed in a dose-dependent manner characterized by phosphatidylserine exposure (Fig. 1
A), changes in cell volume and granularity (Fig. 1B
), DNA fragmentation (Fig. 1C
), reduction of metabolic activity (Fig. 1D
),and ultrastructural changes (Fig. 1E
). Time analyses revealed that though Bcl-2 overexpression slightly delayed the onset of apoptosis, this effect was lost by incubating cells for longer than 24 h.
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2. TC-A-induced mitochondrial signals and activation of multiple caspases
Analysis of the signal cascade triggered by TC-A revealed that TC-A acted on mitochondria and reduced mitochondrial transmembrane potential 
m in the C7H2-VC cells in a dose- and time-dependent manner. Again, Bcl-2 overexpression delayed but did not block TC-A-mediated loss of m. Cleavage of Bid and Bax could be inhibited by preincubation of cells with the pan-caspase inhibitor zVAD-fmk, suggesting that their processing occurred in a caspase-dependent manner. Applying immunoblot analyses to identify the caspases involved, we observed activation of caspase-3, -7, -8, and -9 after TC-A treatment. The importance of caspases in this cell death program was further underscored by the inhibition of TC-A-induced cell death by zVAD-fmk. Although activation of caspase-8 was also found in Jurkat cells, in a Jurkat cell line lacking caspase-8 we observed sensitivity toward apoptosis induction by TC-A similar to the wt caspase-8-expressing cell line. These data argue against an important role of caspase-8 in TC-A-induced apoptosis.
3. Up-regulation of ER stress proteins is specific for TC-A-induced apoptosis
Preincubation of C7H2 or Jurkat cells with cycloheximide (CHX), an inhibitor of protein synthesis, efficiently reduced sensitivity to TC-A-induced apoptosis. By screening for potential inducer(s) of TC-A apoptosis using a cDNA microarray of 200 genes known to be involved in apoptosis, we observed TC-A-triggered up-regulation of 4 transcription factors and 6 members of the heat shock protein family. The strong increase of heat shock proteins (HSP), especially of HSP70 (>35-fold), suggested the activation of a program known as endoplasmic reticulum (ER) -induced stress. The up-regulation of HSP70 was confirmed on protein level by flow cytometric (Fig. 2
A) and immunoblot analyses (Fig. 2B
). Analyzing the protein expression levels of HSP110 and grp78, we found HSP110 significantly up-regulated in TC-A-treated cells (Fig. 2C
) whereas grp78 protein expression remained almost unchanged, confirming the data from the microarray analysis. Three other proapoptotic agents with distinct mechanisms of action—the agonistic anti-Fas antibody CH11 (Fig. 2A, B
), vincristine, a vinca alkaloid leading to cytoskeletal changes, and fludarabine phosphate, a purine analog involved in the inhibition of DNA synthesis—failed to up-regulate HSP70 (Fig. 2D
).
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5. Characterization of the ER stress response triggered by TC-A
We raised the question of whether TC-A-induced apoptosis might be driven by caspase-12 activation, the central caspase involved in ER stress signaling. In C7H2-VC or Jurkat cells stimulated with TC-A, a decrease in the specific signal for procaspase-12 could be detected. We next analyzed whether caspase-12 processing after TC-A treatment occurs upstream or downstream of other caspases. In C7H2-VC cells pretreated with zVAD-fmk, the processing of caspase-12 could be inhibited, which suggests that caspases other than caspase-12 are activated upstream and these are involved in the processing of caspase-12.
CONCLUSION
In the T-ALL cell line model analyzed, TC-A proved to be an efficient inducer of apoptosis. The molecular events of TC-A-induced apoptosis proceeded in cells overexpressing Bcl-2 or lacking caspase-8 and comprised the activation of effector molecules involved in mitochondrial and ER signaling. A hypothetical model for the TC-A-induced signaling pathway is presented in Fig. 3
.
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The role of Bcl-2 expression in the modulation of chemosensitivity of tumor cells is controversial. Evidence has been provided that Bcl-2 expression levels did not correlate with chemosensitivity of tumor cell lines in most cell lines tested. However, retrospective analyses of samples from ovarian cancer patients revealed an inverse relation between Bcl-2 levels and initial responses to chemotherapy, and identified Bcl-2 as independent prognostic factor. In tumors of the lymphatic system, no correlation of Bcl-2 expression alone but of Bcl-2/Bax ratios with drug-induced apoptosis has been found. Antisense strategies demonstrated successful in increasing chemosensitivity of tumor cells lines in vitro or in murine xenograft models. Drugs that inhibit the molecular interaction of Bcl-2 and its proapoptotic 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 anti-tumor therapy.
The data presented here imply that TC-A might fulfill such criteria. 1) We found that after TC-A treatment, Bax was cleaved into a 18 kDa fragment in a caspase-dependent manner. An early amino-terminal cleavage of Bax in etoposide-treated Jurkat cells has been described recently. In that study it was found that the processing of the full-protein Bax to a 18 kDa fragment occurred in a caspase-independent manner, but was mediated by calpain. Bax/p18 colocalized to mitochondria that 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. Such conformational changes in the carboxyl terminus of Bax have been reported to be triggered by Bid leading to the insertion of Bax into mitochondrial membranes. Our immunofluorescence studies of Bax and cytochrome c oxidase revealed the association of Bax to mitochondria in TC-A-treated cells.
We observed that TC-A treatment led to the activation of caspases that was indispensable for apoptosis induction. On the other hand, sensitivity to apoptosis by TC-A was independent of the caspase-8 expression status of the T-ALL tumor cell lines used. The activation of caspase-8 has been identified as a common principle in death receptor- and drug-mediated cell death. 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 correlated with N-MYC gene amplification. The latter is associated with a more aggressive form of this tumor characterized by the frequent occurrence of drug resistance. Reintroduction of caspase-8 into neuroblastoma cell lines resensitized these cells to drug-induced apoptosis. 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.
Using the cDNA microarray technique, we observed up-regulation of 6 members of the heat shock protein family after TC-A treatment, pointing to the induction of ER stress by TC-A. Similar to the cellular response to DNA damage, an intrinsic apoptotic pathway is triggered. This pathway is initiated by the activation of caspase-12, a caspase associated with the ER and mediating ER stress in a Ca2+- and calpain-dependent manner. Consistent with induction of an irreversible ER stress response by TC-A, we observed the cleavage of procaspase-12. Two types of proteases have recently been described to link ER stress and caspase-12 activation. 1) In a study by Nakagawa et al., deprivation of oxygen- and glucose-induced caspase-12 activation in glial cells was mediated by calpain. 2) An alternative activation signal was found to involve caspase-7, which, after ER stress, translocates from the cytosol to the surface of the ER and associates with procaspase-12 leading to its activation. We hypothesize that in the TC-A-mediated signaling pathway, caspase-12 is activated downstream of caspase-3 and -7. The exact mechanism, however, by which TC-A induces caspase-12 activation in T-ALL cells and its sequence in the signal cascade remains to be determined.
The massive up-regulation of HSP70 observed in TC-A-treated cells suggests that ER functions are heavily disturbed and, as a result, HSP70 is induced to limit cellular damage by its chaperone function. HSP70 was found to be highly expressed in human tumor tissue and in tumor cell lines. Together with the ability of HSP70 to protect cells from a wide range of apoptotic and necrotic stimuli, this underscores the relevance of this heat shock protein in tumorigenesis. 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.
We conclude that TC-A might represent an attractive anti-tumor agent since it induces stressful death via mitochondrial and ER signaling that is inhibited by neither aberrant expression/function of important regulators of death receptor- and drug-induced apoptosis nor survival proteins involved in tumorigenesis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0020fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.02-0020fje 
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