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Induction of Apoptosis by Enediyne Antibiotic Calicheamicin 0II

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Induction of Apoptosis by Enediyne Antibiotic Calicheamicin 0II Oncogene (2003) 22, 9107–9120 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc ORIGINAL PAPERS Induction of apoptosis by enediyne antibiotic calicheamicin 0II proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner Aram Prokop1,4, Wolf Wrasidlo2,4, Holger Lode2, Ralf Herold1, Florian Lang3,5,Gu¨ nter Henze1, Bernd Do¨ rken3, Thomas Wieder3,5,6 and Peter T Daniel*,3,6 1Department of Pediatric Oncology/Hematology, University Medical Center Charite´, Humboldt University of Berlin, Berlin 13353, Germany; 2Department of General Pediatrics, University Medical Center Charite´, Humboldt University of Berlin, Berlin 13353, Germany; 3Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charite´, Humboldt University of Berlin, Berlin 13125, Germany Calicheamicin 0II is a member of the enediyne class of Keywords: calicheamicin; apoptosis; caspase-3; Bax; antitumor antibiotics that bind to DNA and induce Bcl-2; cytochrome c; mitochondria; BJAB; Jurkat; apoptosis. These compounds differ, however, from con- HCT116 ventional anticancer drugs as they bind in a sequence- specific manner noncovalently to DNA and cause sequence-selective oxidation of deoxyriboses and bending of the DNA helix. Calicheamicin is clinically employed as Introduction immunoconjugate to antibodies directed against, for example, CD33 in the case of gemtuzumab ozogamicin. In contrast to necrosis, apoptosis is a morphologically Here, we show by the use of the unconjugated drug that and biochemically defined form of cell death that plays a calicheamicin-induced apoptosis is independent from role under different physiological conditions, such as death-receptor/FADD-mediated signals. Moreover, cali- cell turnover, the immune response or embryonic cheamicin triggers apoptosis in a p53-independent manner development. In addition, the apoptotic program may as shown by the use of p53 knockout cells. Cell death be triggered by various, unphysiological death stimuli, proceeds via activation of mitochondrial permeability for example, ionizing radiation (Belka et al., 2000, transition, cytochrome c release and activation of 2001), chemotherapeutic anticancer drugs (Engels et al., caspase-9 and -3. The overexpression of Bcl-xL or Bcl-2 2000; Wieder et al., 2001a) and by natural compounds strongly inhibited calicheamicin-induced apoptosis. (Wieder et al., 2001b). So far, three major pathways of Knockout of Bax abrogated cell death after calicheamicin apoptotic cell death have been defined: (i) the death treatment. Thus, the activation of mitochondria and receptor-mediated signalling cascade (Daniel et al., execution of cell death occur through a fully Bax- 2001), (ii) the mitochondrial pathway involving the dependent mechanism. Interestingly, caspase inhibition apoptosome (Daniel, 2000; Rudner et al., 2001), and (iii) by the pancaspase-inhibitor zVAD-fmk interfered with the endoplasmic reticulum-dependent stress response mitochondrial activation by calicheamicin. This places (Belka and Budach, 2002; Rudner et al., 2002). caspase activation upstream of the mitochondria and In previous studies, we demonstrated that inactivation indicates that calicheamicin-triggered apoptosis is en- of apoptotic pathways results in both apoptotic hanced through death receptor-independent activation of resistance in vitro and clinical resistance to anticancer the caspase cascade, that is, an amplification loop that is treatment (Mrozek et al., 2003; Gu¨ ner et al., 2003; required for full activation of the mitochondrial pathway. Sturm et al., 2001; Sturm et al., 2003). One of the main Oncogene (2003) 22, 9107–9120. doi:10.1038/sj.onc.1207196 regulatory steps of apoptotic cell death is controlled by the ratio of anti- and proapoptotic members of the Bcl-2 family of proteins function as a rheostat and determine the susceptibility to apoptosis (for reviews, see, Daniel, 2000; Daniel et al., 2003). For example, the over- expression of antiapoptotic Bcl-2 family members that *Correspondence: PT Daniel, Molecular Hematology and Oncology, University Medical Center Charite´ , Campus Berlin-Buch, Humboldt can tilt the rheostat towards survival, thereby confering University of Berlin, Berlin 13125, Germany drug resistance, at least in some cellular tumor model 4These authors contributed equally to the present study systems (Rudner et al., 2001; Belka and Budach, 2002; 5Current address: Department of Physiology I, Eberhard-Karls Radetzki et al., 2002; von Haefen et al., 2003). On the University Tubingen, Tu¨ bingen 72076, Germany 6These authors share senior authorship other hand, forced expression of proapoptotic Bax or Received 9 May 2003; revised 28 August 2003; accepted 10 September Bak or, for example, the BH3-only protein Nbk/Bik, is 2003 sufficient to increase the sensitivity of malignant cells to Calicheamicin activates mitochondria via caspases and Bax A Prokop et al 9108 apoptosis and to overcome drug resistance (Hemmati Results et al., 2002; Radetzki et al., 2002; von Haefen et al., 2002). Interestingly, these in vitro data are in line with Dose-dependent induction of apoptosis by calicheamicin signalling analyses performed in primary carcinoma, WII lymphoma and leukemia cells. Upon activation of the mitochondria, cell death proceeds via the formation of Depending on their concentration, many cytostatic the mitochondrial apoptosome and initiation of the substances cause necrotic, membrane damaging as et al caspase cascade. Consequently, loss of proapoptotic Bax well as apoptotic cell death (Wieder ., 1998; et al results in the failure of cancer cells to activate caspase-3 Friedrich ., 2001). We therefore determined the in vivo (Prokop et al., 2000). unspecific, cytotoxic effect of calicheamicin WII (further Calicheamicin is a naturally occurring hydrophobic designated as calicheamicin) in BJAB cells by the enediyne antibiotic that was isolated in Micromonospora determination of LDH release into the culture echinospora calichensis (Maiese et al., 1989). Calichea- medium. As shown in Figure 1a, calicheamicin did micin and other naturally occurring enediynes exhibit not significantly reduce viability of BJAB cells after significant activity against Gram-positive and -negative 24 h of incubation at concentrations p100 pM, thereby indicating that a necrosis-like mechanism, for bacteria. In addition, calicheamicin exerts a potent example, through direct membrane damage, does not antitumor activity and has been shown to induce apoptotic cell death in the picomolar range. Calichea- play a role for its death-inducing potency. After 48 h, however, 25% of the cells showed LDH release, which micin binds to the minor groove of the DNA helix, can be attributed to secondary necrosis that follows preferentially to the 30 ends of oligopurine tracts, and apoptosis in vitro. Calicheamicin potently induced DNA causes sequence-selective oxidation of deoxyribose and fragmentation in up to 60% of the cells. Apoptosis bending of the DNA helix by an induced-fit mechanism induction was concentration-dependent with a half- of DNA target recognition (Salzberg and Dedon, 2000). maximum concentration of 10 pM (Figure 1b), and In addition, calicheamicin and other naturally occurring DNA fragmentation was already observed after 24 h enediyne antibiotics, including esperamicin, neocarci- (Figure 1c). The percentage of cells showing loss of nostatin, kedarcidin and dynemicin constitute a unique membrane integrity at 48 h (Figure 1a) corresponds well class of reactive compounds, which can undergo aromatization to produce biradicals. Interestingly, to the number of cells showing DNA fragmentation at 24 h (Figure 1c). Thus, DNA fragmentation precedes phosphodiester bond breakage of DNA was shown to membrane damage induced in calicheamicin-induced be dispensable for the apoptosis-inducing activity (Hiatt et al., 1994). Furthermore, prevention of apoptosis was cells. This indicates that calicheamicin induces apoptotic cell death in BJAB cells and this is followed by observed in analogs that were electronically stabilized to secondary necrosis. impair aromatic rearrangements and generation of biradicals (Hiatt et al., 1994). Thus, apart from enforcing a conformational change in the DNA Calicheamicin-induced apoptosis occurs independently of structure and the induction of DNA strand breaks, CD95/Fas signalling additional signalling events appear to be critically involved in the activation of apoptosis by calicheamicin To investigate the involvement of CD95/Fas or other and its analogs. death receptor systems in calicheamicin-induced cell In clinical anticancer therapy, the enediyne calichea- death, we employed BJAB cells overexpressing a micin is currently successfully employed as an immuno- dominant-negative FADD (FADD-dn) mutant. Both conjugate to antibodies serving as vehicles for the mock and FADD-dn transfectants express endogenous targeting of calicheamicin to tumor cells (Linenberger FADD (27 kDa), while the FADD-dn transfectants also et al., 2001; van der Velden et al., 2001). So far, the show a lower band representing the truncated FADD- mechanisms of apoptosis induction by these compounds dn (Wieder et al., 2001a). To provide a positive control remains enigmatic. Paradoxically, the induction of for the biological activity of the FADD-dn, we apoptotic cell death in PC12 cells by neocarcinostatin functionally characterized the FADD-dn-overexpressing was shown to be potentiated in cells overexpressing the BJAB cells. Vector-transfected
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