Apoptosis is a fundamental biological process used to eliminate unwanted cells in a multicellular organism. An increasing number of regulatory proteins have been identified that either promote or inhibit apoptosis. For tumors to arise, apoptosis must be blocked in the transformed cells, for example by mutational overexpression of anti-apoptotic proteins, which represent attractive target proteins for molecular therapy strategies. In a functional yeast survival screen designed to select new anti-apoptotic mammalian genes, we have identified the chromosomal high-mobility group box-1 protein (HMGB1) as an inhibitor of yeast cell death induced by the pro-apoptotic Bcl-2 family member Bak. The C-terminal 33 amino acids of HMGB1 are dispensable for this inhibitory function. HMGB1 is also able to protect mammalian cells against different death stimuli including ultraviolet radiation, CD95-, TRAIL-, Casp-8-, and Bax-induced apoptosis. We found high HMGB1 protein levels in human primary breast carcinoma. Hmgb1 RNA levels are changing during different stages of mouse mammary gland development and are particularly low during lactation and involution. These data suggest that HMGB1 may participate in the regulation of mammary gland apoptosis and that its high expression level promotes tumor growth because of its anti-apoptotic properties.

Background: High mobility group box 1 (HMGB1) is a non-histone chromosomal protein implicated in a variety of biologically important processes, including transcription, DNA repair, V(D)J recombination, differentiation, and development. Overexpression of HMGB1 inhibits apoptosis, arguing that the molecule may act as an antiapoptotic oncoprotein. Indeed, increased expression of HMGB1 has been reported for several different tumour types. In this study, we analysed human colon carcinoma for HMGB1 as well as for c-IAP2 expression levels. c-IAP2 is an antiapoptotic protein which may be upregulated as a consequence of nuclear factor kB (NFkB) activation via HMGB1. Methods: A comparative genomic hybridisation (CGH) database comprising 1645 cases from different human tumour types was screened to detect cytogenetic changes at the HMGB1 locus. Immunohistochemical staining of human colon tissue microarrays and tumour biopsies, as well as western blot analysis of tumour lysates, were performed to detect elevated HMGB1 and c-IAP2 expression in colon carcinomas. The antiapoptotic potential of HMGB1 was analysed by measuring caspase activities, and luciferase reporter assays and quantitative polymerase chain reaction analysis were employed to confirm NFkB activation and c-IAP2 mRNA upregulation on HMGB1 overexpression. Results: According to CGH analysis, the genomic locus containing the HMGB1 gene was overrepresented in one third (35/96) of colon cancers. Correspondingly, HMGB1 protein levels were significantly elevated in 90% of the 60 colon carcinomas tested compared with corresponding normal tissues evaluable from the same patients. HMGB1 increased NFkB activity and led to co-overexpression of the antiapoptotic NFkB target gene product c-IAP2 in vitro. Furthermore, increased HMGB1 levels correlated with enhanced amounts of c-IAP2 in colon tumours analysed by us. Finally, we demonstrated that HMGB1 overexpression suppressed caspase-9 and caspase-3 activity, suggesting that HMGB1 interferes with the apoptotic machinery at the level of apoptosomal caspase-9 activation. Conclusions: We identified in vitro a molecular pathway triggered by HMGB1 to inhibit apoptosis via c-IAP2 induction. Our data indicate a strong correlation between upregulation of the apoptosis repressing HMGB1 and c-IAP2 proteins in the pathogenesis of colon carcinoma.

The anti-apoptotic molecule Aven was originally identified in a yeast two-hybrid screen for Bcl-x L -interacting proteins and has also been found to bind Apaf-1, thereby interfering with Apaf-1 self-association during apoptosome assembly. Aven is expressed in a wide variety of adult tissues and cell lines, and there is increasing evidence that its overexpression correlates with tumorigenesis, particularly in acute leukemias. The mechanism by which the anti-apoptotic activity of Aven is regulated remains poorly understood. Here we shed light on this issue by demonstrating that proteolytic removal of an inhibitory N-terminal Aven domain is necessary to activate the anti-apoptotic potential of the molecule. Furthermore, we identify Cathepsin D (CathD) as the protease responsible for Aven cleavage. On the basis of our results, we propose a model of Aven activation by which its N-terminal inhibitory domain is removed by CathD-mediated proteolysis, thereby unleashing its cytoprotective function.

The ecdysone-inducible mammalian expression system is frequently used for inducible transgene expression in vitro and in vivo. Here, we describe a strong antiapoptotic effect of ecdysone analogs in the human colon carcinoma cell line RKO, which is in contrast to published data that ecdysteroids do not influence mammalian cell physiology. Inhibition of Fas ligand-and TNF-related apoptosis-inducing ligand-induced apoptosis by muristerone A occurs at the level of caspase-8 activation and is neutralized by phosphatidylinositol-3-kinase/Akt, protein kinase C and mitogen-activated protein kinase inhibitors. Microarray, Northern and Western blot analysis revealed that incubation of RKO cells with muristerone A leads to changes in gene expression levels, including an upregulation of bcl-x L mRNA and protein levels. Our data imply that ecdysteroids and ecdysone mimics can induce and/or repress gene transcription in RKO and other mammalian cells, thereby influencing the apoptotic behavior. Therefore, the ecdysone-inducible mammalian expression system may not be suitable for the analysis of apoptosisrelated genes.

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GIST and histogenetically related cells including interstitial cells of Cajal (ICCs), ‘fibroblast-like cells’ (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription—polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SC stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

The cellular homologues of the viral anti-apoptotic v-FLIP proteins exist as a long (c-FLIP L ) and a short (c-FLIP S ) splice variant. While c-FLIP S and v-FLIP are composed solely of two death effector domains, c-FLIP L contains an (inactive) caspase-like domain in addition to these two death effector domains, thereby structurally resembling proCaspase-8. Both c-FLIP L and c-FLIP S suppress apoptosis by inhibiting Caspase-8 activation, although at different levels of pro-Caspase-8 processing. To analyze the consequences of deregulated c-FLIP S expression in vivo, we established lck FLIP Stransgenic mice overexpressing the transgene in thymocytes and in mature T cells. As expected, CD95L-induced apoptosis was impaired in lck FLIP S -transgenic T cells, indicating the functionality of the FLIP S transgene. Remarkably, activation-induced cell death of transgenic T cells was unaffected, despite the observed inhibition of CD95-induced T cell death. Thymic and splenic cell numbers as well as CD4/CD8 cellularity were normal in lck FLIP S -transgenic animals, which in contrast to CD95-deficient mice do not accumulate Thy1 + B220 + CD4 -CD8 -peripheral T cells. c-FLIP S overexpression leads to a significant decrease in activation-induced T cell proliferation in vitro. Despite the capacity of FLIP S to inhibit CD95-induced apoptosis, T cell lymphomagenesis is not observed in lck FLIP S -transgenic mice. Interestingly, the Vb8 + memory T cell pool is enlarged upon staphylococcal enterotoxin B injections, suggesting a specific in vivo function for FLIP S in the maintenance of restimulated T cells. IntroductionAmong many anti-apoptotic regulatory proteins, the Caspase-8-inhibitory FLIP molecule has been studied intensively. Originally detected in different viruses, c-FLIP was eventually identified as the cellular homologue of the v-FLIP proteins ([1, 2] and references herein). Several spliced isoforms of c-FLIP have been characterized, two of which are expressed as proteins: c-FLIP L , the full-length 55-kDa form of c-FLIP, comprises two Nterminal death effector domains (DED) and an enzymatically inactive caspase domain, thereby structurally resembling . c-FLIP S is a shorter 26-kDa form of c-FLIP and, like v-FLIP, contains only the two DED.A functional pro-apoptotic death-inducing signaling complex (DISC) is usually assembled by recruitment of the adaptor protein FADD to the activated CD95 receptor via death domain interactions [3]. Due to binding of their DED, FADD then associates Caspase-8 Molecular immunologyThe first two authors contributed equally to this work. [2,5]. Recruitment of both c-FLIP L and c-FLIP S to the activated intracellular CD95 receptor complex by FADD has been demonstrated, and both proteins are able to inhibit CD95-induced apoptosis as well as cell death mediated by other death receptors [6]. It has even been suggested that c-FLIP S rather than c-FLIP L may be the prime inhibitor of CD95-mediated apoptosis in T cells [7]. Nevertheless, a recent study showed that whereas high levels of c-FLIP L o...

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Key words: FADD-DN; death receptor; cell cycle; oncogene cooperationApoptosis as a special form of programmed cell death has been recognized as a fundamental process both throughout embryogenesis and in the adult organism to maintain tissue homeostasis and remove dangerous or surplus cells without inflammation. 1 For a cancer to occur and progress, apoptosis must be inhibited as the tumor cells are constantly exposed to a variety of apoptotic stimuli, e.g., overexpression of apoptosis-inducing oncogenes, hypoxia or lack of survival signals after detachment of metastatic cells from the tumor mass (anoikis). 2 Furthermore, conventional treatment of cancer by chemotherapy and irradiation is based on the selective induction of apoptosis in tumor cells, and tumor resistance to such treatment is a result of the inhibition of drug-or radiation-induced apoptosis. 3 The extrinsic apoptotic pathway is triggered by so-called death receptors, which together with their ligands belong to the TNF/ TNFR superfamily. The death receptor subfamily consists of CD95/Fas/Apo-1, TNFR1, DR3 and the TRAIL receptors DR4/ DR5, DR6, NGFR and EDAR. 4 Death receptors contain in their intracellular part a specific protein-binding motif, the DD, which mediates homophilic interactions with other DD-bearing proteins. FADD is essential for death receptor-induced apoptosis as it is used as an adaptor protein by several DD-containing death receptors. [5][6][7][8][9][10][11][12][13][14][15] In the activated CD95 receptor, FADD binds with its N-terminal DD to the CD95 DD. FADD then recruits the apical caspase-8 via another protein-binding domain in its C terminus, the DED. Within this assembled intracellular receptor complex (DISC 16 ), caspase-8 becomes activated ("induced proximity" 17 ) and starts to proteolyse its targets.Death receptor-mediated cell killing is normally induced by the death receptor ligands and plays an important role during physiological cell death as well as in different antitumor strategies (e.g., cytotoxic T-cell killing 18 ). Consequently, several mutations have been identified in tumors which lead to impairment of death receptor-induced apoptosis. 1,19 Examples include overexpression of the inhibitory FLIP proteins in melanomas, 20 transcriptional downregulation of caspase-8 mRNA by gene deletion or promoter methylation 21 and CD95 mutations in different cancers. [22][23][24] Direct proof of the tumor-suppressing proapoptotic activity of the CD95 receptor has been obtained by in vivo cooperation experiments. Tumor development in L-myc transgenic mice is accelerated when mice are bred onto a CD95-deficient lpr background. 25 Interference with FADD functionality leads to the expected impairment of death receptor killing. 26 -30 Surprisingly, however, not only cell death but also T-cell proliferation is strongly inhibited in lck FADD-DN transgenic mice 28 -30 and in rag1 -/-k.o. animals reconstituted with a FADD -/-k.o. T-cell compartment. 26 The influence of such a FADD-dependent signaling pathway on proliferation is not re...