A novel member of the IB family, human IB-, was identified by a differential screening approach of apoptosis-sensitive and -resistant tumor cells. The protein consists of 6 ankyrin repeats at its COOH terminus and shares about 30% identity with other IB members. IB-associates with both the p65 and p50 subunit of NF-B and inhibits the transcriptional activity as well as the DNA binding of the transcription factor. Interestingly, IB-is localized in the nucleus where it aggregates in matrix-associated deacetylase bodies, indicating that IB-regulates nuclear NF-B activity rather than its nuclear translocation from the cytoplasm. IBexpression itself was regulated by NF-B, suggesting that its activity is controlled in a negative feedback loop. Unlike classical IB proteins, IB-was not degraded upon cell stimulation. Treatment with tumor necrosis factor-␣, interleukin-1␤, and lipopolysaccharide induced a strong induction of IB-transcripts. Expression of IB-was detected in different tissues including lung, liver, and in leukocytes but not in the brain. Suppression of endogenous IB-by RNA interference rendered cells more resistant to apoptosis, whereas overexpression of IB-was sufficient to induce cell death. Our results, therefore, suggest that IB-functions as an additional regulator of NF-B activity and, hence, provides another control level for the activation of NF-B-dependent target genes.NF-B is an evolutionarily conserved pleiotropic transcription factor that plays a crucial role in many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis (Refs. 2-6; for review, see Ref. 1). The mammalian NF-B/Rel family consists of RelA (p65), RelB, c-Rel, p50, and p52 that bind as homo-or heterodimers at B sites in the DNA of their target genes. This combinatorial diversity contributes to the regulation of a distinct but overlapping set of genes, in that the individual dimers have distinct preferences for different B sites that they can bind with distinguishable affinity and specificity. In addition, whether the transcription of a target gene is activated or repressed depends on the dimer combination. While RelA/ p50, RelA/c-Rel, RelB/p50, and RelB/p52 heterodimers are transcriptional activators, p50/p50 and p52/p52 homodimers generally repress transcription (7-9). Regulation of the great diversity of genes requires a precise control of NF-B, which is achieved by various mechanisms of posttranslational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. Dysregulation of NF-B activation results in a wide range of human disorders including inflammatory and neurodegenerative diseases (10, 11) and different types of cancer (4, 12).NF-B activity is also tightly regulated by interaction with IB proteins (13). Characteristic for all IB proteins is a domain of multiple ankyrin repeats that bind to the conserved Rel homology domain of NF-B proteins. Various members of the IB family target different NF-B complexes, e.g....

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We have shown previously that ionizing radiation (IR) induces a persistent G 2 -M arrest but not cell death in MCF-7 breast carcinoma cells that harbor functional p53 but lack caspase-3. In the present study, we investigated the mechanisms of apoptosis resistance and the roles of p53, caspase-3, and cell cycle arrest in IR-induced apoptosis. The methylxanthine caffeine and the staurosporine analog UCN-01, which can inhibit ATM and Chk kinases, efficiently abrogated the IR-induced G 2 -M arrest and induced mitochondrial activation as judged by the loss of the mitochondrial membrane potential and the release of cytochrome c and Smac/ Diablo. However, despite these proapoptotic alterations, cell death and activation of the initiator caspase-9 were not induced in MCF-7 cells but were interestingly only observed after reexpression of caspase-3. Sensitization to IR-induced apoptosis by caffeine or UCN-01 was abrogated neither by cycloheximide nor by pifithrin-␣, an inhibitor of the transcriptional activity of p53. Furthermore, suppression of p53 by RNA interference could not prevent caffeine-and IR-induced mitochondrial alterations and apoptosis but resulted in an even more pronounced G 2 -M arrest. Collectively, our results clearly show that the resistance of MCF-7 cells to IR-induced apoptosis is caused by two independent events; one of them is a caffeine-or UCN-01-inhibitable event that does not depend on p53 or a release of the G 2 -M arrest. The second event is the loss of caspase-3 that surprisingly seems essential for a fully functional caspase-9 pathway, even despite the previous release of mitochondrial proapoptotic proteins.

Recent data suggest that a-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect a-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on a-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of a-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, a-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that a-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.

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