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....

Taxane derivatives such as paclitaxel elicit their antitumor effects at least in part by induction of apoptosis, but the underlying mechanisms are incompletely understood. Here, we used different cellular models with deficiencies in key regulators of apoptosis to elucidate the mechanism of paclitaxel-induced cell death. Apoptosis by paclitaxel was reported to depend on the activation of the initiator caspase-10; however, we clearly demonstrate that paclitaxel kills murine embryonic fibroblasts (MEFs) devoid of caspase-10 as well as human tumor cell lines deficient in caspase-10, caspase-8, or Fas-associating protein with death domain. In contrast, the lack of Apaf-1 or caspase-9, key regulators of the mitochondrial pathway, not only entirely protected against paclitaxel-induced apoptosis but could even confer clonogenic survival, depending on the cell type and drug concentration. Thus, paclitaxel triggers apoptosis not through caspase-10, but via caspase-9 activation at the apoptosome. This conclusion is supported by the fact that Bcl-2-overexpressing cells and Bax/Bak doubly-deficient MEFs were entirely resistant to paclitaxel-induced apoptosis. Interestingly, also the single knockout of Bim or Bax, but not that of Bak or Bid, conferred partial resistance, suggesting a particular role of these mediators in the cell-death pathway acti- IntroductionPaclitaxel (Taxol; Calbiochem, Darmstadt, Germany) is one of the most effective antitumor drugs and is approved for chemotherapy of a variety of human malignancies, including breast, ovarian and non-small cell lung cancer. 1 Like other taxane derivatives, paclitaxel binds to the ␤-subunit of tubulin, thus preventing microtubule depolymerization that is required for proper mitosis and cell division. During mitosis, the spindle checkpoint ensures proper chromosome segregation by blocking the anaphase onset until all chromosomes are attached to microtubules and tension across the kinetochores is generated. Paclitaxel inhibits the dynamic instability of the mitotic spindle, leading to impaired chromosome alignment. Consequently, the cells become arrested by the spindle checkpoint at the G 2 /M phase and then die eventually by an apoptotic pathway, called mitotic catastrophe. 2 Cells that escape this pathway can be also arrested by a second, postmitotic checkpoint, which is activated by aberrant division of cells with multipolar spindles, and leads to cell death in polyploid cells following paclitaxel treatment. 3 Although both p53-dependent and -independent mechanisms have been implicated in cell death during aberrant mitosis, the exact mechanisms of paclitaxelinduced apoptosis are currently unknown.Apoptosis is largely controlled by a family of aspartate-specific cysteine proteases, called caspases, that function as initiators and executioners of the apoptotic process. 4,5 Caspases are activated by two major signaling routes, the extrinsic death receptor and the intrinsic mitochondrial pathway, that both depend on the formation of large multiprotein complexes. 4,6 Initia...

The tumor suppressor protein p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. Recently, it was shown that during apoptosis p53 can specifically translocate to mitochondria, where it physically interacts with and inactivates prosurvival Bcl-2 proteins. In the present study, we therefore investigated the role of mitochondrial translocation of p53 for the stress response of tumor cells. In various cell lines, DNA damage induced by either ionizing irradiation or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by ionizing radiation. Irradiated cells became senescent, although irradiation triggered a functional p53 response and induced expression of p21, Bax, and Puma. Interestingly, even the targeted expression of p53 to mitochondria was insufficient to launch apoptosis, whereas overexpression of wild-type p53 induced Bax activation and apoptotic alterations. Together, these results suggest that, in contrast to previous reports, mitochondrial translocation of p53 does not per se lead to cell death and that this might constitute a mechanism that contributes to the resistance of tumor cells to ionizing radiation-induced apoptosis.

The photosynthetic chloroplast mutant G64 of Chlamydomonas reinhardtii was shown to contain a single point mutation within the 5' region of the psbD gene encoding the D2 protein of the photosystem II reaction center. The mutation affects the sequence element TATAATAT which has previously been hypothesized to function as the psbD promoter. Run-on analysis confirmed that transcription of psbD in the mutant was reduced to approximately 10% of the wild-type level. However, psbD mRNA accumulated to approximately 35%, despite the prominent decrease in RNA synthesis. This suggests that RNA-stabilization effects can compensate to some extent for a reduction in transcriptional activity. Interestingly, a direct correlation between transcript levels and the accumulation of the psbD gene product, the D2-protein, was observed in G64. The data suggest that posttranscriptionally acting regulatory factors determine the rate-limiting steps of chloroplast psbD gene expression.