A novel human member of the Bcl-2 family was identified, Bcl-B, which is closest in amino acid sequence homology to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2 homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted carboxyl-terminal transmembrane (TM) domain. The BCL-BmRNA is widely expressed in adult human tissues. The Bcl-B protein binds Bcl-2, Bcl-X_L, and Bax but not Bak. In transient transfection assays, Bcl-B suppresses apoptosis induced by Bax but not Bak. Deletion of the TM domain of Bcl-B impairs its association with intracellular organelles and diminishes its anti-apoptotic function. Bcl-B thus displays a unique pattern of selectivity for binding and regulating the function of other members of the Bcl-2 family.

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)–expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X_L. Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule–1 (ICAM-1) and vascular cell adhesion molecule–1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.

The common neurotrophin receptor, p75^NTR, has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-κB. However, the mechanisms by which p75^NTR initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75^NTR and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75^NTR was mapped to distinct regions in p75^NTR. Furthermore, TRAF4 interacted with dimeric p75^NTR, whereas TRAF2 interacted preferentially with monomeric p75^NTR. TRAF2-p75^NTR, TRAF4-p75^NTR, and TRAF6-p75^NTR interactions modulated p75^NTR-induced cell death and NF-κB activation with contrasting effects. Coexpression of TRAF2 with p75^NTRenhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75^NTR. TRAF4 also inhibited the NF-κB response, whereas TRAF2 and TRAF6 enhanced p75^NTR-induced NF-κB activation. These results demonstrate that TRAF family proteins interact with p75^NTR and differentially modulate its NF-κB activation and cell death induction.

The Drosophila seven in absentia ( sina ) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin‐conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53‐inducible gene in a myeloid leukemia cell line. To explore the function of human SINA‐homologous (Siah) proteins, expression plasmids encoding Siah‐1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG‐1, a ubiquitin‐like Hsp70/Hsc70‐regulating protein, is a negative regulator of Siah‐1A. Siah‐1A was identified as a BAG‐1‐binding protein via yeast two‐hybrid methods. Specific interaction of BAG‐1 with Siah‐1A was also demonstrated by in vitro binding experiments using glutathione S ‐transferase fusion proteins and co‐immunoprecipitation studies. Siah‐1A‐induced growth arrest in 293 and GM701 cells was abolished by co‐transfection of wild‐type BAG‐1 with Siah‐1A but not by a C‐terminal deletion mutant of BAG‐1 that fails to bind Siah‐1A. Over‐expression of BAG‐1 significantly inhibited p53‐induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG‐1 also prevented growth arrest following UV‐irradiation‐induced genotoxic injury without interfering with accumulation of p53 protein or p21waf‐1 expression. BAG‐1 functions downstream of p53‐induced gene expression to inhibit p53‐mediated suppression of cell growth, presumably by suppressing the actions of Siah‐1A. We suggest that Siah‐1A may be an important mediator of p53‐dependent cell‐cycle arrest and demonstrate that Siah‐1A is directly inhibited by BAG‐1.

Ischemia-reperfusion (IR) injury induces endoplasmic reticulum (ER) stress and cell death. Bax Inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses cell death and that is abundantly expressed in both liver and kidney. We explored the role of BI-1 in protection from ER stress and IR injury by using bi - 1 knockout mice, employing models of transient hepatic or renal artery occlusion. Compared to wild-type bi-1 mice, bi - 1 knockout mice subjected to hepatic IR injury exhibited these characteristics: ( i ) increased histological injury; ( ii ) increased serum transaminases, indicative of more hepatocyte death; ( iii ) increased percentages of TUNEL-positive hepatocytes; ( iv ) greater elevations in caspase activity; and ( v ) more activation of ER stress proteins inositol-requiring enzyme 1 and activating transcription factor 6 and greater increases in expression of ER stress proteins C/EBP homologous protein and spliced XBP-1 protein. Moreover, hepatic IR injury induced elevations in bi - 1 mRNA in wild-type liver, suggesting a need for bi - 1 gene induction to limit tissue injury. Similar sensitization of kidney to ER stress and IR injury was observed in bi - 1 ^−/− mice. We conclude that bi - 1 provides endogenous protection of liver and kidney from ER stress and IR injury. Analysis of components of the bi - 1 -dependent pathway for protection from IR injury may therefore reveal new strategies for organ preservation.