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 ubiquitinconjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-

B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

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.

We have identified three new tumor necrosis factorreceptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein.

). CD40L and Bryostatin decreased both spontaneous and drug-induced apoptosis in most B-CLL specimens tested. Apoptosis resistance was associated with CD40L-and Bryostatin-induced elevations in the anti-apoptotic Bcl-2 family protein Mcl-1. CD40L also induced striking increases in the levels of the anti-apoptotic protein Bcl-X L in B-CLLs. CD40L stimulated increases in the surface expression of CD40, CD54, CD69, CD70, CD80 and CD95, whereas Bryostatin induced expression of CD40, CD54, CD69 and CD95 but not the co-stimulatory molecules CD70 and CD80. Despite elevations in the expression of CD95 (Fas), anti-Fas antibodies failed to induce apoptosis of CD40L-and Bryostatin-treated B-CLL cells. This Fasresistance was associated with increased expression of the Fas-antagonist Flip in CD40L-treated, and with elevations in the caspase inhibitor XIAP in Bryostatin-treated B-CLLs. The potential anti-apoptotic properties of CD40L and Bryostatin should be taken into consideration when employing these agents in clinical trials involving patients with B-CLL.

The ATPase Inhibitory Factor 1 (IF1) is an inhibitor of the mitochondrial H+-ATP synthase that regulates the activity of both oxidative phosphorylation (OXPHOS) and cell death. Here, we have developed transgenic Tet-On and Tet-Off mice that express a mutant active form of hIF1 in the hepatocytes to restrain OXPHOS in the liver to investigate the relevance of mitochondrial activity in hepatocarcinogenesis. The expression of hIF1 promotes the inhibition of OXPHOS in both Tet-On and Tet-Off mouse models and induces a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK. Expression of the transgene significantly augmented proliferation and apoptotic resistance of carcinoma cells, which contributed to an enhanced diethylnitrosamine-induced liver carcinogenesis. Moreover, the expression of hIF1 also diminished acetaminophen-induced apoptosis, which is unrelated to differences in permeability transition pore opening. Mechanistically, cell survival in hIF1-preconditioned hepatocytes results from a nuclear factor-erythroid 2-related factor (Nrf2)-guided antioxidant response. The results emphasize in vivo that a metabolic phenotype with a restrained OXPHOS in the liver is prone to the development of cancer.

Inhibitor of apoptosis proteins (IAPs) 3 are a family of anti-apoptotic proteins characterized by the presence of baculoviral IAP repeat (BIR) domains (for view, see Ref. 1). IAPs modulate apoptosis by binding and inactivating caspases (2). Several IAPs contain E2 binding RING domains and also operate as E3 ligases that catalyze attachment of ubiquitin to protein substrates. This E3 ligase activity can be directed either toward caspases and caspase regulators or toward signal-transducing proteins primarily involved in activation of NF-B and Jun N-terminal kinase.The human genome includes eight genes that encode BIR-containing proteins. Among these are cIAP1 and cIAP2, which are highly homologous in amino acid sequence and domain structure, containing (from the N to C terminus) three tandem BIR domains followed by a CARD and RING. The cIAP1 and cIAP2 proteins are unique among the BIRfamily proteins in their ability to form complexes with TRAF-family adapter proteins involved in TNF receptor signaling (3-5). These members of the IAP family are known to induce ubiquitination and proteasome-dependent degradation of the TRAFs to which they bind (e.g. TRAF1, TRAF2), but the structural basis for this phenomenon is largely unknown.In addition to binding certain caspases (6), cIAP1 and cIAP2 are reported to bind SMAC, a mitochondrial protein that competes with caspases for binding to IAPs when released into the cytosol (7). In the IAP-family member, XIAP, the binding site for SMAC has been mapped to the third BIR domain (BIR3), where it competes for binding to caspase-9 (8). However, the site on cIAP1 and cIAP2, where SMAC binds has not been previously elucidated, and its relation to the sites required for binding to other targets such as TRAF1 and TRAF2 have not been heretofore defined.Here, we undertook a structure-function analysis of cIAP1 to compare the binding sites for TRAF2 and SMAC and to reveal the functional consequences of disruptions in these binding sites. Our findings indicate that TRAF2 binds the BIR1 domain, whereas SMAC binds the BIR3 domain of cIAP1. The integrity of these binding sites on BIR1 and BIR3 is required for cIAP1-mediated ubiquitination of TRAF2 and SMAC, respectively, revealing the basis for differential targeting of protein substrates by this E3 ligase. We also show evidence of BIR1-dependent modulation of TRAF2-mediated regulation of NF-B. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single IAP-family protein can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains. MATERIALS AND METHODSPlasmids, Mutagenesis, Recombinant Proteins-A cDNA clone encoding cIAP1 (ID 627116) was obtained from IMAGE consortium through ResGen (Invitrogen) and PCR-subcloned into the pcDNA3-FLAG expression vector. Deletion or point mutations were introduced by PCR or using the QuikChange site-directed mutagen...

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.