Juan M. Zapata

P53-INDUCIBLE HUMAN HOMOLOGUE OF DROSOPHILA SEVEN IN ABSENTIA (SIAH) INHIBITS CELL GROWTH : SUPPRESSION BY BAG-1

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.

Bryostatin and CD40-ligand enhance apoptosis resistance and induce expression of cell survival genes in B-cell chronic lymphocytic leukaemia.

Modulating signal transduction pathways represents a promising approach for altering the biological behaviour of haemopoietic malignancies. B‐cell chronic lymphocytic leukaemia (B‐CLL) cells were treated in vitro with CD40‐ligand (CD40L) (CD154) or the protein kinase C modulator Bryostatin‐1, exploring the effects on: (a) sensitivity to apoptosis induction by chemotherapeutic drugs (fludarabine, dexamethasone) or anti‐Fas antibody; (b) expression of apoptosis‐regulatory proteins (Bcl‐2, Bcl‐X, Mcl‐1, Bax, Bak, BAG‐1, Flip, XIAP); (c) expression of cell surface co‐stimulatory antigens (CD80 [B7.1]; CD54 [ICAM‐1]; CD70); and (d) expression of immune modulatory receptors (CD27, CD40, CD95 [Fas]). 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‐XL 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 Fas‐resistance 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.

TRAF Family Proteins Interact with the Common Neurotrophin Receptor and Modulate Apoptosis Induction

The common neurotrophin receptor, p75NTR, 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 p75NTR initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75NTR and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75NTR was mapped to distinct regions in p75NTR. Furthermore, TRAF4 interacted with dimeric p75NTR, whereas TRAF2 interacted preferentially with monomeric p75NTR. TRAF2-p75NTR, TRAF4-p75NTR, and TRAF6-p75NTR interactions modulated p75NTR-induced cell death and NF-κB activation with contrasting effects. Coexpression of TRAF2 with p75NTRenhanced 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 p75NTR. TRAF4 also inhibited the NF-κB response, whereas TRAF2 and TRAF6 enhanced p75NTR-induced NF-κB activation. These results demonstrate that TRAF family proteins interact with p75NTR and differentially modulate its NF-κB activation and cell death induction.

A Diverse Family of Proteins Containing Tumor Necrosis Factor Receptor-associated Factor Domains

We have identified three new tumor necrosis factor-receptor 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. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH2 termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of bindingin vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-κB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-κB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.

Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.

TNFR-Associated Factor Family Protein Expression in Normal Tissues and Lymphoid Malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.

Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases

Inhibitor of apoptosis proteins (IAPs) regulate apoptosis primarily by inhibiting caspase-family proteases. However, many IAPs also possess E3 ligase (ubiquitin-protein isopeptide ligase) activities implicated in both caspase-dependent and -independent functions of these proteins. Here, we compared the structural features of cIAP1 responsible for its interactions with two known target proteins, TRAF2 and SMAC. The N-terminal (BIR1) and C-terminal (BIR3) BIR domains of cIAP1 were determined to be necessary and sufficient for binding TRAF2 and SMAC, respectively. Mutational analysis of the BIR1 and BIR3 domains identified critical residues required for TRAF2 and SMAC binding. Using these mutants, cIAP1-mediated ubiquitination of TRAF2 and SMAC in vitro was determined to be correspondingly dependent on intact binding sites on BIR1 and BIR3. Because TRAF2 regulates NF-κB activation, the effects of cIAP1 on TRAF2-mediated induction of NF-κB transcriptional activity were studied using reporter gene assays. Expression of a fragment of cIAP1 encompassing the three BIR domains (but not full-length cIAP1) greatly enhanced TRAF2-induced increases in NF-κB activity, providing a convenient assay for monitoring BIR-dependent effects of cIAP1 on TRAF2 in cells. BIR1 mutants of the BIR1-3 fragment of cIAP1 that failed to bind TRAF2 lost the ability to modulate NF-κB activity, demonstrating a requirement for BIR1-mediated interactions with TRAF2. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single cIAP 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.

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.

Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors

The expression of several apoptosis-regulating genes was evaluated in 9 human breast cancer cell lines, 2 immortalized human mammary epithelial lines, 1 normal breast tissue biopsy, and 3 primary breast tumors, using a multiple antigen detection (MAD) immunoblotting method. The anti-apoptotic proteins Bcl-2, Bcl-XL, Mcl-1, and BAG-1 were present at immunodetectable levels in 7, 10, 10, and 9 of the 11 lines. Comparing these 11 cell lines among themselves revealed that steady-state levels of Bcl-2, Bcl-XL, Mcl-1, and BAG-1 were present at relatively higher levels in 4, 6, 5, and 5 of the lines, respectively. In contrast, the pro-apoptotic proteins Bax and Bak were detected in all 11 cell lines, and were present at relatively higher levels in 10 and 5 of the 11 lines, respectively. The Interleukin-1β converting enzyme (ICE) homolog CPP32 (Caspase-3) was expressed in 10/11 breast cell lines. High levels of p53 protein, indicative of mutant p53, were found in 8 of the 11 lines and correlated inversely with Bax expression (p=0.01). Bcl-2 and BAG-1 protein levels were positively correlated (p = 0.03). Immunoblot analysis of primary adenocarcinomas revealed expression of the anti-apoptotic proteins Bcl-2, Bcl-XL, Mcl-1, and BAG-1, as well as the pro-apoptotic proteins Bax, Bak, and CPP32, in at least 2 of the 3 tumors examined. Immunohistochemical analysis was also performed for all of these proteins using 20 paraffin-embedded breast cancer biopsy specimens that all contained residual normal mammary epithelium in combination with both invasive cancer and carcinoma in situ. All of these apoptosis-regulating proteins were detected in primary breast cancers, though the percentage of immunopositive tumor cells varied widely in some cases. Comparisons of the intensity of immunostaining in normal mammary epithelium and invasive carcinoma suggested that Bcl-2 immunointensity tends to be lower in cancers than normal breast epithelium (p=0.03), whereas CPP32 immunointensity was generally higher in invasive cancers (p < 0.0001). Taken together, the results demonstrate expression of multiple apoptosis-modulating proteins in breast cancer cell lines and primary tumors, suggesting complexity in the regulation of apoptosis in these neoplasms of mammary epithelial origin.

Differential Requirements for Tumor Necrosis Factor Receptor-associated Factor Family Proteins in CD40-mediated Induction of NF-κB and Jun N-terminal Kinase Activation

CD40 is a member of the tumor necrosis factor receptor family that mediates a number of important signaling events in B-lymphocytes and some other types of cells through interaction of its cytoplasmic (ct) domain with tumor necrosis factor receptor-associated factor (TRAF) proteins. Alanine substitution and truncation mutants of the human CD40ct domain were generated, revealing residues critical for binding TRAF2, TRAF3, or both of these proteins. In contrast to TRAF2 and TRAF3, direct binding of TRAF1, TRAF4, TRAF5, or TRAF6 to CD40 was not detected. However, TRAF5 could be recruited to wild-type CD40 in a TRAF3-dependent manner but not to a CD40 mutant (Q263A) that selectively fails to bind TRAF3. CD40 mutants with impaired binding to TRAF2, TRAF3, or both of these proteins completely retained the ability to activate NF-κB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-κB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Δ32), mediated NF-κB induction through a mechanism that was suppressible by co-expression of TRAF6(ΔN), a dominant-negative version of TRAF6, but not by TRAF2(ΔN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(ΔN) did not impair JNK activation by CD40(Δ32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-κB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.