Efficient expression of bioactive murine IL12 as a self-processing P2A polypeptide driven by inflammation-regulated promoters in tumor cell lines.

Interleukin 12 (IL12) is a heterodimeric proinflammatory cytokine that has shown promise as an anticancer agent. However, despite encouraging results in animal models, clinical trials involving IL12 have been unsuccessful due to toxic side effects associated with its systemic administration, prompting investigation into new delivery methods to confine IL12 expression to the tumor environment. In this study we used the self-cleaving property of the 2A peptide to express both codon-optimized murine IL12 subunits (muIL12opt) as a self-processing polypeptide (muIL12opt-P2A). We cloned muIL12opt-P2A driven by different inflammation-induced lentiviral expression systems to transduce murine tumor cell lines commonly employed in syngeneic tumor models. We confirmed the inducibility of these systems in vitro and in vivo and demonstrated the successful expression of both IL12 subunits and the release of bioactive IL12 upon proinflammatory stimulation in vitro. Therefore, IL12 release driven by these inflammation-regulated expression systems might be useful not only to address the impact of IL12 expression in the tumor environment but also to achieve a local IL12 release controlled by the inflammation state of the tumor, thus avoiding toxic side effects associated with systemic administration.

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco.

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.

Interstock-induced mechanism of increased growth and salt resistance of orange (Citrus sinensis) trees.

Interstocks improve the growth and salt resistance of lemon (Citrus limon (L.) Burm. f.) trees, but their effects on orange (Citrus sinensis (L.) Osbeck) trees are unknown. We grew 'Cleopatra' mandarin (CM) seedlings, budded trees of 'Salustiano' orange (SAO) on CM, 'Valencia Late' orange (VLO) on CM (VLO/CM), and interstock trees VLO/SAO/CM in pots of sand watered with nutrient solution containing 5 (control) or 50 mM NaCl for 12 weeks. Plants were harvested on six successive occasions and the time trends in relative growth rate (RGR) and its components were estimated by fitting a Richards function regression to the harvest data. At low and high salinities, the VLO/SAO/CM combination had higher mean RGR than VLO/CM. Under control conditions, the increase in RGR caused by the interstock was the result of an increase in leaf mass fraction (LMF; leaf dry mass/plant dry mass ratio). Increases in net assimilation rate on a leaf mass basis (NARm) and LMF contributed equally to the increase in RGR in saline conditions, their growth response coefficients being 0.52 and 0.48, respectively. The structural modifications, specific leaf area (SLA) and leaf area ratio (LAR; leaf area:plant dry mass ratio), had a slight influence on the reduction in RGR by salinity. However, NARm had a large influence on RGR, except in CM. The interstock-induced mechanism increased biomass allocation to the assimilatory organs and, under saline conditions, increased Cl- and Na+ allocations to roots. Thus, the flux of ions to the leaves was either delayed or reduced or both. The dilution of imported ions by foliar growth reduced ion concentrations in leaves, resulting in higher NARm, which together with higher LMF, increased RGR.

Improvement in growth and salt resistance of lemon (Citrus limon) trees by an interstock-induced mechanism.

Interstocks can reduce toxic ion accumulations in leaves of budded citrus trees, but the mechanism is not understood. We grew sour orange (Citrus aurantium L.; SO) seedlings, budded trees of 'Salustiano' orange (Citrus sinensis L. Osbeck; SAO) on SO, 'Verna' lemon (Citrus limon L. Burm. f; VL/SO) and interstock trees (VL/SAO/SO) in pots of sand watered with nutrient solution containing 5 (control) or 50 mM NaCl (saline treatment) for 12 weeks. Plants were harvested in six successive harvests and time trends in relative growth rate (RGR) and its components were estimated by fitting a Richards function regression to the harvest data. The VL/SAO/SO trees in saline conditions had higher mean RGR than VL/SO trees in control conditions. Increases in both net assimilation rate on a leaf mass basis (NARw) and leaf mass fraction (LMF) contributed equally to a twofold increase in RGR of VL/SAO/SO trees in saline conditions. In control conditions, the increase in RGR caused by the interstock had growth response coefficients of GRC(NARw) = 0.20 and GRC(LMF) = 0.80. Structural modifications-specific leaf area, leaf area ratio and LMF-had a slight influence on the salt-induced changes in RGR, whereas NARw had a large influence. Salinity decreased root mass fraction (RMF) and increased stem mass fraction (SMF). In contrast, the interstock decreased SMF and increased LMF and RMF. The VL/SAO/SO trees had the highest RMF and proportionally higher Cl- and Na+ allocations in roots than the other plant types. In saline conditions, reductions in leaf ion transport rate and dilution of imported ions by foliar growth nearly halved ion accumulations in leaves of VL/SAO/SO trees.

Bag1 is a regulator and marker of neuronal differentiation.

Bag 1 acts as a co-chaperone for Hsp70/Hsc70. We report here that stable over-expression of Bag1 in immortalized neuronal CSM14.1 cells prevents death following serum deprivation. Bag1 over-expression slowed the proliferative rate of CSM14.1 cells, resulted in increased levels of phospo-MAP kinases and accelerated neuronal differentiation. Immunocytochemistry revealed mostly nuclear localization of Bag1 protein in these cells. However, during differentiation in vitro, Bag1 protein shifted from predominantly nuclear to mostly cytosolic in CSM14.1 cells. To explore in vivo parallels of these findings, we investigated Bag1 expression in the developing mouse nervous system using immunohistochemical methods. Early in brain development, Bag1 was found in nuclei of neuronal precursor cells, whereas cytosolic Bag1 staining was observed mainly after completion of neuronal precursor migration and differentiation. Taken together, these findings raise the possibility that the Bag1 protein is expressed early in neurogenesis in vivo and is capable of modulating neuronal cell survival and differentiation at least in part from a nuclear location.

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.

Dynamics of glyphosate and aminomethylphosphonic acid in a forest soil in Galicia, north-west Spain.

Residues of the herbicide glyphosate (N-phosphonomethylglicine) and its main metabolite, aminomethylphosphonic acid (AMPA), were determined in a forest soil in north-west Spain, previously treated with 5 and 8 l ha(-1) of glyphosate. Both products were monitored in the solid and liquid soil phases for an 8-week period after the treatment. Soil samples were extracted by KH2PO4. Concentrated extracts and liquid phase samples were derivatized with 9-fluorenylmethyl chloroformate (FMOC) before determination by HPLC using an anion exchange column and spectrofluorometric detection. The treated soil peaked at 6.9 microg g(-1) of glyphosate, whereas soil water samples peaked at 0.74 microg ml(-1) of glyphosate. One month after the treatment, both glyphosate and AMPA concentrations in soil and water samples were almost negligible. AMPA peaked at 0.77 microg ml(-1) in soil water samples. Glyphosate and AMPA exhibited high vertical mobility in the treated soil, quickly reaching high concentrations in subsurface horizons where the degradation is slower.

Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer.

Fas-associated phosphatase-1 (FAP-1) is a protein-tyrosine phosphatase that binds the cytosolic tail of Fas (Apo1, CD95), presumably regulating Fas-induced apoptosis. Elevations of FAP-1 protein levels in some tumor cell lines have been correlated with resistance to Fas-induced apoptosis. To explore the expression of FAP-1 in ovarian cancer cell lines and archival tumor specimens, mouse monoclonal and rabbit polyclonal antibodies were generated against a FAP-1 peptide and recombinant FAP-1 protein. These antibodies were used for immunoblotting, immunohistochemistry, and flow-cytometry analysis of FAP-1 expression in the Fas-sensitive ovarian cancer lines HEY and BG-1, and in the Fas-resistant lines OVCAR-3 FR and SK-OV-3. All methods demonstrated high levels of FAP-1 in the resistant lines OVCAR-3 FR and SK-OV-3, but not in the Fas-sensitive lines HEY and BG-1. Furthermore, levels of FAP-1 protein also correlated with the amounts of FAP-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction analysis. FAP-1 protein levels were investigated by immunoblotting in the National Cancer Institute's panel of 60 human tumor cell lines. Although FAP-1 failed to correlate with Fas-resistance across the entire tumor panel, Fas-resistance correlated significantly with FAP-1 expression (P: < or = 0.05) and a low Fas/FAP-1 ratio (P: < or = 0.028) in ovarian cancer cell lines. FAP-1 expression was also evaluated in 95 archival ovarian cancer specimens using tissue-microarray technology. FAP-1 was expressed in nearly all tumors, regardless of histological type or grade, stage, patient age, response to chemotherapy, or patient survival. We conclude that FAP-1 correlates significantly with Fas resistance in ovarian cancer cell lines and is commonly expressed in ovarian cancers.

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 NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in 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-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB 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.

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.

Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia.

Compounds that inhibit protein kinases are currently undergoing clinical evaluation for the treatment of a variety of malignancies. The kinase inhibitors flavopiridol and 7 hydroxy-staurosporine (UCN-01) were examined for their effects on B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro (n = 49). Flavopiridol and UCN-01 induced concentration-dependent apoptosis of most B-CLL samples tested, with greater than 50% cell killing occurring at concentrations of less than 1 mcmol/L, and with flavopiridol displaying more potent activity than UCN-01. Flavopiridol (0.1 mcmol/L) and UCN-01 (1 mcmol/L) also induced striking decreases in the levels of the antiapoptosis proteins Mcl-1, X-linked inhibitor of apoptosis (XIAP), and BAG-1 in nearly all cases of B-CLL and of Bcl-2 in approximately half of B-CLL specimens evaluated. In contrast, expression of the proapoptotic proteins Bax and Bak was not significantly influenced by these kinase inhibitors. Flavopiridol-induced decreases in the levels of antiapoptosis proteins Mcl-1 and XIAP preceded apoptosis and were not substantially affected by the addition of caspase inhibitors to cultures. In contrast, UCN-01-stimulated decreases in antiapoptosis proteins were slower, occurred concurrently with apoptosis, and were partially prevented by caspase inhibitors. The findings suggest that flavopiridol and UCN-01 induce apoptosis of B-CLL cells through different mechanisms. The potent apoptotic activities of flavopiridol and UCN-01 against cultured B-CLL cells suggest that they may be effective as single agents in the treatment of B-CLL or for sensitizing B-CLL cells to conventional cytotoxic drugs. (Blood. 2000;96:393-397)

The Drosophila tumor necrosis factor receptor-associated factor-1 (DTRAF1) interacts with Pelle and regulates NFkappaB activity.

A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family was identified in Drosophila. DTRAF1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of DTRAF1 binding to different members of the human TNF receptor family showed that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-beta receptor. DTRAF1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, DTRAF1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, DTRAF1 did not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increased NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction was partially inhibited by DTRAF1. Mutants of DTRAF1 lacking the N-terminal region inhibited NFkappaB induction by either TRAF2 or TRAF6. DTRAF1 specifically associated with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and DTRAF1 individually were unable to induce NFkappaB in a human cell line, co-expression of Pelle and DTRAF1 resulted in significant NFkappaB activity. Interactions of DTRAF1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution.

Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase.

Polypeptides encoded by plastid ndh genes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H(2)O(2). By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgare L.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H(2)O(2) in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O(2). When FeSP is a component of cytochrome b(6).f complex or of the same Ndh complex, O(2) may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O(2) concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities.

Peroxidase activity in Aloe barbadensis commercial gel: probable role in skin protection.

A basic peroxidase (EC 1.11.1.7) (pl around 9.0) has been identified in commercial gel of Aloe barbadensis. In vivo, the activity is localised in the vascular system of inner aqueous leaf parenchyma. Some relevant properties of this basic peroxidase of Aloe have been investigated in leaf extract and in commercial gel where it is notably stable. The acid optimum pH (5.0) for activity and the low KM for H2O2 (0.14 mM) suggest that, when topically applied, Aloe peroxidase may scavenge H2O2 in skin surface.

CD40-mediated activation of Ig-Cgamma1- and Ig-cepsilon germ-line promoters involves multiple TRAF family proteins.

CD40 plays a critical role in immunoglobulin (Ig) class switching in B cells, but the molecular events involved remain poorly understood. Using CD40 mutants with impairments in their ability to bind selected TNF receptor-associated factor (TRAF) family proteins, we observed that CD40-mediated transcriptional induction of the germ-line Ig-Cgamma1- and Ig-Cepsilon promoters was markedly reduced by mutations that prevent TRAF2, TRAF3, TRAF5 or TRAF6 binding. Moreover, co-expression of trans-dominant inhibitory forms of TRAF2, 3, 5 or 6 with wild-type CD40 also suppressed induction of these promoters. Overexpression of TRAF2 or TRAF6 was sufficient to induce transcription of the C(H) promoters through an NF-kappaB-dependent mechanism. In contrast, TRAF3 and TRAF5 failed to induce these promoters, implying a more indirect role for these TRAF family members. Altogether, the results demonstrate a non-redundant role for multiple TRAF in the signal transduction pathways by which CD40 induces transcription of germ-line C(H) promoters. Since C(H) germ-line transcription represents an obligatory step in Ig class switching in B cells, these findings suggest that interference with the functions of any of these TRAF might provide a means of preventing class switching for therapeutic purposes.

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.

Differential requirements for tumor necrosis factor receptor-associated factor family proteins in CD40-mediated induction of NF-kappaB 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-kappaB and Jun N-terminal kinase (JNK), implying that CD40 can stimulate TRAF2- and TRAF3-independent pathways for NF-kappaB and JNK activation. A carboxyl-truncation mutant of CD40 lacking the last 32 amino acids required for TRAF2 and TRAF3 binding, CD40(Delta32), mediated NF-kappaB induction through a mechanism that was suppressible by co-expression of TRAF6(DeltaN), a dominant-negative version of TRAF6, but not by TRAF2(DeltaN), implying that while TRAF6 does not directly bind CD40, it can participate in CD40 signaling. In contrast, TRAF6(DeltaN) did not impair JNK activation by CD40(Delta32). Taken together, these findings reveal redundancy in the involvement of TRAF family proteins in CD40-mediated NF-kappaB induction and suggest that the membrane-proximal region of CD40 may stimulate the JNK pathway through a TRAF-independent mechanism.

Impairment of the proapoptotic activity of Bax by missense mutations found in gastrointestinal cancers.

We have reported previously that codon 169 of the proapoptotic gene BAX is a mutational hot spot in gastrointestinal cancer. Two different mutations were found in this codon, replacing the wild-type threonine by alanine or methionine. To compare the proapoptotic activity of these Bax mutants with wild-type Bax, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induced a dose-dependent decrease in the viability of cells transfected with wild-type BAX, but this loss of viability was inhibited in cells transfected with BAX mutants. Furthermore, muristerone A induced morphological changes characteristic of apoptosis, including cell shrinkage, rounding, formation of apoptotic bodies, detachment and nuclear condensation and fragmentation, in cells transfected with wild-type BAX. These hallmarks of apoptosis were clearly diminished in cells transfected with BAX mutants. Mutation of threonine 169 did not affect the binding of Bax to Bax, Bcl-2, or Bcl-X(L). These results demonstrate that missense mutations at codon 169 of BAX are functional because they inhibit its apoptotic activity. This is the first report of the functional significance of missense mutations in BAX, or any other proapoptotic member of the Bcl-2 family, in primary human tumors.