Control of cell cycle entry and apoptosis in B lymphocytes infected by Epstein-Barr virus.
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Infection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0 and G1 phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-kappaB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-kappaB during the time course of infection. (+info)
Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak.
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The sequenced gammaherpesviruses each contain a single viral bcl-2 homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2 homologs and cellular bcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2. (+info)
Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland.
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Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis. (+info)
Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations.
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Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore-the apoptotic pore-large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole. (+info)
Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia.
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Caspase-9 is critical for cytochrome c (cyto-c)-dependent apoptosis and normal brain development. We determined that this apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca2+ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis. (+info)
Role of BAX mutations in mismatch repair-deficient colorectal carcinogenesis.
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BAX gene mutations occur in approximately 50% of RER+ colorectal cancers. To determine the role of these mutations in tumour progression we analysed multiple different tumour sites from RER+ colorectal cancers for BAX mutations. Sixty colorectal carcinomas were analysed for microsatellite instability at loci BAT-26, L-myc, TGF betaRII, D13S160 and D2S123. Twelve out of 60 tumours (20%) were RER+. Forty-five different tumour sites from the 12 RER+ carcinomas were analysed for BAX mutations at the [(G)8] tract in exon 3. Six out of 12 (50%) RER+ tumours showed BAX mutations, four of which showed a homogenous pattern of such mutations detected in all tumour sites. In the other two cases, BAX mutations were present in some but not all tumour sites sampled from the same patient. In contrast, TGF betaRII mutations were found in 9/12 cases (75%) and in each of these were present in all the sampled sites. Two cases showed neither BAX nor TGF betaRII mutation. These data suggest that mutations in TGF betaRII may occur at a very early stage in tumour progression, perhaps in the founder clone. BAX mutations, however, are clearly not necessary for formation of the founder clone and can occur for the first time later in tumour progression. (+info)
Mechanisms of apoptosis induced by the synthetic retinoid CD437 in human non-small cell lung carcinoma cells.
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The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase. (+info)
HPV16 E6 oncoprotein inhibits apoptosis induced during serum-calcium differentiation of foreskin human keratinocytes.
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Transfection of human papillomavirus (HPV) 16 E6 oncogene into foreskin primary human keratinocytes (PHKs) causes the formation of colonies of viable cells resistant to serum-calcium differentiation. To define the stage of keratinocyte differentiation inhibited by E6, we examined the response of PHKs to serum and calcium with respect to parameters of both growth and differentiation. The effect of HPV16 E6 was evaluated by infection with recombinant retroviruses encoding the E6 protein. Results of these studies indicated that terminal differentiation of cultured foreskin keratinocytes, triggered by serum and calcium, is a progressive process (2-3 weeks) that ends with cell death with characteristics of apoptosis. Human keratinocyte terminal differentiation was accompanied by time-related changes in the expression of cellular proteins involved in the control pathways of apoptosis, including downregulation of Bcl-2 and p53 and upregulation of Bax, which coincided with the appearance of morphological signs of apoptosis. E6 expression did not override the differentiation-associated G1 arrest or prevent the induction of squamous differentiation-specific markers, transglutaminase 1 and involucrin. E6 expression led, however, to a significant reduction in cell stratification and cell death by apoptosis, which correlated with prolonged expression of Bcl-2 and reduced elevation of Bax levels that occurred concomitant with a complete loss of p53. The data argue that E6 inhibits terminal differentiation of foreskin PHKs through inhibition of their differentiation-induced apoptotic program. (+info)