Fas ligand--caught between Scylla and Charybdis.
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Animals↗
Biomedical subjects
A Strasser
Publications and source records attributed to A Strasser.
Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells.
IL-7 receptor-deficient (IL-7R(-/-)) mice are lymphopenic as a result of defective cell production at early steps in both B and T lymphopoiesis. In the bone marrow, there is an incomplete block in B cell development at the transition from the pro-B to the pre-B cell stage. As a consequence, peripheral lymphoid organs of IL-7R(-/-) mice contain abnormally low numbers of mature surface (s) Ig-expressing B cells and this is accompanied by a relative increase in immature sIg- B cells. Transgenic expression of the anti-apoptotic protein Bcl-2 in IL-7R(-/-) mice rescues the defect in T cell development and in mature T cell function. The present report shows that constitutive expression of Bcl-2 is incapable of rescuing B lymphopoiesis in IL-7R(-/-) mice but can enhance survival of those mature B cells which escape the developmental arrest. Thus the essential role of IL-7R signaling in B lymphoid cells cannot be replaced by Bcl-2, indicating that in B lymphopoiesis IL-7R signaling is necessary for promoting cell division and/or for inhibiting a Bcl-2-insensitive pathway to apoptosis.
Animals↗
Rapid hybridoma screening method for the identification of monoclonal antibodies to low-abundance cytoplasmic proteins.
Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.
Animals↗
Does "death receptor" signaling play a role in tumorigenesis and cancer therapy?
Physiological cell death, known as apoptosis, is an evolutionarily conserved process that is required for normal development and function of multicellular organisms. Abnormalities in cell death control are implicated as a cause or contributing factor in a range of diseases, including cancer, autoimmunity, and degenerative disorders. Importantly, the propensity of a cell to undergo apoptosis is one of the determinants of the sensitivity of tumor cells to antineoplastic therapy. Apoptosis can be triggered by stress-induced signals that arise from within the doomed cell or by signals that are elicited by binding of extracellular "death ligands" to their "death receptors." Cysteine proteases have been recognized as essential effectors of all pathways to apoptosis. Experiments with transgenic mice and gene knockout mice have shown that different caspases and their adaptor molecules are needed for "death receptor" signaling and apoptotic pathways elicited by cytokine withdrawal, DNA damage, or corticosteroids. These differences allow the pathways to be regulated by distinct inhibitors. It has been published that chemotherapeutic drugs and gamma-radiation induce apoptosis by "death ligand"-mediated activation of "death receptors," but this model has been challenged. Our review discusses this controversy in the light of current knowledge of the molecular control of apoptosis.
Animals↗
Effect of a bcl-2 transgene on production and localization of precursor B cells in mouse bone marrow.
Many B cell precursors die while differentiating in mouse bone marrow. To ascertain the mechanisms involved in this process, populations of B lineage cells and their tissue localization were analyzed in bone marrow of transgenic mice overexpressing the apoptosis inhibitor, Bcl-2. Immunofluorescence labeling and mitotic arrest were used to quantitate the number and proliferative activity of mu- pro-B cells (terminal deoxynucleotidyl transferase [TdT]+B220-, TdT+B220+, and TdT-B220+); pre-B cells (cmu+); and B cells (smu+). Mature B cells (IgM+IgD+) were increased 16- to 20-fold. In addition, immature B lymphocytes (IgM+IgD-/low), representing newly formed cells, were increased three- to sixfold, whereas pre-B cells and late pro-B cells were increased 30 to 60% in production rate. Earlier pro-B cells expressing TdT were unaffected. In spleen, both mature and immature B cells were greatly increased, but cells of precursor phenotype were few and TdT+ cells were absent. The in vivo location of B cells was examined by autoradiography using light and electron microscopy after intravenous injection of 125I-labeled antibodies. B lineage cells (B220+) were increased throughout bone marrow, often within dilated venous sinusoids, particularly in subosteal regions. Many intravascular and perisinusoidal cells were IgDhigh mature B lymphocytes. In contrast, many other IgM+ and IgDlow immature B lymphocytes clustered extravascularly around the central venous sinus. Plasma cells with distended endoplasmic reticulum were numerous. These findings provide evidence that, in addition to expanding the recirculating pool of B cells entering bone marrow from the blood stream, high levels of Bcl-2 can inhibit some of the apoptosis occurring during B cell differentiation, thereby expanding populations of B lymphopoietic precursor cells within the bone marrow parenchyma.
Animals↗
The CD2-scl transgene alters the phenotype and frequency of T-lymphomas in N-ras transgenic or p53 deficient mice.
Abnormal expression of SCL (TAL-1/TCL5) occurs in the majority of paediatric cases of acute T-cell lymphoblastic leukemia (T-ALL). Unexpectedly however, transgenic mice carrying scl coupled to the human T-cell specific CD2 enhancer (CD2-scl) did not spontaneously develop T-cell lymphomas despite high levels of scl expression in their thymocytes. Analogous to other transgenic models of lymphomagenesis, it is likely that additional genetic abnormalities are required to cooperate with scl to trigger lymphomagenesis. Two possible candidates are the p53 and N-ras genes which are mutated in some cases of T-ALL, particularly in relapsed disease. Therefore, we examined lymphomagenesis in the progeny of CD2-scl mice crossed with N-ras transgenic mice or p53 deficient. Surprisingly, the frequency of lymphomas in the p53 nullizygous or N-ras transgenic mice was not enhanced by expression of the scl transgene. In fact, expression of scl in both genetic backgrounds paradoxically reduced the frequency of thymic lymphomas and, at least in the p53 nullizygous mice, shifted the pattern of organ involvement to the peripheral lymphoid organs. In contrast, CD2-scl transgene expression accelerated lymphomagenesis in p53 heterozygous mice. These data suggest that the collaborative effects of scl with N-ras or p53 vary according to the developmental stage of the T-cell.
Alleles↗
Enforced Bcl-2 expression inhibits antigen-mediated clonal elimination of peripheral B cells in an antigen dose-dependent manner and promotes receptor editing in autoreactive, immature B cells.
The mechanisms that establish immune tolerance in immature and mature B cells appear to be distinct. Membrane-bound autoantigen is thought to induce developmental arrest and receptor editing in immature B cells, whereas mature B cells have shortened lifespans when exposed to the same stimulus. In this study, we used Emu-bcl-2-22 transgenic (Tg) mice to test the prediction that enforced expression of the Bcl-2 apoptotic inhibitor in B cells would rescue mature, but not immature, B cells from tolerance induction. To monitor tolerance to the natural membrane autoantigen H-2Kb, we bred 3-83mudelta (anti-Kk,b) Ig Tg mice to H-2(b) mice or to mice expressing transgene-driven Kb in the periphery. In 3-83mudelta/bcl-2 Tg mice, deletion of autoreactive B cells induced by peripheral Kb antigen expression in the liver (MT-Kb Tg) or epithelia (KerIV-Kb Tg), was partly or completely inhibited, respectively. Furthermore, Bcl-2 protected peritoneal B-2 B cells from deletion mediated by acute antigen exposure, but this protection could be overcome by higher antigen dose. In contrast to its ability to block peripheral self-tolerance, Bcl-2 overexpression failed to inhibit central tolerance induced by bone marrow antigen expression, but instead, enhanced the receptor editing process. These studies indicate that apoptosis plays distinct roles in central and peripheral B cell tolerance.
Animals↗
The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumourigenesis and resistance to chemotherapy.
Cell production within an organ is determined by the rate of immigration, proliferation, differentiation, emigration and death of cells. Abnormalities in any one of these processes will disturb normal control of cell production, thereby eliciting hyperplasia can be an early event in neoplasia. Cell death, apoptosis, is a physiological process responsible for removing unwanted cells. It is used in multi-cellular organisms for tissue remodelling during embryogenesis, regulation of cell turnover and as a defence strategy against invading pathogens. In this review article we describe the role of the bcl-2/ced-9 gene family in cancer and discuss the general implications of defects in the apoptosis program for tumourigenesis and resistance of cancer cells to chemotherapy in light of current knowledge of the molecular mechanisms of cell death.
Apoptosis↗
Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo.
B lymphocyte differentiation proceeds through a series of alternating stages of proliferative expansion interspersed with noncycling stationary phases during which cells undergo either positive selection or apoptotic cell death. The molecular control of cell cycle progression and that of apoptosis appear to be interconnected. Overexpression of Bcl-2 in lymphocytes or fibroblasts antagonizes apoptosis and delays their transition from the quiescent state into the cell cycle. We have undertaken a systematic analysis of the impact of bcl-2 transgene expression on cell cycle distribution and turnover rate of developing B lymphocytes in normal mice and in mutant animals in which B cell differentiation is arrested at the pro-B/pre-BI or the pre-BII stage. These experiments revealed that overexpression of Bcl-2 reduces proliferation and slows turnover of B cells at all stages of development. This demonstrates that Bcl-2 can retard transition of B cells between the quiescent and the cycling state regardless of the mitogenic stimulus and the differentiation stage. The implications of these results for the normal control of B lymphopoiesis and for lymphomagenesis are discussed.
Animals↗
The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry.
The Bcl-2 family of proteins regulate apoptosis, some antagonizing cell death and others facilitating it. It has recently been demonstrated that Bcl-2 not only inhibits apoptosis but also restrains cell cycle entry. We show here that these two functions can be genetically dissociated. Mutation of a tyrosine residue within the conserved N-terminal BH4 region had no effect on the ability of Bcl-2 or its closest homologs to enhance cell survival and did not prevent heterodimerization with death-enhancing family members Bax, Bak, Bad and Bik. Neither did this mutation override the growth-inhibitory effect of p53. However, on stimulation with cytokine or serum, starved quiescent cells expressing the mutant proteins re-entered the cell cycle much faster than those expressing comparable levels of wild-type proteins. When wild-type and Y28 mutant Bcl-2 were co-expressed, the mutant was dominant. Although R-Ras p23 has been reported to bind to Bcl-2, no interaction was detectable in transfected cells and R-Ras p23 did not interfere with the ability of Bcl-2 to inhibit apoptosis or cell cycle entry. These observations provide evidence that the anti-apoptotic function of Bcl-2 is mechanistically distinct from its inhibitory influence on cell cycle entry.
3T3 Cells↗
Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice.
Signals from cytokine and antigen receptors play crucial roles during lymphocyte development. Mice lacking interleukin-7 receptor are lymphopenic, due to a defect in cell expansion at an early stage of differentiation, and the few mature T cells that develop in IL-7R-/- animals are functionally impaired. Both defects were rescued completely by overexpression of the anti-apoptosis protein Bcl-2. T cell progenitors lacking antigen receptor molecules are also blocked in differentiation and die, presumably because they fail to receive a positive signal via their pre-T cell receptor. Surprisingly, Bcl-2 did not promote survival or differentiation of T cells in rag-1-/- mice. These results provide evidence that blocking apoptosis is the essential function of IL-7R during differentiation and activation of T lymphocytes and that pre-TCR signaling blocks a pathway to apoptosis that is insensitive to Bcl-2.
Animals↗
Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts.
Binding of CD95 (Fas/APO-1) by its ligand (CD95L) commonly induces apoptosis. Apoptosis of activated T cells, induced by CD95L expressed in the rodent testis, has been proposed to be the mechanism of immune privilege [Bellgrau, D., Gold, D., Selawry, H., Moore, J., Franzusoff, A. & Duke, R. C. (1995) Nature (London) 377, 630-632]. To test whether CD95L could protect pancreatic islet grafts from rejection, we made transgenic mice expressing murine CD95L on their islet beta cells and transplanted fetal pancreata under the kidney capsules of allogeneic animals. Expression of CD95L failed to protect the grafts from rejection. However, transgenic mice developed a granulocytic infiltration in their pancreata. These results demonstrate a pro-inflammatory function of CD95L and suggest that expression of CD95L may not be sufficient to protect organ allografts.
Animals↗
Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death.
Apoptosis is the physiological process by which unwanted cells in an organism are killed. Bcl-2, a membrane-bound cytoplasmic protein, is an effective inhibitor of apoptotic cell death induced by many cytotoxic agents. Survival-promoting homologues of Bcl-2 include its close relative, Bcl-xL and the 19 kD protein encoded by the E1B gene of adenoviruses. Whether these proteins are functionally equivalent and whether they can antagonise all or only some pathways to apoptosis is unresolved. We have carried out a systematic comparison of Bcl-2, Bcl-xL and adenovirus E1B19kD activity, using several cell lines and a range of cytotoxic conditions. High levels of expression of each of these proteins inhibited apoptosis induced by growth factor deprivation or treatment with gamma-radiation, glucocorticoid and various cytotoxic drugs. In contrast, none of them could effectively counter apoptosis induced via the TNF receptor or Fas/APO-1 (CD95). Biochemical analysis revealed that all three proteins can associate with Bax and Bak, members of the Bcl-2 protein subfamily that can facilitate apoptosis. The results provide evidence that Bcl-2, Bcl-xL and adenovirus protein E1B19kD are indistinguishable in their ability to regulate the cell death effector machinery.
Adenovirus E1B Proteins↗
Lymphomas and plasmacytomas in transgenic mice involving bcl2, myc and v-abl.
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Animals↗
Hypoxia modulates free radical formation in brain microvascular endothelium.
Although free radical species (ROS; i.e., .O2-. .OH.H2O2) among other mediators, may be involved in altering the blood-brain barrier (BBB), little is known about the endogenous ability of cerebromicrovascular endothelium to generate ROS. This study examines the capacity of rat endothelial cells (RBEC) to produce ROS in normoxia and hypoxia/reoxygenation. Cultured RBEC were exposed to an oxygen-depleted atmosphere (containing 95% N2 and 5% CO2) for 4 hr at 37 degrees C and air (10 min) at room temperature to simulate "ischemia/reperfusion". Nitroblue tetrazolium (NBT) reduction [formation of nitroblue formazan (NBF)] served as a marker for the production of ROS. The release of lactate dehydrogenase (LDH) and [3H]arachidonic acid (AA) was used to assess cellular integrity. RBEC exposed to hypoxia/reoxygenation produced up to 59% greater NBF formation than controls without affecting the LDH or AA release. The production of ROS was calcium-dependent and not affected by AA or its metabolites. The findings indicate that the RBEC can produce superoxide dismutase (SOD)-inhibitable ROS which are augmented by hypoxia/reoxygenation. It is suggested that in vivo cerebromicrovascular endothelium may contribute to the formation of ROS and play a role in ischemic brain edema.
Animals↗
The effect of nitric oxide inhibition on ischemic brain edema.
The involvement of nitric oxide (NO) in the development of ischemic cytotoxic edema was investigated by inhibiting nitric oxide synthase (NOS) activity with N omega-nitro-L-arginine (NLA). Bilateral carotid artery occlusion (15 min) alone or with release (15 and 60 min) served as a model for edema induction. NLA, N omega-nitro-D-arginine methyl ester (D-NAME) or Ringer's solution were administered 4 hr prior to ischemia or sham operation. Treatment with a stable nitroxide radical, 4-hydroxy-2,2, 6,6-tetramethylpiperidine-L-oxyl (TPL), was used to assess free radical involvement in edema. Accumulation of tissue water was evaluated by measuring specific gravity (SG) of brain cortex and histological examination. There was a greater reduction of cortical SG in early reperfusion (15 min) and a lesser decrease in SG (60 min later) in NLA-than in D-NAME- or Ringer's-treated gerbils. The NLA effect was confirmed by histological examination of the brain tissue. TPL treatment (pre- and postischemic) ameliorated the formation of edema to the same degree as NLA. The findings indicate a biphasic NLA modulation of cytotoxic edema most likely mediated through absence or presence of NO-derived free radicals.
Animals↗
Overexpression of the bcl-2 oncogene in the mouse pre-B cell line SPGM-1 protects from apoptosis, but does not affect blocked B-lineage differentiation and lineage switch towards macrophage like cells.
The CD5(+) mouse pre-B cell line SPGM-1 is able to undergo lineage conversion towards a macrophage like cell type. Although one of the kappa-light chain alleles is rearranged in V-Jkappa4 configuration, no protein is expressed nor could the expression be induced. Infection with and expression of the human bcl-2 gene protected SPGM-1 cells from apoptosis, allowing the rearrangement of their second kappa-allele in V-Jkappa5 configuration. mRNA transcripts of the V-Jkappa regions were detected only in SPGM-1 x bcl-2 cells, but not in SPGM-1, while kappa-light chain protein was not found in any of the cell lines. The myeloid differentiation potential of SPGM-1 cells was not affected by overexpression of the human bcl-2 gene. Upon appropriate stimulation, SPGM-1xbcl-2 cells became enlarged, adhered to the plastic surfaces and lost their immunoglobulin mu heavy chain expression.
Journal Article↗
Establishment of multipotential and antigen presenting cell lines derived from myeloid leukemias in GM-CSF transgenic mice.
In this study neonatal mice expressing a GM-CSF transgene (GMT mice), and their normal littermate controls, were infected with Moloney murine leukemia virus (MoMLV) to examine in vivo tumorigenesis. By 200 days, all of the GMT mice had died whereas median survival had not been reached in the littermates (P < 0.0003). Thymomas developed in 32% of GMT mice and were more frequently CD4+CD8+ (83%) compared to the CD4+CD8- phenotype seen in 90% of thymomas developing in MoMLV-infected littermate mice. A primitive myeloid leukemia was induced in 21% of GMT mice, but none of the littermates. To characterize further the nature of the leukemic cells, a factor-dependent cell line (DGM36) was derived. DGM36 cells were tumorigenic, capable of differentiation to neutrophils, macrophages and eosinophils, and contained a partial deletion of chromosome 2. A subline arose spontaneously that was factor-independent and produced GM-CSF in an autocrine manner (IGM36 cells). Stimulation of the IGM36 cells with TNF alpha and IFNgamma resulted in increased expression of B7-1, class I MHC and class II MHC and consequent presentation of antigen in allogeneic MLRs. IGM36 cells thereby satisfy many of the criteria of dendritic cells and consequently may be used to examine antigen presentation by leukemic cells. This is the first report of primary myeloid leukemias arising in GMT mice and documents the derivation of a multipotential, autocrine leukemic cell line with dendritic cell characteristics.
Animals↗