Neither macromolecular synthesis nor myc is required for cell death via the mechanism that can be controlled by Bcl-2.

Expression of c-myc and macromolecular synthesis have been associated with physiological cell death. We have studied their requirement for the death of factor (interleukin-3)-dependent cells (FDC-P1) bearing an inducible bcl-2 expression construct. FDC-P1 cells expressing bcl-2 turned off expression of c-myc when deprived of interleukin-3 but remained viable as long as bcl-2 was maintained. A subsequent decline in Bcl-2 allowed the cells to undergo apoptosis directly from G0, in the absence of detectable c-myc expression. Thus c-myc expression may lead to apoptosis in some cases but is not directly involved in the mechanism of physiological cell death that can be controlled by Bcl-2. The macromolecular synthesis inhibitors actinomycin D and cycloheximide triggered rapid cell death of FDC-P1 cells in the presence of interleukin-3, but the cells could be protected by Bcl-2. Thus, the cell death machinery can exist in a quiescent state and can be activated by mechanisms that do not require synthesis of RNA or protein.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.     

The super anti-apoptotic factor Bcl-xFNK constructed by disturbing intramolecular polar interactions in rat Bcl-xL

A powerful artificial anti-apoptotic factor will be useful for medical applications of the future therapies for many diseases by prolonging survival of sick cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among alpha-helix structures of Bcl-x(L). The resultant mutant Bcl-x(L), named Bcl-xFNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, Bcl-xFNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea, and trichostatin A), or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-x(L). Furthermore, the transfectants of bcl-xFNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-x(L). In addition, Bcl-xFNK showed marked anti-apoptotic activity in Chinese hamster ovary and Jurkat cells deprived of serum. Thus, Bcl-xFNK may be the first mutant generated by site-directed mutagenesis of Bcl-x(L) with a gain-of-function phenotype. Interestingly, Bcl-xFNK was found to allow interleukin-3-dependent FDC-P1 to grow without interleukin-3, but not BaF/3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat, the p42/p44 mitogen-activated protein kinase was activated by 2 to 5 times, but not in those of BaF/3 and Chinese hamster ovary. Bcl-xFNK might gain a new function to activate the mitogen-activated protein kinase in a cell-type specific manner. The findings of this study suggest that the central alpha5-alpha6 pore-forming region of anti-apoptotic factor Bcl-x(L) has a pivotal role in suppressing apoptosis.     

Effects of Endogenous Epidermal Growth Factor Receptor Signaling on DNA Synthesis and ERK Activation in a Cytokine-Dependent Hematopoietic Cell Line

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5.12. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.     

Apoptosis in the liver and its role in hepatocarcinogenesis

Apoptosis seems to be the predominant type of active cell death in the liver (type I), while in other tissues cells may die via biochemically and morphologically different pathways (type II, type III). Active cell death is under the control of growth factors and death signals. In the liver, endogenous factors, such as transforming growth factor 1 (TGF-1), activin A, CD95 ligand, and tumor necrosis factor (TNF) may be involved in induction of apoptosis. Release and action of these death factors seems to be triggered by exogenous signals such as withdrawal of hepato-mitogens, food restriction, etc.During stages of hepatocarcinogenesis, not only DNA synthesis but also apoptosis gradually increase from normal to preneoplastic to adenoma and carcinoma tissue. Also, in human carcinomas, birth and death rates of cells are several times higher than in surrounding liver. (Pre)neoplastic liver cells are more susceptible than normal hepatocytes to stimulation of cell replication and of cell death. Consequently, tumor promoters may act as survival factors, i.e., inhibit apoptosis preferentially in preneoplastic and even in malignant liver cells, thereby stimulating selective growth of (pre)neoplastic lesions. On the other hand, regimens favoring apoptosis and lowering cell replication may result in selective elimination of (pre)neoplastic cell clones from the liver. Finally, we have studied the first stage of carcinogenesis, namely the appearance of putatively initiated cells after a single dose of the genotoxic carcinogen N-nitrosomorpholine (NNM). Most of these cells were found to be eliminated by apoptosis, suggesting that initiation, at the organ level, can be reversed at least partially by preferential elimination of initiated cells. These events may be regulated by autocrine or paracrine actions of survival factors.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of Drug-Induced Apoptosis by Survival Factors in PC12 Cells

Abstract: Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2 . We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum-deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl-2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl-2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.     

Environmental chemical-induced pro/pre-B cell apoptosis: analysis of c-Myc,p27Kip1, and p21WAF1 reveals a death pathway distinct from clonal deletion

Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants that suppress the immune system in part by inducing pro/pre-B cell apoptosis. The PAH-induced death signaling pathway resembles the signaling cascade activated during clonal deletion and modeled by B cell receptor cross-linking or by dexamethasone exposure of immature surface Ig(+) B cells in that apoptosis is mediated by NF-kappa B down-regulation. Because a PAH-induced, clonally nonrestricted deletion of B cells would have important implications for B cell repertoire development, the nature of the PAH-induced intracellular death signal was studied further. Particular emphasis was placed on the roles of growth arrest and c-Myc, p27(Kip1), and p21(WAF1) expression, because all of these elements contribute to clonal deletion. As in clonal deletion models, and as predicted by the down-regulation of NF-kappa B, PAH-induced death of pro/pre-B cells was at least partially dependent on c-Myc down-regulation. Furthermore, whereas dexamethasone induced a G(0)/G(1) cell cycle arrest, PAH had no effect on pro/pre-B cell growth, indicating that growth arrest and apoptosis occur by separable signaling pathways in this early phase of B cell development. Finally, in contrast to clonal deletion, PAH-induced pro/pre-B cell death was not dependent on p27(Kip1) or p21(WAF1) up-regulation but did coincide with p53 induction. These results distinguish the PAH-induced apoptosis pathway from that activated during clonal deletion and indicate that signaling cascades leading to growth arrest and/or apoptosis in pro/pre-B cells differ from those active at later B cell developmental stages.     

Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism

Cycloheximide (CHX) can contribute to apoptotic processes, either in conjunction with another agent (e.g. tumor necrosis factor-alpha) or on its own. However, the basis of this CHX-induced apoptosis has not been clearly established. In this study, the molecular mechanisms of CHX-induced cell death were examined in two different human T-cell lines. In T-cells undergoing CHX-induced apoptosis (Jurkat), but not in T-cells resistant to the effects of CHX (CEM C7), caspase-8 and caspase-3 were activated. However, the Fas ligand was not expressed in Jurkat cells either before or after treatment with CHX, suggesting that the activation of these caspases does not involve the Fas receptor. To determine whether CHX-induced apoptosis was mediated by a Fas-associated death domain (FADD)-dependent mechanism, a FADD-DN protein was expressed in cells prior to CHX treatment. Its expression effectively inhibited CHX-induced cell death, suggesting that CHX-mediated apoptosis primarily involves a FADD-dependent mechanism. Since CHX treatment did not result in the induction of Fas or FasL, and neutralizing anti-Fas and anti-tumor necrosis factor receptor-1 antibodies did not block CHX-mediated apoptosis, these results may also indicate that FADD functions in a receptor-independent manner. Surprisingly, death effector filaments containing FADD and caspase-8 were observed during CHX treatment of Jurkat, Jurkat-FADD-DN, and CEM C7 cells, suggesting that their formation may be necessary, but not sufficient, for cell death.     

Autoimmune MRL/lpr mice sera contain IgG with interleukin 3-like activity

The spleen cells, thymocytes, and bone marrow cells of autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice do not constitutively produce interleukin 3 (IL-3), but these mice had IL-3-like activity in their sera. MRL/lpr sera supported the growth of the IL-3-dependent cell lines FDC-P2 and DA-1 but not the growth of IL-2-dependent T-572 cells. This IL-3-like activity increased with age. Biochemical analysis of the MRL/lpr sera by anion-exchange chromatography, gel filtration on a Superose 12 column, the binding to protein-A and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the serum factor with the IL-3-like activity was not IL-3 itself but was associated with IgG. Flow cytometric analysis also showed that the serum level of the Ig capable of binding FDC-P2 cells was high in MRL/lpr but not in MRL/+ mice. We suggest that IL-3 is not responsible for lymphoid hyperplasia, contrary to a previous report; rather auto-antibodies directed toward the IL-3 receptor may act pathogenically in MRL/lpr mice.     

The variation in Fas localization and the changes in Fas expression level upon stimulation with growth factors

Although Fas (APO-1/CD95) is well known as a death receptor, its stimulation occasionally fails to induce apoptosis in malignant cells. On the contrary, Fas is reported to advance the cell cycle in cancer cells. Therefore, we investigated roles of Fas in cell growth and apoptosis using human lung cancer cell lines. Fas was localized in the cytoplasm in exponentially growing cells, whereas only confluent cells expressed Fas on the cell membrane. A stimulation of confluent cells by either of EGF, IGF-I or VEGF induced once a decrease in Fas expression level and its sequential recovery. Fas expression levels in confluent cells were negatively correlated with cell doubling times (r=0.757, p=0.0088). Fas remained on the cell membrane of IgM-treated cells even after the growth factor stimulation, leading to apoptosis with abnormal mitosis, whereas the same stimulation induced Fas internalization in IgG(1)-treated cells. From these results, we suggest that Fas remaining on the cell membrane amplifies to induce apoptosis. Conversely, Fas internalization may enable cancer cells to escape from apoptosis. Our results suggest that growth factor may contribute to the resistance of cancer cells to Fas-mediated apoptosis in an autocrine or paracrine fashion.     

Sensitization of AIDS-Kaposi's sarcoma cells to Apo-2 ligand-induced apoptosis by actinomycin D

Kaposi's sarcoma (KS) is the most frequent malignancy associated with HIV infection (AIDS-KS), a complication that leads to high mortality and morbidity. AIDS-KS cells are resistant to killing by chemotherapeutic drugs/NK cells and Fas-induced apoptosis, suggesting that the acquisition of antiapoptotic characteristics by AIDS-KS cells may contribute to their prolonged survival. Apo-2 ligand (Apo-2L)/TNF-related apoptosis-inducing ligand, a new member of the TNF family, has been identified as an apoptosis-inducing molecule. In this study we examined the sensitivity of 10 different AIDS-KS isolates to Apo-2L-mediated cytotoxicity. AIDS-KS cells were relatively resistant to Apo-2L; however, Apo-2L and actinomycin D (Act D) used in combination synergistically potentiated the induction of cell death in nine of the 10 isolates. Apo-2L induced apoptosis in >80% of AIDS-KS cells pretreated with Act D. The caspase inhibitors, zIETD-fmk and zDEVD-fmk, inhibited apoptosis in AIDS-KS by sApo-2L, suggesting that caspase 3-like and caspase 8 or 10 activities are essential for Apo-2L-mediated apoptosis. Act D treatment of AIDS-KS cells markedly and selectively down-regulated Bcl-xL expression, while the expressions of decoy receptors 1 and 2, Bax, cellular FLICE (Fas-associated death domain protein-like IL-1-converting enzyme) inhibitory protein, FADD (Fas-associated death domain protein), procaspase 8, and p53 were not affected. These findings suggest the possible involvement of Bcl-xL in Act D-induced sensitization of AIDS-KS cells to Apo-2L-mediated apoptosis. Furthermore, Act D did not sensitize PBMC or fibroblast cells to Apo-2L. Thus, Apo-2L and Act D used in combination may be of therapeutic value in the treatment of AIDS-KS.     

Haemopoietic cell growth factor mediates cell survival via its action on glucose transport

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC-P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC-P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2-deoxyglucose by FDC-P2 cells is stimulated by HCGF in a dose-dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L-glucose uptake by FDC-P cells. These results suggest that HCGF acts to maintain FDC-P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.     

Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium.

A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.     

Binase possesses a selective cytotoxic action on kit-transformed precursors of myeloid cells

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.