It has been shown that cytochrome c is released from mitochondria during apoptosis, activates pro-caspase CPP32 (caspase III), and induces DNA fragmentation in mixtures of cytosolic extracts and isolated nuclei. To establish whether cytochrome c can primarily induce apoptosis in intact cells, we used direct electroporation of cytochrome c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of micromolar external concentrations of cytochrome c rapidly induced apoptosis (2 to 4 hours) that was concentration-dependent, did not affect mitochondrial transmembrane potential, and was independent of cell growth. Only certain isoforms of cytochrome c were apoptogenic; yeast cytochrome c and other redox proteins were inactive. Cytochrome c-induced apoptosis was dependent on heme attachment to the apo-enzyme and was completely abolished by caspase inhibitors. Nonapoptogenic isoforms of cytochrome c did not compete for apoptogenic cytochrome c. Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2 overexpression and expression of an activated MAP-kinase-kinase (MAP-KK), cytochrome c induced apoptosis in the presence of IL-3 signaling, bcl-2 over-expression, expression of activated MAP-KK, and the combined antiapoptotic action of all three. Cytochrome c also induced apoptosis in the leukemic cell line WEHI 3b. However, human HL60 and CEM cells were resistant to cytochrome c-induced apoptosis. HL60 cells did not electroporate, but CEM cells were efficiently electroporated. Our studies with IL-3-dependent cells confirm that the apoptogenic attributes of cytochrome c are identical in intact cells to those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of bcl-2 and mitochondria, but that other cells are resistant to its apoptogenic activity. The system described offers a novel, simple approach for investigating regulation of apoptosis by cytochrome c and provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.

Animals↗

PubMed1997

Energy metabolism during apoptosis. Bcl-2 promotes survival in hematopoietic cells induced to apoptose by growth factor withdrawal by stabilizing a form of metabolic arrest.

We have investigated cell metabolism during apoptosis in the murine interleukin-3 (IL-3)-dependent cell line Bo and two derivative clones (B14 and B15) overexpressing human bcl-2a. On removal of IL-3, Bo cells underwent apoptosis within 8 h, whereas B14 and B15 cells were resistant for at least 24 h. Metabolically, Bo, B14, and B15 cells were indistinguishable from each other. All were insensitive to mitochondrial poisons, derived ATP entirely by glycolysis, and maintained similar mitochondrial membrane potentials measured by rhodamine-123 fluorescence with or without IL-3. All virtually ceased glycolysis and production of lactic acid on IL-3 withdrawal but maintained intracellular [ATP] until in Bo cultures the cells began to apoptose. B14 and B15 cells became glycolytically arrested but maintained stable ATP levels during protection from apoptosis. Depletion of intracellular ATP by uncoupling the mitochondrial ATPase with 2,4-dinitrophenol or carbonyl cyanide p-trifluoromethoxyphenylhydrazone induced apoptosis in Bo cells with or without IL-3, but not in B14 or B15 cells. bcl-2-overexpressing cells were recoverable with high plating efficiency even after prolonged exposure to 2,4-dinitrophenol. We conclude that IL-3 withdrawal leads to arrest of energy metabolism in which ATP levels are maintained. In Bo cells this is followed by apoptosis, whereas in bcl-2-overexpressing cells this state is stably prolonged. ATP depletion is a strong apoptotic signal which overrides IL-3 signaling in normal cells but is ineffective in bcl-2-overexpressing cells. Prolonged metabolic arrest and resistance to ATP depletion facilitated by bcl-2 are both reversible. Persistent reversible metabolic dormancy would provide cells with a survival advantage in nonsustainable environments (e.g. hypoxia or substrate lack) and suggests a mechanism for the survival advantage displayed by cells overexpressing bcl-2.

Animals↗

PubMed1997

Redox regulation of apoptosis in interleukin-3-dependent haemopoietic cells: absence of alteration in both mitochondrial membrane potential (delta psi(m)) and free radical production during apoptosis induced by IL3 withdrawal.

Apoptosis in haemopoietic cells has been linked to the production of free radicals and reduction in mitochondrial transmembrane potential, delta psi(m). We have found that apoptosis in murine IL3-dependent haemopoietic cells induced by IL3 withdrawal lowers a principal source of anti-oxidant (reduced glutathione) but does not increase free radicals nor affect delta psi(m). Increased free radicals are, however, produced during IL3 signalling. Artificially increasing intracellular free radicals by depleting internal glutathione mimics IL3 signalling in that cells maintain shape and are transiently stimulated into S-phase in the absence of IL3. Conversely, depletion of free radicals by anti-oxidants accelerates apoptosis both in the presence and absence of IL3. Our studies suggest that redox conditions rather than free radicals or mitochondrial membrane potential per se may play a significant role in maintaining cell and replicative integrity, and in determining apoptosis in haemopoietic cells.

Apoptosis↗

PubMed1997

Concordant expression of class II and class III CD34 epitopes on haemopoietic cells in leukapheresis and cord blood samples with CD45/CD34 dual staining.

We used flow cytometry with CD45/CD34 dual antibody labelling to investigate the relative expression of class II and class III CD34 epitopes on haemopoietic progenitor cells (HPC) in 22 leukapheresis and cord blood samples. There was a close correlation between class II (QBEnd-10) and class III (clone 581) CD34+ cells (R2= 0.975, ratio of class III to class II CD34+ count 1.24 +/- 0.04). The linear relationship between class II and III CD34 epitopes on HPC suggests that the choice of antibody class is not a source of random variation in the quality assurance of CD34+ cell enumeration.

Antibodies, Monoclonal↗

PubMed1992

Haematopoietic stem cell lines activate novel enhancer-dependent expression of reporter DNA immediately after transfection by mechanisms involving interleukin 3 and protein kinase C.

Approaches to analysing gene regulation in haematopoietic stem cells are limited by their low concentration and rapid cell death outside of a trophic marrow environment. We have used interleukin 3 (IL3)-dependent cell lines as stem-cell models to investigate gene regulation during signal transduction by growth factors. We report that expression of the bacterial chloramphenicol acetyl transferase reporter gene linked via the weak thymidine kinase promoter to known upstream enhancer regions required for expression of the proliferation-dependent proto-oncogene c-fos occurs almost immediately (within 2 h) after transfection. Expression is stimulated by IL3 or activation of protein kinase C. Our findings indicate that IL3-dependent cell lines possess an extremely rapid transcription mechanism for introduced DNA, which if also present in normal cells may be usefully used to analyse gene regulation during signal transduction leading to growth and differentiation by haematopoietic growth factors.

Alkaloids↗

PubMed1990

Lymphocytes, lymphokines, and hematopoiesis.

It may be conclusively said that lymphocytes (T cells) produce an array of very powerful hematopoietic peptides, at least one of which (IL-3) appears to be exclusive. All are able to induce proliferation of stem cells of varying commitment, and in combination can reproduce virtually the entire sequence of hematopoietic cell production from the earliest stem cell, for example the HPP stem cells, which responds to T lymphokines in vitro or IL-3 in vivo, to a fully differentiated myeloid cell. Many T lymphokines can be demonstrated to exert highly significant changes in both hematopoiesis and lymphopoiesis in vivo. Thus, IL-3 suppresses NK development, thought to be partly responsible for hybrid resistance, as well as augmenting Ig responses [perhaps through an action on pre-B cells] and stimulating hematopoiesis. The effect on hematopoiesis is probably direct, because infused IL-3 is readily detectable when bound to the surfaces of cells both in the marrow and spleen. It is therefore somewhat surprising that the regulatory role of T cells themselves is less well defined. This arises variously from the availability of purified or recombinant products that replace crude T cell supernates, the complexity of analyzing a multicell system in which very many growth factors cannot be controlled for experimentally, and a certain lag in understanding the basic biology of T cells. This last is perhaps due to the historical recognition of the T cell as an immunocyte, which had led it to be principally investigated in an immunological context. The advent of molecular genetics has resulted in a reappraisal of the interrelationships between many cells, and growth-factor technology has been led to the realization that immune reactivity is perhaps only one function of the T-cell system, and that the classic concept of "immune surveillance" is only a part of a perhaps much wider "surveillance" system in which peptide growth factors and T cells play an exceedingly important role. A pertinent observation concerns abrogation of renal allograft rejection in rats by preoperation injection of donor blood. Although the allografts survive well, the graft is infiltrated by allospecific and nonspecific cytotoxic T cells and expresses donor class I and II antigens in a manner equal to that of a rejecting graft. What, therefore, is the role of lymphokines in this situation--Which of them are expressed, do they activate target cells, and what is the network that controls the macrophages and dendritic cells involved in allograft rejection?(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Colony-stimulating factors: future prospects.

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Animals↗

PubMed1989

Novel characteristics of a 33KDa protein (pp33) rapidly phosphorylated in IL3 dependent cells by stimulation with IL3.

We report the novel properties of a 33 KDa cellular protein rapidly phosphorylated by stimulation of growth by IL3 in IL3 dependent lines. Although pp33 is readily soluble in SDS, SDS-solubilised pp33 is insoluble in non-ionic detergents and is excluded from electrophoretic analysis (IEF, NEPHGE) employing such detergents. Native pp33 is not extracted by non-ionic detergents with or without cation chelation. pp33 is concentrated in a cell fraction containing endoplasmic reticulum where it is associated with a specific trypsin-sensitive degredative enzyme, active at 4 degrees. Its unusual characteristics and kinetics of phosphorylation suggest pp33 may be a novel molecule, explain its absence in studies elsewhere where non-ionic detergent extraction has been exclusively used and suggest it is intimately related to the signal transduced by IL3.

Amino Acids↗

PubMed1988

Rapid phosphorylation of a specific 33-kDa protein (p33) associated with growth stimulated by murine and rat IL3 in different IL3-dependent cell lines, and its constitutive expression in a malignant independent clone.

Protein phosphorylation mediated by murine IL3 and other factors has been studied in two different IL3-dependent lines, AC2 and 123. In both lines, responses to rat recombinant IL3 are enhanced or induced by growth in rat spleen lymphocyte conditioned medium. Growth stimulation by murine and rat IL3, by rat lymphokine(s), and by ATP in ATP-responsive cells is closely associated with the rapid (2-4 min) phosphorylation of a 33-kDa protein (p33) in all the cells examined. p33 phosphorylation is not stimulated by another lymphokine, IL4, nor by TPA or calcium ionophore alone, which are unable to stimulate growth by themselves, and is independent of serum. p33 phosphorylation is inhibited by trifluoperazine, an inhibitor of calcium-calmodulin, but is less sensitive to inhibition by H7, an inhibitor of protein kinase c, in AC2 cells. A spontaneous IL3-independent clone of AC2 (AC-) has been isolated. AC- cells are aggressively leukemic, do not produce detectable IL3, but phosphorylate p33 constitutively where it is associated with a particulate cell fraction. It is suggested that p33 is a common intermediate molecule involved in signal transduction by the various ligands which result in growth stimulation and that its constitutive phosphorylation may play a key role in the maintenance of the leukemic state.

Adenosine Triphosphate↗

PubMed1988

Cell stimulation by interleukin 3 is a novel process of signal transduction: the rolling cell cycle and its implications for leukemic transformation.

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Cell Cycle↗

PubMed1987

Cell stimulation by haemopoietic lymphokines.

Haemopoietic cells appear to respond to two distinct classes of growth factors, cell-bound molecules and lymphokines. Stimulation by lymphokines can be modelled by IL-3-dependent cell lines, and evidence is presented that such stimulation may represent a novel system of cell signalling unrelated to those where stimulation is a progression from a resting cell state into DNA synthesis and mitosis. The concept of a "rolling" cycle is introduced, and discussed in relationship to recent results suggesting that phosphorylation of a specific 33 kDa protein may be part of the control mechanism. Based on responses to different IL-3s, it is suggested that one function of lymphokines is to modulate responses to others by mechanisms other than regulation of receptor expression.

Animals↗

PubMed1987

Relationship of factor-induced proliferation to respiratory status in marrow progenitor cells.

We have examined the respiratory status of murine and human marrow progenitor cells proliferating in response to growth factors. Proliferation of fresh marrow cells, which are almost entirely entering terminal differentiation, depends on oxidative phosphorylation as determined by oxygen uptake, lactic acid production, and effect of mitochondrial poisons which within minutes completely prevent incorporation of thymidine. However, marrow progenitors responding to growth factors in in vitro assays can proliferate in the presence of high concentrations of mitochondrial poisons, with mixed granulocyte/macrophage colonies being almost refractory to inhibition. We suggest that marrow anaerobiosis may have a physiological significance and may be of relevance to the aberrant respiration of leukemic cells which are considered to be derived from progenitor populations.

Anaerobiosis↗

PubMed1986

Kinetics of responses of IL3 dependent lines to growth factors: implications for the assay and mechanism of cell promotion.

We have investigated the promotion of two IL3 dependent cell lines by IL3 and a rat factor derived from stimulated spleen cells. Both lines respond to purified IL3, but do not respond to IL2, GM-CSF contained in lung conditioned medium nor to Human IL2. Both lines respond to a factor in rat lymphocyte conditioned media as determined by a standard assay used to detect IL3. However, 123 cell line cells continue to proliferate whereas AC2 cells decline, due to different sensitivities of the lines to rat factor. 123 cells can be re-cloned in the rat factor and exhibit enhanced responses to IL3. The changing kinetics of cell promotion during the first 48 hours question the validity of the standard assay which is widely used, and the specificity of the mechanism by which the cells are promoted.

Animals↗

PubMed1983

Studies on the in vivo production of a lymphokine activity, interleukin 3 (IL-3) elaborated by lymphocytes and a myeloid leukaemic line in vitro and the fate of IL-3 dependent cell lines.

Interleukin 3 (IL-3) is produced constitutively by WEHI-3b leukaemic cells and stimulated lymphoid cell populations in vitro. We have investigated the in vivo production of IL-3 in mice rendered leukaemic with WEHI-3b cells and mice stimulated by acute graft versus host disease (GVHD). In leukaemic mice, IL-3 was not found in serum or sonicates of 18-day spleens or bone marrow, although cells from the leukaemic organs were fully competent to elaborate IL-3 in vitro. Further, elaboration of IL-3 by WEHI cells in vitro was not affected by co-culture with normal haemopoietic cells. However, intracellular IL-3 was detected in leukaemic nodules isolated from the liver. Inhibitors specific for IL-3 were not found, although liver-cell conditioned medium and leukaemic nodule sonicates contained potent non-specific inhibitors of cell growth. At 21 days, intracellular IL-3 was also present in spleens and correlated with the presence of non-specific inhibitors. In GVHD, no evidence for IL-3 elaboration in vivo was found, nor did lymphoid populations affected by GVHD spontaneously elaborate it in vitro; however, their competence to produce it was unaffected, as IL-3 was elaborated on subsequent mitogen stimulation in vitro. We also investigated the recovery and circulation of in vitro 111Indium-labelled IL-3 dependent cells after injection in vivo and the half-life of semi-purified IL-3. Dependent cells were not recovered after injection into irradiated recipients, although the cells recirculated for at least 24 hours. Inability to recover dependent cells was explicable on general cytotoxicity which masked potential recovery. The serum half-life of injected partially purified material with IL-3 activity was short (less than 30 min). We conclude that the elaboration of IL-3 by leukaemic WEHI-3b is not an in vitro artifact and these results are discussed in relationship to other growth factors and the leukaemic state, and the origin of IL-3 dependent lines.

Animals↗