Insulin-like growth factor I (IGF-I) stimulates proliferation but also increases caspase-3 activity, Annexin-V binding, and DNA-fragmentation in human MG63 osteosarcoma cells: co-activation of pro- and anti-apoptotic pathways by IGF-I.

Insulin-like growth factor-I (IGF-I) was found to promote proliferation, cell survival, and inhibition of apoptosis. But in some instances, IGF-I was found to mildly induce apoptosis, i. e. Fas-mediated apoptosis in human MG63 osteosarcoma cells. In the present study, we intended to further investigate IGF-I dependent pathways leading either to proliferation and cell survival or to cell death. MG63 osteosarcoma cells were treated with serum free medium alone or in combination with IGF-I, a neutralizing antibody against the human IGF-I receptor (alphaIR-3) or non-immune control IgG (1) for two to six days. We investigated cell survival (cell count), proliferation (CD71-FACS), apoptosis (Annexin-V-FACS, Caspase-3 activity, PCD) and anti-apoptosis (112-Ser Bad phosphorylation), and regulation of IGF-I receptor surface expression (IGF-I receptor-FACS). We found that IGF-I treatment (48 h) stimulated cell growth and proliferation, but also mildly induced apoptosis. IGF-I activated specific apoptotic pathways (Caspase-3 activation, Annexin-V binding and DNA degradation), as well as anti-apoptotic signals (Bad phosphorylation at serine 112). alphaIR-3 blocked cell proliferation, strongly induced apoptosis, and inhibited Bad-phosphorylation. Thus, IGF-I treatment overall resulted in increased tumour cell mass, despite a detectable stimulation of apoptosis; in other words proliferation exceeded cell death. If IGF-I was first added on day 0, 2, or 4 of serum free culture, we found decreasing IGF-I specific effects on proliferation and apoptosis. In parallel, we found a down-regulation of IGF-I receptors (FACS) by serum withdrawal, which was partly reversed if IGF-I was added. Therefore receptor number might have an impact on IGF-I function in MG63 cells. In conclusion, co-activation of apoptosis and proliferation by IGF-I might result in higher cell turnover in MG63 osteosarcoma cells. Furthermore, in sarcomas or carcinomas showing clinical association to IGF-I levels and malignancy, IGF-I dependent apoptosis and proliferation could be a significant mechanism of malignant tumour growth.     

Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors.

Interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) exert their biological functions through acting on a specific receptor which consists of a ligand-specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL-3/GM-CSF-mediated signals, including c-myc induction, resulted in the abrogation of DNA synthesis, however, IL-3 still protected cells from apoptotic cell death. Conversely, a C-terminal truncated form of the GM-CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf-1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM-CSF. Consequently, cells died by apoptosis in the presence of GM-CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long-term proliferation in concert with GM-CSF. These results indicate that IL-3 and GM-CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long-term cell proliferation requires the activation of both pathways.     

Activation of metalloproteinases and their association with integrins: an auxiliary apoptotic pathway in human endothelial cells

Anchorage of cells to the extracellular matrix and integrin-mediated signals play crucial roles in cell survival. We have previously shown that during growth factor deprivation-induced apoptosis in human umbilical vein endothelial cells (HUVECs), key molecules in focal adhesions and adherens junctions are cleaved by caspases. In this study we provide evidence for a selective upregulation of cell-associated matrix metalloproteinases (MMPs). We observe a physical association of MMP2 with beta1 and alphav integrins, which increased three- to fourfold during apoptosis and is dependent upon integrin beta1 levels and activation state. Both enforced activation of beta1 integrin by a specific antibody and inhibition of MMPs protect HUVECs from apoptosis. We hypothesize that, prior to the commitment to apoptosis, 'inside-out' signals initiated by the apoptotic stimulus alter cell shape together with the activation states and/or the availability of integrins, which promote matrix-degrading activity around dying cells. This 'auxiliary' apoptotic pathway may interrupt ECM-mediated survival signaling, and thus accelerate the efficient execution of the cell death program.     

Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action

Prominent blood and tissue eosinophilia is clinically manifested in a number of inflammatory states, particularly in allergic diseases. Corticosteroids are the most effective anti-inflammatory drugs used in the treatment of eosinophilic disorders, including bronchial asthma. Their beneficial effects result, among others, from (i) the suppression of the synthesis and the effects of eosinophil survival factors, (ii) the direct induction of eosinophil apoptosis and (iii) the stimulation of their engulfment by professional phagocytic cells. Failure of steroids to propagate apoptotic signals and to promote eosinophil clearance may explain the corticoresistance observed in a proportion of asthmatic patients. Accordingly, studies on the intracellular mechanisms involved in eosinophil corticosensitivity and resistance may provide a valuable tool for identifying new and selective molecular targets to therapeutically resolve otherwise persistent eosinophilic inflammation. In this review, the intracellular cascade of events involved in corticosteroid-mediated eosinophil apoptotic death is discussed and compared to the signalling pathway governing this process in the established model of dexamethasone-induced thymocyte apoptosis.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.     

Dual role for TGF-beta1 in apoptosis

The exposure of cells to TGF-beta1 can trigger a variety of cellular responses including the inhibition of cell growth, migration, differentiation and apoptosis. TGF-beta1-regulated apoptosis is cell type and context-dependent, indeed TGF-beta1 provides signals for both cell survival or apoptosis. The molecular mechanisms underlying the role of TGF-beta1 in apoptosis remains unclear. The proteins that primarily mediate the intracellular signaling of TGF-beta1 are the members of the Smad family. Nevertheless, TGF-beta1 signaling can also cooperate with the death receptor apoptotic pathway (Fas, TNF), with the intracellular modulators of apoptosis JNK and p38 MAP kinases, Akt, NF-kappaB, and with the mitochondrial apoptotic pathway mediated by members of the Bcl-2 family. Moreover, the involvement of TGF-beta1 in the production of oxidative stress and in preventing the inflammatory processes required for the clearance of apoptotic bodies is further evidence of its integration into apoptotic pathways. The interaction and balance between different stimuli provides the basis for the pro- or anti-apoptotic output of TGF-beta1 signaling in a given cell.     

Signal antonymy: a mechanism for apoptosis induction

The unfolding of the developmental programme and the organization of multicellular organisms require that cell numbers in differentiating and differentiated tissues are regulated. This is done by two distinct processes : control of cell proliferation and differentiation to a post-mitotic stage; and control of survival in post-mitotic cells. It is argued that elimination of cells by programmed cell death (PCD), which operates in both cases, is regulated by distinct mechanisms: PCD in post-mitotic cells corresponds to 'death-by-default' of (counter apoptotic) survival signals (Raff, 1992), while apoptosis in cycling cells, or in resting cells submitted to proliferative signals, results from antonymy in signalling pathways, i.e. a situation where a cell simultaneously engages into incompatible pathways of proliferation and cell cycle arrest. Antonymy arises in cells irreversibly committed to either proliferation or arrest and responding to a contradictory signal. In turn, the irreversible commitment arises by uncoupling of signal transduction from co-ordinated pathways (as in transformed cells with constitutive expression of growth-associated genes or in terminally differentiated post-mitotic cells).     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Role of apoptosis in congenital hematologic disorders and bone marrow failure

Apoptosis, the cell's intrinsic death program, plays a critical role in the regulation of tissue homeostasis, especially in cell systems with a high turnover rate such as hematopoiesis. Imbalances between survival, proliferation and death of precursor cells or mature cells may result in accelerated loss or impaired output or uncontrolled polyclonal or monoclonal expansion and may pave the way to the development of leukemia. Congenital hematologic disorders are characterized by disturbed growth control of hematopoietic cells. In the previous years, it has become clear that deregulated apoptosis contributes or is even a key determinator of the pathophysiology of diseases such as lymphoproliferation, aplastic anemia or chronic neutropenia. Hematopoietic growth factors have been shown not only to stimulate proliferation of hematopoietic stem cells and committed precursor cells, but also to act as survival factors protecting developing precursor cells from apoptotic signals. The molecular delineation of pathways of apoptosis signaling or survival in hematopoietic cells is expected to provide tools for molecular understanding of the pathophysiology of congenital and acquired hematopoietic disorders and to identify targets for therapeutic intervention strategies.     

The novel primary response gene MyD118 and the proto-oncogenes myb, myc, and bcl-2 modulate transforming growth factor beta 1-induced apoptosis of myeloid leukemia cells.

Cell numbers are regulated by a balance among proliferation, growth arrest, and programmed cell death. A profound example of cell homeostasis, controlled throughout life, is the complex process of blood cell development, yet little is understood about the intracellular mechanisms that regulate blood cell growth arrest and programmed cell death. In this work, using transforming growth factor beta 1 (TGF beta 1)-treated M1 myeloid leukemia cells and genetically engineered M1 cell variants, the regulation of growth arrest and apoptosis was dissected. Blocking of early expression of MyD118, a novel differentiation primary response gene also shown to be a primary response gene induced by TGF beta 1, delayed TGF beta 1-induced apoptosis, demonstrating that MyD118 is a positive modulator of TGF beta 1-mediated cell death. Elevated expression of bcl-2 blocked the TGF beta 1-induced apoptotic pathway but not growth arrest induced by TGF beta 1. Deregulated expression of either c-myc or c-myb inhibited growth arrest and accelerated apoptosis, demonstrating for the first time that c-myb plays a role in regulating apoptosis. In all cases, the apoptotic response was correlated with the level of MyD118 expression. Taken together, these findings demonstrate that the primary response gene MyD118 and the c-myc, c-myb, and bcl-2 proto-oncogenes interact to modulate growth arrest and apoptosis of myeloid cells.     

Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells.

The death of hemopoietic cells on withdrawal of CSF occurs by a mechanism known as apoptosis characterized by the early degradation of chromatin into oligonucleosome-length fragments. Insulin-like growth factor I plays a pivotal role in the regulation of somatic cell growth as a mediator of growth hormone action. Animals with low levels of circulating IGF-I are more vulnerable to infections and have diminished immune responses. To analyze the possibility of a regulatory role of IGF-I on hemopoiesis and determine its mechanism of action, we have studied the effect of this growth factor on the survival and proliferation of two IL-3-dependent hemopoietic cell lines and in IL-3-responsive primary cultures of bone marrow-derived mast cells. In IL-3-depleted cultures, IGF-I prevented DNA fragmentation and apoptotic cell death. Insulin at high concentration had a weak protective action and IGF-II was inactive in suppressing apoptosis in these IL-3-dependent hemopoietic cells. Cell proliferation was also stimulated by IGF-I in the absence of other hemopoietic growth factors although it was a weak mitogen when compared with IL-3. These results indicate that circulating or locally produced IGF-I may promote survival of both the steady state hemopoietic precursor population and cytokine-producing cells and could therefore regulate hemopoiesis acting in a concerted manner with other CSF.     

Polyamine depletion induces apoptosis through mitochondria-mediated pathway

Polyamines, namely putrescine, spermidine, and spermine, are essential for cell survival and proliferation. A decrease in intracellular polyamine levels is associated with apoptosis. In this study, we used inhibitors of polyamine biosynthesis to examine the effect of polyamine depletion. A combination of inhibitors of ornithine decarboxylase, S-adenosylmethionine decarboxylase, or spermidine synthase decreased intracellular polyamine levels and induced cell death in a WEHI231 murine B cell line. These cells exhibited apoptotic features including chromatin condensation and oligonucleosomal DNA fragmentation. Addition of exogenous polyamines reversed the observed features of apoptotic cell death. Similar effects were also observed in other cell lines: a human B cell line Ramos and a human T cell line Jurkat. Depletion of polyamines induced activation of caspase-3 and disruption of the mitochondrial membrane potential (Delta psi m). Inhibition of caspase activities by an inhibitor prevented the apoptotic nuclear changes but not Delta psi m disruption induced by polyamine depletion. Overexpression of Bcl-xl, an anti-apoptotic Bcl-2 family protein, completely inhibited Delta psi m disruption, caspase activation, and cell death. These results indicate that the depletion of intracellular polyamines triggers the mitochondria-mediated pathway for apoptosis, resulting in caspase activation and apoptotic cell death.     

Polyol pathway-dependent osmotic and oxidative stresses in aldose reductase-mediated apoptosis in human lens epithelial cells: role of AOP2

Aldose reductase (AR) has been implicated as a major contributor to the pathogenesis of diabetic cataracts. AR activation generates osmotic and oxidative stresses via the polyol pathway and induces cell death signals. Antioxidant protein 2 (AOP2) protects cells from oxidative stress. We investigated the effect of AR overexpression on polyol accumulation and on hyperglycemic oxidative stress and osmotic stress, as well as the effects of these stresses on human lens epithelial cell (hLEC) survival. hLECs overexpressing the AR became apoptotic during hyperglycemia and showed elevated levels of intracellular polyols. Glutathione and AOP2 levels were significantly decreased in these cells. Interestingly, supply of AOP2 and/or the AR inhibitor fidarestat protected the cells against hyperglycemia-induced death. Overexpression of AR increased osmotic and oxidative stresses, resulting in increased apoptosis in hLECs. Because AOP2 protects hyperglycemia-induced hLEC apoptosis, this molecule may have the potential to prevent hyperglycemia-mediated complications in diabetes.     

Caspase inhibition during apoptosis causes abnormal signalling and developmental aberrations in Drosophila

Programmed cell death or apoptosis plays an important role in the development of multicellular organisms and can also be induced by various stress events. In the Drosophila wing imaginal disc there is little apoptosis in normal development but X-rays can induce high apoptotic levels, which eliminate a large fraction of the disc cells. Nevertheless, irradiated discs form adult patterns of normal size, indicating the existence of compensatory mechanisms. We have characterised the apoptotic response of the wing disc to X-rays and heat shock and also the developmental consequences of compromising apoptosis. We have used the caspase inhibitor P35 to prevent the death of apoptotic cells and found that it causes increased non-autonomous cell proliferation, invasion of compartments and persistent misexpression of the wingless (wg) and decapentaplegic (dpp) signalling genes. We propose that a feature of cells undergoing apoptosis is to activate wg and dpp, probably as part of the mechanism to compensate for cell loss. If apoptotic cells are not eliminated, they continuously emit Wg and Dpp signals, which results in developmental aberrations. We suggest that a similar process of uncoupling apoptosis initiation and cell death may occur during tumour formation in mammalian cells.     

Hypoxia-ischemia-induced apoptotic cell death correlates with IGF-I mRNA decrease in neonatal rat brain.

Hypoxia-ischemia induces apoptotic and necrotic cell death, which results partially from persistent alterations in cellular energy homeostasis. Insulin-like growth factor I (IGF-I) is an anabolic pleiotrophic factor required by developing neurons for their optimal proliferation, differentiation, and survival. To determine how cell death and changes in IGF-I gene expression relate to the extent of hypoxia-ischemia, we evaluated the time course of apoptosis in a neonatal hypoxia-ischemia model in relation to the cellular distribution of IGF-I and IGFBP5 mRNA. Severe hypoxia-ischemia results in an immediate decrease in neuronal IGF-I and IGFBP5 mRNA. The decrease in neuronal IGF-I mRNA was concurrent with an increase in the number of apoptotic cells. It is conceivable that the immediate decrease in IGF-I gene expression may contribute to the impending neuronal death and selective vulnerability of myelinogenesis during the perinatal period.     

Phosphatidylinositol 3-kinase signaling to Akt mediates survival in isolated canine islets of Langerhans

The isolation of islet cells from the pancreas by enzymatic digestion causes many of these cells to undergo apoptosis. The aim of this work was to investigate the role of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling in mediating the survival of isolated islets. Insulin-like growth factor-1 (IGF-I) was examined as a potential culture media supplement that could rescue isolated islets from their apoptotic fate. Western blot analysis demonstrated that Akt phosphorylation peaks 20 h after routine islet isolation. PI3-K inhibition with wortmannin abolished both basal and IGF-I-mediated Akt phosphorylation. IGF-I did not increase survival of isolated islets under normal conditions but it did have a protective effect against cytokine (TNF-alpha, IL-1beta, INF-gamma)-mediated cell death. The protective effect of IGF-I against cytokine-stimulated apoptosis was blocked by wortmannin. In addition, inhibition of basal levels of PI3-K activity caused a 31% decrease in islet survival, as shown by MTT assay. These results demonstrate that the PI3-K/Akt pathway mediates survival of isolated islets of Langerhans.     

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.     

Xenopus death receptor-M1 and -M2, new members of the tumor necrosis factor receptor superfamily, trigger apoptotic signaling by differential mechanisms

Signaling through the tumor necrosis factor receptor (TNFR) superfamily can lead to apoptosis or promote cell survival, proliferation, and differentiation. A subset of this family, including TNFR1 and Fas, signals cell death via an intracellular death domain and therefore is termed the death receptor (DR) family. In this study, we identified new members of the DR family, designated xDR-M1 and xDR-M2, in Xenopus laevis. The two proteins, which show high homology (71.7% identity), have characteristics of the DR family, that is, three cysteine-rich domains, a transmembrane domain, and a death domain. To elucidate how members of xDR-M subfamily regulate cell death and survival, we examined the intracellular signaling mediated by these receptors in 293T and A6 cells. Overexpression of xDR-M2 induced apoptosis and activated caspase-8, c-Jun N-terminal kinase, and nuclear factor-kappaB, although its death domain to a greater extent than did that of xDR-M1 in 293T cells. A caspase-8 inhibitor potently blocked this apoptosis induced by xDR-M2. In contrast, xDR-M1 showed a greater ability to induce apoptosis through its death domain than did xDR-M2 in A6 cells. Interestingly, a general serine protease inhibitor, but not the caspase-8 inhibitor, blocked the xDR-M1-induced apoptosis. These results imply that activation of caspase-8 or serine protease(s) may be required for the xDR-M2- or xDR-M1-induced apoptosis, respectively. Although xDR-M1 and xDR-M2 are very similar to each other, the difference in their death domains may result in diverse signaling, suggesting distinct roles of xDR-M1 and xDR-M2 in cell death or survival.     

New developments on the TNFα-mediated signalling pathways

TNFα (tumour necrosis factor α) is an extensively studied pleiotropic cytokine associated with the pathogenesis of a variety of inflammatory diseases. It elicits a wide spectrum of cellular responses which mediates and regulates inflammation, immune response, cell survival, proliferation and apoptosis. TNFα initiates its responses by binding to its receptors. TNFα-induced effector responses are mediated by the actions and interactions among the various intracellular signalling mediators in the cell. TNFα induces both survival and apoptotic signal in a TRADD (TNF receptor-associated DD)-dependent and -independent way. The signals are further transduced via a variety of signalling mediators, including caspases, MAPKs (mitogen-activated protein kinases), phospholipid mediators and miRNA/miR (microRNA), whose roles in specific functional responses is not fully understood. Elucidating the complexity and cross talks among signalling mediators involved in the TNFα-mediated responses will certainly aid in the identification of molecular targets, which can potentially lead to the development of novel therapeutics to treat TNFα-associated disorders and in dampening inflammation.