Apoptotic Programmed Cell Death Research Articles

Programmed cell death (PCD). Apoptosis, autophagic PCD, or others?

The occurrence of cell death as a physiological event in multicellular organisms has been known for more than 150 years; in 1972 the term apoptosis was introduced on morphological grounds. However, accumulating evidence suggests that programmed cell death (PCD) is not confined to apoptosis, but that cells use different pathways for active self-destruction as reflected by different morphology: condensation prominent, type I or apoptosis; autophagy prominent, type II; etc. Autophagic PCD appears to be a phylogenetically old phenomenon; it may occur in physiological and disease states. We have studied the relation between morphological and biochemical events during autophagic and apoptotic PCD in human mammary, lymphoblast, and colon cancer cells using electron microscopy and proteom analysis. We find that autophagic cell death (type II) PCD includes degradation of Golgi apparatus, polyribosomes, and endoplasmic reticulum, which precedes nuclear destruction. Intermediate and microfilaments are largely preserved; presumably the cytoskeleton is required for autophagocytosis. Apoptosis (type I) PCD is characterized by condensation of cytoplasm and preservation of organelles; cytoskeletal elements disintegrate in early stages. Either type of PCD involves synthesis of distinct proteins. Finally, both types of PCD share features some of a cell's stress response (e.g., translocation of hsp90). In conclusion our findings support the concept that autophagic cell death is a separate pathway of PCD distinctly different from "classical" apoptosis. However, autophagic and apoptotic PCD should not be considered as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in a cell's response to changes of environmental conditions, both physiological or pathological.

Structure/Function Analysis of p55 Tumor Necrosis Factor Receptor and Fas-associated Death Domain: EFFECT ON NECROSIS IN L929sA CELLS

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.

SPI-1 transforming properties depend upon specifically activated forms of the EPOR.

Friend erythroleukemia induced in mice by the spleen focus forming virus (SFFV) is a multi-step process. The pre-leukemic phase of the disease results from the abnormal activation of the Erythropoietin (Epo) receptor by the gp55 env gene product of SFFV. Later in disease progression, the emergence of leukemic clones is associated with recurrent genetic events, in particular the activation of the expression of SPI-1, an ETS family transcriptional regulator. We show here that the expression of either SPI-1 or GP55 with the mouse EPOR in avian primary erythroblasts only marginally affects their normal Epo-induced terminal differentiation. In contrast, the co-expression of GP55 and SPI-1 resulted in inhibition of Epo-induced differentiation of EPOR-expressing erythroblasts, promoting instead their proliferation. Co-expression of SPI-1 and GP55 also inhibited the apoptotic cell death program normally induced in response to Epo withdrawal. This cooperation between SPI-1 and GP55 to induce primary erythroblast transformation suggests that progression of Friend erythroleukemia critically depends upon inter-dependent interactions between the molecular events specific of the early and late phase of the disease.

In vivo study of motoneuron death induced by nerve injury in mice deficient in the caspase 1/ interleukin-1β-converting enzyme

The apoptotic cell death program is orchestrated by members of the caspase family. Among these caspases, several in vitro and in vivo reports indicate that the interleukin-1β-converting enzyme (or caspase 1) may be involved in neurodegenerative processes. In view of these findings, and in order to characterize the role of the interleukin-1β-converting enzyme in mediating or modulating cell death processes in vivo, we have investigated the effects of its deletion on motoneuron survival after a facial nerve transection in newborn and adult interleukin-1β-converting enzyme knock-out mice. During the postnatal period of development, when facial motoneurons are highly vulnerable to axotomy, we did not observe any significant effect of the interleukin-1β-converting enzyme-deletion on the percentage of cell death in the lesioned nuclei. In addition, the spontaneous cell death characteristic of the postnatal period was not altered in knock-out mice. In contrast, in adult knock-out mice, a significant reduction (16%) in the number of surviving facial motoneurons was observed six weeks after axotomy. We therefore conclude that the interleukin-1β-converting enzyme does not appear to be critical for cell death during the postnatal period but may favor motoneuron survival during adulthood. Given the key role of caspase 3 in neuronal apoptosis during embryonic development of the central nervous system, we also investigated the role of this caspase in cell death following axotomy. Combined immunofluorescence revealed that, at least during the postnatal period, axotomized motoneurons that have apoptotic nuclear morphologies were immunopositive for the active form of caspase 3. Double-stained cells could be also observed on the unlesioned side. These results strongly suggest that caspase 3 may be involved in both the postnatal spontaneous- and axotomy-induced facial motoneuron death processes. Similar results were obtained in interleukin-1β-converting enzyme-deficient and wild-type mice, indicating that the interleukin-1β-converting enzyme may not be required for caspase 3 activation.

Overexpression of human α-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine neurons in the substantia nigra and the progressive loss of these neurons. Recently, mutations in the α-synuclein gene have been identified in early-onset familial PD, and α-synuclein has been shown to be a major component of LB in all patients. Yet, the pathophysiological function of α-synuclein remains unknown. In this report, we have investigated the toxic effects of adenovirus-mediated α-synuclein overexpression on dopamine neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell line – the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27). Adenovirus-transduced cultures showed high-level expression of α-synuclein within the cells. Overexpression of human mutant α-synuclein (Ala 53Thr) selectively induced apoptotic programmed cell death of primary dopamine neurons as well as N27 cells. The mutant protein also potentiated the neurotoxicity of 6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human α-synuclein was not directly neurotoxic but did increase cell death after 6-OHDA. Overexpression of wild-type rat α-synuclein had no effect on dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that overexpression of human mutant α-synuclein directly leads to dopamine neuron death, and overexpression of either human mutant or human wild-type α-synuclein renders dopamine neurons more vulnerable to neurotoxic insults.

Impairment of rat thymocyte differentiation and functions by neonatal capsaicin treatment is associated with induction of apoptosis

The direct and indirect interaction between the nervous system and its transmitters with the immune system was evaluated in the rat by using the neurotoxin capsaicin (Caps). In the present study we investigated the effect of Caps administration to neonatal rats on thymocyte subpopulation distribution and functions at different times after treatment. Caps treatment results in a marked reduction of thymus weight and cellularity. As shown by immunofluorescence and FACS analysis, profound depletion of double negative (DN), double positive (DP), and single positive (SP) CD4 + cells was already evident at day 7 after treatment and persisted until day 28. Reduced numbers of SP CD8 + cells were observed only at later time points. Analysis of TCR phenotype indicates that CD5 + TCR γ/δ + are particularly sensitive to neonatal Caps treatment. Caps-induced thymocyte depletion was associated with reduced proliferation in response to T cell mitogens. Moreover, in situ TUNEL reaction and agarose gel electrophoresis indicate that neonatal Caps treatment induces apoptosis of thymus cells. Morphological analysis reveals the presence of apoptotic cells in the subcapsular thymus cortical region. Overall our results suggest that Caps when administered at birth, profoundly affects T cell differentiation, likely through its ability to activate apoptotic cell death program.

C-Jun and Bax: regulators of programmed cell death in developing neurons.

Introduction Apoptotic programmed cell death occurs extensively during the development of the mammalian nervous system and approx. 50% of the neurons formed by neurogenesis die during embryonic and early postnatal development. This death is required for the formation of appropriate connections between neurons and their targets and for morphogenetic processes such as neural tube closure [1,2]. In addition, there is increasing evidence that cell death by apoptosis may contribute to the pathology of human neurodegenerative disease [3]. Sympathetic neurons have proved to be a particularly useful model system for in vitro studies of the molecular mechanisms of neuronal apoptosis. These cells normally die by apoptosis between days 3 and 7 of postnatal development in the rat and require nerve growth factor (NGF) for survival at that time [4]. When deprived of N G F in culture, sympathetic neurons undergo a form of cell death that has the classic features of apoptosis: rates of RNA and protein synthesis decrease, the cytoplasm and nucleus shrink, mitochondria release cytochrome c into the cytoplasm, caspases are activated and required for cell death, chromatin condenses and the DNA is degraded into oligonucleosomal fragments, phosphatidylserine redistributes from the inner to the outer surface of the plasma membrane, neurites fragment and the cells ultimately detach from their substrate [S-131. Interestingly, NGF-withdrawal-induced death is blocked by the inhibition of transcription or translation, suggesting that new gene expression and protein synthesis may be required for cell death to occur [S]. The goal of our research has been (1) to identify pro-apoptotic genes that are induced in sympathetic neurons after the withdrawal of NGF, (2) to understand

Implication of calpain in caspase activation during B cell clonal deletion.

In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI-231 B cell lymphoma model, anti-IgM crosslinking triggers activation of caspase-7 independently of caspase-8, followed by apoptosis. Two main mechanisms for caspase-7 activation have been proposed: (i) caspase-8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase-9. Here we report that caspase-7 activation induced by BCR crosslinking is independent of caspase-8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell-free system, the S-100 fraction of anti-IgM-treated WEHI-231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase-7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation.

Requirement for protein kinase C in reactive oxygen species-induced apoptosis of vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) apoptosis is a component of a variety of cardiovascular diseases and may be related to reactive oxygen species (ROS). This study was designed to determine the role of protein kinase C (PKC) in ROS-induced VSMC apoptosis. Rat aortic VSMCs were exposed to H(2)O(2), and the nature of cell death was characterized in the absence or presence of different PKC inhibitors. The results demonstrate that exposure of VSMCs to H(2)O(2) led to a dose-dependent (25 to 100 micromol/L) and time-dependent (peak at 2 minutes) activation of PKC. Among the PKC isoforms alpha, beta, delta, epsilon, and zeta, only PKC-alpha and PKC-epsilon were found to change their intracellular distribution on H(2)O(2) treatment. Apoptosis was the predominant form of cell death when PKC had been activated by H(2)O(2) alone or by H(2)O(2) in the presence of 50 nmol/L phorbol 12-myristate 13-acetate. In contrast, necrosis became the predominant form of cell death when PKC had been downregulated by prolonged exposure to 200 nmol/L phorbol 12,13-dibutyrate or inhibited by 50 nmol/L staurosporine, 100 nmol/L calphostin C, or 30 micromol/L H-7. In addition, caspase-3 was activated in H(2)O(2)-induced VSMC apoptosis but not when PKC was downregulated or inhibited. Inhibition of caspase-3 by 50 micromol/L Ac-DEVD-CHO could significantly attenuate H(2)O(2)-induced apoptosis and was not associated with the induction of necrosis. We conclude that in VSMCs, PKC converts the ROS-induced signals from necrotic cell death to the activation of an apoptotic cell death program. These data imply a novel and important role of PKC for the pathogenesis of such vascular diseases as atherosclerosis, restenosis, and hypertension.

Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury.

Traumatic spinal cord injury often results in complete loss of voluntary motor and sensory function below the site of injury. The long-term neurological deficits after spinal cord trauma may be due in part to widespread apoptosis of neurons and oligodendroglia in regions distant from and relatively unaffected by the initial injury. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death program. Caspase-3 cleaves several essential downstream substrates involved in the expression of the apoptotic phenotype in vitro, including gelsolin, PAK2, fodrin, nuclear lamins and the inhibitory subunit of DNA fragmentation factor. Caspase-3 activation in vitro can be triggered by upstream events, leading to the release of cytochrome c from the mitochondria and the subsequent transactivation of procaspase-9 by Apaf-1. We report here that these upstream and downstream components of the caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in oligodendroglia adjacent to and distant from the injury site. Given these findings, targeting the upstream events of the caspase-3 cascade has therapeutic potential in the treatment of acute traumatic injury to the spinal cord.

Protein kinase C targeting in antineoplastic treatment strategies.

Neoplastic cell survival is governed by a balance between pro-apoptotic and anti-apoptotic signals. Noteworthy among several anti-apoptotic signaling elements is the protein kinase C (PKC) isoenzyme family, which mediates a central cytoprotective effect in the regulation of cell survival. Activation of PKC, and subsequent recruitment of numerous downstream elements such as the mitogen-activated protein kinase (MAPK) cascade, opposes initiation of the apoptotic cell death program by diverse cytotoxic stimuli. The understanding that the lethal actions of numerous antineoplastic agents are, in many instances, antagonized by cytoprotective signaling systems has been an important stimulus for the development of novel antineoplastic strategies. In this regard, inhibition of PKC, which has been shown to initiate apoptosis in a variety of malignant cell types, has recently been the focus of intense interest. Furthermore, there is accumulating evidence that selective targeting of PKC may prove useful in improving the therapeutic efficacy of established antineoplastic agents. Such chemosensitizing strategies can involve either (a) direct inhibition of PKC (e.g., following acute treatment with relatively specific inhibitors such as the synthetic sphingoid base analog safingol, or the novel staurosporine derivatives UCN-01 and CGP-41251) or (b) down-regulation (e.g., following chronic treatment with the non-tumor-promoting PKC activator bryostatin 1). In preclinical model systems, suppression of the cytoprotective function(s) of PKC potentiates the activity of cytotoxic agents (e.g., cytarabine) as well as ionizing radiation, and efforts to translate these findings into the clinical arena in humans are currently underway. Although the PKC-driven cytoprotective signaling systems affected by these treatments have not been definitively characterized, interference with PKC activity has been associated with loss of the mitogen-activated protein kinase (MAPK) response. Accordingly, recent pre-clinical studies have demonstrated that pharmacological disruption of the primary MEK-ERK module can mimic the chemopotentiating and radiopotentiating actions of PKC inhibition and/or down-regulation.

Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.

The importance of free radical homeostasis and apoptosis in normal and diseased hearts and their interrelationships are poorly defined. We tested whether reactive oxygen species can trigger apoptosis in cardiomyocytes, and we explored the underlying pathways. A cell culture model of isolated cardiac cells and different reactive oxygen species (ROS)-generating systems were used. Apoptosis became evident when cardiomyocytes were exposed to either H2O2 or superoxide anion (O2-). Both H2O2- and O2--induced apoptosis of cardiomyocytes were associated with an increase in p53 protein content, whereas protein levels of Bax and Bcl-2 were unaltered. H2O2, but not O2-, induced an increase in the protein content of Bad. Furthermore, H2O2 elicited translocation of Bax and Bad from cytosol to mitochondria, where these factors formed heterodimers with Bcl-2, which was followed by the release of cytochrome c, activation of CPP32, and cleavage of poly(ADP-ribose) polymerase. Interestingly, this pathway was not activated by O2-. Instead, O2- used Mch2alpha to promote the apoptotic pathway, as revealed by the activation of Mch2alpha and the cleavage of its substrate, lamin A. Taken together, these results indicate that ROS may play an important pathophysiological role in cardiac diseases characterized by apoptotic cell death and suggest that different ROS-induced activations of the apoptotic cell death program in cardiomyocytes involve distinct signaling pathways.

The role of Fas and related death receptors in autoimmune and other disease states

The Fas receptor, also known as APO-1 or CD95, has emerged as a key initiator of apoptotic programmed cell death in a variety of cell types. CD4 + T cells are unique in their ability to commit “suicide” by stimulating their own Fas receptors with secreted or membrane-bound Fas ligand. This takes place in the setting of repeated stimulation with T-cell antigens and is thought to be a mechanism for controlling the expansion of T cells during viral infections and autoimmune disease states. T cells can also trigger apoptosis in B cells, macrophages, and other cell types through Fas ligand. These interactions negatively regulate the immune system but can also contribute to immunopathology, as occurs in Fas-mediated damage of target tissues in hepatitis and other organ-specific autoimmune diseases. The dual role of Fas in the immune response complicates the understanding of its role in disease states and may limit its potential as a therapeutic target. Despite the many roles of Fas in immunoregulation, findings in experimental mouse strains and human patients with genetic deficiencies in the Fas pathway have shown that the main result of disrupting this pathway in vivo is systemic autoimmunity and a predisposition toward lymphoid malignancies. The role of Fas in various cell types and the lessons we have learned from Fas-deficient patients with the autoimmune lymphoproliferative syndrome will be discussed. (J Allergy Clin Immunol 1999;103:729-38.)

Apoptosis: Mechanisms of life and death in the immune system

When normal cells die, it is almost always by the process of apoptosis, which is a morphologic description. Some cells die readily and are rapidly replaced; others live as long as we do. Because apoptotic morphology is similar from cell to cell, researchers have embarked on an intense search for a common underlying biochemical process. There is an apoptotic mechanism or cell death program, which is expressed in embryos and throughout life. Most tissues, and especially the skin, gut, and immune system, depend on well-ordered apoptosis and cell replacement. Apoptosis can fail to occur, as in the newly described autoimmune lymphoproliferative syndrome, or be turned on pathologically, as in degenerative diseases. We would like to induce apoptosis in dangerous cells or block it to preserve valuable cells. We need to know more about how apoptosis is regulated and what we can do to influence the process. (J Allergy Clin Immunol 1999;103:548-54.)

Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells.

Physiological levels of laminar shear stress completely abrogate apoptosis of human endothelial cells in response to a variety of stimuli and might therefore importantly contribute to endothelial integrity. We show here that the apoptosis-suppressive effects of shear stress are mediated by upregulation of Cu/Zn SOD and NO synthase. Shear stress-mediated inhibition of endothelial cell apoptosis in response to exogenous oxygen radicals, oxidized LDL, and tumor necrosis factor-alpha was associated with complete inhibition of caspase-3-like activity, the central effector arm executing the apoptotic cell death program in endothelial cells. Shear stress-dependent upregulation of Cu/Zn SOD and NO synthase blocks activation of the caspase cascade in response to apoptosis-inducing stimuli. These findings establish the upregulation of Cu/Zn SOD and NO synthase by shear stress as a central protective cellular mechanism to preserve the integrity of the endothelium after proapoptotic stimulation.

Bcl-2 Transcription from the Proximal P2 Promoter Is Activated in Neuronal Cells by the Brn-3a POU Family Transcription Factor

The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.

Apoptosis in cellular compartments of rat spinal cord after severe contusion injury.

Following a controlled, severe contusion lesion to the lower thoracic spinal cord in adult rats, we found that apoptosis occurred in cells located in both gray and white matter. This suggested that both nonneuronal cells, including astrocytes, oligodendroglia and microglia, as well as neurons, might participate in programmed cell death (PCD) following spinal cord injury (SCI). Determination of which cell populations participate, and the kinetics and extent of their involvement might reveal new paradigms for approaches to therapy. Consequently, we assessed the functional deficit, comparing a comprehensive locomotor rating scale (LRS) with the inclined plane test at various times after injury. Using standard histology, along with cell-specific markers, we assessed PCD in different spinal cord segments using several parameters of apoptosis. Our results indicate that hind limb motor function was lost at day 1, and then only gradually and ineffectively (about 10-15%) recovered over the next month. Evidence for increased cell number was present for astrocytes and microglia beginning at day 1 after injury. Over the postinjury time period, apoptotic cells appeared (from day 1 to 14), and peaked (in terms of apoptotic index) on day 3. About one-third were microglia, whereas neurons, both large and small, also underwent apoptosis, again peaking at day 3. However, neurons continued to die and were not replaced by proliferation, so that at day 7, three times as many neurons (as a percentage) underwent PCD compared with the glial compartment. Oligodendrocytes also underwent apoptosis, with a biphasic curve, both at days 3 and 14 following injury. Thus, in addition to immediate, passive necrosis, delayed and apoptotic PCD also occurred in all cell populations in severely injured spinal cord.

Cell death in the avian blastoderm: resistance to stress-induced apoptosis and expression of anti-apoptotic genes.

We investigated the expression of an apoptotic cell death program in blastodermal cells prior to gastrulation and the susceptibility of these cells to stress-induced cell death. A low frequency (3.1%) of apoptotic blastodermal cells was observed in Hoechst 33342-vitally stained cytological preparations of complete blastoderms from unincubated eggs. These cells showed the stereotypic features of apoptosis including a progression of nuclear changes, cell shrinkage and blebbing, and the formation of apoptotic bodies. Prolonged storage of eggs at 12 degrees C induced apoptosis in blastodermal cells (14%). A modest amount of apoptosis (10%) was also induced at the heat shock temperature of 48 degrees C, but not at 45 degrees C. Etoposide and other potent cytotoxic drugs failed to induce apoptosis in the blastodermal cells after 4 h of exposure. Progressively more apoptosis was induced at 8 and 24 h, but it did not exceed 35% of the cells. We detected transcripts for the anti-apoptotic genes bcl-2, bcl-xL, and hsp70. The developmental expression of these genes, especially hsp70, correlated with the delayed and limited stress-induction of apoptosis. These studies reveal the capacity of pre-streak blastodermal cells to engage in apoptosis and their relative resistance to stress conditions. This may be due to the prominent expression of hsp70 and/or multiple cell death genes which primarily antagonize cell death.

Requirement for DCP-1 caspase during Drosophila oogenesis.

Caspases, a class of cysteine proteases, are an essential component of the apoptotic cell death program. During Drosophila oogenesis, nurse cells transfer their cytoplasmic contents to developing oocytes and then die. Loss of function for the dcp-1 gene, which encodes a caspase, caused female sterility by inhibiting this transfer. dcp-1- nurse cells were defective in the cytoskeletal reorganization and nuclear breakdown that normally accompany this process. The dcp-1- phenotype suggests that the cytoskeletal and nuclear events in the nurse cells make use of the machinery normally associated with apoptosis and that apoptosis of the nurse cells is a necessary event for oocyte development.

NGF exhibits a pro-apoptotic activity for human vascular smooth muscle cells that is inhibited by TGFβ1

Apoptosis of vascular smooth muscle cells (SMCs) has been described in culture and also during remodelling of the artery following injury. However, the mediators that regulate apoptosis in SMCs are unknown. Because neurotrophins, a family of related polypeptide growth factors, including nerve growth factor (NGF) and its cognate receptor TrkA have been shown to be strongly expressed in atherosclerotic lesions, the present study was undertaken to evaluate in vitro, the activity of NGF with regard to apoptosis of confluent cultures of human aortic SMCs. We report here that NGF induced apoptosis of SMCs in a dose-dependent manner. This effect was detected from the concentration of 1 ng/ml and reached a maximum at 100 ng/ml. The concentration that induced a half-maximum effect was 8.8 ng/ml. The pro-apoptotic activity of NGF was time dependent and was significant after 3 h of incubation. The pro-apoptotic activity of NGF was blocked in a dose-dependent manner by K-252a, an inhibitor of TrkA tyrosine phosphorylation, suggesting that a NGF/TrkA signal transduction pathway could activate apoptotic cell death programs in human SMCs. Significantly, NGF-induced apoptosis was inhibited by wortmannin and PD 98059, showing that both PI3 kinase and MEK kinase were involved. At a NGF concentration that strongly induced apoptosis (100 ng/ml), TGFβ1 which has been identified several times as a protective factor, dose dependently inhibited the pro-apoptotic effect of NGF. The IC50 value was 1.5 ng/ml. These results indicate that, at least in vitro, TGFβ1 can inhibit the pro-apoptotic activity of NGF for SMCs therefore suggesting that TGFβ1 has the capacity to diminish the deleterious consequences of an excitotoxic or ischemic injury that might occur during atherogenesis or following angioplasty.