Cleavage of β-Catenin and Plakoglobin and Shedding of VE-Cadherin during Endothelial Apoptosis: Evidence for a Role for Caspases and Metalloproteinases

Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell–matrix and cell–cell contacts. In this study, we show that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. β-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding α-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved β-catenin from lysates of apoptotic cells does not bind to endogenous α-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell–cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this “shedding” of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of β-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival.

Apoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Inhibited by Association with Rb Protein

Rb protein inhibits both cell cycle progression and apoptosis. Interaction of specific cellular proteins, including E2F1, with Rb C-terminal domains mediates cell cycle regulation. In contrast, the nuclear N5 protein associates with an Rb N-terminal domain with unknown function. The N5 protein contains a region of sequence similarity to the death domain of proteins involved in apoptotic signaling. We demonstrate here that forced N5 expression potently induces apoptosis in several tumor cell lines. Mutation of conserved residues within the death domain homology compromise N5-induced apoptosis, suggesting that it is required for normal function. Endogenous N5 protein is specifically altered in apoptotic cells treated with ionizing radiation. Furthermore, dominant interfering death domain mutants compromise cellular responses to ionizing radiation. Finally, physical association with Rb protein inhibits N5-induced apoptosis. We propose that N5 protein plays a role in the regulation of apoptosis and that Rb directly coordinates cell proliferation and apoptosis by binding specific proteins involved in each process through distinct protein binding domains.

Role of Akt and c-Jun N-terminal Kinase 2 in Apoptosis Induced by Interleukin-4 Deprivation

We have shown previously that interleukin-4 (IL-4) protects TS1αβ cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1αβ cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1αβ survival is independent of Bcl-2, Bcl-x, or Bax.

T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.

Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21waf1 with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

Nitric oxide inhibits tumor necrosis factor-α-induced apoptosis by reducing the generation of ceramide

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-α (TNF-α) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-α, however, ceramide potentiated, whereas NO inhibited, TNF-α-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-α-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.

Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.

Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

Evolutionary conservation of apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from Spodoptera frugiperda Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome c pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the Drosophila cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.

The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout (DNA-PKcs−/−) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs−/− mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm−/− mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.

Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.

The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1

Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1β. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34cdc2 on the mitotic apparatus, and is phosphorylated on Thr34 by p34cdc2-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr34 resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34cdc2-cyclin B1 and suggest that survivin phosphorylation on Thr34 may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.