Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

Programmed cell death 50 (and beyond).

In the 50 years since we described cell death as 'programmed,' we have come far, thanks to the efforts of many brilliant researchers, and we now understand the mechanics, the biochemistry, and the genetics of many of the ways in which cells can die. This knowledge gives us the resources to alter the fates of many cells. However, not all cells respond similarly to the same stimulus, in either sensitivity to the stimulus or timing of the response. Cells prevented from dying through one pathway may survive, survive in a crippled state, or die following a different pathway. To fully capitalize on our knowledge of cell death, we need to understand much more about how cells are targeted to die and what aspects of the history, metabolism, or resources available to individual cells determine how each cell reaches and crosses the threshold at which it commits to death.

Programmed cell death and apoptosis--where it came from and where it is going: from Elie Metchnikoff to the control of caspases.

The story of cell death began with the origins of cell biology, including important observations by Elie (Ilya) Metchnikoff, who realized that phagocytes engulfed dying cells. Most of the early studies were observational. By the middle of the 20th C, researchers were beginning to explore how cells died, had recognized that cell death was a physiologically controlled process, that the most common mode of death ("shrinkage necrosis", later apoptosis) was tightly controlled, and were speculating whether lysosomes were "suicide bags". Just prior to 1990 several discoveries led to rapid expansion of interest in the field and elucidation of the mechanisms of apoptosis. Closer to the beginning of the 21st C comprehensive analysis of the molecules that controlled and effected apoptosis led to the conclusion that autophagic processes were linked to apoptosis and could serve to limit or increase cell death. Today, realizing that knowledge of the components of cell death has not yet produced pharmaceuticals of therapeutic value, research is turning to questions of what metabolic or other mechanisms indirectly control the activation or suppression of the cell death positive feedback loop. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later"

Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos.

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.

Programmed cell death and apoptosis: origins of the theory.

Interest in the study of apoptosis grew with the recognition that it is a highly regulated process. Such a change in attitude allowed the intellectual and technical breakthroughs that led to the explosive development of this subject.

Cell death in the heart.

The role of apoptosis in cardiac disease remains controversial. Much of the apoptosis detected, by chemical or molecular means, reflects inflammatory reaction and responding blood cells rather than myocytes, though their apoptosis in situ may exacerbate a bad situation, and their direct action against myocytes has not been excluded definitely. Myocyte apoptosis may reflect end-stage cardiac failure rather than causing it. If this is the case, then preventing apoptosis so that the cells can undergo necrosis does not accomplish much. Apoptosis is a consistent and important finding in many forms of cardiovascular disease. As determined by ultra-structure, apoptosis is common in cardiomyocytes, fibroblasts, vascular endothelial cells, and smooth muscle cells in cardiovascular disease of many origins. (62) Even though smooth muscle cells in atheromatous plaques appear to be necrotic,l it is likely that this is an evolved situation of apoptotic cells that were not removed. Given the prevalence of apoptotic processes in diseased heart and the very limited capacity of this organ to repair itself, (56) it is appropriate and justified to continue to explore the significance of apoptosis in cardiac disease and, above all, to explore the use of antiapoptotic agents in acute situations. Researchers must pay explicit attention to how they document cell death and in what tissues or cells it occurs. Otherwise, clinicians risk being deluded by preservation of morphology in nonfunctional cells and by confusion of what happened and where death occurred in the sequence of causality. Cell death in the heart is a matter of substantial theoretical and practical concern. A major problem in analyzing it is that, although apoptosis may be demonstrated easily in myocytes, particularly embryonic myocytes, under conditions of culture, interpretation is much more complex in an intact organ. The first issue is one of timing. In situations of severe, acute loss of cells, such as in an infarct, apoptotic cells may not be cleared rapidly and may progress to a more oncotic or necrotic morphology. Second, in situations of inflammation, biochemical or molecular techniques may confound apoptosis of inflammatory cells with apoptosis of myocytes. Third, priorities in the sequence of apoptosis differ between large, generally nonmitotic cells with massive cytoplasm (as differentiated myocytes) and small mitotic cells in culture, which usually are studied. The appearance and many markers of physiological cell death may differ from the most widely recognized forms of apoptosis, including late collapse of the nucleus and primacy of lysosomal or other proteases as opposed to caspases. Investigators should always strive to establish multiple criteria for apoptosis, with good documentation of timing and cell type. When these factors are taken into consideration, it seems that aggressive action against apoptosis may be of value in acute situations, such as infarct, in which buying short increments of time may reduce damage. In more chronic situations, much of the apoptosis detected derives from invading lymphocytes, mast cells, or other cells relating to inflammation. The apoptosis of these cells may exacerbate an already difficult situation, and intervention may prove of value. Otherwise, apoptosis of myocytes is more typically an end-stage situation, and it is more fruitful to alleviate the problem before this stage is reached.

Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation.

UVB irradiation can induce apoptotic, necrotic, and differentiation pathways in normal human keratinocytes. The present study was undertaken to determine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fragmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low doses of UVB, 5-20 mJ/cm2, increase the levels of apoptosis as shown by increased levels of fragmented DNA, Fas, PARP, and FasL protein, and the number of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm2) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell viability is decreased as measured by DNA synthesis (BrdU labeling) neutral red uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and keratin K10, are also observed to decrease with increasing UVB dose. These changes are accompanied by a further increase in DNA fragmentation. We conclude that low doses of UVB (5-20 mJ/cm2) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm2) of UVB produce a direct necrotic effect and inhibit terminal differentiation.

Gender differences: the perspective from biology.

The remarkable thing about sexual differentiation is its diversity. That males are the heterogametic sex, larger than females, more aggressive than females, and the 'non-default' mode of sexual differentiation are concepts not valid throughout most of the animal kingdom. Sex chromosomes are characteristic only of land animals. In birds, the heterogametic sex is female and the sex chromosomes are not related to those of mammals. External factors such as temperature determine sex in lower vertebrates, and there is no similarity among sex-determining genes of different species. The somatic origin of the sex-determining genes and sex chromosomes forces us to ask: what are the other functions of these genes? Because of the obviousness of the sex chromosomes and hormones we may have focused too little on the somatic effects of sex.

The induction of terminal differentiation markers by the cAMP pathway in human HaCaT keratinocytes.

The terminal differentiation of human epidermal keratinocytes is a complex morphological and biochemical shift from a mitotically active cell to an inert protein cross-linked envelope. This transition is a clearly predetermined cell death mechanism, but it is unlike many other programmed cell deaths in that it is not apoptotic. To explore and contrast the mechanism by which keratinocytes are committed to differentiation rather than apoptosis, we focused on the cyclic adenosine monophosphate (cAMP) signaling pathway using selective modulators of intracellular cAMP levels. Markers of differentiation were assayed by Western blotting. Raising intracelluar cAMP levels by treating HaCaT cells with forskolin, a diterpene, or with isobutylmethylxanthine, a phosphodiesterase inhibitor, and isoproterenol, a beta-adrenergic receptor agonist that selectively activates adenylate cyclase, increased the levels of the differentiation markers keratin K1 and K10, involucrin and transglutaminase. H89 and KT5720, both inhibitors of cAMP-dependent protein kinase, suppressed the expression of keratins K1 and K10. These observations are in line with the defined role for cAMP in the control of keratinocyte differentiation.

Rearrangement of the tubulin and actin cytoskeleton during programmed cell death in Drosophila salivary glands.

During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.

Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands of Manduca sexta.

Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae, homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4-day period during larva-to-pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end-labeling of single strand breaks, and pulsed-field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coupled with synthesis of a glycine-rich protein, reminiscent of silk-like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell-independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. It is at this period that unequivocal nucleosomal ladders are seen and end-labeling in situ or electrophoretic techniques document single on double-strand breaks, respectively. DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instor, raising the possibility of potential limitations built into the cells before their final collapse.