cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

The role of reaper-dependent apoptosis in radiation-induced life-span alterations in Drosophila melanogaster

This is the result of studying on a problem of radio-induced ageing. From the point of view of radiation genetics it is perspective to investigate influence of low doze irradiation on individuals with the mutant phenotypes that allows to assume a role of separate genes and mechanisms controllable by them in determination of life span and ageing. The role of a reaper-dependent way of apoptosis regulation in the induced change of life span is shown. The assumption is put forward that apoptosis has the important role during ageing an animal organism.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.     

The influence of low doze gamma-irradiation on life span of Drosophila mutants with defects of DNA damage sensation and repair

It was shown that life span of wild type strain Canton-S increased after low doze gamma-irradiation. It was revealed the decrease of life span after irradiation in Drosophila mutants with defects of DNA damage sensation and repair genes mei-9 and mei-41, both in homozygous and heterozygous lines. In mei-41 line males' lives longer than females in contrast to other lines.     

Effect of stress on the life span of the yeast Saccharomyces cerevisiae

A correlation is known to exist in yeast and other organisms between the cellular resistance to stress and the life span. The aim of this study was to examine whether stress treatment does affect the generative life span of yeast cells. Both heat shock (38 degrees C, 30 min) and osmotic stress (0.3 M NaCl, 1 h) applied cyclically were found to increase the mean and maximum life span of Saccharomyces cerevisiae. Both effects were more pronounced in superoxide dismutase-deficient yeast strains (up to 50% prolongation of mean life span and up to 30% prolongation of maximum life span) than in their wild-type counterparts. These data point to the importance of the antioxidant barrier in the stress-induced prolongation of yeast life span.     

Long-lived worms and aging

Several investigators have generated long-lived nematode worms (Caenorhabditis elegans) in the past decade by mutation of genes in the organism in order to study the genetics of aging and longevity. Dozens of longevity assurance genes (LAG) that dramatically increase the longevity of this organism have been identified. All long-lived mutants of C. elegans are also resistant to environmental stress, such as high temperature, reactive oxygen species (ROS), and ultraviolet irradiation. Double mutations of some LAGs further extended life span up to 400%, providing more insight into cellular mechanisms that put limits on the life span of organisms. With the availability of the LAG mutants and the combined DNA microarray and RNAi technology, the understanding of actual biochemical processes that determine life span is within reach: the downstream signal transduction pathway may regulate life span by up-regulating pro-longevity genes such as those that encode antioxidant enzymes and/or stress-response proteins, and down-regulating specific life-shortening genes. Furthermore, longevity could be modified through chemical manipulation. Results from these studies further support the free radical theory of aging, suggest that the molecular mechanism of aging process may be shared in all organisms, and provide insight for therapeutic intervention in age-related diseases.     

Endothelial cells regulate p53-dependent apoptosis of neural progenitors after irradiation

Endothelial cells represent an important component of the neurogenic niche and may regulate self-renewal and differentiation of neural progenitor cells (NPCs). Whether they have a role in determining the apoptotic fate of NPCs after stress or injury is unclear. NPCs are known to undergo p53-dependent apoptosis after ionizing radiation, whereas endothelial cell apoptosis after irradiation is dependent on membrane acid sphingomyelinase (ASMase) and is abrogated in sphingomyelin phosphodiesterase 1 (smpd1)- (gene that encodes ASMase) deficient mice. Here we found that p53-dependent apoptosis of NPCs in vivo after irradiation was inhibited in smpd1-deficient mice. NPCs cultured from mice, wild type (+/+) or knockout (−/−), of the smpd1 gene, however, demonstrated no difference in apoptosis radiosensitivity. NPCs transplanted into the hippocampus of smpd1−/− mice were protected against apoptosis after irradiation compared with those transplanted into smpd1+/+ mice. Intravenous administration of basic fibroblast growth factor, which does not cross the blood–brain barrier, known to protect endothelial cells against apoptosis after irradiation also attenuated the apoptotic response of NPCs. These findings provide evidence that endothelial cells may regulate p53-dependent apoptosis of NPCs after genotoxic stress and add support to an important role of endothelial cells in regulating apoptosis of NPCs after injury or in disease.     

A Genetic Pathway Conferring Life Extension and Resistance to Uv Stress in Caenorhabditis Elegans

A variety of mechanisms have been proposed to explain the extension of adult life span (Age) seen in several mutants in Caenorhabditis elegans (age-1: an altered aging rate; daf-2 and daf-23: activation of a dauer-specific longevity program; spe-26: reduced fertility; clk-1: an altered biological clock). Using an assay for ultraviolet (UV) resistance in young adult hermaphrodites (survival after UV irradiation), we observed that all these Age mutants show increased resistance to UV. Moreover, mutations in daf-16 suppressed the UV resistance as well as the increased longevity of all the Age mutants. In contrast to the multiple mechanisms initially proposed, these results suggest that a single, daf-16-dependent pathway, specifies both extended life span and increased UV resistance. The mutations in daf-16 did not alter the reduced fertility of spe-26 and interestingly a daf-16 mutant is more fertile than wild type. We propose that life span and some aspects of stress resistance are jointly negatively regulated by a set of gerontogenes (genes whose alteration causes life extension) in C. elegans.     

Monocyte chemotactic protein-induced protein 1 (MCPIP1) suppresses stress granule formation and determines apoptosis under stress

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.     

Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis

Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, gamma-irradiation, and H(2)O(2). SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.     

Xanthine oxidase is a peroxynitrite synthase: newlyidentified roles for a very old enzyme

Abstract

Several investigators have generated long-lived nematode worms (Caenorhabditis elegans) in the past decade by mutation of genes in the organism in order to study the genetics of aging and longevity. Dozens of longevity assurance genes (LAG) that dramatically increase the longevity of this organism have been identified. All long-lived mutants of C. elegans are also resistant to environmental stress, such as high temperature, reactive oxygen species (ROS), and ultraviolet irradiation. Double mutations of some LAGs further extended life span up to 400%, providing more insight into cellular mechanisms that put limits on the life span of organisms. With the availability of the LAG mutants and the combined DNA microarray and RNAi technology, the understanding of actual biochemical processes that determine life span is within reach: the downstream signal transduction pathway may regulate life span by up-regulating pro-longevity genes such as those that encode antioxidant enzymes and/or stress-response proteins, and down-regulating specific life-shortening genes. Furthermore, longevity could be modified through chemical manipulation. Results from these studies further support the free radical theory of aging, suggest that the molecular mechanism of aging process may be shared in all organisms, and provide insight for therapeutic intervention in age-related diseases.