IGFBP-3 and apoptosis—a licence to kill?

Insulin-like growth factor binding protein (IGFBP)-3, the major carrier of insulin-like growth factors (IGFs) in the circulation, was first isolated and characterised over a decade ago. More recently, IGFBP-3 has been assigned a role as a putative death-promoting factor, a function that appears, under certain circumstances, to be independent of its IGF-binding ability. This review examines the current evidence for a pro-apoptotic function for IGFBP-3 and speculates on its physiological significance.     

Inhibition of NF-κ B activity and cFLIP expression contribute to viral-induced apoptosis

Virus-induced activation of nuclear factor-kappa B (NF-B) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-B is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-B activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-B regulation, where NF-B activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-B activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-B is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-B also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-B-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-B activation in reovirus-infected cells. Further, inhibition of NF-B activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-B and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.     

Downregulation of mTORC1 and Mcl-1 by lipid-oversupply contributes to islet β-cell apoptosis and dysfunction

Pancreatic β-cell apoptosis is a key feature of diabetes and can be induced by chronic exposure to saturated fatty acids (FAs). However, the underlying mechanisms remain poorly understood. We presently evaluated the role of Mcl-1 and mTOR in mice fed with high-fat-diet (HFD) and β-cells exposed to the overloaded palmitic acid (PA). Compared with normal-chow-diet (NCD)-fed mice, HFD group showed impaired glucose tolerance after two months. Along with the diabetes progression, pancreatic islets first became hypertrophic and then atrophic, the ratio of β-cell:α-cell increased in the islets of four months HFD-fed mice while decreased after six months. This process was accompanied by significantly increased β-cell apoptosis and AMPK activity, and decreased Mcl-1 expression and mTOR activity. Consistently, glucose-induced insulin secretion dropped. In terms of mechanism, PA with lipotoxic dose could activate AMPK, which in turn inhibited ERK-stimulated Mcl-1^Thr163 phosphorylation. Meanwhile, AMPK blocked Akt activity to release Akt inhibition on GSK3β, followed by GSK3β-initiated Mcl-1^Ser159 phosphorylation. The context of Mcl-1 phosphorylation finally led to its degradation by ubiquitination. Also, AMPK inhibited the activity of mTORC1, resulting in a lower level of Mcl-1. Suppression of mTORC1 activity and Mcl-1 expression positively related to β-cell failure. Alteration of Mcl-1 or mTOR expression rendered different tolerance of β-cell to different dose of PA. In conclusion, lipid oversupply-induced dual modulation of mTORC1 and Mcl-1 finally led to β-cell apoptosis and impaired insulin secretion. The study may help further understand the pathogenesis of β-cell dysfunction in case of dyslipidemia, and provide promising therapeutic targets for diabetes.     

TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.     

Yeast as a model to study apoptosis?

Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms.The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, . . .) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis.Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics).This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.     

Sensitization of multiple myeloma and B lymphoma lines to dexamethasone and γ-radiation-induced apoptosis by CD40 activation

We examined the effects of CD40 activation with dexamethasone (Dex) or ⁶⁰Co--irradiation on the growth of malignant B cells in vitro, using the human multiple myeloma (MM) cell line, XG2, and the B lymphoma Daudi cell line as models. Both lines are resistant to Dex and irradiation; 10^–7M Dex or 10 Gy of -irradiation induced only minimal growth arrest and apoptosis of the cells. Treatment of the cells with the agonistic anti-CD40 monoclonal antibody 5C11 partially inhibited the proliferation of the Daudi cells; XG2 underwent apoptosis. XG2 is an Interleukin-6 (IL-6)-dependent myeloma cell line and CD40 activation blocked XG2 in the G1 phase of the cell cycle, in a manner similar to the effect of IL-6 deprivation. Daudi was blocked in the G2/M phase after treatment with the agonistic CD40 mAb 5C11. Furthermore, the activation of CD40 on Daudi and XG2 enhanced their sensitivity to dexamethasone-and -irradiation -induced growth arrest and apoptosis. CD40 activation stimulated both anti-apoptotic Bcl-XL and pro-apoptotic Bax mRNA synthesis in the Daudi cell line; CD40 activation increased the Bax mRNA level but had no effect on the Bcl-XL mRNA level in the XG2 cell line. Apoptosis in both cell lines was associated with an increasing ratio of Bax-to-Bcl-XL both in mRNA and in protein levels. It is concluded that use of the anti-CD40 mAb 5C11 either by itself or in combination with chemotherapy and/or radiotherapy may have significant therapeutic potential.Z-H. Zhou and Qin Shi are equally contributed to this article.     

Acetylcholinesterase is associated with apoptosis in β cells and contributes to insulin-dependent diabetes mellitus pathogenesis

Acetylcholinesterase (AChE) expression is pivotal during apoptosis. Indeed, AChE inhibitors partially protect cells from apoptosis. Insulin-dependent diabetes mellitus (IDDM) is characterized in part by pancreatic β-cell apoptosis. Here, we investigated the role of AChE in the development of IDDM and analyzed protective effects of AChE inhibitors. Multiple low-dose streptozotocin (MLD-STZ) administration resulted in IDDM in a mouse model. Western blot analysis, cytochemical staining, and immunofluorescence staining were used to detect AChE expression in MIN6 cells, primary β cells, and apoptotic pancreatic β cells of MLD-STZ-treated mice. AChE inhibitors were administered intraperitoneally to the MLD-STZ mice for 30 days. Blood glucose, plasma insulin, and creatine levels were measured, and glucose tolerance tests were performed. The effects of AChE inhibitors on MIN6 cells were also evaluated. AChE expression was induced in the apoptotic MIN6 cells and primary β cells in vitro and pancreatic islets in vivo when treated with STZ. Induction and progressive accumulation of AChE in the pancreatic islets were associated with apoptotic β cells during IDDM development. The administration of AChE inhibitors effectively decreased hyperglycemia and incidence of diabetes, and restored plasma insulin levels and plasma creatine clearance in the MLD-STZ mice. AChE inhibitors partially protected MIN6 cells from the damage caused by STZ treatment. Induction and accumulation of AChE in pancreatic islets and the protective effects of AChE inhibitors on the onset and development of IDDM indicate a close relationship between AChE and IDDM.     

A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy

Background

Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture.

Results

A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8.

Conclusions

The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.     

Dose–response and threshold effects in cytotoxicity and apoptosis

Cell death can occur as an active, programmed event in response to cytotoxic injury or to endogenous growth limiting factors; the latter serve to maintain homeostasis of cell number in tissues. Cells seem to use different pathways for programmed death, as reflected by their different morphology and different biochemistry. Severe cell damage leading to incapacitation of essential cell functions such as ATP synthesis or the maintenance of membrane potential may lead to “necrosis”. In any event, the incidence and rate of cell death increase with increasing signal intensity. Cytotoxic injury requires a certain number of primary insults; cell death will therefore occur only beyond a definable threshold. Growth factor control of cell death is receptor-mediated with dose–response relations including threshold phenomena follow the general principles of receptor kinetics. The occurrence of programmed cell death during the stages of carcinogenesis introduces a reversible component into this disease. Therefore, there may exist thresholds of dose or durations of exposure to certain carcinogens below which irreversible disease is not generated.     

Does yeast shmooing mean a commitment to apoptosis?

Treatment of yeast Saccharomyces cerevisiae with alpha-pheromone has been reported to lead to massive apoptosis of cells finding no conjugation partner [Severin FF, Hyman AA. Pheromone induces programmed cell death in S. cerevisiae. Curr Biol 2002;12:R233-5]. We report here that this effect is not common in yeast. Using different yeast strains, we demonstrate that identical treatment results in a low mortality even after prolonged treatment with the pheromone. These findings are followed by a general discussion of the biological relevance of apoptosis in yeast.     

Diacylglycerol kinase η colocalizes and interacts with apoptosis signal-regulating kinase 3 in response to osmotic shock

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.     

Virus infections and apoptosis: Lessons from HIV

Many viruses establish life-long infections in their natural host with few if any clinical manifestations. The relationship between virus and host is a dynamic process in which the virus has evolved the means to coexist by reducing its visibility, while the host immune system attempts to suppress and eliminate infection without damage to itself. We are now beginning to understand that viruses can employ a variety of strategies to evade host immune responses. These include escape from T cell recognition, resistance to     

Free radical–induced megamitochondria formation and apoptosis

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H₂O₂, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H₂-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G₁ phase, and apoptotic sub-G₁ populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H₂O₂ and O₂⁻ from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.