Inhibition of p38 kinase reveals a TNF-alpha-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line

TNF-alpha transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-kappaB-mediated transactivation, without affecting the nuclear translocation of NF-kappaB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.     

Osmotic stress sensitizes naturally resistant cells to TNF-alpha-induced apoptosis

Most cells are naturally resistant to TNF-alpha-induced cell death and become sensitized when NF-kappaB transactivation is blocked or in the presence of protein synthesis inhibitors that prevent the expression of anti-apoptotic genes. In this report we analyzed the role of osmotic stress on TNF-alpha-induced cell death. We found that it sensitizes the naturally resistant HeLa cells to TNF-alpha-induced apoptosis, with the involvement of an increase in the activity of several kinases, the inhibition of Bcl-2 expression, and a late increase on NF-kappaB activation. Cell death occurs regardless of the enhanced NF-kappaB activity, whose inhibition produces an increase in apoptosis. The inhibition of p38 kinase, also involved in NF-kappaB activation, significantly increases the effect of osmotic stress on TNF-alpha-induced cell death.     

Calmodulin Inhibitor-induced Apoptosis was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calmodulin is known to transduce Ca²⁺ signals by interacting with specific target proteins. In order to determine the role of calmodulin in regulating neuronal survival and death, we examined, whether calmodulin inhibitors induce caspase-dependent apoptotic cell death, and whether glycogen synthase kinase-3 is involved in calmodulin inhibitor-induced cell death in PC12 cells. W13, a calmodulin specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation of fragmentation. Glycogen synthase kinase-3 inhibitors prevented calmodulin inhibitor-induced apoptosis. In addition, nerve growth factor and cycloheximide, a protein synthesis inhibitor, completely blocked cell death. Moreover, caspase-3 activation was accompanied by calmodulin inhibitor-induced cell death and inhibited by nerve growth factor. These results suggest that calmodulin inhibitors induce caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Chelation of Extracellular Calcium-Induced Cell Death was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

Identification of p54(nrb) and the 14-3-3 Protein HS1 as TNF-alpha-inducible genes related to cell cycle control and apoptosis in human arterial endothelial cells

TNF-alpha plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-alpha induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-alpha on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-alpha cytotoxicity, presumably by NF-kappaB mediated induction of protective genes. However, the cytoprotective genes involved in NF-kappaB dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-alpha inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-alpha-induced expression of the RNA binding protein p54(nrb) and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-alpha mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, p21(cip1) and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-alpha induced gene expression patterns mediating the prosurvival effect of TNF-alpha in endothelial cells.     

Apoptosis: live or die--hard work either way!

This review presents a brief overview of the cell's apoptotic machinery, including specific and indirect death signals. Specific death signals are transferred via death ligands, death receptors, and their intracellular signalling pathways. Indirect death signals cumulate a wide range of stimuli that potentially harm survival of cells. These include intercalating drugs, irradiation or altered intracellular signalling. Herein, a focal point is the mitochondrial control of specific death enzymes--so called caspases--by members of the pro-apoptotic Bax and BH3 subfamily or the anti-apoptotic Bcl-2 subfamily. While the initiation of cell death happens through a variety of signalling systems, the activation of caspases plays a pivotal role in the progression towards the final morphologic findings in cells undergoing apoptosis. Caspases appear to directly cleave and inactivate substrates that are clinical for the maintenance of cell structure and function but also regulate the activity of other enzymes that induce the apoptotic phenotype within the cell. The insulin-like growth factors (IGFs) are potent proliferation factors and potently inhibit apoptosis acting via the ubiquitously expressed IGF-I receptor. Within IGF-I receptor signalling, key to the inhibition of apoptosis are the RAS/RAF/mitogen-activated protein (MAP)-kinase pathway and the PI 3'-kinase pathway. To give an example of high clinical relevance of apoptosis within endocrine disorders, apoptotic death of pancreatic beta cells in type 1 diabetes disease and the involvement of IGF-II in beta cell survival and beta cell function is discussed in detail. Finally, further understanding of signalling systems that are involved in proliferation or in apoptosis might provide novel tools to treat or even heal disorders like type I diabetes.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

p53 Proteasomal Degradation: Poly-Ubiquitination is Not the Whole Story

Interactions between survival pathways and apoptotic cascades play a determinant role in the maintenance of neoplastic clone proliferation and impaired response to apoptosis. Recently, we established a novel interplay between the NF-kappaB survival- and p53 death- pathways in a tumor model system that represents the most common extranodal lymphoid cell neoplasia, MALT (Mucosa Associated Lymphoid Tissue) lymphoma. MALTs are genetically characterized by the t(11;18)(q21;q21) chromosomal translocation that results in API2/MALT1 fusion products. It was shown that distinct API2/MALT1 chimeric proteins function as oncogenes that bilaterally confer a proliferative advantage to the neoplastic clone by activating the NF-kappaB signaling pathway and also inhibiting p53 mediated cell death. Here, we demonstrate that API2/MALT1 mediated inhibition of apoptosis is p53 specific, as distinct API2/MALT1 fusion proteins fail to protect cells from FAS induced cell death. Furthermore, we demonstrate that API2/MALT1 mediated NF-kappaB activation does not alter p53 protein levels or subcellular localization suggesting a post-translational or indirect mechanism of p53 deregulation.     

Survival pathways regulating the apoptosis induced by tumour necrosis factor-alpha in primary cultured bovine endothelial cells

The aim of the present study was to identify biochemical pathways driving the resistance of endothelial cells to apoptosis induced by tumour necrosis factor-alpha (TNF). (1) Although nuclear factor-kappa B (NF-kappaB) was activated by TNF, its inhibition by MG-132 failed to sensitize these cells. (2) The activation of protein kinase C (PKC) by phorbol ester completely abolished the TNF-induced cell death. (3) The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (Wo) triggered apoptosis and enhanced the TNF-induced cell death. (4) The MEK inhibitor PD98059 did not affect the TNF-induced apoptotic process. (5) The p38 is activated by TNF and its inhibition by SB203580 sensitized the cells to TNF. This is correlated with the inhibition of phosphorylation of heat-shock protein of 27 kDa (HSP27).These results indicate that TNF activates NF-kappaB, which does not drive any anti-apoptotic response, and p38, which plays an anti-apoptotic function probably through HSP27 phosphorylation. Moreover, PKC and PI3K are involved in the control of survival pathways.