Claus Reimertz

Gene expression during ER stress–induced apoptosis in neurons induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of ischemic and neurodegenerative disorders. Treatment of human SH-SY5Y neuroblastoma cells with tunicamycin, an inhibitor of protein glycosylation, rapidly induced the expression of target genes of the unfolded protein response. However, prolonged treatment also triggered a delayed, caspase-dependent cell death. Microarray analysis of gene expression changes during tunicamycin-induced apoptosis revealed that the Bcl-2 homology domain 3-only family member, Bcl-2 binding component 3/p53 upregulated modulator of apoptosis (Bbc3/PUMA), was the most strongly induced pro-apoptotic gene. Expression of Bbc3/PUMA correlated with a Bcl-xL–sensitive release of cytochrome c and the activation of caspase-9 and -3. Increased expression of Bbc3/PUMA was also observed in p53-deficient human cells, in response to the ER stressor thapsigargin, and in rat hippocampal neurons after transient forebrain ischemia. Overexpression of Bbc3/PUMA was sufficient to trigger apoptosis in SH-SY5Y neuroblastoma cells, and human cells deficient in Bbc3/PUMA showed dramatically reduced apoptosis in response to ER stress. Our data suggest that the transcriptional induction of Bbc3/PUMA may be sufficient and necessary for ER stress–induced apoptosis.

Apoptosis induced by proteasome inhibition in cancer cells : predominant role of the p53/PUMA pathway

The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.

The amyloid precursor protein protects PC12 cells against endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress is believed to play an important role in neurodegenerative disorders such as Alzheimers disease. In the present study, we investigated the effect of the human amyloid precursor protein (APP) on the ER stress response in PC12 cells. Tunicamycin, an inhibitor of N‐glycosylation, rapidly induced the expression of the ER‐resident chaperone Bip/grp78, a known target gene of the unfolded protein response. Prolonged treatment with tunicamycin (≥ 12 h) resulted in the activation of executioner caspases 3 and 7. Interestingly, PC12 cells overexpressing human wild‐type APP (APPwt) showed increased resistance to tunicamycin‐induced apoptosis compared with empty vector‐transfected controls. This neuroprotective effect was significantly diminished in cells expressing the Swedish mutation of APP (KM670/671NL). Similar effects were observed when ER stress was induced with brefeldin A, an inhibitor of ER‐to‐Golgi protein translocation. Of note, APP‐mediated neuroprotection was not associated with altered expression of Bip/grp78 or transcription factor C/EBP homologous protein‐10 (CHOP/GADD153), suggesting that APP acted either downstream or independently of ER‐to‐nucleus signaling. Our data indicate that APP plays an important physiological role in protecting neurons from the consequences of prolonged ER stress, and that APP mutations associated with familial Alzheimers disease may impair this protective activity.

Ca2+-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1)

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro‐apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease‐activating factor‐1 (APAF‐1). This complex activates procaspase‐9 in the presence of dATP, resulting in caspase‐mediated execution of apoptotic neuron death. Many forms of Ca2+‐mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca2+ induced prominent degradation of APAF‐1 in human SH‐SY5Y neuroblastoma cells and in a neuronal cell‐free apoptosis system. Loss of APAF‐1 correlated with a reduced ability of cytochrome c to activate caspase‐3‐like proteases. Ca2+ induced the activation of calpains, monitored by the cleavage of full‐length α‐spectrin into a calpain‐specific 150‐kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF‐1 degradation also occurred via calpain‐independent pathways. Our data suggest that Ca2+ inhibits caspase activation during Ca2+‐mediated neuron death by triggering the degradation of the cytochrome c‐binding protein APAF‐1.

Dlk/ZIP kinase-induced apoptosis in human medulloblastoma cells: requirement of the mitochondrial apoptosis pathway

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression.

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide‐induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (ρ– cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2‐ or C8‐ceramide induced cell death in D283 control cells, while ρ– cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro‐apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and ρ– cells. Overexpression of the antiapoptotic protein Bcl‐xL failed to inhibit the toxic effect of C2‐ceramide in D283 control cells, and no significant increase in caspase‐3‐like protease activity could be detected during the death process. Despite this, C2‐ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in ρ– cells. Our data indicate that the apoptosis‐inducing effect of C2‐ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.

The death associated protein (DAP) kinase homologue Dlk/ZIP kinase induces p19ARF- and p53-independent apoptosis

Dlk/ZIP kinase is one of five members of the death associated protein (DAP) kinase family. DAP kinase is able to induce apoptosis in a p19ARF/p53-dependent manner. We elucidated the potential role of the p19ARF/p53 pathway in Dlk/ZIP kinase-triggered cell death. Overexpression of a constitutively pro-apoptotic form of Dlk/ZIP kinase induced apoptosis in rat fibroblast cells which express wild-type p19ARF and p53. Cell death was characterised by apoptotic membrane blebbing, mitochondrial depolarisation, cytochrome c release and activation of caspase-3. However, Dlk/ZIP kinase-triggered cell death was also observed in p19ARF-deficient and p53-deficient mouse fibroblast cells. Quantitative analysis revealed that the status of p53 had no major influence on cellular susceptibility to Dlk/ZIP kinase-triggered cell death. Loss of p53 did not prevent Dlk/ZIP kinase-induced mitochondrial membrane depolarisation and release of cytochrome c. Furthermore, overexpression of Dlk/ZIP kinase did not lead to an increased expression of pro-apoptotic p53 target genes in either cell line. These data suggest that Dlk/ZIP kinase is able to trigger the mitochondrial apoptosis pathway independent of the p19ARF/p53 signalling pathway.

AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of Bcl-xL and Bcl-2 in MCF7 cells

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.

Erratum to “The death associated protein (DAP) kinase homologue Dlk/ZIP kinase induces p19ARF- and p53-independent apoptosis” [European Journal of Cancer, 39 (2003) 249–256]☆

D. Kogel*, C. Reimertz, H. Dusmann, P. Mech, K.H. Scheidtmann, J.H.M. Prehn Interdisciplinary Center for Clinical Research (IZKF), Research Group ‘‘Apoptosis and Cell Death’’, Westphalian Wilhelms-University, D-48149 Munster, Germany Institute of Genetics, University of Bonn, D-53117 Bonn, Germany Department of Pharmacology and Toxicology, Faculty of Medicine, Westphalian Wilhelms-University, D-48149 Munster, Germany