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Biomedical subjects

G Häcker

Publications and source records attributed to G Häcker.

At least 19 recordsLinked to original sources
PubMed2006

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis.

Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

Amino Acid Sequence↗
PubMed2003

Functional evaluation of the apoptosome in renal cell carcinoma.

Renal cell carcinoma (RCC) responds very poorly to chemo- or radiotherapy. Renal cell carcinoma cell lines have been described to be resistant to apoptosis-inducing stimuli and to lack caspase expression. Here, we provide a structural and functional assessment of the apoptosome, the central caspase-activating signalling complex and a candidate for apoptosis-inactivating mutations. Cells from RCC cell lines and clinical samples isolated from RCC patients were included. Apoptosome function was measured as quantitative activation of caspases in protein extracts. In all five cell lines and in 19 out of 20 primary clear cell RCC samples, the expression of apoptosome components and caspase activation appeared normal. Of the four nonclear cell RCC that could be included, both oncocytomas gave no response to cytochrome c (in one case, no Apaf-1 was detected), one chromophobe RCC lacked caspase-9 and failed to activate caspase-3 in response to cytochrome c, and one papillary RCC showed good caspase activation despite the lack of caspase-7. Experiments utilising a peptide derived from Smac/DIABLO gave no indication that inhibitor of apoptosis proteins might exert an inhibiting effect in primary clear cell RCC. Thus, the apoptosome signalling complex is intact in human (clear cell) RCC, and an apoptosis defect must be located at other, probably upstream, sites.

Apoptosis↗
PubMed2003

Characterization of antiapoptotic activities of Chlamydia pneumoniae in infected cells.

C. pneumoniae has the capacity to inhibit apoptosis. Here we provide further insight into how infection with C. pneumoniae affects the apoptotic signal transduction in infected human epitheloid cells. For assessing changes in the apoptotic response, intact cells and cell extracts were used. These results show that C. pneumoniae is able to interfere with the host cell's apoptotic apparatus at least at two steps in the signal transduction.

Apoptosis↗
PubMed2002

Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma.

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome c-induced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis.

Apoptosis↗
PubMed2001

Characterization of antiapoptotic activities of Chlamydia pneumoniae in human cells.

Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in atherosclerosis. Here we show that infection with C. pneumoniae protects HeLa human epithelioid cells against apoptosis induced by external stimuli. In infected HeLa cells, apoptosis induced by staurosporine and CD95-death-receptor signaling was strongly reduced. Upon treatment with staurosporine, generation of effector caspase activity, processing of caspase-3 and caspase-9 and cytochrome c redistribution were all profoundly inhibited in cells infected with C. pneumoniae. Bacterial protein synthesis during early infection was required for this inhibition. Furthermore, cytochrome c-induced processing and activation of caspases were inhibited in cytosolic extracts from infected cells, suggesting that a C. pneumoniae-dependent antiapoptotic factor was generated in the cytosol upon infection. Infection with C. pneumoniae failed to induce significant NF-kappaB activation in HeLa cells, indicating that no NF-kappaB-dependent cellular factors were involved in the protection against apoptosis. These results show that C. pneumoniae is capable of interfering with the host cell's apoptotic apparatus at probably at least two steps in signal transduction and might explain the propensity of these bacteria to cause chronic infections in humans.

2,4-Dinitrophenol↗
PubMed2000

Baculovirus P35 protein does not inhibit caspase-9 in a cell-free system of apoptosis.

The P35 protein derived from the baculovirus Autographa californica NPV has been characterized as an inhibitor of apoptotic cell death in a great number of organisms and situations. This potential has been further mapped to the capacity of P35 to inhibit all caspases investigated. Here we show that P35 does not inhibit caspase-9 activity in a cell-free system of mammalian caspase activation. In cell extracts, cytochrome c addition led to the activation of caspase-9, -3 and -7. When cytosolic extract from cells expressing P35 was added, caspase-9-mediated maturation of caspase-3 proceeded normally but caspase-3-mediated further events were prevented, such as complete processing of caspase-3, processing of caspase-7 and the appearance of DEVD-cleaving activity. The P35 protein from Bombyx mori NPV, which has been reported to have a much weaker anti-apoptosis activity in vivo, was found also to have significant caspase-3-inhibiting activity. These data suggest that P35 evolved specifically to inhibit effector rather than initiator caspases.

Apoptosis↗
PubMed2000

Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors.

Members of the caspase family of proteases are important in the implementation of apoptotic cell death. These caspases are intracellularly activated upon a death stimulus, and exhibit a distinctive proteolytic activity which transmits a death signal and readily detected by measuring the cleavage of synthetic substrates in cell extracts. In this report, we show that apoptosis-associated caspase activation can be recorded not only in cell lysates but also in intact lymphoid cells with commercially available peptides which are either biotinylated or carry an amino-methylcoumarin (AMC) group. Incubation of intact cells induced to undergo apoptosis with Ac-Asp-Glu-Val-Asp-AMC (DEVD-AMC) leads to the release of AMC in amounts very similar to the amounts released when cell extracts are prepared and incubated with DEVD-AMC. This release can be detected by a fluorescence read-out and is blocked by caspase-inhibitors such as Ac-DEVD-cho or Z-VAD-fmk. Similarly, labelling of intact cells with the biotinylated peptides Tyr-Val-Ala-Asp-cmk (YVAD-cmk) or YVAD-faom permits the detection of active caspases by affinity blotting and the detection of apoptotic cells by FACS analysis. These methods enable the investigator to detect at the single-cell level those cells which have activated their caspases and to evaluate such activation without the need for lysis of the cells.

Amino Acid Chloromethyl Ketones↗
PubMed2000

The morphology of apoptosis.

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.

Animals↗
PubMed1999

Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells.

Activation of antigen-presenting cells (APCs) by invariant constituents of pathogens such as lipopolysaccharide (LPS) or bacterial DNA (CpG-DNA) initiates immune responses. We have analyzed the mitogen-activated protein kinase (MAPK) pathways triggered by CpG-DNA and their significance for cytokine production in two subsets of APCs, i.e. macrophages and dendritic cells (DCs). We found that CpG-DNA induced extracellular signal-regulated kinase (ERK) activity in macrophages in a classic MEK-dependent way. This pathway up-regulated tumor necrosis factor production but down-regulated interleukin (IL)-12 production. However, in DCs, which produce large amounts of IL-12, CpG-DNA and LPS failed to induce ERK activity. Consistent with a specific negative regulatory role for ERK in macrophages, chemical activation of this pathway in DCs suppressed CpG-DNA-induced IL-12 production. Overall, these results imply that differential activation of MAP kinase pathways is a basic mechanism by which distinct subsets of innate immune cells regulate their effector functions.

Animals↗
PubMed1999

Cytochrome c is dispensable for fas-induced caspase activation and apoptosis.

Cytochrome c is thought to play an important role in the initiation of apoptosis following its release from mitochondria. It is controversial whether such release is also involved in caspase activation and apoptotic cell death after ligation of the cell surface molecule Fas. We addressed this issue by investigating cells from the human cell lines Jurkat and SKW6 which had been treated with the inhibitor of the mitochondrial F0/F1-ATPase, oligomycin. Oligomycin-treatment led, over a wide range of concentrations, to ATP-depletion and, at similar concentrations, abrogated the appearance of caspase-3-like activity caused by stauroporine. Electroporation of cytochrome c protein into intact cells induced caspase activation in both cell lines and significant nuclear apoptosis in Jurkat cells. In ATP-depleted cells, electroporation of cytochrome c induced neither caspase activation nor nuclear fragmentation. Fas-induced caspase activation and nuclear apoptosis, however, were unaffected by the depletion of ATP. Thus, cytochrome c is unlikely to be an important factor in Fas-induced cell death.

Adenosine Triphosphate↗
PubMed1999

Extent and limitation of the control of nuclear apoptosis by DNA-fragmenting factor.

During apoptosis, changes to the nucleus of the dying cell include DNA degradation and structural collapse. These changes are accomplished by caspase-mediated cleavage of DNA-fragmenting factor DFF45, an inhibitor of the effector molecule DFF40. DFF45 and, more efficiently, a mutant lacking one caspase-cleavage site (DFF45m) inhibited nuclear changes in a cell-free system when apoptosis was initiated by adding caspase-3 to cell extracts. In primary tissues from several mammalian species, human caspase-3 activated and human DFF45m blocked nuclear apoptosis demonstrating evolutionary conservation of this step. However, DFF45m did not significantly inhibit DNA-fragmenting activity in extracts from staurosporine-treated cells from the human cell line Jurkat. In extracts from normal Jurkat cells, DFF45m blocked caspase-triggered DNA cleavage efficiently only if added within a short time of the addition of the caspase. At later time points, this inhibition by DFF45m was strongly reduced in efficiency while Zn2+ still completely blocked DNA fragmentation. These results demonstrate the evolutionary conservation of a linear pathway in apoptosis and suggest the existence of more complex events as final effector machinery.

Animals↗
PubMed1999

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology.

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Inhibition of nuclear factor kappa B and induction of apoptosis in T-lymphocytes by sulfasalazine.

1. Chronic inflammatory diseases have been shown to be associated with NF-kappaB activation and impaired apoptosis of immune cells. The aim of the present study was to investigate if sulfasalazine and its colonic metabolites 5-aminosalicylic acid (5ASA) and sulfapyridine affect NF-kappaB/Rel activation and viability of T-lymphocytes. 2. Sulfasalazine inhibits NF-kappaB/Rel activation in the murine T-lymphocyte cell line RBL5 using electrophoretic mobility shift assays. In transfection assays sulfasalazine treatment for 4 h inhibits kappaB-dependent transcription with an IC50 value of approximately 0.625 mM. 3. Higher doses or prolonged treatment result in cell death of T-lymphocytes in a dose- and time-dependent manner. Cell death is caused by apoptosis as judged by DNA fragmentation, annexin V and Apo 2.7 staining. Induction of apoptosis is a fast event with 50% apoptotic cells after a 4 h incubation with 2.5 mM sulfasalazine. The ED50 value for apoptosis induction after 24 h treatment was approximately 0.625 mM. 4. In contrast, 5ASA and sulfapyridine neither inhibit NF-kappaB/Rel activation nor induce apoptosis in T-lymphocytes at doses up to 5.0 mM. 5. These results demonstrate that sulfasalazine, but not 5ASA or sulfapyridine, strongly inhibits NF-kappaB activation and potently induces apoptosis in T-lymphocytes. Inhibition of NF-kappaB/Rel activation and subsequent clearance of activated T-lymphocytes by apoptosis might thus explain the beneficial effects of sulfasalazine in the treatment of chronic inflammatory disorders.

Animals↗
PubMed1999

Uncouplers of oxidative phosphorylation can enhance a Fas death signal.

Recent work suggests a participation of mitochondria in apoptotic cell death. This role includes the release of apoptogenic molecules into the cytosol preceding or after a loss of mitochondrial membrane potential DeltaPsim. The two uncouplers of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2, 4-dinitrophenol (DNP) reduce DeltaPsim by direct attack of the proton gradient across the inner mitochondrial membrane. Here we show that both compounds enhance the apoptosis-inducing capacity of Fas/APO-1/CD95 signaling in Jurkat and CEM cells without causing apoptotic changes on their own account. This amplification occurred upstream or at the level of caspases and was not inhibited by Bcl-2. The effect could be blocked by the cowpox protein CrmA and is thus likely to require caspase 8 activity. Apoptosis induction by staurosporine in Jurkat cells as well as by Fas in SKW6 cells was unaffected by CCCP and DNP. The role of cytochrome c during Fas-DNP signaling was investigated. No early cytochrome c release from mitochondria was detected by Western blotting. Functional assays with cytoplasmic preparations from Fas-DNP-treated cells also indicated that there was no major contribution by cytochrome c or caspase 9 to the activation of effector caspases. Furthermore, an increase of rhodamine-123 uptake into intact cells, which has been explained by mitochondrial swelling, occurred considerably later than the caspase activation and was blocked by Z-VAD-fmk. These data show that uncouplers of oxidative phosphorylation can presensitize some but not all cells for a Fas death signal and provide information about the existence of separate pathways in the induction of apoptosis.

2,4-Dinitrophenol↗
PubMed1998

Activation of caspase-3-like enzymes in non-apoptotic T cells.

The activation of the caspase family of cysteine proteases is a key step in the implementation of apoptotic cell death leading to further downstream effects such as DNA fragmentation. In cultured tumor cells, caspase activity appears only when cells are undergoing apoptosis. Here we show that human and murine T lymphocytes acquire high intracellular activities of cell death-specific caspases upon activation by mitogens and IL-2 without evidence that apoptosis is proceeding. The highest activity is seen when cells are mitogen activated for 3 days. On a per cell basis, caspase activity in activated T cells is much higher than in tumor cells induced to undergo apoptosis. In the presence of exogenously added IL-2 cells stay alive and maintain a high level of caspase activity while IL-2 withdrawal results in cell death and decline of caspase activity. Caspase activity can also be measured in extracts from spleen and lymph nodes from mice injected with superantigen. While in tumor cell lines caspase activity correlates with cleavage of poly(ADP)-ribose polymerase (PARP) and DNA fragmentation, in activated T cells cleavage products of cellular PARP can be detected whereas DNA fragmenting activity appears only upon IL-2 withdrawal which coincides with cell death. These data show that caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. Furthermore, they provide a molecular correlate for the high susceptibility of activated T cells for apoptosis.

Animals↗
PubMed1997

Inhibition of etoposide-induced apoptotic events by azide.

Azide is reported to be an inducer of necrotic rather than apoptotic cell death. Using various parameters of cell death, we demonstrate here that azide is capable of completely inhibiting apoptosis induced by VP-16/etoposide. Azide was found systematically to protect Jurkat cells against VP-16-induced effects as determined by an array of biochemical and morphological parameters. The cells were able partially to recover proliferative activity following removal of the drugs. We conclude that azide inhibits apoptosis induced by VP-16 at an early point in the apoptotic pathway and that inhibition of apoptosis might be the mechanism of necrosis-induction by azide.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Requirements for proteolysis during apoptosis.

The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.

Animals↗