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Biomedical subjects

K Schulze-Osthoff

Publications and source records attributed to K Schulze-Osthoff.

At least 55 records · Page 3Linked to original sources
PubMed2001

Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (DeltaPsi(m)) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/DeltaTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial DeltaPsi(m) breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

Apoptosis↗
PubMed2000

Enhancement of T cell receptor signaling by a mild oxidative shift in the intracellular thiol pool.

Exposure of T cells to the macrophage products hydrogen peroxide (HP) or L-lactate (LAC) was previously shown to enhance IL-2 production and to modulate glutathione (GSH) status. We now found that 50 microM HP and 30 mM LAC enhanced strongly the transcription from the IL-2 promoter in Jurkat T cells after stimulation with anti-CD28 together with or without anti-CD3 but not with anti-CD3 Abs alone. Therefore, we used anti-CD3 plus anti-CD28-stimulated cells to investigate the effect of the GSH reductase inhibitor 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the signal cascade. BCNU enhanced the transcription to a similar extent as HP or LAC. Lowering the intracellular GSH/GSH disulfide ratio by BCNU, HP, or NO resulted in all cases in the fulminant enhancement of Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2. Jun-N-terminal kinase and NF-kappaB activation was enhanced through pathways involving Rac, Vav1, PKCTheta, p56(lck), p59(fyn), and IkappaB kinases. In a cell-free system, the autophosphorylation of rFyn was stimulated by GSH disulfide but not by HP. These findings suggest that the oxidation of the cellular thiol pool may play a role as an amplifying mechanism for TCR/CD3 signals in immune responses.

Antibodies, Monoclonal↗
PubMed2000

Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis.

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Adenocarcinoma↗
PubMed2000

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells.

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.

Animals↗
PubMed2000

The cardiac Fas (APO-1/CD95) Receptor/Fas ligand system : relation to diastolic wall stress in volume-overload hypertrophy in vivo and activation of the transcription factor AP-1 in cardiac myocytes.

BACKGROUND: Fas (APO-1/CD95) is a transmembrane receptor belonging to the tumor necrosis factor receptor superfamily. Cross-linking of Fas by Fas ligand (FasL), a tumor necrosis factor-alpha-related cytokine, promotes apoptosis and/or transcription factor activation in a highly cell-type-specific manner. The biological consequences of Fas activation in cardiomyocytes and the regulation of Fas and FasL abundance in the myocardium in vivo remain largely unknown. METHODS AND RESULTS: As shown by immunohistochemistry, Fas was expressed on the sarcolemma of cardiomyocytes in left ventricular tissue sections. Moreover, FasL was constitutively expressed in the myocardium and in isolated cardiomyocytes, as revealed by reverse transcription polymerase chain reaction and Western blotting. Left ventricular abundance of Fas but not FasL was upregulated in a rat model of compensated volume-overload hypertrophy and was closely related to diastolic but not systolic wall stress as determined by MRI. Cardiomyocyte apoptosis was not enhanced in volume-overload hypertrophy despite the increased expression of Fas and the presence of FasL in the myocardium. Moreover, injection of mice with an agonistic anti-Fas antibody promoted hepatocyte but not cardiomyocyte apoptosis in vivo. Stimulation of isolated cardiomyocytes with recombinant FasL promoted an activation of the transcription factor AP-1 as shown by electrophoretic mobility shift assays but did not induce cell death. CONCLUSIONS: Fas and FasL are constitutively expressed in the myocardium and in cardiomyocytes. Myocardial expression of Fas is closely related to diastolic loading conditions in vivo. Signaling pathways emanating from Fas are coupled to an activation of the transcription factor AP-1 in cardiomyocytes.

Animals↗
PubMed2000

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells.

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.

Adenosine Triphosphate↗
PubMed2000

Differential role of caspase-8 and BID activation during radiation- and CD95-induced apoptosis.

Activation of the CD95 death receptor as well as ionizing radiation induces apoptotic cell death in human lymphoma cells. The activation of caspases is a hallmark of apoptosis induction irrespective of the apoptotic trigger. In contrast to death receptor signaling, the exact mechanisms of radiation-induced caspase activation are not well understood. We provide evidence that both, radiation and CD95 stimulation, induce the rapid activation of caspase-8 and BID followed by apoptosis in Jurkat T-cells. To analyse the relative position of caspase-8 within the apoptotic cascade we studied caspase activation and apoptosis in Jurkat cells overexpressing Bcl-2 or Bcl-xL. Caspase-8 activation, pro-apoptotic BID cleavage and apoptosis in response to radiation were abrogated in these cells, while the responses to CD95 stimulation were only partially attenuated by overexpression of Bcl-2 family members. In parallel, the breakdown of the mitochondrial transmembrane potential (DeltaPsim) in response to radiation was inhibited by overexpression of Bcl-2/Bcl-xL Jurkat cells genetically deficient for caspase-8 were found to be completely resistant towards CD95. However, radiation-induced apoptotic responses in caspase-8-negative cells displayed only a modest reduction. We conclude that ionizing radiation activates caspase-8 and BID downstream of mitochondrial damage suggesting that, in contrast to CD95, both events function as executioners rather than initiators of the apoptotic process.

Apoptosis↗
PubMed2000

Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization.

Endothelial cells play an important regulatory role in inflammatory responses by upregulating various proinflammatory gene products including cytokines and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor-alpha (TNF). In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-inducible genes in human umbilical arterial endothelial cells. Following mRNA isolation of non-stimulated and TNF-stimulated cells, cDNAs of both populations were prepared and subtracted by suppression PCR. Sequencing of the enriched cDNAs identified 12 genes differentially expressed including vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, interleukin-8 and IkappaBalpha, an inhibitor of the transcription factor nuclear factor-kappaB. Interestingly, also syntenin, a PDZ motif-containing protein which binds to the cytoplasmic domain of syndecans, was identified by SSH. Time course studies using RT-PCR analysis confirmed that all genes were differentially expressed and rapidly induced by TNF. Our data reveal that SSH is a powerful technique of high sensitivity for the detection of differential gene expression in primary arterial endothelial cells.

Carrier Proteins↗
PubMed2000

IL-10 induces apoptosis in human monocytes involving the CD95 receptor/ligand pathway.

The cytokine IL-10 exerts potent immunosuppressive and anti-inflammatory effects, although the mechanisms of this action remain largely unknown. In the present study, we investigated the effects of IL-10 in human peripheral blood monocytes. We were able to demonstrate that IL-10 dose- and time-dependently triggers apoptosis in these cells as detected by annexin-V staining, the nick end labeling (TUNEL) procedure, electron microscopy and analysis of DNA laddering. IL-10-induced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death and, in addition, the proteolytic activation of caspase-8 was observed. Since caspase-8 has been implicated as a regulator of apoptosis mediated by death receptors, we investigated a potential involvement of the CD95 receptor/ligand system. Indeed, treatment of monocytes with IL-10 induced a dose-dependent up-regulation of CD95 receptor and ligand expression on the monocyte surface. Furthermore, a CD95 ligand-neutralizing antibody significantly inhibited IL-10-induced apoptosis. In summary, our data show that IL-10 triggers monocyte apoptosis involving the CD95 system via an autocrine or paracrine process. Therefore, at least part of the anti-inflammatory properties of IL-10 may involve induction of apoptosis in monocytes.

Animals↗
PubMed2000

Differential gene expression in synovium of rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the major types of arthritis. Although both diseases are characterized by joint destruction, their etiologies are different. To get insights into pathophysiological pathways, we used the suppression subtractive hybridization (SSH) method to identify differentially expressed genes in RA. DNA sequencing identified 12 gene products including cytoskeletal gamma-actin and extracellular matrix components such as fibronectin, collagen III alpha(1), and superficial zone protein. Interferon gamma-inducible genes such as a novel thiol reductase, two genes of unknown function (HSIFNIN4, RING3), and annexin II were also found. Two genes encoded proteins involved in proliferation such as elongation factor 1 alpha and the granulin precursor. Furthermore, the protease cathepsin B and synovial phospholipase A2 group IIA were detected by SSH. To confirm the differential expression of the genes, we performed RT-PCR analyses of RA and OA synovial tissues. Compared to OA patients, 9 of the 12 genes were overexpressed in RA, suggesting that SSH is a powerful tool for the detection of differential gene expression in synovial tissues. Further characterization of the gene products may help to identify pathophysiological mechanisms in arthritic diseases.

Adult↗
PubMed2000

Mesalazine inhibits activation of transcription factor NF-kappaB in inflamed mucosa of patients with ulcerative colitis.

OBJECTIVES: The salicylate mesalazine is commonly used for the treatment of inflammatory bowel diseases, yet its precise mechanism of action is unknown. Because transcription factor NF-kappaB plays an important role in inflammatory bowel diseases, we investigated the effects of mesalazine therapy on NF-kappaB activation in patients with ulcerative colitis. METHODS: A total of 20 patients with moderately active ulcerative colitis received mesalazine for 8 wk. Biopsies were taken before and after drug administration and analyzed for NF-kappaB activation using an antibody specific for active NF-kappaB. RESULTS: In biopsies of active ulcerative colitis but not in noninflamed mucosa, activation of NF-kappaB was detected predominantly in macrophages. Mesalazine therapy resulted, in a strong abrogation of NF-kappaB activation in situ. CONCLUSIONS: Our results suggest that the therapeutic properties of mesalazine rely at least in part on the inhibition of NF-kappaB activation, resulting in the suppression of proinflammatory gene expression in the inflamed mucosa.

Adult↗
PubMed1999

The tyrosine kinase lck is required for CD95-independent caspase-8 activation and apoptosis in response to ionizing radiation.

Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.

Antibodies↗
PubMed1999

P2X7/P2Z purinoreceptor-mediated activation of transcription factor NFAT in microglial cells.

ATP is released from neurons and other cell types during several physiological and stress conditions under which it exerts various biological effects upon binding to purinoreceptors. A rather peculiar purinoreceptor called P2X7/P2Z is expressed on microglial and other myeloic cells. Although increasing evidence implicates an important role for P2Z in inflammatory processes, little information exists about underlying signaling pathways. Here, we report that in N9 microglial cells, extracellular ATP potently activates nuclear factor of activated T cells (NFAT), a central transcription factor involved in cytokine gene expression. ATP activated NFAT rapidly (within 1 min), whereas activation of nuclear factor kappaB was much delayed, with strikingly distinct kinetics. During ATP stimulation, both NFAT-1 and NFAT-2 were activated by a calcineurin-dependent pathway that required the influx of extracellular calcium ions. Based on the pharmacological profile, NFAT activation was specifically mediated by P2Z and not by other purinoreceptors. N9 cells that lacked P2Z but still expressed P2Y purinoreceptors failed to respond to NFAT activation. We conclude that P2Z-mediated NFAT activation may represent a novel mechanism by which extracellular ATP can modulate early inflammatory gene expression within the nervous and immune system.

Adenosine Triphosphate↗