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Henning Walczak

Publications and source records attributed to Henning Walczak.

29 records · Page 2Linked to original sources
PubMed2003

In chronic pancreatitis, widespread emergence of TRAIL receptors in epithelia coincides with neoexpression of TRAIL by pancreatic stellate cells of early fibrotic areas.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptosis. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL-R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL-R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptosis were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-gamma. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner.

Apoptosis↗
PubMed2003

Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by removing the resistance-mediating block of effector caspase maturation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-kappaB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-kappaB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

Apoptosis↗
PubMed2003

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes.

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-kappaB, and bcl-x(L) was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

Adult↗
PubMed2002

T cells require TRAIL for optimal graft-versus-tumor activity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that exhibits specific tumoricidal activity against a variety of tumors. It is expressed on different cells of the immune system and plays a role in natural killer cell-mediated tumor surveillance. In allogeneic hematopoietic-cell transplantation, the reactivity of the donor T cell against malignant cells is essential for the graft-versus-tumor (GVT) effect. Cytolytic activity of T cells is primarily mediated through the Fas-Fas ligand and perforin-granzyme pathways. However, T cells deficient for both Fas ligand and perforin can still exert GVT activity in vivo in mouse models. To uncover a potential role for TRAIL in donor T cell-mediated GVT activity, we compared donor T cells from TRAIL-deficient and wild-type mice in clinically relevant mouse bone-marrow transplantation models. We found that alloreactive T cells can express TRAIL, but the absence of TRAIL had no effect on their proliferative and cytokine response to alloantigens. TRAIL-deficient T cells showed significantly lower GVT activity than did TRAIL-expressing T cells, but no important differences in graft-versus-host disease, a major complication of allogeneic hematopoietic cell transplantation, were observed. These data suggest that strategies to enhance TRAIL-mediated GVT activity could decrease relapse rates of malignancies after hematopoietic cell transplantation without exacerbation of graft-versus-host disease.

Animals↗
PubMed2002

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Sensitive and real-time determination of H2O2 release from intact peroxisomes.

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H(2)O(2): an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H(2)O(2) turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H(2)O(2) generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H(2)O(2) can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H(2)O(2) forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H(2)O(2) in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the beta-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H(2)O(2) from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H(2)O(2) metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.

Animals↗
PubMed2002

Lack of tumor necrosis factor-related apoptosis-inducing ligand but presence of its receptors in the human brain.

Apoptosis mediated by members of the tumor necrosis factor (TNF)-nerve growth factor superfamily plays a crucial role in the interaction of the nervous and the immune system. On the one hand, it is involved in the defense mechanisms of the brain, the immune privilege. On the other hand, it is involved in the induction of glial-neuronal cell death in neuroinflammatory diseases. Here, we show that in contrast to the other known death ligands, TNF-related apoptosis-inducing ligand (TRAIL) is not constitutively expressed in the human brain, whereas both apoptosis-mediating and apoptosis-blocking TRAIL receptors are found on neurons, astrocytes, and oligodendrocytes. Thus, the brain differs from other immune-privileged organs, such as the placenta, with the TRAIL receptor-TRAIL system not being part of the immune privilege of the brain. Conversely, this death receptor-ligand system might well play an important role in T cell-mediated autoimmune diseases of the CNS such as multiple sclerosis.

Antibody Specificity↗
PubMed2002

TRAIL enhances thymidine kinase/ganciclovir gene therapy of neuroblastoma cells.

The clinical benefit of suicide gene therapy of tumors has been marginal, mostly due to the low gene transfer efficiency in vivo. The death-inducing ligand, TRAIL, effectively kills many tumor cell types, while sparing most normal tissues. We hypothesized that TRAIL may enhance HSV thymidine kinase/ganciclovir (TK/GCV) gene therapy of tumor cells by augmenting both target and bystander cell kill. Human SH-EP neuroblastoma cells expressing TK as well as bystander cells were effectively killed by apoptosis, and their clonogenicity was ablated following GCV. Human TRAIL enhanced TK/GCV-induced cell death and decreased clonogenicity of TK-expressing cells and also of bystander cells. Cooperation between TRAIL and TK/GCV depended both on caspase activation and on mitochondrial apoptogenic function because both the broad-spectrum caspase inhibitor zVAD.fmk and overexpression of Bcl-2 decreased enhancement of cell kill by TRAIL. Facilitation of TRAIL signalling by up-regulation of TRAIL receptors did not contribute to enhancement because cell surface expression of the agonistic TRAIL receptors 1 and 2 was not increased by TK/GCV. In conclusion, the concerted activation of caspases and the mitochondrial amplification of caspase activation by TK/GCV may explain the cooperative effect of TK/GCV and TRAIL on the kill of neuroblastoma cells. Because combined treatment also augmented the bystander cell kill, the addition of TRAIL may increase the efficacy of TK/GCV gene therapy of neuroblastoma.

Adenoviridae↗
PubMed2002

TRAIL and its receptors in the colonic epithelium: a putative role in the defense of viral infections.

BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and induces apoptosis by cross-linking either of the 2 TRAIL receptors containing a death domain (TRAIL-R1 or TRAIL-R2). TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. The aim of this study was to investigate the expression and function of TRAIL and its receptors in normal colonic epithelium. METHODS: TRAIL and TRAIL receptor expression was studied by reverse-transcriptase polymerase chain reaction and immunohistochemistry. TRAIL sensitivity of epithelial cells was determined in vitro. RESULTS: Normal colonic epithelial cells express TRAIL, TRAIL-R1, TRAIL-R2, and TRAIL-R4. Interestingly, TRAIL and TRAIL-R2 are coexpressed mostly in the luminal surface epithelium. Despite the expression of apoptosis-mediating TRAIL receptors, the normal colonic crypt epithelium is completely resistant to TRAIL-induced apoptosis in vitro. Using an infection model with restricted human cytomegalovirus gene expression or productive adenovirus infection in the colon carcinoma cell line Caco-2, we show that TRAIL sensitivity of colonic epithelial cells is induced on virus infection along with an up-regulation of TRAIL-R1 and TRAIL-R2 on the cell surface. CONCLUSIONS: We conclude that the TRAIL system may play a role in the early elimination of virus-infected epithelial cells in the normal gut.

Adenoviridae Infections↗
PubMed2002

Expression of TRAIL and TRAIL receptors in colon carcinoma: TRAIL-R1 is an independent prognostic parameter.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells and may be involved in protection from metastases. TRAIL receptor (TRAIL-R) 1 and TRAIL-R2, but not TRAIL-R3 and TRAIL-R4, mediate apoptosis. We examined the expression of TRAIL and its receptors in normal and neoplastic colon epithelium, and studied its correlation with prognosis in colon cancer. EXPERIMENTAL DESIGN: Immunohistochemistry was performed on normal colon mucosa (n = 10), colon adenomas (n = 20), and R0-resected Unio Internationale Contra Cancrum stage II/III colon carcinomas (n = 129). Disease-free survival was examined by Kaplan-Meier estimates and the log-rank test. Prognostic factors were determined by multivariate Cox-analysis. RESULTS: In normal colon mucosa, TRAIL and TRAIL-R2 were expressed mostly in the surface epithelium, whereas TRAIL-R1 and TRAIL-R4 were detected all along the crypt axis. In adenomas, this expression pattern was mostly retained, although some adenomas also neoexpressed TRAIL-R3. In carcinomas, the expression of TRAIL and TRAIL receptors was much more variable. TRAIL, TRAIL-R2, TRAIL-R3, and TRAIL-R4 expression did not correlate statistically with disease-free survival (multivariate analysis: P = 0.54, P = 0.67, P = 0.45, and P = 0.69, respectively), but TRAIL-R1 expression was significantly associated with disease-free survival in colon cancer (multivariate analysis: P = 0.003). CONCLUSIONS: TRAIL-R1 is an independent prognostic factor in R0-resected Unio Internationale Contra Cancrum stage II/III colon cancer.

Adenocarcinoma↗