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Biomedical subjects

K M Debatin

Publications and source records attributed to K M Debatin.

At least 55 records · Page 3Linked to original sources
PubMed2000

c-Jun-dependent CD95-L expression is a rate-limiting step in the induction of apoptosis by alkylating agents.

Mouse 3T3 fibroblasts derived from fetuses lacking c-Jun were used to define an essential role of c-Jun, a main component of the transcription factor AP-1, in the cellular response to the alkylating agent methyl methanesulfonate (MMS). MMS represents the most potent and selective activator of the stress-induced kinases JNK/SAPK and p38, resulting in very efficient induction of c-Jun hyperphosphorylation and c-jun transcription. This agent induced apoptosis with high efficiency in wild-type cells but not in c-jun(-/-) cells. Resistance to apoptosis was accompanied by impaired expression of CD95 ligand (CD95-L), a well-known inducer of apoptosis. The addition of recombinant CD95-L restored apoptosis sensitivity in c-jun(-/-) fibroblasts. MMS-induced apoptosis in wild-type fibroblasts or human lymphocytes was strongly reduced by neutralizing CD95-L antibodies or transdominant negative FADD, confirming the importance of CD95 signalling in MMS-induced apoptosis. The loss-of-function approach in fibroblasts allowed the identification and dissection of c-Jun-dependent and -independent processes upstream or downstream of CD95 activation. We have found that c-Jun can act as a proapoptotic regulator in cells exposed to DNA damage via induction of CD95-L. Once activated, CD95-induced death signalling is not affected by the loss of c-Jun, demonstrating that only the initiation and not the execution of stress-induced apoptosis depends on c-Jun.

Activating Transcription Factor 2↗
PubMed1999

Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells.

Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is based on a substrate that contains only aspartate residues linked to rhodamine 110. With this and similar substrates, we are able to detect intracellular caspase activation by flow cytometry after apoptosis induction in intact hematopoetic cell lines.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Betulinic acid: a new cytotoxic agent against malignant brain-tumor cells.

Malignant brain tumors are the most common solid tumors in children. The overall prognosis for this group of patients is still poor, emphasizing the importance of more effective therapies. Betulinic acid (Bet A) has been described as a novel cytotoxic compound active against melanoma and neuroblastoma cells. Here we report that Bet A was active against medulloblastoma and glioblastoma cell lines. In addition, Bet A exerted cytotoxic activity against primary tumor cells cultured from patients in 4 of 4 medulloblastoma-tumor samples tested and in 20 of 24 glioblastoma-tumor samples. Since a small percentage of primary-glioblastoma-tumor cells (4/24) did not respond to Bet-A treatment, resistance to Bet A might occur. Induction of apoptosis by Bet A involved mitochondrial perturbations, since inhibition of the mitochondrial permeability transition by the mitochondrion-specific inhibitor bongkrekic acid (BA) reduced Bet-A-induced apoptosis. In addition, mitochondria undergoing Bet-A-induced permeability transition triggered DNA fragmentation in isolated nuclei. Cytochrome c was released from mitochondria of Bet-A-treated cells, and might be involved in activation of caspases. Following treatment with Bet A, caspase-8, caspase-3 and PARP were proteolytically processed. Inhibition of caspase cleavage by the broad-range caspase inhibitor zVAD.fmk strongly reduced Bet-A-induced apoptosis, indicating that apoptosis was mediated by activation of caspases. Since Bet A did not exhibit cytotoxicity against murine neuronal cells in vitro, these findings suggest that Bet A may be a promising new agent for the treatment of medulloblastoma and glioblastoma cells that clearly warrants further pre-clinical and clinical evaluation.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases.

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1999

CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons.

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Animals↗
PubMed1999

Early effects of antiretroviral combination therapy on activation, apoptosis and regeneration of T cells in HIV-1-infected children and adolescents.

OBJECTIVES: To assess the relationship between apoptosis, activation and regeneration of T cells, and viral replication in paediatric patients with HIV-1 infection during antiretroviral therapy (ART). DESIGN: In 15 HIV-1-positive children and adolescents sequential blood samples were obtained during 16 episodes of ART using combinations of nucleosidic HIV-1 reverse transcriptase (RT)-inhibitors and HIV-1 protease inhibitors or non-nucleosidic RT-inhibitors. METHODS: We assessed sensitivity of freshly isolated peripheral blood T cells towards spontaneous, anti-CD95- and anti-CD3-induced apoptosis and activation before and after 6-8 weeks of ART. Expression of CD95, CD45RA, CD45RO and CD62L on CD4 and CD8 T cells and of CD34 on mononuclear cells was studied by multiparameter flow cytometry before and after 10-12 weeks of ART. RESULTS: ART caused a significant increase in absolute lymphocyte and CD4 T cell counts (P < 0.03 and P < 0.02, respectively) and a decrease in both anti-CD95- and anti-CD3-induced apoptosis of CD4 and CD8 T cells to near normal levels even in patients without complete suppression of viral replication. A significant reduction in the percentage of CD95 (but not of CD95high) CD4 T cells was observed (P < 0.005). Resting/naive cells contributed significantly (P < 0.03) to the rise in CD4 T cells especially in infants and young children. CONCLUSIONS: Different mechanisms may contribute to early T cell reconstitution in HIV-1-infected children and adolescents during ART: decreased activation-induced apoptosis leading to increased survival of circulating primed/memory T cells; decreased activation-induced naive-to-memory shift increasing the frequency of circulating resting/naive T cells; increased input of haematopoietic progenitor cells from the bone marrow into the thymus and decreased intrathymic T cell death leading to an increased thymic output of naive T cells.

Adolescent↗
PubMed1999

FK506 prevents stroke-induced generation of ceramide and apoptosis signaling.

Ceramide is a key mediator of apoptosis during the cellular stress response which is also involved in stroke-induced death. Transient occlusion of the middle cerebral artery (MCA) in rats led to a strong generation of ceramide as measured in thalamus and entorhinal cortex of the ischemic brain tissue. Enhanced levels of ceramide may be involved in apoptosis signaling following stroke since exogenously added synthetic C2-ceramide increased expression of c-jun and the death-inducing ligands (DILs) CD95-L, TRAIL and TNF-alpha in neuroblastoma cells. DILs in turn mediated death via binding to their respective receptors as concluded from diminished apoptosis upon blocking of the common pathway by dominant negative FADD. C2-ceramide induced both necrosis and apoptosis in a concentration-dependent manner corresponding to the situation present in the ischemic brain. The immunosuppressant FK506 inhibited the release of ceramide, expression of CD95-L and apoptosis in an in vitro and in vivo model for ischemia/reperfusion. These data suggest that ceramide is a crucial initiator of death, e.g., by induction of DILs following stroke.

Animals↗
PubMed1999

MycN sensitizes neuroblastoma cells for drug-induced apoptosis.

Amplification of the MYCN gene is found in a large proportion of neuroblastoma and considered as an adverse prognostic factor. To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs we used a human neuroblastoma cell line harboring tetracycline-controlled expression of MycN. Neither conditional expression of MycN alone nor low drug concentrations triggered apoptosis. However, when acting in concert, MycN and cytotoxic drugs efficiently induced cell death. Apoptosis depended on mitochondrial permeability transition and activation of caspases, since the mitochondrion-specific inhibitor bongkrekic acid and the caspase inhibitor zVAD-fmk almost completely abrogated apoptosis. Loss of mitochondrial transmembrane potential and release of cytochrome c from mitochondria preceded activation of caspase-8 and caspase-3 and cleavage of PARP. CD95 expression was upregulated by treatment with cytotoxic drugs, while MycN cooperated with cytotoxic drugs to increase sensitivity to CD95-induced apoptosis and enhancing CD95-L expression. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression, while Bcl-2 and Bcl-X(L) protein levels remained unchanged. Since amplification of MYCN is usually associated with a poor prognosis, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.

Apoptosis↗
PubMed1999

JNK/SAPK activity is not sufficient for anticancer therapy-induced apoptosis involving CD95-L, TRAIL and TNF-alpha.

We report here that stress stimuli such as gamma-irradiation or the anticancer drug doxorubicin activate expression of the death-inducing ligands (DILs) CD95-L, TNF-alpha and TRAIL. Apoptosis induced by gamma-irradiation or doxorubicin engages a FADD- and caspase-dependent apoptosis pathway which is inhibited by dominant negative FADD or the caspase inhibitor zVAD. zVAD did not prevent activity of JNK/SAPKs in response to doxorubicin suggesting that JNK/SAPK activity is independent of death receptor triggering during cellular stress-induced apoptosis. In addition, JNK/SAPKs remained activated by doxorubicin in resistant cell lines in which cleavage of caspases and apoptosis was not observed. These data uncouple JNK/SAPK activation and apoptosis signaling and indicate that cellular stress-induced apoptosis involves signaling via DILs which is paralleled by activation of JNK/SAPKs. Activation of these kinases may contribute e.g., to the expression of molecules involved in apoptosis but is not sufficient for induction of the apoptosis program following cellular stress.

Antineoplastic Agents↗
PubMed1999

Angiotensin I converting enzyme and angiotensinogen gene polymorphisms related to 24-h blood pressure in paediatric type I diabetes mellitus.

UNLABELLED: The aim of this study was to evaluate two putative predictive genetic markers for hypertension in children and adolescents with diabetes mellitus. Ambulatory blood pressure measurements were performed in 199 patients with type I diabetes mellitus (mean age 16.5 years, mean duration of diabetes 7.7 years) and compared to those of 1141 healthy children. The local allele frequencies were established based on a control population consisting of 181 healthy subjects. The allele frequencies of the angiotensinogen gene M235T polymorphism was nearly identical in insulin-dependent diabetes mellitus patients (MM 33%, MT 51%, TT 16%) and controls (MM 35%, MT 49%, TT 16%). In contrast, the genotype distribution of the angiotensin I converting enzyme gene insertion/deletion (I/D) polymorphism was different between patients with type I diabetes mellitus (DD 26%, ID 49%, II 25%) and the control group (DD 37%, ID 44%, II 19%) (P=0.04). Relative nocturnal systolic and diastolic pressures in patients with diabetes were higher than in healthy age- and height-matched controls; no association was found with the angiotensinogen gene M235T polymorphism. Relative nocturnal diastolic blood pressure was higher in patients homozygous for the I allele of the angiotensin I converting enzyme gene. CONCLUSION: Nocturnal systolic and diastolic blood pressure is higher in patients with type I diabetes than in healthy children. The formerly described, but controversial, association of the M235T polymorphism with arterial hypertension could not be confirmed in this study.

Adolescent↗
PubMed1999

Expression of CD69 on T-cells from HIV-1-infected children and adolescents increases with increasing viral load.

We evaluated the use of a whole-blood assay that measures spontaneous and activation-induced CD69 expression on peripheral blood T-cells in vitro for assessment of T-cell function in HIV-1-infected paediatric patients. Heparinized venous blood from 28 HIV-1 positive children and adolescents and 23 healthy controls was incubated for 4 h with or without 5 microg/ml phytohaemagglutinin (PHA). Thereafter, analysis of CD69 expression on CD4+ and CD8+ T-cells was done by flow cytometry; simultaneously we determined CD4+ T-cell counts and plasma HIV-1 viral load. Neither spontaneous nor PHA-induced CD69 expression differed significantly between HIV-1 positive patients and healthy controls. However, T-cells from HIV-1 positive patients with plasma HIV-1 viral load levels above 70x10(3) copies/ml showed a higher spontaneous CD69 expression than T-cells from patients with lower plasma viral load levels in different stages of the disease. Antiretroviral treatment in four patients reduced spontaneous CD69 expression in CD4+ T-cells and PHA-induced CD69 expression in CD4+ and CD8+ T-cells significantly after 8 weeks of therapy. Conclusion Spontaneous and activation-induced expression of the early (activation) antigen CD69 on peripheral blood T-cells does not distinguish HIV-1 positive patients from HIV-1 negative healthy controls and is not correlated with peripheral blood CD4+ T-cell counts. This test may not be a reliable marker for functional T-cell deficiency during early stages of HIV disease. Increased spontaneous as well as PHA-induced CD69 expression on T-cells from HIV-1-infected children and adolescents in vitro may rather reflect HIV-induced pre-activation of T-cells in vivo.

Adolescent↗
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

Definition of a critical T cell threshold for prevention of GVHD after HLA non-identical PBPC transplantation in children.

The aim of this study was to reduce the rate of graft failure after HLA non-identical stem cell transplantation by using G-CSF mobilized CD34+ peripheral blood progenitor cells (PBPC), either in combination with bone marrow or as single grafts. To prevent GVHD, PBPC were highly purified, resulting in a 5 to 6 log T cell depletion. In additon to T cell depletion no further GVHD prophylaxis was used. We transplanted 23 pediatric patients with life-threatening malignant or non-malignant hematological disorders, who had no available matched donor. Engraftment was obtained in 18 of 21 evaluable patients. Five patients developed acute GVHD of grade II and III, which became chronic in four cases and was fatal in four. The use of highly purified PBPC allowed the exact quantification of residual T cells in the grafts and a strict correlation between the residual T cell load and the development of GVHD was observed: patients with GVHD had received numbers of T cells between 8 and 20 x 104/kg, whereas patients without GVHD were grafted with T cell numbers ranging from 0.7 to 6.0 x 104/kg. We therefore clearly demonstrate that a residual T cell content of <5 x 104/kg is safe for prevention of GVHD after HLA non-identical PBPC transplantation in children.

Adolescent↗
PubMed1999

JNK/SAPK activity contributes to TRAIL-induced apoptosis.

We report here that JNK/SAPKs are activated by TRAIL in parallel to induction of apoptosis in human T and B cell lines. Death signaling as well as JNK/SAPK activation by TRAIL in these cells is FADD- and caspase-dependent since dominant-negative FADD or the caspase inhibitor zVAD prevented both, apoptosis and JNK/SAPK activity. JNK/SAPK activity in response to triggering of CD95 by an agonistic antibody (alphaAPO-1) was also diminished by dominant-negative FADD or zVAD. Correspondingly, a cell line resistant to alphaAPO-1-induced death exhibited crossresistance to TRAIL-induced apoptosis and did not upregulate JNK/SAPK activity in response to TRAIL or alphaAPO-1. Inhibition of JNK/SAPK activity, by stably transfecting cells with a dominant-negative JNKK-MKK4 construct, reduced apoptosis in response to TRAIL or alphaAPO-1. Therefore, activation of JNK/SAPKs by TRAIL or alphaAPO-1 occurs downstream of FADD and caspases and contributes to apoptosis in human lymphoid cell lines.

Antibodies↗
PubMed1999

Induction of CD95 ligand and apoptosis by doxorubicin is modulated by the redox state in chemosensitive- and drug-resistant tumor cells.

Induction of CD95 ligand (CD95-L) may contribute to drug-induced apoptosis in chemosensitive leukemias and solid tumors. Here we report that induction of CD95-L and apoptosis by doxorubicin in leukemic and neuroblastoma cells is regulated by the redox state and reactive oxygen species (ROS). Preincubation of chemosensitive cells with antioxidants such as N-acetyl-cysteine (NAC) or glutathione (GSH), significantly reduced doxorubicin-induced apoptosis, hyperexpression of ROS, loss of mitochondrial membrane potential (DeltaPsim) and upregulation of CD95-L expression. Doxorubicin-resistant cells exhibited higher levels of GSH in comparison to chemosensitive cells and were deficient in hyperproduction of ROS, loss of DeltaPsim and upregulation of CD95-L in response to cytotoxic drugs. Downregulation of intracellular GSH concentrations reversed deficient drug-induced hyperproduction of ROS and CD95-L upregulation. In addition, overexpression of Bcl-XL in CEM cells blocked doxorubicin-triggered ROS and CD95-L expression. These findings suggest that induction of CD95-L by cytotoxic drugs is modulated by the cellular redox state and mitochondria derived ROS.

Acetylcysteine↗
PubMed1999

Induction of apoptosis by IFNgamma in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit.

The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferongamma (IFNgamma) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNgamma through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNgamma-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNgamma stimulation, (iii) was at least partially inhibited by using blocking F(ab')2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNgamma are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNgamma may have a significant potential in the therapy of neuroblastoma in vivo.

Apoptosis↗