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Biomedical subjects

Klaus-Michael Debatin

Publications and source records attributed to Klaus-Michael Debatin.

At least 37 records · Page 2Linked to original sources
PubMed2005

Tumor necrosis factor-related apoptosis-inducing ligand-mediated proliferation of tumor cells with receptor-proximal apoptosis defects.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might represent a future cytotoxic drug to treat cancer as it induces apoptosis in tumor cells without toxicity in animal trials. We recently described that in contrast to apoptosis, TRAIL mediates tumor cell survival and proliferation in certain tumor cells. Here we studied the effect of TRAIL on 18 cell lines and 53 primary leukemia cells and classified these tumor cells into four groups: TRAIL, anti-DR4 or anti-DR5 induced apoptosis in group A cells, whereas they had no effect on group 0 cells and mediated proliferation in group P cells. To our surprise, TRAIL induced simultaneous apoptosis and proliferation in group AP cells. More than 20% of all cells tested belonged to group P and showed TRAIL-mediated proliferation even in the presence of certain cytotoxic drugs but not inhibitors of nuclear factor-kappaB. Transfection with B-cell leukemia/lymphoma protein 2 transformed group A cells into group 0 cells, whereas transfection with Fas-associated polypeptide with death domain (FADD)-like interleukin-1-converting enzyme-inhibitory protein (FLIP) transformed them into group AP cells. Loss of caspase-8 or transfection of dominant-negative FADD transformed group A cells into group P cells. Taken together, our data suggest that proliferation is a frequent effect of TRAIL on tumor cells, which is related to receptor-proximal apoptosis defects at the level of the death-inducing signaling complex and should be prevented during antitumor therapy with TRAIL.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Caspase-8L expression protects CD34+ hematopoietic progenitor cells and leukemic cells from CD95-mediated apoptosis.

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.

Antigens, CD↗
PubMed2005

CD95/Apo-1/Fas: independent cell death induced by doxorubicin in normal cultured cardiomyocytes.

Doxorubicin is a commonly used cytotoxic drug for effective treatment of both solid tumors and leukemias, which may cause severe cardiac adverse effects leading to heart failure. In certain tumor cells, doxorubicin-induced cell death is mediated by death receptors such as CD95/Apo-1/Fas. Here we studied the role of death receptors for doxorubicin-induced cell death in primary neonatal rat cardiomyocytes and the embryonic cardiomyocytic cell line H9c2.1. Doxorubicin-induced cell death of cardiomyocytes was associated with cleavage of caspases 3 and 8, a drop in mitochondrial transmembrane potential, and release of cytochrome c. Doxorubicin-treated cardiomyocytes secreted death-inducing ligands into the culture supernatant, but remained resistant toward cell death induction by death receptor triggering. In contrast to the chelator dexrazoxane, blockade of death receptor signaling by stable overexpression of transdominant negative adapter molecule FADD did not inhibit doxorubicin-induced cell death. Our data suggest that cultured cardiomyocytes secrete death-inducing ligands, but undergo death receptor-independent cell death upon exposure to doxorubicin.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Growth inhibition of murine neuroblastoma cells by c-myc with cell cycle arrest in G2/M.

c-myc and N-myc belong to the myc family of proteins that plays an important role in cell proliferation, differentiation and apoptosis. The N-myc gene is amplified in aggressive neuroblastoma and c-myc is overexpressed in many lymphomas and cancers. However, c-myc has not been implied in tumorigenesis or progression of neuroblastoma. We therefore investigated the so far unknown effects of c-myc overexpression on the aggressiveness of neuroblastoma cells with single copy N-myc. c-myc overexpression in serum-deprived murine NXS2 neuroblastoma cells led to cell cycle progression and massive apoptosis, causing a net decrease of viable cells. In serum-replete medium c-myc caused NXS2 cells to arrest in G2/M. Furthermore, c-myc decreased clonogenic growth of neuroblastoma cells. Taken together, these data suggest that c-myc attenuates the malignant phenotype of NXS2 neuroblastoma cells. Thus, although c-myc increased NXS2 tumor mass in vivo, c-myc appears to have decreased malignant potency in neuroblastoma cells compared to N-myc. This may be one reason why c-myc does not play a role in neuroblastomagenesis.

Animals↗
PubMed2005

Resveratrol-mediated sensitisation to TRAIL-induced apoptosis depends on death receptor and mitochondrial signalling.

Natural food products such as resveratrol have gained considerable attention as cancer chemopreventive agents. In the present study, we investigated the potential of resveratrol to overcome the resistance of tumour cells against TRAIL. While resveratrol enhanced TRAIL-induced apoptosis through G1 cell cycle arrest and survivin depletion, resveratrol failed to sensitise cells with high expression levels of Bcl-2 or FADD-DN. Interestingly, overexpression of Bcl-2 or FADD-DN did not interfere with resveratrol-mediated cell cycle arrest or survivin depletion, but blocked release of cytochrome c and Smac from mitochondria into the cytosol, enhanced caspase activation and apoptosis upon combined treatment with resveratrol and TRAIL indicating that overexpression of Bcl-2 or FADD-DN decoupled the effect of resveratrol on the cell cycle and apoptosis. Similarly, cell cycle arrest at G1 using the cell cycle specific inhibitor mimosine or downregulation of survivin expression by antisense oligonucleotides failed to enhance TRAIL-induced apoptosis in Bcl-2- or FADD-DN-transfected cells. Likewise, inhibition of caspase activity using the broad range caspase inhibitor zVAD.fmk did not interfere with resveratrol-mediated cell cycle arrest and survivin depletion, while blocking apoptosis upon combined treatment with resveratrol and TRAIL. Thus, resveratrol is a potent sensitiser for TRAIL in certain tumours. However, it may be ineffective in others, e.g. in tumours with enhanced Bcl-2 expression or defective death receptor signalling.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Sensitization for anticancer drug-induced apoptosis by betulinic Acid.

We previously described that betulinic acid (BetA), a naturally occurring pentacyclic triterpenoid, induces apoptosis in tumor cells through the mitochondrial pathway. Here, for the first time, we provide evidence that BetA cooperated with anticancer drugs to induce apoptosis and to inhibit clonogenic survival of tumor cells. Combined treatment with BetA and anticancer drugs acted in concert to induce loss of mitochondrial membrane potential and the release of cytochrome c and Smac from mitochondria, resulting in activation of caspases and apoptosis. Overexpression of Bcl-2, which blocked mitochondrial perturbations, also inhibited the cooperative effect of BetA and anticancer drugs, indicating that cooperative interaction involved the mitochondrial pathway. Notably, cooperation of BetA and anticancer drugs was found for various cytotoxic compounds with different modes of action (e.g., doxorubicin, cisplatin, Taxol, VP16, or actino-mycin D). Importantly, BetA and anticancer drugs cooperated to induce apoptosis in different tumor cell lines, including p53 mutant cells, and also in primary tumor cells, but not in human fibroblasts indicating some tumor specificity. These findings indicate that using BetA as sensitizer in chemotherapy-based combination regimens may be a novel strategy to enhance the efficacy of anticancer therapy, which warrants further investigation.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

TSH receptor mutation V509A causes familial hyperthyroidism by release of interhelical constraints between transmembrane helices TMH3 and TMH5.

Mutations of the human thyrotrophin receptor (TSH-R) are a cause of thyroid adenomas and hyperthyroidism. Here we study mechanisms of receptor activation in a genomic TSH-R variant V509A located in transmembrane helix (TMH) 3, which we identify in a family with congenital hyperthyroidism, multiple adenomas and follicular thyroid cancer. Using molecular modelling and dynamic simulation, we predicted the release of amino acid residue A593 (located opposite in domain TMH5) from a tight 'knob-and-hole' interaction with TMH3, physiologically constrained in the native receptor state by the bulky side chain of V509. To experimentally validate this concept, we generated mutant TSH-R expression constructs for functional in vitro studies. TSH-R mutant V509A showed a 2.8-fold increase in basal cAMP production, confirming constitutive TSH-R activation. The addition of a second site suppressor mutant A593V to TSH-R V509A resulted in the normalization of basal cAMP release, and the dose-responsiveness to TSH ligand was maintained. These data thus demonstrate that TSH-R V509A activation is caused by the release of TMH3-TMH5 interhelical constraints, while the native TSH-R conformation is re-stabilized by the introduction of a spacious valine residue at position 593. In conclusion, we delineate a novel mechanism of constitutive TSH-R activation, leading to thyroid hyperfunction and neoplasia.

Adenoma↗
PubMed2005

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and VHL-haplotype analysis in patients with presumable congenital erythrocytosis.

BACKGROUND AND OBJECTIVES: Congenital erythrocytoses or polycythemias are rare and heterogeneous. A homozygous mutation (C598T->Arg200Trp) in the von Hippel-Lindau (VHL) gene was originally identified as the cause of the endemic Chuvash polycythemia. Subsequently this and other mutations in the VHL gene were also detected in several patients of different ethnic origin. Haplotype analyses of the VHL gene suggested a common origin for the Chuvash-type mutation. DESIGN AND METHODS: Thirty-four patients with presumable congenital erythrocytosis due to an unknown underlying disorder were examined for VHL gene mutations and VHL region haplotypes. RESULTS: Four patients were homozygous and one patient heterozygous for the Chuvash-type mutation. One additional patient presented a previously not described heterozygous mutation G311->T VHL in exon 1. The haplotype analyses were in agreement with recently published data for three of the four patients with homozygous mutations as well as for the patient with a heterozygous Chuvash-type mutation. One patient of Turkish origin with homozygous Chuvash-type mutation had a haplotype not previously found in individuals with Chuvash-type mutation. INTERPRETATION AND CONCLUSIONS: These results confirm that mutations in the VHL gene are responsible for a substantial proportion of patients with congenital erythrocytoses. Erythrocytoses due to a C598->T mutation of the VHL gene are not geographically restricted. The majority of patients with Chuvash polycythemia share a common VHL gene haplotype. The different haplotype in one of the patients with Chuvash-type mutation indicates that this mutation was not spread only from a single founder but developed independently in other individuals.

Adolescent↗
PubMed2004

Exploiting death receptor signaling pathways for tumor therapy.

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. Tipping the balance between cell death and proliferation in favor of cell survival may result in tumor formation. Moreover, current cancer therapies, e.g. chemotherapy, gamma-irradiation, immunotherapy or suicide gene therapy, primarily exert their antitumor effect by triggering an evolutionary conserved apoptosis program in cancer cells. For example, death receptor signaling has been implied to contribute to the efficacy of cancer therapy. Thus, failure to undergo apoptosis in response to anticancer therapy because of defects in death receptor pathways may result in resistance. Further insights into the mechanisms regulating apoptosis in response to anticancer therapy and how cancer cells evade cell death may provide novel opportunities for targeted therapeutics. Thus, agents designed to selectively activate death receptor pathways may enhance the efficacy of conventional therapies and may even overcome some forms of cancer resistance.

Apoptosis↗
PubMed2004

Cooperation of betulinic acid and TRAIL to induce apoptosis in tumor cells.

We previously reported that the TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand)-induced death signal requires amplification by mitochondria in certain cell types, for example, in type II cells. Here, we provide for the first time evidence that the natural compound betulinic acid (BetA) cooperated with TRAIL to induce apoptosis in tumor cells. Through functional complementation, simultaneous stimulation of the death receptor pathway by TRAIL and the mitochondrial pathway by BetA resulted in complete activation of effector caspases, apoptosis and inhibition of clonogenic survival. BetA and TRAIL cooperated to trigger loss of mitochondrial membrane potential and release of cytochrome c and Smac from mitochondria. Also, combination treatment with BetA and TRAIL resulted in increased cleavage of caspase-8 and Bid indicating that activation of effector caspases may feed back in a positive amplification loop. Importantly, the combination treatment with BetA and TRAIL cooperated to induce apoptosis in different tumor cell lines and also in primary tumor cells, but not in normal human fibroblasts indicating some tumor specificity. Since most human cancers represent type II cells, triggering the mitochondrial pathway by BetA may be a novel approach to enhance the efficacy of TRAIL-based therapies, which warrants further investigation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Sensitization for anticancer drug-induced apoptosis by the chemopreventive agent resveratrol.

Current attempts to improve the survival of cancer patients largely depend on strategies to target tumor cell resistance. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we report that resveratrol acts as potent sensitizer for anticancer drug-induced apoptosis by inducing cell cycle arrest, which in turn resulted in survivin depletion. Concomitant analysis of cell cycle and apoptosis revealed that pretreatment with resveratrol resulted in cell cycle arrest in S phase and apoptosis induction preferentially out of S phase upon subsequent drug treatment. Likewise, cell cycle arrest in S phase by cell cycle inhibitors enhanced drug-induced apoptosis. Resveratrol-mediated cell cycle arrest sensitized for apoptosis by downregulating survivin expression through transcriptional and post-transcriptional mechanisms. Similarly, downregulation of survivin expression using survivin antisense oligonucleotides sensitized for drug-induced apoptosis. Importantly, downregulation of survivin and enhanced drug-induced apoptosis by resveratrol occurred in various human tumor cell lines irrespective of p53 status. Thus, this combined sensitizer (resveratrol)/inducer (cytotoxic drugs) concept may be a novel strategy to enhance the efficacy of anticancer therapy in a variety of human cancers.

Anticarcinogenic Agents↗
PubMed2004

Compound heterozygous and homozygous mutations of the TSHbeta gene as a cause of congenital central hypothyroidism in Europe.

BACKGROUND: Thyroid hormones are crucial for normal growth and central nervous system development. In recent years, germline variants of the TSHbeta subunit gene have been identified as a cause of congenital TSH deficiency. METHODS: We performed a genetic and clinical study in children from four European countries diagnosed with congenital isolated central hypothyroidism. RESULTS: TSHbeta gene analysis revealed compound heterozygosity for 145C-->T (Q49X) and 313delT (C105Vfs114X) in 1 infant and homozygous mutation 313delT (C105Vfs114X) in 5 patients. Although all presented with typical symptoms of hypothyroidism, diagnosis and treatment was delayed until 3-5 months in 5 of 6 patients. In a longitudinal sibpair analysis, thyroxine substitution initiated immediately after birth was effective to prevent developmental delay and growth retardation. CONCLUSION: Clinical awareness is required to detect hypothyroidism due to TSHbeta mutations, which is not identified by TSH-based newborn screening. TSHbeta variants C105Vfs114X and Q49X are the most frequent cause of this severe disorder in Europe, now for the first time observed in compound heterozygous state.

Child↗
PubMed2004

Mutation analysis of the apoptotic "death-receptors" and the adaptors TRADD and FADD/MORT-1 in osteosarcoma tumor samples and osteosarcoma cell lines.

Apoptosis is a key mechanism of the organism that regulates embryogenesis and development, maintains homeostasis of the immune system and removes potentially hazardous cells. A dysregulation of apoptosis signaling may thus disturb the balance of cell survival and cell death, leading to the development of several diseases including cancer. In order to determine whether osteosarcomas display an increased frequency of genetic alterations that affect apoptosis signaling, we analyzed the death domains of the death receptor genes CD95/Fas/Apo1, TNFR1, DR3/Apo3/WSL-1/LARD/TRAMP, DR5/TRAIL-R2/TRICK2/KILLER, DR6 and the complete coding sequences of the death receptor gene DR4/TRAIL-R1 and the genes of the adaptors TRADD and FADD/MORT-1. The investigation included 15 osteosarcoma tumor samples, 3 osteosarcoma cell lines (SAOS-2, HOS and MG63) and peripheral blood from 20 donors as controls. We were able to identify 4 different sequence variations within the DR4 gene located on exons 3, 4, 5 and 10 (death-domain). No alterations have been detected in the other genes or exons investigated. Except the sequence variant affecting exon 4, the alterations were homozygous in 15% of the tumor samples and cell lines, whereas the same alterations found in the control group were heterozygous or even not detectable. Three out of 4 alterations are located in the receptor's extracellular cysteine rich domain, which contains the ligand binding area and 1 on exon 10 coding for the death-domain. They may thus exert influence on ligand-receptor interactions and subsequent apoptosis induction. Our findings suggest that homozygous genetic alterations within the DR4 gene may be implicated in the formation of osteosarcoma.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Death receptors in chemotherapy and cancer.

Apoptosis, the cell's intrinsic death program, is a key regulator of tissue homeostasis. An imbalance between cell death and proliferation may result in tumor formation. Also, killing of cancer cells by cytotoxic therapies such as chemotherapy, gamma-irradiation or ligation of death receptors is predominantly mediated by triggering apoptosis in target cells. In addition to the intrinsic mitochondrial pathway, elements of death receptor signaling pathways have been implied to contribute to the efficacy of cancer therapy. Failure to undergo apoptosis in response to anticancer therapy may lead to resistance. Also, deregulated expression of death receptor pathway molecules may contribute to tumorigenesis and tumor escape from endogenous growth control. Understanding the molecular events that regulate apoptosis induced by anticancer therapy and how cancer cells evade apoptosis may provide new opportunities for pathway-based rational therapy and for drug development.

Animals↗
PubMed2004

Rescue of death receptor and mitochondrial apoptosis signaling in resistant human NSCLC in vivo.

Non small cell lung carcinoma (NSCLC) is a highly lethal malignancy that often becomes resistant to chemotherapy. To determine whether alterations in apoptotic signaling might contribute to such resistance, we established in vitro and in vivo models for sensitive and resistant human NSCLC. We found that resistance is due to multiple defects found in expression of CD95-L, CD95 and members of the Bcl-2 and IAP family, as well as caspase-8, -9 and -3 as examined by immunohistochemistry, Western blot analysis, gene array analysis and functional assays. Failure to activate death receptor, as well as mitochondrial apoptosis signaling, points to a central role of caspases. To restore apoptosis signaling we transfected NSCLC xenografts on nude mice with caspase-8 and -9. This treatment strongly induced apoptosis per se and sensitized the tumors to cisplatin-induced cell death. Thus, these findings indicate that re-expression of caspases might be an effective strategy to restore sensitivity for chemotherapy in NSCLC in vivo.

Animals↗
PubMed2004

Apoptosis pathways in cancer and cancer therapy.

Activation of apoptosis pathways is a key mechanism by which cytotoxic drugs kill tumor cells. Also immunotherapy of tumors requires an apoptosis sensitive phenotype of target cells. Defects in apoptosis signalling contribute to resistance of tumors. Activation of apoptosis signalling following treatment with cytotoxic drugs has been shown to lead to activation of the mitochondrial (intrinsic) pathway of apoptosis. In addition, signalling through the death receptor (extrinsic) pathways, contributes to sensitivity of tumor cells towards cytotoxic treatment. Both pathways converge finally at the level of activation of caspases, the effector molecules in most forms of cell death. In addition to classical apoptosis, non-apoptotic modes of cell death have recently been identified. Mechanisms to overcome apoptosis resistance include direct targeting of antiapoptotic molecules expressed in tumors as well as re-sensitization of previously resistant tumor cells by re-expression of caspases and counteracting apoptotis inhibitory molecules such as Bcl-2 and molecules of the IAP family of endogenous caspase inhibitors. Molecular insights into regulation of apoptosis and defects in apoptosis signalling in tumor cells will provide novel approaches to define sensitivity or resistance of tumor cells towards antitumor therapy and provide new targets for rational therapeutic interventions for future therapeutic strategies.

Apoptosis↗
PubMed2004

Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol.

Survivin is a member of the inhibitor of apoptosis proteins that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we discovered a novel function of the chemopreventive agent resveratrol: resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin depletion. Concomitant analysis of cell cycle, survivin expression, and apoptosis revealed that resveratrol-induced G(1) arrest was associated with down-regulation of survivin expression and sensitization for TRAIL-induced apoptosis. Accordingly, G(1) arrest using the cell cycle inhibitor mimosine or induced by p21 overexpression reduced survivin expression and sensitized cells for TRAIL treatment. Likewise, resveratrol-mediated cell cycle arrest followed by survivin depletion and sensitization for TRAIL was impaired in p21- deficient cells. Also, down-regulation of survivin using survivin antisense oligonucleotides sensitized cells for TRAIL-induced apoptosis. Importantly, resveratrol sensitized various tumor cell lines, but not normal human fibroblasts, for apoptosis induced by death receptor ligation or anticancer drugs. Thus, this combined sensitizer (resveratrol)/inducer (e.g., TRAIL) strategy may be a novel approach to enhance the efficacy of TRAIL-based therapies in a variety of human cancers.

Anticarcinogenic Agents↗