Publications by authors named "Csaba Mahotka"

29 Publications

  • Page 1 of 1

Nucleolin promotes Wnt signaling in human hematopoietic stem/progenitor cells.

Leukemia 2019 04 22;33(4):1052-1054. Epub 2019 Feb 22.

Medical Faculty, Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University, Düsseldorf, Germany.

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http://dx.doi.org/10.1038/s41375-019-0401-4DOI Listing
April 2019

Nucleolin promotes execution of the hematopoietic stem cell gene expression program.

Leukemia 2018 08 8;32(8):1865-1868. Epub 2018 Mar 8.

Institute of Pathology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.

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http://dx.doi.org/10.1038/s41375-018-0090-4DOI Listing
August 2018

The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling.

Cell Commun Signal 2017 05 2;15(1):16. Epub 2017 May 2.

Institute of Pathology, Heinrich Heine University Hospital, Medical Faculty, Moorenstrasse 5, 40225, Düsseldorf, Germany.

Background: Renal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain).

Methods: Expression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student's t-test.

Results: Regarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells.

Conclusion: Taken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols.
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http://dx.doi.org/10.1186/s12964-017-0170-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414156PMC
May 2017

Discovery of Novel, Potent, and Specific Cell-Death Inducers in the Jurkat Acute Lymphoblastic Leukemia Cell Line.

ChemMedChem 2015 Oct 12;10(10):1700-6. Epub 2015 Aug 12.

Department of Pathology, Medical Faculty, Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf (Germany).

The limited clinical efficacy of many cancer therapeutics has initiated intense research efforts toward the discovery of novel chemical entities in this field. In this study, 31 hit candidates were selected from nearly 800,000 database compounds in a ligand-based virtual screening campaign. In turn, three of these hits were found to have (sub)micromolar potencies in proliferation assays with the Jurkat acute lymphatic leukemic cell line. In this assay, the three hits were found to exhibit higher potency than clinically tested cell-death inducers (GDC-0152, AT-406, and birinapant). Importantly, antiproliferative activity toward non-cancer peripheral blood mononuclear cells (PBMCs) was found to be marginal. Further biological characterization demonstrated the cell-death-inducing properties of these compounds. Biological testing of hit congeners excluded a nonspecific, toxic effect of the novel structures. Altogether, these findings may have profound relevance for the development of clinical candidates in tumor therapy.
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http://dx.doi.org/10.1002/cmdc.201500245DOI Listing
October 2015

Expression of TRAIL-splice variants in gastric carcinomas: identification of TRAIL-γ as a prognostic marker.

BMC Cancer 2013 Aug 12;13:384. Epub 2013 Aug 12.

Department of Surgery A, Heinrich Heine University and University Hospital Duesseldorf, Moorenstrasse 5, 40225 Duesseldorf, Germany.

Background: TNF-related apoptosis inducing ligand (TRAIL) belongs to the TNF-superfamily that induces apoptotic cell death in a wide range of neoplastic cells in vivo as well as in vitro. We identified two alternative TRAIL-splice variants, i.e. TRAIL-β and TRAIL-γ that are characterized by the loss of their proapoptotic properties. Herein, we investigated the expression and the prognostic values of the TRAIL-splice variants in gastric carcinomas.

Methods: Real time PCR for amplification of the TRAIL-splice variants was performed in tumour tissue specimens and corresponding normal tissues of 41 consecutive patients with gastric carcinoma. Differences on mRNA-expression levels of the TRAIL-isoforms were compared to histo-pathological variables and correlated with survival data.

Results: All three TRAIL-splice variants could be detected in both non-malignant and malignant tissues, irrespective of their histological staging, grading or tumour types. However, TRAIL-β exhibited a higher expression in normal gastric tissue. The proapoptotic TRAIL-α expression was increased in gastric carcinomas when compared to TRAIL-β and TRAIL-γ. In addition, overexpression of TRAIL-γ was associated with a significant higher survival rate.

Conclusions: This is the first study that investigated the expression of TRAIL-splice variants in gastric carcinoma tissue samples. Thus, we provide first data that indicate a prognostic value for TRAIL-γ overexpression in this tumour entity.
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http://dx.doi.org/10.1186/1471-2407-13-384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751299PMC
August 2013

Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

J Biol Chem 2011 Jun 10;286(22):19576-88. Epub 2011 Apr 10.

Institute of Pathology, Heinrich Heine University, University Hospital, Medical Faculty, Düsseldorf, Germany.

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-β and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-β and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-β and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-β and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-β/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-β and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.
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http://dx.doi.org/10.1074/jbc.M110.196311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103337PMC
June 2011

Rb and nucleolin antagonize in controlling human CD34 gene expression.

Cell Signal 2011 Aug 1;23(8):1358-65. Epub 2011 Apr 1.

Department of Pediatric Oncology, Hematology and Clinical Immunology, Center for Child and Adolescent Health, Heinrich Heine University, Düsseldorf, Germany.

Retinoblastoma protein (Rb) controls cell proliferation, differentiation, survival and gene expression and it has a central role in the signaling network that provides a cell cycle checkpoint in the G1 phase of the cell cycle. Studies in mice have shown that Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment and it acts as a critical regulator of hematopoietic stem and progenitor cells under stress. In human hematopoiesis, the CD34 protein is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution, and CD34 has a role in the differentiation of hematopoietic cells. Here we find that, in CD34-positive hematopoietic cells, Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin to provide a mechanism that links expression of endogenous CD34 to cell cycle progression. Our study suggests a direct involvement of Rb in the transcriptional program of human CD34-positive hematopoietic stem/progenitor cells, thus providing further insights into the molecular network relevant to the features of these cells.
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http://dx.doi.org/10.1016/j.cellsig.2011.03.018DOI Listing
August 2011

UVA-induced phenoxyl radical formation: A new cytotoxic principle in photodynamic therapy.

Free Radic Biol Med 2010 Sep 7;49(6):1129-37. Epub 2010 Jul 7.

Department of Plastic and Reconstructive Surgery, Hand Surgery, and Burn Center, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany.

Psoralens are regularly used in therapy in combination with ultraviolet A light irradiation (PUVA) to treat skin diseases such as psoriasis, vitiligo, and mycosis fungoides. PUVA therapy is also used within the scope of extracorporeal photopheresis to treat a variety of diseases that have a suspected involvement of pathogenic T cells, including rejection of organ transplants, graft-vs-host disease, cutaneous T cell lymphoma, and autoimmune disorders. Because psoralens are the only photosensitizers used in PUVA therapies and are considered to be responsible for a number of side effects, the identification of alternative drugs is of practical interest. Here we investigated the impact of activated Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic vitamin E analog lacking the phytyl tail, as an alternative photoactivatable agent with T cell cytotoxic properties. Despite the well-known antioxidative capacity of Trolox, we found that at low UVA doses and in the presence of supraphysiological concentration of nitrite, a natural constituent of human skin, this compound selectively enhances radical-mediated cytotoxicity toward T cells but not toward human skin fibroblasts, keratinocytes, or endothelial cells. The cytotoxic mechanism comprises a reaction of Trolox with photo-decomposition products of nitrite, which leads to increased Trolox phenoxyl radical formation, increased intracellular oxidative stress, and a consecutive induction of apoptosis and necrosis in fast proliferating T cells. Thus, the identified UVA/nitrite-induced phenoxyl radical formation provides an opportunity for a new cytotoxic photodynamic therapy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.06.031DOI Listing
September 2010

Regulation of p53 isoform expression in renal cell carcinoma.

Front Biosci (Elite Ed) 2010 Jun 1;2:1042-53. Epub 2010 Jun 1.

Institute of Pathology, Heinrich-Heine University Hospital, 40225 Duesseldorf, Germany.

Differential expression of p53 isoforms might participate in the marked resistance towards conventional chemotherapy of renal cell carcinomas (RCCs). Therefore, we analysed their differential expression and regulation in RCCs. RCCs expressed a more p53 activating isoform pattern during tumor initiation and progression, in vivo. In vitro, two cell lines exhibiting a similar sensitivity towards Topotecan-induced cell death revealed a similar induction of p53 target genes but strongly differed in their extent of apoptosis. Furthermore, they strongly differed in their basal expression patterns and differential regulation of the isoforms. In conclusion, our study examined for the first time the differential expression and regulation of all p53 isoforms in a tumor in vivo. Furthermore, novel results in our in vitro studies show that p53 isoforms are strongly differentially regulated by chemotherapy in RCCs and that expression and regulation of so-called "p53-target genes" are obviously at least in part regulated by other transcription factors. In addition, our original findings show that p53 isoform expression in RCC cell lines is of minor importance for sensitivity towards chemotherapy.
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http://dx.doi.org/10.2741/e162DOI Listing
June 2010

PIDDosome expression and the role of caspase-2 activation for chemotherapy-induced apoptosis in RCCs.

Cell Oncol 2010 ;32(1-2):29-42

Institute of Pathology, Heinrich-Heine University Hospital, Düsseldorf, Germany.

Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs) of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro.

Methods: The analyses were performed by semiquantitative real-time PCR, Western Blot and Caspase-2 Assay.

Results: Our in vivo results showed an overall decrease in proapoptotic caspase-2L expression during tumour progression due to an increase in the relative share of caspase-2S mRNA in total caspase-2 mRNA expression. Furthermore, an increase in the expression of PIDD and RAIDD could be observed. In contrast, antiapoptotic BCL-2 expression increased only during early tumour stages, whereas expression decreased in pT3 RCCs. In vitro, caspase-2 activation in RCC cell lines coincidenced with sensitivity of tumour cells towards Topotecan-induced apoptosis. However, inhibition of caspase-2 could not prevent Topotecan-induced apoptosis. Interestingly, Topotecan-resistance could be overcome by the apoptosis-sensitizing drug HA14-1.

Conclusion: Our study confirms the concept of a shift towards a more antiapoptotic transcriptional context during tumour progression in RCCs. Furthermore, it shows that caspase-2 participates in chemotherapy-induced apoptosis in RCCs although it is not mandatory for it. Additionally, inhibition of antiapoptotic BCL-2 family members might provide a possible way to overcome chemotherapy resistance of RCCs.
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http://dx.doi.org/10.3233/CLO-2009-0492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619288PMC
June 2010

Photolytically generated nitric oxide inhibits caspase activity and results in AIF-mediated cell death.

J Mol Med (Berl) 2010 Mar 22;88(3):279-87. Epub 2009 Oct 22.

Institute of Molecular Medicine, Research Group Immunobiology, Heinrich-Heine-University Duesseldorf, Universitaetsstrasse 1, Duesseldorf, Germany.

In human skin tissue nitrite is found at relatively high concentrations and represents the main source for cutaneous non-enzymatic nitric oxide (NO) formation during UVA exposure due to photolytical decomposition. Since NO has been repeatedly shown to act pro- as well as anti-apoptotic we here studied the effects of UVA irradiation on human keratinocytes in the presence of nitrite. We show that UVA-induced nitrite photodecomposition effectively inactivated caspase activity. In parallel, we observed in human skin keratinocytes, UVA-irradiated in the presence of nitrite, a proteolytic processing of apoptosis-inducing factor (AIF) followed by translocation from the mitochondrion into the nucleus. This translocation resulted in a characteristic apoptotic nuclear phenotype, which differs from the known nuclear phenotype of caspase-mediated chromatin condensation and apoptotic body formation. Interestingly both, AIF translocation and AIF-induced nuclear phenotype changes can be inhibited by NO scavengers, demonstrating the distinct role of nitrite-derived NO in the observed processes. This mode of UVA-induced apoptosis is AIF-dependent and NO-mediated and strongly depends on the presence of nitrite, abundantly present in skin tissue. Thus, photolysis of nitrite in the skin appears to represent an important backup mechanism, which ensures removal of UVA-damaged cells even in the absence of caspase activation.
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http://dx.doi.org/10.1007/s00109-009-0551-3DOI Listing
March 2010

Stem cell divisions controlled by the proto-oncogene BMI-1.

J Stem Cells 2009 ;4(3):141-6

Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University Medical Center, Düsseldorf, Germany.

Divisions of somatic stem cells are required for the maintenance and regeneration of normal tissues, while divisions of cancerous stem cells likely underlie the existence of certain malignant diseases. Studies of recent years suggest that molecular mechanisms governing stem cell self-renewal can be subverted in tumorigenesis to maintain cancerous growth. This is exemplified by the proto-oncogene BMI-1 that is involved in the maintenance of somatic stem cells and in carcinogenesis within the same tissues. BMI-1 interferes with the central cellular tumor suppressor pathways linked to retinoblastoma protein (Rb) and p53. These signaling pathways control the cell cycle, cell differentiation, cellular senescence and cell death. While the roles of the pathways associated with Rb and p53 in cancer are broadly established, further elucidation thereof in stem cells might have implications in cancer research, stem cell biology and regenerative medicine.
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April 2010

HA14-1 is able to reconstitute the impaired mitochondrial pathway of apoptosis in renal cell carcinoma cell lines.

Cell Oncol 2008 ;30(5):419-33

Institute of Pathology, Heinrich-Heine-University Hospital, 40225 Düsseldorf, Germany.

Renal cell carcinomas (RCCs) exhibit a marked resistance towards apoptosis. Although most apoptotic stimuli converge at the level of the mitochondria, little is known about the mitochondrial apoptosis pathway in renal cell carcinomas. The aim of the present study, therefore, was to investigate the functionality of the mitochondrial apoptosis pathway in renal cell carcinoma cell lines by exposure to TRAIL, etoposide, HA14-1 and betulinic acid activating the mitochondria by different mechanisms. Sensitivity to TRAIL-induced apoptosis correlated with cleavage of the initiator caspase-8, but the mitochondrial apoptosis pathway was not induced. Similarly, etoposide and betulinic acid could not induce mitochondrial damage. In contrast, HA14-1 was able to activate mitochondrial apoptosis, thereby demonstrating functionally inducible signalling pathways downstream of the mitochondria. The intactness of the pathways upstream of the mitochondria was shown by pretreatment of TRAIL-sensitive cell lines with HA14-1, which could reconstitute TRAIL-induced mitochondrial damage and resulted in a synergistic apoptosis induction. Our results demonstrate that the apoptotic pathways upstream and downstream of the mitochondria are intact and inducible in renal cell carcinoma cell lines. However, resistance towards mitochondrial apoptosis is located on the level of the mitochondria themselves.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618985PMC
http://dx.doi.org/10.3233/clo-2008-0438DOI Listing
November 2008

Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC.

Apoptosis 2008 Jul;13(7):938-49

Institute of Pathology, Heinrich-Heine University Hospital, Moorenstrasse 5, 40225 Duesseldorf, Germany,

Activation of the initiator-caspase, caspase-8 is under tight control of multiple antiapoptotic regulators including ARC, cFlip(S), cFlip(L) and PED/PEA-15. Since there is little data regarding the expression of caspase-8 and its antiapoptotic regulators in human tumours in vivo, we analysed their expression in renal cell carcinomas (RCCs) to identify which of these genes might be crucial for the well known impaired apoptosis and--as a result--resistance towards chemotherapy and ionizing radiation of RCCs. Caspase-8, cFlip(S), cFlip(L) and PED/PEA-15 mRNA expression was significantly increased only in early stages of RCCs compared to non-neoplastic renal tissue. In contrast, ARC mRNA expression was significantly increased in RCCs of all stages without differences between the tumour stages and grades. Importantly, the relative mRNA expression ratio between ARC and caspase-8 was significantly increased during carcinogenesis and tumour progression. In contrast, the relative mRNA expression ratio between cFlip(S), cFlip(L) or PED/PEA-15 and caspase-8 remained constant during all tumour stages. In conclusion, our analysis revealed that ARC is the only caspase-8 inhibiting regulator being constantly overexpressed in RCCs. Furthermore, the balance between antiapoptotic ARC and proapoptotic caspase-8 is the only one to be disturbed during carcinogenesis and tumour progression of RCCs. This inhibition of Caspase-8 might therefore be one example for the multiple antiapoptotic functions of ARC in RCCs possibly contributing to the marked resistance of RCCs towards radio- and chemotherapy and reflects a shift of gene expression towards a more antiapoptotic context in RCCs.
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http://dx.doi.org/10.1007/s10495-008-0225-6DOI Listing
July 2008

Paclitaxel (Taxol)-induced apoptosis in human epithelioid sarcoma cell lines is enhanced by upregulation of CD95 ligand (FasL/Apo-1L).

J Cancer Res Clin Oncol 2008 Jun 12;134(6):689-95. Epub 2007 Dec 12.

Institute of Pathology, Heinrich Heine-University, Moorenstr. 5, Bldg. 14.79, 40225 Duesseldorf, Germany.

Purpose: Metastasizing epithelioid sarcoma (ES) is an extremely aggressive tumor, because conventional chemotherapy and irradiation are largely ineffective. Here, we analyzed the impact of the CD95-mediated drug-induced apoptosis in ES cell lines.

Methods: The effects of paclitaxel (Taxol) and 5-FU were determined by MTT assay. The extent of apoptosis was analyzed by light microscopy and Annexin V staining (flow cytometry). The expression of death receptors and ligands was defined by RT-PCR, Western blotting and flow cytometry.

Results: All cell lines expressed CD95, but not the CD95 ligand. The CD95 activation resulted in apoptosis and cell death in all cell lines. Both paclitaxel and 5-FU are able to trigger apoptosis, and furthermore, to upregulate CD95, whereas only paclitaxel increases CD95 ligand expression. Neutralizing antibodies directed against CD95 ligand effectively inhibited paclitaxel-induced cell death, thereby providing evidence for a direct involvement of the CD95 system in paclitaxel-induced apoptosis.

Conclusions: Concomitant upregulation of CD95 receptor and ligand may significantly enhance the response of ES to anticancer drugs. As evident from the differential response of our clonal ES subpopulations to paclitaxel and 5-FU, effective activation of the CD95 system depends on intrinsic properties of both the chemotherapeutic agent and target cell population.
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http://dx.doi.org/10.1007/s00432-007-0340-8DOI Listing
June 2008

The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

Free Radic Biol Med 2007 Sep 31;43(5):818-29. Epub 2007 May 31.

Department of Plastic and Reconstructive Surgery, Hand Surgery, and Burn Center, University Hospital of the RWTH-Aachen, Pauwelsstr. 30, D-52074 Aachen, Germany.

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.
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http://dx.doi.org/10.1016/j.freeradbiomed.2007.05.030DOI Listing
September 2007

Survivin and its prognostic significance in pediatric acute B-cell precursor lymphoblastic leukemia.

Haematologica 2007 Aug 20;92(8):1043-50. Epub 2007 Jul 20.

Clinic for Pediatric Oncology, Hematology and Immunology, University of Duesseldorf, Duesseldorf, Germany.

Background And Objectives: Impaired apoptosis, mediated by members of the inhibitor of apoptosis proteins (IAP) family such as survivin, is thought to contribute to leukemic cell survival. In contrast to low expression of survivin in normal differentiated adult tissues, very high levels of survivin have been described in a number of different tumors. Overexpression of survivin was found to correlate with poor prognosis in a variety of cancers including hematologic malignancies. To date, however, there is no information available on the prognostic role of survivin in pediatric precursor B-cell acute lymphocytic leukemia (BCP-ALL), the most frequent malignancy in childhood.

Design And Methods: In a retrospective study including 66 pediatric patients we analyzed the impact of survivin protein levels on outcome in BCP-ALL.

Results: Survivin overexpression, with an up to ten-fold increase of the normal level, was detected in 65% of the leukemic samples in contrast to negligible expression in non-malignant hematopoietic cells. Despite considerable variety of expression levels in ALL cells, there was no association of survivin levels with established risk factors. However, patients suffering relapse of disease or death had significantly higher survivin expression than those with a favorable outcome. Overexpression of survivin is a significant prognostic marker for 3 year relapse free, event-free and overall survival, again independent of the established prognostic factors in ALL, such as age and leukocyte count at diagnosis as assessed in multivariate analysis.

Interpretation And Conclusions: Overexpression of survivin in BCP-ALL identifies patients with a high risk of early relapse. Upon confirmation in a prospective analysis, survivin expression may, in the future, serve to further refine treatment stratification with intensification of therapy in those patients prone to relapse.
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http://dx.doi.org/10.3324/haematol.10675DOI Listing
August 2007

Differential gene expression in anticancer drug- and TRAIL-mediated apoptosis in renal cell carcinomas.

Apoptosis 2007 Sep;12(9):1645-57

Institute of Pathology, Heinrich Heine University Hospital, Moorenstrasse 5, 40225 Duesseldorf, Germany.

Renal cell carcinomas (RCC) exhibit marked differences in susceptibility towards anticancer drug- and TRAIL-induced apoptosis. However, the underlying mechanisms determining apoptosis-sensitivity or -resistance are not well understood. The purpose of this study was to compare gene expression patterns induced by DNA-damage- and death receptor-induced apoptosis and to detect differentially expressed genes responsible for differences in apoptosis-susceptibility. Therefore, we performed a comparative cDNA-array analysis in an apoptosis-resistant and an apoptosis-sensitive RCC cell line. In the sensitive cell line an upregulation of multiple E2F1- and p53-inducible proapaptotic and cell-cycle regulating target genes by Topotecan as well as TRAIL was observed. Interestingly, several antiapoptotic NFkappaB-dependent target genes were also induced. In the resistant cell line, however, only a small number of E2F1-, p53- and NFkappaB-dependent target genes were differentially regulated. Conclusively, anticancer drug- as well as TRAIL-sensitivity go along with an upregulation of multiple proapoptotic genes. In contrast, the mechanisms of apoptosis-resistance are-at least in part-located upstream of gene induction and seem not to depend upon upregulation of de-novo-synthesized antiapoptotic genes. Conclusively, the proapoptotic stimuli are confronted with a cellular context which allows apoptosis to be conducted-in the sensitive cell line-or not-in the resistant cell line.
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http://dx.doi.org/10.1007/s10495-007-0064-xDOI Listing
September 2007

Disturbed expression of the apoptosis regulators XIAP, XAF1, and Smac/DIABLO in gastric adenocarcinomas.

Diagn Mol Pathol 2007 Mar;16(1):1-8

Institute of Pathology, Heinrich-Heine University Hospital, Duesseldorf, Germany.

Dysregulation of apoptosis plays an important role in carcinogenesis and tumor progression. Whereas x-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of apoptosis, its antagonists second mitochondria-derived activator of caspases/direct IAP binding protein with low PI (Smac/DIABLO), and XIAP-associated factor 1 (XAF1) promote apoptosis. To explore the relevance of XIAP, Smac/DIABLO, and XAF1 for carcinogenesis and tumor progression, we analyzed 46 primary gastric adenocarcinomas and non-neoplastic gastric mucosa samples by quantitative real-time polymerase chain reaction. XIAP, Smac/DIABLO, and XAF1 expression was found in all non-neoplastic gastric mucosa samples and all adenocarcinomas. XIAP expression levels did not change between non-neoplastic gastric mucosa and adenocarcinomas or between carcinomas of early and advanced stages. Although Smac/DIABLO expression was significantly (P=0.01) higher in carcinomas, the ratio of XIAP to Smac/DIABLO expression remained stable between non-neoplastic mucosa and carcinomas. XAF1 expression had the tendency to decrease from non-neoplastic mucosa to advanced adenocarcinomas. Importantly, the ratio of XIAP to XAF1 expression significantly (P=0.03) increased from non-neoplastic mucosa to adenocarcinomas and the increase was even higher in carcinomas of advanced stage (P=0.01). Moreover, expression of the XAF1 splice variants differing in the zinc-finger domain essential for XIAP-binding was analyzed and revealed a significant higher (P=0.03) variant-2/variant-1 ratio in advanced carcinomas. In conclusion, an increased expression ratio of XIAP to XAF1 in combination with a disturbed expression of the XAF1 splice variants could be shown in gastric adenocarcinomas. These marked imbalances probably result in an impaired ability for XAF1 to antagonize the effects of XIAP thereby contributing to apoptosis-resistance and generating an important growth advantage.
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http://dx.doi.org/10.1097/01.pdm.0000213471.92925.51DOI Listing
March 2007

TRAIL-R4-beta: a new splice variant of TRAIL-receptor 4 lacking the cysteine rich domain 1.

Biochem Biophys Res Commun 2006 Oct 14;349(1):115-21. Epub 2006 Aug 14.

Department of General and Visceral Surgery, Heinrich Heine University Duesseldorf, Germany.

Transcriptional modification by alternative splicing is known to be involved in the regulation of programmed cell death. Recently, alternative splice variants of the TNF-related apoptosis inducing ligand (TRAIL/APO2L) and of the death receptor TRAIL-R2/DR5 have been identified. In this study, we report the identification of a novel alternative splice variant of the decoy receptor with a truncated death domain TRAIL-R4 lacking exon 3, which we designated TRAIL-R4-beta. As revealed by BLAST search we identified the genomic organisation of the TRAIL-R4 gene which consists of 9 exons. Loss of exon 3 resulted in the truncation of the first complete cysteine rich domain 1 which is known to be involved in ligand-receptor-complex. In conclusion, alternative splicing might be involved in functional fine-tuning of TRAIL-induced programmed cell death.
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http://dx.doi.org/10.1016/j.bbrc.2006.08.031DOI Listing
October 2006

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Eur J Cancer 2006 Jan 4;42(2):264-74. Epub 2006 Jan 4.

Institute of Pathology, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf, Germany.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.
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http://dx.doi.org/10.1016/j.ejca.2005.10.019DOI Listing
January 2006

Paclitaxel/Taxol sensitivity in human renal cell carcinoma is not determined by the p53 status.

Cancer Lett 2005 May 10;222(2):165-71. Epub 2004 Nov 10.

Institute of Pathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany.

In this study, we analyzed the role of the p53 status for paclitaxel/Taxol sensitivity in renal cell carcinomas (RCCs) of the clear cell type. Using immunohistochemistry, nuclear p53 accumulation could not be correlated to the paclitaxel/Taxol sensitivity. DNA sequencing detected a p53 gene mutation in two out of eight RCC cell lines, i.e. in exon 8 (cell line clearCa-6), and in exon 9 (cell line clearCa-5). No correlation, however, was found between the p53 status of our RCC cell lines and their paclitaxel/Taxol sensitivity as indicated by the IC50 values. However, paclitaxel-induced growth inhibition in paclitaxel-sensitive RCC cell lines was accompanied by an increase in apoptosis, irrespective of their p53 status. Although CD95 up-regulation was observed in renal cell carcinoma with wild-type p53 upon paclitaxel treatment, paclitaxel-induced apoptosis itself is triggered independently from the CD95 system. In conclusion, the p53 status cannot predict paclitaxel/Taxol sensitivity in RCC cell lines of the clear cell type.
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http://dx.doi.org/10.1016/j.canlet.2004.09.045DOI Listing
May 2005

Survivin splice variants regulate the balance between proliferation and cell death.

Oncogene 2005 Mar;24(12):1994-2007

Center for Childhood Cancer, Columbus Children's Research Institute, The Ohio State University, Columbus, OH, USA.

Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DeltaEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.
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http://dx.doi.org/10.1038/sj.onc.1208350DOI Listing
March 2005

UVB radiation-mediated expression of inducible nitric oxide synthase activity and the augmenting role of co-induced TNF-alpha in human skin endothelial cells.

J Invest Dermatol 2004 Nov;123(5):950-7

Research Group Immunobiology, Heinrich-Heine-University of Dusseldorf, Dusseldorf, Germany.

Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-alpha expression. In HUDEC addition of TNF-alpha results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-alpha- dependent mechanism which essentially contributes to increased iNOS activity.
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http://dx.doi.org/10.1111/j.0022-202X.2004.23422.xDOI Listing
November 2004

XIAP expression is an independent prognostic marker in clear-cell renal carcinomas.

Hum Pathol 2004 Aug;35(8):1022-8

Institute of Pathology, University Hospital, Düsseldorf, Germany.

Deregulation of apoptosis plays an important role in carcinogenesis, tumor progression, and resistance to chemotherapy. XIAP is considered to be the most potent caspase inhibitor of all known IAP (inhibitor of apoptosis) family members. To explore the relevance of XIAP for progression and prognosis in renal cell carcinomas (RCCs) of the clear-cell type, we analyzed XIAP protein expression in formalin-fixed tissue from 145 clear-cell RCCs by immunohistochemistry. XIAP protein expression was found in 95% of clear-cell RCCs. A significant increase of XIAP expression became evident from well (G1) to poorly (G3) differentiated clear-cell RCCs (P < 0.0001) and from low (pT1) to advanced (pT3) tumor stages (P = 0.0016). Log-rank test showed a significant inverse correlation (P = 0.0174) between XIAP expression and tumor aggressiveness as indicated by patients' survival. Most important, multivariate Cox regression analysis revealed that XIAP expression is an independent prognostic parameter (P = 0.018) in clear-cell RCCs. Our results suggest an important role for XIAP-mediated inhibition of apoptosis during progression of clear-cell RCCs and introduce XIAP expression as a new independent prognostic marker in this tumor type.
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http://dx.doi.org/10.1016/j.humpath.2004.03.011DOI Listing
August 2004

The presence of nitrite during UVA irradiation protects from apoptosis.

FASEB J 2003 Dec 2;17(15):2342-4. Epub 2003 Oct 2.

Institute of Molecular Medicine, Research Group Immunobiology, Heinrich-Heine-University of Düsseldorf, 40225 Düsseldorf, Germany.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.
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http://dx.doi.org/10.1096/fj.03-0359fjeDOI Listing
December 2003

Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma.

Int J Cancer 2003 May;104(5):568-78

Urologische Klinik, Heinrich-Heine-Universität Düsseldorf, Germany.

In many common cancers such as transitional cell carcinoma (TCC), specific genes are hypermethylated, whereas overall DNA methylation is diminished. Genome-wide DNA hypomethylation mostly affects repetitive sequences such as LINE-1 retrotransposons. Methylation of these sequences depends on adequate expression of DNA methyltransferase I (DNMT1) during DNA replication. Therefore, DNMT1 expression relative to proliferation was investigated in TCC cell lines and tissue as well as in renal carcinoma (RCC) cell lines, which also display hypomethylation, as indicated by decreased LINE-1 methylation. Cultured normal uroepithelial cells or normal bladder tissue served as controls. In all tumor cell lines, DNMT1 mRNA as well as protein was decreased relative to the DNA replication factor PCNA, and DNA hypomethylation was present. However, the extents of hypomethylation and DNMT1 downregulation did not correlate. Reporter gene assays showed that the differences in DNMT1 expression between normal and tumor cells were not established at the level of DNMT1 promoter regulation. Diminished DNMT1:PCNA mRNA ratios were also found in 28/45 TCC tissues but did not correlate with the extent of DNA hypomethylation. In addition, expression of the presumed de novo methyltransferases DNMT3A and DNMT3B mRNAs was investigated. DNMT3B overexpression was observed in about half of all high-stage TCC (DNMT3B vs. tumor stage, chi(2): p = 0.03), whereas overexpression of DNMT3A was rarer and less pronounced. Expression of DNMT3A and DNMT3B in most RCC lines was higher than in TCC lines. Our data indicate that DNMT1 expression does not increase adequately with cell proliferation in bladder cancer. This relative downregulation probably contributes to hypomethylation of repetitive DNA but does not determine its extent alone.
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http://dx.doi.org/10.1002/ijc.10988DOI Listing
May 2003

Revisiting an old antimicrobial drug: amphotericin B induces interleukin-1-converting enzyme as the main factor for inducible nitric-oxide synthase expression in activated endothelia.

Mol Pharmacol 2002 Oct;62(4):936-46

Research Group Immunobiology, Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany.

We have investigated the impact of the widely used antifungal agent Amphotericin B (AmB) on cytokine activated aortic endothelial cells (AEC) and their inflammatory response as monitored by cytokine and inducible nitric-oxide synthase (iNOS) expression as well as high-output nitric oxide synthesis. Because both blood-borne infections and systemically administered drugs will first encounter vessel lining endothelial cells, this cell type represents an important participant in innate immune reactions against xenobiotics. Culturing cytokine-activated AEC in the presence of 1.25 microg/ml AmB, a concentration equivalent to serum levels during patient treatment, we find increases in iNOS promoter activity up to 120%, in iNOS mRNA or protein expressions by factors of up to 3.5 +/- 1.1, and in iNOS activity of up to 180% compared with cells with cytokines only. In parallel, a strong increase in endothelial interleukin (IL)-1beta-converting enzyme (ICE) and IL-1beta expression and activity was observed. Specific inhibition of ICE activity or IL-1beta functionality significantly reduces expression and activity of the iNOS to control values. Because ICE activity is essential for the endogenous synthesis of active IL-1beta, ICE overexpression represents the key signal in the AmB-induced and IL-1beta-mediated effects on iNOS activity. In summary, in endothelial cells, AmB strongly augments cytokine-induced iNOS expression and activity by increasing the expression and activity of the ICE. This adjuvant activity for augmented endogenous cytokine processing adds to the efficacy of the antimycotic activity of AmB. Furthermore, our data underline the relevance of the endothelial iNOS as a potent effector of the innate immune system.
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http://dx.doi.org/10.1124/mol.62.4.936DOI Listing
October 2002

Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas.

Int J Cancer 2002 Jul;100(1):30-6

Institute of Pathology, Heinrich Heine-University, Duesseldorf, Germany.

Survivin, a novel member of the inhibitor of apoptosis protein (IAP) family, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor progression and treatment with anticancer drugs. Recently, we identified 2 novel alternative splice variants of survivin, survivin-2B and survivin-Delta Ex3, which differ in their antiapoptotic properties. Survivin-2B has lost its antiapoptotic potential and may act as a naturally occurring antagonist of antiapoptotic survivin and survivin-Delta Ex3. Because the in vivo expression of these splice variants in human cancer has not been analyzed so far, 57 renal cell carcinomas (RCCs) were explored using quantitative reverse transcriptase polymerase chain reaction. We found that all RCCs express survivin-Delta Ex3, survivin-2B and survivin, the latter being the dominant transcript. When we compared early and intermediate stages with late stages of clear cell RCCs, no significant changes in the expression levels of survivin and survivin-Delta Ex3 became evident. However, a significant decrease was observed for the mRNA ratio between survivin-2B and survivin in late tumor stages (p = 0.036). Chromophilic/papillary RCCs, which are known to be less aggressive than clear cell RCCs, did not show significantly lower expression levels of antiapoptotic survivin and survivin-Delta Ex3, compared with stage-adjusted clear cell RCCs. Our study demonstrates for the first time in vivo expression of functionally different survivin variants and suggests a role of these survivin splice variants in the progression and clinical behavior of human RCCs.
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http://dx.doi.org/10.1002/ijc.10450DOI Listing
July 2002