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1.
Mol Cell ; 37(2): 159-71, 2010 Jan 29.
Article in English | MEDLINE | ID: mdl-20122399

ABSTRACT

The Wilms' tumor suppressor protein WT1 functions as a transcriptional regulator of genes controlling growth, apoptosis, and differentiation. It has become clear that WT1 can act as an oncogene in many tumors, primarily through the inhibition of apoptosis. Here, we identify the serine protease HtrA2 as a WT1 binding partner and find that it cleaves WT1 at multiple sites following the treatment of cells with cytotoxic drugs. Ablation of HtrA2 activity either by chemical inhibitor or by siRNA prevents the proteolysis of WT1 under apoptotic conditions. Moreover, the apoptosis-dependent cleavage of WT1 is defective in HtrA2 knockout cells. Proteolysis of WT1 by HtrA2 causes the removal of WT1 from its binding sites at gene promoters, leading to alterations in gene regulation that enhance apoptosis. Our findings provide insights into the function of HtrA2 in the regulation of apoptosis and the oncogenic activities of WT1.


Subject(s)
Mitochondrial Proteins/physiology , Serine Endopeptidases/physiology , WT1 Proteins/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Binding Sites , Gene Expression Regulation , HeLa Cells , High-Temperature Requirement A Serine Peptidase 2 , Humans , Mice , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/genetics , Promoter Regions, Genetic , RNA Interference , Serine Endopeptidases/genetics
2.
Arch Toxicol ; 92(6): 2119-2135, 2018 06.
Article in English | MEDLINE | ID: mdl-29589053

ABSTRACT

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.


Subject(s)
Apoptosis/drug effects , DNA Damage , Doxorubicin/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Hydroxyurea/pharmacology , WT1 Proteins/metabolism , Animals , Apoptosis/genetics , Caspases/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , DNA Replication/drug effects , Fallopian Tubes/drug effects , Female , Humans , K562 Cells , Mice, Knockout , Primary Cell Culture , WT1 Proteins/genetics
3.
Bioorg Med Chem Lett ; 27(12): 2659-2662, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28479198

ABSTRACT

The ATM- and Rad3-related (ATR) kinases play a key role in DNA repair processes and thus ATR is an attractive target for cancer therapy. Here we designed and synthesized sulfilimidoyl- and sulfoximidoyl-substituted analogs of the sulfone VE-821, a reported ATR inhibitor. The properties of these analogs have been investigated by calculating physicochemical parameters and studying their potential to specifically inhibit ATR in cells. Prolonged inhibition of ATR by the analogs in a Burkitt lymphoma cell line resulted in enhanced DNA damage and a substantial amount of apoptosis. Together our findings suggest that the sulfilimidoyl- and sulfoximidoyl-substituted analogs are efficient ATR inhibitors.


Subject(s)
Imines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Sulfones/pharmacology , Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , DNA Damage , Dose-Response Relationship, Drug , Humans , Imines/chemical synthesis , Imines/chemistry , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazines/chemical synthesis , Pyrazines/chemistry , Structure-Activity Relationship , Sulfones/chemical synthesis , Sulfones/chemistry
4.
Sci Rep ; 13(1): 18550, 2023 10 29.
Article in English | MEDLINE | ID: mdl-37899458

ABSTRACT

Neuronal ceroid lipofuscinosis 6 (CLN6) is a rare and fatal autosomal recessive disease primarily affecting the nervous system in children. It is caused by a pathogenic mutation in the CLN6 gene for which no therapy is available. Employing an untargeted metabolomics approach, we analyzed the metabolic changes in CLN6 subjects to see if this system could potentially yield biomarkers for diagnosis and monitoring disease progression. Neuronal-like cells were derived from human fibroblast lines from CLN6-affected subjects (n = 3) and controls (wild type, n = 3). These were used to assess the potential of a neuronal-like cell-based metabolomics approach to identify CLN6 distinctive and specific biomarkers. The most impacted metabolic profile is associated with sphingolipids, glycerophospholipids metabolism, and calcium signaling. Over 2700 spectral features were screened, and fifteen metabolites were identified that differed significantly between both groups, including the sphingolipids C16 GlcCer, C24 GlcCer, C24:1 GlcCer and glycerophospholipids PG 40:6 and PG 40:7. Of note, these fifteen metabolites were downregulated in the CLN6 disease group. This study is the first to analyze the metabolome of neuronal-like cells with a pathogenic mutation in the CLN6 gene and to provide insights into their metabolomic alterations. This could allow for the development of novel biomarkers for monitoring CLN6 disease.


Subject(s)
Membrane Proteins , Neuronal Ceroid-Lipofuscinoses , Child , Humans , Membrane Proteins/metabolism , Neuronal Ceroid-Lipofuscinoses/metabolism , Lipid Metabolism , Metabolomics , Glycerophospholipids , Sphingolipids , Biomarkers/metabolism
5.
Diagnostics (Basel) ; 13(17)2023 08 30.
Article in English | MEDLINE | ID: mdl-37685353

ABSTRACT

Gaucher disease (GD) is a rare autosomal recessive disorder arising from bi-allelic variants in the GBA1 gene, encoding glucocerebrosidase. Deficiency of this enzyme leads to progressive accumulation of the sphingolipid glucosylsphingosine (lyso-Gb1). The international, multicenter, observational "Lyso-Gb1 as a Long-term Prognostic Biomarker in Gaucher Disease"-LYSO-PROOF study succeeded in enrolling a cohort of 160 treatment-naïve GD patients from diverse geographic regions and evaluated the potential of lyso-Gb1 as a specific biomarker for GD. Using genotypes based on established classifications for clinical presentation, patients were stratified into type 1 GD (n = 114) and further subdivided into mild (n = 66) and severe type 1 GD (n = 48). Due to having previously unreported genotypes, 46 patients could not be classified. Though lyso-Gb1 values at enrollment were widely distributed, they displayed a moderate and statistically highly significant correlation with disease severity measured by the GD-DS3 scoring system in all GD patients (r = 0.602, p < 0.0001). These findings support the utility of lyso-Gb1 as a sensitive biomarker for GD and indicate that it could help to predict the clinical course of patients with undescribed genotypes to improve personalized care in the future.

6.
Ann Med ; 53(1): 1787-1796, 2021 12.
Article in English | MEDLINE | ID: mdl-34658264

ABSTRACT

BACKGROUND: Hereditary Transthyretin-Related Amyloidosis, a clinically heterogeneous autosomal dominant disease caused by pathogenic variants in the TTR gene, is characterized by the deposition of insoluble misfolded protein fibrils. The diagnosis, especially in non-endemic areas, is typically delayed by 4-5 years; a misdiagnosis due to clinical heterogeneity is common. The study objective was to define the prevalence of Hereditary Transthyretin-Related Amyloidosis in patients with polyneuropathy and/or cardiomyopathy of no obvious aetiology. METHOD: A multicenter observational "Epidemiological analysis for the hereditary Transthyretin-Related AMyloidosis"-TRAM study was performed in Germany, Austria, and Switzerland. RESULTS: A total of 5141 participants were recruited by 50 neurologic and 27 cardiologic specialized centres. Genetic analysis demonstrated a 1.1% Hereditary Transthyretin-Related Amyloidosis positivity rate among patients with polyneuropathy and/or cardiomyopathy of not obvious aetiology. Twenty-one various TTR variants (TTR-positive) were identified. Body Mass Index was lower in the TTR-positive patients as an indicator for the involvement of the autonomic nervous system; the age of onset of clinical manifestations was higher in TTR-positive patients. There were no other genotype-phenotype correlations or the prevalence of specific clinical manifestations in TTR-positive patients. CONCLUSIONS: Our data support the fact that Hereditary Transthyretin-Related Amyloidosis is underdiagnosed in polyneuropathy and cardiomyopathy patients. Routine implementation of genetic testing is recommended in patients with unexplained polyneuropathy and/or cardiomyopathy to accelerate the earlier diagnosis and the time-sensitive treatment initiation.KEY MESSAGESMore than 5.000 participants with CM and/or PNP of no obvious aetiology were recruited in the observational "Epidemiological analysis for the hereditary Transthyretin-Related AMyloidosis" TRAM study and screened for pathogenic TTR variants.The study demonstrated >1% of patients with CM and/or PNP of unclear aetiology are positive for a pathogenic TTR variant.Routine genetic testing is recommended in patients with unexplained CM and/or PNP to accelerate the initial diagnosis and timely treatment initiation.


Subject(s)
Amyloid Neuropathies, Familial/genetics , Cardiomyopathies/epidemiology , Polyneuropathies/epidemiology , Prealbumin/genetics , Amyloid Neuropathies, Familial/complications , Amyloid Neuropathies, Familial/diagnosis , Amyloid Neuropathies, Familial/epidemiology , Cardiomyopathies/diagnosis , Cardiomyopathies/genetics , Genetic Testing , Humans , Polyneuropathies/diagnosis , Polyneuropathies/etiology
7.
Sci Rep ; 9(1): 8755, 2019 06 19.
Article in English | MEDLINE | ID: mdl-31217499

ABSTRACT

Most deaths from breast cancer result from tumour recurrence, which is typically an incurable disease. Down-regulation of the pro-apoptotic tumour suppressor protein prostate apoptosis response-4 (PAR-4) is required for breast cancer recurrence and resistance to chemotherapy. Recent advances in the analysis of apoptotic signalling networks have uncovered an important role for activation of caspase-8 following DNA damage by genotoxic drugs. DNA damage induces depletion of IAP proteins and causes caspase-8 activation by promoting the formation of a cytosolic cell death complex. We demonstrate that loss of PAR-4 in triple negative breast cancer cell lines (TNBC) mediates resistance to DNA damage-induced apoptosis and prevents activation of caspase-8. Moreover, loss of PAR-4 prevents DNA damage-induced cIAP1 depletion. PAR-4 functions downstream of caspase-8 by cleavage-induced nuclear translocation of the C-terminal part and we demonstrate that nuclear translocation of the C-terminal PAR-4 fragment leads to depletion of cIAP1 and subsequent caspase-8 activation. Specifically targeting cIAP1 with RNAi or Smac mimetics (LCL161) overcomes chemo-resistance induced by loss of PAR-4 and restores caspase-8 activation. Our data identify cIAP1 as important downstream mediator of PAR-4 and we provide evidence that combining Smac mimetics and genotoxic drugs creates vulnerability for synthetic lethality in TNBC cells lacking PAR-4.


Subject(s)
Drug Resistance, Neoplasm , Inhibitor of Apoptosis Proteins/metabolism , Neoplasm Proteins/metabolism , Receptors, Thrombin/metabolism , Signal Transduction , Triple Negative Breast Neoplasms/metabolism , Ubiquitin-Protein Ligases/metabolism , Caspase 8/genetics , Caspase 8/metabolism , DNA Damage , Female , Humans , Inhibitor of Apoptosis Proteins/genetics , MCF-7 Cells , Neoplasm Proteins/genetics , Receptors, Thrombin/genetics , Thiazoles/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Ubiquitin-Protein Ligases/genetics
8.
Methods Mol Biol ; 1510: 11-22, 2017.
Article in English | MEDLINE | ID: mdl-27761810

ABSTRACT

Aberrant histone deacetylase (HDAC) activity often correlates with neoplastic transformation and inhibition of HDACs by small molecules has emerged as a promising strategy to treat hematological malignancies in particular. Treatment with HDAC inhibitors (HDACis) often prompts tumor cells to undergo apoptosis, thereby causing a caspase-dependent cleavage of target proteins. An unexpectedly large number of proteins are in vivo caspase substrates and defining caspase-mediated substrate specificity is a major challenge. In this chapter we demonstrate that the hematopoietic transcription factor PU.1 becomes cleaved after treatment of acute myeloid leukemia (AML) cells with the HDACis LBH589 (panobinostat) or MS-275 (entinostat). To define caspase specificity for PU.1, an in vitro caspase assay including caspases 1-10 with in vitro-translated PU.1 is described in detail.


Subject(s)
Antineoplastic Agents/pharmacology , Caspase 8/genetics , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Benzamides/pharmacology , Blotting, Western/methods , Caspase 8/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Enzyme Activation , HEK293 Cells , HL-60 Cells , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Indoles/pharmacology , K562 Cells , Panobinostat , Proteolysis , Proto-Oncogene Proteins/metabolism , Pyridines/pharmacology , Substrate Specificity , Trans-Activators/metabolism , Transfection
9.
J Neuroimmunol ; 129(1-2): 97-105, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12161025

ABSTRACT

Due to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain-Barré syndrome, specific immunotherapies aim at modifying T cell responses. Use of truncated mutants of the neuritogenic peptide of myelin basic protein (MBP) has been shown to anergize autoreactive T cells and to reverse experimental autoimmune encephalitis (EAE). To establish a rationale basis for the use of altered peptide ligands (APLs) in the treatment of autoimmune diseases we designed a set of N- and C-terminally truncated mutants of the minimal experimental autoimmune neuritis (EAN) inducing bovine P2 (bP2) (60-70) peptide and compared them for the ability to induce immune responses and T cell receptor (TCR) cell signaling. Truncated peptides bound to MHC class II molecules and induced TCR internalization and expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) with decreasing potency. None of the shortened mutants elicited a proliferative response in P2-specific T cells. Stimulation of these antigen-specific T cells with peptide bP2(62-69) using antigen presenting cells (APCs) prepulsed with bP2(60-70) resulted in a significant decrease of the proliferative response. In agreement with the observed effects on T cell activation, analysis of TCR signaling demonstrated a lack of CD3 epsilon phosphorylation and MAPK activation. Moreover, repeated injection of bP2(62-69) significantly slowed progression of adoptive transfer EAN (AT-EAN). Taken together, these findings strongly suggest that peptide bP2(62-69) can favorably modulate the antigen-induced response of neuritogenic T cells.


Subject(s)
Autoimmune Diseases of the Nervous System/drug therapy , Chemotaxis, Leukocyte/drug effects , Myelin P2 Protein/chemistry , Peptide Fragments/chemistry , T-Lymphocytes/drug effects , Animals , Autoimmune Diseases of the Nervous System/immunology , Autoimmune Diseases of the Nervous System/physiopathology , Cell Division/drug effects , Cell Division/immunology , Cells, Cultured , Chemotaxis, Leukocyte/immunology , Female , Histocompatibility Antigens Class II/drug effects , Histocompatibility Antigens Class II/immunology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Ligands , Molecular Weight , Myelin P2 Protein/immunology , Myelin P2 Protein/pharmacology , Peptide Fragments/immunology , Peptide Fragments/pharmacology , Protein Binding/drug effects , Protein Binding/immunology , Protein Structure, Tertiary/physiology , Rats , Rats, Inbred Lew , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes/immunology
10.
Oncotarget ; 5(10): 2988-98, 2014 May 30.
Article in English | MEDLINE | ID: mdl-24931006

ABSTRACT

The tumor suppressor protein prostate apoptosis response-4 (PAR-4) is silenced in a subset of human cancers and its down-regulation serves as a mechanism for cancer cell survival following chemotherapy. PAR-4 re-expression selectively causes apoptosis in cancer cells but how its pro-apoptotic functions are controlled and executed precisely is currently unknown. We demonstrate here that UV-induced apoptosis results in a rapid caspase-dependent PAR-4 cleavage at EEPD131G, a sequence that was preferentially recognized by caspase-8. To investigate the effect on cell growth for this cleavage event we established stable cell lines that express wild-type-PAR-4 or the caspase cleavage resistant mutant PAR-4 D131G under the control of a doxycycline-inducible promoter. Induction of the wild-type protein but not the mutant interfered with cell proliferation, predominantly through induction of apoptosis. We further demonstrate that TNFα-induced apoptosis leads to caspase-8-dependent PAR-4-cleavage followed by nuclear accumulation of the C-terminal PAR-4 (132-340) fragment, which then induces apoptosis. Taken together, our results indicate that the mechanism by which PAR-4 orchestrates the apoptotic process requires cleavage by caspase-8.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis/physiology , Caspase 8/metabolism , Neoplasms/metabolism , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , Microscopy, Confocal , Mutagenesis, Site-Directed , Tumor Necrosis Factor-alpha/metabolism
11.
FEBS J ; 280(5): 1330-43, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23305266

ABSTRACT

ADP-ribosylation is a post-translational modification that regulates various physiological processes, including DNA damage repair, gene transcription and signal transduction. Intracellular ADP-ribosyltransferases (ARTDs or PARPs) modify their substrates either by poly- or mono-ADP-ribosylation. Previously we identified ARTD10 (formerly PARP10) as a mono-ADP-ribosyltransferase, and observed that exogenous ARTD10 but not ARTD10-G888W, a catalytically inactive mutant, interferes with cell proliferation. To expand on this observation, we established cell lines with inducible ARTD10 or ARTD10-G888W. Consistent with our previous findings, induction of the wild-type protein but not the mutant inhibited cell proliferation, primarily by inducing apoptosis. During apoptosis, ARTD10 itself was targeted by caspases. We mapped the major cleavage site at EIAMD406↓S, a sequence that was preferentially recognized by caspase-6. Caspase-dependent cleavage inhibited the pro-apoptotic activity of ARTD10, as ARTD10(1-406) and ARTD10(407-1025), either alone or together, were unable to induce apoptosis, despite catalytic activity of the latter. Deletion of the N-terminal RNA recognition motif in ARTD10(257-1025) also resulted in loss of pro-apoptotic activity. Thus our findings indicate that the RNA recognition motif contributes to the pro-apoptotic effect, together with the catalytic domain. We suggest that these two domains must be physically linked to stimulate apoptosis, possibly targeting ARTD10 through the RNA recognition motif to specific substrates that control cell death. Moreover, we established that knockdown of ARTD10 reduced apoptosis in response to DNA-damaging agents. Together, these findings indicate that ARTD10 is involved in the regulation of apoptosis, and that, once apoptosis is activated, ARTD10 is cleaved as part of negative feedback regulation.


Subject(s)
Apoptosis , Caspases/metabolism , DNA Damage/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Cell Proliferation , Cells, Cultured , Humans , Immunoenzyme Techniques , Immunoprecipitation , Monocytes/cytology , Monocytes/metabolism , Mutagenesis, Site-Directed , Mutation/genetics , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics
12.
Cell Cycle ; 9(13): 2508-14, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20543571

ABSTRACT

Wilms' tumour is a paediatric malignancy of the kidneys and is one of the most common solid childhood cancers. The Wilms' tumour 1 protein (WT1) is a transcription factor that can either activate or repress genes involved in growth, apoptosis and differentiation. It is frequently mutated or aberrantly expressed in Wilms' tumour, where the wild type protein would normally act as a tumour suppressor. Several studies, however, have found that wild type WT1 acts as an oncogene in adult tumours, primarily through the inhibition of apoptosis. The expression of WT1 correlates with the aggressiveness of several adult cancers, and its continued expression following treatment is indicative of a poor outcome.We recently found that the treatment of tumour cell lines with cytotoxic drugs leads to the cleavage of WT1 by the serine protease HtrA2. HtrA2 binds to a specific region of WT1, the suppression domain, and then cleaves WT1 at multiple sites. The HtrA2-mediated proteolysis of WT1 leads to its removal from gene promoter regions and changes in gene expression. Cleavage of WT1 by HtrA2 enhances apoptosis. This event is advantageous to the treatment of adult tumours where WT1 acts as an oncogene. However, when WT1 is acting as a tumour suppressor in paediatric malignancies, proteolysis by HtrA2 would be antagonistic to therapy.


Subject(s)
Mitochondrial Proteins/metabolism , Serine Endopeptidases/metabolism , WT1 Proteins/metabolism , Amino Acid Motifs , Apoptosis , Cell Nucleus/enzymology , High-Temperature Requirement A Serine Peptidase 2 , Humans , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Processing, Post-Translational , WT1 Proteins/chemistry , Wilms Tumor/metabolism , Wilms Tumor/pathology , Wilms Tumor/therapy
13.
Biochem Soc Trans ; 36(Pt 4): 629-31, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18631130

ABSTRACT

The Wilms' tumour-suppressor gene (WT1), encodes a zinc-finger transcription factor that is critical for the development of several organs, including the kidneys, gonads and spleen. Despite its identification as a tumour suppressor that plays a crucial role in the formation of a paediatric malignancy of the kidneys (Wilms' tumour), it has also emerged as an oncogenic factor influencing proliferation and apoptosis in a large variety of adult cancers. This review focuses on new insights into WT1's role in early development and its potential oncogenic role in adult cancer.


Subject(s)
Apoptosis , WT1 Proteins/metabolism , Animals , Gene Expression Regulation, Developmental , Humans , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , WT1 Proteins/genetics
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