Theo M. Luider

Bio: Theo M. Luider is an academic researcher from Erasmus University Rotterdam. The author has contributed to research in topics: Mass spectrometry & Medicine. The author has an hindex of 46, co-authored 191 publications receiving 7253 citations. Previous affiliations of Theo M. Luider include Instituto Nacional de Saúde Dr. Ricardo Jorge & University of Texas Southwestern Medical Center.

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

Abstract: DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PKCS. We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.

Proteomic Analysis of Exosomes Isolated from Human Malignant Pleural Effusions

Abstract: Exosomes are membrane vesicles from endosomal origin secreted by various cells such as hematopoietic, epithelial, and tumor cells. Exosomes secreted by tumor cells contain specific antigens potentially useful for immunotherapeutic purposes. Our aim was to determine if exosomes are present in human cancerous pleural effusions and to identify their proteomic content. Exosomes were purified by sucrose gradient ultracentrifugation, and electron microscopy was used to check both concentration and purity of exosomes. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and protein bands were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blotting. Exosomes were present in pleural fluid obtained from patients suffering from mesothelioma (n = 4), lung cancer (n = 2), breast cancer (n = 2), and ovarian cancer (n = 1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction-involved proteins were present. Proteins not previously reported were identified (SNX25, BTG1, PEDF, thrombospondin 2). Different types of immunoglobulins and complement factors were abundantly present in the sucrose fractions containing exosomes. Exosome-directed specificity of these immunoglobulins was not observed. In conclusion, sucrose gradient ultracentrifugation allows isolation of exosomes from malignant pleural effusions. However, pleural fluid proteins and especially immunoglobulins are coisolated and may hamper the use of exosomes isolated from malignant effusion for immunotherapy programs.

Proteomic Analysis of Exosomes Secreted by Human Mesothelioma Cells

Abstract: Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.

Recommendations for Biomarker Identification and Qualification in Clinical Proteomics

Abstract: Clinical proteomics has yielded some early positive results-the identification of potential disease biomarkers-indicating the promise for this analytical approach to improve the current state of the art in clinical practice. However, the inability to verify some candidate molecules in subsequent studies has led to skepticism among many clinicians and regulatory bodies, and it has become evident that commonly encountered shortcomings in fundamental aspects of experimental design mainly during biomarker discovery must be addressed in order to provide robust data. In this Perspective, we assert that successful studies generally use suitable statistical approaches for biomarker definition and confirm results in independent test sets; in addition, we describe a brief set of practical and feasible recommendations that we have developed for investigators to properly identify and qualify proteomic biomarkers, which could also be used as reporting requirements. Such recommendations should help put proteomic biomarker discovery on the solid ground needed for turning the old promise into a new reality.

Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

Abstract: Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

Membrane vesicles as conveyors of immune responses

Abstract: In multicellular organisms, communication between cells mainly involves the secretion of proteins that then bind to receptors on neighbouring cells But another mode of intercellular communication - the release of membrane vesicles - has recently become the subject of increasing interest Membrane vesicles are complex structures composed of a lipid bilayer that contains transmembrane proteins and encloses soluble hydrophilic components derived from the cytosol of the donor cell These vesicles have been shown to affect the physiology of neighbouring recipient cells in various ways, from inducing intracellular signalling following binding to receptors to conferring new properties after the acquisition of new receptors, enzymes or even genetic material from the vesicles This Review focuses on the role of membrane vesicles, in particular exosomes, in the communication between immune cells, and between tumour and immune cells

Patterns of Somatic Mutation in Human Cancer Genomes

Abstract: AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore PL02-05 All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

The somatic genomic landscape of glioblastoma

Abstract: We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.