Papers by Jan Van Groningen
iScience, 2022
Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary ... more Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca2+]i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.
Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The S... more Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas the SSX proteins function as transcriptional corepressors and are associated with the polycomb complex. The domains involved in these opposite transcriptional activities are retained in the SS18-SSX fusion proteins. Here, we set out to determine the direct transcriptional consequences of conditional SS18-SSX2 fusion protein expression using complementary DNA microarray-based profiling. By doing so, we identified several clusters of SS18-SSX2–responsive genes, including a group of genes involved in cholesterol synthesis, which is a general characteristic of malignancy. In addition, we identified a group of SS18-SSX2–responsive genes known to be specifically deregulated in primary synovial...
Molecular Immunology, 2013
iScience, 2021
Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary ... more Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca2+]i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca2+]i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.
SLAS DISCOVERY: Advancing the Science of Drug Discovery
Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising ne... more Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well...
Experimental and Molecular Therapeutics
American Journal Of Pathology
From a differential mRNA display comparing a nonand a highly metastasizing human melanoma celi li... more From a differential mRNA display comparing a nonand a highly metastasizing human melanoma celi line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma celi lines of a larger paneL The encoded protein, MEMD, is identical to activated leukocyte celi adhesion molecule (ALCAM), a recently identified ligand of CD6. ALCAM is involved in homophylic (ALCAM-ALCAM) and heterophylic (ALCAM-CD6) celi adhesion interactions. We have studied MEMD/ ALCAM celi-cell interactions between human melanoma cells. The expression of this cell adhesion molecule not only correlates with enhanced metastatic properties and with aggregational behavior of h melanoma cells as tested by FACS analysis, but transfection experiments also make clear that MEMD/AL-CAM expression is essential for cell-cell interaction of the investigated human melanoma cells. As the melanoma celi lines analyzed are all CD6 negative, these results strongly suggest that MEMDI/ALCAM is an adhesion molecule mediating homophylic clustering of melanoma cells. MEMD/ALCAM expression is not restricted to subsets of leukocytes and melanoma cells, it can also be found in healthy organs and in several other mallgnant tumor celi lines. Besides, MEMD/ ALCAM is also expressed in cultured endothelia1 cells, pericytes and melanocytes, in xenografts derived from the radial and vertical growth phase and in 4 of 13 melanoma metastasis lesions. The potential role is discussed of MEMD/ALCAM mediated cell-cell interactions in migration of mobile cells (ie, activated leukocytes, metastasizing tumor cells) through tissues.
Biological chemistry, 1997
We describe the characterization of recombinant clones for the human transcription factor CCAAT/e... more We describe the characterization of recombinant clones for the human transcription factor CCAAT/enhancer binding protein alpha (hC/EBP alpha). The intronless hC/EBP alpha gene is almost 90% homologous to its rat and mouse counterparts. The gene copies of more distant species are less conserved, but the alignment reveals a striking homology in five regions, of which four may be involved in transactivation functions while the fifth concerns the carboxy-terminal bZip sequences (basic region and leucine zipper) mediating sequence specific DNA-binding. In addition to the usual expression sites, significant transcript levels were detected in the epidermal compartment of human skin and in rat aorta by northern analysis. The presence of hC/EBP alpha is further documented by immunohistochemical analysis of human skin biopsies and cultured keratinocytes showing the nuclear presence of the protein, notably in the suprabasal layers of the epidermis and in human keratinocytes induced to differen...
Immunology, 1983
Lymphoid cell populations in various organs of the carp Cyprinus carpio L. were investigated usin... more Lymphoid cell populations in various organs of the carp Cyprinus carpio L. were investigated using a series of mouse monoclonal antibodies raised against carp thymocytes and carp serum Ig. Clones have been designated as Ig+T+, Ig+T- or Ig-T+ on the basis of the reactivity with thymocytes and serum Ig in the enzyme-linked immunosorbent assay (ELISA) screening. Their reaction to the lymphoid organs of carp was investigated on cryostat sections and cytocentrifuge slides using immunoperoxidase techniques. IG+T+ clones could be subdivided into those that stained reticular fibres around blood vessels in various organs (R+) and those that did not (R-). The former stained most thymocytes and most peripheral lymphocytes as well as plasma cells whereas the latter did not stain cortical thymocytes and some peripheral lymphocytes. IG+T- clones were negative for thymocytes but positive for plasma cells and a certain population of peripheral lymphocytes. Ig-T+ clones reacted similarly to Ig+T+R- ...
Journal of immunological methods, Jan 28, 2015
The complement system is an essential component of our innate immunity, both for the protection a... more The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24h following reperfusion, which returned to base-line at 3 and 7days post reperfusion. Whe...
Virus Research, 1987
A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a mo... more A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a molecular weight of approximately 104 kDa (P104) was compared to the P85 encoding prototype clone of ST-FeSV. Analysis of chimeric genes constructed with the viral oncogenes of the two clones indicated that the variant clone coded for a larger polyprotein than the prototype clone because of genetic differences in its 3' portion. Comparative DNA sequence analysis revealed that one nucleotide just upstream of the termination condon TGA in the prototype proviral DNA was deleted from the variant clone resulting in a 468-bp larger open reading frame. Furthermore, it appeared that the U3 regions of the long terminal repeats (LTRs) of the variant clone contained an insertion of 71 bp as compared to the LTRs of the prototype clone. In addition, both clones differed also from each other with respect to genetic sequences deleted from their env gene regions.
Proceedings of the National Academy of Sciences, 2001
PLoS ONE, 2009
Background: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bo... more Background: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/ or CDH1(FZR1), its exact role in cell cycle control still remains to be established. Methodology/Principal Findings: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a wellknown cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed. Conclusions/Significance: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.
PLoS ONE, 2010
Klaas M e d e n d o rp 1, Lilian V re e d e 1, Jan J. M. van G ro n in g e n 1, Lisette H e tte r... more Klaas M e d e n d o rp 1, Lilian V re e d e 1, Jan J. M. van G ro n in g e n 1, Lisette H e tte rs c h ijt1, Linda B rugm ans1, P atrick A. M. Jansen2, W ilh e lm in a H. van den H u rk 1, D ie d e rik R. H. d e B ru ijn 1, A d G eurts van Kessel1*
Oncogene, 2001
A recurrent chromosomal abnormality associated with a subset of papillary renal cell carcinomas i... more A recurrent chromosomal abnormality associated with a subset of papillary renal cell carcinomas is t(X;1)(p11;q21). This translocation leads to the formation of two fusion genes, TFE3PRCC and the reciprocal product PRCCTFE3. Both fusion genes are expressed in t(X;1)-positive renal cell carcinomas and contain major parts of the coding regions of the parental transcription factor PRCC and TFE3 genes, respectively. To ®nd out whether these fusion genes possess transforming capacity, we transfected NIH3T3 and rat-1 cells with the fusion products, either separately or combined. When using soft agar assays, we observed colony formation in all cases. NIH3T3 cells transfected with PRCCTFE3 or PRCCTFE3 together with TFE3PRCC yielded the highest colony forming capacities. Examination of other characteristics associated with malignant transformation, i.e., growth under low-serum conditions and formation of tumors in athymic nude mice, revealed that cells transfected with PRCCTFE3 exhibited all these transformation-associated characteristics. Upon transfection of the fusion products into conditionally immortalized kidney cells, derived from the proximal tubules of an H-2Kb-tsA58 transgenic mouse, and consecutive incubation under non-permissive conditions, growth arrest was observed, followed by dierentiation except for those cells transfected with PRCCTFE3. Therefore, we conclude that PRCCTFE3 may be the t(X;1)-associated fusion product that is most critical for the development of papillary renal cell carcinomas. Oncogene (2001) 20,
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Papers by Jan Van Groningen