D Verwoerd

PamGene, Amsterdamo, North Holland, Netherlands

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Publications (27)200.93 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: With current techniques, it remains a challenge to assess coregulator binding of nuclear receptors, for example, the estrogen receptor alpha (ERα). ERα is critical in many breast tumors and is inhibited by antiestrogens such as tamoxifen in cancer therapy. ERα is also modified by acetylation and phosphorylation that affect responses to the antiestrogens as well as interactions with coregulators. Phosphorylation of ERα at Ser305 is one of the mechanisms causing tamoxifen resistance. Detection of resistance in patient samples would greatly facilitate clinical decisions on treatment, in which such patients would receive other treatments such as aromatase inhibitors or fulvestrant. Here we describe a coregulator peptide array that can be used for high-throughput analysis of full-length estrogen receptor binding. The peptide chip can detect ERα binding in cell and tumor lysates. We show that ERα phosphorylated at Ser305 associates stronger to various coregulator peptides on the chip. This implies that ERαSer305 phosphorylation increases estrogen receptor function. As this is also detected in a breast tumor sample of a tamoxifen-insensitive patient, the peptide array, as described here, may be applicable to detect tamoxifen resistance in breast tumor samples at an early stage of disease and contribute to personalized medicine.
    Molecular Cancer Therapeutics 02/2012; 11(4):805-16. · 5.60 Impact Factor
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    ABSTRACT: Anti-estrogen resistance is a major clinical problem in the treatment of breast cancer. In this study, fluorescence resonance energy transfer (FRET) analysis, a rapid and direct way to monitor conformational changes of estrogen receptor alpha (ERalpha) upon anti-estrogen binding, was used to characterize resistance to anti-estrogens. Nine different anti-estrogens all induced a rapid FRET response within minutes after the compounds have liganded to ERalpha in live cells, corresponding to an inactive conformation of the ERalpha. Phosphorylation of Ser(305) and/or Ser(236) of ERalpha by protein kinase A (PKA) and of Ser(118) by mitogen-activated protein kinase (MAPK) influenced the FRET response differently for the various anti-estrogens. PKA and MAPK are both associated with resistance to anti-estrogens in breast cancer patients. Their respective actions can result in seven different combinations of phospho-modifications in ERalpha where the FRET effects of particular anti-estrogen(s) are nullified. The FRET response provided information on the activity of ERalpha under the various anti-estrogen conditions as measured in a traditional reporter assay. Tamoxifen and EM-652 were the most sensitive to kinase activities, whereas ICI-182,780 (Fulvestrant) and ICI-164,384 were the most stringent. The different responses of anti-estrogens to the various combinations of phospho-modifications in ERalpha elucidate why certain anti-estrogens are more prone than others to develop resistance. These data provide new insights into the mechanism of action of anti-hormones and are critical for selection of the correct individual patient-based endocrine therapy in breast cancer.
    Molecular Cancer Therapeutics 06/2007; 6(5):1526-33. · 5.60 Impact Factor
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    ABSTRACT: Rho GTPases are crucial regulators of the actin cytoskeleton and they play a role in the control of membrane trafficking. In contrast to the close family members RhoA and RhoC, RhoB localises to endosomes and delays epidermal growth factor receptor traffic. Here, we show that activated RhoB induces the peripheral distribution of endosomes, which align along subcortical actin stress fibres and are surrounded by an actin coat. The Diaphanous-related formin, Dia1, is recruited to endosomes by activated RhoB. Dia1 is required for the formation of the actin coat around endosomes downstream of RhoB, connecting membrane trafficking with the regulation of actin dynamics.
    Journal of Cell Science 07/2005; 118(Pt 12):2661-70. · 5.88 Impact Factor
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    ABSTRACT: Using a novel approach that detects changes in the conformation of ERα, we studied the efficacy of anti-estrogens to inactivate ERα under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERα by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERα-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERα. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIα, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIα converts tamoxifen from an ERα inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).
    Cancer Cell 01/2004; 5(6):597-605. · 24.76 Impact Factor
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    ABSTRACT: Estrogen receptor-mediated transcription is enhanced by overexpression of G1/S cyclins D1, E or A in the presence as well in the absence of estradiol. Excess of G1/S cyclins also prevents the inhibition of transactivation of estrogen receptor (ER) by the pure antiestrogen ICI 182780. Cyclin D1 mediates this transactivation independent of complex formation to its CDK4/6 partner. This raises the possibility that overexpression of G1/S cyclins renders growth of ER-positive breast cancer hormone-independent and resistant to treatment with antiestrogens. Transient transfection of ER-positive breast cancer cell lines T47D and MCF7 with G1/S cyclins could overcome the growth arrest induced by ICI 182780 treatment. The ability of various cyclin D1 mutants to overcome the ICI 182780 mediated growth arrest corresponded with their ability to stimulate cyclin A- and E2F- promoter based reporter activities in the presence of ICI 182780. Transfection of a mutant cyclin D1 (cyclin D1-KE) that was unable to bind CDK4 and was reported to transactivate ER in the presence of ICI 182780, could not stimulate proliferation in ICI 182780 treated cells. On the other hand, cyclin D1-LALA, which is unable to stimulate ERE transactivation, could overcome the ICI 182780 cell cycle arrest. Furthermore, transient transfection of T47D cells using cyclin D1 together with a catalytic inactive mutant of CDK4 (CDK4-DN) indicated that the observed effect is due to binding to CDK inhibitors. However, a moderate, sixfold overexpression of cyclin D1 in stably transfected MCF7 cells did not overcome the ICI 182780 mediated growth arrest. These results indicate that CDK-independent transactivation of the estrogen receptor by cyclin D1 is by itself, not sufficient to result in estradiol-independent growth of breast cancer cells, whereas a vast overexpression of G1/S cyclins is able to do so, most likely by capturing of CDK inhibitors.
    Oncogene 12/2002; 21(53):8158-65. · 7.36 Impact Factor
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    ABSTRACT: MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.
    The Journal of Immunology 08/2001; 167(2):884-92. · 5.52 Impact Factor
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    ABSTRACT: MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH(4)Cl-sensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.
    Proceedings of the National Academy of Sciences 09/1999; 96(18):10326-31. · 9.74 Impact Factor
  • A Tulp, D Verwoerd, J Neefjes
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    ABSTRACT: Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.
    Electrophoresis 04/1999; 20(3):438-44. · 3.26 Impact Factor
  • A Tulp, D Verwoerd, J Neefjes
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    ABSTRACT: This paper describes electromigration of complexes, consisting of two or more proteins and non-covalently associated peptides. Relatively small complexes (Mr < 1000000) can be resolved in sieving matrices. Large complexes are separated in free liquid systems. Examples of separation are given using native gels, denaturing gels and special formats thereof: blue native PAGE and gels incorporating a transversal temperature gradient. Both preparative and analytical applications are discussed as well as separations leading to mechanistic models of protein interaction. Carrier-free electrophoresis is represented by capillary zone electrophoresis, free-flow electrophoresis and density gradient electrophoresis. Emphasis is put on the free liquid separation of clathrin-coated vesicles and proteasomes.
    Journal of chromatography. B, Biomedical sciences and applications 02/1999; 722(1-2):141-51.
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    ABSTRACT: We have studied the degradation of the free major histocompatibility complex (MHC) class II beta subunit in the ER. Domain swapping experiments demonstrate that both the intra- and extracellular domain determine the rate of degradation. Recently, it has been shown that some ER-retained proteins are exported from the ER by the translocon followed by deglycosylation and degradation in the cytosol by proteasomes. Degradation of the beta chain follows a different route. The proteasome is involved but inhibition of the proteasome by lactacystin does not result in deglycosylation and export to the cytosol. Instead, the beta chain is retained in the ER implying that extraction of the beta chain from the ER membrane requires proteasome activity. Surprisingly, brefeldin A accelerates the degradation of the beta chain by the proteasome. This suggests that various processes outside the ER are involved in ER-degradation. The ER is the site from where misfolded class II beta chains enter a proteasome-dependent degradation pathway.
    Journal of Cell Science 09/1998; 111 ( Pt 15):2217-26. · 5.88 Impact Factor
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    ABSTRACT: We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.
    Electrophoresis 07/1998; 19(8-9):1288-93. · 3.26 Impact Factor
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    ABSTRACT: A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.
    Electrophoresis 07/1998; 19(7):1171-8. · 3.26 Impact Factor
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    ABSTRACT: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide 'editor', favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive. The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM. HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.
    Current Biology 01/1998; 7(12):950-7. · 9.49 Impact Factor
  • A Tulp, D Verwoerd, A Benham, J Neefjes
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    ABSTRACT: Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.
    Electrophoresis 01/1998; 18(14):2509-15. · 3.26 Impact Factor
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    ABSTRACT: Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.
    European Journal of Immunology 10/1997; 27(9):2426-35. · 4.97 Impact Factor
  • A Tulp, D Verwoerd, A A Hart
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    ABSTRACT: A density gradient electrophoresis apparatus made of Perspex (7 cm, O 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill-defined Ampholines, the cost of these well-defined amphoters is low.
    Electrophoresis 06/1997; 18(5):767-73. · 3.26 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.
    Advances in experimental medicine and biology 02/1997; 417:171-4. · 1.83 Impact Factor
  • Immunology Letters - IMMUNOL LETT. 01/1997; 56:370-370.
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    ABSTRACT: Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.
    The Journal of Cell Biology 12/1996; 135(3):611-22. · 10.82 Impact Factor
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    ABSTRACT: MHC class II molecules associate with peptides in the endocytic pathway. Different endosomal locations for peptide loading of class II molecules, varying from early endosomes (EE) to lysosomes, have been assigned on the basis of subcellular fractionation experiments. We have determined the intracellular location of HLA-DM, a molecule that supports peptide loading of class II molecules, by separating vesicles from the melanoma cell line Mel JuSo on the basis of buoying density and surface charge. In both fractionations, HLA-DM co-fractionated with a lysosomal compartment containing beta-hexosaminidase (beta-hex) activity and not with endosomes. Further analysis showed that HLA-DM mainly co-fractionated with a sub-lysosomal structure characterized by a relative low density and containing both pro- and mature cathepsin D and MHC class II molecules. Fluid phase markers first enter this compartment before entering high-density lysosomes that contain exclusively mature cathepsin D, some HLA-DM and no detectable MC class II molecules. Finally we determined the intracellular location of neutral and acidic peptidases. Whereas neutral peptidase activity was detected in the endoplasmic reticulum and/or plasma membrane fractions, acidic peptidase activity exclusively migrated at the position of HLA-DM containing lysosomal vesicles. Our results show that class II molecules co-migrate with HLA-DM, pro- and mature cathepsin D, beta-hex and acidic peptidase activity. HLA-DM, cathepsin d and class II molecules were not observed at the position of EE. Our data suggest that HLA-DM-mediated peptide loading of class II molecules occurs in a lysosomal subcompartment.
    International Immunology 06/1996; 8(5):625-40. · 3.14 Impact Factor

Publication Stats

1k Citations
200.93 Total Impact Points

Institutions

  • 2012
    • PamGene
      Amsterdamo, North Holland, Netherlands
  • 1992–2007
    • Netherlands Cancer Institute
      Amsterdamo, North Holland, Netherlands
  • 1995
    • Academisch Centrum Tandheelkunde Amsterdam
      • Field of Oral Biochemistry
      Amsterdam, North Holland, Netherlands