Alexander Griekspoor

A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Peptide Diffusion, Protection, and Degradation in Nuclear and Cytoplasmic Compartments before Antigen Presentation by MHC Class I

Antigenic peptides generated by the proteasome have to survive a peptidase-containing environment for presentation by MHC class I molecules. We have visualized the fate and dynamics of intracellular peptides in living cells. We show that peptides are distributed over two different but interconnected compartments, the cytoplasm and the nucleus, and diffuse rapidly through and between these compartments. Since TAP is excluded from the nuclear face of the nuclear envelope, nuclear peptides have to leave the nucleus to contact TAP. Thereby, these peptides encounter cytosolic peptidases that degrade peptides within seconds unless bound to chromatin. Since peptide degradation is far more efficient than translocation, many peptides will be lost for antigen presentation by MHC class I molecules.

PKA-induced resistance to tamoxifen is associated with an altered orientation of ERα towards co-activator SRC-1

Resistance to tamoxifen is observed in half of the recurrences in breast cancer, where the anti‐estrogen tamoxifen acquires agonistic properties for transactivating estrogen receptor α (ERα). In a previous study, we showed that protein kinase A (PKA)‐mediated phosphorylation of serine 305 (S305) of ERα results in resistance to tamoxifen. Now, we demonstrate that phosphorylation of S305 in ERα by PKA leads to an altered orientation between ERα and its coactivator SRC‐1, which renders the transcription complex active in the presence of tamoxifen. This altered orientation involves the C‐termini of ERα and SRC‐1, which required a prolonged AF‐1‐mediated interaction. This intermolecular reorientation as a result of PKA‐mediated phosphorylation of ERα‐S305 and tamoxifen binding provides a unique model for resistance to the anticancer drug tamoxifen.

MHC class I alleles and their exploration of the antigen-processing machinery

Summary:  At the cell surface, major histocompatibility complex (MHC) class I molecules present fragments of intracellular antigens to the immune system. This is the end result of a cascade of events initiated by multiple steps of proteolysis. Only a small part of the fragments escapes degradation by interacting with the peptide transporter associated with antigen presentation and is translocated into the endoplasmic reticulum lumen for binding to MHC class I molecules. Subsequently, these newly formed complexes can be transported to the plasma membrane for presentation. Every step in this process confers specificity and determines the ultimate result: presentation of only few fragments from a given antigen. Here, we introduce the players in the antigen processing and presentation cascade and describe their specificity and allelic variation. We highlight MHC class I alleles, which are not only different in sequence but also use different aspects of the antigen presentation pathway to their advantage: peptide acquaintance.

Visualizing the action of steroid hormone receptors in living cells

Transcription controlled by Steroid Hormone Receptors (SHRs) plays a key role in many important physiological processes like organ development, metabolite homeostasis, and response to external stimuli. Understandably, the members of this family have drawn a lot of attention from the scientific community since their discovery, four decades ago. Still, after many years of research we are only beginning to unravel the complex nature of these receptors. The pace at which we do has improved significantly in recent years with the discovery of genetically encoded fluorescent probes, and the accompanying revival of biophysical approaches that allow more detailed study of SHRs. Here, we will look into the different aspects of SHR signalling, and discuss how biophysical techniques have contributed to visualizing their function in their native context, the living cell.

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body

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 DMβ and the class II-associated invariant chain, the DOβ cytoplasmic tail contains potential lysosomal targeting signals. The DOβ signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DOβ 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.

B Cell Receptor-Mediated Internalization of Salmonella: A Novel Pathway for Autonomous B Cell Activation and Antibody Production

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4+ T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

Cryo‐Immunogold Electron Microscopy

This unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids. This unit is accompanied by eleven videos that illustrate many of the procedures used in this unit.

Classification of anti-estrogens according to intramolecular FRET effects on phospho-mutants of estrogen receptor α

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 α (ERα) 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 ERα in live cells, corresponding to an inactive conformation of the ERα. Phosphorylation of Ser305 and/or Ser236 of ERα by protein kinase A (PKA) and of Ser118 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 ERα where the FRET effects of particular anti-estrogen(s) are nullified. The FRET response provided information on the activity of ERα 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 ERα 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. [Mol Cancer Ther 2007;6(5):1526–33]