http://repub.eur.nl/res/aut/12764/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/20683/ 2010-07-01T00:00:00Z Gene-engineered T cell therapy represents a promising strategy to treat cancers. To enable pre-selection of patients sensitive to this type of treatment we have setup and validated a T cell activation assay to test antigen expression on patient-derived tumor tissues. Chimeric antibody-based receptor (CAR) directed against CAIX, currently used in a clinical trial to treat RCC patients, was used as a model receptor. Primary human T cells expressing CAIX CAR were able to respond to CAIX-positive but not CAIX-negative tumor tissue and showed an increased production of IFNγ, TNFα, IL-10 and IL-4, but not IL-2 or IL-5. Tumor tissue driven responses of primary T cells were paralleled by NFAT activation measured in CAR-transduced Jurkat T cells, which was shown to be triggered in a CAR and antigen-specific manner. Next, the reporter gene assay was applied to two independent PSMA CARs, which both mediated NFAT activation in response to tumor tissue. Taken together, a sensitive and donor-independent assay was established to measure T cell activation upon exposure to patient-derived tumor tissue, which may facilitate pre-selection of patients for clinical adoptive T cell therapy. http://repub.eur.nl/res/pub/21878/ 2010-01-01T00:00:00Z In human adenoviruses (HAdV), 240 copies of the 14.3-kDa minor capsid protein IX stabilize the capsid. Three N-terminal domains of protein IX form triskelions between hexon capsomers. The C-terminal domains of four protein IX monomers associate near the facet periphery. The precise biological role of protein IX remains enigmatic. Here we show that deletion of the protein IX gene from a HAdV-5 vector enhanced the reporter gene delivery 5 to 25-fold, specifically to Coxsackie and Adenovirus Receptor (CAR)-negative cell lines. Deletion of the protein IX gene also resulted in enhanced activation of peripheral blood mononuclear cells. The mechanism for the enhanced transduction is obscure. No differences in fiber loading, integrin-dependency of transduction, or factor-X binding could be established between protein IX-containing and protein IX-deficient particles. Our data suggest that protein IX can affect the cell tropism of HAdV-5, and may function to dampen the innate immune responses against HAdV particles. http://repub.eur.nl/res/pub/14456/ 2008-09-15T00:00:00Z In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage- independent growth of these cells. PN T2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin β3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed. http://repub.eur.nl/res/pub/35945/ 2007-06-01T00:00:00Z BACKGROUND. Specificity of transgene expression is important for safety during gene therapeutical applications. For prostate cancer, transcriptional targeting has been applied but was hampered by loss of specificity and low activity. We constructed a small chimeric promoter for high and prostate-specific transgene expression from adenoviral vectors. METHODS. A chimeric promoter, composed of the prostate-specific antigen (PSA) enhancer and the rat probasin promoter, was cloned into an adenoviral vector and its activity was compared to vectors containing conventional prostate-specific promoters and the constitutive Cytomegalovirus (CMV) promoter in in vitro and in vivo prostate cancer models. RESULTS. The chimeric PSA-probasin promoter was the most active prostate-specific promoter reaching up to 20% of CMV promoter activity while maintaining prostate-specificity. CONCLUSIONS. The chimeric PSA-probasin promoter is a small promoter that can be utilized in viral vectors for high prostate-specific transgene expression. http://repub.eur.nl/res/pub/8888/ 1998-01-01T00:00:00Z Glycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and FSH-R2. The splice variant FSH-R1 differs from the full-length FSH receptor mRNA by the inclusion of a small extra exon between exons 9 and 10. FSH-R2 lacks the first three base pairs of exon 4, contains an extra exon between exons 4 and 5, and has an extended 3'-untranslated region. According to the predicted open reading frames, both mRNAs encode truncated FSH receptor proteins, consisting of the entire extracellular domain (FSH-R1) or the amino-terminal half of the extracellular domain (FSH-R2), and are expressed at a low level in testes and ovaries. The levels of expression of the FSH-R1 and FSH-R2 mRNAs in the gonads show a constant ratio to the expression level of the full-length FSH receptor mRNA. Furthermore, in vitro co-expression of either one of the truncated proteins with the full-length FSH receptor in COS1 cells did not affect signal transduction through the full-length FSH receptor. The absence of a function of the truncated FSH receptors in FSH signal transduction in vitro, and the lack of differential regulation of the alternative transcripts, indicate that there is no clear function for alternative splicing of the FSH receptor pre-mRNA in the postnatal testis and the cycling adult ovary. http://repub.eur.nl/res/pub/22564/ 1996-06-26T00:00:00Z Among the many hormones that are produced by the anterior pituitary gland, luteinizing hormone (LH, lutropin), follicle-stimulating hormone (FSH, follitropin), and thyroidstimulating hormone (TSH, thyrotropin) form the separate family of so-called glycoprotein hormones (reviewed by Oharib el al., 1990). These hormones consist of two glycosylated subunits, a and p, which are associated through non-covalent interactions. The a-subunit is identical for all glycoprotein hormones, whereas the p-subunit is hormone specific. The gonadotropins, LH and FSH, are the key regulators of testis and ovary function, and are synthesized in cells called the gonadotrophs of the pituitary gland. TSH, which regulates thyroid function, is produced in the thyrotrophs. In primates and horses, a fourth glycoprotein hormone exists, chorionic gonadotropin (CO), which is synthesized in the placenta during pregnancy, and is structurally and functionally related to LH (Oharib el al., 1990).