http://repub.eur.nl/res/aut/3638/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/25537/ 2011-04-06T00:00:00Z RNA interference (RNAi) is widely used as a screening tool for the identification of host genes involved in viral infection. Due to the limitation of raw small interfering RNA (siRNA), we tested two commonly used short hairpin RNA (shRNA) lentiviral libraries to identify host factors involved in hepatitis C virus (HCV) infection. It was found that these shRNA library vectors caused non-specific disturbance of HCV replication that was not due to toxicity or interferon response, but related to the high shRNA levels disturbing the endogenous microRNA biogenesis. The high shRNA levels achieved with these vectors reduced the levels of mature microRNAs, including miR-122 known to promote HCV replication. Our findings extend the caution of potential off-target effects of lentiviral shRNA libraries which appear unsuitable to screen microRNA regulated phenotypes, such as HCV replication. http://repub.eur.nl/res/pub/28584/ 2010-04-01T00:00:00Z Immunosuppression considerably affects hepatitis C virus (HCV) recurrence and the outcome of antiviral treatment after liver transplantation. Recent findings have suggested that the calcineurin inhibitor tacrolimus (Tac), unlike cyclosporine A (CsA), interferes with the antiviral activity of interferon-α (IFN-α) in vitro. The aim of this study was to more extensively investigate the effects of calcineurin inhibitors on IFN-α signaling and antiviral activity in subgenomic and infectious HCV models. Treatment with Tac and CsA did not affect Huh7 cell proliferation at doses of 10 to 500 ng/mL; however, it completely inhibited T cell proliferation. In contrast to previous reports, Tac had no effect on IFN-α-stimulated reporter gene expression, even at the dose of 5 μg/mL. Furthermore, in Huh7 subgenomic HCV replicon cells, treatment with Tac had no significant effect on the suppression of viral replication by IFN-α. In the infectious HCV model, treatment with IFN-α effectively inhibited both viral RNA replication and de novo production of virus particles, and neither was attenuated at any concentration of Tac. CsA had no significant effect on IFN-α-stimulated reporter gene expression; however, as shown previously, a combination of CsA (at 500 ng/mL and higher) and IFN-α resulted in enhanced inhibition of viral replication in both the subgenomic and infectious HCV models. In conclusion, our study shows no evidence that Tac or CsA interferes with IFN-α-mediated inhibition of HCV replication and virion production in vitro. Therefore, no further mechanistic arguments have been found to break the clinical controversy about the choice of calcineurin inhibitors during posttransplantation antiviral therapy. http://repub.eur.nl/res/pub/15102/ 2005-05-01T00:00:00Z OBJECTIVE: Loss of expression of progesterone receptors (PR) in endometrial cancer is related to a more invasive and metastatic phenotype. In this study we aim to investigate whether selective loss of PRA or PRB affects the invasive capacity of endometrial cancer cells. METHODS: cDNA microarrays were performed to compare gene expression profiles of a set of endometrial cancer sub-cell lines expressing PRA and/or PRB. In vitro invasion assays were performed to assess whether differences in gene expression between the lines were reflected by their invasive behavior. RESULTS: It was observed that cell lines that express only PRA express higher levels of cadherins, and show a lower level of invasion compared to cell lines that express PRB. When cadherin function was inhibited in exclusively PRA-expressing cell lines, an increase of in vitro invasion was observed. In support of these findings, it was observed that in higher grade and more invasive endometrial cancer, expression of E-cadherin decreased. CONCLUSIONS: These results indicate that relative loss of PRA during progression of endometrial cancer can have a negative impact on cadherin expression, which may lead to development of a more metastatic phenotype. http://repub.eur.nl/res/pub/13437/ 2004-10-15T00:00:00Z Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed. http://repub.eur.nl/res/pub/10130/ 2003-01-01T00:00:00Z Tibolone, a synthetic steroid acting in a tissue-specific manner and used in hormone replacement therapy, is converted into three active metabolites: a Delta(4) isomer (exerting progestogenic and androgenic effects) and two hydroxy metabolites, 3 alpha-hydroxytibolone (3 alpha-OH-tibolone) and 3beta-OH-tibolone (exerting estrogenic effects). In the present study an endometrial carcinoma cell line (Ishikawa PRAB-36) was used to investigate the progestogenic properties of tibolone and its metabolites. This cell line contains progesterone receptors A and B, but lacks estrogen and androgen receptors. When tibolone was added to the cells, complete conversion into the progestogenic/androgenic Delta(4) isomer was observed within 6 d. Furthermore, when cells were cultured with tibolone or when the Delta(4) isomer or the established progestagen medroxyprogesterone acetate was added to the medium, marked inhibition of growth was observed. Interestingly, 3 beta-OH-tibolone also induces some inhibition of growth. These growth inhibitions were not observed in progesterone receptor-negative parental Ishikawa cells, and progestagen-induced growth inhibition of PRAB-36 cells could readily be reversed using the antiprogestagen Org-31489. Upon measuring the expression of two progesterone-regulated genes (fibronectin and IGF-binding protein-3), tibolone, the Delta(4) isomer and medroxyprogesterone acetate showed similar gene expression regulation. These results indicate that tibolone, the Delta(4) metabolite, and to some extent 3 beta-OH-tibolone exert progestogenic effects. Tibolone and most likely 3 beta-OH-tibolone are converted into the Delta(4) metabolite. http://repub.eur.nl/res/pub/10230/ 2003-01-01T00:00:00Z PURPOSE: In endometrial cancer, loss of progesterone receptors (PR) is associated with more advanced disease. This study aimed to investigate the mechanism of action of progesterone and the loss of its receptors (PRA and PRB) in development of endometrial cancer. EXPERIMENTAL DESIGN: A 9600-cDNA microarray analysis was performed to study regulation of gene expression in the human endometrial cancer subcell line Ishikawa PRAB-36 by the progestagen medroxy progesterone acetate (MPA). Five MPA-regulated genes were selected for additional investigation. Expression of these genes was studied by Northern blot and by immunohistochemistry in Ishikawa subcell lines expressing different PR isoforms. Additionally, endometrial cancer tissue samples were immunohistochemically stained to study the in vivo protein expression of the selected genes. RESULTS: In the PRAB-36 cell line, MPA was found to regulate the expression of a number of invasion- and metastasis-related genes. On additional investigation of five of these genes (CD44, CSPG/Versican, Tenascin-C, Fibronectin-1, and Integrin-beta 1), it was observed that expression and progesterone regulation of expression of these genes varied in subcell lines expressing different PR isoforms. Furthermore, in advanced endometrial cancer, it was shown that loss of expression of both PR and E-cadherin was associated with increased expression CD44 and CSPG/Versican. CONCLUSION: The present study shows that progestagens exert a modulatory effect on the expression of genes involved in tumor cell invasion. As a consequence, loss of PR expression in human endometrial cancer may lead to development of a more invasive phenotype of the respective tumor. http://repub.eur.nl/res/pub/9214/ 1999-01-01T00:00:00Z 17Beta-hydroxysteroid dehydrogenase-3 (17betaHSD3) deficiency is an autosomal recessive form of male pseudohermaphroditism caused by mutations in the HSD17B3 gene. In a nationwide study on male pseudohermaphroditism among all pediatric endocrinologists and clinical geneticists in The Netherlands, 18 17betaHSD3-deficient index cases were identified, 12 of whom initially had received the tentative diagnosis androgen insensitivity syndrome (AIS). The phenotypes and genotypes of these patients were studied. Endocrine diagnostic methods were evaluated in comparison to mutation analysis of the HSD17B3 gene. RT-PCR studies were performed on testicular ribonucleic acid of patients homozygous for two different splice site mutations. The minimal incidence of 17betaHSD3 deficiency in The Netherlands and the corresponding carrier frequency were calculated. Haplotype analysis of the chromosomal region of the HSD17B3 gene in Europeans, North Americans, Latin Americans, Australians, and Arabs was used to establish whether recurrent identical mutations were ancient or had repeatedly occurred de novo. In genotypically identical cases, phenotypic variation for external sexual development was observed. Gonadotropin-stimulated serum testosterone/androstenedione ratios in 17betaHSD3-deficient patients were discriminative in all cases and did not overlap with ratios in normal controls or with ratios in AIS patients. In all investigated patients both HSD17B3 alleles were mutated. The intronic mutations 325 + 4;A-->T and 655-1;G-->A disrupted normal splicing, but a small amount of wild-type messenger ribonucleic acid was still made in patients homozygous for 655-1;G-->A. The minimal incidence of 17betaHSD3 deficiency in The Netherlands was shown to be 1: 147,000, with a heterozygote frequency of 1:135. At least 4 mutations, 325 + 4;A-->T, N74T, 655-1;G-->A, and R80Q, found worldwide, appeared to be ancient and originating from genetic founders. Their dispersion could be reconstructed through historical analysis. The HSD17B3 gene mutations 326-1;G-->C and P282L were de novo mutations. 17betaHSD3 deficiency can be reliably diagnosed by endocrine evaluation and mutation analysis. Phenotypic variation can occur between families with the same homozygous mutations. The incidence of 17betaHSD3 deficiency is 0.65 times the incidence of AIS, which is thought to be the most frequent known cause of male pseudohermaphroditism without dysgenic gonads. A global inventory of affected cases demonstrated the ancient origin of at least four mutations. The mutational history of this genetic locus offers views into human diversity and disease, provided by national and international collaboration. http://repub.eur.nl/res/pub/8595/ 1993-01-01T00:00:00Z Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.