Search

Gene expression profiling of ovarian carcinoma tissues has shown an increase of four-fold expression of SORT1 gene. Sortilin 1 (NTR-3) is a 95-100 kDa protein normally expressed in heart, brain, placenta, skeletal muscle, spinal cord, thyroid, and testis. However, its expression has never been reported in normal ovary. Here, we report expression of sortilin 1 in ovarian carcinoma tissues both at gene and protein levels. Sortilin 1 was expressed in all ovarian carcinoma patients (n=15) as well as ovarian carcinoma cell lines (n=5) regardless of their phenotypic characteristics. Non-malignant ovaries (n=6) did not express sortilin 1. The molecular basis for this ectopic expression is not yet clear. Our results showed a major cell surface expression of sortilin 1 rather than ER-Golgi compartment where it is mainly expressed. This finding may introduce sortilin 1 as a novel tumor marker for diagnosis of ovarian carcinoma and may signify its therapeutic value in targeted therapy.

Introduction The cells expressing Indoleamine 2, 3-dioxygenase (IDO) in feto-maternal interface mediate tryptophan catabolism, hence protect allogeneic fetus from lethal rejection by maternal immune responses. In this study, we report immuno-localization of IDO+ cells in murine reproductive tract and placenta throughout mouse pregnancy by immunohistochemistry. Materials and Methods Syngeneic pregnant mice were examined for vaginal plug to discover about their state of pregnancy. A total of three pregnant mice were examined at each stage.The examination was further confirmed by the detection of sperm in vaginal smear. On the gestational days of 2nd, 12th and 18th, the uterus and oviduct were removed and expression of IDO was investigated in the endometrium, placenta and oviduct by immunohistochemistry. Results Our results showed that IDO is expressed consistently in feto-maternal interface throughout pregnancy. In endometrium, expression of IDO was predominantly confined to luminal and glandular epithelial cells. Cells at junctional and labyrinth zones of placenta showed strong IDO immunoreactivity as well. Conclusion Expression of IDO at the protein level in reproductive tract of pregnant mice during entire periods of gestation points to its potential protective role in maintenance of pregnancy. In our knowledge this is the first report of expression of IDO in feto-maternal phase during murine pregnancy.

Comprehensive integration site analysis for monitoring the clonal dynamics in clinical gene therapy has revealed that the insertion of the therapeutic retroviral vector (RV) can deregulate and even substantially activate neighboring genes leading to selection advantage and clonal outgrowth. Strikingly, 7 out of 10 Wiskott Aldrich Syndrome (WAS) gene therapy patients developed acute leukemia driven by gene corrected cell clones aberrantly expressing LMO2, MDS1 or MN1. This indicates that RV-mediated activation of adjacent regions cannot only influence the fate of hematopoietic stem cells but also cause clonal dominance up to leukemia. To identify novel regulators of benign hematopoiesis we established a systematic selection strategy using the total genomic integration site dataset of normal and highly polyclonal clinical blood and bone marrow samples. In this thesis, the unique integration site (IS) dataset within a cohort of 10 WAS gene therapy patients was systematically analyzed to select for candidate genes involved in the regulation of hematopoiesis. Initially, a total of 12.887 unique IS in vicinity of 3.267 genes were identified. Next, we selected all genes with at least 10 different IS within a 200 kb window around the gene (n=588). To enrich for genes with increased probability of transcriptional activation we then chose those genes with at least 10 IS within a 50kb window around the transcriptional start site (n=424). After stringent exclusion of all genes located within gene clusters 32 candidate genes were identified. To evaluate the hematopoietic activity of gene corrected cell clones, their contribution to blood cell formation within four years post gene therapy was monitored. We observed that these clones were detectable 15 to 93 times in a total of 102 individually analyzed patient samples, demonstrating long term activity of these hematopoietic stem cell clones. Interestingly, 20 out of the 32 highest ranked genes such as EVI1, CCND2 and LMO2 are known hematopoietic key regulators, strongly validating our selection strategy. After identification of 12 novel hematopoietic regulatory candidate genes, the top five ranked genes, ZNF217, LRRC33, PLCB4, EVL and IRF2BPL were chosen for further functional analysis in murine hematopoietic primary cells. To evaluate the endogenous expression of candidate genes in murine hematopoietic stem and progenitor cells global transcriptome datasets from purified populations were evaluated. We observed that all five selected candidate genes were expressed in at least one out of the five analyzed hematopoietic stem and progenitor cell populations which may point to an important role in the respective cell fraction. In order to validate our selection II strategy and to further investigate whether the chosen candidate genes play roles in hematopoiesis, we performed various in vitro and in vivo assays. We tested the effect of the selected candidate genes on proliferation, differentiation, and cytokine independency as well as for their influence on long term multilineage reconstitution and self-renewal after murine bone marrow transplantation. The first candidate gene, ZNF217, is a zinc finger protein known as a transcription factor. To analyze the impact of ZNF217 on transcriptional activity, global gene expression profiling in hematopoietic cells was performed. We observed that 337 out of 422 genes were significantly downregulated and that they are mainly involved in cellular movement indicating that ZNF217 plays a role in hematopoietic cell migration. Since ZNF217 is known as a proto-oncogene in breast and ovarian carcinoma we evaluated its effect on growth factor independency of hematopoietic cells. Interleukin 3 (IL3)-dependent cells overexpressing ZNF217 acquired the capacity to survive and form colonies in the absence of IL3 suggesting a transforming role for ZNF217 in hematopoietic cells. The second candidate LRRC33 resembles the protein structure of Toll-like receptor (TLR) proteins which are involved in innate immunity. The overexpression of LRRC33 and TLR4 decreased the activity of NF-κB in vitro when stimulated with bacterial lipopolysaccharide. This may point to an inhibitory role of LRRC33 in NF-kB signaling, an important pathway for maintaining stem cell integrity. Transplanted LRRC33-overexpressing LSK (Lin-Sca-1+cKit+) cells gave rise to a 1.3-6.7-fold lower ratio of T-cells and in contrast a 1.1-7.2-fold higher amount of donor derived macrophages in secondary recipients compared to control mice. To study the function of the third candidate gene on hematopoiesis constitutive PLCB4 knockout mice were obtained. This phospholipase has been shown to be important for brain development but has not been linked to hematopoiesis so far. Preliminary results indicate that PLCB4 deficient mice have a reduced LSK cell fraction compared to age matched littermates within the first 18 days after birth. These results demonstrate that clinical integration site datasets can be used to identify regulatory genes of hematopoiesis. Here, we identified ZNF217 as a driver of hematopoietic transformation applying the established selection strategy. In total, we could show that four out of five candidate genes play a role in hematopoiesis and they will be further evaluated for their stem cell regulatory potential. Systematic identification of novel regulatory genes in meta-datasets derived from a larger number of gene therapy studies and subsequent validation in vitro and in vivo will allow to gain new insights into the biology of post-embryonic hematopoiesis.