The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy

Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.

Retroviral-mediated gene transfer of the human erythropoietin gene into a factor-dependent cell line, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.

Prokineticin ligands and receptors are expressed in the human fetal ovary and regulate germ cell expression of COX2

CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear.

OBJECTIVE: To investigate expression and localization of the PROK ligands, receptors and their downstream transcriptional targets in the human fetal ovary.

SETTING: This study was conducted at the University of Edinburgh.

PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses.

DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting and immunohistochemistry. Functional studies were performed using a human germ tumour cell line (TCam-2) stably transfected with PROKR1.

RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 weeks) than at earlier gestations (8-11 and 14-16 weeks). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localised to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signalling in the human fetal ovary. PROK1 treatment of a germ cell line stably-expressing PROKR1 resulted in ERK phosphorylation, and elevated COX2 expression.

CONCLUSIONS: Developmental changes in expression and regulation of COX2 and pERK by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.

Prior knowledge transfer across transcriptional data sets and technologies using compositional statistics yields new mislabelled ovarian cell line

Here, we describe gene expression compositional assignment (GECA), a powerful, yet simple method based on compositional statistics that can validate the transfer of prior knowledge, such as gene lists, into independent data sets, platforms and technologies. Transcriptional profiling has been used to derive gene lists that stratify patients into prognostic molecular subgroups and assess biomarker performance in the pre-clinical setting. Archived public data sets are an invaluable resource for subsequent in silico validation, though their use can lead to data integration issues. We show that GECA can be used without the need for normalising expression levels between data sets and can outperform rank-based correlation methods. To validate GECA, we demonstrate its success in the cross-platform transfer of gene lists in different domains including: bladder cancer staging, tumour site of origin and mislabelled cell lines. We also show its effectiveness in transferring an epithelial ovarian cancer prognostic gene signature across technologies, from a microarray to a next-generation sequencing setting. In a final case study, we predict the tumour site of origin and histopathology of epithelial ovarian cancer cell lines. In particular, we identify and validate the commonly-used cell line OVCAR-5 as non-ovarian, being gastrointestinal in origin. GECA is available as an open-source R package.