International conference for healthcare and medical students 2011 dublin, ireland. 4-5 november 2011. Abstracts.

withdrawn: O13 Abstract withdrawn BMC Proceedings 2012, 6(Suppl 4):O13 O14 Inhibition of IGF signalling pathway in MDA-MB-231 triple negative breast cancer cells S Chandran, JH Harmey, S Toomey School of Medicine, Royal College of Surgeons in Ireland; Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland BMC Proceedings 2012, 6(Suppl 4):O14 Introduction: Triple-negative breast cancer (TNBC) is characterised by the absence of estrogen receptor (ER), progesterone receptor (PR) and the HER-2 receptor. TNBC is typically associated with a poor prognosis due to aggressive tumour phenotypes, partial response to chemotherapy, and current lack of clinically validated targeted therapies. Insulin-like growth factors (IGFs) stimulate cell proliferation and promote cell survival via receptor phosphorylation and activation of adaptor proteins such as mitogen-activated protein kinase (MAPK), and Akt. The overall aim of this project was to characterise the expression and activation of the IGF signalling pathway in the MDA-MB-231 TNBC cell line. Methods: Expression of steroid and growth hormones and activation of the IGF signalling pathway in MDA-MB-231 cells was analysed by western blotting, while expression of PAPP-A was detected by PCR. The effect of stimulation with IGF1 or inhibition of EGFR/IGF1R tyrosine kinase activity on proliferation was determined by the MTS cell proliferation assay. Proliferation was expressed relative to untreated controls, and data was analysed by ANOVA with Tukey’s multiple comparison post hoc test. Results: MDA-MB-231 cells express epidermal growth factor receptor (EGFR) and low levels of HER3, and were confirmed as negative for ER, PR and HER2. IGFBP4 inhibits IGF1 activity but cleavage by pregnancy associated plasma protein A (PAPP-A) protease releases active IGF1. MDA-MB-231 cells express high levels of insulin-like growth factor binding protein 4 (IGFBP4), and low levels of PAPP-A. Moreover, MDA-MB-231 cells express type I IGF1 receptor and proteins along the IGF signalling cascade; namely, Erk and Akt. The presence of phosphorylated forms of these proteins shows activation of the IGF1R signal transduction pathway in MDA-MB-231 cells. Proliferation was increased by IGF1 (E3R) (recombinant IGF1, resistant to binding by IGFBPs). Inhibition of EGFR tyrosine kinase activity or IGF1R tyrosine kinase activity inhibited proliferation of MDA-MB-231 cells and a similar effect was observed with dual inhibitors of PI3K/mTOR or Akt/ P70S6K. Conclusions: These results suggest that the IGF1 signalling pathway is activated in MDA-MB-231 TNBC cells. Therefore, inhibition of the IGF1R and/ or its downstream targets may be of benefit in the treatment of TNBC. O15 Producing and evaluating a novel lentiviral vector for b-thalassaemia gene therapy Y Bokinni, V Kao, M García-Gómez, M Antoniou King’s College London, School of Medicine, Department of Medical and Molecular Genetics, UK E-mail: [email protected] BMC Proceedings 2012, 6(Suppl 4):O15 Introduction: The b-haemoglobinopathies are of the most prevalent inherited disorders worldwide. b-thalassaemia is a single gene disorder affecting the b-globin gene, thus resulting in a lack or depleted availability of b-globin for formation of haemoglobin. b-thalassaemia has become a target for gene therapy based treatments in hope of a cure, or significant phenotype amelioration. The technique aims to treat the haematopoietic stem cells (HSC) of patients with the viral vectors ex vivo, in the hope of significant b-globin mRNA transcript production on HSC erythroid differentiation post re-transplantation. Numerous investigations have been conducted in the use of Lentiviral vectors harbouring human b-globin transcription units, only one of which has proceeded into clinical trials (Cavazzana-Calvo, Payen et al. 2010). The ultimate aim of all ‘construct’ designs is to present significant phenotype amelioration with an average of one vector copy number (VCN) per HSC. Methods: Antoniou’s group have recently devised a number of “GLOBE” constructs based on previous functional studies with the inclusion of regions physiologically present within the endogenous b-globin gene, previously deemed insignificant, and therefore, omitted from to all known published constructs to date. Previous observations with gammaretroviral vectors had determined the inclusion of the full b-globin 2nd intron to be highly detrimental to viral production and quality (Leblouch, 1994). Based on recent findings, the inclusion of a transcriptional terminator region and full 2nd intron have been added, yielding the latest generation of Lentiviral vector constructs (GLOBE-2 and GLOBE-4). The aim of this project was to produce these Lentiviral vectors and conduct a comparative expression analysis via transducing Murine erythroleukaemia cells. Results and conclusions: Average viral titres obtained for the GLOBE-2 and GLOBE-4 constructs were 7.2x107 and 5x107 viral particles (vp)/ml respectively, incurring a 31% variance despite a 600bp difference in size. BMC Proceedings 2012, Volume 6 Suppl 4 http://www.biomedcentral.com/bmcproc/supplements/6/S4 Page 4 of 29