Molecular profiling of breast cancer cell lines containing amplified fibroblast growth factor receptors and Analysing lymphocyte populations and CD52 intensity of stem cell transplant bags and during early immune reconstitution following allogeneic stem cell transplantation.

摘要

Project 1: Fibroblast Growth Factor Receptor (FGFR) genes 1 and 2 are amplified in a subset of breast cancers. This can lead to overexpression of the receptor at the extracellular membrane resulting in increased ligand independent dimerization and subsequent aberrant signalling involved in the development and progression of the tumour. A SILAC experiment using SU5402 and Dasatinib, FGFR and Src inhibitors respectively was performed on CAL120 breast cancer cells following validation of the function and effectiveness of the inhibitors via western blotting. Increasing our knowledge of the phosphorylation events involved in the signalling pathways affected in breast cancers will produce a molecular map of specific breast cancers allowing more specialised treatment. 340 proteins were found with significantly decreased levels of phosphorylation when cells were treated with SU5402, with MAPK pathway repression. Dasatinib treatment reduced 131 phospho-proteins and suggested components of Src directed cytoskeleton assembly necessary for FGFR transport. Testing of separate FGFR and Src inhibitor effectiveness was also achieved as well as method development of the phosphoenrichment stage of the SILAC experiment. Duplicate SILAC experiments need to be performed to increase the data set and validate findings.ududProject 2: Haematopoietic stem cell transplantation is a last resort treatment to cure many haematological malignancies. Stem cells collected from a donor are infused into the patient following chemo/radiotherapy treatment. The graft verses leukaemia response is essential for remission to occur but is likely to result in a graft versus host disease (GvHD) side effect. Campath, a CD52 specific monoclonal antibody, is used as a GvHD preventative measure as it reduces the T lymphocytes responsible for the effect. Comparison of the immune system cell populations present in the transplanted cells have not been tested to see if they are the same as a healthy donor, or how they change during the first two weeks post-transplantation. Our findings indicate that the ratios of T, B and NK lymphocytes are different in the transplanted cells compared to healthy blood and that CD8 and CD4 T cells reach a roughly 1:1 ratio. CD52 intensity was also dimmed in all cell types before transplantation, although campath was still able to reduce cell numbers, affecting T and B lymphocytes with higher CD52 densities than NK cells. Post-transplantation NK cells become the dominant lymphocyte cell population conferring a GvL response and restricting GvHD.ud