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Title: Investigation of multimolecular complexes and signalling in chronic myeloid leukaemia (CML)
Author: Patel, Hetal
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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Chronic Myeloid Leukaemia (CML) arises as a consequence of the expression of a chimaeric fusion protein; p210BCR-ABL. Many publications report that p210BRR-ABL forms complexes with multiple cytoplasmic proteins which affect signalling pathways demonstrated in cell lines or transduced cells. This has been necessary because primary haemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL. We have identified that the degradative enzymes located in the cell lysosomes and have demonstrated substantial inhibition of the p210BCR-ABL-degradative activity by high pH lysis conditions. We show to the best of our knowledge, the first set of data demonstrating expression and immunoprecipitation of p210BCR-ABL and co-immunoprecipitation of adaptor proteins CBL, CRKL and GRB2. The degradative activity also affects ABL protein, preventing analysis of protein complexes of normal ABL but using the high pH lysis we have shown that normal ABL complexes with GRB2 potentially mediated via BCR or a direct association with ABL, this is different from some cell line data published which implies that proteins that complex with p210BCR-ABL do not complex with normal ABL. We also analysed complexes in two Ph-negative, acute lymphocytic leukaemia (ALL) cell lines, KG1a and HL60 cells and found ABL forms complexes with CBL, CRKL and GRB2 similar to CML. This data suggests that adaptor proteins which complex with p210BCR-ABL also form complexes with ABL in Ph- leukaemic cells and some of them form complexes in non-leukaemic cells. Thus, there may be CML-specific, leukaemia-associated and normal interactions with ABL proteins. Using confocal microscopy and a junction specific anti-BCR-ABL (b2a2) antibody we analysed the subcellular distribution of p210BCR-ABL and have shown that p210BCR-ABL is arranged in discrete foci in the cell cytoplasm in various CML cell lines and primary CML cells. Many studies have implicated CRKL as an important target of p210BCR-ABL that can be used as an indirect indicator of p210BCR-ABL protein tyrosine kinase activity. When we analysed co-localisation of p210BCR-ABL and CRKL, we found that CRKL also formed foci. However, the CRKL and p210BCR-ABL foci were completely or partially associated or separate in different regions of the same cell. Since CRKL in CML is phosphorylated by association with p210BCR-ABL, these data imply that binding of CRKL and p210BCR-ABL maybe in a state of dynamic equilibrium. Heterogeneity of protein complexes from patients in blast crisis (BC) was observed, where the p210BCR-ABL-CBL complex was absent in one CML BC patient. Effects of imatinib on protein complexes were analysed and we found that p210BCR-ABL-CRK1 and p210BCr-ABL- CBL complex dissociates over time of treatment however, the GRB2 remains in complex within the 24 hour treatment period which maybe a potential target in imatinib resistant CML cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available