Bureau, E.A.; (2008) Investigation of the role of haematopoietic cell kinase in normal and leukaemic haematopoiesis. Doctoral thesis , University of London.

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Abstract

Acute myeloid leukaemia (AML) is characterised by an accumulation of immature blasts that fail to fully differentiate. Leukaemia is organised as a hierarchy, which is maintained by leukaemic stem cells (LSC). To identify molecular differences between normal haematopoietic stem cells (HSC) and LSC, we performed microarray analysis and found that haematopoietic cell kinase (HCK) is overexpressed in LSC. Thus, by knocking-down HCK in leukaemic cells or overexpressing it in stem cell enriched fractions, we should be able to evaluate its role in leukaemogenesis. Since LSC are difficult to culture in vitro, we started to validate HCK silencing in leukaemic cell lines, Mono-mac-6 (MM6), U937 and Fujioka/P31, which highly express HCK. In all cell lines studied, no more than 50% silencing could be achieved, even when a short-hairpin anti-HCK was cloned into a lentiviral backbone to follow the long-term effect of HCK silencing. Decrease in kinase activity was confirmed using kinase assay and phospho-specific antibody recognising the activated HCK kinase. We show that HCK silencing does not affect the cell cycle, proliferation, differentiation or apoptosis of the cell lines. However, using methylcellulose assay, we observe a significant change in MM6 colony morphology caused by a decrease in their migration properties. Moreover, using phospho-flow cytometry, G-CSF and GM-CSF signal transduction towards STAT5 could be proven to occur via HCK in MM6, but not in Fujioka/P31 or U937 cells. HSC enriched umbilical cord blood cells were also transduced with a lentivirus encoding p59HCK. Overexpression of p59HCK in these cells led to their enhanced erythroid differentiation at the expense of myeloid differentiation underlined by the activation of c-Raf, ERK and STATS. Overall, this study can be used as a preliminary set up for further investigation of the role of HCK in normal human stem cells and in primary AML samples in vivo.

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