The role of ceramide in growth arrest and terminal differentiation of human leukaemia cells

The processes of proliferation, differentiation, and apoptosis are tightly regulated during haemopoiesis with aberrations resulting in the uncontrolled clonal growth of non-functional haemopoietic cells, a disease state known as leukaemia. Approaches that target cellular growth and differentiation pathways to alter the proliferative potential of leukaemia cells while limiting cytotoxicity to healthy cells have recently undergone a revival in clinical interest. Known as differentiation therapy, the immature cancer cells undergo terminal differentiation culminating in the ultimate removal of these cells by apoptosis. The sphingomyelin signalling pathway, and its second messenger ceramide, has emerged as an important regulator of cellular growth, differentiation and apoptosis. Whilst numerous studies have addressed the apoptogenic properties of ceramide signalling, very few have investigated the mechanisms underlying ceramide mediated terminal differentiation. The aim of this study was to investigate the potential for the ceramide signalling pathway to be utilised in differentiation therapy for the treatment of human leukaemia. A range of haemopoietic cell lines were treated with the synthetic ceramide analogue, C2-ceramide. Growth inhibition, cell cycle arrest, activation of the retinoblastoma (Rb) tumour suppressor protein, expression of the cyclin-dependent kinase inhibitor p21aPI/Waf 1 and apoptosis were shown to be common outcomes of ceramide treatment. Myeloid cells exposed to ceramide differentiated into a mature phenotype. Taken together, these results show that ceramide is inducing terminal differentiation in the treated cell lines. Phosphatases have a well characterised function in Rb activation and their potential as an effector mechanism for ceramide-induced Rb activation and differentiation was examined. Inhibitor studies revealed that ceramide-mediated Rb activation resulted from protein phosphatase activity, and that the contribution of p2Cip 1/Waft was not significant, at least in the early phases of cell cycle arrest. Interestingly, inhibition of protein phosphatase activity did not impact on the ability of ceramide signalling to induce myeloid cell differentiation. This lead to the conclusion that Rb activation and ceramide-induced differentiation occur by two independent pathways. Gene expression profiling using a cDNA filter array was performed to elucidate the involvement of genetic mechanisms involved in the ceramide response. Genes that prevent progression through the cell cycle and genes involved in differentiation were upregulated whereas the expression of proliferation-related genes were downregulated. These findings are consistent with the hypothesis that ceramide reduces the proliferative potential of leukaemic cells by inducing terminal differentiation. This thesis presents a comprehensive study into the biological effects of the ceramide signalling pathway, and has in part defined the molecular mechanisms responsible for ceramide-induced growth arrest and differentiation. Ceramide was shown to mediate the terminal differentiation of leukaemia cells into mature, nondividing cells that die by apoptosis. Manipulation of the ceramide-signalling pathway may present a novel therapeutic target for the treatment of diseases that lack control of growth and differentiation processes.