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Abstract

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.

Item Type:	Thesis - PhD
Authors/Creators:	Connor, Charles Edward
Keywords:	Leukemia, Cancer, Ceramides
Copyright Holders:	The Author
Copyright Information:	Copyright 2004 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).
Additional Information:	Thesis (Ph.D.)--University of Tasmania, 2004. Includes bibliographical references
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