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UBC Theses and Dissertations

The cellular and functional role of melanotransferrin in melanomas Tian, Mei Mei

Abstract

Melanoma, one of the deadliest forms of skin cancers, is a malignant tumor derived from abnormal proliferation of epidermal melanocytes, and its formation involves a series of events leading to altered cellular properties and rapid proliferation. High cellular iron content has been connected to the development of cancers in human. One way iron may affect cancer development and progression is by altering cell growth and proliferation. Iron has been proposed to promote progression from G₁ to S phase of the cell cycle through activation of ribonucleotide reductase during DNA synthesis. Iron is normally absorbed by the enterocyte of the duodenum where it is transported throughout the body via serum transferrin. However, the existence of transferrin-transferrin receptor independent iron transport pathways has been shown. Such pathways are thought to play critical roles in non-transferrin bound iron overload in diseases such as hemochromatosis and Alzheimer’ s disease, though the molecular details of these pathways remain obscure. Many genes are up-regulated to aid the abnormal proliferation of cancer cells and subsequent invasion of tissues. In the case of melanoma, the expression of melanotransferrin (p97), an iron binding molecule, is elevated. In this thesis, the cellular and functional role of glycosyiphosphatidylinositol (GPI)-anchored p97 in melanoma was investigated. The first aim of this thesis was to perform detailed investigation of the internalization pathway of GPI-anchored p97 in melanoma cells. Although many GPI anchored proteins have been studied previously, there is still an on-going debate whether caveolae-dependent or clathrin-dependent pathways are involved. Using confocal immunofluorescence microscopy and quantitative immunoelectron microscopy, iron bound GPI-anchored p97 was shown to be internalized via a caveolae vesicles dependent endocytotic pathway. In addition, endosomal disruption studies demonstrated that the intracellular trafficking of the GPI-anchored p97 in melanoma cells is endosomal dependent. The studies performed in this thesis demonstrate that GPI-anchored p97 protein can mediate the iron uptake in melanoma cells. As GPI-anchored p97 is highly expressed in melanoma cells, this thesis also examined the functional role of GPI anchored p97 in melanoma cells and tumors. The expression of GPI-anchored p97 in melanoma cells through over-expression and down-regulation techniques. The results demonstrated that the GPI-anchored p97 promote melanoma cell proliferation, melanin production and secretion in vitro and melanoma tumor growth in vivo. Taken together, this thesis sheds new light on the relationship between GPI-anchored p97, melanoma development and cellular iron uptake.

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