Iron (“Fe”) is a fundamental element required by all cells for growth and normal physiological processes. Rapidly proliferating cells have higher iron requirements than quiescent cells. In humans, iron is thought to be provided by the binding of iron to the major serum iron-transporting protein, transferrin (“Tf”). Transferrin bound to iron can bind as a complex to the transferrin receptor expressed on the plasma membrane of most cells. After binding, the iron-transferrin-transferrin receptor complex remains membrane bound and is concentrated and then taken into the cell via endocytotic vesicles. Within the cell, the endosomes become acidified and the iron is released from the complex. The iron-free apotransferrin molecules remain bound to the transferrin receptor and are recycled to the cell surface where they are released to participate further in the uptake of more iron. Disruption of blood circulation deprives the cell of oxygen and iron and may result in cell death. Deposition of iron from cell death, for example in ischemic injury, may result in the generation of highly reactive and toxic superoxide or hydroxyl free radicals which can result in further tissue damage. Accordingly, the abundance of iron and its availability can greatly alter survival of damaged tissues. Rapidly proliferating cells, such as malignant cells, have an increased requirement for iron and must possess efficient mechanisms to obtain iron. Limiting the ability of malignant cells to acquire iron may provide a method of killing tumour cells or modulating their uncontrolled cell growth.

Iron is rarely found in the blood plasma in the free state since it is highly toxic. Transferrin, one of the major proteins in blood plasma, serves mainly to mop up free iron and to shuttle it among the organs of the body in a soluble non-toxic form. The established mechanism by which cells acquire iron and transferrin involves binding to the cell surface oriented transferrin receptor (“TR”) which binds to iron-loaded transferrin and internalizes the complex by the mechanism of receptor mediated endocytosis (“RME”). Since normal blood plasma levels of transferrin are high and about 99% of iron in the plasma is bound to it, iron uptake is believed to be regulated by the level of TR expression. Any free iron generally circulates in the form of low molecular weight complexes such as citrate and certain amino acids or in association with other plasma proteins such as albumin. High levels of free iron are usually only found in the plasma upon release from dying cells or during iron overload disorders such as haemochromatosis, thalassaemia and atransferrinemia.

Based on studies where cells were grown in serum free (hence transferrin-free) media and in cases of iron overload disorders it has become evident that some cells are able to obtain iron by mechanisms other than the RME pathway. Although cellular iron uptake has been shown to be mediated mainly by the transferrin receptor, a non-transferrin-mediated pathway has been implicated for iron incorporation into leukemic cells, HeLa cells, hepatocytes and melanoma cells.

The protein p97, also known as melanotransferrin, was one of the first cell surface antigens associated with the human skin cancer melanoma. p97 is a monomeric membrane-associated protein with a molecular mass of 97,000 Daltons and has been suggested as a melanoma specific marker. As well as being associated with the cell surface of melanomas and some other tumours and cell lines, p97 has also been found in certain fetal tissue and, more recently on normal endothelial cells of the human liver.

The primary structure of p97, deduced from its mRNA sequences indicates that it belongs to a group of closely related iron binding proteins found in vertebrates. This family includes serum transferrin, lactoferrin and avian egg white ovotransferrin. Human p97 and lactotransferrin share 40% sequence homology, however, in contrast to

the other molecules of the transferrin family, p97 is the only one which is directly associated with the cell membrane.The deduced sequence of p97 has, in addition to a transferrin-like domain, a hydrophobic segment at its C-terminal which is thought to allow the molecule to be inserted into and held in the plasma membrane.

Detergent-solubilized p97 has been reported to bind iron. However, the role of p97 in iron transport is far from clear. Iron binding to p97 at the plasma membrane has not been demonstrated and, despite numerous studies, no evidence of a role for p97 in iron-mediated transport has been obtained to date. Recent studies have concluded that p97 does not play a role in iron transport. The physiological role of p97 in normal and malignant cells has thus not been determined.

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