The acquisition of invasive potential by cancer cells is considered to play a central role in tumor progression, as it enables such cells to disseminate from the primary site of growth to distant organs. We have previously demonstrated that cell adhesion, rearrangement of extracellular matrix components, assembly of cytoskeleton networks, cell geometry and locomotion are all closely interrelated properties affecting tumor cell metastasis. Since cell motility is a prerequisite for tumor cell invasion, the first step in metastasis, it is critical to explore the mechanisms governing cell motility and to identify factors that regulate this process. Recent studies have demonstrated that certain tumor cells, including melanoma, sarcoma and carcinoma cells produce and secrete, an autocrine motility factor (AMF), a 55 kDa cytokine, and respond to it via a cell surface receptor designated gp78. Here we report on the isolation and purification of the mouse and human melanoma AMFs and the generation of an antibody against AMF. AMF was found to be not only a highly potent motogen, but also a mitogen exhibiting differential growth stimulation on normal and tumor cells. The cellular response to AMF is mediated by G- protein(s), inositol triphosphate production, arachidonic acid metabolism, receptor phosphorylation probably involving PKC and receptor-ligand internalization. The AMF receptor was purified, cloned and mapped to human chromosome 16 at band p21. Gp78, like the receptors of many growth factors has at least three distinct domains with specific function: an extracellular N-terminal ligand-binding domain, a transmembrane anchoring domain and a cytoplasmic C-terminal domain which undergoes phosphorylation in response to ligand binding and may contain a kinase region, regulatory sequences and sites responsible for interaction with intracellular substrates. Gp78 is not randomly distributed on the cell surface, but rather is localized at distinct poles, controlling the directional motility of migratory cells and its expression in formal cells is cell- contact regulated. The preliminary results suggest that AMF may be involved in an """"""""autocrine loop"""""""" providing motile and growth advantages to invading cells.
The aim of this project is to continue to study the role of AMF and its receptor in vitro (motility) and in vivo (hematogenous spread). In particular it is proposed: l) to clone the gene coding for AMF and to chromosomally map it; 2) to continue to study the effect of AMF-gp78 interaction on cell migration and proliferation; 3) to continue to study signal transduction pathways of motility and proliferation stimulation by AMF; 4) to clone the gene coding for the murine gp78 from normal and tumor cell cDNA libraries; 5) to transfect specific sense and anti-sense gp78 coding sequences into normal and tumor cells and analyze its expression in relation to cell-contact regulation, motility and metastasis. It is expected that these studies will allow a better understanding of the role of the AMF-gp78 interaction in cell kinesis and proliferation and their relevance to invasion and metastasis.
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