There are two major aspects of tumor progression: cell proliferation and cell-cell-matrix interactions. During the process of tumor cell dissemination, adhesive mechanisms are rate limiting Integrin receptors on tumor cells have been shown to be involved in various steps of the metastatic cascade i.e., adhesion endothelium and spreading on matrices, as well as in cellular migration. Among the different receptors, beta1 as well as beta3 integrins have been implicated in tumor cell-ECM interactions and as a determinant of the metastatic phenotype. The cytoplasmic domains of integrins are connected to cytoskeletal proteins forming a fibronexus with an extracellular matrix ligand. The attachment point of surface membrane and ECM (focal adhesions) contains the matrix ligand, its receptors(s), as well as cytoskeletal proteins. Both the receptors and the associated proteins can be phosphorylated - either by PTK or PKC - and this phosphorylation alters their function. Experimental data suggest, that outside - in signalling through integrins may involve PTK while inside - out signalling may involve PKC. PKC as well as some tyrosine kinases have been localized to focal adhesions suggesting that PKC and/or PTK activation/translocation may serve as an effector element of the signaling pathway involved in cell-ECM interaction. In our previous work we have found that the 12-lipoxygenase (12 LOX) metabolite of arachidonic acid i.e., 12-(s)-HETE was capable of activating PKC, inducing reversible cytoskeletal rearrangement, increase beta3-integrin expression and stimulate tumor cell motility. We suggest, that 12(S)-HETE is part of an integrin-coupled signaling pathway in tumor cells and plays an important role in their dissemination during metastasis. Therefore, the aims of the present study are: (1) To study the coupling of 12(S)-HETE to beta1/beta3 integrins in melanoma cells (2) To study the involvement of G proteins in integrin-mediated activation of 12-LOX in melanoma cells (3) To study the role of PKC in the integrin- coupled signalling pathway involving 12-LOX in melanoma cells (4) To study lysosomal enzyme release and motility of tumor cells after specific activation of beta1/beta3 integrins in melanoma cells (5) To study the involvement of G-proteins, 12-LOX and PKC in dissemination of melanoma cells. Tumor cell ECM interactions will be defined as integrin mediated adhesion and spreading on fibronectin and subendothelial matrix, cathepsin B release and tumor cell motility. In vivo metastasis formation will be defined as lung colonization (following i.v. injection) and liver colonization. Tumor cells to be used are: B16a murine melanoma and human melanomas HT 168, HT 168-MI, HT 18.
