With the recent advances in primary site control and control of recurrent disease, metastatic disease remains one of the principle impediments to improved cancer treatment. The metastatic cascade is a complex multi-step process which involves homotypic and heterotypic interactions among tumor cells and host cells, in addition to tumor cell adhesion to matrix proteins. Tumor cells possess a variety of phenotypic characteristics which enable them to complete the metastatic process and form a new lesion. However, some steps of the metastatic cascade may be rate limiting and possibly targeted for therapeutic intervention. Most, if not all, steps of the metastatic cascade involve cell surface receptors such as the integrins. A number of integrin adhesion receptors have been found on various human and rodent tumor cell lines. Among them, are the vitronectin receptor alpha-v-beta-3 and alpha-II-b-beta-3. The expression of alpha-II- b-beta-3 was originally thought to be confined to platelets and megakaryocyte lineage cells. Recently, we have demonstrated, by Southern blotting, Northern blotting and immunoprecipitation, the presence of alpha- II-b-beta-3 in several human and murine tumor lines. This study is aimed to determine cDNA sequences of tumor cell alpha-II-b and beta-3 by synthesizing the first strand cDNA by reverse transcription and using the polymerase chain reaction technique to amplify the alpha-II-b and beta-3 cDNAs and sequence the PCR products. Next, the alpha-II-b-beta-3 receptor involvement in phenotypic traits indicative of metastic potential such as adhesion to and spreading on endothelium, platelets and lung colony formation will be determined, in an attempt to establish a possible correlation between alpha-II-beta-3 expression in tumor cells and metastasis. To further define the role of alpha-II-beta-3, murine tumor cell lines have been transfected with anti-alpha-II-b and/or anti-alpha- beta-3 constructs. The alpha-II-b-beta-3 negative cells will be tested for their ability to undergo interactions necessary for metastasis (e.g., platelet aggregation, adhesion, spreading and lung colonization). Moreover, the relative contribution of the cytoadhesin integrins (i.e., alpha-II-b-beta-3 and alpha-v-beta-3) to tumor cell adhesion to and spreading on matrix ligands and endothelium, tumor cell induced platelet aggregation will be studied. A tumor cell line which expresses alpha-II-b but not alpha-v (i.e., B 16 amelanotic melanoma) and another cell line (Lewis lung carcinoma) which expresses both the alpha-II-b and alpha-v will be used to compare the relative contribution of each integrin to metastatic potential. The anti-sense constructs against alpha-II-b and/or alpha-v will be introduced into these cell lines in these comparison experiments prior to adhesion, platelet aggregation and lung colony forming assays. In addition, the cytokines TGF-beta, tumor necrosis factor and interleukin 1, as well as the eicosanoid 12-hydroxyeicosatetraenoic acid, which have been demonstrated to regulate the expression of some integrins, will be studied for their effects on transcriptional and/or translational control of alpha- II-b-beta-3 and alpha-v-beta-3 expression and on their functions. Cytokines and/or eicosanoid effect on integrin function may be regulated by phosphorylation or increase in their association with the cellular cytoskeleton. The phosphorylation pattern of alpha-II-b, alpha-v and beta- 3 and the receptor association with cell skeleton will be examined. In this proposal, we will use a comprehensive approach including molecular, biochemical, and cell biological techniques to study the relative contribution of cytoadehesin integrins (i.e., alpha-II-b-beta-3 and alpha- v-beta-3 ) to cellular functions correlated with high metastatic potential.
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