The goal of the proposed research is to characterize precisely the role of the plasminogen activator (PA) system in tumor cell invasiveness and metastasis. In addition to the effects of urokinase (UK) and tissue plasminogen activator (t-PA), potential modulating influences of the PA inhibitors PAI-1 and PAI-2 on the malignant phenotype will be investigated. Inhibitory anti-UK antibodies have been shown by others to enhance tumor cell invasiveness in vitro. In addition, the ability of B16 melanoma cells to colonize the lungs of mice after intravenous administration is decreased by pretreatment with anti-UK antibodies. However, a convincing demonstration of the role of UK in primary tumor growth and spontaneous metastasis in vivo is lacking. Moreover, the role of other components of the PA system in tumor development and progression has not been characterized. This proposal will utilize mammalian expression vectors to achieve constitutive expression of exogenous cDNAs encoding UK, t-PA, PAI- 1, and PAI-2 in human and rodent tumor cell lines. Exogenous PAs will be expressed in tumor cells not expressing endogenous PA to determine their specific role in tumor cell behavior. Rodent and human cell lines will be injected into syngeneic animals and nude mice respectively. Subcutaneous and intramuscular routes of injection will e used to examine the effects of transfection on local tumor growth as well as spontaneous metastasis to lungs, liver, and spleen. Tail vein injection will also be used to provide a quantitative model for the late stages of the metastatic process. As a first step in accomplished these goals, cDNAs for t-PA and UK have been expressed in B16 mouse melanoma clones of well-characterized metastatic capacity. Synthesis and secretion of PAs have been confirmed by Western blot analysis and functional assays. In vivo behavior will be investigated in C57BL/6 mice. Controls will include individual B16 clones and pooled cultures transfected with the expression vector only. A similar approach will be used to determine the role of the PA inhibitors. In vitro models will be developed in order to investigate the mechanisms for effects observed observed in vivo. Vectors employing """"""""antisense"""""""" sequences will also be used to inhibit endogenous synthesis of UK and t-PA. Antisense constructs will be tested in the t-PA- secreting Bowers melanoma cell line a the UK-secreting T24 bladder carcinoma cell line. If initial experiments suggest a functional role for PAs or PAIs, subsequent studies will utilize the expression of deletion mutants to assess the importance of specific protein domains to the malignant phenotype. A precise understanding of the role of PAs and PAIs in malignancy many ultimately have therapeutic implications. For example, appropriate pharmacologic manipulation of fibrinolysis might be useful to decrease the likelihood of hematogenous metastasis at the time of initial surgery for a localized primary tumor.
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