UP-REGULATION OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR BY ENDOGENOUS AND EXOGENOUS HIV-1 TAT PROTEIN IN TUMOR-CELL LINES DERIVED FROM BK VIRUS TAT-TRANSGENIC MICE

Objective: To demonstrate that Tat modulates the plasminogen-dependent proteolytic activity of tumour cell lines derived from BK virus (BKV) /tat-transgenic mice by affecting the production of plasminogen activa tors (PA) and the PA inhibitor (PAI)-1 and to demonstrate that this oc curs through mechanism(s) that are distinct from those responsible for transactivating activity of extracellular Tat. Design and methods: To assess whether endogenous Tat is responsible for PA activity in T53 a denocarcinoma cells, cell cultures were transfected with antisense Tat cDNA and evaluated for cell-associated PA activity by a plasmin chrom ogenic assay. The assay was also used to evaluate PA activity in T53 c ells and T111 leiomyosarcoma cells stimulated by extracellular Tat. Th e type(s) of PA produced were identified by sodium dodecyl sulphate-po lyacrylamide gel electrophoresis zymography. The levels of PAI-1 were evaluated by Western blotting. Tat transactivating activity was measur ed by a chloramphenicol acetyltransferase (CAT) enzyme-linked immunoso rbent assay in HL3T1 cells containing integrated copies of an HIV-1 lo ng terminal repeat (LTR)-CAT plasmid. Results: Transfection of T53 cel ls with antisense Tar cDNA results in the decrease of Tat production a nd PA activity. Exogenously added Tat increases PA levels in T53 and i n T111 cells. PA activity was identified as urokinase-type PA (uPA). T at also increases the production of PAI-1 in T111 but not in T53 cells . Chloroquine and heparin have different affects on the LTR-CAT-transa ctivating and the PA-inducing activities of Tat. The fusion protein gl utathione-S-transferase-Tat and the mutant Tat-1e, lacking the second Tat exon, cause LTR-CAT transactivation without stimulating uPA upregu lation. Conclusions: Tat affects the fibrinolytic activity of tumour c ell lines derived from BKV/tat-transgenic mice by modulating the produ ction of both uPA and PAI-1 via autocrine and paracrine mechanisms of action. The capacity of Tat to modulate the plasminogen-dependent prot eolytic activity of these tumour cell lines may contribute to their me tastatic potential. The uPA-inducing activity of Tat depends upon spec ific biological and structural features of the Tat protein that are di stinct from those responsible for its LTR-CAT-transactivating activity , suggesting distinct mechanisms of induction for the two biological r esponses.