The process of tumor metastasis is complex and probably involves the actions of several proteolytic enzymes which are regulated at several levels temporally in the metastatic process. uPA is among pivotal enzymes involved in the metastatic process through its ability to activate plasminogen to plasmin. The activity of uPA in tumor cells is probably regulated by the binding of uPA with the inhibitor PAI-1. Preliminary data from a human osteosarcoma model comprised of cell lines showing varying abilities to form tumors and metastasize in athymic mice has shown that the activity of membrane bound uPA rather than the level of secreted uPA correlates with the invasive and metastatic behavior of the cells. One of the cell lines, AD110, is tumorigenic but not metastatic. This cell line secretes and binds higher levels of PAI-1 than another cell line, KRIB which is highly tumorigenic and metastatic and which secretes and binds very low levels of PAI-1. Both cell lines bind equivalent amounts of uPA. Studies have been undertaken to evaluate the transcriptional regulation of PAI-1 in these two cell lines as well as in the parental HOS cell line which is nontumorigenic and nonmetastatic in athymic mice. By Southern blot technique using HEL299, a diploid human embryonic cell line as a control for gene copy number, we have found that the gene encoding PAI-1 is single copy in the three cell lines. Serial 5' deletions of the promoter for PAI-1 linked to the chloramphenicol acetyl-transferase (CAT) reporter gene reveal that full basal promoter activity in all of the cell lines resides within the first 100 bases and is probably conferred by an AP-1/ CRE like element at -60. Experiments are underway to evaluate the role of PKC in basal promoter activity of the PAI-1 gene in HOS, AD110, and KRIB.
