Osteosarcoma is an aggressive malignancy of bone or soft tissue that frequently metastasizes to lung. In order to study the invasive and metastatic properties of human osteosarcoma, we have used previously described human osteosarcoma (HOS) cell lines that differ in their biologic behavior in athymic mice. The parental cell line, HOS, derived from a tumor of the distal femur, is nontumorigenic and nonmetastatic in athymic mice. Two subclones, KRIB and AD110, were derived following transformation of HOS by v-Ki-ras or by a mutated c-Ha-ras oncogene respectively. KRIB cells show the greatest tumorigenicity and metastatic potential while AD110 cells are tumorigenic but not metastatic in athymic mice. We have examined these three cell lines for steady state mRNA, protein levels, and cell surface expression of urokinase plasminogen activator (uPA), urokinase receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1). While HOS cells express low levels of steady state mRNA for uPA and secrete and bind low levels of uPA, both AD110 and KRIB secrete higher, approximately equivalent levels of uPA and show higher fluorescence intensity when examined for binding of uPA by flow cytometry using either a monoclonal antibody specific for pro-uPA or a monoclonal antibody specific for a B chain epitope. In addition, both AD110 and KRIB show similar fluorescence intensity when examined for expression of uPAR. AD110 cells express approximately 3-fold more PAI-1 mRNA or protein than either HOS or KRIB. AD110 cells show higher fluorescence intensity when examined by flow cytometry using a monoclonal antibody capable of detecting PAI-1 in complex with uPA. Previous experiments quantitating uPA activity by the plasminogen dependent fibronectin degradation assay showed that KRIB had the highest cell associated uPA activity while AD110 and HOS showed consistently lower levels. These data suggest that endogenously synthesized PAI-1 is secreted and bound by AD110 cells resulting in the modulation of uPA activity with a consequent reduction in metastases in athymic mice.
