Expression of bone formation-related molecules in a newly established protein-independent osteosarcoma

Protein-independent cells are useful fur analysis of proteins that are prod uced by the cells themselves without any consideration of exogenous protein s. This experimental protein-independent tumor system provides new biology of the autonomous nature of neoplastic cells during their evolution. We est ablished a Dunn protein-free osteosarcoma (DPF) sell line, which was derive d from parental fetal calf serum (FCS)-dependent murine Dunn osteosarcoma ( DOS) cells. The DPF cells grew in a chemically defined protein-free medium at the high seeding density of 1x10(4) cells/well of a 96-well-plate with a similar doubling time to that of cells growing in the presence of FCS, whi le the cells did not grow at a density lower than 1x10(3)/well. Furthermore , addition of conditioned medium stimulated the growth in a dose-dependent manner. In contrast, DOS did not grow in the protein-free condition at all. Morphological examination revealed that DPF cells exhibited a more round s hape than DOS cells. RT-PCR analysis exhibited the augmentation of the RNA message of bone morphogenetic protein-4 (BMP-4) and osteocalcin in DPF cell s. Enhanced expression of BMP-4 protein was also demonstrated by immunoblot analysis. Furthermore, alkaline phosphatase (ALP) activity was higher in D PF cells, indicating that bone-formation related molecules may be overexpre ssed in protein-independent osteosarcoma cells. These results suggest that putative growth factors may play a role in the DPF cell growth in an autocr ine fashion, and the acquisition of autonomous growth independent of exogen ous proteins may be coupled to the osteogenic differentiation.