Expression of bone formation-related molecules in a newly established protein-independent osteosarcoma.
Protein-independent cells are useful for analysis of proteins that are produced by the cells themselves without any consideration of exogenous proteins. This experimental protein-independent tumor system provides new biology of the autonomous nature of neoplastic cells during their evolution. We established a Dunn protein-free osteosarcoma (DPF) cell line, which was derived 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, while 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 shape than DOS cells. RT-PCR analysis exhibited the augmentation of the RNA message of bone morphogenetic protein-4 (BMP-4) and osteocalcin in DPF cells. Enhanced expression of BMP-4 protein was also demonstrated by immunoblot analysis. Furthermore, alkaline phosphatase (ALP) activity was higher in DPF cells, indicating that bone-formation related molecules may be overexpressed in protein-independent osteosarcoma cells. These results suggest that putative growth factors may play a role in the DPF cell growth in an autocrine fashion, and the acquisition of autonomous growth independent of exogenous proteins may be coupled to the osteogenic differentiation.
Yanagawa
T
T
Department of Orthopedic Surgery, Gunma University Faculty of Medicine, Gunma 371-8511, Japan.
Watanabe
H
H
Shinozaki
T
T
Nakajima
T
T
Takagishi
K
K
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D051379:Mice; D008809:Mice, Inbred C3H; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C525809:BMP4 protein, human; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D015675:Osteocalcin; D000469:Alkaline Phosphatase