Katsumasa Amitani

Solubilization and concentration of a bone-inducing substance from a murine osteosarcoma.

The osteogenic factor from murine osteosarcoma was soluble in 4M guanidine-HCl. The precipitate that appeared after dialysis of the guanidine solution against deionized water showed a higher osteogenetic activity than did the initial dry powder of tumor tissue. Electron-microscopically, this bone-inducing fraction seems to be homogenous. Neither a fibrous nor collagenous cross-banded structure was observed in the fraction. Since the chemical analysis of the fraction also showed a relatively low content of hydroxyproline, the osteogenic factor is probably not collagen.

Bone induction by lyophilized osteosarcoma in mice.

Bone formation was induced by iso- and home-implantation of lyophilized mouse osteosarcoma, in which an osteogenic activity is observed. The inducing factor is present exclusively in osteosarcoma, such as BF and Gardner, while not in non-osteogenic tumors, such as Ehrlich ascites tumor (2N & 4N) or mouse mammary carcinoma. The production of bone was rapid and extensive. It usually occurred within 21 days, and the time required for osteogenesis was almost the same as that reported in previous studies.

Studies on a factor responsible for new bone formation from osteosarcoma in mice

The bone inducing factor derived from BF osteosarcoma was purified in the following manner. Step 1. The sarcoma, grown in CBA mice, was excised and lyophilized. Step 2. The powder was washed with chilled acetone. Step 3. The acetone-treated powder was then homogenized with chilled distilled water. Step 4. Washing with 0.15M KCl. Step 5. The precipitate was incubated in 0.2 N NH2OH, pH 7.0, for 48 h at 25°. After Step 5, the bone-forming activity showed a slight increase; however, the factor remained insoluble. The properties of the factor were as follows. The factor is relatively heat stable; the osteogenic activity survived the treatment at 75° for 15 min or at 55° for 19 h. The activity was easily lost by mechanical shaking. Incubation with DNase, RNase, neuraminidase, chondroitinase ABC and β-galactosidase left the osteogenic activity intact, but treatment with either pronase or collagenase destroyed this activity. The results suggest that the factor may be a protein. The activity was seen with the lyopholized BF osteosarcoma cells (without matrix), and it is probable that the factor was exclusively synthesized in the cells. The bone formation, observed across a millipore filter when living BF osteosarcoma enclosed in a millipore chamber was implanted in mice, suggests the synthesis and secretion of the factor from the cells.

Partial Purification of Bone-inducing Substances from a Murine Osteosarcoma

Bone-inducing substance from a murine osteosarcoma (BFO osteosarcoma) was partially purified by biochemical procedures, i.e., extraction by 4M Gu-HCl, fractionation by ethanol, precipitation in neutral buffer solution with low ionic strength and two steps of gel filtration. The yield of the final preparation was 2 mg from 10 g (wet weight) of BFO osteosarcoma. The preparation constantly induced ectopic bone in situ when implanted into allogenic mice. SDS polyacrylamide electrophoresis revealed that the biologically active fraction contained two distinct substances, the molecular weights of which were 19,500 and 22,500, respectively. Further purification of the bone-inducing substance is now in progress.

Characteristics of Osteosarcoma Cells in Culture

Osteosarcoma cells (BFO cell line) were successfully maintained in tissue culture for 3 years. BFO cells showed 100 per cent tumorigenicity by the isologous implantation, and almost the same histological features as the original BF osteosarcoma. BFO cells synthesize and secrete large quantities of alkaline phosphatase both in vitro (cell culture) and in vivo (tumor bearing mice). BFO cells showed a suppression in synthesizing the osteoinductive factor in vitro, but regained the capacity to synthesize it when implanted back into an isologous host. The cells showed rapid growth, a serum requirement, and no contact inhibition. Doubling time was 8.6 hours in a logarithmic growth phase. Cell cycle analyses by pulse labeling of 3H-thymidine was performed after synchronization of the cells by double treatment with an excess thymidine.

Establishment and alkaline phosphatase activity of clonal cell lines of murine osteosarcomas. A preliminary study.

Clonal cell lines of murine osteosarcomas were established and have been maintained in vitro for over a year. By implanting these cultured cells into mice, osteosarcomas, whose histological picutres were exactly the same as those of the original tumors, were easily reproduced. The cultured cells of murine osteosarcomas contain an extremely high level of alkaline phosphatase activity. The cells also secrete a great amount of extracellular alkaline phosphatase in culture media.

Biochemical stability of a bone-inducing substance from murine osteosarcoma.

Devitalized cultured cells of a murine osteosarcoma preserved strong osteogenic activity even after incubation in neutral and acidic buffer at 37 degrees for six days. This observation suggested that an osteosarcoma-derived osteogenic factor was considerably stable to endogenous proteases, or that osteosarcomas do not synthesize enzymes for degradation of the osteogenic factor. EDTA decalcification or extraction of the osteosarcoma at 37 degrees for six days had no apparent effect on the osteogenic factor. Disulfide-bond reducing agents (2-mercaptoethanol and dithiothreitol) completely inactivated the osteogenic activity and in the presence of guanidine HCl, the loss of activity was irreversible by reoxidation. There findings support the view that disulfide-bonds in the molecular structure of the osteogenic factor are essential for the biological activity.