SummaryWe examined structure and expression of the p53 and Rb genes in a C3HOS transplantable mouse model of osteosarcoma. The results were compared to analogous studies conducted with five human osteosarcoma cell lines. The p53 gene was found rearranged in the mouse tumour. The rearrangement mapped to the first intron region of the p53 gene and as a result, no p53 expression could be detected in C3HOS tumours. Using p53 genomic probes, we have detected the same rearrangement in the original radiation-induced tumour and the various clones that were isolated from it. Deletion and rearrangement of the p53 gene were also found in three out of five of the human osteosarcoma cell lines . No p53 expression could be detected in these three cell lines. In the affected human osteosarcoma cell lines, the rearrangement involved the first intron region. In addition, the mouse tumour was analysed for structural and expression changes in the Rb and the c-myc genes. Normal expression of both genes were detected in the murine tumour. Only one (Saos-2) human osteosarcoma cell line exhibited gross structural alteration in the retinoblastoma gene. The results suggest that the inactivation of p53 may be an important step in the development of osteosarcomas, and that a rearrangement affecting the first intron is common in osteosarcomas.

Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR-GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4–20 spots while some of the OTR diffuses throughout the nucleoplasm.The behaviour and kinetics of OTR-GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR-positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand-dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand-dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G-protein-coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

Plasmin cleaves osteocalcin at a site within its carboxyl end, thus creating an N-midterminal 1-43 and a short C-terminal 44 -49 peptides. The products of the cleavage were identified by matrix assisted laser desorption ionization time of flight mass spectrophotometry and by reversed phase high performance liquid chromatography followed by N-terminal sequence determination. When separated by sodium dodecyl sulfide-polyacrylamide gel electrophoresis in the presence of reducing agents, large (LF; N-midterminal) and a small molecular weight (SF; C-terminal) fragments can be identified. The major cleavage site involves arg43-arg44 amino acid residues, and the resulting 44 -49 C-terminal fragment appears as a slow migrating band on native gels (SFnat). Elevated levels of calcium ion inhibit the plasmin-mediated lysis of osteocalcin. Plasmin-mediated cleavage of osteocalcin occurs both in solution and when bound to hydroxyapatite. Both osteocalcin cleavage products detach from the hydroxyapatite substrate. Diisopropyl fluorophosphate-inhibited plasmin does not displace osteocalcin from the hydroxyapatite surface. Previously, the C-terminal pentapeptide has been shown to be chemotactic for bone cells while bone particles lacking osteocalcin were resistant to bone resorption. We therefore hypothesize that the plasminmediated digestion of free and hydroxyapatite-bound osteocalcin could play a role in the regulation of bone

The p53 gene undergoes rearrangement in a high percentage of osteosarcomas, resulting in loss of its expression. A p53-null murine osteosarcoma cell line F6 was transfected with either a wild-type or a mutant p53 gene. Stably transfected cell lines were obtained, and their differentiation capabilities were compared in vitro with the parental cell line. Alkaline phosphatase and osteocalcin expression were measured as early and late differentiation markers, respectively. Induction of alkaline phosphatase expression was not affected by the presence of either p53 gene, whereas osteocalcin expression was seen in cells containing the wild-type p53 gene but not in the parental p53-null or mutant-expressing cell lines. That the induction of osteocalcin was intrinsically dependent on the presence of wild-type p53 was also indicated by the use of a temperature-sensitive Val 135 p53 mutant at 32 degrees C; predominant expression of p53 in the wild-type conformation resulted in osteocalcin expression. While the wild-type p53 gene could suppress tumor formation in vivo, the tumors expressing the mutant p53 gene grew two to three times as large as the tumors that did not express p53. Therefore, the absence of end-point differentiation in bone due to p53 rearrangements may contribute to the maintenance of the tumorigenic phenotype in osteosarcomas.