Expression of the Basic Fibroblast Growth Factor and Detection of Simian Virus 40 Footprints in Human Osteosarcoma Cell Lines.

Human osteosarcoma cell lines, the non-mineralizing MG63 and mineralizing TE85 cells were investigated in order to verify the role of the basic Fibroblast Growth Factor (bFGF) in modulating their differentiative responses. Extracellular matrix production was evaluated by studying glycosaminoglycan (GAG), collagen and proteoglycan (PG) synthesis. Osteocalcin, RUNX2 and FGF gene expressions were studied by RT-PCR analysis and Northern blotting, whereas FGF receptors were quantified by the binding assay. The results showed a less differentiated stage of MG63 compared to TE85 cells. Indeed, MG63 cells showed enhanced differentiate parameters such as proliferation, GAG synthesis, proteoglycan mRNA expression, bFGF receptor number and bFGF transcript, while procollagen expression and alkaline phosphatase activity were significantly reduced. Moreover, in the two human osteosarcoma cell lines RUNX2 gene was differentially spliced and differently modulated by bFGF. Since bFGF induced de-differentiative effects in TE85 increasing cell proliferation and reducing procollagen and osteocalcin production, we suggest that the less differentiated stage of MG63 could be related to their constitutive bFGF over-production and to the greater bFGF receptor expression. In parallel experiments, carried out in 15 human primary osteosarcoma cell lines and 5 normal bone tissue specimens used as control, footprints of the oncogenic simian virus 40 (SV40) were investigated. Three different coding sequences of the viral oncogene T antigen (Tag) were analyzed by PCR and filter hybridization with specific oligoprobes. SV40 Tag N- and C-terminal coding sequences were found in 8 out of 15 primary human osteosarcoma cell lines. None of the DNA samples from 5 normal bone tissues was SV40-positive. SV40 Tag expression, investigated at mRNA level, was revealed in two out of five SV40 Tag cell lines. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive human primary osteosarcoma cell lines, confirmed the SV40 specificity and indicated that PCR products had a sequence indistinguishable from SV40 DNA wild type strain 776. The data support a putative role of SV40, as co-factor, in the onset/progression of human bone tumors. Works are in progress to explore the intriguing hypothesis that SV40 acts in the onset/progression of bone cancer by modulating the FGF expression, thus generating an autocrine/paracrine loop responsible of the alterations in the extracellular matrix production and transformation.