|
Connective Tissue Oncology Society |
|
 |
| 2001 CTOS Annual Meeting Posters— Biology ANCHORAGE INDEPENDENT
GROWTH AND PROLONGED SURVIVAL OF PRIMARY HUMAN OSTEOBLASTS OVER-EXPRESSING
THE MET RECEPTORS BY MEANS OF TRANSDUCTION WITH LENTIVIRAL VECTORS OBJECTIVE: Osteoblast proliferation and differentiation is achieved through the integrated effects of several signalling molecules, which switch on receptor-mediated events and activate gene transcription. Each of the molecules controlling critical steps might play a role in the genesis of osteoblast-derived tumours, including receptor and non-receptor tyrosine kinases. Fragmentary data suggest that among these receptors, those of the MET family might also be involved in bone development and in osteoblast transformation. The MET oncogene-encoded tyrosine kinase receptor and its ligand Hepatocyte Growth Factor (HGF) ordinarily constitute a paracrine signalling system where cells of mesenchymal origin produce the ligand, which binds to receptors expressed on cells of epithelial origin. However, during mouse development HGF and met are both expressed in tissues of mesenchymal origin and regulate migration of myoblast precursors. We previously showed that the MET receptor is aberrantly over-expressed in a number of human osteosarcomas producing the ligand, suggesting that overexpression with or without autocrine activation of MET receptors might contribute to transformation of osteoprogenitor cells. METHODS: To demonstrate the transforming potential of the MET oncogene in human osteoblasts, first we are constructing an ex-vivo model using primary 2D cultures of human osteoblasts stably expressing the MET receptors by means of transduction with Lentiviral vectors. Bicistronic Lentiviral vectors carrying either wild-type or mutation-activated (METY1235D) MET cDNA followed by IRES (internal ribosomal entry site) and GFP-encoding sequences were used to transduce human osteoblasts. IRES sequences will ensure the concomitant expression of the MET receptor and the reporter protein, allowing the detection and the selection in vitro and in vivo of transduced cells. The human primary cultures transduced were from bone fragments and consisted of approximately 90% cells showing osteoblast phenotype. The latter was assessed with alkaline phosphatase assay and alizarine red staining of cells growing in differentiating medium. RESULTS: Propagated transduced osteoblasts showed a peculiar morphology and behaviour: cells were spindle-shaped and small, did not show cell contact inhibition in monolayer cultures and form colonies in soft agar. Different levels of MET expression were detectable in osteoblast clones and correlated with the ability of the cells to form colonies in agar. MET expressing osteoblasts grew for more than 30 passages in culture, corresponding to approximately 150 days. After 100 days the expression of the transgene declined but the level of MET receptor expression remained elevated. Most of the MET receptor stably over-expressed in long-term cultures was due to activation of the endogenous MET gene transcription. CONCLUSION: These data show that over-expression of the MET oncogene in human primary osteoblast due either to transgene transduction or reactivation of endogenous MET expression confers to cells of mesenchymal origin the ability to grow in an anchorage independent fashion and to survive longer than the parental line. Further experiments will assess if the prolonged survival of osteoblasts depends on a block of apoptosis or to inactivation of regulated Rb control or to a telomer lengthening activity.
|
