Posters— Basic Science/Biology

P16INK4A GENE AND OSTEOSARCOMA

M.S.Benassi, L.Molendini, G.Gamberi, M.Merli, P.Ragazzini, G.Magagnoli and P.Picci. (Laboratory of Cancer Research, Rizzoli Institute, 40100Bologna, Italy)

INTRODUCTION: The p16INK4A putative tumor suppressor gene expresses two different mRNA transcripts, alpha and beta. The alfa transcript p16INK4a, a cyclin D/cdk4 inhibitor, prevents inactivation of the retinoblastoma protein (pRb). The beta transcript p14ARF, protects p53 protein by binding with MDM2, and up-regulates the p21 protein. AIM: In order to define the role of the p16INK4A gene in human osteosarcoma (OS), we studied gene status and expression of the molecules involved in p16 regulatory system.

MATERIALS AND METHODS: 35 high-grade formalin-fixed, paraffin embedded and frozen OS samples were studied by immunohistochemistry (IHC) and immunoblot. Exons 1 and 2, 5’CpG island methylation of the p16INK4A gene and semiquantitative analysis of the CDK4 gene were performed by specific PCR.

RESULTS: IHC analysis revealed an increased expression of p16 protein in 14/35 OS. p16 staining intensity ranged from moderate to strong. Of these, 6 with pRb positive expression, simultaneously showed cdk4 overexpression (6X) and CDK4 gene amplification (8X-10X). No deletions or mutations were found in p16INK4A gene, but 9 p16-negative OS showed 5’CpG methylation. A weak to moderate expression of p14ARF protein was detected in 15/35 OS; of these, 11 had active p53 and 10 immunoreacted to p21protein.

CONCLUSIONS: In conclusion, these results suggest that two important cell cycle regulatory pathways are involved in OS pathogenesis.