Alteration of prb/p16/cdk4 regulation in human osteosarcoma

Cell-cycle regulation depends on a fine balance between cyclin-cyclin-dependent kinase complexes and a family of kinase inhibitors that bind cyclin-cdk complexes and block their activity. To investigate the role of mechanisms regulating cell-cycle progression in human osteosarcomas (OS), pRb/p16/cdk4 expression was analyzed in 39 high-grade OS; 19 of these developed metastasis during follow-up. Positive reaction for functional pRB was shown by 18/39 (46%) OS, while 21/39 (54%) were negative. A higher probability of metastasis was seen in patients with negative pRb expression (p <0.05). Furthermore, while functional pRb and DI expression are inversely associated to metastasis occurrence, the presence of DI /cdk4 complex in our study was related to poor prognosis. We found that 10/18 pRb-positiye and 14/21 pRb-negative tumors were p 16-positive. No significant correlation was found between pRb and pi6 expression. On the other hand, high cdk4 levels in pie-positive tumors as compared with pie-negative tumors resulted in a positive association between pi6 and cdk4 expression (Chi squared = 5.98; p = 0.01). No extensive p16INK4A genomic alterations were found in tumors lacking pie-protein expression. To determine which mechanisms are involved in the down-regulation of p16 protein, the methylation status of the p16INK4 gene was evaluated on the 15 pie-negative tumors: 8 samples showed 5′ CpG-island methylation; 4/8 had a complete methylation status, while in the remaining 4 the gene was only partially methylated. These data confirm the role of the pRb/p16/cdk4 pathway in OS development.