Analysis of the proportion of p53 bound to mdm-2 in cells with defined growth characteristics.

A critical parameter affecting cell growth properties is the relative levels of the p53 tumor suppressor protein and the mdm-2 oncoprotein. Because mdm-2 overexpression is observed in several types of human cancers and its physical association with p53 appears essential for down-regulating p53 activity the proportion of p53 bound to mdm-2 was examined in four types of cells with divergent growth properties: (1) Growth arrested cells (Al) expressing high levels of wild-type p53 activity; (2) Tumorigenic cells (3T3DM) expressing high levels of mdm-2; (3) Immortalized non tumorigenic cells (Swiss3T3 and Balb/c3T3); and (4) Normal murine fibroblasts. In Al cells, greater than 78% of the p53 was not bound to mdm-2, demonstrating that excess free p53 is available for cell cycle arrest. In 3T3DM cells 100% of the p53 was bound to mdm-2 and these cells were unable to support p53-mediated transactivation, a p53 function essential for cell growth inhibition. In Swiss3T3 cells 75% of the p53 was bound to mdm-2. In Balb/c3T3 cells and normal cells no detectable mdm-2 was bound to p53. Since free p53 was detected in several of these cell lines the possibility that mdm-2 is completely titrated by p53 was investigated. However, free mdm-2 was present in all these cells. Phosphorylation of p53 does not appear to control complex formation since the free and the mdm-2-bound form of p53 from Al cells had identical phosphorylation maps. These data suggest that a high proportion of p53 bound to mdm-2 is observed in some cells with a more transformed phenotype and that p53-mdm-2 complex formation is controlled by a posttranslational event other than p53 phosphorylation.