The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2

Loss of function of the tumour suppressor gene p53 is a key event in most human cancers. Although usually occurring through mutation, in some tumour types this appears to be achieved via an indirect mechanism involving inappropriate expression of a functional inhibitor, mdm2, which binds to the transactivation domain of p53. This interaction offers an ideal potential target for novel cancer therapies. However, therapeutic specificity may depend on the extent to which this p53-inhibitory action of mdm2 is also required by normal cells. Transgenic data have already established that mdm2 is needed to prevent embryonic lethality, but the situation in adult cells is still unclear. Here we show that micro-injection of normal human fibroblasts with an antibody directed against the p53-binding domain of mdm2 induces expression of p53-responsive genes, and furthermore results in p53-dependent growth arrest. We conclude that normal cell proliferation can be dependent on negative regulation of p53 by mdm2, a finding which raises an important note of caution for mdm2-directed cancer therapies.     

Mdm2 association with p53 targets its ubiquitination

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.     

p53 and mdm2 are expressed independently during cellular proliferation

We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.