The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser 6 , Ser 15 , Ser 20 , Ser 37 , Ser 46 , and Ser 392 ) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo.

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo-and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMXoverexpressing cancers.T he tumor suppressor p53 is a powerful growth-suppressive and proapoptotic protein tightly controlled by its negative regulators: murine double minute (MDM)2 and MDMX (1, 2). These proteins bind p53 with their structurally similar N-terminal domains and effectively inhibit p53 transcriptional activity (1, 3). They both possess a RING (really interesting new gene) domain in their C termini, but it is only functional in MDM2, which serves as a specific E3 ligase and main regulator of p53 stability (4, 5). Despite its RING domain, MDMX does not have an intrinsic ligase activity and does not affect directly p53 stability (6). However, MDMX can enhance ligase activity of MDM2 toward p53 by forming MDM2/MDMX heterodimers (7,8). It has been reported that the MDM2/MDMX complex is responsible for polyubiquitination of p53, whereas MDM2 alone primarily induces monoubiquitination (9). Targeted disruption of MDM2/MDMX heterocomplexes is embryonic-lethal in mice, suggesting that complex formation is essential for p53 regulation in vivo (10). On the other hand, MDM2 can also ubiquitinate MDMX and is, therefore, responsible for its stability as well (11,12). MDM2 is a transcriptional target of p53, and both proteins form an autoregulatory feedback loop by which they mutually control their cellular levels (13).The functional relationship between MDM2 and MDMX is still being refined at the molecular level, but it is well established that these two negative regulators play a critical role in controlling p53 tumor-suppressor function in normal cells (2,14). This is why they are frequently overproduced through gene amplification and/or overexpression in tumors that retain wildtype p53 (14). Therefore, antagonizing the binding of MDM2 and MDMX to p53 is expected to restore p53 function and may offer a strategy for cancer therapy (15). Recently identified small-molecule inhibitors of the p53-MDM2 interaction have validated this approach, and the first pharmacological MDM2 antagonists ar...

The tumor suppressor p53 is often referred to as "the guardian of the genome" because of its central role in the cellular response to oncogenic stress and prevention of tumor development. Mutations of p53 in acute myeloid leukemia (AML) are rare but resistance to chemotherapy has been reported because of the deregulation of the p53 signaling and differentiation pathways. It is known that the interaction of the vitamin D metabolite 1,25-dihydroxyvitamin D 3 (1,25D) with its functional vitamin D receptor leads to differentiation, G 1 arrest, and increased cell survival in p53-null AML cells. However, there are no reports on the effect of 1,25D in leukemia cells expressing wild-type p53. Here, we examine vitamin D signaling in AML cells MOLM-13 and OCI-AML3 expressing wild-type p53 in the presence and absence of the MDM2 antagonist nutlin-3. We find that 1,25D alone induces monocytic differentiation in these cell lines similar to that seen in p53-null AML cells, suggesting that the presence of wild-type p53 is compatible with activation of vitamin D signaling. Combination of nutlin-3a with 1,25D accelerated programmed cell death, likely because of enhanced nutlin-induced upregulation of the proapoptotic PIG-6 protein and downregulation of antiapoptotic BCL-2, MDMX, human kinase suppressor of Ras 2, and phosphorylated extracellular signal-regulated kinase 2.