RESUMO
Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12). Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.
Assuntos
Dosagem de Genes , Neoplasias/etiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Neoplasias da Mama/química , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Transformação Celular Neoplásica , Humanos , Camundongos , Neoplasias/química , Neoplasias/patologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/farmacologia , Células-Tronco/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.