Mdm2 phosphorylations : Characterization and applications

Sammanfattning: Cancer is a widespread disease. In spite of all research resulting in new improved therapies, there are still many tumors that cannot be cured. To oppose that, also a preventive research approach is important with goals such as decreasing the human exposure to carcinogens. An early step in that process could be to find biomarkers of exposure and to develop mechanistically informative test systems. The aim of this study was to characterize Mdm2 phosphorylations in order to investigate their potential applications in preventive research. p53 is a tumor suppressor protein that mediates cell cycle stop and apoptosis in response to cellular stress such as DNA damage. There is a strong coupling between p53 and cancer since it is mutated or inactivated in the majority of tumors. Mdm2 is a key regulator of p53 that induces proteasomal degradation of p53, through ubiquitination. p53 induces transcription of Mdm2, and together p53 and Mdm2 form an autoregulatory feedback loop. The activity of Mdm2 is regulated by phosphorylations. It has been shown that phosphorylation of Mdm2 at Ser166 promotes degradation of p53 and that Akt mediates this phosphorylation. Thus TCDD, which has a tumor promoting effect, induces phosphorylation of Mdm2 at Ser166 and thereby attenuates p53 response to DNA damage. Mdm2 is also phosphorylated by ATM at Ser395, a residue within the 2A10 epitope, upon DNA damage. This phosphorylation is important for DNA damage induced p53 stabilization. Also other proteins such as DNA-PK, c-Abl and ATR are involved in Mdm2 phosphorylation and thereby also have indirect effects on p53. We demonstrate that Mdm2 phosphorylation at Ser166 is mediated via MEK-ERK, and not by Akt, in hepatocytes. We also show that inhibition of Akt activation induces phosphorylation of Mdm2 at Ser166 via MEK-ERK in hepatocytes. This results in downstream effects such as attenuated p53 and p21 responses to DNA damage. Our data suggest Akt as a negative regulator of Mdm2 in these cells and propose a hepatocyte specific regulation of Mdm2 phosphorylation at Ser166. We also find that low concentrations of genotoxic compounds such as mitomycin C, etoposide, 5-fluorouracil and benzo[a]pyrene induce phosphorylation of Mdm2 at the 2A10 epitope. No p53 accumulation was observed at these low doses. Low concentrations of BP also induce Mdm2 2A10 phosphorylation in human lymphoblasts. These data indicate that Mdm2 could be used as a sensitive marker for certain types of genotoxicity and a potential use of Mdm2 as a marker for human exposure of genotoxic agents. The possibility to use Mdm2 as a biological marker was further strengthened as we identified two different patterns of proteins associated to chromatin induced by polycyclic aromatic hydrocarbons, where a loss of Mdm2 binding to chromatin seemed to indicate severe and non-repairable DNA damage. In conclusion, this study show a liver specific regulation of Mdm2 phosphorylation at Ser166 and that Mdm2 phosphorylation at the 2A10 epitope could be used as a tool in detecting and characterizing certain types of genotoxicity.