Xenobiotics-induced phosphorylations of MDM2

The aim of the present study was to characterize the Mdm2 and p53 responses induced by DNA damaging xenobiotics and how these responses can be modified by non-genotoxic xenobiotics. During control conditions cellular levels of p53 are low. The level is regulated by Mdm2 and Mdm2 and p53 forms an autoregulatory loop. When DNA is damaged, the p53-Mdm2 feedback loop is perturbed, and the cellular levels of p53 are increased so that p53 can regulate the transcriptional activity of proteins mediating responses such as apoptosis or cell cycle arrest. DNA damage may induce posttranslational modifications on p53 and Mdm2, such as phosphorylation or dephosphorylation induced by different kinases. These posttranslational modifications may modify the functions of Mdm2 and p53.

This study focuses on Mdm2 phosphorylation within the epitopes of antibody 2A 10, and phosphorylation of Mdm2 on Ser 166. Both TCDD and the cholesterol lowering drugs statins were shown to attenuate the p53 stabilization in response to DNA damaging agents in HepG2 cells and in rodents. TCDD is a persistent, bioaccumulating pollutant. It is a carcinogen without being genotoxic.

The detailed mechanisms for cancer induction are unknown, however. Statins are drugs used to treat hypercholesterolemia, and have also anticarcinogenic properties. The attenuation of p53 DNA damage response was associated with Mdm2 phosphorylation on Ser166. Thus, statins and TCDD induces Mdm2 Ser166. phosphorylation and attenuates p53 stabilization in response to DNA damage in HepG2 cells. TCDD also attenuated p53 stabilization in response to DNA damage in rats. As statin-induced Mdm2 Ser166. phosphorylation was attenuated by rapamycin (an inhibitor of MTOR) and Mdm2 Ser166. phosphorylation occurred in parallel to MTOR) phosphorylation, it is likely that MTOR) induced Ser166. phosphorylation.

Our findings thus suggests that mTOR is one of the kinases inducing Mdm2 Ser166. phosphorylation. TCDDand statin-induced attenuation of p53 response might interfere with the cells ability to handle genotoxic agents. TCDD has been associated with cancer, especially liver cancer in rodents. Statins, on the other hand, rather seems to have anti care i nogenic properties. However, if statins are used together with genotoxic substances, attenuation of p53 response could affect its capacity to kill cells.

Mdm2 phosphorylation within the epitopes of antibody 2A10 occurred at much lower concentrations of genotoxic substances benzo[a]pyrene, BPDE, mitomycinC and etoposide than those inducing p53 stabilization. This suggests that Mdm2 can be used as a marker for certain types of DNA damage. Mdm2 2A 10 phosphorylation induced by lower doses than those inducing p53 stabilization did not occur after exposure to dibenzo[a,l]pyrene and its metabolite DBPDE.

We suggest that Mdm2 phosphorylated within the 2A10 epitopes might be involved in DNA repair of BPDE adducts, since BPDE induces 2A10 phosphorylation and DNA repair while DBPDE do not. Mdm2 has been shown to interact with proteins involved in DNA repair such as DNA polymerase epsilon and Nbs I. Mdm2 also partly localize to double stranded DNA breaks in response to IR irradiation. Mdm2 2A10- and p53 phosphorylation in response to DNA damage were shown to be induced by different signalling pathways.

List of scientific papers

I. Paajarvi G, Viluksela M, Pohjanvirta R, Stenius U, Hogberg J (2005). TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents. Carcinogenesis.
https://doi.org/10.1093/carcin/bgh289

II. Paajarvi G, Roudier E, Crisby M, Hogberg J, Stenius U (2005). HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage. FASEB J. 19(3): 476-8.
https://doi.org/10.1096/fj.04-2745fje

III. Pääjärvi G, Jernström B, Stenius U, Seidel A. (2004). Exposure of mammalian cells to diol epoxides from benzo[a]pyrene and dibenzo[a,l]pyrene and effects on Mdm2. Polycyclic Aromatic Compounds. 24: 537-548.
https://doi.org/10.1080/10406630490471636

IV. Pääjärvi G, Jernström B, Seidel A, Stenius U (2006). Anti-diol epoxide from benzo[a]pyrene induces transient Mdm2 phosphorylation/chromatin binding, while anti-diol epoxide from dibenzo[a,l]pyrene induces a persistent p53 Ser15 phosphorylation. [Submitted]

V. Malmlöf M, Pääjärvi G, Högberg J, Stenius U (2006). Benzo[a]pyrene and other DNA damaging compounds, but not dibenzo[a,l]-pyrene, induce Mdm2 alterations and chromatin binding; Mdm2 as a sensitive marker for genotoxicity. [Manuscript]