Interaction of human NAD(P)H:quinone oxidoreductase 1 (NQO1) with the tumor suppressor protein p53 in cells and cell-free systems.
NAD(P)H:quinone oxidoreductase 1 (NQO1) has been proposed to stabilize p53 via a redox mechanism involving oxidation of NAD(P)H as a consequence of the catalytic activity of NQO1. We report that treatment of HCT-116 human colon carcinoma cells with the NQO1 inhibitor ES936 had no effect on the levels of p53 protein. ES936 is a mechanism-based inhibitor of NQO1 that irreversibly blocks the catalytic function of the enzyme. This suggests that a redox mechanism involving NQO1-mediated NAD(P)H oxidation is not responsible for the stabilization of p53. We also examined the ability of the NQO1 protein to associate with p53 using co-immunoprecipitation experiments. Results from these experiments demonstrated co-immunoprecipitation of NQO1 with p53 and vice versa. The association between p53 and NQO1 was not affected by treatment of HCT-116 cells with ES936, demonstrating that the association was not dependent on the catalytic activity of NQO1. A comparison of isogenic HCT-116 p53+/+ and HCT-116 p53-/- cells demonstrated an interaction of NQO1 and p53 only in the p53+/+ cells. Experiments performed in an in vitro transcription/translation system utilizing rabbit reticulocyte lysates confirmed the interaction of NQO1 and p53. In these experiments a full-length p53 coding region was used to express p53 in the presence of recombinant NQO1 protein. An association of p53 and NQO1 was also observed in primary human keratinocytes and mammary epithelial cells. In studies where mdm-2 co-immunoprecipitated with p53, no association of mdm-2 with NQO1 was observed. These data demonstrate an association between p53 and NQO1 that may represent an alternate mechanism of p53 stabilization by NQO1 in a wide variety of human cell types.
Anwar
Adil
A
Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver 80262, USA.
Dehn
Donna
D
Siegel
David
D
Kepa
Jadwiga K
JK
Tang
Luo Jia
LJ
Pietenpol
Jennifer A
JA
Ross
David
D
D002474:Cell-Free System; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000032:analysis / Q000737:chemistry*; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D051736:Proto-Oncogene Proteins c-mdm2; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry*
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2