15-Deoxy-Δ 12,14-prostaglandin J 2 stabilizes, but functionally inactivates p53 by binding to the cysteine 277 residue

15-Deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), a representative cyclopentenone prostaglandin, has many interesting biological effects. In this study, treatment of human breast cancer cells (MCF-7) with 15d-PGJ 2 led to accumulation of p53 protein. However, the p53 DNA binding and its transcriptional activity were significantly reduced. 15d-PGJ 2 directly modified p53 as verified by reacting recombinant p53 with biotinylated 15d-PGJ 2. 9,10-Dihydro-15-deoxy-Δ 12,14-prostaglandin J 2 lacking the electrophilic α,Β-unsaturated functionality failed to inhibit p53 DNA binding as well as to modify p53. Moreover, by conducting an in vitro [35 S]-labeled p53 translation assay, we identified cysteine 277 as a putative site of p53 modification by 15d-PGJ 2. The DNA-binding ability of a mutant p53 in which cysteine 277 was substituted by alanine was virtually unaffected by 15d-PGJ 2. Likewise, p53 binding activity of biotinylated 15d-PGJ 2 was abolished in mutant cells. In addition, cells expressing wild-type p53 exhibited p53 protein stability to a greater extent than mutant C277A cells. In conclusion, 15d-PGJ 2 can undergo nucleophilic addition to p53, presumably at the cysteine 277 residue, rendering this tumor suppressor less susceptible to proteasomal degradation. © 2010 Macmillan Publishers Limited All rights reserved.