Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines
Mohd. A. Yusuf at al.
Free Radic Biol Med.
Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity- purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethiol-treated rp53 only under non-reducing conditions. Untreated HCT116 cells showed low levels of glut-p53 which increased markedly after H2O2, diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after passing cells to oxidant-free media, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed abundant presence of glut-p53 in luminal epithelium, a site well-known to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. Availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.