Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.
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 the 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 nonreducing conditions. Untreated HCT116 cells showed low levels of glut-p53, which increased markedly after H(2)O(2), diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after cells were passed into oxidant-free medium, 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 an 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. The availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.
Yusuf
Mohd A
MA
Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
Chuang
Trinette
T
Bhat
G Jayarama
GJ
Srivenugopal
Kalkunte S
KS
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity; D002460:Cell Line; D002477:Cells / Q000378:metabolism*; D003545:Cysteine / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005978:Glutathione / Q000276:immunology / Q000378:metabolism*; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011817:Rabbits; D016159:Tumor Suppressor Protein p53 / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D016159:Tumor Suppressor Protein p53; D005978:Glutathione; D003545:Cysteine