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Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.

ABSTRACT: 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.

SUBMITTER: Yusuf MA

PROVIDER: S-EPMC2914855 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.

Yusuf Mohd A MA Chuang Trinette T Bhat G Jayarama GJ Srivenugopal Kalkunte S KS

Free radical biology & medicine 20100625 5

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 ...[more]

PMID: 20600834

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Project description:IntroductionSustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy.MethodsWe generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine.ResultsHER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation.ConclusionThese data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.

Dataset Information

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.

Publications

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines.

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