GRIM-19 disrupts E6/E6AP complex to rescue p53 and induce apoptosis in cervical cancers

PLoS One. 2011;6(7):e22065. doi: 10.1371/journal.pone.0022065. Epub 2011 Jul 12.

Abstract

Background: Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis.

Methodology/principal findings: The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model.

Conclusion/significance: The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / metabolism*
  • Apoptosis*
  • Cell Line, Tumor
  • Cell Proliferation
  • DNA-Binding Proteins / metabolism*
  • Down-Regulation
  • Female
  • G1 Phase
  • Humans
  • Mice
  • NADH, NADPH Oxidoreductases / metabolism*
  • Oncogene Proteins, Viral / metabolism*
  • Protein Binding
  • Protein Stability
  • Resting Phase, Cell Cycle
  • Tumor Suppressor Protein p53 / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination
  • Uterine Cervical Neoplasms / metabolism*
  • Uterine Cervical Neoplasms / pathology*
  • Uterine Cervical Neoplasms / ultrastructure

Substances

  • Apoptosis Regulatory Proteins
  • DNA-Binding Proteins
  • E6 protein, Human papillomavirus type 18
  • Oncogene Proteins, Viral
  • Tumor Suppressor Protein p53
  • NADH, NADPH Oxidoreductases
  • GRIM19 protein, human
  • UBE3A protein, human
  • Ubiquitin-Protein Ligases