Restoration of wild-type conformation and activity of a temperature-sensitive mutant of p53 (p53(V272M)) by the cytoprotective aminothiol WR1065 in the esophageal cancer cell line TE-1.
The aminothiol WR1065, the active metabolite of the cytoprotector amifostine, exerts its antimutagenic effects through free-radical scavenging and other unknown mechanisms. In an earlier report, we showed that WR1065 activates wild-type p53 in MCF-7 cells, leading to p53-dependent arrest in the G(1) phase of the cell cycle. To determine whether WR1065 activates p53 by modulating protein conformation, we analyzed its effects on p53 conformation and activity in the esophageal cancer cell line TE-1. This cell line contains a mutation in codon 272 of p53 (p53(V272M), with methionine instead of a valine), conferring temperature-sensitive properties to the p53 protein. At the nonpermissive temperature (37 degrees C), p53(V272M) adopts the mutant p53 conformation (nonreactive with the antibody PAb1620), does not bind specifically to DNA, and is not activated in response to DNA-damaging treatment. However, treatment with 0.5-4 mM WR1065 partially restored wild-type conformation at 37 degrees C, stimulated DNA binding activity, and increased the expression of p53 target genes WAF-1, GADD45, and MDM2, leading to cell-cycle arrest in G(1). These results suggest that WR1065 activates p53 through a mechanism distinct from DNA-damage signaling, which involves modulation of p53 protein conformation.
North
Sophie
S
Group of Molecular Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Pluquet
Olivier
O
Maurici
Daniela
D
El-Ghissassi
Fatiha
F
Hainaut
Pierre
P
D002453:Cell Cycle / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism*; D016158:Genes, p53; D006801:Humans; D008624:Mercaptoethylamines / Q000494:pharmacology*; D009154:Mutation; D011487:Protein Conformation; D012334:RNA, Neoplasm / Q000096:biosynthesis; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D013696:Temperature; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008624:Mercaptoethylamines; D012334:RNA, Neoplasm; D011837:Radiation-Protective Agents; D016159:Tumor Suppressor Protein p53; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane