u-0126 and 2-(2-hydroxyethylsulfanyl)-3-methyl-1-4-naphthoquinone

We previously showed that prolonged and strong ERK phosphorylation induced by Compound 5 (Cpd 5), a Cdc25A protein phosphatase inhibitor, was involved in its mechanism of cell growth inhibition. To study the relationship between ERK phosphorylation and cell growth inhibition, we used Cpd 5 as a tool to investigate ERK-regulated c-Myc expression in Hep3B hepatoma cells. We found that ERK phosphorylation caused by Cpd 5 induced c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and DNA binding ability, and this inhibition was antagonized by ERK kinase (MEK) inhibitor U-0126, implying that the ERK pathway was involved in regulating c-Myc expression. Since the participation of c-Myc protein in transcription requires its dimerization with Max protein, we examined the Myc-Max association in Cpd 5-treated cells and found that Cpd 5 suppressed Myc-Max dimerization. Transfection of Hep3B cells with mutated ERK (T188A/Y190F), which has lost its dual-phosphorylation sites, attenuated the actions of Cpd 5 on Myc-Max association. To further demonstrate whether Myc phosphorylation by Cpd 5-induced ERK activation was able to directly regulate c-myc gene expression, a chromatin immunoprecipitation (ChIP) assay was used to examine the binding of phospho-Myc to the c-myc promoter region. We found that phospho-Myc induced by Cpd 5 had lost its ability to bind to the c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that an increase in c-Myc phosphorylation in response to prolonged ERK phosphorylation negatively auto-regulates c-Myc gene expression, leading to the suppression of its target gene expression and cell cycle block.

Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Liver Neoplasms; Nitriles; Oligonucleotide Array Sequence Analysis; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction; Transcription, Genetic; Transfection; Vitamin K