Mechanism of diepoxybutane-induced p53 regulation in human cells

Abstract

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a known mutagen and human carcinogen and possesses multisystems organ toxicity. We previously reported the elevation of p53 in human TK6 lymphoblasts undergoing DEB-induced apoptosis. In this study, we have characterized the DEB-induced p53 accumulation and investigated the mechanisms by which DEB regulates this p53 accumulation. The elevation of p53 levels in DEB-exposed TK6 lymphoblasts and human embryonic lung (HEL) human fibroblasts was found to be largely due to the stabilization of the p53 protein. DEB increased the acetylation of p53 at lys-382, dramatically reduced complex formation between p53 and its regulator protein mdm2 and induced the phosphorylation of p53 at serines 15, 20, 37, 46, and 392 in human lymphoblasts. A dramatic increase in phosphorylation of p53 at serine 15 in correlation to total p53 levels was observed in DEB-exposed Ataxia Telangiectasia Mutated (ATM) proficient human lymphoblasts as compared to DEB-exposed ATM-deficient human lymphoblasts; this implicates the ATM kinase in the elevation of p53 levels in DEB-exposed cells. Collectively, these findings explain for the first time the mechanism by which p53 accumulates in DEB-exposed cells and contributes to the understanding of the molecular toxicity of DEB and BD.

FIGURE 1.

Dose dependent elevation of cellular p53 levels in DEB-exposed human cells. TK6 lymphoblasts (Panel A) and HEL human fibroblasts (Panel B) were exposed to various concentrations of DEB as shown and incubated for 24 h. Cellular p53 levels were analyzed by the western blot technique, using anti-p53 antibody. The quantity of protein retained on the blot was determined by quantifying GAPDH levels for each sample. Relative normalized p53 levels shown in graphical representation were obtained after densitometer tracing, followed by normalization to the corresponding GAPDH levels for each sample. A representative of three experiments is shown.

FIGURE 2.

Role of p53 synthesis in DEB-induced cellular p53 elevation in human cells. TK6 lymphoblasts (Panel A) and HEL cells (Panel B) were exposed to vehicle alone or 10 μM DEB. At 24 h post-DEB exposure, cells were pulse labeled with ³⁵S-methionine-³⁵S-cysteine for 45 min. Labeled protein extracts (~10 × 10⁶ counts per minute (cpm)) were immunoprecipitated using an anti-p53 antibody or control antibody, and the immunoprecipitates were fractionated by SDS-PAGE. Labeled p53 protein bands were detected by autoradiography and quantitated by densitometric analysis. A graphical representation of the relative rate of p53 protein synthesis was obtained by comparing the quantity of immunoprecipitated p53 in unexposed and exposed cells after normalization of each value to the density of a corresponding immunoprecipitated representative non-specific band shown as the control.

FIGURE 3.

Elevation of p53 mRNA levels in DEB-exposed TK6 lymphoblasts. TK6 cells were exposed to vehicle alone, 10 or 20 μM DEB (in duplicates), and incubated for 24 h. Total RNA was extracted using the Trizol reagent, and steady state p53 mRNA levels were determined by northern blot analysis using a ³²P labeled probe against the human p53 gene. The filters were then stripped and re-probed with ³²P-labeled 18S rRNA to normalize for the quantity of RNA retained on the blot. The determined relative normalized p53 mRNA levels in control and DEB-exposed TK6 cells are graphically represented.

FIGURE 4.

Stability of the p53 protein increases in DEB-treated human cells. TK6 lymphoblasts (Panel A) and HEL fibroblasts (Panel B) were exposed to vehicle alone or 10 μM DEB. At 24 h postexposure, 75 μg/mL of cycloheximide was added to the cells, and extractions were performed at various times (as shown). Cellular p53 and GAPDH levels in protein extracts were determined by western blot analysis, and quantitation was performed by densitometry tracing of the protein bands. The percent normalized p53 levels were determined by dividing the p53 to GAPDH band density ratio for each sample to the p53 to GAPDH band density ratio at 0 time point, and multiplying by 100. After graphic representation, the half-life of the p53 was determined as the time corresponding to the 50% normalized p53 value.

FIGURE 5.

Effect of DEB exposure on p53–mdm2 complex formation in human lymphoblasts. TK6 lymphoblasts were exposed to vehicle alone or 10 μM DEB for 24 h. Cell lysates (200 μg of protein) were subjected to immunoprecipitation using mouse control IgG or anti-p53 antibody. (A) The resulting immunoprecipitates (IP) and non-immunoprecipitated extracts (no IP, 100 μg of protein) were analyzed by western blot analysis using antibodies against p53 and mdm2. (B) A graphical representation of the relative mdm2/p53 ratio in whole cell lysates (no IP) and p53 immunocomplexes (IP) of control-unexposed and DEB-exposed cells is shown.

FIGURE 6.

Acetylation of p53 protein in response to DEB exposure in TK6 cells. Cells were exposed to (A). various concentrations of DEB for 24 h or (B) 10 μM DEB for various times, as shown. Acetyl-p53-(lys³⁸²) levels were determined by western blot analysis, using an antibody specific for acetyl-p53-(lys³⁸²) protein. The blots were stripped and reprobed with antibodies against p53 and GAPDH. (C) To determine the specificity of the anti-acetyl-p53-(lys³⁸²) antibody, extracts from vehicle alone, 10 μM and 20 μM DEB-treated TK6 lymphoblasts as well as mock and HCMV-infected HEL fibroblasts were analyzed by the western blot analysis.

FIGURE 7.

Role of ATM kinase in mediating the phosphorylation and stability of p53 in response to DEB exposure. (A) ATM-proficient GM02254 and (B) ATM-deficient GM01526 cells were simultaneously exposed to vehicle alone, 10 μM, or 20 μM DEB for 24 h (top panels) and 48 h postexposure (bottom panels). Phospho-p53-(ser¹⁵) levels in the protein extracts were determined by the western blot technique, using specific antibody. The blots were then stripped and reprobed with anti-p53 and anti-GAPDH antibodies. Protein levels were normalized by using GAPDH levels.