1,2,3,4-Diepoxybutane-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells

Abstract

1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

Figure 1

Concentration-dependent formation of DNA–protein cross-links in human fibrosarcoma (HT1080) cells treated with DEB. Cells were treated with 0–2 mM DEB for 3 h, and chromosomal DNA containing cross-linked proteins was isolated by modified phenol/chloroform extraction in the presence of proteasome inhibitors. Proteins (from 26 μg DNA) were released from DNA by thermal hydrolysisin the form of protein-guanine conjugates, separated by 12% SDS-PAGE, and visualized by staining with SimplyBlue SafeStain (A). Densitometric analysis of protein bands: normalized band intensity values were obtained by subtracting the values observed in controls. Error bars represent the standard error from two independent experiments (B).

Figure 2

SDS-PAGE analysis of samples employed in the proteomics studies of DEB-induced DNA-protein cross-linking. HT1080 cells (~10⁷) were treated with 0 (lanes 2, 3, and 4) or 2 mM DEB (lanes 6, 7, and 8) for 3 h. Following modified phenol/chloroform extraction of DNA in the presence of proteasome inhibitors and thermal hydrolysis to release proteins, the cross-linked proteins were separated by 12% SDS-PAGE and visualized by staining with SimplyBlue SafeStain. Proteins present in the 10–250 kDa molecular weight range were excised from the gel, subjected to in-gel tryptic digestion, and analyzed by HPLC–ESI⁺–MS/MS.

Figure 3

Representative HPLC–ESI⁺–MS/MS spectra of tryptic peptides used in the identification of DPCs involving nucleolin (A) and histone-H4 (B).

Figure 4

GO annotations for proteins involved in DEB-induced DPC formation in human HT1080 cells: (A) cellular distributions, (B) molecular functions, (C) biological processes, and (D) disease associations. The number of proteins falling into each category is labeled on the charts.

Figure 5

Western blot analysis of DEB-induced DPCs in HT1080 cells. Following treatment with 0, 0.05, 0.5, 1.0, or 2.0 mM DEB, DNA and covalently cross-linked proteins were isolated by phenol/chloroform extraction. Proteins (from 100 μg DNA) were released by thermal hydrolysis, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Western blotting was performed using primary antibodies specific for PARP, nucleolin, and GAPDH. (A) Densitometric analysis of protein bands: normalized band intensity values were obtained by subtracting the values observed in controls. Error bars represent the standard error from two independent experiments (B).

Figure 6

HPLC-ESI⁺-MS/MS analysis of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) conjugates in total proteolytic digests. HT1080 cells were exposed to 0, 0.1, 0.5, 1.0, or 2.0 mM DEB for 3 h. Following extraction of DPC-containing chromosomal DNA, equal DNA amounts from each sample were subjected to thermal and enzymatic hydrolysis to release amino acid-nucleobase conjugates. The samples were subjected to offline HPLC to enrich for Cys-N7G-BD prior to HPLC-ESI⁺-MS/MS analysis. Quantification of Cys-N7G-BD was accomplished using isotope dilution with Cys-¹⁵N₅-N7G-BD. Shown are extracted ion chromatograms corresponding to HT1080 cells incubated in the absence of DEB (negative control) and samples treated with 2 mM DEB (A) and concentration dependent formation of Cys-N7G-BD in DEB-treated HT1080 cells (B).

Figure 7

Venn diagrams showing the overlaps between proteins that form cross-links to chromosomal DNA in DEB-treated HT1080 cells (2 mM) with proteins that form DPCs in the presence of mechlorethamine (25μM) (A) and proteins that form DPCs in the presence of cisplatin (100 μM) (B).

Scheme 1

Metabolic activation of 1,3-butadiene to 1,2,3,4-diepoxybutane (DEB) and the formation of DNA-protein cross-links by DEB.

Scheme 2

Strategy for the isolation and analysis of DPCs from DEB-treated mammalian cell cultures. Genomic DNA is isolated from control and treated cells using modified phenol-chloroform extraction in the presence of proteasome inhibitors. DPCs are released form the DNA backbone using thermal hydrolysis and are characterized by MS based proteomics, western blotting, and HPLC-ESI-MS/MS of amino acid-nucleobase conjugates in enzymatic digests.