Exposure of human lymphocytes to bis-(2-chloroethyl)sulfide solubilizes truncated and intact core histones

Abstract

Bis-(2-chloroethyl)sulfide (BCES) is a radiomimetic, bifunctional alkylating agent that cross-links DNA, disrupts higher-order nuclear structure and selectively kills rapidly proliferating cell types. While chemically fractionating primary, human lymphocytes after challenge with cytotoxic doses of BCES, we detected a 12 900 M r polypeptide in 1.0 M NaCl extracts of exposed cells that was markedly increased compared to controls. By computer-aided image analysis of polyacrylamide gels, it was detected as early as 4 h following 1 mM BCES and increased approximately 10-fold by 24 h. Two other polypeptides of 16 320 and 16 970 M r also were increased measurably at 24 h following BCES exposure. Altered polypeptides were found in 28 of 28 separate lymphocyte preparations ranging in cell density from 5 · 10 6/ml to 6 · 10 7/ml. They were not present if cells were killed with equimolar concentrations of a different cytotoxic agent, chlorovinyl-dichloroarsine (lewisite). Appearance of the polypeptides was unaffected by sulfhydryl reducing agents or pretreatment of cells with the protein synthesis inhibitor, cycloheximide. Micro sequencing resulted in a perfect match of the 12 900 M r polypeptide amino terminus with residues 19–27 of histone H2B. This corresponds to the exact site of H2B cleavage obtained when intact nucleosomes are treated with chymotrypsin. Sequence data from the other two altered polypeptides identified them as intact histone H2B and histone H3. Lymphocyte genomic DNA integrity also was assessed after BCES exposure and found to undergo extensive fragmentation typical of cellular necrosis. We speculate that exposure of isolated cells to BCES disrupts nucleosome structure by mechanism(s) that involve abnormal removal and perhaps proteolysis of core histones.