EXPOSURE OF HUMAN-LYMPHOCYTES TO BIS-(2-CHLOROETHYL)SULFIDE SOLUBILIZES TRUNCATED AND INTACT CORE HISTONES

Bis-(2-chloroethyl)sulfide (BCES) is a radiomimetic, bifunctional alky lating agent that cross-links DNA, disrupts higher-order nuclear struc ture and selectively kills rapidly proliferating cell types. While che mically fractionating primary, human lymphocytes after challenge with cytotoxic doses of BCES, we detected a 12900 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 appr oximately 10-fold by 24 h. Two other polypeptides of 16320 and 16970 M (r), also were increased measurably at 24 h following BCES exposure. A ltered polypeptides were found in 28 of 28 separate lymphocyte prepara tions 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 d ifferent cytotoxic agent, chlorovinyl-dichloroarsine (lewisite). Appea rance of the polypeptides was unaffected by sulfhydryl reducing agents or pretreatment of cells with the protein synthesis inhibitor, cycloh eximide. Micro sequencing resulted in a perfect match of the 12900 M(r ) polypeptide amino terminus with residues 19-27 of histone H2B. This corresponds to the exact site of H2B cleavage obtained when intact nuc leosomes are treated with chymotrypsin. Sequence data from the other t wo altered polypeptides identified them as intact histone H2B and hist one H3. Lymphocyte genomic DNA integrity also was assessed after BCES exposure and found to undergo extensive fragmentation typical of cellu lar necrosis. We speculate that exposure of isolated cells to BCES dis rupts nucleosome structure by mechanism(s) that involve abnormal remov al and perhaps proteolysis of core histones.