PROTEASE IN NORMAL HUMAN EPIDERMAL-KERATINOCYTES

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-meth ylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroet hyl sulfide (sulfur mustard HD). The results obtained by using a chrom ozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 mu M GEES, 100 mu M HN2 or HD, and 16 hours. The mustard-stimulated proteas e was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 mu M) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM) or a protein synthesis inhibitor cyclohex imide (10 mu M) in the extracellular medium. These results suggest tha t one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2 + and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated similar or equal to 70- 80 KDa protein band that was associated with protease activity which w as inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stim ulated protein (protease) may serve as a diagnostic tool for mustard e xposure as well as an assay for screening prospective antivesicant pro tease inhibitor drugs.