tosylphenylalanyl-chloromethyl-ketone and Leukemia--Myeloid

We investigated the effect of various protease inhibitors on several tumor necrosis factor (TNF)-mediated cellular responses. Treatment of a human myelogenous leukemia cell line, ML-1a, with TNF in the presence of cycloheximide triggers endonucleolytic activity and apoptotic cell death within 90 minutes. The general serine protease inhibitor diisopropyl fluorophosphate (DFP) and the chymotrypsin-like protease inhibitor N-tosyl-L-lysyl chloromethyl ketone (TPCK) completely abrogated TNF-induced DNA fragmentation and the formation of apoptotic bodies. However, 13 other protease inhibitors, including serine protease inhibitors, did not. The addition of TPCK to cells 30 minutes after TNF treatment completely inhibited the cytokine action, indicating that TPCK-sensitive proteases are not involved in the early stages of signal transduction. TNF is cytotoxic and induces differentiation in ML-1a cells after a 3-day incubation. TPCK had no effect on the TNF-induced cytotoxicity and differentiation, indicating that TPCK-sensitive proteases are specific for DNA fragmentation. TPCK also blocked TNF-induced activation of nuclear factor (NF)-kappa B. The dose-response and the time-course of the inhibitor, however, indicated that the site of action of TPCK for NF-kappa B activation and for DNA fragmentation are quite distinct. Therefore, we conclude that TNF activates two distinct TPCK-sensitive pathways, one leading to apoptosis and the other to NF-kappa B activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Apoptosis; Base Sequence; Cell Differentiation; Cycloheximide; Cytotoxicity, Immunologic; DNA Damage; Ethers, Cyclic; Gene Expression Regulation, Leukemic; Humans; Isoquinolines; Leukemia, Myeloid; Molecular Sequence Data; Neoplasm Proteins; NF-kappa B; Okadaic Acid; Piperazines; Protease Inhibitors; Tosylphenylalanyl Chloromethyl Ketone; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans.

Topics: Blood Proteins; Chromatography, Affinity; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Granulocytes; Humans; Hydrogen-Ion Concentration; Kinetics; Leukemia, Myeloid; Leukocytes; Neutrophils; Tosylphenylalanyl Chloromethyl Ketone