title: Protease inhibitors differentially regulate tumor necrosis factor-induced apoptosis, nuclear factor-kappa B activation, cytotoxicity, and differentiation.
abstract: 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.
authors.affiliation: Department of Molecular Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: M
authors.initials: M
authors.lastname: Higuchi
authors.forename: S
authors.initials: S
authors.lastname: Singh
authors.forename: H
authors.initials: H
authors.lastname: Chan
authors.forename: B B
authors.initials: BB
authors.lastname: Aggarwal
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004249:DNA Damage; D004988:Ethers, Cyclic / Q000494:pharmacology; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007951:Leukemia, Myeloid / Q000473:pathology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D019319:Okadaic Acid; D010879:Piperazines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D004988:Ethers, Cyclic; D007546:Isoquinolines; D016328:NF-kappa B; D009363:Neoplasm Proteins; D010879:Piperazines; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D019319:Okadaic Acid; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide