Mechanism of target cytolysis by peptide defensins. Target cell metabolic activities, possibly involving endocytosis, are crucial for expression of cytotoxicity.
In a previous study, potent tumor cytolysis mediated by human neutrophil peptide defensins occurred slowly over 3 to 15 h. Because these kinetics suggested a requirement for target cell metabolic processes before tumor killing could be realized, the mechanism of lysis by these purified peptides was further investigated. 125I-labeled defensin bound extensively to peptide-sensitive K562 targets with biphasic kinetics. Binding was inhibited in parallel with cytotoxicity when both assays were performed at low temperature or in the presence of FCS. The albumin content of serum could account for the inhibitory effects of FCS. Cytotoxicity was also antagonized by agents that interfered with target cell energy metabolism (azide and 2-deoxyglucose), the cytoskeletal apparatus (cytochalasin B and dihydrocytochalasin B), lysosomal function (NH4Cl and chloraquin), or calmodulin-mediated activities (trifluoperazine). FCS also completely removed membrane-bound defensin when it was added after 5 min of binding at 37 degrees C. However, significantly less defensin was removed when FCS was added at later time points after binding was initiated. Cytochalasin B and azide/2-deoxyglucose did not prevent binding of defensin to targets but it significantly inhibited the development of FCS resistance in membrane-bound peptide. However, these two classes of inhibitors acted during distinct time windows: cytochalasin-sensitive events were complete by 1 h, whereas azide/2-deoxyglucose continued to be inhibitory when added as late as 2 h after defensins. These latter data indicated that critical energy-dependent events continue after the cytochalasin-sensitive phase has been completed. The results suggest that defensin-mediated cytotoxicity requires initial binding of defensin molecules to targets and subsequent cytoskeletal- and energy-dependent translocation or internalization. Although the defensins are low m.w. peptides, the initial processes required for their cytotoxic activity resemble those of more complex bacterial, plant and mammalian cytotoxins.
Lichtenstein
A K
AK
Department of Medicine, VA Wadsworth-UCLA Medical Center 90073.
Ganz
T
T
Nguyen
T M
TM
Selsted
M E
ME
Lehrer
R I
RI
D000818:Animals; D001790:Blood Physiological Phenomena; D001798:Blood Proteins / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D003571:Cytochalasin B / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D023082:Defensins; D004705:Endocytosis; D004734:Energy Metabolism / Q000187:drug effects; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009504:Neutrophils / Q000502:physiology*; D011485:Protein Binding; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism
D001798:Blood Proteins; D023082:Defensins; D009363:Neoplasm Proteins; D003571:Cytochalasin B