title: Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA.
abstract: The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
authors.forename: S J
authors.initials: SJ
authors.lastname: Dewitte-Orr
authors.forename: J R
authors.initials: JR
authors.lastname: Zorzitto
authors.forename: L P
authors.initials: LP
authors.lastname: Sutton
authors.forename: N C
authors.initials: NC
authors.lastname: Bols
mesh_terms: D015075:2-Aminopurine / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000633:toxicity*; D003609:Dactinomycin / Q000633:toxicity*; D004587:Electrophoresis, Agar Gel; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D017686:Oncorhynchus mykiss / Q000276:immunology*; D011070:Poly I-C / Q000633:toxicity; D012330:RNA, Double-Stranded / Q000633:toxicity*
chemical_list: D012330:RNA, Double-Stranded; D003609:Dactinomycin; D015075:2-Aminopurine; D003513:Cycloheximide; D011070:Poly I-C