Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA.
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.
Dewitte-Orr
S J
SJ
Department of Biology, University of Waterloo, Waterloo, ON, N2L 3G1, Canada.
Zorzitto
J R
JR
Sutton
L P
LP
Bols
N C
NC
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*
D012330:RNA, Double-Stranded; D003609:Dactinomycin; D015075:2-Aminopurine; D003513:Cycloheximide; D011070:Poly I-C