Programmed cell death (apoptosis) of mouse fibroblasts is induced by the topoisomerase II inhibitor etoposide.
The mechanism by which etoposide, a topoisomerase II inhibitor, killed replicating mouse L929 fibroblasts was investigated. Etoposide at 10 microM killed 70% of the cells within 4 days, a result that was accompanied by DNA fragmentation. A characteristic "ladder" pattern of DNA fragmentation was confirmed by agarose gel electrophoresis. Simultaneous exposure of the cells to 10 microM etoposide plus 1 microM cycloheximide reduced both the extent of cell killing and the fragmentation of DNA. Delayed addition of cycloheximide protected cells only if cycloheximide was added 1-6 hr after exposure to etoposide. When added 6-24 hr after treatment with etoposide, cycloheximide lost the ability to protect cells. Cell growth was completely inhibited by either etoposide or cycloheximide. Furthermore, DNA synthesis was inhibited by either etoposide or cycloheximide within 6 hr. Protein synthesis, however, was not inhibited by etoposide. Thus, the ability of cycloheximide to protect cells correlated with inhibition of protein synthesis, rather than inhibition of DNA synthesis. A 1-hr exposure to 2.5 mM N-methyl-N-nitrosourea similarly inhibited DNA synthesis within 6 hr. without affecting protein synthesis. However, no loss of viability accompanied N-methyl-N-nitrosourea treatment. Thus, an imbalance between protein synthesis and DNA synthesis cannot explain the cell killing by etoposide. H-7, a protein kinase C inhibitor, prevented the cell killing and DNA fragmentation, whereas aurintricarboxylic acid, an endonuclease inhibitor, reduced the extent of DNA fragmentation but did not have an effect on cell killing. The data document that the killing of replicating mouse fibroblasts by etoposide represents an example of programmed cell death (apoptosis) that depends on protein synthesis. Although protein synthesis is required during the first 24 hr of exposure to etoposide, cell death is delayed until several days later.
Mizumoto
K
K
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Rothman
R J
RJ
Farber
J L
JL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D005047:Etoposide / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D007739:L Cells / Q000166:cytology / Q000187:drug effects; D008770:Methylnitrosourea / Q000494:pharmacology; D051379:Mice; D010879:Piperazines / Q000494:pharmacology; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D000477:Alkylating Agents; D007546:Isoquinolines; D010879:Piperazines; D011500:Protein Synthesis Inhibitors; D059005:Topoisomerase II Inhibitors; D008770:Methylnitrosourea; D005047:Etoposide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004247:DNA; D003513:Cycloheximide; D011493:Protein Kinase C