Inhibition of puromycin-induced apoptosis in breast cancer cells by IGF-I occurs simultaneously with increased protein synthesis.
The objective of the following work was to study the apoptosis inducing effect of puromycin in MCF-7 breast cancer cells and compare this effect with cycloheximide and emetine, 2 other inhibitors of protein synthesis. We also wished to investigate if the apoptosis modulating effect of insulin-like growth factor-1 (IGF-1) was similar for the 3 inhibitors. An immunological assay, quantifying mono- and oligonucleosome fragments and morphological criteria after nuclear staining, were used to study apoptosis. Protein synthesis was measured by incorporation of 3H-leucine in the cells, and solution hybridization and Western blot were performed to estimate IGF-I receptor m-RNA and IGF-I receptor protein respectively. Puromycin at 0.5 microg/ml induced a high level of apoptosis in MCF-7 breast cancer cells, although there was still a non-negligible amount of synthesized protein. In the case of cycloheximide and emetine, apoptosis occurred when protein synthesis was almost completely blocked. IGF-1 at a concentration of 10 ng/ml significantly reduced the level of apoptosis induced by puromycin, emetine, or cycloheximide. We also noticed a parallel increase in 3H-leucine incorporation when apoptosis induced by puromycin was lowered as an effect of IGF-1, in contrast to cycloheximide and emetine where IGF-1 reduced the apoptosis level without increasing the 3H-leucine incorporation. At a higher concentration of puromycin (5.7 microg/ml), which blocked protein synthesis, IGF-1 at 10 ng/ml did not reduce apoptosis. The level of IGF-1 receptor m-RNA was not influenced by the use of a concentration of puromycin (0.5 microg/ml) inducing a high degree of apoptosis. These results suggest, that reduction of puromycin-induced apoptosis by IGF-1 occurs simultaneously with increased protein synthesis, in contrast to emetine and cycloheximide. Furthermore it would appear that puromycin-induced apoptosis is not caused by reduced levels of IGF-1 receptors.
Söderlund
G
G
Division of Oncology, University Hospital, Linköping, Sweden. jgf2679q@post.utfors.se
Haarhaus
M
M
Chisalita
S
S
Arnqvist
H J
HJ
D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004640:Emetine / Q000494:pharmacology; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011691:Puromycin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D017526:Receptor, IGF Type 1 / Q000032:analysis / Q000235:genetics
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011691:Puromycin; D007334:Insulin-Like Growth Factor I; D003513:Cycloheximide; D017526:Receptor, IGF Type 1; D004640:Emetine