Protein Synthesis Is Required for Caspase Activation and Induction of Apoptosis by Bisphosphonate Drugs
- 1Department of Medicine and Therapeutics, University of Aberdeen Medical School, Foresterhill, Aberdeen, AB25 2ZD, UK (F.P.C., H.L.B., M.J.R.), and 2Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Sheffield S10 2RX, UK (F.P.C., R.G.G.R.)
Abstract
The exact mechanisms of action of antiresorptive bisphosphonate drugs remain unclear, although they may inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. These drugs also cause apoptosis in J774 macrophages, probably as a consequence of inhibition of protein prenylation. However, the molecular pathways that lead to apoptosis are not known. In some cells, apoptosis induced by statins (other inhibitors of protein prenylation) is dependent on protein synthesis. The aim of this study was to further characterize the kinetics and biochemical features of bisphosphonate-induced apoptosis, including the dependence on protein synthesis. Alendronate-induced apoptosis in J774 cells occurred after ∼16 hr of treatment, although shorter exposures to the drug followed by incubation in bisphosphonate-free medium also committed cells to apoptosis. The appearance of apoptotic cells was associated with the appearance of caspase-3-like activity. Apoptosis induced by bisphosphonate or mevastatin was found to be dependent on protein synthesis because cycloheximide inhibited chromatin condensation, DNA fragmentation and activation of caspase-3-like protease or proteases. Protein synthesis was required for events that lead to commitment to apoptosis but not for the execution phase because cycloheximide did not prevent apoptosis when added ≥15 hr after the start of alendronate treatment. Furthermore, staurosporine-induced caspase-3-like activity and apoptosis in J774 cells could not be prevented by cycloheximide. These observations demonstrate that activation of caspase-3-like proteases and inhibition of commitment to apoptosis by cycloheximide are common features of apoptotic cell death induced by inhibitors of protein prenylation such as bisphosphonates.
Footnotes
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Send reprint requests to: Dr. F. P. Coxon, Department of Medicine and Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail:f.p.coxon{at}abdn.ac.uk
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This work was funded by a grant from the Medical Research Council (Realising Our Potential Award), UK. M.J.R. was a recipient of the J. G. Graves Medical Research Fellowship. H.L.B. is supported by a studentship from the National Association for the Relief of Paget’s Disease.
- Abbreviations:
- BP
- bisphosphonate
- ALN
- 4-amino-1-hydroxy-butylidene-1,1-bisphosphonate (alendronate)
- PAM
- 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (pamidronate)
- PBS
- phosphate-buffered saline
- CHX
- cycloheximide
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
- DAPI
- 4,6-diamidino-2-phenylindole
- STP
- staurosporine
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
- Ac-DEVD-AMC
- N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
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- Received May 7, 1998.
- Accepted June 19, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
