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Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes
1Laboratoire de Chimie Organique des Substances Naturelles, URA CNRS no. 31, Strasbourg, France.
Abstract:
Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed.
Insights
Certain cholesterol derivatives (oxysterols) trigger apoptosis, a form of programmed cell death, in lymphoma and thymocytes. This process requires new protein synthesis, suggesting a role in thymus involution.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Oxysterols, oxygenated cholesterol derivatives, exhibit immunosuppressive and anti-proliferative effects.
- They are known to inhibit T-cell responses.
- Specific oxysterols like 25-hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) can induce cell death.
Purpose of the Study:
- To investigate the mechanism of cell death induced by oxysterols in RDM4 murine lymphoma and mouse thymocytes.
- To determine if oxysterol-induced cell death exhibits characteristics of apoptosis.
- To explore the role of macromolecular synthesis in this process.
Main Methods:
- Agarose gel electrophoresis to detect DNA fragmentation.
- Flow cytometry with propidium iodide staining to quantify DNA content and apoptosis.
- Treatment with cycloheximide and actinomycin D to assess the requirement for macromolecular synthesis.
Main Results:
- Oxysterol treatment induced apoptosis in RDM4 lymphoma cells and thymocytes.
- DNA fragmentation and altered DNA fluorescence (detected by flow cytometry) confirmed apoptosis.
- Inhibition of protein synthesis (cycloheximide) and RNA synthesis (actinomycin D) reduced oxysterol-induced apoptosis, indicating a requirement for macromolecule synthesis.
Conclusions:
- 25-OHC and 7.25-OHC induce programmed cell death (apoptosis) in lymphoma and thymocytes.
- Oxysterol-induced apoptosis is dependent on the synthesis of new macromolecules.
- These findings suggest a potential role for oxysterols in thymus involution during development.