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Induction of programmed cell death in a dorsal root ganglia X neuroblastoma cell line
M D Linnik1, M D Hatfield, M D Swope
1Department of Pharmacology, Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215-6300.
Abstract:
Growth factor-dependent neurons die when they are deprived of their specific growth factor. This "programmed" cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD.
Insights
Serum deprivation triggers programmed cell death (PCD) in neurons, requiring protein synthesis and leading to DNA fragmentation. This study models neuronal PCD using F-11 cells, revealing metabolic activation alongside cell death.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Growth factor-dependent neurons undergo programmed cell death (PCD) upon growth factor withdrawal.
- PCD is a regulated process requiring macromolecular synthesis, distinct from necrosis.
Purpose of the Study:
- To investigate the molecular mechanisms and temporal sequence of events during neuronal PCD.
- To utilize the F-11 neuronal cell line as a model for serum deprivation-induced PCD.
Main Methods:
- Serum deprivation of F-11 cells.
- Inhibition of protein synthesis using cycloheximide.
- Assessment of DNA cleavage via nucleosome ladders.
- Mitochondrial function evaluation using MTT reduction.
- Cell viability assessment with propidium iodide staining.
Main Results:
- Protein synthesis inhibition occurred within 8 hours of serum deprivation.
- DNA cleavage into nucleosome ladders was evident by 24 hours and inhibited by cycloheximide.
- Mitochondrial function remained intact; cells showed signs of metabolic activation.
- No significant cell death was observed within 24 hours, but cell shrinkage and propidium iodide uptake increased after 48 hours.
Conclusions:
- Serum deprivation effectively induces PCD in the F-11 neuronal cell line.
- The study provides insights into the sequence of events, including protein synthesis requirement and metabolic changes, during neuronal PCD.
- The F-11 cell line serves as a valuable model for studying the intracellular mechanisms of PCD.