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Expression of c-jun and c-fos in apoptotic cells after DNA damage
S R Kumari1, R Alvarez-Gonzalez
1Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, USA.
Abstract:
Apoptosis was induced in HeLa cells by exposure to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for various time intervals (up to 120 min). Apoptotic death was confirmed by the microscopic observation of cell blebbing, cell granulation, and cell aggregation. Cells also showed loss of phospholipid symmetry as judged by immunofluorescent microscopy with fluorescently labeled phosphatidyl serine-specific annexin V. In addition, staining of cells with ethidium bromide showed the presence of genomic DNA apoptotic bodies. The protein expression levels of c-jun and c-fos increased in DNA-damaged HeLa cells after MNNG treatment in a time-dependent fashion. Although the levels of c-fos increased rapidly during the first 30 min and remained high for 2 hr, the increase in c-jun expression was more gradual and slower (60-120 min) after MNNG treatment. These results are consistent with the conclusion that c-fos is important in the initial stages (commitment phase) of apoptosis and c-jun is involved in the late stages (execution phase) of apoptosis induced with alkylating agents.
Insights
N-methyl-N-nitro-N-nitrosoguanidine (MNNG) induces apoptosis in HeLa cells, characterized by distinct cellular changes and DNA fragmentation. The study reveals c-fos involvement in early apoptosis stages and c-jun in later stages.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis is a crucial process for cellular homeostasis and development.
- Understanding the molecular mechanisms of apoptosis is vital for disease research.
- Alkylating agents like MNNG are known inducers of DNA damage and apoptosis.
Purpose of the Study:
- To investigate the temporal expression patterns of c-fos and c-jun during MNNG-induced apoptosis in HeLa cells.
- To elucidate the roles of c-fos and c-jun in different phases of apoptosis.
Main Methods:
- HeLa cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for varying durations.
- Apoptosis was assessed via microscopy (cell blebbing, granulation, aggregation), Annexin V staining for phospholipid symmetry, and ethidium bromide staining for DNA fragmentation.
- Protein expression levels of c-jun and c-fos were analyzed using methods likely involving Western blotting or similar techniques.
Main Results:
- MNNG treatment induced characteristic apoptotic morphological changes and DNA fragmentation in HeLa cells.
- Annexin V staining confirmed loss of phospholipid symmetry, a hallmark of early apoptosis.
- c-fos protein levels increased rapidly within 30 minutes and remained elevated, suggesting a role in the initial apoptosis stages.
- c-jun protein levels showed a more gradual increase between 60-120 minutes, indicating involvement in later apoptosis stages.
Conclusions:
- MNNG effectively induces apoptosis in HeLa cells through DNA damage.
- c-fos appears to be a key mediator in the commitment phase of apoptosis.
- c-jun plays a significant role in the execution phase of apoptosis induced by alkylating agents like MNNG.