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Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry
Published on: September 1, 2019
Human fibroblasts undergo oxidative stress-induced apoptosis without internucleosomal DNA fragmentation
P Formichi1, E Radi, C Battisti
1Department of Neurological and Behavioural Sciences, University of Siena, Siena, Italy.
Journal of Cellular Physiology
|April 29, 2006
Summary
Human fibroblasts undergo apoptosis when exposed to oxidative stress. However, they exhibit only high molecular weight DNA fragmentation, not the typical DNA ladder, indicating unique cell death pathways.
Area of Science:
- Cell Biology
- Biochemistry
Background:
- Apoptosis is a crucial biological process.
- Fibroblasts are widely used cell models.
- Understanding apoptosis in fibroblasts is vital for disease research.
Purpose of the Study:
- To assess the reliability of human fibroblasts as a cell model for apoptosis studies.
- To investigate fibroblast response to oxidative stress.
Main Methods:
- Treatment of normal human fibroblasts with hydrogen peroxide (H2O2) and 2-deoxy-D-ribose (dRib).
- Assessment of apoptosis using cell counting, chromatin condensation, phosphatidylserine exposure, caspase activation, mitochondrial membrane potential, TUNEL assay, flow cytometry (sub-G1, PI staining), and agarose gel electrophoresis.
Main Results:
- Fibroblasts treated with dRib and H2O2 showed clear signs of apoptosis, including caspase activation and PS exposure.
- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay detected high molecular weight (HMW) DNA fragments.
- No significant sub-G1 peak by flow cytometry or DNA laddering (low molecular weight fragments) by gel electrophoresis was observed.
Conclusions:
- Human fibroblasts exhibit apoptosis with HMW DNA fragmentation but lack internucleosomal DNA fragmentation.
- This suggests unique apoptotic characteristics of fibroblasts, irrespective of the stimulus.
- Multiple methods are necessary to accurately assess apoptosis across different cell types.
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