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Transcription abnormalities potentiate apoptosis of normal human fibroblasts
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, ULP, Illkirch, France.
Background:
Apoptosis is a natural process by which damaged and potentially tumorigenic cells are removed. Induction of apoptosis is important in chemotherapy aimed at eliminating cancer cells. We address the mechanisms by which this process can be triggered in cells that are recalcitrant to cell death induced by DNA-damaging agents.
Materials And Methods:
Normal human fibroblasts and lymphoblasts, and fibroblasts with defined genetic changes, were treated with DNA-damaging agents and inhibitors of transcription. Western blotting was used to study the expression of some of the key factors involved in the response to DNA damage and the induction of apoptosis, namely, p53, p21WAFI,Cip1, Mdm2, Bax, and CD95 (Fas/APO1). Apoptosis was followed by various criteria, including DNA fragmentation, specific proteolysis, cell morphology, viability, and FACS scan for sub-G1 cells.
Results:
Normal human fibroblasts were more resistant than lymphoblasts to DNA damage-induced apoptosis. The DNA-damaging agents mitomycin C and cisplatin induced rapid apoptosis of fibroblasts with defects in the repair of transcribed DNA, compared with wild-type cells or those with defects in overall genome repair. Short-term treatment with inhibitors of RNA polymerase II transcription, actinomycin D, and alpha-amanitin induced rapid cell death of normal fibroblasts. These results show that there is a link between defective transcription and apoptosis. Treatments and genetic backgrounds that favored apoptosis were associated with efficient and prolonged induction of p53 and often altered or imbalanced expression of its downstream effectors p21WAFI,Cip1 and Mdm2, whereas there were no changes in Bax or CD95 (Fas/APO1).
Conclusion:
Transcription inhibitors increase p53 levels and are better inducers of apoptosis than DNA-damaging agents in some cell types. Apoptosis might be triggered by blocked polymerases and/or faulty expression of downstream effectors.
Insights
Transcription inhibitors trigger apoptosis more effectively than DNA-damaging agents in some cells by increasing p53 levels. This suggests blocked polymerases or faulty downstream effectors can induce programmed cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Apoptosis is crucial for removing damaged cells and is a key target in cancer chemotherapy.
- Understanding mechanisms to induce apoptosis in resistant cancer cells is vital for effective treatment.
Purpose of the Study:
- To investigate the mechanisms of apoptosis induction in cells resistant to DNA-damaging agents.
- To explore the role of transcription inhibition in triggering apoptosis.
Main Methods:
- Utilized normal human fibroblasts and lymphoblasts, including genetically modified cells.
- Treated cells with DNA-damaging agents and transcription inhibitors (actinomycin D, alpha-amanitin).
- Analyzed key apoptosis-related protein expression (p53, p21WAFI,Cip1, Mdm2, Bax, CD95) via Western blotting and assessed apoptosis through multiple methods.
Main Results:
- Fibroblasts with defects in transcribed DNA repair underwent rapid apoptosis upon treatment with mitomycin C and cisplatin.
- Transcription inhibitors rapidly induced cell death in normal fibroblasts.
- Apoptosis correlated with increased p53 levels and altered expression of downstream effectors like p21WAFI,Cip1 and Mdm2.
Conclusions:
- Transcription inhibitors can be potent inducers of apoptosis, surpassing DNA-damaging agents in certain cell types.
- Blocked polymerases and/or dysregulated expression of downstream effectors may trigger apoptosis.