Related Experiment Videos
Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent
1Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Germany.
Abstract:
In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3. We also observed cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor-proximal caspase-8 was not activated after methylation. Also, inhibition of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis. Overall, the data indicate that O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.
Insights
Methylating agents induce apoptosis in tumor cells lacking DNA repair. This cell death pathway involves Bcl-2 decline, caspase activation, and DNA double-strand breaks, independent of Fas signaling.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Death Pathways
Background:
- Methylating agents are used in cancer therapy, generating O6-methylguanine (O6MeG) DNA lesions.
- DNA repair protein alkyltransferase (MGMT) proficient (MGMT+) and deficient (MGMT-) cells exhibit different responses to these agents.
Purpose of the Study:
- To elucidate the molecular mechanism of cell death induced by methylating agents.
- To compare apoptosis induction in MGMT+ and MGMT- isogenic cell lines.
Main Methods:
- Comparison of isogenic MGMT+ and MGMT- cell lines.
- Analysis of apoptosis markers including Bcl-2, caspases (caspase-9, caspase-3, caspase-8), cytochrome c, and Bad.
- Assessment of DNA double-strand breaks and mismatch repair involvement.
Main Results:
- MGMT- cells showed hypersensitivity to methylation-induced cell killing, characterized by late-onset apoptosis (>48h).
- Apoptosis involved Bcl-2 decline, cytochrome c release, hypophosphorylation of Bad, and activation of caspase-9 and caspase-3, independent of Fas/Fas ligand.
- O6MeG-induced apoptosis requires mismatch repair and is preceded by DNA double-strand breaks in MGMT- cells.
Conclusions:
- O6MeG induces apoptosis via secondary DNA double-strand breaks, triggering the intrinsic pathway involving Bcl-2 and caspases.
- Mismatch repair is essential for O6MeG-induced apoptosis.
- The apoptotic pathway is independent of Fas/Fas ligand and p53 in these mutated cells.