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Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in
J Tatsumi-Miyajima1, J H Küpper, H Takebe
1Institute of Radiation Genetics, Faculty of Medicine, Kyoto University, Japan.
Abstract:
In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of gamma-radiation. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.
Insights
Poly(ADP-ribose) polymerase (PARP) inhibition enhances DNA damage effects. Overexpressing the PARP DNA-binding domain increases cell sensitivity to genotoxic agents and potentiates mutagenesis, highlighting PARP
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- Poly(ADP-ribose) polymerase (PARP) activation is an early cellular response to DNA damage in eukaryotes.
- Poly(ADP-ribosyl)ation plays a role in maintaining genomic integrity and stability.
Purpose of the Study:
- To investigate the biological functions of poly(ADP-ribosyl)ation.
- To examine the effect of inhibiting poly(ADP-ribosyl)ation on cellular responses to genotoxic stress.
Main Methods:
- Established stable transfectants (COM3 cells) overexpressing the PARP DNA-binding domain conditionally.
- Utilized a shuttle-vector assay to assess mutagenesis after exposure to N-methyl-N-nitrosourea.
- Assessed cytotoxicity after alkylation treatment and gamma-radiation.
Main Results:
- Overexpression of the PARP DNA-binding domain potentiated the cytotoxicity of alkylation and gamma-radiation.
- Carcinogen-induced gene amplification, a marker of genomic instability, was enhanced by PARP DNA-binding domain overexpression.
- PARP inhibition potentiated the mutagenicity of N-methyl-N-nitrosourea in COM3 cells.
Conclusions:
- Poly(ADP-ribosyl)ation is crucial for maintaining genomic integrity and stability under genotoxic stress.
- Inhibition of PARP activity can sensitize cells to DNA-damaging agents and increase mutation frequency.
