Accelerated Repair and Reduced Mutagenicity of DNA Damage Induced by Cigarette Smoke in Human Bronchial Cells Transfected with E.coli Formamidopyrimidine DNA Glycosylase

Mara Foresta,Donatella Vecchio,Alberto Izzotti,Sebastiano La Maestra,Guido Frosina,Rosanna Micale,Alessandro Poggi,Robert W Sobol

doi:10.1371/journal.pone.0087984

Abstract

Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na+K+-ATPase locus (ouar) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells’ capacity to repair damaged DNA.

Highlights

Cigarette smoke (CS) is a self-inflicted damaging agent associated with high risk of developing chronic-degenerative diseases including cancer, obstructive pulmonary disease, and cardiovascular diseases
We report here that expression of formamidopyrimidine DNA glycosylase (FPG) in human lung cells stably enhances DNA repair of damage induced by cigarette smoke condensate (CSC) and reduces its mutagenicity
The studies described here have been performed in transformed, immortal adenocarcinoma cells in order to achieve permanent expression of Enhanced Green Fluorescent Protein (EGFP)-FPG and investigate its effects on Condensate from cigarette smoke (CSC) mutagenicity at a target locus [Na+/K+ ATPase]

Summary

Introduction

Cigarette smoke (CS) is a self-inflicted damaging agent associated with high risk of developing chronic-degenerative diseases including cancer, obstructive pulmonary disease, and cardiovascular diseases. Major mutagenic components of CS are polycyclic aromatic hydrocarbons (PAH), aromatic amines and N-nitrosamines that produce adducts on DNA. Those adducts are repaired in human cells by a network of DNA repair pathways including the nucleotide excision repair (NER) and the DNA base excision repair (BER) pathways. The 30.2 kDa formamidopyrimidine DNA glycosylase (FPG) is a BER protein that directly removes the damaged base and subsequently cleaves the resulting AP site by its associated b,d AP lyase activity [5,6]. We report here that expression of FPG in human lung cells stably enhances DNA repair of damage induced by cigarette smoke condensate (CSC) and reduces its mutagenicity

Methods

Results

Conclusion