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 (oua(r)) 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.
Subject(s)
Bronchi/metabolism , DNA Damage , DNA Repair , DNA-Formamidopyrimidine Glycosylase/biosynthesis , Escherichia coli Proteins/biosynthesis , Escherichia coli/enzymology , Tobacco Smoke Pollution/adverse effects , Bronchi/pathology , Cell Line, Tumor , DNA-Formamidopyrimidine Glycosylase/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Female , Humans , Middle Aged , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , TransfectionABSTRACT
We have previously shown that DNA repair of oxidized bases (either purines or pyrimidines) is inefficient in cells from patients with Cockayne syndrome (cs), a rare developmental and neurological genetic disorder. Here, we show for the first time that resolution of ionizing radiation (IR)-induced pH2AX foci, an indicator of DNA double-strand breaks, is significantly delayed in IR-treated cells belonging to the B complementation group of cs (csb). Using alkaline single-cell gel electrophoresis, which predominantly detects single-strand breaks, we further demonstrate elevated DNA breakage in csb cells early after irradiation. Both the delayed resolution of pH2AX foci and the early DNA breakage of csb cells were partially complemented by expression of wild-type CSB protein. Hence, the csb mutation impairs resolution of pH2AX foci and causes DNA fragility, broadening the spectrum of lesions whose processing is altered in this syndrome.
Subject(s)
Cockayne Syndrome/metabolism , DNA Breaks/radiation effects , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , Fibroblasts/radiation effects , Histones/metabolism , Cell Line , Child, Preschool , Cockayne Syndrome/pathology , DNA Helicases/genetics , DNA Repair , DNA Repair Enzymes/genetics , Female , Fibroblasts/metabolism , Fluorescent Antibody Technique, Direct , Humans , Kinetics , Mutation , Poly-ADP-Ribose Binding ProteinsABSTRACT
Repair of the oxidized purine 8-oxo-7,8-dihydro-2'-deoxyguanosine is inefficient in cells belonging to both complementation groups A and B of Cockayne syndrome (CS), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that both CS-A and CS-B cells are also defective in the repair of 5-hydroxy-2'-deoxycytidine (5-OHdC), an oxidized pyrimidine with cytotoxic and mutagenic properties. The defect in the repair of oxidatively damaged DNA in CS cells thus extends to oxidized pyrimidines, indicating a general flaw in the repair of oxidized lesions in this syndrome. The defect could not be reproduced in in vitro repair experiments on oligonucleotide substrates, suggesting a role for both CS-A and CS-B proteins in chromatin remodeling during 5-OHdC repair. Expression of Escherichia coli formamidopyrimidine DNA glycosylase (FPG) or endonuclease III complemented the 5-OHdC repair deficiency. Hence, the expression of a single enzyme, FPG from E. coli, stably corrects the delayed removal of both oxidized purines and oxidized pyrimidines in CS cells.
Subject(s)
Cockayne Syndrome/metabolism , DNA-Formamidopyrimidine Glycosylase/metabolism , Deoxycytidine/analogs & derivatives , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Adolescent , Aged, 80 and over , Cell Line, Transformed , Child, Preschool , Chromatin Assembly and Disassembly/genetics , Cockayne Syndrome/genetics , Cockayne Syndrome/therapy , DNA Repair-Deficiency Disorders/genetics , DNA-Formamidopyrimidine Glycosylase/genetics , Deoxycytidine/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Escherichia coli Proteins/genetics , Female , Humans , Male , TransfectionABSTRACT
It has been reported that cancer stem cells may contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. We have examined DNA repair in five stem and nonstem glioma cell lines. The population doubling time was significantly increased in stem compared with nonstem cells, and enhanced activation of Chk1 and Chk2 kinases was observed in untreated CD133(+) compared with CD133(-) cells. Neither DNA base excision or single-strand break repair nor resolution of pH2AX nuclear foci were increased in CD133(+) compared with CD133(-) cells. We conclude that glioma stem cells display elongated cell cycle and enhanced basal activation of checkpoint proteins that might contribute to their radioresistance, whereas enhanced DNA repair is not a common feature of these cells.
Subject(s)
Brain Neoplasms/genetics , DNA Repair , Glioblastoma/genetics , Neoplastic Stem Cells/physiology , AC133 Antigen , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Apoptosis/physiology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Checkpoint Kinase 1 , Checkpoint Kinase 2 , DNA Damage , Enzyme Activation , Glioblastoma/metabolism , Glioblastoma/pathology , Glycoproteins/biosynthesis , Glycoproteins/genetics , Humans , Karyotyping , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Peptides/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolismABSTRACT
Repair of the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoGua) is inefficient in cells belonging to the B complementation group of Cockayne syndrome (CS-B), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that cells belonging to the A complementation group (CS-A) are also defective in repair of 8-oxoGua and we demonstrate that expression of the Escherichia coli formamidopyrimidine DNA glycosylase (FPG) completely corrects the repair deficiency in both CS-A and CS-B cells. Phenotypically, CS-A cells are normally sensitive to toxicity and micronuclei induced by the oxidizing agent potassium bromate. CS-B cells display sensitivity to elevated concentrations of potassium bromate but this is not compensated by FPG expression, suggesting toxicity of lesions that are not FPG substrates. The data indicate that 8-oxoGua is not a major toxic and clastogenic lesion in CS cells.
