8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines.
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AbstractSeveral lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.
CitationMutat. Res. 2011, 718 (1-2):62-67
SponsorsThis work was supported by a grant from the Associazione Italiana per le Ricerche sul Cancro (Italy; G.M., E.D.) of the Progetto Integrato Oncologia, Regione Toscana- Ministero della Salute “Identiﬁcation of population risk proﬁles as an approach to cancer prevention” and of the Environmental Cancer Risk Nutrition and Individual Susceptibility project (G.M.), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No.513943).