8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines.
Several 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.
Mazzei
Filomena
F
Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
Guarrera
Simonetta
S
Allione
Alessandra
A
Simonelli
Valeria
V
Narciso
Laura
L
Barone
Flavia
F
Minoprio
Anna
A
Ricceri
Fulvio
F
Funaro
Ada
A
D'Errico
Mariarosaria
M
Vogel
Ulla
U
Matullo
Giuseppe
G
Dogliotti
Eugenia
E
D002460:Cell Line; D002472:Cell Transformation, Viral; D015925:Cryopreservation; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism*; D004260:DNA Repair; D015870:Gene Expression; D056726:Genetic Association Studies; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D004854:Herpesvirus 4, Human; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000378:metabolism*
C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human