title: Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity.
abstract: As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.
authors.affiliation: Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Zijno
authors.forename: Paola
authors.initials: P
authors.lastname: Porcedda
authors.forename: Francesca
authors.initials: F
authors.lastname: Saini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Allione
authors.forename: Bruno
authors.initials: B
authors.lastname: Garofalo
authors.forename: Francesca
authors.initials: F
authors.lastname: Marcon
authors.forename: Simonetta
authors.initials: S
authors.lastname: Guarrera
authors.forename: Valentina
authors.initials: V
authors.lastname: Turinetto
authors.forename: Valentina
authors.initials: V
authors.lastname: Minieri
authors.forename: Ada
authors.initials: A
authors.lastname: Funaro
authors.forename: Riccardo
authors.initials: R
authors.lastname: Crebelli
authors.forename: Claudia
authors.initials: C
authors.lastname: Giachino
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Matullo
mesh_terms: D002460:Cell Line; D002461:Cell Line, Transformed / Q000528:radiation effects; D002472:Cell Transformation, Viral; D002869:Chromosome Aberrations; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase; D056726:Genetic Association Studies; D006657:Histones / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000528:radiation effects*; D008214:Lymphocytes / Q000528:radiation effects; D010766:Phosphorylation
chemical_list: C468783:H2AX protein, human; D006657:Histones