title: A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes.
abstract: The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.
authors.affiliation: Health Science Research Institute Germans Trias i Pujol, Badalona, Barcelona, Spain. pmendez00@gmail.com
authors.forename: Pedro
authors.initials: P
authors.lastname: Mendez
authors.forename: Miquel
authors.initials: M
authors.lastname: Taron
authors.forename: Teresa
authors.initials: T
authors.lastname: Moran
authors.forename: Marco A
authors.initials: MA
authors.lastname: Fernandez
authors.forename: Gerard
authors.initials: G
authors.lastname: Requena
authors.forename: Rafael
authors.initials: R
authors.lastname: Rosell
mesh_terms: D002478:Cells, Cultured; D015925:Cryopreservation; D004260:DNA Repair; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D015203:Reproducibility of Results