Abstract 3095: The host cell reactivation (HCR) assay for measuring DNA repair capacity (DRC) in cancer research after 20 years; re-evaluation and lessons learned
Authors: Jaime L. Matta, Luisa Morales, Heidi Acosta, Julie Dutil, Alvaro Monteiro, Carolina Alvarez-Garriga, Manuel Bayona
Journal: Cancer Research (2012)

Abstract The HCR assay was developed by Athas et al. 1991 to measure DRC. DNA can be transiently transfected when introduced into a cell. When damaged, its expression will depend on the repair capacity of the host cell. A non-replicating plasmid expression vector (pCMVluc, 4,863 bp) containing a luciferase reporter gene is used and damaged by UVC exposure. It level of expression is a direct measure of the total repair capacity of lymphocytes, primarily of their nucleotide excision repair. The HCR assay has been used in molecular epidemiological studies of cancer (e.g. Wei et al. 1993, Spitz et al. 2001, Matta et al. 2003, Ramos et al. 2004). Qiao et al. 2002 published a thorough discussion of the development and extensive validation of this assay. We present a critical evaluation of this assay based on 12 years of experience conducting large scale skin and breast cancer (BC) population studies. This includes the appraisal of the validity and reliability or reproducibility of the assay, transfection efficiencies using data from a BC case control study and commercial cell lines. When 100 women (i.e., 50 BC cases and 50 controls combined) were compared, a calculation of a Pearson's correlation of 0.983 and 0.980 Spearman's rho correlation coefficient (97% determination coefficient) were both significant (p &amp;lt;0.001). The fitted regression line showed a high predictability with a narrow 95% mean prediction confidence interval. Duplicates from the 50 cases had values of 0.995 and 0.981 for the Pearson's and Spearman's correlation respectively with a 99% determination coefficient (p &amp;lt; 0.001). The fitted regression line showed the same high predictability. Similar results were obtained for the duplicates from the 50 controls (0.975 and 0.969 for the Pearson's and Spearman's correlation respectively (95% determination coefficient and mean prediction confidence interval, p &amp;lt; 0.001). The 90 duplicates from the commercial cell lines were 0.989 and 0.994 for the Pearson's and Spearman's correlation respectively (98% determination coefficient, p &amp;lt; 0.001, 95% mean prediction confidence interval). This assay is highly reliable and has little variation or minimal random error as it was found during an assessment of the reliability or repeatability. Recently the HCR assay has been substantially modified (Mendez et al. 2011) in order to improve some of its limitations. We found stable transfection efficiencies using an assay (Dual-GloR®) based on the combined use of the Firefly and Renilla luciferases as co-reporters. The high coefficient of variation (C.V.∼23%) obtained in over 1,000 assays is due to the inherent multi-step nature of the HCR as well as due to biological variability. Grossman and Wei (1994) concluded that the HCR assay can distinguish the intra-assay variation and inter-assay variation by being able to maintain the ranks of samples measured in triplicate from multiple cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3095. doi:1538-7445.AM2012-3095