Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

Abstract

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

“…It was also observed that the neutral comet assay provided a stronger correlation with clonogenicity at higher doses (4 Gy and 6 Gy) than at 2 Gy, which may be attributed to a lower occurrence of strand breaks at 2 Gy. For all the cell lines, the DNA repair kinetics, as measured using either neutral or alkaline comet assay, failed to strongly correlate with the radiosensitivity, which is in agreement with some of the earlier reports [24] but not with other studies [12]. Though the residual DNA damage, as measured by the neutral comet assay, is able to segregate the most radioresistant and radiosensitive cell lines, it is not able to grade all the cell types according to their radiosensitivity.…”

“…Attempts have been made in the past to correlate DNA damage with the clonogenic survival of the tumor cells using either an alkaline or a neutral comet assay. A good correlation between DNA damage, as measured by the comet assay and clonogenic survival has been observed in some studies [10][11][12]. However, this correlation was not observed in other studies [13,14], suggesting for more number of studies in this direction for the potential application of the comet assay in determining the radiosensitivity of tumor cells.…”

Dubowitz Syndrome Is a Complex Comprised of Multiple, Genetically Distinct and Phenotypically Overlapping Disorders

“…The neutral comet assay measures the level of unrepaired DNA in a cell [14]–[16]. Recent studies have shown a correlation between double-strand DNA post-irradiation damage, as measured by NCA, and the colony survival assay [14].…”

“…Recent studies have shown a correlation between double-strand DNA post-irradiation damage, as measured by NCA, and the colony survival assay [14]. Longer, undamaged DNA fragments will remain predominantly in the “head” of the comet, while smaller, heavily damaged DNA fragments will migrate more quickly and form the tail of the comet.…”

“…The NCA assay was performed as previously described [14]. Briefly, LCLs from patients, ATM -deficient and wild type individuals were exposed to 15 Gy of ionizing radiation using a Mark IV series (Co-137) irradiator at a dose rate of 4.21 Gy/min.…”