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.

FIG. 1

Radiosensitivity of the radiosensitive LCL panel. Radiosensitivity was based on the colony survival assay using previously described ranges (5). Fourteen of the radiosensitive LCLs scored within a radiosensitive range (survival <21%); five scored in an ‘intermediate’ range (21–36%). In all experiments, each radiosensitive LCL was tested with an AT and wild-type LCL internal control. Error bar for each radiosensitive LCL represents the mean ± SD of three experiments. *SE were determined in a previous study (5) using 104 AT and 29 wild-type LCLs.

FIG. 2

Neutral comet assay measuring the effects of DNA damage 5 h after 15 Gy. Note that all but two radiosensitive LCLs (RS07, RS15) had “AT-like” reduced DNA repair efficiency. Error bars for all LCLs tested represent means ± SD of three experiments, normalized to the average tail moments of five different wild-type LCLs, WT1 to 5 (first bar). Inset 1: Representative data from WT1 and AT3 LCLs demonstrates the kinetics of tail moment 30 min and 5 h postirradiation. Tail moment on the y-axis is the measurement of pixel fluorescence in the form of arbitrary units. The tail moment values of the nonirradiated cells and cells 5 h postirradiation were used to calculate the average percentage return to baseline (%RTB) represented in the bar graph. Inset 2: Representative photomicrographs of comet tails (WT1 and RS18) at 5 h after damage.

FIG. 3

Radiation-induced γ-H2AX focus formation. Panel A: Cells were harvested 24 h after irradiation, stained for γ-H2AX detection (green), counterstained with DAPI (blue), and scored for nuclei with >4 foci. One hundred nuclei were counted per sample (31). WT1 and 5 and AT3 and 4 cells were used as controls. Panel B: Normalization of γ-H2AX focus formation 24 h postirradiation to nonirradiated (spontaneous) focus formation in panel A. Error bars represent means ± SD. N = 3. Inset: representative data for RS50.

FIG. 4

Kinetics of SMC1 phosphorylation after 10 Gy. Normal repair (Pattern I): pSMC1-s966 phosphorylation peaks at 1 h postirradiation; minimal response (pattern II): absence of pSMC1-s966; intermediate response (pattern III): pSMC1-s966 peaks at 4 h postirradiation; late response (pattern IV): pSMC1-s966 peaks at 24 h postirradiation. Native SMC1 was used as a loading control. All experiments were performed three times using WT3 and AT4 controls in each immunoblot. Inset: Qualitative interpretive summary of responses (see text for details).

FIG. 5

Intra-S-phase cell cycle checkpoint. RDS was performed using cells that were labeled with [¹⁴C]dThd for 24 h, subsequently exposed to radiation, and then chased with [³H]dThd for 1 h. Cells were fixed and the [³H]dThd/[¹⁴C]dThd ratio was calculated by scintillation (23). Each experiment was performed in triplicate. Error bars represent means ± SD of three experiments. *Represents the radiosensitive LCLs with an AT-like RDS response.

FIG. 6

Mitochondrial respiratory function. Resazurin reduction was impaired in 13 of 19 radiosensitive LCLs. Panel A: Thirteen radiosensitive LCLs exhibited a reduced (R) AT-like rate of resazurin reduction. Panel B: Six radiosensitive LCLs exhibited a wild-type (WT)-like rate. Cells were incubated with resazurin (3 μM) in 96-well microtiter plates under constant growth conditions in replicates of six for each condition (32). All LCLs were tested in three independent experiments. Wells containing only resazurin and medium served as experimental controls. Error bars represent means ± SD. N = 3. WT4 and AT1 LCLs were used as further experimental controls. Radiosensitive LCLs exhibited either an AT-like reduced (R) or WT-like (W) response.