Direct use of the comet assay to study cell cycle distribution and its application to study cell cycle-dependent DNA damage formation

Marcin Kruszewski 1 2 3 Teresa Iwanen ko 2 3 Eugeniusz K. Machaj 0 2 6 Tomasz Odak 2 5 Maria Wojewo dzka 2 3 Lucyna Kapka-Skrzypczak 1 2 4 Zygmunt Pojda 0 2 6 0 Department of Cellular Engineering, Maria Skodowska-Curie Memorial Cancer Center and Institute of Oncology , 5 W.K. Roentgena Street, 02-781, Warszawa, Poland 1 Indepenent Laboratory of Molecular Biology, Institute of Rural Health , 2 Jaczewskiego Street, 20-090, Lublin, Poland 2 Biological Dosimetry, Institute of Nuclear Chemistry and Technology , 16 Dorodna Street, 03-195, Warszawa, Poland . Tel: 3 Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology , 16 Dorodna Street, 03-195, Warszawa, Poland 4 Department of Public Health, University of Information Technology and Management in Rzeszow , 2 Sucharskiego Street, 35-225, Rzeszow, Poland 5 Polish Stem Cell Bank , 2/41 Grzybowska Street, 00-131, Warszawa, Poland 6 Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology , 4 Kozielska Street, 01-163, Warszawa, Poland The comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring the induction and repair of DNA damage in individual cells. However, one of the drawbacks of the assay is the bias caused by changes in the ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. In this paper, we validate the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle and can be used to assign single cells to specific cell cycle phases. To validate the approach, we used a very homogenous blood mononuclear CD341 cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. An analysis of the cell cycle distribution revealed that the 15 comet intensity classes and the 100 comets usually analyzed in a typical comet experiment are sufficient to obtain a reliable cell cycle distribution comparable with the results obtained by the flow cytometry for the same cell population. The effect of the cell cycle position on the results obtained by the comet assay for proliferating and non-proliferating cell populations irradiated with 3 Gy of X-radiation is also discussed. Introduction Since its development, the comet assay or single cell gel electrophoresis has proven to be a versatile and sensitive method of measuring DNA damage in individual cells. Initially dedicated to the detection of DNA double-strand breaks (DSBs), over the last 2 decades the assay has been modified at various stages to allow for the assessment of various kinds of damage including double- and single-strand breaks, base damage, DNA-protein cross-links, thymine dimers, 6-4 photoproducts and bulky adducts by use of lesion-specific antibodies or repair enzymes (for the recent reviews, see (15)). Today the assay is a well-established, extensively used, simple, money and time effective tool to assess DNA damage and repair quantitatively and qualitatively in individual cell populations. The assay has been applied in many fields of fundamental and applied biology and medicine, such as in DNA damage and repair studies, genotoxicity testing, molecular epidemiology and human and environmental biomonitoring. One potential drawback of the comet assay is the bias caused by changes in ability of cells to repair DNA damage in different cell cycle phases. Whereas the bias seems less important when G0 peripheral blood lymphocytes are studied, it might cause problems when proliferating cells are investigated. The cell cycle-dependent repair of DNA DSB via homologous recombination or non-homologous end joining is a well-established phenomenon based on the availability of the homologous chromatid during DNA repair (69); however, it has recently been shown that other types of DNA damage, such as cis-platininduced interstrand cross-links (10,11) or H2O2-induced oxidative DNA damage (12), might also be repaired by alternative DNA repair pathways during different cell cycle phases. The other problem occurs if two sets of cells with different population distribution are given two different treatments, and difference between comet results is falsely attributed to the difference between treatments, but in fact results from the difference in population distribution. McArt et al. (13) suggested also that comet assay results may be biased, when measuring low amounts of DNA breaks, by the differences in initial level of DNA damage between cells in different cell cycle phase. The authors questioned the use of asynchronous cells in studies where low amounts of strand breaks are being observed, particularly if analyzed without unbiased sampling techniques. The first attempts to use the comet assay to assess the cell cycle-dependent DNA damage were made by Olive and Banath (14,15). The authors using an elutriator or the FACS cell sorter to fractionate cells according to the cell cycle phases and measured the induction and repair of DNA damage. However, such an approach is laborious and requires special instrumentation. We (16,17) and others (18,19) proposed that comet results could be directly used to assess cell cycle-dependent DNA damage and its repair. This approach is based on the assumption that the total comet fluorescence intensity corresponds to the position of the cell in the cell cycle (an assumption similar to that underlying flow cytometric analysis) and that these data can be used to assign single cells to specific cell cycle phases. In this paper, we validate the approach by using a very homogenous blood mononuclear cell population in G0 phase (unstimulated) or stimulated to enter the cell cycle. The effect of the cell cycle position on the results obtained by the comet assay in proliferating and non-proliferating cell is discussed. Materials and methods All chemicals used were from SigmaAldrich (USA), unless otherwise indicated. Cell isolation, stimulation and irradiation CD34 cells were isolated from cord blood samples, collected from full-term normal deliveries and were diluted 1:1 with phosphate-buffered saline (PBS) (Gibco, USA). Subsequently, mononuclear cells were isolated by centrifugation on Ficoll; 1.077 g/ml at 400 g for 40 min. The mononuclear cells were collected, washed twice in IMDM (Gibco) supplemented with 10 % fetal calf serum (Cytogen, USA) and resuspended in PBS with the addition of 0.5 % human serum albumin (Biomed, Poland). The CD34 fraction was isolated immunomagnetically using MS MiniMACS columns and the CD34 Direct Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer recommendations. In brief, after adding FcR Blocking Reagent, cells were labeled with MACS CD34 Microbeads for 30 min at 612 C. Subsequently, the labeled cells were enriched by passing the cell suspension throu (...truncated)