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Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay.
MedLine Citation:
PMID:  22870978     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.
Authors:
Peggy L Olive; Judit P Banáth; Ralph E Durand
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Radiation research     Volume:  178     ISSN:  1938-5404     ISO Abbreviation:  Radiat. Res.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-08-08     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0401245     Medline TA:  Radiat Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  AV35-42     Citation Subset:  IM; S    
Affiliation:
British Columbia Cancer Research Centre, 601 W. 10th Ave., Vancouver, B.C., Canada V5Z 1L3.
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