A comparison of cellular DNA repair rates of different cell lines using the TUNEL assay

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Cells possess several DNA repair systems to counteract the damage produced by endogenous and exogenous agents. Repair of DNA damage is significant in preventing genetic modifications involved in the progression of carcinogenesis. Inefficient repair systems facilitate an increased risk of developing cancer, supporting the prospective study of DNA repair in relation to the onset of cancer. The aim of this project was to develop a simple and accurate method of detecting DNA damage and measuring repair rate. Identification of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is normally applied to investigate active cell death (apoptosis). Using this staining technique, we developed a method of measuring cellular DNA repair rates. We selected H₂0₂ as our primary damaging agent because of its reported ability to induce a large number of single-strand breaks (ssb>36000/cell) without impairing cell survival. (Int J Radiat Biol. 2000 Jan: 67-75). After damaging various lymphocyte cancer cell lines with H₂0₂, cells were allowed to repair in media at 37°C for 0, 5, 15, 30, or 60 minutes. Following the different repair incubation time points, the cells were fixed with 4% paraformaldahyde and then stained using TUNEL, followed by flow cytometry. The TUNEL repair assay was conducted on different mononuclear leukocyte cell lines, which revealed specific trends in DNA repair rates among the various cell lines. The Raji cell line with no repair time (0 minutes) had a damage population at 12.8 percent. As the time of repair increased, the damage population continued to decrease, reaching a percentage of 11.4 at a 15 minute repair time, and 8.4 at a 60 minute repair time. By analyzing the equation of the graphed line, the slope, or repair rate, was determined to be -.07 for Raji cells. The U937 cell line had a damage population of 4.7 percent at 0 minutes, 2.3 at 15 minutes, and 1.9 at 60 minutes, resulting in an overall repair rate of -.03 for U937 cells. Our results from the flow cytometer revealed a significant decrease in the amount of DNA damaged as the time for repair increased, indicating that TUNEL is an accurate method of determining and measuring DNA repair. This technique will be useful in further analysis of DNA repair and its contribution to the progression of cancer.