The comet assay (single-cell electrophoresis) is a comparably new method for measurement of DNA strand breakage, primarily in eukaryotic cells. Its main advantage is that it works on the single-cell level, making it possible to detect heterogeneity in DNA damage induction and repair. Here, it has been used in conjunction with other strand break methods and its relationship to them has been investigated.
The direct-acting alkylating agent ethylene oxide was examined for induction and repair of DNA single- (ssb) and double-strand (dsb) breaks. It was concluded that the dsb that were observed were at least partly formed during the processing of the samples, and that they consisted of unidentified heat-labile sites. The comet assay was used to exclude the possibility of heterogeneous repair, and it also showed induction of alkali-labile sites.
In a methodological study of the comet assay, differences in the fraction of DNA that migrates during alkaline and neutral pH versions of the assay were shown, and that it consisted of fragments in the former and relaxed looped domains in the latter. Building upon this experience, another study was conducted where the neutral version of the comet assay was compared to the anomalous viscosity time dependence method, and it was concluded that both methods respond not only to strand breaks, but also to changes in chromatin conformation.
The dependence of DNA dsb repair on the direct and indirect effects was studied after irradiation with gamma rays and accelerated ions in the LET range of 40 to 125 keV/mm. The relative contributions of the direct and indirect effects were modified through addition of 2 M DMSO to the cells during irradiation. It was found that this abolished the fast phase after low-LET irradiation, but to a lesser degree after higher LET:s. A model is presented that explains the results in terms of chromatin of different compactness.
The structures observed in a neutral version of single-cell electrophoresis were used, together with a pulse-labelling protocol, to study the attachment of replicating DNA to the nuclear matrix, during and after the S-phase. Changes after the end of the S-phase were detected, that cannot have been dependent on retardation of replication forks. Also, the attachments in early-replicating chromatin were dependent on the metabolic status of the cells.
Three lung cancer cell lines of different radiosensitivity, which also had different grades of spontaneous apoptosis, were investigated with respect to ssb repair. The fraction of cells that did not display apoptotic fragmentation displayed slow ssb repair in a fraction that correlated to the degree of apoptosis. It is hypothesised that this fraction consists of cells in an early stage of apoptosis.
Stockholm: Stockholm University, 2000. , 52 p.