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INVESTIGATION OF DNA DAMAGE AND TELOMERE FRAGILITY
BY COMET-FISH TECHNIQUE IN DIFFERENT CELL LINES
G.G. Hovhannisyan
Department of Genetics and Cytology, Yerevan State University, Yerevan, Armenia
The purpose of present study was the detection of the involvement of telomeric
repeats in DNA damage induced by cytostatic bleomycin (BLM). The investigation was
performed with Comet-FISH, combination of Comet-assay (single cell gel electrophoresis)
with FISH (Fluorescent In Situ Hybridization) technique. This approach permits to detect in
the individual cell simultaneously the total DNA damage and specific DNA sequences. To
visualise telomer damages telomere-specific PNA probes were used. BLM effect was studied
on three cell lines: HT 1080 (derived from a human fibrosarcoma), CCRF-CEM (derived
from a human T-cell acute lymphocytic leukemia) and CHO (Chinese hamster ovary cells).
The number of telomere signals and the localisation of the signals (at comet head or tail) were
recorded. Comet image analysis and Comet-FISH evaluation was performed by the Komet 4
software package (Kinetic imaging, UK. It was shown that the level of DNA migration is
increased in all cell types in dependence on BLM dose. CHO cells were shown to be most
sensitive to the agent. The same cell line demonstrated the highest level of telomeric signals
in the comet tail. The relation between amount of DNA and the number of telomeric signals
outside the nucleus varied between the examined cell types. In CCRF-CEM cells this relation
was equal to 2.62±0.55, in CHO cells - 2.06±0.33, and in HT1080 cells - 0.73±0.28. Nuclei
of intact cells shown mainly produced slightly damaged comets with a few telomeres outside
of the head. Thus, the results suggest applicability of Comet-FISH technique to estimate the
sensitivity in malignant cells to chemoterapeutics and to reveal the involvement of specific
genomic regions in the genetic damage. This study was supported by ISTC grant A-301.2.