Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.
The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.
Hovhannisyan
G
G
Institute of Human Genetics, University of Erlangen-Nuremberg, D-91054 Erlangen, Germany.
Rapp
A
A
Arutyunyan
R
R
Greulich
K O
KO
Gebhart
E
E
D000818:Animals; D001761:Bleomycin / Q000494:pharmacology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D020552:Comet Assay; D006224:Cricetinae; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016685:Mitomycin / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000032:analysis*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D009153:Mutagens; D020135:Peptide Nucleic Acids; D001761:Bleomycin; D016685:Mitomycin; D004247:DNA