Comet-FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage.
For the optimal use of anticancer drugs a knowledge of the whole spectrum of side-effects is required. A potential hazard, so far only scarcely investigated, is uncontrolled effects of drugs such as bleomycin (BLM) and mitomycin C (MMC) on telomere shortening in non-cancerous tissues of the treated person. For the first time, directly labelled telomere-specific peptide nucleic acid (PNA) hybridization probes were applied in comet-FISH to detect DNA fragmentation on an intermediate scale. The effects of BLM and MMC were measured in peripheral blood cells of three human volunteers, following ex vivo incubation. Fragmentation of telomeres and subtelomeric regions was highly specifically detected by the comet-FISH assay, a combination of the comet assay and fluorescence in situ hybridization. As a technical detail, the effects of the hybridization procedure have been studied on the level of single comets. Image analysis before and after the hybridization process reveals a small decrease in the detected fragmented DNA, probably due to diffusion of small fragments. It could not only be shown that both drugs actually induce breaks in telomere-associated DNA, but also that the comet-FISH technique, as a quantitative approach, is a useful tool for the detection and evaluation of the role of sequence-specific DNA damage after mutagenic action. The breakage frequency for DNA of or adjacent to telomeric repeats was found to be proportional to that of the total DNA, which hints at random induction of DNA breaks by BLM and MMC. In terms of therapy, the results indicate that no over- or under-proportional effects on telomeres of BLM or MMC need be expected.
Arutyunyan
Rouben
R
Institute of Human Genetics, Schwabachanlage 10, D-91054 Erlangen, Germany.
Gebhart
Erich
E
Hovhannisyan
Galina
G
Greulich
Karl Otto
KO
Rapp
Alexander
A
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D002467:Cell Nucleus / Q000648:ultrastructure; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000648:ultrastructure; D004249:DNA Damage; D053938:DNA Fragmentation; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016685:Mitomycin / Q000494:pharmacology*; D009153:Mutagens; D009693:Nucleic Acid Hybridization; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D020135:Peptide Nucleic Acids; D016615:Telomere / Q000648:ultrastructure; D013997:Time Factors
D000903:Antibiotics, Antineoplastic; D009153:Mutagens; D019384:Nucleic Acid Synthesis Inhibitors; D020135:Peptide Nucleic Acids; D001761:Bleomycin; D016685:Mitomycin; D004247:DNA