Comet–FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage

Mutagenesis vol. 19 no. 5 pp. 403--408, 2004 doi:10.1093/mutage/geh049 Comet--FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage Rouben Arutyunyan1,2, Erich Gebhart1, Galina Hovhannisyan2, Karl Otto Greulich3 and Alexander Rapp3,4 1 Institute of Human Genetics, Schwabachanlage 10, D-91054 Erlangen, Germany, 2Department of Genetics and Cytology, Yerevan State University, Yerevan 375049, Armenia and 3Institute for Molecular Biotechnology, Department for Single Cell and Single Molecule Techniques, Beutenbergstrasse 11, D-07745 Jena, Germany 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. Introduction By their specific repeat structure and their ability to form Tloops (Griffith et al., 1999), telomeres not only build up and stabilize the ends of chromosomes (Day et al., 1993; Knight and Flint, 2000) but, under normal conditions, also protect chromosomes from natural damage. Telomere erosion or loss, on the other hand, destabilizes the human genome and is an early event in DNA damage-induced apoptosis (Ramirez et al., 2003). If chemical mutagens, in particular cytostatics, were able to erode telomeres, this would interfere with the natural processes of cellular senescence and/or malignant 4 transformation. Since it is known that the sub-telomeric chromosomal regions contain genes in high density, a knowledge of the susceptibility of these chromosomal regions to breakage is of general interest. First reports on the action of ionizing radiation indicated telomeres as points of mutagenic attack (Slijepcevic et al., 1998; Boei et al., 2000). In Chinese hamster cell lines (CHO and CHE) radiomimetic drugs have been shown to induce telomeric damage involved in chromosome breakage and recombination (Bolzan et al., 2001). Telomere shortening has recently been reported in patients undergoing chemotherapy (Schroder et al., 2001; Lee et al., 2003). Telomere breakage sensitivity is also interesting, since DNA structure is known to influence DNA damage and repair mechanisms. These have mainly been studied up to now following radiation-induced damage. In addition, telomeric repeats in damaged DNA can also be considered as signalling breaks in the adjacent generich subtelomeric DNA. One way to measure sequence specific DNA fragmentation on an intermediate scale (10--800 kb) is the comet--FISH technique. This assay is a combination of the comet assay with fluorescence in situ hybridization. Hybridization is performed on electrophoretically separated DNA of a single cell, embedded in agarose. Up to now several hybridization procedures have been described, including those of DNA whole chromosome painting probes (Rapp et al., 1999, 2000), gene-specific probes (McKelvey-Martin et al., 1998; Schaeferhenrich et al., 2003) and centromere probes. Due to the limited hybridization efficiency on agarose embedded cells detection has up to now been dependent on signal enhancing steps, such as antibody cascades or enzyme-enhanced reactions. The application of peptide nucleic acid (PNA) probes has so far only been discussed theoretically as a possible way to overcome this limitation. The use of telomere-specific PNA probes now allows a highly sensitive and reliable detection of telomeric DNA. Therefore, it was considered to also be suited to the detection of damaged DNA closely associated with telomere repeats in ‘comets’. A further question that has not been addressed up to now is whether the hybridization procedure alters the results of overall DNA damage. Since this technique contains several washing steps and high temperature stringency washes it is possible that small fragments are lost from the comet tail and therefore total DNA damage is underestimated. As mutagens we used two classic cytostatics: the radiomimetic bleomycin (BLM) and the recombinogen mitomycin C (MMC). The antibiotic bleomycin is an S phase-independent radiomimetic antitumoral agent with unique genotoxic properties (Povirk, 1996). The drug is a free radical-based DNAdamaging agent which induces a mixture of strand breaks and abasic sites by highly specific, concerted free radical attack on deoxyribose moieties in both DNA strands. Anderson To whom correspondence should be addressed. Tel: 149 3641 656401; Fax: 149 3641 656410; Email: Mutagenesis vol. 19 no. 5 ß UK Environmental Mutagen Society 2004; all rights reserved. 403 R.Arutyunyan et al. et al. (1997) mentioned that BLM is known to react through oxygen radical mechanisms. A major determinant of BLMinduced damage is the permeability of the cell membrane and the presence of BLM hydrolase, which inactivates BLM. On the other hand, MMC was selected because it mainly induces DNA--DNA interstrand crosslinks (Yang and Wang, 1999) and has been used in previous comet assay as well as comet--FISH studies. Although comet assay results for MMC-treated cells are difficult to interpret, since DNA fragmentation is overlaid by DNA crosslinking effects, we have included this drug to compare the telomere comet--FISH data with recently published data on single copy genes (McKenna et al., 2003). It was shown that a concentration 100--800 mM MMC added to human whole blood led to an increase in DNA migration in the comet assay (Pfuhler and Wolf, 1996). As found by McKenna et al. (2003) with comet-- (...truncated)