Leukaemia-specific chromosome damage detected by comet with fluorescence in situ hybridization (comet-FISH)

Abstract

Acute myeloid leukaemia (AML) is associated with exposure to benzene and treatment with chemotherapeutic agents. It is thought to arise from damage to specific regions of DNA, resulting in chromosome rearrangements or loss. For instance, a deletion on the long arm of chromosome 5 [e.g. del(5q31)] is common in AML patients previously treated with alkylating agents, such as melphalan, or exposed to benzene. Translocations of the MLL gene at 11q23 are frequently observed in AML arising from treatment with topoisomerase II inhibitors, such as etoposide. Our goal was to determine whether or not breakage at 5q31 and 11q23 is selectively induced by these chemical agents. To address this question, the comet assay combined with fluorescence in situ hybridization (comet-FISH) was used to detect DNA breakage in the specific chromosomal regions in an in vitro model. TK6 lymphoblastoid cells were exposed to melphalan, etoposide or the benzene metabolite, hydroquinone (HQ), at various concentrations. HQ, melphalan and etoposide induced DNA breaks at both 5q31 and 11q23 chromosome regions in a dose-dependant manner. However, HQ produced significantly more DNA damage at 5q31 than at 11q23. Etoposide produce slightly more DNA damage at 11q23 and melphalan had a somewhat greater effect at 5q31, but not significantly so. Thus, HQ and melphalan act similarly, perhaps explaining some similarities between benzene- and alkylating agent-induced AML. Comet-FISH also appears to be a useful approach for detecting and comparing damage to specific chromosome regions of significance in leukaemogenesis.

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells

“…Furthermore, etoposide is also known to cause secondary malignancies including therapy-related AML (Felix, 2001). Etoposide-induced DNA damage leads to chromosomal breakages and translocations involving both 5q31 and 11q23, which are specific chromosome regions of significance in leukemogenesis (Escobar et al, 2007;Felix et al, 2006). Therefore, mangiferin may present chemopreventive activity against therapy-related AML, which needs further study.…”

A Retrospective Study on Second Primary Malignancies Incidence in Regional Cancer Institute

“…In addition, the treatment used for the first malignancy has resulted in damage to specific regions of DNA with chromosome rearrangement or loss, responsible for tumorigenesis. (16) The new technologies available could analyze various genetic changes such as punctiform mutations, loss of heterozygosity or genetic instability. Microsatellite instability (MSI) has been noticed that occur more frequently in cases of multiple primary malignancies than in sporadic cancers.…”

New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair

“…Examples of Comet-FISH applied to DNA damage and repair include the use of a probe for human chromosome 1 in doxorubicin-treated human lymphocytes [36], various heterochromatic and euchromatic chromosomal domain probes in the plant Vicia faba treated with endonucleases [37], whole chromosome probes in UVA-irradiated human lymphocytes [38], whole chromosome paints for measurement of DNA breaks in human oropharyngeal mucosa cells exposed to benzo[a]pyrene-diol epoxide [39], peptide nucleic acid (PNA) probes to detect telomeric repeats in peripheral blood cells treated with bleomycin or mitomycin C [40], the HER-2 and p53 loci in breast cancer cells [41], telomeres in human leukocytes treated with bleomycin and cisplatin [42], the Ret, c-Abl and Trp53 loci in peripheral blood cells to study gene fragmentation resulting from X-ray irradiation of mice [43], the 5q31 and 11q23 loci, susceptible to breakage in acute myeloid leukemia [44], and the APC, KRAS and p53 regions in human colon cells treated with agents thought to be relevant to the etiology of colon cancer [45]. Some probes used for FISH in the examples cited above were purchased from commercial sources as DNA fragments [36,39,41,44] or PNA [40,42] pre-labeled with fluorescent dyes to allow direct detection, or with digoxygenin, which requires a second step with enzyme-coupled anti-digoxygenin [38]. In some cases the probes were made in-house by PCR, using digoxygenin-11-dUTP and amplification with 3 rounds of antibodies [37], or labeling PCR fragments with biotin by nick-translation and detection with FITC-avidin [43].…”