title: Induction of a whole chromosome loss by colcemid in human cells elucidated by discrimination between FISH signal overlap and chromosome loss.
abstract: Aneuploidy is a change in the number of chromosomes and an essential component in tumorigenesis. Therefore, accurate and sensitive detection of aneuploidy is important in screening for carcinogens. In vitro micronucleus (MN) assay has been adopted in the recently revised International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S2 guideline and can be employed to predict both clastogenic and aneugenic chromosomal aberrations in interphase cells. However, distinguishing clastogens and aneugens is not possible using this assay. The Organization for Economic Co-operation and Development (OECD) guideline TG487 therefore recommends the use of centromere/kinetochore staining in micronuclei to differentiate clastogens from aneugens. Here, we analyzed numerical changes of a specific chromosome in cytokinesis-blocked binucleated cells by fluorescence in situ hybridization (FISH) using the specific centromere probe in human lymphoblastoid TK6 cells treated with aneugens (colcemid and vincristine) or clastogens (methyl methanesulfonate [MMS] and 4-nitroquinoline-1-oxide [4-NQO]). Colcemid and vincristine significantly increased the frequencies of nondisjunction and loss of FISH signals, while MMS and 4-NQO slightly increased only the frequency of loss of FISH signals. The loss of FISH signals of a specific chromosome from two to one per nucleus implies either a loss of a whole chromosome or an overlap of two signals. To distinguish a chromosome loss from signal overlap, we investigated the number of FISH signals and the fluorescent intensity of each signal per nucleus using a probe specific for whole chromosome 2 in binucleated TK6 cells and primary human lymphocytes treated with colcemid and MMS. By discriminating between chromosome loss and FISH signal overlap, we revealed that colcemid, but not MMS, induced a loss of a whole chromosome in primary lymphocytes and TK6 cells.
authors.affiliation: Drug Safety Research Laboratories, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan; Laboratory of Radiation Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Akihiro
authors.initials: A
authors.lastname: Wakata
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Aoki
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamae
authors.forename: Seiji
authors.initials: S
authors.lastname: Kodama
mesh_terms: D024422:Aneugens / Q000494:pharmacology*; D000782:Aneuploidy; D002478:Cells, Cultured; D002869:Chromosome Aberrations / Q000139:chemically induced; D020090:Chromosome Segregation / Q000187:drug effects; D002889:Chromosomes, Human, Pair 2 / Q000187:drug effects / Q000235:genetics; D002894:Chromosomes, Human, Pair 4 / Q000187:drug effects / Q000235:genetics; D003703:Demecolcine / Q000494:pharmacology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008214:Lymphocytes / Q000187:drug effects; D015162:Micronucleus Tests; D009153:Mutagens / Q000494:pharmacology; D015203:Reproducibility of Results
chemical_list: D024422:Aneugens; D009153:Mutagens; D003703:Demecolcine
keywords: Aneuploidy; Chromosome loss; DNA fragmentation; FISH; Micronucleus; Whole chromosome painting probe