INVOLVEMENT OF CHROMOSOMES 7, 18 AND X IN MITOMYCIN C-INDUCED MICRONUCLEI
Hovhannisyan GG1,2*, Mkrtchyan H1,2, Liehr T2, Aroutiounian R1
*Corresponding Author: Dr. Galina G. Hovhannisyan, Department of Genetics and Cytology, State University, Biological Faculty, 1 Alex Manoukian Street, Yerevan 375025, Armenia; Tel.: +374-10552354; Fax: +374-10554641; e-mail: hovgalina@list.ru
page: 45

MATERIALS AND METHODS

  Cytogenetics. Cell suspensions of peripheral blood lymphocytes of one healthy human female and of one healthy male were used. Standard protocols using mito­mycin C (MMC) for MN-induction were applied [16,17]. In short, heparinized whole blood was added to RPMI 1640 medium (1:10) containing 10% fetal bovine serum, 1% penicillin/streptomycin and 10 mg/mL phytohemag­glutinin. After 22 h of cultivation, MMC was added at a final concentration 0.1 mg/mL. Forty-four hours later, the cultures were supplemented with cytochalasin B (3 mg/ mL) to achieve cytokinesis block. Total incubation time was 72 h at 37C. Hypotonic treatment was performed for 3 min in cold 0.075 M KCl at +4C. This procedure preserves the cytoplasm, which is required for recognition of cell borders so that MN can be assigned to their corresponding main nucleus. Fixation was done twice in methanol/acetic acid (3:1).           

   Molecular Cytogenetics. Fluorescence in situ hybridization was performed according to standard procedures [18]. One thousand and two hundrend and two female MN were hybridized and evaluated by a three-color-FISH probe set consisting of centromeric probes (cep) for chromosomes 7 (SpectrumRed; Abbott/Vysis, Abbott GmbH & Co. KG, Wiesbaden, Germany), 18 (SpectrumGreen; Abbott/Vysis) and X (SpectrumOrange; Abbott/Vysis). The positions of MN on the slides were recorded for their further analysis by whole chromosome probes (wcp). In a second round of hybridization, the nuclei/MN were hybridized with the wcp for 7 (SpectrumGreen), 18 (Diethyl­aminocoumarine) and X (SpectrumOrange); the three probes were prepared as in [19]. Only 916 of the 1202 MN were evaluated with the wcp probes. The remaining cells could not be relocated due to cell/MN loss on the slides during the second hybridization, since repeated treatment with pepsin and high temperature induces slight destruction of cell material. Nine hundred and sixteen male MN were evaluated only with the wcp X probe.




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