Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding.
Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X-rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non-cycling cell fraction; and a distinctive, non-cycling G-/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst 33258-ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non-cycling cell fraction in the control culture to 29% (X-rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3-aminobenzamide, this aberrant cell population increased significantly after X-ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non-cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non-cycling and cycling cell fractions, this subpopulation with non-stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of non-cycling cells represents damaged cells with altered dye binding properties.
Department of Human Genetics, University of Wuerzburg, Federal Republic of Germany.
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M
Kubbies
D000328:Adult; D001549:Benzamides / Q000494:pharmacology*; D001562:Benzimidazoles; D006690:Bisbenzimidazole; D001761:Bleomycin / Q000494:pharmacology*; D002478:Cells, Cultured; D002843:Chromatin / Q000032:analysis / Q000187:drug effects*; D004249:DNA Damage; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects*; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology*
D001549:Benzamides; D001562:Benzimidazoles; D002843:Chromatin; D005456:Fluorescent Dyes; D008937:Mitomycins; D001761:Bleomycin; D016685:Mitomycin; C025160:3-aminobenzamide; D006690:Bisbenzimidazole