Kinetochore immunofluorescence in micronuclei: a rapid method for the in situ detection of aneuploidy and chromosome breakage in human fibroblasts.
We have developed a rapid and simple immunodetection assay for the in situ identification of aneuploidy in mitotic fibroblasts. Kinetochore (centromere)-containing micronuclei can be detected easily and rapidly by immunofluorescence. The action of colchicine and its derivatives on the mitotic spindle apparatus of mammalian cells induces chromosome lag and aneuploidy. The treatment of normal human fibroblasts with Colcemid resulted in increased levels of micronuclei. Using an immunofluorescence stain (scleroderma CREST antiserum, biotinylated goat antihuman IgG and streptavidin-Texas Red) to detect the presence of kinetochores, it was observed that 90% of the Colcemid-induced micronuclei contained one or more fluorescent bodies (kinetochores). Cultured skin fibroblasts from a patient with ataxia telangiectasia (AT), which is a chromosome breakage syndrome, were used as a control. The AT fibroblasts exhibited elevated levels of spontaneous micronuclei when compared with normal fibroblasts, and 85% of these micronuclei were kinetochore-negative. This finding supports the hypothesis that the majority of spontaneous micronuclei in AT cells arise from chromosome breakage. The spontaneous micronucleus frequencies for 8 strains of human fibroblasts were in the order of 0.5-2%. Spontaneous levels of kinetochore-positive micronuclei were measured for these 8 strains; in 5 of the strains, about 25% of the micronuclei were kinetochore-positive, and in the other 3 strains approximately 50% of the micronuclei were kinetochore-positive. These data suggest that genetic factors may play a role in the control of the spontaneous levels of chromosome breakage and/or segregation errors which result in aneuploidy.
Hennig
U G
UG
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Rudd
N L
NL
Hoar
D I
DI
D000782:Aneuploidy; D001323:Autoantibodies / Q000276:immunology; D002478:Cells, Cultured; D002503:Centromere / Q000648:ultrastructure*; D002875:Chromosomes / Q000648:ultrastructure*; D003703:Demecolcine / Q000494:pharmacology; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006801:Humans; D015162:Micronucleus Tests; D008940:Mitotic Index; D008941:Spindle Apparatus / Q000648:ultrastructure*
D001323:Autoantibodies; D003703:Demecolcine