Sequence of centromere separation: kinetochore formation in induced laggards and micronuclei.

Mouse L-cells were treated with bis-benzimidazole derivative (Hoechst 33258), caffeine and bleomycin in order to study genesis of laggards and micronuclei and formation of kinetochores as revealed by antikinetochore antibody staining. Apparently, the Hoechst 33258-induced decondensation experienced by the A:T-rich pericentric heterochromatin does not extend into the centromeric region and does not affect formation, physical appearance or function of kinetochores. The laggards induced by Hoechst 33258 are generally whole chromosome laggards which have antikinetochore antibody binding sites. These kinetochore-carrying laggards were seen to lie outside the spindle region in cells untreated with spindle inhibitors or hypotonic and stained differentially for spindle and chromosomes. Some micronuclei did not show kinetochore dots indicating their origin in acentric chromosome fragments. When cells were treated with caffeine or bleomycin, both types of micronuclei, namely those carrying kinetochores and those generated by acentric fragments, were seen. These results are interesting in that caffeine prevents the rejoining of chromosome breaks and one would expect only kinetochore-less micronuclei in caffeine-treated cells. It may mean that caffeine also induces aneuploidy. The L-cells carry minichromosomes which are no more than a pair of kinetochore dots. Such chromosomes, though detectable by antikinetochore antibody staining, may be missed in routine, acid-fixed, Giemsa-stained preparations.