M-phase specific centrosome-microtubule alterations induced by the fungicide MBC in human granulosa cells

Abstract

The mitostatic action of the commonly used fungicide methyl 2-benzimidazolecarbamate (MBC) was evaluated in primary cultures of human ovarian granulosa cells with respect to the organization and stability of spindle microtubules and mitotic centrosomes. MBC caused metaphase arrest and abnormal chromosome organization following a 3-15 h treatment at a concentration of 30 microM. While microtubules were retained in MBC-treated cells, alterations in spindle shape and microtubule composition were noted. Exposure to MBC resulted in an increased number of spindle poles associated with chromosomes displaced from the metaphase plate. A gradual increase from tri- to multipolar spindles was noted with prolonged treatment although a relatively constant fraction (50%) of bipolar spindles was maintained. In non-dividing cells, MBC had no effect on microtubule organization. Analysis of mitotic figures by immunofluorescence microscopy showed a reduction in interpolar and astral microtubules in response to MBC treatment while acetylated kinetochore microtubules were retained and their plus-ends were attached to metaphase chromosomes. In multipolar spindles, analysis of microtubule organizing centers (MTOCs) with antisera to stable centrosomal markers (SPJ and 5051) revealed that only poles associated with displaced chromosomes retained these markers. In contrast, transient centrosome markers (NuMA and centrophilin) were localized to all poles of multipolar spindles. Since MBC alters centrosome organization during mitosis, the results suggest that one mechanism of action of this agent is impairment of spindle microtubule dynamics at the centrosome.