Organization of mitotic apparatus poles in etoposide-treated CHO-K1 cells

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Analyzed in this study is the organization of mitotic spindle poles in CHO-K1 cells dividing after treatment with etoposide (1 h, 25 μM). At various periods after the treatment, we studied the following: (1) the distribution of γ-tubulin in mitotic cells by immunofluorescent staining, (2) the level of post-translational modification of α-tubulin in spindle microtubules by immunoelectron microscopy, and (3) the ultrastructure of mitotic apparatus poles by standard electron microscopy. 48 h after the addition of etoposide, disturbances in the ultrastructure of mitotic spindle poles were observed in etoposide-treated CHO-K1 cells with both bipolar and with multipolar mitotic apparatuses. The increased number of centrioles was unevenly distributed between the mitotic spindle poles; some centrioles did not take an obvious part in the mitotic spindle organization and differed in their number of outgrowing microtubules. Most centrioles were without fibrillar halos. Immunoelectron microscopy showed the differences in the staining of the poles of a multipolar spindle within one cell with antibodies to tyrosinated α-tubulin, whereas the staining of cells with antibodies to acetylated α-tubulin did not reveal such differences. Immunofluorescence staining for γ-tubulin also indicated differing organizations of poles in the same spindle. Our data findings provided the first evidence that the pattern of immunostaining and ultrastructure of mitotic apparatus poles can differ in cells dividing at various time periods after the action of etoposide.

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