Abnormal Organization of Mitotic Spindles in Cultured Mammalian Cells after X-Irradiation as Visualized with Anti-Tubulin Antibody

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Abstract
Rounded mitotic cells of a mouse melanoma cell line B16-C2W were harvested by gentle pipetting at different times after x-irradiation, and their mitotic spindles were visualized by indirect immunofluorescence using antibody against tubulin. At the first mitosis after irradiation with 1, 000R, 91 % of total rounded cells displayed mitotic spindles and 36 % of them showed gross abnormalities. The prominent abnormal features were multiple fluores-cent spots with or without association with spindle microtubules, and partial absence of fluorescence of the microtubules at the site of the lagging chromo-somes. As the irradiated cells progressed into the second or third mitosis, fractions of cells with clumped microtubules or those lacking spindles markedly increased. About 80 % reduction in the figures of anaphase and telophase, and scattered chromosomes on Giemsa staining suggested the prolonged arrest of cells at abnormal metaphase. Dead cells without dye-exclusion ability were selectively found among rounded cells after the second division rather than spread interphase cells. A good correlation was obtained between the fraction of the dead cells and the fractions of cells having spindle abnormality for 4 time-points after irradiation with 1, 000R. These results suggest that the mitotic cells with extensive spindle abnormalities die at that rounded state without the completion of the mitosis.