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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.

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