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We found previously that short-term (3 and 6 h) exposure to ethanol (100 and 200 mM) induced the transient arrest of L929 cells at the G2+M phase. To identify the exact site blocked during the G2+M phase, we carried out flow cytometry and microscopic analysis with asynchronous L929 cells exposed to ethanol (12.5-330 mM) for 3, 6 or 24 h. Flow cytometry (the simultaneous analysis of cellular DNA and cyclin B1 content) revealed that the percentage of 4c (tetraploid) cells with a high level of cyclin B1 increased after continuous 6 h exposure to ethanol (&gt; or =82.5 mM) and decreased after 24 h exposure, which supports the idea of a transient M-phase block. To determine the sub-M phase of 4c cells with high levels of cyclin B1 based on spindle microtubules and their karyotype, we viewed immunofluorescent images by double staining with Hoechst 33258 (bis-benzimide trihydrochloride) for DNA and with fluorescein isothiocyanate-labelled antibody for cyclin B1 or beta-tubulin. A 6 h exposure to intermediate concentrations (50-100 mM) of ethanol increased the number of early-anaphase cells, compared with the control, suggesting an inhibition of the elongation of polar microtubules. Both 6 and 24 h exposure to higher concentrations (100-200 mM) of ethanol increased metaphase cells, indicating an arrest at the spindle assembly checkpoint and suggesting an inhibition of the shortening of kinetochore microtubules and/or the degradation of cyclin B . Moreover, 6 h exposure to 330 mM ethanol increased round, probably early-prophase, cells, suggesting inhibition of the formation of spindle microtubules. Thus, it is likely that higher concentrations of ethanol affect the elongation, contraction, and formation of the spindle microtubules of L929 cells dose-dependently and also disrupt the correlation between microtubule organization and the synthesis and degradation of cyclin B1, thereby delaying the progress of karyokinesis, which may lead to an ethanol-induced G2+M block.

FLOW CYTOMETRIC AND FLUORESCENCE MICROSCOPIC ANALYSIS OF ETHANOL-INDUCED G2+M BLOCK: ETHANOL DOSE-DEPENDENTLY DELAYS THE PROGRESSION OF THE M PHASE