Alcohol and Alcoholism Vol. 34, No. 3, pp. 300-310, 1999
© 1999 Medical Council on Alcoholism
FLOW CYTOMETRIC AND FLUORESCENCE MICROSCOPIC ANALYSIS OF ETHANOL-INDUCED G2+M BLOCK: ETHANOL DOSE-DEPENDENTLY DELAYS THE PROGRESSION OF THE M PHASE
Department of Legal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
Received 24 August 1998; first review notified 5 November 1998; accepted 30 November 1998
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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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 (
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 ß-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 B1. 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. |
INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Cultured cells are instrumental in examining the effects of chemicals on cell proliferation. Ethanol suppresses cell proliferation (Higgins, 1987
; Cook et al., 1990a; Adickes et al., 1990, 1993; Davies and Cox, 1991; Devi et al., 1993; Mikami et al., 1997) and DNA synthesis (Guerri et al., 1990; Snyder et al., 1992; Adickes et al., 1993; Devi et al., 1993; Wimalasena, 1994) in cell cultures. An accumulation of cells in the G0/G1 phase of the cell cycle (a G0/G1 block) after ethanol exposure has been demonstrated by many investigators (Higgins, 1987; Guerri et al., 1990; Cook et al., 1990a,b; Cook and Keiner, 1991; Mikami et al., 1997). The G0/G1 block partly explains the ethanol-induced reduction in cell proliferation and DNA synthesis. In addition, we found a transient G2+M block after short-term exposures (3 and 6 h) to high concentrations (100 and 200 mM) of ethanol, which was released after continuous 24 h exposure (Mikami et al., 1997). However, it remains to be elucidated how the transient arrest takes place and whether it contributes to the suppression of cell proliferation.
The cell-cycle progression is regulated by the association of cyclins with protein kinases, and is perturbed by various agents through the inhibition of the synthesis and/or degradation of cyclins. Cyclin B is increasingly expressed in cells during the G2 and M phases, reaching its highest level in the metaphase, and then degrades before entry into or during anaphase (Evans et al., 1983). Cyclin B is a subunit protein of M-phase promoting factor (MPF), a cyclin B-p34cdc2 complex (Labbé et al., 1989a; Gautier et al., 1990). The dephosphorylated form of MPF promotes the cell cycle into the M phase (Gautier et al., 1989; Labbé et al., 1989b); the degradation of cyclin B then inactivates MPF terminating the M phase (Murray et al., 1989). It should be possible to determine the exact subphase within the ethanol-induced G2+M block by estimation of the cellular cyclin B content.
Moreover, at the transition between interphase and mitosis, the cytoskeletal architecture changes dynamically: microtubules are transformed from a cytoplasmic network into the mitotic spindle and actin filaments form the contraction ring. Ethanol administration affects the dynamics of rat hepatic microtubules (Matsuda et al., 1979; Jennett et al., 1989) and hepatic cytokeratins (French et al., 1987; Eckert and Yeagle, 1996). The teratogenetic effect of ethanol in relation to the fetal alcohol syndrome (Jones et al., 1973; Clarren and Smith, 1978) can be explained by ethanol-induced structural changes in the microtubules and microfilaments of cultured salamander neural cells (Hassler and Moran, 1986) and in the intermediate filaments (glial fibrillary acidic protein and vimentin) of cultured rat radial glia (Vallés et al., 1996) and astrocytes (Renau-Piqueras et al., 1989). These results suggest that cytoskeletal alterations are associated with the ethanol-induced G2+M block.
Thus, in the present study, we attempted to elucidate the mechanism of the ethanol-induced G2+M block by carrying out simultaneous analyses of cellular DNA and cyclin B1 contents using flow cytometry as well as fluorescence microscopy by double staining with a fluorescent dye for DNA staining and a fluorescein isothiocyanate (FITC)-labelled antibody for cyclin B1 or ß-tubulin.
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MATERIALS AND METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Cell culture and ethanol exposure
L929 cells were obtained from the Japanese Cancer Research Resources Bank (JCRB, Tokyo, Japan) and maintained according to a previously described procedure (Mikami et al., 1997
). The cells used for morphological observation and flow cytometry were inoculated in Petri dishes (diameter 60 mm, Nunclon, Nunc, Naperville, IL, USA) in 3 ml of the cell suspension at a concentration of 1.2 x 105 cells/ml. The cells used for fluorescence microscopy were inoculated onto gelatin-coated Lab-Tek® Chamber Slides (Permanox® slide, Nunc). The cells were exposed to ethanol (12.5, 20.6, 50, 82.5, 100, 165, 200 and 300 mM; Wako, Osaka, Japan) for 3, 6, or 24 h, according to the procedure described previously (Mikami et al., 1997).
4Flow cytometry
The cells were trypsinized and washed with cold phosphate-buffered saline (PBS) by centrifugation (4°C, 150 g, 5 min). The cells were resuspended in 70% cold ethanol and fixed overnight at 4°C. The fixed cells were washed with cold PBS by centrifugation (4°C, 300 g, 5 min). The final cell pellet was incubated with a monoclonal anti-cyclin B1 antibody (Pharmingen, San Diego, CA, USA) in 1% BSA/PBS overnight at 4°C. The cells were washed and then incubated with a FITC-conjugated anti-mouse IgG antibody (Pharmingen) in PBS containing 1% BSA and 2% RNase (Wako) at room temperature for 30 min. After the cells were washed and incubated with 0.5 ml of 2% RNase/PBS at 37°C for 10 min, they were filtered through a nylon mesh sheet (300 mesh/in.). To the filtrate, 0.05 ml of 100 µg/ml propidium iodide (PI, Sigma, St Louis, MO, USA)/PBS was added. The stained suspension was shielded from light and stored on ice until measurement.
Flow cytometric measurements were performed on samples containing ~10 000–20 000 cells with a FACScanTM flow cytophotometer (Becton Dickinson, San Jose, IL, USA) equipped with a 488-nm argon laser.
Phase contrast microscopy
Living cells in Petri dishes were photographed through a phase contrast inverted microscope (TM300, Nippon Kogaku Co., Tokyo, Japan) and classified by cell shape. More than 1000 cells were counted for each concentration of ethanol.
Fluorescence microscopy
The cells inoculated on Chamber Slides were fixed in methanol at –20°C for 10 min for cyclin B1 detection. The cells for microtubule detection were fixed with a 10% formaldehyde solution containing 0.5% Triton X-100, 1 mM EGTA and 1 mM MgC12 at room temperature for 10 min and further fixed with the same solution without Triton X-100 for 5 min (Weber et al., 1975; Saitoh et al., 1988). The fixed cells were washed with cold PBS and incubated overnight with a monoclonal anti-cyclin B1 (Pharmingen) or anti-ß-tubulin antibody (Sigma) in 1% BSA/PBS at 4°C. They were washed and incubated with a FITC-conjugated anti-mouse IgG antibody (Pharmingen) in 1% BSA/PBS in the dark at room temperature for 1 h. The cells were then washed and stained with 0.2 µg/ml Hoechst 33258 (bis-benzimide trihydrochloride, Sigma) at room temperature for 10 min. The slide was washed and mounted in glycerol.
The cells were viewed on a fluorescence microscope using epi-illumination (BX60, Olympus Optics, Tokyo, Japan) and photographed on Tmax film (Kodak, ASA 400). In addition, the immunofluorescent image (TMD300, Nippon Kogaku Co.) of tubulin was viewed on an image processor (ARGUS-20, Hamamatsu Photonics Co., Hamamatsu, Japan) through a cooled charge-coupled-device camera (C5985, Hamamatsu Photonics Co.), and the digitized image was analysed with a personal computer to count sub-M phase cells by scoring a minimum of 500 cells for each concentration of ethanol.
Statistics
Statistical analyses were performed by one factor factorial analysis of variance (one-way ANOVA) for ethanol concentration. Results were considered statistically significant at P < 0.05.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Flow cytometry
The results of the simultaneous measurement of the DNA content (PI) and cyclin B1 content (FITC) of control L929 cells by flow cytometry are shown in Fig. 1A
. The cells with a 2c (diploid) amount of DNA (R1) consisted of G0/G1-phase and early S-phase cells, and had low levels of cyclin B1 (Figs 1A, C). The cells with a 4c (tetraploid) amount of DNA (R3) consisted of G2+M-phase and late S-phase cells, most of which had much more cyclin B1 than cells in other phases (Figs 1A, C). The peak level in the histogram of cyclin B1 content for the 4c cells (R3) was twice that for the 2c cells (R1) (Fig. 1C). For convenience, we refer to the peak level in the histogram of cyclin B1 content for the 2c cells (R1) as the standard cyclin B1 level (1x) of the cell population, to the 2c cells enclosed by a square in Fig. 1A as semi-G0/G1-phase cells, to the 4c cells with more than the standard cyclin B1 level as semi-G2+M phase cells (>1x), and to those with more than the standard level but less than twice the standard level as semi-G2+M cells (1–2x) and so on (Fig. 1A).
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Although we used eight different concentrations of ethanol in the exposure experiments, as described in Materials and methods, we represented the measured values of the flow cytometric analyses of cyclin B1 by grouping the ethanol levels into three classes (12.5–20.6–50 mM, 82.5–100–165 mM, and 200–330 mM). Such groupings might manifest the dose-dependent effects of ethanol more clearly, as shown by the small deviation within the grouped values (data not given).
The percentage of semi-G2+M cells (>1x) increased dose-dependently at 3 h (Fig. 2B; 200–300 mM, P < 0.05 vs control) and at 6 h (Fig. 2B; 82.5–165 mM, P < 0.05; 200–330 mM, P < 0.0001), except when the concentration of ethanol was low (12.5–50 mM). In contrast, the percentage of semi-G0/G1 cells decreased at high concentrations of ethanol at 6 h (Fig. 2A; 200– 330 mM, P < 0.01).
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, 1–2x; 
, 2–3x; 
In addition, the histogram of cyclin B1 content for ethanol-exposed semi-G2+M cells (cf. Fig. 1C
; R3) shifted dose-dependently to higher values than those of the control after exposure to higher concentrations of ethanol (more than 82.5 mM) for 3 and 6 h (data not shown). The mean cyclin B1 content of semi-G2+M cells (>1x) tended to increase dose-dependently, though statistical significance was observed only for semi-G2+M cells with more than twice the standard cyclin B1 level after 6 h exposure to 200–330 mM ethanol (Table 1).
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A prolonged (24 h) exposure to ethanol dose-dependently decreased the mean cyclin B1 level (Table 1
) and percentage (Fig. 2B) of semi-G2+M cells. In contrast, the percentage of semi-G0/G1 cells increased dose-dependently (Fig. 2A). These three parameters all showed statistically significant differences when the ethanol dose was more than 82.5 mM. Moreover, the number of 2c cells with higher levels of cyclin B1 about twice as high as the standard level, which were named supra-semi-G0/G1 cells (Fig. 1A), increased at 24 h with high ethanol doses (Table 2; 200–330 mM vs control; P < 0.01).
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Cell shape
A continuous 6 h exposure to higher concentrations of ethanol increased the number of some specific morphological-types of L929 cells. The number of spindle-shaped cells (Fig. 3
; large arrow) increased in 100 and 165 mM ethanol (Fig. 3B; Table 3) with a significant increase at the latter dose (Table 3). However, the number of round cells (Fig. 3; small arrow) for a dose of 330 mM was triple that for lower concentrations (Fig. 3C; Table 3). The number of coupled daughter cells just after cell division (Fig. 3; asterisk) increased at a dose of 330 mM ethanol (Table 3). In addition, the number of apoptosis-like shrunken cells (Fig. 3C; arrow head) increased in a range of doses over 100 mM, and was at least three times greater for a dose of 330 mM than for a dose of less than 82.5 mM (Table 3).
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Fluorescence microscopy
Cyclin B. The cytoplasm of L929 cells was visualized by FITC immunofluorescence using an antibody against cyclin B1 (Fig. 4
). Well spread-out interphase cells (Figs 4A–C; a) revealed very weak cytoplasmic fluorescence due to cyclin B1. The cytoplasmic fluorescence of two daughter cells was slightly stronger than that of interphase cells (Fig. 4B; b). The cytoplasmic fluorescence of cyclin B1 became stronger with cell shape in the order: longer spindle-shaped (Fig. 4A; c); shorter spindle-shaped (Figs 4A, B; d); round (Fig. 4B; e); and egg-shaped (Figs 4A, B; f, g) cells, which correspond to G2, prophase, prometaphase, and metaphase/early anaphase cells, respectively, judging from their DNA when viewed by double staining. The FITC immunofluorescence of cytoplasm in metaphase (Fig. 4B; f), anaphase (Figs 4A, B; g, h) and telophase (Fig. 4C; i) cells was stronger than that of prophase cells (Figs 4A, B; d). After the completion of cytokinesis, it seemed that the cytoplasmic fluorescence of cyclin B1 decreased rapidly as the distance between daughter cells increased (Fig. 4B; b).
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Continuous 6 and 24 h exposures to less than a 200 mM concentration of ethanol did not induce any marked changes in the staining pattern of cyclin B1 in each cell-cycle phase. However, 6 h exposure to 330 mM ethanol increased the number of round cells with an increased level of cyclin B1, which are probably a karyotype of early prophase (Figs 4D, E
; j).
Microtubules.
Immunofluorescence microscopy with FITC bound to anti-ß-tubulin antibody revealed a cytoplasmic network of microtubules permeating the control cell at interphase (Figs 5A, B; a). In addition, in control cells with mitosis-related shapes, the microtubules of the spindle (Figs 5A–D; b–f) and intercellular bridge (Figs 5A, B; g, h) were stained more strongly than the cytoplasmic ones of interphase cells (Figs 5A, B; a). The midbody of L929 cells was not stained with ß-tubulin antibody (Fig. 5B; h).
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The total percentage of M-phase cells from prometaphase through telophase, other than for prophase cells, tended to increase at doses of 50 and 200 mM of ethanol, but decreased at a dose of 330 mM of ethanol at both 6 and 24 h, compared with the control (Table 4
). A 6 h exposure to 50, 100, and 200 mM of ethanol and 24 h exposure to 12.5, 100, and 200 mM of ethanol tended to increase the percentage of metaphase cells compared with the control, but 6 h exposure to 330 mM of ethanol tended to decrease it (Table 4). When compared to controls, 50–100 mM ethanol tended to increase the percentage of early-anaphase cells at 6 h, but did not do so at 24 h (Table 4). Higher concentrations of ethanol (more than 82.5 mM) tended to alter the cytoplasmic microtubules of interphase cells so that they gathered in the central region of the cell (Fig. 5E; i); the number of such cells and the degree of gathering increased in a dose-dependent manner (data not shown). The microtubules in round, probably early-prophase, cells, which appeared after exposure to 330 mM ethanol and contained a high level of cyclin B1 (Fig. 4D; j), gathered around the nucleus, but did not seem to form mitotic spindles (Figs 5E, F; j).
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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We have succeeded in identifying the specific site of the ethanol-induced cell-cycle block throughout the G2 and M phases by the simultaneous flow cytometric analysis of cellular cyclin B1 content and cellular DNA content in combination with phase contrast and fluorescence microscopy. We found that higher concentrations (100–200 mM) of ethanol caused a metaphase block at both 6 and 24 h, as demonstrated by the increased incidence of metaphase shapes at these time intervals (Table 4
), and of semi-G2+M cells with high levels of cellular cyclin B1 at 6 h (Table 1, Fig. 2B). This metaphase block persisted up to 24 h (Table 4). Since the metaphase–anaphase transition is one of the cell-cycle checkpoints (Hartwell and Weinert, 1989), this checkpoint might be more sensitive to ethanol than any other sub-M phases.
Fluorescence microscopy revealed that the percentage of control M-phase cells, from prometaphase through telophase, which contained very high levels of cyclin B1 (Figs 4A–C), was about 2% at 6 h (Table 4). These cells might correspond to an upper half region of control >4x semi-G2+M cells in the dot plot by flow cytometry (Fig. 1A), since the percentage of control >4x semi-G2+M cells was about 4% at 6 h (Fig. 2B). In addition, the intensity of cyclin B1 fluorescence in control L929 cells at anaphase (Figs 4A, B; g, h) and telophase (Fig. 4C; i) was still almost the same as that at metaphase (Fig. 4B; f). The degradation of cyclin B is thought to start at the initiation of anaphase (Murray et al., 1989), and the cyclin B1 fluorescence of HeLa S3 at anaphase is reported to be very poor (Sherwood et al., 1994). In L929 cells, the degradation of cyclin B1 also probably starts at that time, but the degradation rate may be slow.
Some investigations demonstrated that the metaphase arrest induced by colcemid (a microtubule inhibitor) in HeLa S3 cells (Kung et al., 1990), by vinblastine (a mitotic blocker) in MOLT-4 leukaemic cells (Gong et al., 1993, 1995) and by ALLN (N-acetylleucylleucylnorleucinal, a neutral cysteine protease inhibitor) in Chinese hamster ovary (CHO) cells (Sherwood et al., 1993) was accompanied by high levels of cyclin B expression, which indicates an inhibition of cyclin B degradation. It therefore seems likely that higher concentrations of ethanol (100–200 mM) also inhibit cyclin B degradation in L929 cells, thus increasing the number of metaphase cells with high levels of cyclin B1. The ethanol-induced dysfunction of cyclin B1 degradation was also demonstrated by the increase in the supra-semi-G0/G1 phase cell population with an intermediate cyclin B1 level after 24 h exposure to high concentrations (200–330 mM) of ethanol (Table 2); the cells might separate, even though ethanol suppressed the completion of cyclin B1 degradation.
On the other hand, intermediate concentrations (50–100 mM) of ethanol induced a cell-cycle block one step after metaphase, at early anaphase (anaphase A), at 6 h (Table 4). The highest concentration (330 mM) of ethanol, however, increased the percentage of round cells (Table 3) with a high level of cyclin B1 (Table 1, Figs 2B, 4D, E) and a nuclear membrane, but without a spindle (Figs 5E, F), which suggests a block at the step before metaphase, probably in early prophase. In this early-prophase block, an increase in the cellular content of cyclin B1 may disconnect to the cell-cycle progression, which has also been observed in aphidicolin-treated CHO cells (Kung et al., 1993) and m-AMSA (4'-[9-acridinylamino]-3-methanesulphon-m-anisidide)-treated MOLT-4 cells (Gong et al., 1995). Aphidicolin (an inhibitor of DNA polymerase 
) induced an accumulation of cyclin B, though it inhibited DNA synthesis, thus preventing the progress of cells into the G2 and M phases. m-AMSA (a DNA topoisomerase II inhibitor) induced a prophase block with elevated levels of both cyclin A and cyclin B.
In addition to the M-phase block, there must be an ethanol-induced G2 block. For example, 6 h exposure to 200 mM ethanol increased the population of G2+M-phase cells by about one and a half times, compared with the control in our previous study (control, about 20%; 200 mM ethanol, about 30%) (Mikami et al., 1997) and also the population of M-phase cells, from prometaphase through telophase (Table 4; control, about 2%; 200 mM ethanol, about 3%). So, 6 h exposure to 200 mM ethanol almost universally increased the number of both G2- and M-phase cells and blocked the cell cycle throughout the G2 phase and telophase. Exposure to other concentrations of ethanol might have a similar effect, although the specific stage where the blockage occurred was slightly different depending on the concentration, as demonstrated in Table 4.
The increase in the percentage of 1–2x semi-G2+M cells (4c cells with a low level of cyclin B1) after 6 h exposure to 200–330 mM ethanol (Fig. 2B) also suggests a G2 (or late-S) and/or late-telophase block. The G2 block might be caused by failure of the initiation of cyclin B1 synthesis in spite of the completion of DNA synthesis. A late-telophase block is also likely, because an increase in the percentage of coupled daughter cells just after cytokinesis (Fig. 3; Table 3) was observed by phase contrast microscopy at a dose of 330 mM.
It is most likely that higher concentrations of ethanol inhibit degradation of cyclin B1 dose-dependently and the highest doses even inhibit its synthesis in some cells. However, it is unknown whether the inhibition of the degradation and/or synthesis of cyclin B1 is a cause or a result of the G2+M block due to ethanol exposure. A clue to answering this question may lie in the following observations: high concentrations (82.5 mM) of ethanol seemed to affect the cytoplasmic microtubules of interphase cells, causing them to gather in the centre (Fig. 5E), and to increase the number of spindle-shaped cells (Table 3, Fig. 3B); the highest concentration of ethanol (330 mM) might cause the cells to become round (Fig. 3C) and probably inhibits the formation of a mitotic spindle (Figs 5E, F), thus blocking cells at early prophase, despite high levels of cyclin B1 (Table 1; Figs 2B, 4D, E). Higher concentrations (100–200 mM) of ethanol probably inhibit the shortening of kinetochore microtubules, causing a transient block at metaphase (Table 4), whereas intermediate concentrations (50–100 mM) of ethanol probably suppress the elongation of polar microtubules, causing a transient block at early anaphase (anaphase A). All these observations suggest that ethanol exposure induces the dysfunction of microtubules.
Many investigations have been performed on the effects of ethanol on the cytoskeleton. Some investigators reported that the metabolite of ethanol, acetaldehyde, decreases the number of hepatic microtubules (Baraona et al., 1975; Matsuda et al., 1979; Kawahara et al., 1987), acetaldehyde being covalently bound to tubulin (Jennett et al., 1989). However, others did not observe these effects (Berman et al., 1983; Kawahara et al., 1989), partly due to differences in conditions, such as the amount of ethanol given, sampling timing etc. The ethanol-induced alteration in microtubules may be associated not only with their content, but also with their polymerization dynamics, such as elongation and shortening, as demonstrated by the immunofluorescent view of microtubules in the present study (Table 4).
With regard to the association of cyclin B-p34cdc2 complex with microtubules, it has been found that the interaction between cyclin B and microtubule-associated proteins regulates microtubule assembly and dynamics (Ookata et al., 1995), and that intact metaphase microtubules are necessary for the degradation of cyclin B (Kubiak et al., 1993). The dynamic interaction between cyclin B and intact microtubules seems to ensure the progression of the M phase. If the microtubules are disarranged, the increase and decrease in cyclin B content may be unconnected to the cell-cycle progression. Thus, it is likely that ethanol disturbs the microtubule assembly of L929 cells so as to damage not only the formation and function of the mitotic spindle, but also the organized correlation between microtubules and the synthesis and degradation of cyclin B1 in a dose-dependent manner, thereby delaying the progress of karyokinesis, which may lead to an ethanol-induced G2+M block.
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ACKNOWLEDGEMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The authors would like to thank Dr Masamichi Ishizaki for his help in operating the fluorescence microscope and Dr Kouji Kameyama for his suggestions on statistical analysis.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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* Author to whom correspondence should be addressed.
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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