Department of Legal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
Received 24 August 1998; in revised form 5 November 1998; accepted 30 November 1998
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ABSTRACT |
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INTRODUCTION |
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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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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 00020 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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The percentage of semi-G2+M cells (>1x) increased dose-dependently at 3 h (Fig. 2B; 200300 mM, P < 0.05 vs control) and at 6 h (Fig. 2B
; 82.5165 mM, P < 0.05; 200330 mM, P < 0.0001), except when the concentration of ethanol was low (12.550 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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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 5AD
; bf) 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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DISCUSSION |
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Fluorescence microscopy revealed that the percentage of control M-phase cells, from prometaphase through telophase, which contained very high levels of cyclin B1 (Figs 4AC), 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 (100200 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 (200330 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 (50100 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 12x semi-G2+M cells (4c cells with a low level of cyclin B1) after 6 h exposure to 200330 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 (100200 mM) of ethanol probably inhibit the shortening of kinetochore microtubules, causing a transient block at metaphase (Table 4
), whereas intermediate concentrations (50100 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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FOOTNOTES |
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