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THE ALTERATION of MITOTIC EVENTS by IONOPHORE A23187 and CARBONYL CYANIDE N-CHLOROPHENYLHYDRAZONE

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THE ALTERATION of MITOTIC EVENTS by IONOPHORE A23187 and CARBONYL CYANIDE N-CHLOROPHENYLHYDRAZONE J. Cell Sci. 7S, 347-355 (1985) 347 Printed in Great Britain © The Company of Biologists Limited 1985 THE ALTERATION OF MITOTIC EVENTS BY IONOPHORE A23187 AND CARBONYL CYANIDE n-CHLOROPHENYLHYDRAZONE MICHAEL L.ZIEGLER, JESSE E. SISKEN* AND SHARANJIT VEDBRATf Department of Medical Microbiology and Immunology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, U.SA. SUMMARY A large quantity of published work indicates that calcium ions may be involved in the regulation of mitotic events and recent reports suggest that the onset of chromosome movement is dependent upon a transient increase in free cytosolic calcium ions. In this paper we examine the effects of two agents known to perturb intracellular calcium pools on mitosis in HeLa cells. These were the calcium-selective ionophore A23187 and carbonyl cyanide n-chlorophenylhydrazone (CCCP), which is a protonophoric inhibitor of oxidative phosphorylation. Owing to a stimulation of glycolysis, the latter agent does not decrease intracellular ATP in HeLa but does cause mitochondria to release calcium ions. Our data show that, at low concentrations, both agents prolong metaphase but differ in their effects on anaphase and cytokinesis. Studies with chlorotetracycline, a commonly used probe for membrane-associated calcium, verify that these agents do affect calcium pools under the conditions of our experiments. The data presented are consistent with the idea that increased cytosolic calcium levels can directly or indirectly affect mitotic events but, contrary to other suggestions, cause a prolongation of metaphase, i.e. they delay the onset of chromosome movement. INTRODUCTION The role of calcium in cell division has been a subject of considerable interest for many years. A substantial quantity of published work suggests that calcium ions may play a regulatory role in mitotic events and that the availability of these ions is itself regulated, both temporally and spatially, during the division process (e.g. see Hepler & Palevitz, 1974; Harris, 1975; Rebhun, 1977; Silver, Cole & Cande, 1980; Sisken, 1980; Salmon & Segal, 1980; Wolniak, Hepler & Jackson, 1980, 1983; Kiehart, 1981; Izant, 1983). The exact role of these ions, their source(s) and the mechanisms by which they are regulated remain open questions. In previous experiments we showed that treatment of HeLa cells with low levels of nicotine can delay the onset of chromosome movement (i.e. prolong metaphase) and accelerate the rate of furrowing once it begins (VedBrat, Sisken & Anderson, 1979). Since nicotine stimulates muscle contraction and calcium-dependent stimulus— • Author for correspondence. f Present address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, N.Y., U.S.A. Key words: calcium ions, mitosis, ionophores, metaphase, HeLa cells. 348 M. L. Ziegler, J. E. Sisken and S. VedBrat secretion coupling by releasing calcium from intracellular pools (e.g. see Weiss, 1968; Tjalve & Papov, 1973), we suggested that nicotine might have similar effects on HeLa cells and that this release could be responsible for the changes in duration of mitotic events (VedBrat et al. 1979). In order to gain a better understanding of the nicotine effect and of the role of calcium in cell division, we have studied the effects of two other agents that release calcium ions (Ca2+) from intracellular sources: carbonyl cyanide n-chlorophenyl- hydrazone (CCCP), which causes release of Ca2+ from mitochondria and the calcium- selective ionophore A23187. Both cause changes in mitotic parameters in HeLa cells when used at non-toxic levels and both drastically reduce fluorescence in cells stained with the fluorescent chelate probe, chlorotetracycline (CTC), a commonly used probe for membrane-associated Ca2+ (e.g. see Caswell, 1979). MATERIALS AND METHODS HeLa (GEY) cells (Microbiological Associates) were grown in plastic T-flasks in Eagle's minimal essential medium (MEM) with Hanks' salts supplemented with 10% calf serum and 2mM- glutamine. In some cases penicillin (100 units/ml) and neomycin (0-l /ig/ml) were also added to the medium. For time-lapse cinemicrography, cells were scraped or trypsinized from the inner surfaces of the flasks, injected into Rose chambers and allowed to grow for 16—48 h. The medium was then removed from the chambers and replaced with a warm sample of treatment or control medium. Control cultures were run in parallel with or in tandem to treated cultures. Time-lapse photography began immediately after the initiation of treatment at the rate of two frames per min for up to 33 h using methods previously described (Sisken, 1964). Frame-by-frame analyses of the films were done on an analytical projector (Photo Instrumentation Corp. Burbank, Calif.). Criteria for determining durations of mitotic stages were as follows. Metaphase was judged to begin when chromosomes were first observed to be aligned on the equator of the mitotic spindle as seen in lateral view, and to end when chromatids began anaphase movement. The time from beginning of chromatid movement to the initiation of cytokinesis was taken as an approximation of anaphase since it was previously shown that in cultured human amnion cells, cytokinesis began, under several different experimental conditions, when the chromatids had moved approximately 85 % of their final distance apart (Sisken, 1973). The beginning of cytokinesis was identified as that point at which dimpling of the cell surface could first be clearly seen and its completion was taken as that point when the separation of the two daughter cells, with the exception of the intercellular bridge, was completed. The data were analysed using a statistical model that takes into account both intra- and interexperiment variances (VedBrat et al. 1979). A23187 (obtained as a gift from Eli Lilly Co. or purchased from Calbiochem) was dissolved in dimethylsulphoxide (DMSO) and diluted to 10~4M in 85% DMSO, 15% distilled water. This stock was added to media to give final concentrations of 1-0 or 2'0//MA23187 and 0-85 or 1-7% DMSO, respectively. Separate control experiments indicated that these concentrations of DMSO had no effects on the mitotic parameters we measured. CCCP (Sigma) was dissolved in ethanol to give a 5 X 10~3 M stock solution and diluted appropriately in media. CTC (Sigma) was prepared fresh for each experiment and dissolved in distilled water to give a stock solution of 1 -0 mM. For staining with CTC, cells were incubated in Rose chambers for at least 24 h in complete culture medium. They were then rinsed twice and incubated for 1 h in MEM containing 10 /iM-CTC, 1 % calf serum, 20mM-HEPES buffer (pH7-l) and, for experimentals, either A23187 or CCCP. Fluorescence microscopy was performed with a Leitz microscope fitted with an epi-illumination system. A Leitz D filter cube was used along with a 400 nm narrow band pass filter in the excitation pathway to enhance the selectivity for the calcium chelate (see Fabiato & Fabiato, 1979). For doubling time measurements, cells were seeded in replicate plastic flasks with 5xlO4 cells/25 cm2 flask. Cells were grown for 4 days in the MEM described above or in MEM containing Calcium affectors and mitotic events 349 CCCP. At the end of this time, cells were trypsinized and counted with a haemocytometer. Doubling times were calculated from average cell counts from two flasks. RESULTS A23187 is a divalent cation ionophore whose biological effects are related to its capacity to bind to cell membranes and allow calcium to diffuse through them along concentration gradients. The specific membranes most affected by this agent and the direction of flow of calcium is dependent upon cell type, A23187 and exogenous Ca2+ concentrations and the duration of treatment. For example, Babcock, Chen, Yip & Lardy (1979) found that at low concentrations, the agent released Ca2+ from internal stores and caused Ca2+ efflux from liver cells and bull sperm, while at higher concentrations it stimulated uptake of exogenous Ca +. Jensen & Rasmussen (1977), on the other hand, concluded that in human peripheral lymphocytes the initial effect was to stimulate uptake of Ca2+ through the plasma membrane followed by a time- dependent redistribution of the ionophore leading to an efflux of calcium from the cell. The effects of 1-0 and 2-0^iM-A23187 on the durations of metaphase, anaphase and cytokinesis in HeLa cells are presented in Table 1 and Fig. 1, which show that each phase of mitosis responded differently. Treatment with this ionophore caused an average increase of 24-33 % in the duration of metaphase and, in the same cells, a 21-33% decrease in the duration of cytokinesis. These effects were essentially Table 1. The effects ofA23187 on the duration of mitotic stages Metaphase Anaphase Cytokinesis mean duration mean duration mean duration Drug (M) ±S.E.(min) ±S.E.(min) ±S.E.(min) None 109 25-4± 1-91 109 5-15±0-31 109 3-12 ±0-20 81 19-2 ±2-02 84 4-73 ±0-31 84 2-96 ±0-20 27 18-9 ±2-75 38 4-83 ±0-33 40 2-91 ±0-21 73 20-6 ±2-07 81 5-20 ±0-31 81 3-01 ±0-21 79 21-5 ±2-03 95 5-20 ±0-31 100 3-04 ±0-20 99 22-5 ±1-94 100 4-99 ±0-31 100 2-97 ± 0-20 Avg. 486 21-7 ±0-85 507 502 ±0-13 514 300 ±008 A23187 79 26-2 ±2-03 79 5-35 ±0-31 79 215 ±0-21 (1X1O"6M) 121 27-5 ±1-87 121 5-43 ±0-31 121 1-87 ±0-20 Avg. 210 26-9 ±1-38 200 5-39 ±0-22 200 2-00 ±0-15 P = 0-01 P = 0-18 P= 0-001 A23187 53 28-4 ±2-24 67 5-89 ±0-32 67 2-29 ±0-21 (2X10~6M) 45 28-0 ±2-34 50 5-33 ±0-32 50 2-35 ±0-21 40 30-2 ±2-42 50 5-05 ±0-32 50 2-45 ±0-21 Avg.
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