Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 498-502, February 1975

Colchicine Affects Kinetics of Concanavalin A-Mediated Agglutination of Hepatoma Cells and Plasma Membranes from Liver and Hepatoma Cells (plant lectin/cell surface/microtubules/membrane fluidity)

JIRO NAKAMURA AND HIROSHI TERAYAMA* Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan

Communicated by Howard A. Bern, November 5, 1974 Concanavalin A-mediated agglutination ABSTRACT reactions of hepatoma cells [AH-130 F(N)J and isolated liver cells, as well as of plasma membranes prepared from the liver and hepatoma, were investigated kinetically together with the effect of colchicine upon them. Concanavalin Amediated agglutination of hepatoma cells at 250 proceeded with biphasic kinetics (first and second stages of agglutination), while no appreciable agglutination of liver cells was observed in the presence of concanavalin A. The plasma membranes from the liver and hepatoma cells were similarly agglutinated with concanavalin A at 250 but not at 00. The concanavalin A-mediated membrane agglutination proceeded with monophasic kinetics and was incomplete. Colchicine inhibited preferentially the second stage of concanavalin A-mediated agglutination of hepatoma cells, but it did not affect the concanavalin A-mediated agglutination of plasma membranes of both types of cells.

sented for the possible involvement of the intracellular level of ATP in controlling the fluidity of cell membranes and the agglutination of cells by Con A (14). Under the conditions of low intracellular ATP content, transformed cells grown at both high and low cell densities are agglutinated by Con A, while normal cells grown only at a high cell density are agglutinated by Con A. The recent study (15) on the lectinmediated agglutination of subcellular fractions of normal cells seems to be very interesting partly because it shows that the plasma membranes isolated from normal cells are agglutinated by lectin in spite of the known inagglutinability of normal cells and partly because such a cell-free model system seems to be useful for analyzing the possible involvement of ATP, microtubules, microfilaments, or other factors in lectinmediated agglutination. However, no information has been obtained concerning to what extent the Con A-mediated agglutination of plasma membranes may be related to the cell agglutination. In the present study we have investigated the kinetics of Con A-mediated agglutination reactions of ascites hepatoma AH-130 F(N) cells and isolated rat liver cells in comparison with the plasma membranes prepared from normal liver and hepatoma cells.

Concanavalin A (Con A), like other plant lectins, is known in general to agglutinate preferentially transformed cells (1-3). Recent studies, however, have indicated that the different sensitivity to the lectin-mediated agglutination between the normal and the transformed cells does not reflect a difference in the number of lectin-binding sites on the cell membranes (4-9). Original hypotheses, assuming the lack or the masking of lectin-binding sites on the normal cell membranes, have thus been replaced with a new theory (5, 7, 10, 11), assuming less fluidity of the normal cell membranes as compared with the transformed ones. The theory is mainly based on the electronmicroscopic observations on the difference in topographical distribution of the lectin-binding sites on the cell membranes between the two types of cells, although the mechanisms of the possible difference in the membrane fluidity between them are not fully understood. The normal cells become sensitive to the lectin-mediated agglutination after protease treatment (2-5, 12). In the preceding paper (13) we have reported that the cell coat acid mucopolysaccharide(s) liberated from cell surfaces by mild protease treatment differ markedly in the pattern of molecular species between the normal liver and the ascites hepatomas and also to some extent among the different strains of ascites hepatomas. These observations might suggest the possibility that the cell coat acid mucopolysaccharide might be responsible for the apparent difference in the fluidity of the cell membranes. On the other hand, evidence has also been pre-

METHODS AND MATERIALS

Preparation of A.scites Hepatoma Cells. Ascites hepatoma, AH-130 F(N) cells (free-cell type without forming islands) were harvested from the peritoneal cavity of Donryu strain rats inoculated with the cells 3 days before they were killed. The hepatoma cells were washed repeatedly with cold phosphate-buffered saline (10 mM Na, K-phosphate buffer at pH 7.2-0.15 M NaCl), each time by centrifugation at 2000 rpm for several minutes and finally suspended in cold phosphate-buffered saline at the concentration of about 1.5 X 106 cells per ml and used for Con A-mediated agglutination tests. Preparation of Isolated Liver Cell Suspension. Male, Wistar rats weighing 150 g were starved for 18 hr and killed by decapitation. The liver was perfused in situ with 30 ml of warm

(370) 27 mM sodium citrate in Ca2+-free Locke solution through the vena cava. The excised liver was gently homogenized with 10% polyethyleneglycol (molecular weight 4000) in a loose-fitting homogenizer with a rubber pestle according to Jacob and Bhargava (16) at room temperature to achieve cellular dispersion. The cellular suspension filtered once through four layers of gauze, was centrifuged at 2000 X g for several minutes at 40, and the pellet was washed twice

Abbreviations: Con A, concanavalin A; phosphate-buffered saline, 10 mM phosphate-150 mM NaCl (pH 7.2); AH, ascites hepatoma. * To whom reprint requests should be addressed. 498

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FIG. 1. Dose-response of Con A-mediated agglutination of AH-130 F(N) cells. The washed cells were suspended in cold phosphate-buffered saline (1.4 X 106 cells per ml). One milliliter of the cell suspension was preincubated at 00 or 25° for 30 min; Con A was then added at concentrations as indicated (0100 ug/ml). Incubation was continued at 00 or at 250, respectively, for another 30-min period. The percentage of free cells (ordinate) and agglutination score (indicated at the top of the figure) were observed after incubation. For detail, see Methods and Materials. 0 and 0 indicate incubation at 250 and 00, respectively.

with phosphate-buffered saline and finally suspended in the same medium at about 1 X 106 cells per ml. Preparation of Isolated Plasma Membranes from Liver and AH-i80 F(N) Cells. The rat liver plasma membranes used in the present study were prepared either by the method of Berman et al. (17) with a little modification or by the method of Ray (18). The plasma membrane fraction that sedimented at the interface between d = 1.16 and d = 1.18 sucrose layers was collected, washed three times with phosphate-buffered saline, and suspended in the same medium at a concentration of 0.2-0.3 mg of protein per ml. The hepatoma plasma membranes were prepared by the method of Emmelot and Bos (19) with a little modification. The membrane pellet at the interface between d 1.16 and d 1.18 was washed once with 1 mM sodium bicarbonate, washed three times with phosphate-buffered saline, and finally suspended in phosphatebuffered saline at the concentration of 0.3 mg of protein per ml. =

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Con A-Mediated Agglutination Tests. Fifty Al of Con A dissolved in cold phosphate-buffered saline at designated concentrations were added to 1 ml of cell or plasma

membrane suspension in cold phosphate-buffered saline. Then the mixture was incubated at 25° under gentle shaking for designated periods of time. Incubation was stopped by putting the tubes containing the reaction mixture in icewater. It has been confirmed in preliminary experiments that the cooling of cells can prevent the further advancement of agglutination. To measure the cellular agglutination, we took aliquots of the cooled mixture, placed them on a hematocytometer, and observed them under a phase contrast microscope (X150). Degree of cell agglutination was expressed in terms of the agglutination score as well as of the decrease in free cells. The following five grades of agglutination score were adopted according to Wray and Walborg, Jr. (20); 0 (no significant agglutination), +1 (at least 2 clumps of 5-15 cells), +2 (at least 2 clumps of 15-30 cells), +3 (clumps of more

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FIG. 2. Kinetics of Con A-mediated agglutination of AH-130 F(N) cells. The cells, suspended in cold phosphate-buffered saline (1.4 to 1.6 X 106 cells per ml), were preincubated with Con A (40 ,g/ml) at 00 for 20 min and then incubated at 250 for various periods of time, as indicated, to observe the cell agglutination. o and 0 correspond to experiments with two different batches of the hepatoma cells. X indicates the kinetics of cell agglutination when Con A (40 jig/ml) was added to the cells preincubated at 250 for 20 min.

than 30 cells but with smaller clumps or single cells still present), and +4 (clumps of more than 100 cells with only few smaller clumps or single cells visible). For agglutination of plasma membranes, the membrane suspension, incubated for designated periods of time, was cooled in ice-water for about 3 min, then centrifuged at 150 X g for 30 see to remove larger clumps. The absorbance of the supernatant at 650 nm was measured in a B3eckman DB spectrophotometer. Chemicals. Con A (grade A) and colchicine were purchased from Calbiochem, Inc. (Los Angeles, California, U.S.A.) and Merck (Darmstadt, Germany), respectively. RESULTS

Dose-Response of Con A-Mediated Agglutination of AH-130 F(N) Cells. AH-130 F(N) cells (1.5 X 106 cells per ml), suspended in cold phosphate-buffered saline containing various concentrations of Con A (0-100 ug of Con A/ml), were incubated at 250 for 30 min. Percentages of free cells and agglutination scores measured at the end of 30 min of incubation are shown in Fig. 1. The percentage of free cells tends to decrease sharply as the concentration of Con A increases, becoming only a few % at the Con A concentration of 30 pAg/ml or larger. The agglutination score of +4 can be achieved at the Con A concentration larger than 20 /ug/ml. It should be noted that no appreciable cell agglutination occurred when the cells were incubated at 00 with Con A or incubated at 25° without Con A. Kinetics of Con 1-.Mfediated Agglutination of AH-180 F(N) Cells. AH-130 F(N) cells (1.5 X 101 cells per ml), suspended in cold phosphate-buffered saline containing 40 jug of Con A per ml were preincubated at 00 for 30 min and then transferred into a constant temperature bath regulated at 250. Aliquots of the incubation mixtures were removed at designated intervals of time after the onset of incubation at 250 to observe the cell agglutination. The kinetics of Con A-mediated cell agglutination thus obtained is shown in Fig. 2. The cells appear to start to aggregate after a lag of about 5 min. This first state of cell agglutination, however, appears to be finished

Cell Biology: Nakamura and Terayama

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Colchicine affects kinetics of concanavalin a-mediated agglutination of hepatoma cells and plasma membranes from liver and hepatoma cells.

Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 498-502, February 1975 Colchicine Affects Kinetics of Concanavalin A-Mediated Agglutination of Hepatoma...
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