Change in cell-to-dish contact of cultured mammalian cells induced by concanavalin A

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research 89 (1974) 121-126

CHANGE IN CELL-TO-DISH CONTACT OF CULTURED MAMMALIAN CELLS INDUCED BY CONCANAVALIN A C. SAT0 and K. TAKASAWA-NISHIZAWA Department

of Experimental Radiology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan

SUMMARY After incubation with Con A, cultured melanoma cells B16-C2W stuck firmly to the dish wall and could be detached neither with trypsin and pronase treatment nor with EDTA treatment, whereas the control cells were easily released from the dish wall by the same treatments. This effect became evident within 5 min of incubation at 37°C with Con A in a greater concentration than 5 &ml. Resistance to trypsinization arose more rapidly as the temperature increased up to 22°C and changed substantially around 15°C. By addition of a-methyl-n-mannoside which combines specifically with Con A, the resistant cells became de novo susceptible to trypsinization within 5 min of incubation at 37°C. The reversing effect of the inhibitor was also temperature-dependent. The appearance of resistance to trypsinization was observed without divalent cations (Cae+ and Mge+), but was inhibited by pretreatment with 1O-4M 2,4-dinitrophenol. The temperature-dependence and the concentration of Con A required for agglutination of freed cells was the same as for induction of resistance to trypsinization.

Agglutination of malignant cells by concanavalin A (Con A) [l] is now explained by structural change of the cell surface. The clustering of agglutinin-binding sites has been demonstrated in several systems by electron microscopy using ferritin-conjugated ConA [2] or peroxidase-bound ConA [3], and immunofluorescence technique [4, 51. Changes in the surface distribution of electrical charge were also found to be induced by Con A [6]. In the present experiment we investigated the effect of Con A on cell-todish contact of cultured melanoma cells and found that ConA-treatment converted the cell surface to a state resistant to proteolytic enzymes and EDTA at the Con A concentration and the temperature suitable to cell agglutination. By means of counting the detached cells after trypsinization, the effects

of ConA concentration, temperature and some inhibitors on the change of cell membrane induced by Con A were more quantitatively studied than by checking the cell agglutination. MATERIAL

AND METHODS

A mouse melanoma cell line B16-C2W [7] was used. The cells were maintained in vitro in Ham’s F12 medium supplemented by 10% fetal calf serum. About 5 x 10” cells were plated in Falcon plastic dishes of 6 cm diameter and used for the experiment after overnight incubation. Following treatment with Con A at various conditions, the cells were washed twice with Hanks balanced salt solution (HBSS), and then incubated in 0.1% trypsin and 0.01 % EDTA in phosphate-buffered saline without Mga+ and Ca2+ (PBS -) for 4 min at 37°C. The number of cells detached from the dish wall by gentle pipetting was counted with a hemocytometer, and was compared with the number of cells released by the similar trypsinization of control dishes without the Con A treatment. Exptl Cell Res 89 (1974)

122 Sate and Takasawa-Nishizawa

Fig. 1. (a) Control cells treated with 0.1 % trypsin and 0.01% EDTA for 4 min at 37°C; (b) cells trypsinized after the incubation with 20 ,ug/ml Con A for 30 min at 37°C; (c) cells trypsinized after incubation with 20 &ml Con A for 30 min at 3°C.

RESULTS After incubation of the untreated cells in 0.1 % trypsin and 0.01% EDTA for 4 min at 37°C the cells rounded up and became Exptl Cell Res 89 (1974)

completely detached from the dish wall by gentle pipetting. The ConA-treated cells, on the other hand, adhered to the dish wall very firmly, keeping their fibrous pseudopods, and could not be detached by intense

Con A effects on cell surface

123

0.6..

aa!

0.1

“‘-i 0.5

I

” 5

: IO

”

50

“‘i

100

200

Fig. 2. Abscissa: Con A cont. @g/ml); ordinate: fraction of cells detached by trypsinization. Cells were treated with various concentrations of ConA for 1 h at 3l”C, and then trypsinized for cell counting. Vertical range bars represent 1 S.D. for 6 to 13 measurements.

0-l 0

; IO

: 20

: 30

i 40

: 50

Fig. 4. Abscissa: incubation time (min) with 20 pg/ml ConA; ordinate: fraction of cells detached by trypsinization. Effect of incubation temperature with ConA on time course of appearance of resistance to trypsinization. Vertical range bars represent 1 S.D.

pipetting after trypsinization. Only the dead cells (stainable by trypan blue) and mitotic cells were detached. A phase-contrast pic- between the concentration of 10 and 20 ,ug/ ml, and remained constant above 50 pug/ml. ture is shown in fig. 1. The time course of the expression of reFig. 2 illustrates the relationship between the number of cells released and the Con A sistance to trypsinization after administraconcentration. The cells were preincubated tion of Con A at 37°C is shown in fig. 3. with various concentrations of Con A in With 20 pg/ml Con A the decrease in dephosphate-buffered saline for 1 h at 37”C, tached cell numbers was evident as early as and then trypsinized for the cell count. 5 min, progressed with time and reached a Substantial resistance to trypsinization was maximum at 20 min. Faster progress was observed with Con A concentrations higher observed with 100 ,ug/ml Con A. To examine than 10 pg/ml. The effect varied markedly whether this effect is specifically due to Con A, a-methyl mannoside (a-MM) was used. a-MM is known to combine with Con A and to prevent cell agglutination by Con A [9]. After treatment of cells with 20 ,ug/ml Con A for 30 min at 37°C the cells were washed and then incubated in 0.2 N crMM at 37°C for successivecell counting. As indicated in fig. 3 (right), the cells resistant to trypsinization became sensitive within 10 min after administration of K-MM. Fig. 3. Abscissa: incubation time (min) before trypThe time course of the change in cell-tosinization; ordinate: fraction of cells detached. Time course of the appearance of resistance to dish contact was temperature-dependent, as trypsinization after treatment with 20 pg/ml ConA shown in fig. 4. The resistance to trypsiniza(O-O) or lOO,ug/ml ConA(x---x) and itsreversion by a-methyl-mannoside (O-O). a-MM was tion advanced faster with higher temperaadded after the 30 min treatment with 20 pg/ml ture up to 22°C. There was no difference beCon A. Vertical range bars represent 1 SD. Exptl Cell Res 89 (1974)

124 Sato and Takasawa-Nishizawa Table 1. Effect of the preincubation with 2,4dinitrophenol (D NP), cycloheximide and actinomycin D on the fraction of cell population detached by trypsinization after ConA treatment More than 400 cells of 6 dishes each were counted for one measurement. Means kS.D. are given

Q 0

: 10

: 20

: 30

Preincubation time

I 40

Fig. 5. Abscissa: temperature (“C) of treatment with Con A; ordinate: fraction of cells detached. Cells were incubated with 20 pg/ml ConA for 30 min at various temperatures before trypsinization for cell counting. Vertical range bars represent 1 S.D.

tween the 22” and 37°C curves. When the cells were transferred from 3” to 37°C the resistance progressed rapidly thereafter. Fig. 5 indicates the relation of susceptibility to trypsinization and the temperature of treatment with ConA. The cells were incubated in 20 pug/ml ConA for 30 min at various temperatures prior to trypsinization for cell counting. A substantial change appeared between 10” and 15°C. The influence of temperature on the re-

DNP 1O-4 M 1O-6 M Cycloheximide 1 ~ug/ml 10 Mm1 Actinomycin D 10 pg/ml 20 ~ix/ml Control

15 min

3h

0.157+0.026 0.066 * 0.019

0.648kO.114 0.064 * 0.009

0.030 + 0.007 0.042? 0.009

0.066 + 0.012 0.092 kO.021

0.031 i: 0.008 0.034 + 0.011 0.023 + 0.004

0.047 + 0.010 0.049 & 0.009 0.027 k 0.007

versing effect of E-MM is demonstrated in fig. 6. The cells were pretreated with 100 ,ug/ ml Con A for 30 min at 37°C and then incubated in 0.1 N a-MM for 30 min at various temperatures. A sharp rise appeared between 15” and 22°C. To examine the involvement of the meta1.04 bolic process in the change of cell-to-dish contact by Con A, 2,4-dinitrophenol (uncoupler of oxidative phosphorylation), cycloheximide (inhibitor of protein synthesis) and actinomycin D (inhibitor of mRNA synthesis) was utilized. The cells were preincubated with the inhibitor in phosphate-buffered saline and Con A was then added to a final concentration of 50 lug/ml. After 30 min incubation at 37°C the cells were washed and trypsinized for the counting of detached 20 30 40 0 IO cells. As shown in table 1, cycloheximide and Fig. 6. Abscissa: temperature (“C) of incubation with a-methyl mannoside; ordinate: fraction of cells de- actinomycin D did not inhibit the appeartached. ance of resistance to trypsinization by Con A. The effect of temperature on the reversing effect Incorporation of 3H-uridine to trichloroaceof a-MM. Cells were pretreated with 100 pug/ml ConA for 30 min at 37°C and then incubated with tic acid-insoluble fraction of the cells was 0.1 N a-MM for 30 min at various temperatures. inhibited by 10 and 20 pg/ml actinomycin D Vertical range bars represent 1 S.D. Exptl Cell Res 89 (1974)

125

Con A effects on cell surface

Table 4. Agglutination

Table 2. Fraction of cell population detached from dish wall by trypsinization after treatment with 100 ,uglml ConA in phosphatebuffered saline with (PBS+) or without (PBS-) Mg2+ and Ca2f for 30 min at 37°C

of C2 W cells treated with various concentrations of ConA for 10 min at 37°C

More than 400 cells of 6 dishes each were counted for one measurement. Means ) S.D. are given

(30 min)

100

20

10

5

1

0.1

37°C 3°C +a-MM

++ + +

++ -

+

+

i -

-

0.0001 % PBS 100 ,ug/ml (Mg*+, Cae+) PBS (-) Con A (+) ConA

EDTA in PBS (-)

Time

Concentrations of Con A &g/ml)

~-

0.032 *0.017 0.059 kO.016 0.083 kO.020 1 0.987 +0.06 0.951 kO.08

from dish wall. But Con A in PBS (-) with or without EDTA could render the cell surface resistant to trypsinization. Various enzymes to release the cells from to 2 and 1.4 Y0 of control, respectively. Incorporation of 3H-proline to the protein was the dish wall were tested to find out the inhibited by 1 and 10 pug/ml cycloheximide characteristics of the binding between the to 11 and 5 % of control. Preincubation with cell and the dish surface. As indicated in DNP at a concentration greater than lo-* M table 3, none of the tested proteolytic ensubstantially inhibited the change in cell-to- zymes, EDTA and digesting enzymes of acidish contact by ConA. With lo-* M DNP, dic sugars of the surface membrane were not however, 17% of the total cells were dead effective in detaching the cells. The enzyme (stainable by trypan blue) at 3 h incubation, solutions were made with PBS (-) for proteolytic enzymesand EDTA, Hanks balanced and all cells were stainable within 8 h. The requirement of divalent cations (Mg2+, salt solution for neuraminidase (from Vibrio Ca2+) in the change of cell-to-dish contact cholerae, General Biochemicals) and 0.02 M by Con A was neglected by the results indi- Verona1 buffer pH 5.0 for hyaluronidase cated in table 2. Without the divalent cations (from Streptomyces hyalurolyticus nov. sp., and Con A, the cells were easily detached Seikagaku Kogyo), respectively. To examine the correlation of the change in cell-to-dish contact with cell agglutinaTable 3. Fraction of cell population detached tion, the conditions required for cell aggluby treatment of various enzymes after incubatination were checked as shown in table 4. tion with 20 ,ug/ml ConA The cells were washed twice with PBS withMore than 400 cells of 6 dishes each were counted out Ca2+ and Mg2+, incubated in 0.001% for one measurement. Means +S.D. are given EDTA for 10 min, and then detached from the dish wall by pipetting to make the free Treatment at 37°C ConA (+) ConA cell suspension. The cell suspension in the Trypsin, 0.1 % + EDTA plastic dish was treated with various ConA 0.01 %, 4 min 0.03 + 0.01 1 concentrations for microscopic examination. Pronase, 0.02 %, 5 min 0.07_+0.02 1.03kO.09 Protenase, 0.1 %, 10 min 0.03 * 0.01 0.89kO.24 Large aggregates were formed with ConA Neuraminidase, 20 units/ higher than 20 pg/ml at 37”C, but not at ml, 30 min 0.12+0.05 0.6950.18 Hyaluronidase, 10 units/ 3°C. The cells of the aggregatesdispersed 10 ml, 30 min 0.08f0.02 0.35iO.11 min after addition of 0.2 N U-MM. Exptl Cell Res 89 (1974)

126 Sato and Takasawa-Nishizawa

DISCUSSION The characteristics of the Con A-induced resistance to trypsinization were similar to those of cell agglutination by ConA, in terms of the relationship between the ConA concentration, temperature dependence [8], and reversibility with ct-MM [9]. The change in cell-to-dish contact appears to be another expression of the same structural change in the cell surface induced by ConA which causes the change in cell-to-cell contact (agglutination). The rapid change in the susceptibility to trypsinization within 5 min of Con A treatment and the ineffectualness of actinomycin D and cycloheximide seemto rule out the change in the amount of membrane component through metabolic turnover as being the cause of change in the cell-to-dish contact. Temperature dependence has been reported in the cluster formation of Con A receptor sites [4], in cell agglutination by Con A [8], and in the lateral mobility of antigenic sites on cell surface [lo]. At low temperatures, topographic changes of ConAbinding sites do not proceed, although ConA molecules bind to the receptors. Low fluidity of the membrane lipids is thought to be responsible for the lack of movement of the membrane component at low temperatures in the fluid mosaic model of the membrane [ll]. Our present results, showing a drastic change around 15”C, seem to lend additional support to this hypothesis. The mechanism of the cell-to-dish contact is not established as well as is the cell-to-cell contact. The agglutination of the cells is assumed to be due to cross-bridging of the cells by Con A molecules. Involvement of a microtubule function in the cell agglutination or mobility of cell receptors is suggested [12, 131. In our recent experiments, a significant decreasein the net negative charge of the cell surface was deExptl Cell Res 89 (1974)

tected by cell electrophoresis. The lowering of the charge density reduces the electrostatic repulsive forces between cells and favors cell agglutination. With regard to cell adhesion to culture dishes, the importance of cell surface protein, Ca2+ and Mg2+, is evident [ 141,since cells are easily detached when peripheral protein or divalent cations are removed. The resistance to trypsinization induced by ConA might be explained as follows: (1) Cross-bridging of the cell and dish wall by ConA molecule is resistant to trypsinization. (2) Conformation change of the protein molecule itself to become resistant to the proteolytic action of the enzymes. (3) Masking of the protein from trypsin following the redistribution of the protein in the membrane. The inhibition of the appearance of resistance to trypsinization by dinitrophenol [S] might suggest the requirement of energy for movement of the membrane component, or changes in the protein conformation. REFERENCES 1. Burger, M M & Goldberg, A R, Proc natl acad sci US 57 (1967) 359. 2. Nicolson, G L, Nature new biol 233 (1971) 244. 3. Martinex-Palomo, A, Wicker, R & Bernhard, W, Int j cancer 9 (1972) 676. 4. Nicolson, G L, Nature new biol 243 (1973) 218. 5. Inbar, M & Sachs, L, FEBS letters 32 (1973) 124. 6. Yamada, T & Yamada, M, Nature 244 (1973) 297. 7. Claunch, C, ‘Oikawa, A, Tchen, T T & Hu, F, Advances in biology of skin. Vol. 8, The pigmentary system, p. 479. Pergamon Press, Oxford, New York (1969). 8. Inbar, M, Ben-Bassat, H & Sachs, L, Proc natl acad sci US 68 (1971) 2748. 9. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418. 10. Frye, L D & Edidin, M, J cell sci 7 (1970) 319. 11. Singer, S J & Nicolson, G L, Science 175 (1972) 720. 12. Yahara, I & Edelman, G M, Nature 246 (1973) 152. 13. Loor, F, Exptl cell res 82 (1973) 415. 14. Takeichi, M & Okada, T S, Exptl cell res 74 (1972) 51. Received April 17, 1974 Revised version received June 14, 1974