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Regulation of concanavalin A agglutination by the extracellular matrix
Printed in Sweden Copyright 0 1975 by Academic Press, Inc. in any form resrrwd AN rights of reproduction
Experimental Cell Research 92 (I 975) 350-360
REGULATION
OF CONCANAVALIN
A AGGLUTINATION
EXTRACELLULAR
BY THE
MATRIX
PHILIP SKEHAN and SUSAN J. FRIEDMAN Department of Pharmacology, University of Colorado Medical Center, Denver, Cola. 80220, USA
SUMMARY The agglutinability of rat C, glioma cells by concanavalin A (ConA) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maxim urn amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which thev are affected by density and the point at which-they become insensitive to further density increases. Both trypsin and EDTA reduce cell aaalutinabilitv. The similarity in recoverv kinetics between low density cells and cells dissociated-with EDNA or trypsin suggests that-low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Densitydependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrrx inhibitor of ConA agglutinability, while high density cultures produce a matrix promotor.
Cooperative cell interactions requiring either direct contact or the close proximity of neighboring cells play a role in the regulation of cell growth [l-3], metabolism [4], maintenance of normal morphology [3], susceptibility to viral infection [5], and chromosome conformation [6]. We have investigated the possibility that cooperative cell interactions might play a role in regulating cell agglutinability by the plant lectin concanavalin A (ConA), and have found that agglutinability is a function of both population density and the number of contacts which a cell makes with its neighbors. Further examination revealed that the apparent correlation of agglutinability with contact number was coincidental, and that the effective intercellular communication mechanism was the matrix, or micro-exudate, secretedby cells. ExptC Cell Res 92 (1975)
METHODS AND MATERIALS Cells Rat C&glioma cells were maintained in growth medium consisting of MEM (GIBCo F-15) plus 5 % fetal calf serum (GIBCo), and passaged at approximately weekly intervals in Falcon T-75 flasks. For EDTA dissociation, cells were washed three times with calcium-magnesium-free, phosphate-buffered saline (CMF-PBS) containing 0.5 mM EDTA, then incubated 25 min in this same solution. With trvnsin dissociation, cells were washed three times-with CMF-PBS. then incubated for 10 min with 5 ml of 0.25 % crude trypsin. At the end of the dissociation period, a 3-fold excess of growth medium was added, and the cell suspension immediately centrifuged for 10 min at 1200 rpm, washed with 20 ml of growth medium, and resuspended in growth medium at an appropriate cell density. Following dissociation itself, growth medium rather than PBS was employed for subsequent manipulations to minimize cell fragility and lysis caused by EDTA.
Con A agglutination Cells were suspended in growth medium at twice the final concentration desired. ConA solutions were
Regulation of Con A agglutination made up as 2 x stocks in PBS, Immediately-prior to an assay, equal volumes of cell suspension and Con A were pipetted into polypropylene tubes and mixed by pipetting. Pasteur pipets were not used for the final mixing of solutions because of a tendency for cells to adhere to their walls; similarly, polystyrene and glass tubes were avoided for the same reason. Unless otherwise stated, 1.5 ml of mixed solution containing 5 x 10’ total cells were pipetted into each of duplicate 35 mm Falcon tissue culture dishes. The dishes were immediately placed on a gyrotory shaker and rotated for 2.5 min at 60 rpm at 37°C. The dishes were then removed from the shaker and the cells allowed to settle for 2-3 min, by which time they were largely attached to the substratum. Cells were examined with an inverted microscope at a magnification of 200, and the number of single cells and multicellular aggregates determined. The ratio A/S of aggregates (A) to singles (5) was used as an index of changes in cell agglutinability evoked by ConA. 250 cells and aggregates were counted per dish. Five separate fields were examined at specified positions along a diameter of the dish. Random field selection was not employed because of a slight tendency for cells to concentrate in the center of the dishes; similarly, care was taken to count equal numbers of cells plus aggregates in each field screened. Duplicates agreed to within an average of about + 5 %.
Density and recovery experiments Unless otherwise indicated, dissociated cells were plated onto T-75 flasks at low (2.5 x lo8 cells/flask) or high (2 x 10’ cells/flask) density in 30 ml of growth medium and incubated at 37°C for 18 h. For Con A agglutination experiments, cells were then dissociated and washed with growth medium. For matrix studies, the procedure of Terry & Culp [7] was used. Cells were removed from flasks by 30 min incubation with CMF-PBS containing 0.5 mM EDTA. Conditioned flasks were washed three times with growth medium, and used for re-inoculation with freshly dissociated glioma cells. For suspension recovery of cells following dissociation, cells were suspended in growth medium at 5 x lo6 cells/ml in polypropylene tubes and incubated at 37°C. For long-term incubation of cells in suspension, cells were seeded at 2 x 1W or
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2 x 1V per -ml in 60 mm bacterial Petri dishes containing 10 ml of growth medium.
Materials ConA was obtained from the Sigma Chemical Co. and methyl-cc-o-mannopyranoside from P-L Biochemicals Inc.
RESULTS AND DISCUSSION The aggregate ratio method
The extent to which cells will aggregate when agitated by rotation is dependent upon (1) cell density; (2) time of rotation; and (3) rate of rotation. At the onset of aggregation, single cells will collide with one another to form doublets. Later, single cells will collide with small aggregates, and small aggregates will combine with one another to form larger but fewer aggregates. For the ratio (A/S) of aggregates (A) to single cells (S) to be a valid reflection of the extent of cell aggregation, it is necessary that measurements be made under conditions where no large aggregatesare formed. When large aggregates do form, the A/S ratio generally declines even though the extent of cell aggregation is obviously increasing. We have found the A/S ratio to be a reliable index of aggregation only when cell aggregatesare limited in size nearly exclusively to doublets and triplets. For maximal aggregate ratio assay sensitivity, it is desirable to minimize self-aggregation (the aggregation of cells in the absence
Fig. 1. Abscissa: cells/dish; ordinate: ratio (A/S) of cell aggregates (A) to single cells (S). The aggregate ratio as a function of cell density. (A) trypsin-dissociated cells; (g) EDTA-dissociated cells. Cells were prepared as described in Methods and Materials. 1.5 ml of cells were placed in 35 mm tissue culture dishes and rotated at 60 rpm at 37°C for either 2.5 (O-O) or zero min (O-O). Exptl Cell Res 92 (1975)
352 Skehan and Friedman of an agglutinating agent such as ConA). Fig. 1 illustrates the A/S ratio for selfaggregation of EDTA and trypsin-dissociated glioma cells as a function of cell density. At a density of 5 x lo4 cells/l.5 ml/dish, the background level of self-aggregation after 2.5 min of rotation at 60 rpm was nearly identical to self-aggregation in the absence of rotation. Fig. 1 further demonstrates a generally close agreement between duplicates. Breakdown in the validity of the aggregate ratio method can be seen in fig. 1a where the A/S ratio declines for self-aggregation as density is increased from 7.5 x lo5 to 1 x lo6 cells/dish even though visual examination revealed an increase in the extent of aggregation. Fig. 2 shows the A/S ratio for glioma cells as a function of rotation time for both self-
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Fig. 2. Abscissa: time of rotation (min); ordinate: net aggregate ratio. The net aggregate ratio as a function of rotation time for glioma cells dissociated with EDTA (E) and trypsin (T). The net aggregate ratio is the (A/$) value of test samples minus that of controls. In this case, controls are samples that have not been rotated. ---, Self-aggregation for EDTA and trypsin-dissociated cells rotated at 60 rpm at 37°C in 35 mm dishes containing 5 x lo4 cells in 1.5 ml. -, Agglutination of cells under the same conditions but in the presence of a supramaximal concentration of ConA (100 tcglml). Exptl Cell Res 92 (197.5)
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Fig. 3. Abscissa: ConA cont. (up/ml); ordinate: net aggregate ratio. The net aggregate ratio as a function of ConA concentration. Glioma cells were dissociated with either EDTA (O-O) or trypsin (O-O) and rotated at a density of 5 x 10’ cells/l.5 ml/dish at 60 rpm for 2.5 min at 37°C. The net aggregate ratio is obtained by subtracting from test sample (A/s) values the (A/S) value of samples not exposed to ConA.
aggregation (no Con A) and for agglutination by a supramaximal concentration of ConA (100 pg/ml). With trypsin dissociated cells, the A/S ratio in the presence of ConA reaches a maximum with about 2.5 min of rotation, retains this level until about minute 5, then declines because of the fusion of small aggregates to form larger but fewer aggregates. With EDTA-dissociated cells exposed to Con A, maximal agglutination is achieved within 1 min; this value is retained until 7.5 min, then declines. The optimal conditions, and indeed the range of conditions, for which the aggregate ratio assay provides a reliable index of increased cell agglutination evoked by ConA entail extremely low cell density, brief and relatively gentle agitation, and limited cel1 aggregation. For C, glioma cells (figs 1-3) and for both normal and chemically transformed Syrian hamster cells (data not shown), the optimal conditions for a valid aggregate ratio assay are quite different from those
Regulation of ConA agglutination
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Fia. 4. Abscissa: concentration of methvl-a-D-mannop$anoside (mM); ordinate: net aggregate ratio. The inhibition of ConA agglutination of glioma cells by methyl-a-D-mannopyranoside (MMS). Cells were dissociated with EDTA and rotated at a density of 5 x lo* cells/l.5 ml/dish at 60 rpm for 2.5 min at 37°C in the mesence of 25 ualml of ConA (submaximal in &is experiment) &Z various concentrations of MMS.
employed by Benjamin & Burger [8] in calibrating their visual estimation procedure for assessingcell agglutination. Fig. 3 shows a dose-response curve for ConA agglutination of glioma cells. With EDTA dissociation, the dose giving 50 % of maximal effect (C,,) was 10 lug/ml of ConA, while maximum agglutination was obtained with 20 ,ug/ml. As with other tumor lines [9], trypsin reduces the sensitivity of glioma cells to agglutination by submaximal concentrations of ConA. With trypsinized gliomas, the C,, was 20pg/ml, while maximum agglutination occurred with about 40 pg/ml. Interestingly, the final supramaximal level of agglutination was higher with trypsin than with EDTA-dissociated cells. Thus with both cell agglutination by ConA (fig. 3) and cell aggregation in the absence of the lectin (control curves in fig. 2), the maximal increase in the A/S ratio above basal level was greater with trypsinized than with EDTA-dissociated cells.
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Methyl-dr-n-hannopyranoside antagonism The agglutination of cells by Con A is known to be partially reversed by methyl-cc-Dmannopyranoside (MMS). Fig. 4 demonstrates that the elevated glioma A/S ratio evoked by ConA is antagonized by MMS. In this particular experiment, 0.25 mM MMS produced a half-maximal reversal of ConA agglutination, and a concentration of 0.75 mM MMS produced a maximal reversal of 83 %. In a series of seven experiments, supramaximal doses of MSS reversed ConA agglutination of glioma cells by an average of 75%. If MMS is a specific antagonist of ConAinduced cell agglutination, we would not expect this sugar to affect self-aggregation. Should it be found, however, that MMS inhibits self-aggregation as well as agglutination, it would follow that the sugar is not specific for the antagonism of ConA binding alone, and would raise the possibility that agglutination by ConA might be simply the promotion of the same basic adhesive mechanism responsible for self-aggregation. Fig. 5 demonstrates that self-aggregation is indeed inhibited by MMS. In ten separate experiments, a supramaximal level of MMS 1
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Fig. 5. Abscissa: MMS cont. (mM); ordinate: net aggregate ratio. The inhibition of self-aggregation by MMS. Cells were EDTA-dissociated and rotated at 60 rpm for 2.5 min at 37°C at a density of 7.5 x lo5 cells/l.5 ml dish. Exptl Cell Res 92 (1975)
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Skehan and Friedman
(10 mM) inhibited self-aggregation by from 43 to 77 %. The average inhibition was 66 %. This value may be an underestimate. The dose-response curves for MMS inhibition of agglutination were performed with a low concentration of cells which minimized the background level of self-aggregation. The self-aggregation experiments, however, were performed at a considerably higher cell density which produced a much higher background, thereby reducing the signal to noise ratio and probably obscuring at least partially the MMS inhibition of self-aggregation. Since the degree to which MMS antagonizes self-aggregation (66%) is of the same order as its inhibition of ConA-evoked cell agglutination (75 %), the question is raised as to whether MMS antagonizes ConA agglutination by releasing ConA from its surface receptor, the traditional hypothesis, or whether the antagonism arises by an entirely different mechanism. An alternative to the traditional hypothesis %
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Fig. 6. Abscissa: ConA cont. (,ug/ml); ordinate: aggregate ratio (A/S). ConA agglutination. as a function of cell density.. Glioma cells were seeded at densities of 2.5 x 10’ (A), -1 x 10’ (B), 7.5 x 1W (C), 5 x l@ (D), and 1 x lo6 (E’) per T75 flask and incubated for 18 ~h..Cells were then dissociated and assayed for ConA agglutinability. Exptl Cell Res 92 (197.5)
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Fig. 7. Abscissa: average number of contacts between cells. ordinate: (left) max. agglutinahihty (0-0); (right) half-maximal Con A cont. (O-O). Cells were plated at the densities described in fig. 6 and incubated for 18 h. A random selection of 250 cells in each of duplicate flasks was made and the number of contacts between one cell and its neighbors determined. Maximum agglutinability is the net aggregate ratio at supramaximal ConA (50 pg/ml). The half-maximal concentration is that ConA concentration required to produce one half of maximum agglutination.
is that MMS possessesits own unique surface receptor that is distinct from the ConA receptor. This separate MMS receptor might (1) be coupled to two separate sets of surface effector systems, one responsible for selfaggregation and the other for agglutination by Con A, or (2) the agglutination and selfaggregation effector systemsmight be identical, or at least share a common set of essential effector components. One important fact can be deduced from the present experiments as well as from the literature: the inhibition by MMS of ConAinduced agglutination is not a competitive inhibition as some have claimed. This conclusion is predicated by the fact that MMS can only partially antagonize cell agglutination by ConA (fig. 4 and refs [9, lo]). If the process were truly competitive, then antagonism would asymptotically approach 100%
Regulation of Con A agglutination .24 .20
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Fig. 8. Abscissa: time of recovery (hours); ordinate: aggregate ratio (A/S). Recoveryof Con A agglutinability by high and low density cells following tryp& dissociation. Cells were plated out in T75 flasks at high (2 x 10’ cells/ flask, O-O) and low (2.5 x lo6 cells/flask, O-O) density, then incubated for 18 h, dissociated for 5 min with trypsin, and assayed for ConA agglutinability after various periods of recovery in suspension. agglutination with 50 /lg/ml of ConA; ---, baz’ground agglutination in the absence of Con A.
with ircreasing MMS concentration; it does not. Cooperative regulation of Con A agglutinability
The role of cell cooperativity in the ConA agglutination of:C, glioma cells was examined by varying population density. Cells were seeded in T-75 flasks at various densities, and dissociated for ConA agglutination assay 18 h later. During this period little growth occurred, but ample time was afforded for the development of density dependent changes in agglutinability. Fig. 6 demonstrates a direct correlation between Con A agglutinability and cell density up to a seeding density of 1 x 10’ cells/flask (near confluency). Cell density was used as a means of regulating the ability of cells to interact with one another. The potential for such interactions can also be reflected by the number of physical contacts which a cell makes with its neighbors. Fig. 7 shows 24-751808
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a plot of the maximum agglutinability (net aggregate ratio at supramaximal ConA) and the half-maximal concentration (C,,,) of Con A as a function of cell contact number. The average contact number of fully confluent cells was about 5.7, indicating that at confluency C, glioma cells assume a quasihexagonal spacing array. As the average contact number increases, because of increasing cell density, the halfmaximal dose of ConA falls modestly from about 9.5 ,ug/ml to about 7 pg/ml, a 26% decrease. Maximal sensitivity to ConA is achieved at a contact number of abnut 3.5. The net aggregate ratio at supramaximal levels of ConA (50 ,ug/ml) increases with contact number, and more than doubles as the contact number rises from 1.2 to 4.3, where maximum effect occurs. Thus both the half-maximal dose (C,,) and the maximum amplitude of response by glioma cells to ConA are functions of the extent to which cells can interact. They are separate cell parameters, however, and differ both in the extent to which they are affected by cell interactions and the contact number at which they become insensitive to further increases in cell density and contact number. The C!,, ceasesto increase at a contact number corresponding to 60-65 % of the full confluency value; the maximum response amplitude reaches a plateau at about 75 % of the full confluency contact number. Since cell dissociation by trypsin is known to reduce the agglutinability by ConA of tumor cells [9], it is possible that glioma growth at low cell density causes the loss of the same surface agglutination factor(s) removed by trypsin. If this were true, we would expect the recovery kinetics of low and high density cells, following brief dissociation with trypsin, to be parallel and differ only by a time lag component reflecting different initial conditions. If the reduced Exptl Cell Res 92 (1975)
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Fig. 9. Abscissa: ConA cont. (,ug/ml); ordinate: aggregate ratio (A/S). Effects of EDTA (panel A) and trypsin (panel B) upon cell agglutination by Con A. Glioma cells were grown in suspension at 2 x 106/ml for 18 h, then incubated for the time in minutes indicated on the graph with either 0.5 mM EDTA or 0.25 % trypsin and assayed for agglutination by Con A.
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agglutinability of low density cells were the result of some mechanism fundamentally different from the responsible for the loss of agglutinability following brief trypsinization, we might expect low and high density cells to display entirely different and nonparallel recovery kinetics. To test this issue, cells grown in monolayer at high (2 x 10’) or low (2.5 x 106) density (cells/flask) for 18 h were trypsin-dissociated for 5 min, and allowed to recover for various periods of time by incubation in suspension at 37°C. Fig. 8 shows that the recovery kinetics of Con A agglutinability by high and low density cells are essentially parallel, and differ only by a time lag component which reflects the difference in initial starting values. High density cells are fully recovered from dissociation by 3 h, while low density cells required
4 h for recovery. It is likely, therefore, that low density cells lose the same surface component(s) that are removed by trypsin. Although it is well known that trypsinization reduces the ConA agglutinability of tumor cells [9], most investigators appear to have implicitly assumed that EDTA does not have such an effect. Fig. 9 compares the effects of 0.5 mM EDTA and 0.25 % trypsin upon the agglutinability of glioma cells grown in suspension for 18 h prior to use. Both dissociating agents reduce cell agglutinability by ConA. For a given period of incubation, trypsin causesa greater loss of agglutinability. However, in glioma monolayer dissociation, 5-10 min of trypsinization is sufficient for detachment, whereas 20-30 min of EDTA treatment is required. Thus the minimum time for adequate trypsin
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Fig. 10. Abscissa: ConA concentration Q&g/ ml); ordinate: aggregate ratio (A/S). Recovery of Con Aaggfutinabilityfollowing dissoeiaton with (A) 0.5 mM EDTA for 30 min or (B) 0.25 % trypsin for 10 min. Cells were dissociated and allowed to recover in growth medium at 37°C in suspension culture. Con A agglutinability was assayed after 1.( q ), 3 (A), 5 ( l ), and 7 (0) of recovery.
Regulation of Con A agglutination
351
lost the same surface component(s) that are removed by trypsin and EDTA. Anchorage dependence
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Fie. 11. Abscissa: time after mating (hours): ordinate: aggregateratio (A/S). -Loss of aaalutinability following- _plating at low
cell density. Confluent gboma cells were dissociated by 5 min of trypsinization and plated at sparse density (2.5 x 1W cells/T75 flask) in growth medium. At various times thereafter cells were redissociated and assayedfor Con A agglutinability. l , Agglutination with 50 ,ug/ml; 0, background agglutination in the absenceof ConA.
dissociation reduces cell agglutinability less than the minimum dissociation time required with EDTA. Fig. 10 illustrates the recovery of ConA agglutinability following treatment of confluent glioma cultures with 10 min of trypsin and 30 min of EDTA respectively. Cells were incubated in suspension at 37°C during the recovery period to avoid the need for a second dissociation. Trypsinized cells were nearly recovered in their agglutinability by 3 h, and fully recovered by 5 h. Recovery
Cells grown in suspension at high density for 18 h are highly agglutinable by Con A (fig. 9). So too are cells grown in monolayer at high density (fig. 6). Cells grown in monolayer at low density are poorly agglutinable (figs 6, 8) but quickly regain their agglutinability when incubated in suspension (fig. 8). This suggeststhat Con A agglutinability may possessan anchorage dependence. To confirm this possibility, it is necessary to examine the degree of contact interaction between cells grown in suspension for 18 h. C, gliomas were grown at low (2 x lo4 cells/ ml) and high (2 x IO5 cells/ml) density in 60 mm bacterial Petri dishes. When examined 18 h later, 43 % of the cells in low density cultures were singlets not in contact with other cells; the corresponding value for high density cultures was 6%. Only 6 % of low density cells were in clusters of more than 5 cells, while 69 % of the high density cells were in clusters of more than 5 cells. Seventyeight percent of the low density cells were in clusters of 3 or fewer cells, while only
19 % of the high density cells
were in such small aggregates. Despite these substantial differences in the extent of cell contact, the high and low density suspension cultures had identical ConA agglutinabilities. Thus cell contact and density do not appear to regulate the Con A agglutinability of cells grown in suspension. Low density suspension cells have about the same degree of physical from EDTA dissociation followed a similar contact with their neighbours as do low but slightly delayed time course. The similarity between the recovery kinetics density monolayer cells, but have a much of confluent cells following dissociation higher agglutinability. Thus when C, glioma (fig. 10) and the recovery of low density cells cells attach to a substratum they undergo (fig. 8) lends further support to our suggestion some manner of change which makes the that cells grown at low density may have level of their agglutinability sensitive to a reduced agglutinability because they have density-dependent cooperative regulation. Exptl Cell Res 92 (1975)
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The rate at which agglutinability is lost following the replating of confluent monolayer cells at low density is shown by fig. 11. Agglutinability is lost in approx. 4 h. The above results suggest that the surface of glioma cells contains at least one component required for Con A agglutination that has a rapid turnover rate (3-5 h) and can be rapidly produced (also 3-5 h) as well. The fact that this component(s) can be removed by brief EDTA treatment suggests that it may not be covalently bound to the cell surface, while its destruction by trypsin suggests that it may contain a protein moiety. Nature of the cooperative mechanism
The correlation of cell agglutinability with population density and contact number suggests that the differential agglutinability of high and low density monolayer cells might result from a direct contact interaction. However, both the release of conditioned medium factors and the production of extracellular matrix would also correlate with density and therefore with contact number. To distinguish between these three possible mechanisms for the density dependence of Con A agglutinability, cells were seeded at low (2.5 x lo6 cells/flask) or high (2 X 10’ cells/flask) density in T75 flasks and incubated for 18 h. At the end of this time conditioned Table 1. Relative ConA agglutinability
of high and low density glioma cells plated onto control flasks or flasks containing matrix collected from high and low density cultures Cell density Matrix
Low
High
Control Low density matrix High density matrix
1.00 0.92 1.43
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Exptl Cell Res 92 (1975)
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Fig. 12. Abscissa: time of recovery (hours); ordinate:
aggregate of ratio (A/S). Recovery of ConA agglutinability by low density (2.5 x l@ cells/T75 flask) cells plated onto flasks conditioned with matrix from high density cultures (2 x lOr/flask) for 18 h. l , Agglutination by 50 pg/ml of ConA; 0, absence of ConA. Cells plated at low density onto control flasks not containing matrix suffer a loss of agglutinability, as shown in fig. 11.
flasks were prepared by removing cells with EDTA. Terry & Culp [7] have shown that this treatment will remove cells but not matrix from the flasks. Fresh cells from briefly trypsinized confluent cultures were seededat both high and low density onto the conditioned flasks containing matrix from high and low density populations, respectively. Fresh cells were also plated onto control flasks that had not previously been conditioned by cell growth, but which had been pre-incubated with growth medium for 18 h. All cultures were incubated for an additional 18 h before dissociation and testing for Con A agglutinability. Table 1 shows the results of this experiment. The level of agglutination by supramatimal ConA (50 ,ug/ml) of control cells, whether at high or low density, was defined as 1.00. When cells were plated at low density upon matrix produced by low density cultures, there occurred a marginal inhibition of
Regulation of Con A agglutination
agglutinability (0.92 of control value). When cells were plated at low density onto flasks preconditioned by matrix from high density cultures there was a 43 % stimulation of agglutinability. Since the ratio of high to low density cell agglutinability in control flasks was 1.39, the extracellular matrix, or microexudate, deposited by high density cells fully restored the agglutinability of low density cells to the level characteristic of control cultures grown at high density. When cells were plated at high density onto conditioned flasks the same qualitative pattern was observed, but quantitative differences were apparent. The agglutinability of cells plated at high density onto matrix deposited by low density cultures fell to 0.85 % of control value. This level of inhibition is about three times the experimental error of the assay procedure, and is therefore probably significant. When cells were plated at high density onto matrix deposited by high density cultures, there was an 18 % stimulation of ConA agglutinability. The rate at which low density cells regain their agglutinability when plated onto flasks conditioned with matrix from high density cultures is shown in fig. 12. It can be seen that the matrix induced stimulation is complete in about 3 h. From the above data it would appear that the extracellular matrix contains two components which can regulate the level of ConA agglutination. One component promotes agglutinability while the second inhibits it. The efficacy of conditioned media from high and low density flasks was also investigated, and was found to have no effect upon cell agglutination by ConA. Since conditioned medium factors can be ruled out as the mechanism of cooperative promotion of cell agglutination, and since high density matrix can fully restore the agglutinability of low density cells, it is not necessary to postulate
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a role for direct cell contact in the promotion of cell agglutinability. Conclusions
C, glioma cells grown in suspension possess an intrinsically high agglutinability by ConA which is not affected by cell contact or density phenomena. When cells are plated out at low density an anchorage-dependent processoccurs whereby they lose a substantial portion, though not all, of their agglutinability. Thus ConA agglutination would seem to involve at least two cellular components: (1) a residual agglutinability that is apparently not subject to density regulation; (2) a second component of agglutinability that is regulated by density-dependent phenomena. Part of the agglutinability loss by low density cells can be attributed to the production of a matrix inhibitor of agglutination, but this alone does not account for the entire loss of agglutinability. Thus in addition to the matrix inhibitor at least one additional mechanism must contribute to the loss of agglutinability at low cell density. The loss of agglutinability is dependent upon cell anchorage to a substratum, but it is not clear whether it is adhesion per se that is responsible, or whether perhaps some consequence of adhesion. Cells plated out at high density produce matrix that contains a factor which stimulates agglutinability. Recovery kinetics suggest that the surface agglutination moiety regulated by matrix-mediated cooperative cell interactions may be the same component removed by EDTA and trypsin. That EDTA can (presumably) strip this moiety from the cell surface indicates that it is not covalently bonded to other surface structures. Both the loss and reappearance of ConA agglutinability as a result of density manipulation are rapid processes requiring 3-5 h. This suggeststhat the responsible surface molecule Exptl Cell Res 92 (1975)
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(s) has a high rate of both turnover and production. This work was supported by grants from the Milheim Foundation for Cancer Research and The General Research Assistance Fund of the University of Colorado (S. J. F.) and by USPHS postdoctoral research fellowship CA-52464-01 (P. S.).
REFERENCES 1. Yaoi, Y & Kanaseki, T, Nature 237 (1972) 283. 2. Mallucci, L, Wells, V & Young, M R, Nature new biol 239 (1972) 53. 3. Skehan, P & Friedman, S J, Exptl cell res 86 (1974) 237.
Exptl Cell Res 92 (1975)
4. Cunningham, D D, J biol them 247 (1972) 2464. 5. Thorne. H V. J gen virol 18 (1973) 153. 6. Bolund; L, Dariynkiewicz, i &‘Ringertz, N R, Exutl cell res 62 (1970) 76. 7. Terry, A H & C&p, L A, Biochemistry 13 (1974) 414. 8. Benjamen, T L & Burger, M M, Proc natl acad sci US 67 (1970) 929. 9. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418. 10. Rottmann, W L, Walther, B T, Hellerqvist, C G, Umbreit, J & Roseman, S, J biol them 249 (1974) 373.
Received September 19, 1974 Revised version received October 28, 1974
Experimental Cell Research 92 (I 975) 350-360
REGULATION
OF CONCANAVALIN
A AGGLUTINATION
EXTRACELLULAR
BY THE
MATRIX
PHILIP SKEHAN and SUSAN J. FRIEDMAN Department of Pharmacology, University of Colorado Medical Center, Denver, Cola. 80220, USA
SUMMARY The agglutinability of rat C, glioma cells by concanavalin A (ConA) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maxim urn amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which thev are affected by density and the point at which-they become insensitive to further density increases. Both trypsin and EDTA reduce cell aaalutinabilitv. The similarity in recoverv kinetics between low density cells and cells dissociated-with EDNA or trypsin suggests that-low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Densitydependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrrx inhibitor of ConA agglutinability, while high density cultures produce a matrix promotor.
Cooperative cell interactions requiring either direct contact or the close proximity of neighboring cells play a role in the regulation of cell growth [l-3], metabolism [4], maintenance of normal morphology [3], susceptibility to viral infection [5], and chromosome conformation [6]. We have investigated the possibility that cooperative cell interactions might play a role in regulating cell agglutinability by the plant lectin concanavalin A (ConA), and have found that agglutinability is a function of both population density and the number of contacts which a cell makes with its neighbors. Further examination revealed that the apparent correlation of agglutinability with contact number was coincidental, and that the effective intercellular communication mechanism was the matrix, or micro-exudate, secretedby cells. ExptC Cell Res 92 (1975)
METHODS AND MATERIALS Cells Rat C&glioma cells were maintained in growth medium consisting of MEM (GIBCo F-15) plus 5 % fetal calf serum (GIBCo), and passaged at approximately weekly intervals in Falcon T-75 flasks. For EDTA dissociation, cells were washed three times with calcium-magnesium-free, phosphate-buffered saline (CMF-PBS) containing 0.5 mM EDTA, then incubated 25 min in this same solution. With trvnsin dissociation, cells were washed three times-with CMF-PBS. then incubated for 10 min with 5 ml of 0.25 % crude trypsin. At the end of the dissociation period, a 3-fold excess of growth medium was added, and the cell suspension immediately centrifuged for 10 min at 1200 rpm, washed with 20 ml of growth medium, and resuspended in growth medium at an appropriate cell density. Following dissociation itself, growth medium rather than PBS was employed for subsequent manipulations to minimize cell fragility and lysis caused by EDTA.
Con A agglutination Cells were suspended in growth medium at twice the final concentration desired. ConA solutions were
Regulation of Con A agglutination made up as 2 x stocks in PBS, Immediately-prior to an assay, equal volumes of cell suspension and Con A were pipetted into polypropylene tubes and mixed by pipetting. Pasteur pipets were not used for the final mixing of solutions because of a tendency for cells to adhere to their walls; similarly, polystyrene and glass tubes were avoided for the same reason. Unless otherwise stated, 1.5 ml of mixed solution containing 5 x 10’ total cells were pipetted into each of duplicate 35 mm Falcon tissue culture dishes. The dishes were immediately placed on a gyrotory shaker and rotated for 2.5 min at 60 rpm at 37°C. The dishes were then removed from the shaker and the cells allowed to settle for 2-3 min, by which time they were largely attached to the substratum. Cells were examined with an inverted microscope at a magnification of 200, and the number of single cells and multicellular aggregates determined. The ratio A/S of aggregates (A) to singles (5) was used as an index of changes in cell agglutinability evoked by ConA. 250 cells and aggregates were counted per dish. Five separate fields were examined at specified positions along a diameter of the dish. Random field selection was not employed because of a slight tendency for cells to concentrate in the center of the dishes; similarly, care was taken to count equal numbers of cells plus aggregates in each field screened. Duplicates agreed to within an average of about + 5 %.
Density and recovery experiments Unless otherwise indicated, dissociated cells were plated onto T-75 flasks at low (2.5 x lo8 cells/flask) or high (2 x 10’ cells/flask) density in 30 ml of growth medium and incubated at 37°C for 18 h. For Con A agglutination experiments, cells were then dissociated and washed with growth medium. For matrix studies, the procedure of Terry & Culp [7] was used. Cells were removed from flasks by 30 min incubation with CMF-PBS containing 0.5 mM EDTA. Conditioned flasks were washed three times with growth medium, and used for re-inoculation with freshly dissociated glioma cells. For suspension recovery of cells following dissociation, cells were suspended in growth medium at 5 x lo6 cells/ml in polypropylene tubes and incubated at 37°C. For long-term incubation of cells in suspension, cells were seeded at 2 x 1W or
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2 x 1V per -ml in 60 mm bacterial Petri dishes containing 10 ml of growth medium.
Materials ConA was obtained from the Sigma Chemical Co. and methyl-cc-o-mannopyranoside from P-L Biochemicals Inc.
RESULTS AND DISCUSSION The aggregate ratio method
The extent to which cells will aggregate when agitated by rotation is dependent upon (1) cell density; (2) time of rotation; and (3) rate of rotation. At the onset of aggregation, single cells will collide with one another to form doublets. Later, single cells will collide with small aggregates, and small aggregates will combine with one another to form larger but fewer aggregates. For the ratio (A/S) of aggregates (A) to single cells (S) to be a valid reflection of the extent of cell aggregation, it is necessary that measurements be made under conditions where no large aggregatesare formed. When large aggregates do form, the A/S ratio generally declines even though the extent of cell aggregation is obviously increasing. We have found the A/S ratio to be a reliable index of aggregation only when cell aggregatesare limited in size nearly exclusively to doublets and triplets. For maximal aggregate ratio assay sensitivity, it is desirable to minimize self-aggregation (the aggregation of cells in the absence
Fig. 1. Abscissa: cells/dish; ordinate: ratio (A/S) of cell aggregates (A) to single cells (S). The aggregate ratio as a function of cell density. (A) trypsin-dissociated cells; (g) EDTA-dissociated cells. Cells were prepared as described in Methods and Materials. 1.5 ml of cells were placed in 35 mm tissue culture dishes and rotated at 60 rpm at 37°C for either 2.5 (O-O) or zero min (O-O). Exptl Cell Res 92 (1975)
352 Skehan and Friedman of an agglutinating agent such as ConA). Fig. 1 illustrates the A/S ratio for selfaggregation of EDTA and trypsin-dissociated glioma cells as a function of cell density. At a density of 5 x lo4 cells/l.5 ml/dish, the background level of self-aggregation after 2.5 min of rotation at 60 rpm was nearly identical to self-aggregation in the absence of rotation. Fig. 1 further demonstrates a generally close agreement between duplicates. Breakdown in the validity of the aggregate ratio method can be seen in fig. 1a where the A/S ratio declines for self-aggregation as density is increased from 7.5 x lo5 to 1 x lo6 cells/dish even though visual examination revealed an increase in the extent of aggregation. Fig. 2 shows the A/S ratio for glioma cells as a function of rotation time for both self-
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Fig. 2. Abscissa: time of rotation (min); ordinate: net aggregate ratio. The net aggregate ratio as a function of rotation time for glioma cells dissociated with EDTA (E) and trypsin (T). The net aggregate ratio is the (A/$) value of test samples minus that of controls. In this case, controls are samples that have not been rotated. ---, Self-aggregation for EDTA and trypsin-dissociated cells rotated at 60 rpm at 37°C in 35 mm dishes containing 5 x lo4 cells in 1.5 ml. -, Agglutination of cells under the same conditions but in the presence of a supramaximal concentration of ConA (100 tcglml). Exptl Cell Res 92 (197.5)
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Fig. 3. Abscissa: ConA cont. (up/ml); ordinate: net aggregate ratio. The net aggregate ratio as a function of ConA concentration. Glioma cells were dissociated with either EDTA (O-O) or trypsin (O-O) and rotated at a density of 5 x 10’ cells/l.5 ml/dish at 60 rpm for 2.5 min at 37°C. The net aggregate ratio is obtained by subtracting from test sample (A/s) values the (A/S) value of samples not exposed to ConA.
aggregation (no Con A) and for agglutination by a supramaximal concentration of ConA (100 pg/ml). With trypsin dissociated cells, the A/S ratio in the presence of ConA reaches a maximum with about 2.5 min of rotation, retains this level until about minute 5, then declines because of the fusion of small aggregates to form larger but fewer aggregates. With EDTA-dissociated cells exposed to Con A, maximal agglutination is achieved within 1 min; this value is retained until 7.5 min, then declines. The optimal conditions, and indeed the range of conditions, for which the aggregate ratio assay provides a reliable index of increased cell agglutination evoked by ConA entail extremely low cell density, brief and relatively gentle agitation, and limited cel1 aggregation. For C, glioma cells (figs 1-3) and for both normal and chemically transformed Syrian hamster cells (data not shown), the optimal conditions for a valid aggregate ratio assay are quite different from those
Regulation of ConA agglutination
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Fia. 4. Abscissa: concentration of methvl-a-D-mannop$anoside (mM); ordinate: net aggregate ratio. The inhibition of ConA agglutination of glioma cells by methyl-a-D-mannopyranoside (MMS). Cells were dissociated with EDTA and rotated at a density of 5 x lo* cells/l.5 ml/dish at 60 rpm for 2.5 min at 37°C in the mesence of 25 ualml of ConA (submaximal in &is experiment) &Z various concentrations of MMS.
employed by Benjamin & Burger [8] in calibrating their visual estimation procedure for assessingcell agglutination. Fig. 3 shows a dose-response curve for ConA agglutination of glioma cells. With EDTA dissociation, the dose giving 50 % of maximal effect (C,,) was 10 lug/ml of ConA, while maximum agglutination was obtained with 20 ,ug/ml. As with other tumor lines [9], trypsin reduces the sensitivity of glioma cells to agglutination by submaximal concentrations of ConA. With trypsinized gliomas, the C,, was 20pg/ml, while maximum agglutination occurred with about 40 pg/ml. Interestingly, the final supramaximal level of agglutination was higher with trypsin than with EDTA-dissociated cells. Thus with both cell agglutination by ConA (fig. 3) and cell aggregation in the absence of the lectin (control curves in fig. 2), the maximal increase in the A/S ratio above basal level was greater with trypsinized than with EDTA-dissociated cells.
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Methyl-dr-n-hannopyranoside antagonism The agglutination of cells by Con A is known to be partially reversed by methyl-cc-Dmannopyranoside (MMS). Fig. 4 demonstrates that the elevated glioma A/S ratio evoked by ConA is antagonized by MMS. In this particular experiment, 0.25 mM MMS produced a half-maximal reversal of ConA agglutination, and a concentration of 0.75 mM MMS produced a maximal reversal of 83 %. In a series of seven experiments, supramaximal doses of MSS reversed ConA agglutination of glioma cells by an average of 75%. If MMS is a specific antagonist of ConAinduced cell agglutination, we would not expect this sugar to affect self-aggregation. Should it be found, however, that MMS inhibits self-aggregation as well as agglutination, it would follow that the sugar is not specific for the antagonism of ConA binding alone, and would raise the possibility that agglutination by ConA might be simply the promotion of the same basic adhesive mechanism responsible for self-aggregation. Fig. 5 demonstrates that self-aggregation is indeed inhibited by MMS. In ten separate experiments, a supramaximal level of MMS 1
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Fig. 5. Abscissa: MMS cont. (mM); ordinate: net aggregate ratio. The inhibition of self-aggregation by MMS. Cells were EDTA-dissociated and rotated at 60 rpm for 2.5 min at 37°C at a density of 7.5 x lo5 cells/l.5 ml dish. Exptl Cell Res 92 (1975)
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(10 mM) inhibited self-aggregation by from 43 to 77 %. The average inhibition was 66 %. This value may be an underestimate. The dose-response curves for MMS inhibition of agglutination were performed with a low concentration of cells which minimized the background level of self-aggregation. The self-aggregation experiments, however, were performed at a considerably higher cell density which produced a much higher background, thereby reducing the signal to noise ratio and probably obscuring at least partially the MMS inhibition of self-aggregation. Since the degree to which MMS antagonizes self-aggregation (66%) is of the same order as its inhibition of ConA-evoked cell agglutination (75 %), the question is raised as to whether MMS antagonizes ConA agglutination by releasing ConA from its surface receptor, the traditional hypothesis, or whether the antagonism arises by an entirely different mechanism. An alternative to the traditional hypothesis %
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Fig. 6. Abscissa: ConA cont. (,ug/ml); ordinate: aggregate ratio (A/S). ConA agglutination. as a function of cell density.. Glioma cells were seeded at densities of 2.5 x 10’ (A), -1 x 10’ (B), 7.5 x 1W (C), 5 x l@ (D), and 1 x lo6 (E’) per T75 flask and incubated for 18 ~h..Cells were then dissociated and assayed for ConA agglutinability. Exptl Cell Res 92 (197.5)
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Fig. 7. Abscissa: average number of contacts between cells. ordinate: (left) max. agglutinahihty (0-0); (right) half-maximal Con A cont. (O-O). Cells were plated at the densities described in fig. 6 and incubated for 18 h. A random selection of 250 cells in each of duplicate flasks was made and the number of contacts between one cell and its neighbors determined. Maximum agglutinability is the net aggregate ratio at supramaximal ConA (50 pg/ml). The half-maximal concentration is that ConA concentration required to produce one half of maximum agglutination.
is that MMS possessesits own unique surface receptor that is distinct from the ConA receptor. This separate MMS receptor might (1) be coupled to two separate sets of surface effector systems, one responsible for selfaggregation and the other for agglutination by Con A, or (2) the agglutination and selfaggregation effector systemsmight be identical, or at least share a common set of essential effector components. One important fact can be deduced from the present experiments as well as from the literature: the inhibition by MMS of ConAinduced agglutination is not a competitive inhibition as some have claimed. This conclusion is predicated by the fact that MMS can only partially antagonize cell agglutination by ConA (fig. 4 and refs [9, lo]). If the process were truly competitive, then antagonism would asymptotically approach 100%
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Fig. 8. Abscissa: time of recovery (hours); ordinate: aggregate ratio (A/S). Recoveryof Con A agglutinability by high and low density cells following tryp& dissociation. Cells were plated out in T75 flasks at high (2 x 10’ cells/ flask, O-O) and low (2.5 x lo6 cells/flask, O-O) density, then incubated for 18 h, dissociated for 5 min with trypsin, and assayed for ConA agglutinability after various periods of recovery in suspension. agglutination with 50 /lg/ml of ConA; ---, baz’ground agglutination in the absence of Con A.
with ircreasing MMS concentration; it does not. Cooperative regulation of Con A agglutinability
The role of cell cooperativity in the ConA agglutination of:C, glioma cells was examined by varying population density. Cells were seeded in T-75 flasks at various densities, and dissociated for ConA agglutination assay 18 h later. During this period little growth occurred, but ample time was afforded for the development of density dependent changes in agglutinability. Fig. 6 demonstrates a direct correlation between Con A agglutinability and cell density up to a seeding density of 1 x 10’ cells/flask (near confluency). Cell density was used as a means of regulating the ability of cells to interact with one another. The potential for such interactions can also be reflected by the number of physical contacts which a cell makes with its neighbors. Fig. 7 shows 24-751808
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a plot of the maximum agglutinability (net aggregate ratio at supramaximal ConA) and the half-maximal concentration (C,,,) of Con A as a function of cell contact number. The average contact number of fully confluent cells was about 5.7, indicating that at confluency C, glioma cells assume a quasihexagonal spacing array. As the average contact number increases, because of increasing cell density, the halfmaximal dose of ConA falls modestly from about 9.5 ,ug/ml to about 7 pg/ml, a 26% decrease. Maximal sensitivity to ConA is achieved at a contact number of abnut 3.5. The net aggregate ratio at supramaximal levels of ConA (50 ,ug/ml) increases with contact number, and more than doubles as the contact number rises from 1.2 to 4.3, where maximum effect occurs. Thus both the half-maximal dose (C,,) and the maximum amplitude of response by glioma cells to ConA are functions of the extent to which cells can interact. They are separate cell parameters, however, and differ both in the extent to which they are affected by cell interactions and the contact number at which they become insensitive to further increases in cell density and contact number. The C!,, ceasesto increase at a contact number corresponding to 60-65 % of the full confluency value; the maximum response amplitude reaches a plateau at about 75 % of the full confluency contact number. Since cell dissociation by trypsin is known to reduce the agglutinability by ConA of tumor cells [9], it is possible that glioma growth at low cell density causes the loss of the same surface agglutination factor(s) removed by trypsin. If this were true, we would expect the recovery kinetics of low and high density cells, following brief dissociation with trypsin, to be parallel and differ only by a time lag component reflecting different initial conditions. If the reduced Exptl Cell Res 92 (1975)
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Fig. 9. Abscissa: ConA cont. (,ug/ml); ordinate: aggregate ratio (A/S). Effects of EDTA (panel A) and trypsin (panel B) upon cell agglutination by Con A. Glioma cells were grown in suspension at 2 x 106/ml for 18 h, then incubated for the time in minutes indicated on the graph with either 0.5 mM EDTA or 0.25 % trypsin and assayed for agglutination by Con A.
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agglutinability of low density cells were the result of some mechanism fundamentally different from the responsible for the loss of agglutinability following brief trypsinization, we might expect low and high density cells to display entirely different and nonparallel recovery kinetics. To test this issue, cells grown in monolayer at high (2 x 10’) or low (2.5 x 106) density (cells/flask) for 18 h were trypsin-dissociated for 5 min, and allowed to recover for various periods of time by incubation in suspension at 37°C. Fig. 8 shows that the recovery kinetics of Con A agglutinability by high and low density cells are essentially parallel, and differ only by a time lag component which reflects the difference in initial starting values. High density cells are fully recovered from dissociation by 3 h, while low density cells required
4 h for recovery. It is likely, therefore, that low density cells lose the same surface component(s) that are removed by trypsin. Although it is well known that trypsinization reduces the ConA agglutinability of tumor cells [9], most investigators appear to have implicitly assumed that EDTA does not have such an effect. Fig. 9 compares the effects of 0.5 mM EDTA and 0.25 % trypsin upon the agglutinability of glioma cells grown in suspension for 18 h prior to use. Both dissociating agents reduce cell agglutinability by ConA. For a given period of incubation, trypsin causesa greater loss of agglutinability. However, in glioma monolayer dissociation, 5-10 min of trypsinization is sufficient for detachment, whereas 20-30 min of EDTA treatment is required. Thus the minimum time for adequate trypsin
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Fig. 10. Abscissa: ConA concentration Q&g/ ml); ordinate: aggregate ratio (A/S). Recovery of Con Aaggfutinabilityfollowing dissoeiaton with (A) 0.5 mM EDTA for 30 min or (B) 0.25 % trypsin for 10 min. Cells were dissociated and allowed to recover in growth medium at 37°C in suspension culture. Con A agglutinability was assayed after 1.( q ), 3 (A), 5 ( l ), and 7 (0) of recovery.
Regulation of Con A agglutination
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lost the same surface component(s) that are removed by trypsin and EDTA. Anchorage dependence
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Fie. 11. Abscissa: time after mating (hours): ordinate: aggregateratio (A/S). -Loss of aaalutinability following- _plating at low
cell density. Confluent gboma cells were dissociated by 5 min of trypsinization and plated at sparse density (2.5 x 1W cells/T75 flask) in growth medium. At various times thereafter cells were redissociated and assayedfor Con A agglutinability. l , Agglutination with 50 ,ug/ml; 0, background agglutination in the absenceof ConA.
dissociation reduces cell agglutinability less than the minimum dissociation time required with EDTA. Fig. 10 illustrates the recovery of ConA agglutinability following treatment of confluent glioma cultures with 10 min of trypsin and 30 min of EDTA respectively. Cells were incubated in suspension at 37°C during the recovery period to avoid the need for a second dissociation. Trypsinized cells were nearly recovered in their agglutinability by 3 h, and fully recovered by 5 h. Recovery
Cells grown in suspension at high density for 18 h are highly agglutinable by Con A (fig. 9). So too are cells grown in monolayer at high density (fig. 6). Cells grown in monolayer at low density are poorly agglutinable (figs 6, 8) but quickly regain their agglutinability when incubated in suspension (fig. 8). This suggeststhat Con A agglutinability may possessan anchorage dependence. To confirm this possibility, it is necessary to examine the degree of contact interaction between cells grown in suspension for 18 h. C, gliomas were grown at low (2 x lo4 cells/ ml) and high (2 x IO5 cells/ml) density in 60 mm bacterial Petri dishes. When examined 18 h later, 43 % of the cells in low density cultures were singlets not in contact with other cells; the corresponding value for high density cultures was 6%. Only 6 % of low density cells were in clusters of more than 5 cells, while 69 % of the high density cells were in clusters of more than 5 cells. Seventyeight percent of the low density cells were in clusters of 3 or fewer cells, while only
19 % of the high density cells
were in such small aggregates. Despite these substantial differences in the extent of cell contact, the high and low density suspension cultures had identical ConA agglutinabilities. Thus cell contact and density do not appear to regulate the Con A agglutinability of cells grown in suspension. Low density suspension cells have about the same degree of physical from EDTA dissociation followed a similar contact with their neighbours as do low but slightly delayed time course. The similarity between the recovery kinetics density monolayer cells, but have a much of confluent cells following dissociation higher agglutinability. Thus when C, glioma (fig. 10) and the recovery of low density cells cells attach to a substratum they undergo (fig. 8) lends further support to our suggestion some manner of change which makes the that cells grown at low density may have level of their agglutinability sensitive to a reduced agglutinability because they have density-dependent cooperative regulation. Exptl Cell Res 92 (1975)
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The rate at which agglutinability is lost following the replating of confluent monolayer cells at low density is shown by fig. 11. Agglutinability is lost in approx. 4 h. The above results suggest that the surface of glioma cells contains at least one component required for Con A agglutination that has a rapid turnover rate (3-5 h) and can be rapidly produced (also 3-5 h) as well. The fact that this component(s) can be removed by brief EDTA treatment suggests that it may not be covalently bound to the cell surface, while its destruction by trypsin suggests that it may contain a protein moiety. Nature of the cooperative mechanism
The correlation of cell agglutinability with population density and contact number suggests that the differential agglutinability of high and low density monolayer cells might result from a direct contact interaction. However, both the release of conditioned medium factors and the production of extracellular matrix would also correlate with density and therefore with contact number. To distinguish between these three possible mechanisms for the density dependence of Con A agglutinability, cells were seeded at low (2.5 x lo6 cells/flask) or high (2 X 10’ cells/flask) density in T75 flasks and incubated for 18 h. At the end of this time conditioned Table 1. Relative ConA agglutinability
of high and low density glioma cells plated onto control flasks or flasks containing matrix collected from high and low density cultures Cell density Matrix
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Exptl Cell Res 92 (1975)
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Fig. 12. Abscissa: time of recovery (hours); ordinate:
aggregate of ratio (A/S). Recovery of ConA agglutinability by low density (2.5 x l@ cells/T75 flask) cells plated onto flasks conditioned with matrix from high density cultures (2 x lOr/flask) for 18 h. l , Agglutination by 50 pg/ml of ConA; 0, absence of ConA. Cells plated at low density onto control flasks not containing matrix suffer a loss of agglutinability, as shown in fig. 11.
flasks were prepared by removing cells with EDTA. Terry & Culp [7] have shown that this treatment will remove cells but not matrix from the flasks. Fresh cells from briefly trypsinized confluent cultures were seededat both high and low density onto the conditioned flasks containing matrix from high and low density populations, respectively. Fresh cells were also plated onto control flasks that had not previously been conditioned by cell growth, but which had been pre-incubated with growth medium for 18 h. All cultures were incubated for an additional 18 h before dissociation and testing for Con A agglutinability. Table 1 shows the results of this experiment. The level of agglutination by supramatimal ConA (50 ,ug/ml) of control cells, whether at high or low density, was defined as 1.00. When cells were plated at low density upon matrix produced by low density cultures, there occurred a marginal inhibition of
Regulation of Con A agglutination
agglutinability (0.92 of control value). When cells were plated at low density onto flasks preconditioned by matrix from high density cultures there was a 43 % stimulation of agglutinability. Since the ratio of high to low density cell agglutinability in control flasks was 1.39, the extracellular matrix, or microexudate, deposited by high density cells fully restored the agglutinability of low density cells to the level characteristic of control cultures grown at high density. When cells were plated at high density onto conditioned flasks the same qualitative pattern was observed, but quantitative differences were apparent. The agglutinability of cells plated at high density onto matrix deposited by low density cultures fell to 0.85 % of control value. This level of inhibition is about three times the experimental error of the assay procedure, and is therefore probably significant. When cells were plated at high density onto matrix deposited by high density cultures, there was an 18 % stimulation of ConA agglutinability. The rate at which low density cells regain their agglutinability when plated onto flasks conditioned with matrix from high density cultures is shown in fig. 12. It can be seen that the matrix induced stimulation is complete in about 3 h. From the above data it would appear that the extracellular matrix contains two components which can regulate the level of ConA agglutination. One component promotes agglutinability while the second inhibits it. The efficacy of conditioned media from high and low density flasks was also investigated, and was found to have no effect upon cell agglutination by ConA. Since conditioned medium factors can be ruled out as the mechanism of cooperative promotion of cell agglutination, and since high density matrix can fully restore the agglutinability of low density cells, it is not necessary to postulate
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a role for direct cell contact in the promotion of cell agglutinability. Conclusions
C, glioma cells grown in suspension possess an intrinsically high agglutinability by ConA which is not affected by cell contact or density phenomena. When cells are plated out at low density an anchorage-dependent processoccurs whereby they lose a substantial portion, though not all, of their agglutinability. Thus ConA agglutination would seem to involve at least two cellular components: (1) a residual agglutinability that is apparently not subject to density regulation; (2) a second component of agglutinability that is regulated by density-dependent phenomena. Part of the agglutinability loss by low density cells can be attributed to the production of a matrix inhibitor of agglutination, but this alone does not account for the entire loss of agglutinability. Thus in addition to the matrix inhibitor at least one additional mechanism must contribute to the loss of agglutinability at low cell density. The loss of agglutinability is dependent upon cell anchorage to a substratum, but it is not clear whether it is adhesion per se that is responsible, or whether perhaps some consequence of adhesion. Cells plated out at high density produce matrix that contains a factor which stimulates agglutinability. Recovery kinetics suggest that the surface agglutination moiety regulated by matrix-mediated cooperative cell interactions may be the same component removed by EDTA and trypsin. That EDTA can (presumably) strip this moiety from the cell surface indicates that it is not covalently bonded to other surface structures. Both the loss and reappearance of ConA agglutinability as a result of density manipulation are rapid processes requiring 3-5 h. This suggeststhat the responsible surface molecule Exptl Cell Res 92 (1975)
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(s) has a high rate of both turnover and production. This work was supported by grants from the Milheim Foundation for Cancer Research and The General Research Assistance Fund of the University of Colorado (S. J. F.) and by USPHS postdoctoral research fellowship CA-52464-01 (P. S.).
REFERENCES 1. Yaoi, Y & Kanaseki, T, Nature 237 (1972) 283. 2. Mallucci, L, Wells, V & Young, M R, Nature new biol 239 (1972) 53. 3. Skehan, P & Friedman, S J, Exptl cell res 86 (1974) 237.
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4. Cunningham, D D, J biol them 247 (1972) 2464. 5. Thorne. H V. J gen virol 18 (1973) 153. 6. Bolund; L, Dariynkiewicz, i &‘Ringertz, N R, Exutl cell res 62 (1970) 76. 7. Terry, A H & C&p, L A, Biochemistry 13 (1974) 414. 8. Benjamen, T L & Burger, M M, Proc natl acad sci US 67 (1970) 929. 9. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418. 10. Rottmann, W L, Walther, B T, Hellerqvist, C G, Umbreit, J & Roseman, S, J biol them 249 (1974) 373.
Received September 19, 1974 Revised version received October 28, 1974