growth inhibition of transformed cells correlates with their junctional communication with normal...

Cell, Vol. 44, 167-196, January 17, 1966, Copyright 0 1966 by Cell Press

Growth Inhibition of Transformed Cells Correlates with their Junctional Communication with Normal Cells Parmender P Mehta: John S. Bertram,* and Werner R. Loewenstein’ * Department of Physiology and Biophysics University of Miami School of Medicine Miami, Florida 33101 tCancer Research Center of Hawaii University of Hawaii Honolulu, Hawaii 96613

Summary

The growth of various chemically and virally transformed cell types in culture is inhibited when they are in contact with normal cell types. We show that this growth inhibition is contingent on the presence of junctional communication between the normal and transformed cells (heterologous communication), as probed with a 443 dalton microinjected fluorescent tracer. In cell combinations where heterologous communication is weak or absent there is no detectable growth inhibition; the inhibition appears when communication is induced by cyclic AMP-dependent phosphorylation, and only then. In cell combinations where heterologous communication is spontaneously strong, the growth inhibition is present, but it is abolished when the communication is blocked by retinol or mtinoic acid. The cell-to-cell membrane channels of gap junctions are the likely conduits of the signals for this growth control.

Introduction

It is as yet unknown how cells in tissues communicate with each other to control their growth. For controls not involv- ing factors transmitted through the extracellular medium, junctional communication may play a role (Loewenstein, 1979). In this communication the interiors of adjacent cells are directly interconnected by specialized membrane channels (Loewenstein et al., 1978; Loewenstein, 1981; Neyton and Trautman, 1985) which seem to be embodied by the membrane particles of gap junctions (Unwin and Zampighi, 1980; Warner et al., 1984; Zampighi et al., 1965). These junctional channels have an effective diameter of 16-20 A in mammalian cells (Schwartzmann et al., 1981) wide enough to conduct many kinds of low molecular weight cytoplasmic molecules (Loewenstein, 1981) and it is conceivable that growth-controlling signals are among them. Thus, the hypothesis has been ad- vanced that these channels transmit growth-regulating information from cell to cell (Loewenstein, 1966; 1968) and models for the control of cell populations have been pro- posed (Loewenstein, 1969; 1979; Burton, 1971). We have tested this hypothesis in coculture systems in which normal cells interact with transformed cells, inhibiting their growth.

We made use of the phenomenon discovered by Stoker (1964) that the growth of certain transformed cells is ar-

rested when they grow in contact with normal cells (see also Stoker et al., 1966; Borek and Sachs, 1966; Eagle et al., 1968). Cell interaction in this inhibition seems to be di- rect; there is no evidence that a factor in the extracellular medium is involved (Stoker, 1967; Bertram, 1979; Bertram and Faletto, 1985). Inhibition can be quantified when the transformed cells grow as colonies on top of a confluent layer of normal cells (Bertram and Faletto, 1985). Also under these conditions, cell-to-cell communication can be tested by the transfer of microinjected fluorescent tracers between the two classes of cells (Loewenstein and Kanno, 1984). We used the size of the colonies as an index of growth inhibition, and the frequency with which they transferred the 443 dalton fluorescent dye Lucifer Yellow as an index of communication.

We chose cell systems where communication between the normal and the transformed cells (heterologous communication) is weaker than between either the normal or the transformed cells themselves (homologous communication), and where the heterologous communication is amenable to long-term up- and down-regulation. By mod- ulating this communication, we show that it correlates with growth inhibition.

We used two experimental designs. In one, we cocul- tured transformed and normal cell types that exhibit a low level of heterologous communication, and raised that level experimentally by stimulating the cyclic-AMP-dependent mechanism of phosphorylation that regulates the cell-to- cell channels (Azarniaet al., 1981; Flagg-Newton and Loe- wenstein, 1981; Flagg-Newton et al., 1981; Radu et al., 1982; Wiener and Loewenstein, 1983; Loewenstein, 1985). In experiments of the other design, we took cell combinations with a high level of heterologous communication, and blocked that communication with the aid of vitamin A (retinol) or retinoic acid. (Retinoic acid blocks metabolic cooperation between cells; Pitts et al., 1981; Walder and Lijtzelschwab, 1984.) Thus, we asked whether establishment of asufficient heterologous communication will lead to growth inhibition of the transformed cells, and whether disruption of such a communication will release the cells from inhibition.

Results and Discussion

Induction of Communication Leads to Growth Inhibition Figure 1 illustrates a set of experiments designed to en- hance communication between normal and transformed cells. One hundred transformed MCA4B cells were seeded over a confluent layer of normal lOTV2 cells in each dish, and the resulting colonies were examined after 7 days. The partners in this cell combination belong to a class of cells that do not establish heterologous communication readily (Fentiman et al., 1976; Pitts and Burk, 1976). In normal medium very few of the transformed cells injected with the Lucifer tracer passed it on to normal cells; in the example illustrated there was no transfer to any nor-

Cell 100

a b C d

Figure 1. Growth of Transformed MCA4B-TlOT% Cells and Their Junctional Communication with Normal 10T1/z Cells

(I) Colonies (stained) of the transformed cells grown (top row) alone and (second row) in coculture with the normal cells. (a) Control; (b) methyl xanthine, 1O-4 M; (c) Ro 20-1724, 1O-4 M; (d) forskolin, 1O-5 M. The cultures were in the control or drug-containing medium for 7 days, from the time of the transformed cells’seeding (100 cells/dish). (II) Samples of junctional probings with the MW 443 fluorescent dye Lucifer Yellow in the cocultures. (e) Control; (f) forskolin-treated. Transformed cells are marked with fluorescent MW 40,000 dextran, which they endocytose. The arrows indicate the transformed cells injected with Lucifer. From left to right: phase-contrast photomicrograph showing the transformed cells on the confluent normal-cell layer; darkfield showing the dextran marker (concentrated around the nuclei) of the transformed cells; and darkfield showing the spread of Lucifer, after microinjection. In the control (a) Lucifer has spread to transformed cell neighbors but not to normal cells. (The Lucifer-containing neighbor below the extreme left arrow is transformed; its dextran marking shows up very faintly.) In the treated condition (f) Lucifer has spread to normal cells in addition.

mal neighbor (Figure 1, Ile, far right). In this condition the transformed cells grew into numerous and relatively large colonies (Figure 1, la, second row). In medium containing the adenylate-cyclase activator forskolin, however, there was extensive heterologous transfer (Figure 1, Ilf) and there were fewer and much smaller transformed-cell colonies (Figure 1, Id).

This result was typical of the five cell combinations of this sort studied: namely, 1OTX cells in association with

their chemically transformed counterparts MCA, MCA46, and MCA4D and in association with their virally transformed counterpart SV4OC98, and normal 3T3A31 cells in association with the transformed MCA cells. The heterologous communication frequencies in these cell combinations ranged from 0%-12% in normal medium, but the homologous communication frequencies were in all cases close to 100%.

When heterologous communication was so poor, there

yg;-to-Cell Communication and Growth Inhibition

Table 1, Growth Inhibition of Chemically and Virally Transformed Cells upon Induction of Communication with Normal iOTr/2 Cells

Coculture of Transformed Cells with Normal lOT% Transformed Cells Alone

-____ Growth Growth

Experiment Transformed Treatment Communication Inhibition Colony Size’ Inhibition Colony Size* Number Cell Type 64 Frequency (%) (%) (mm2) W) (mmZ)

1 MCA4STlOYz Forskolin 0 0 0 38.0 f 3.4 (51) 0 38.6 k 3.2 (76) 1 50 69 11.6 k 1.5 (64) -17 45.3 f 5.5 (51) 5 60 77 8.9 f 1.1 (80) -33 51.5 f 4.6 (63)

10 100 85 5.6 k 0.7 (65) -41 54.6 A 4.3 (82)

2 Ro 20-l 724 0 0 0 63.4 f 12.3 (10) 0 45.2 f 9.8 (3) 10 36 39 36.6 f 6.7 (10) -45 65.4 f 11.9 (7)

100 41 65 22.4 f 4.0 (10)

3 Methyl Xanthine 0 0 0 66.8 -c 3.9 (75) 0 75.2 f 3.3 (95) 100 33 36 42.7 f 2.4 (143) 6 70.9 f 3.3 (115)

4 SV40-Tl O% Forskolin 0 9 0 97.3 f 5.9 (122) 0 75.1 f 11.4 (42) 1 70 46 52.4 f 3.9 (140) 24 56.8 f 15.4 (34) 5 63 49 49.6 2 4.6 (83) -4 78.3 ” 37.2 (6)

10 100 52 46.7 f 4.0 (125)

5 RO 20-l 724 0 10 0 78.7 f 4.7 (114) 0 34.7 5z 5.5 (17) 10 72 46 41.0 f 3.1 (144) -45 50.1 ” 7.4 (15) 50 a7 49 40.0 f 4.0 (106) -2 35.2 f 6.4 (18)

100 100 52 31.6 f 2.5 (139) -156 88.6 f 12.9 (9)

Communication frequency: the proportion of transformed cells tested exhibiting transfer of fluorescent tracer to normal cells. Growth inhibition: the percent reduction of the mean colony area with respect to the mean colony area of the untreated control (0 vM). * Mean area & SE.; in parenthesis the number of colonies analyzed. In the cocultures, all values of communication frequency and growth inhibition are statistically different from the corresponding values of the untreated controls at confidence levels, P < 0.02 (in most cases < 0.001) and < 0.000001, respectively (standard T test). In the transformed ceils growing alone, the two sole cases with positive values of growth inhibition (experiments number 3 and number 4, 1 vM) are not statistically significant (P > 0.15). The data of communication frequency and colony size for each experiment, in this and the following tables, except for Table 3, are from the same culture dishes. Seedings of transformed cells/dish: Experiment Number 1, 150; Number 2, 175; Numbers 3-5, 200. Colony size and communication frequency determined at: Experiment Numbers 1 and 5, day 7; Numbers 2 and 3, day 10; Number 4, day 8.

was no sign of growth inhibition of the transformed cells; the size of the transformed cell colonies on top of normal cells was not smaller than when they were grown in the absence of normal cells. Upon treatment of the cocultures with forskolin or with the phosphodiesterase inhibitors Ro 20-1724 or isobutyl methyl xanthine, the frequency of communication between the normal and transformed cells in- creased and, hand in hand, the growth of the transformed cell colonies decreased. Both effects were marked and dose-dependent (Figure 1 and Table 1). In the MCA4Bl 1OTVz coculture, for example, 10 ,uM forskolin caused the frequency of communication to rise from 8% to 100% and the colony size to fall from 88 mm2 to 5.8 mm2-a growth inhibition of 85%.

In all of these cell combinations, the growth inhibition also showed itself in the number of transformed-cell colonies; there were fewer colonies discernible after the drug treatments when the cells were allowed to grow for 5-7 days. As an index of inhibition, however, the colony size was useful more generally, because the cloning efficiency in the treated condition was not very different from that in the untreated controls; with longer growth periods, the difference in the number of colonies in the two conditions became smaller as more treated colonies reached a discernible size. With the colony size as an index, we could evaluate the data from growth periods both short and long. The data from the different experiment series in Ta-

ble 1 and Table 2 cover short and long periods (see table legends), which accounts for the variation in the colony areas of the controls. Since within each series the growth periods were always constant, the normalized (%) values of growth inhibition are entirely comparable throughout the present work. Our method, however, tends to underes- timate the inhibition as it measures colonies only above a minimal size, the threshold size of our digitizing system. In the treated condition the number of subthreshold colonies was still abundant after long periods of growth, whereas in the untreated controls virtually all colonies al- ready were above threshold. The method thus biased the mean values of colony size against the trend we were try- ing to establish.

Even so, growth inhibition correlated with heterologous communication in all five cell combinations, regardless of the drug used to raise the level of communication. Figure 2 displays the relationships between growth and communication frequency for the five cell combinations; each plot pools the data from the various drug treatments for a given cell combination. All plots show the same basic feature: a threshold of communication frequency (about 2001640%) where growth becomes inhibited. Moreover, growth inhibition increases with increasing communication frequency.

The relationship between growth inhibition and communication frequency was shown most clearly by an analysis

Cell 190

Table 2. Growth Inhibition of Transformed MCAlO-TlO%T Cells: A Clone that Communicates with Normal IOTB Cells Spontaneously

Transformed Cells Coculture with IOTVz Alone R

Experiment Treatment Communication Colony Size cl Colony Size cp Number V4 Frequency (%) (mm*) (mm2) w

1 Control 0 73 169.5 rt 9.7 (61) 295.7 f 30.5 (27) 43 Forskolin 1 67 156.9 f 9.2 (47) 351.6 f 19.95 (34) 55

10 71 162.4 f 25.0 (17) 342.4 f 17.36 (30) 53

2 Control 0 63 37.1 + 2.9 (76) 125.1 f 9.8 (46) 70 Forskolin 1 00 28.6 f 2.6 (73) 192.6 2 15.7 (40) 65

5 75 31.8 f 3.3 (66) 169.6 f 13.9 (41) 61 10 69 31.9 f 3.6 (54) 167.1 f 10.2 (51) 61

R = 100 (cs- ct)/cs, a measure of growth inhibition using the size of the transformed cell colonies growing alone (cs) as a comparison basis for each condition (instead of the colony size of the untreated control, as in Table 1). R is statistically significant at a confidence level P < 0.0002 in all cases. There is no significant difference between the values of communication frequency in the treated condition and those of the corresponding controls (P b 0.2) or between cl-treated and controls (P B 0.1). Seedings of transformed cells/dish: Experiment Number 1, 200; Number 2, 225. Colony size and communication frequency determined at: Experi- ment Number 1, day 12; Number 2, day 6.

of variance of the individual growth-inhibition values (un- derlying the plotted means) classified by the communication frequency. Such analysis was performed for each cell combination and followed by linear regression analysis. The correlations between growth inhibition and heterologous communication frequency were highly significant in all five cell combinations (even the correlations of the non- zero data alone were significant) (Figure 2, bottom inset).

The drugs had no growth-inhibiting action by themselves. In fact, in the absence of normal cells, the growth of the transformed cells was sometimes enhanced by the drugs (Figure 1 and Table 1).

In this connection we note the results obtained with cell combinations whose heterologous communication proved refractory to the drugs. We encountered three combinations of this sort: MCA-TlOTVz paired with the normally growing rat liver cells, transformed mouse Cl-1D cells paired with 1OTVz cells, and Rous sarcoma virus transformed vole 1T cells paired with 1OTVz. In all three combinations the cyclic-AMP-elevating drugs failed to in- duce detectable heterologous communication and also failed to produce growth inhibition, in agreement with the basic outcome of the work with the drug-responsive cell combinations.

Block of Communication Leads to Growth Disinhibition For the second line of experiments we chose a clone of transformed TlOT% cells, MCAlO-TlOT%, which commu- nicated well with the normal lOTV2 cells in (untreated) coculture. The heterologous communication frequency was quite high; for example, in the experiments of Table 2, this frequency (63%-73%) was not far from that of the cell combinations of Table 1 after drug treatment. Thus, if junctional communication with normal cells mediates growth inhibition, one would expect this transformed clone to grow less in coculture than by itself. Indeed, the colony size was much smaller in cocultures (cl); the growth inhibitions (R) amounted to 43% and 70%, repre- sentative values for coculture of these cell types.

In this case, where a high heterologous communication frequency was present to start with, forskolin did not raise that frequency, nor did it inhibit the growth further than did coculture per se (Table 2). This is the correlation expected if the degree of growth inhibition depended on the degree of heterologous communication.

Next we examined the effect of blockage (uncoupling) of the heterologous junctional communication. An agent capable of long-term uncoupling was needed. We tried the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which has been found to uncouple mammalian cul- tured cells (Yotti et al., 1979; Enomoto et al., 1981; Murray and Fitzgerald, 1979); however, this chemical uncoupled our cells only transiently (at most for a few hours) in the present conditions. Among various other agents tried, retinoids proved the mildest and most useful for long-term uncoupling: retinol at 0.3 PM and retinoic acid at 0.1-l PM, continuously applied for 3 days, reduced the heterologous communication frequency to 0%-10% (Table 3) without affecting the nonjunctional membrane permeance of the junctional tracer (see Experimental Procedures). The treated cells looked healthy, kept growing, and stayed in contact (cell contact, homologous and heterologous, as seen in phase contrast actually seemed to be more extensive; the cells became flatter). The communication frequencies of the more strongly coupled homologous junctions remained close to 100%. (Presumably the homologous junctions contained more channels than the heterologous junctions, both in the untreated and in the treated condition, and the relationship number-of-open- channels/communication-frequency was saturated here. Then, even when the fraction of channels blocked by the retinol compounds in homologous junctions is the same as that in heterologous junctions, the decrement in homologous communication frequency would be smaller or even undetectable by our method.)

Blocking of the heterologous junctions led to growth disinhibition. Table 3 summarizes typical results obtained with the combination of MCAlO and lOTV2 cells treated with retinol or retinoic acid (from the moment the trans-

Cell-to-Cell Communication and Growth Inhibition 191

100

r

(a) MCA TlOT~/lOT~

z -

COhlMUNI;ATION FREQUENCY %

(d)SW C98 TlOT+/lOT+

COMMUNICATION FREQUENCY %

Figure 2. Growth Inhibition vs. Communication Frequency

(b) MCA4B TlOT+/lOT+ (c) MCA4D TiOT+/lOT+

85 80 6

c (e) MCA TlOT+/3T3

0 Control r-l

l Forrkolin 13 RO-20.1724 q Methyl Xonthine

- a b

i e

Srrt P Pt 0 85 0.47 o 20 <O 000001 <OOOOOOI

086 0 65 013 ~0000001 < 0008

0.72 0.53 C’I ~0 000001 < OOOOOOI

062 062 007 <0000001 coo2

0.81 0 58 ~36 <O 000001 < OOOOOO~

The plots pool the data from the untreated controls (open circles) and from the treatments with forskolin (solid circles), Ro 20-1724 (half-solid circles), and methyl xanthine (squares) for each transformed cell/normal cell combination. Plotted are the mean values of growth inhibition (standard error bars) and the communication frequency. The number of cell colonies measured for growth inhibition is given next to the data points. The individual growth-inhibition values categorized by the communication frequency were subjected to an analysis of variance, followed by a linear regression analysis of the relationship of growth inhibition vs. frequency of communication. The analyses were performed for two conditions: including all data (the curves shown) and excluding the data of zero inhibition. The bottom inset lists the slopes of the computed curves (s), the correlation coefficients (r) and the statistical confidence levels (P) of the correlations as computed by analysis of variance. The values with subscript t correspond to the curves excluding the data of zero inhibition.

Table 3. Blocking Communication between MCAIO-TlOTVz and IOTYz Cells by Retinoids

Treatment

CPM)

Control

Retinoic Acid

Retinol

0

0.07 0.7 0.3

Coculture with IOTVz

Communication Frequency (%)

33 (40)

7 (43) 0 (37)

10 (41)

Colony Size

(mm?

15.8 AZ 1.7 (59)

39.7 f 3.2 (40) 45.7 f 3.6 (62)

Transformed Cells Alone

- Colony Size

(mm21

85.8 f 6.8 (48)

60.2 f 3.0 (61)

In parentheses are the number of injection trials and the number of colonies analyzed. The data of communication frequency and colony size in this table are from different experiments. All values in the treated condition are significantly different from those in the control. (Communication frequency: retinoic acid, P d 0.007; retinol, P = 0.02. Colony size: P < 0.0006.) Seedings of transformed cells/dish: 30,000, for communication frequency determination; 150, for colony size determination. Communication frequency and colony size were determined at days 3 and 7. respectively.

formed cells were seeded). The transformed-cell colonies were markedly enlarged compared to those of the untreated controls in the high-communication condition. The transformed cells behaved as if their junctional uncoupling from the normal cells had released them from the growth-inhibiting influence of those cells.

In a variant of these experiments, the retinoids were used on cell combinations with low heterologous communication, like those of Figure 2, and were applied together

with drugs stimulating cyclic-AMP-dependent phosphorylation, such as forskolin, Ro 20-1724, or dibutyryl cyclic AMP? Here an increase of heterologous communication, the usual response to these drugs, did not develop (the retinoids often even abolished whatever little heterologous communication there was); and, importantly, the growth-inhibiting response did not develop. Table 4 shows this for the MCMDMOT% combination.

None of these effects on growth are attributable to an

Table 4. Blocking induction of Heterologous Communication by Retinoids, MCA4D-TlOTM/lOTM

Treatment (MM) Communication Colony Size Frequency (o/o) (mm*)

Control Forskolin Ro 20-l 724 dbcAMP’ Retinoic acid Forskolin + Retinoic

Acid Ro 20-1724 +

Retinoic Acid dbcAMP + Retinoic

Acid

0 12 37.0 2 3.4 (31) 10 67 4.7 f 0.5 (43)

100 79 3.8 f 0.3 (36) 500 74 5.6 r 0.4 (39)

0.7 7 36.9 r 1.9 (52) 13 30.0 r 1.7 (61)

0

0 34.7 e 1.7 (61)

30.8 f 1.4 (64)

* Dibutyryl cyclic AMP. All values of communication frequency and colony size in the treatments with forskolin, Ro 20-1724, and dbcAMP are statistically different from the value of the control (P < 0.0006), but none of the values in treatments combined with retinoic acid (including retinoic acid alone) are (P 2 0.09). The concentrations for the drug combinations are the same as those for each drug alone. Seedings of transformed cells: 175 cells/dish. Colony size and communication frequency were determined at day 8.

action of the retinoids independent of their action on junctional communication. At the concentrations used, the compounds did not increase the MCA-10 colony size when these transformed cells were growing alone (Table 3), nor did they significantly change the colony size of MCA4D cells in coculture with normal cells with very low heterologous communication (Table 4) or when those transformed cells were alone. They also do not affect the growth rate of lOTV2 cells (Bertram, 1983; Mordan and Bertram, 1983).

We do not know by what mechanism the retinoids block communication, but it is not simply by a reduction of the cells’ cyclic AMP levels: retinol did not alter the constitu- tive levels of cyclic AMP in the normal or transformed cells (lOTV2, MCA4D, and MCAlO), although retinoic acid pro- duced a decrease; and, in the presence of either retinol or retinoic acid, the elevations in the cyclic AMP levels of the normal cells caused by Ro 20-1724 or forskolin, al-

though less than in the absence of the retinoids were still more than sixfold. In the transformed cells, Ro 20-1724 and forskolin caused only relatively small elevations and these were not very different in the presence of the retinoids (Table 5).

Models and Conclusions The most important conclusion to emerge from these results is that the growth inhibition of the transformed cells depends on the junctional communication with their normal-cell partners. This is in line with the hypothesis that the cell-to-cell channels transmit cytoplasmic growth-regulating molecules (Loewenstein, 1988) and with its simple corollary that blockage of these channels causes deregu- lation (Loewenstein, 1989). The results reported here are compatible with such molecules being either inhibitory or stimulatory signals. We suggest two models to accommo- date these alternatives.

The basic feature of the models is a pervasive but vari- able connectivity (Figure 3): all cells participating in growth inhibition are in junctional communication with each other; the communication between the normal and transformed cells is weaker than the homologous commu- nications, and its modulation varies the degree of growth regulation.

One of the models (I), the simplest expression of the hypothesis (Loewenstein, 1988), operates with inhibitory signals; here the (growth-arrested) normal cell population is the signal source. The growth-inhibiting signals are conducted by the cell-to-cell channels to the transformed cell partners, and from there are disseminated by the channels throughout the transformed cell population. In this connection it is interesting that the transformed cells inter- acting with the normal cells are arrested at G1 or Go (as determined by flow cytometry; see Experimental Proce- dures), the same stage in the cycle where normal confluent cells are arrested.

The other model (II), leaning on an earlier more general scheme (Loewenstein, 1989; 1979), operates with stimulatory signals. The transformed cells here are the source of growth-stimulating signals which are conducted to the normal cells and diluted by that large population to below threshold concentration for growth stimulation.

Table 5. Cyclic AMP Levels in Normal and Transformed Cells (%) and in Their Culture Medium

1 OT%

Treatment bM) Cells Medium

Control 0 1008 I oob Retinal 0.3 107 58 Retinoic Acid 0.7 107 58 Ro 201724 100 2,532 5,211 Forskolin 10 10,488 10,480 Ro 20-1724 + Retinol 734 4,169 Ro 20-1724 + Retinoic Acid 582 1,667 Forskolin + Retinol 839 2,620 Forskolin + Retinoic Acid 1,154 2,305

pmol/106 cells f S. D.: =1.35 f 0.8; c1.8 f 0.27; e0.96 f 0.02. pmol/lO ml f SD.: b18.33 f 4; d6.32 f 0.6; ‘1.67 f 0.4. Determinations on confluent cells in 10 ml medium 72 hr after treatment.

MCA4

Cells

1 ooc 92 92

114 135 117 138 200 158

Medium

1 OOd 101 101 118

71 114 139 188 182

MCAlO

Cells

1ooe 113 113 161 228 254 237 342 271

Medium

100’ 106 106 199

52 330 233 214

65


PI a- Ts-- -------

i-l --- __- ------___

-I I I ,‘I’, I I r 432101234

Figure 3. Models

1 I I I I I , , ,

432101234

b

- - - - - - - - - - - - - - - - - - - - - - - - - -_-- -

1 I I I , I I , ,

432101234

The center diagrams apply to both models described in the text. They represent two states (a, b) in the connecting network between normal (white) and transformed cells (gray). The bridges symbolize connections (junctional permeabilities) sufficient for effective growth inhibition; homologous connections are diagrammed as solid bridges. The weaker heterologous connections (stippled)-the regulator switches in the network-undergo transition between permeability states(b) sufficient and (a) insufficient for growth inhibition. The plots represent the corresponding steady-state intracellular concentration profiles of growth-inhibiting signals(i) generated by the normal cells (model I) or of growth-stimulating signals(S) generated by the transformed cells (model II): abscissae, cell order; ordinates, signal concentration; T, the concentration thresholds for inhibition or stimulation of growth. Growth inhibition of the transformed cells ensues in state b as i flows into the transformed-cell compartment and the local [i] rises above T,; or, in model II, as S flows into the large normal-cell compartment and [S] fails below T,.

In either model the crucial link in the communication network is the junction between the normal and the transformed cells. It is here where our modulators of communication would be effective in switching on or off growth inhibition of the transformed cells. Thus, in either model establishment of sufficient communication at this junction-that is, the formation or opening of a sufficient number of channels-spontaneously (as in the case of the MCAlO/lUl% coculture) or induced (by activators of cyclic AMP-dependent phosphorylation) would effectively lead to growth inhibition. Conversely, disruption of that communication (as by the retinoids) would lead to disinhibition.

Fusion of many types of normal diploid cells with tumor cells from the same or different species gives rise to hybrids in which the transformed phenotype is sup- pressed, so long as the hybrids retain the chromosomes of the normal parent cell (Harris et al., 1969; Klein et al., 1971; Azarnia and Loewenstein, 1977; Harris, 1961; Howell and Sager, 1961; Stanbridge et al., 1962; Klinger and Shows, 1983; Sager, 1985). The normal cells seem to generate a highly conserved signal capable of suppress- ing the transformed state. It is conceivable that this is the same signal molecule that is transferred here from cell to cell. If so, our results would set constraints on the size of the molecule: it must be small (ltss than about 2000 daltons) to fit through the 16-20 A cell-to-cell channel

(Schwartzmann et al., 1981) and hence cannot be a protein.

Retinoids are known to be potent inhibitors of carcinogen-induced transformation (Sporn and Newton, 1979). In the lOT% line retinol, but not retinoic acid, inhibits methylcholanthrene-induced transformation (Bertram, 1980). This effect is probably unrelated to junctional communication since both compounds reduce communication. However, the effect on junctional communication may explain why, under certain circumstances, retinoic acid enhances transformation in vitro (Bertram, 1980) and tumor development (Forbes et al., 1979; Verma et al., 1980).

Apart from their physiological implications, our findings directly bear on the cancer problem. The results intimate that the growth of cancer cells can be restrained by junctional communication with normal cells. Many of the transformed cells we used form tumors in animals (J. S. B., unpublished). The possibility thus emerges that junctional communication with normal cells plays a part in organismic defenses against the development of tumors, including defenses against latent metastases. The findings also open a possible new avenue for the treatment of cancer by promoting junctional communication between cancer cells and normal ones. Although this approach would not be expected to work with the kind of cancer cells that are

Cell 194

communication-incompetent (Loewenstein, 1979), it might work with the kind that is communication-competent (Furshpan and Potter, 1966; Borek et al., 1969; Sheridan, 1970; Azarnia et al., 1974; Atkinson et al., 1981; Loewen- stein, 1985) and susceptible to enhancement of heterologous communication by chemical treatment.

Experimental Procedures

Cell Culture We used the folllowing cell types: normally growing mouse iOT% cells (passage 9-18) (Reznikoff et al., 1973) 3T3A31 cells (Aaronson and Todaro, 1966) and rat liver cells (A. T. C. C. CRL 1439); methylcholanthrene @CA)-transformed lOT% cells, MCA-TlOT% (Reznikoff et al., 1973), clones 48, 44 and 10; Simian virus 40-transformed SV4OC98- TlOT% cells (Sompayrac and Danna, 1963); mouse Cl-ID cells, a transformed L subline (Dubbs and Kit, 1964; Azarnia et al., 1961); and the Rous sarcoma virus-transformed vole strain IT (Krzyzek et al., 1977). The rat liver cells were epithelioid; all others were fibroblasts.

The cells were grown in Eagle’s basal medium supplemented with 10% heat-inactivated fetal calf serum (Gibco) in 60 mm plastic dishes (Falcon). They were protected from light <500 nm in the experiments with retinol compounds. The normal cells were seeded at 5 x lo4 cells per dish and grown to confluence (7-10 days). The transformed cells were seeded, in fresh medium, on top of the confluent normal cell layer or directly on the plastic dish surface for controls. The stocks were checked to be free of mycoplasma, by means of the staining method with Hoechst 33256 (Chen, 1977).

Communication Assay The fluorescent dye Lucifer Yellow (Sigma) was injected iontophoreti- tally or hydraulically into the cells with the aid of a micropipette. For determination of heterologous junctional communication, the transformed cells at the colony borders were injected and the spread of the Lucifer fluorescence to the cell neighbors was viewed in the micro- scope darkfield. We determined the frequency with which the transformed cells transferred the dye to the normal cells, namely the proportion of the injection trials in which, within 8 min, there was transfer to one or more normal cells (communication frequency). For each individual trial the criterion for communication, thus, was simply yes or no-a criterion that gained by the fact that the transfer, when detectable at all, usually was to at least three normal cells. When there was no heterologous transfer, the presence of homologous transfer (99% frequency) (e.g., Figure le) provided a useful criterion against artifac- tual uncoupling (Socolar and Loewenstein, 1979); cases with neither heterologous nor homologous transfer- < 1% of all cases studied- were rejected. For each communication frequency value, up to 67, but usually 10-18, transformed cells were injected in randomly chosen colonies (1-2 injection trials per colony, 2-3 colonies per culture dish, 2-3 parallel dishes). Ten to eighteen trials provided an adequate sam- ple; despite the larger sampling error, the corresponding communication frequencies were entirely comparable to those based on 60-67 trials, as tested in four experiments on parallel cultures for four experimental conditions. The frequency data in the tables and figures are based on IO-18 trials, unless stated otherwise.

The transformed cells were readily distinguishable from the normal ones by their morphology. To remove all doubts, the transformed cells were marked in some experiment series with rhodamine-conjugated latex beads (Polyscience) or with fluorescein isothiocyanate-conjugated dextran (m 40,000 dalton; Sigma), which the cells endocytosed (Figure 1). To avoid excessive dilution of the markers over the cell generations, the transformed cells were seeded at 20,000-30,000 per dish, and communication and growth were assayed after 3 days. In all other series the cells were seeded at 100-200 cells per dish and the assays were performed after 7-12 days.

The latex and dextran markers are too large to be admitted by the cell-to-cell channels (Schwartzmann et al., 1981) and when care was taken to wash the cells free of nonendocytosed marker before coculture, they did not exchange the marker via the medium. This was ascer- tained in runs in which the transformed-cell partner was labeled with the green fluorescent dextran and the normal partner with the red fluorescent beads. The cell marking did not affect the communication frequency or the growth rate of the cells.

Growth Assay Following the determinations of communication, the cultures were fixed with 10% formaldehyde and stained with 0.2% crystal violet. The areas of the individual transformed-cell colonies were then measured. The colony areas correlate with the number of clonogenic cells in soft agar (Bertram, 1977). For each experiment, the mean colony area of the treated condition was compared with that of the control (Figure 1). The percent reduction of the mean colony area relative to that in the untreated controls provided the measure of growth inhibition. A Ladd Graphical Digitizer coupled to a computer, programmed to integrate areas from the digital coordinates, measured the individual colonies, projected with sixfold magnification. The threshold of the system was set at 0.5 mm2 (about 50100 transformed cells, depending on morphology and packing).

Drugs Ro 20-1724 (Hoffmann-LaRoche) and forskolin (Calbiochem) were dissolved in dimethyl sulfoxide and ethanol, respectively. The final concentration of the solvents in the growth medium was 0.2% and O.l%, respectively; the same concentration was used for the controls. Retinol and retinoic acid (Eastman Kodak) were dissolved in ethanol (final concentration < 0.07%). lsobutyl methyl xanthine (Sigma) was directly dissolved in the medium. Control and drug-containing media were renewed every third day.

Upon treatment with these drugs, the communication frequency rose within a few hours, eventually reaching a plateau. The measurements of communication for the present work were all taken in the plateau phase.

Tests of Nonjunctional Permeability For all the aforegoing drugs we checked, in special runs, that their effects on communication frequency were not caused, indirectly, by al- terations of nonjunctional membrane permeability. This was ascer- tained by photometric measurements (Flagg-Newton et al., 1981) of the rates of loss of Lucifer fluorescence from the cells in the treated and untreated condition. None of the drugs affected these rates at the concentrations used for the experiments.

Cyclic AMP Assay The cyclic AMP content of the cells and medium was measured by ra- dioimmunoassay. The procedures for extraction and quantitation are described elsewhere (Bertram, 1979). The measurements were performed in duplicate on parallel confluent cultures for each experimental series. Intracellular cyclic AMP concentrations were calculated as pmol/106 cells. The number of cells was determined by electronic par- ticle counting (Coulter). For ease of comparison the data are tabulated as percentages of the controls.

Flow Cytometry For flow cytometry, transformed cells labeled with green fluorescent microbeads were seeded at IO5 cells per dish onto confluent mono- layers of 1OTQ cells or on the dish surface. After allowing 4 hr for plat- ing, cultures were treated with Ro 20-1724 or forskolin; after 24 hr in drug-containing medium or drug-free control medium, the cultures were trypsinized, fixed in methanol, and stained with red fluorescent propidium iodite. Cell suspensions were then passed through a flow cytometer and DNA profiles (red fluorescent) were determined only in cells emitting green fluorescence.

Acknowledgments

We thank Dr. Birgit Rose for discussion and advice, Dr. R. C. Duncan for analysis of variance, Mr. T. Lopez for assistance in the measurements of colony areas, and Dr. R Sorter, Hoffmann-LaRoche, for a gift of Ro 20-1724. This work was supported by research grants CA14464 and CA39604 from the National Cancer Institute, U. S. National Insti- tutes of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact.

Received September 16, 1985; revised October 18, 1985


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