J. Cell Sci. I, 297-310 (1966) 297

Printed in Great Britain

GROWTH INHIBITION OF POLYOMA-

TRANSFORMED CELLS BY CONTACT

WITH STATIC NORMAL FIBROBLASTS

M. G. P. STOKER, MOIRA SHEARER AND C. O'NEILLMedical Research Council Experimental Virus Research Unit,Institute of Virology, University of Glasgow

SUMMARY

The growth of polyoma-transformed BHK21 cells was studied in mixed cultures with normalmouse fibroblasts. On coverslips in excess medium, normal fibroblasts undergo one or twodivisions after the cells become contiguous. Py-cell growth was not inhibited by contact withconfluent fibroblasts which were still dividing, but the Py cells were rapidly inhibited aftercontact with fibroblasts which had become static. Further experiments confirmed the earlierview that the inhibitory effect was not due to a general change in the medium but was onlybrought about when cells were in contact or close proximity.

INTRODUCTION

Confluent cultures of freshly isolated normal cells on solid substrates are usuallysubject to two types of inhibition, of movement and of growth. The inhibition ofmovement was originally described by Abercrombie & Heaysman (1954) and isapparently caused by contact between cells. Inhibition of growth on the other hand isa distinct phenomenon which is not always correlated with inhibition of movement.It results in cell numbers reaching a maximum, or saturation density, which isunaffected by replenishment of medium. This has been shown by Levine, Becker,Boone & Eagle (1965), working with human diploid cells, to be accompanied bydepression of synthesis of deoxyribonucleic acid, ribonucleic acid and protein.Sensitivity to inhibition of growth varies with different cell types; for example the3 Tj line of mouse cells (Todaro & Green, 1963) and certain other epithelioid cellsshow a saturation density much lower than that of normal fibroblasts.

Hamster cells transformed by polyoma virus, derived from freshly isolated fibro-blasts of the BHK21 continuous cell line, are not subject to inhibition of movementwhen in contact with one another in crowded cultures. As reported in a previous paper,however, they are subject to inhibition of both growth and movement when they arein contact with normal mouse or hamster fibroblasts in mixed cultures (Stoker, 1964).Contact inhibition of movement between transformed BHK21 cells and normalcells has been confirmed by Ambrose & Shepley (personal communication) usingmicrocinematography, and has been observed independently by Barski & Belehradek(1965) with another type of tumour cell.

This paper reports further investigations on the inhibition of growth of polyoma-

298 M. G. P. Stoker, M. Shearer and C. O'Neill

transformed BHK21 cells which are in contact with normal mouse fibroblasts. It isconcerned in particular with the conditions required for normal cells to exert theinhibitory effect on transformed cells.

METHODS

Cells

Media and culture methods were as described previously (Stoker, 1964) unlessotherwise stated. Mouse embryo cells (designated ME cells) were used after one or twosubcultures. The growth medium was modified Eagle's medium with 10% tryptosephosphate broth and 10% uninactivated calf serum. Cells were suspended in 0-5%trypsin in versene. Two clones of polyoma virus-transformed BHK21 cells (designatedPy cells) were used, both grown for fewer than 50 generations since original transfor-mation and cloning. These two clones behaved similarly and will not be distinguished.

Growth studies with mixed cultures

ME cells from primary and secondary cultures were seeded at different densities in50-mm plastic dishes. For confluent cultures 4 x io6 cells, and for sparse culturesio5 cells.were added per Petri dish (2 x io5 and 5 x io3 per cm2). After 18 h no surfacewas visible in confluent cultures, whereas sparse cultures consisted mainly of baresurface which did not become covered for the duration of the experiments. Countsshowed that half to two-thirds of the added cells had attached and spread. At suc-cessive time intervals the medium was removed and 2 x io5 Py cells were added infresh medium. Control cultures of ME cells were subjected to the same procedures butwithout addition of Py cells.

In the earlier experiments the entire bottom surface of a dish was used with 5 mlmedium, giving a ratio of medium volume to available cell-growth area of 0-3 mlper cm2. Most experiments, however, were done in a higher relative volume of mediumby using glass coverslip cultures in Petri dishes, so that the ratio of medium to cell-growth area was 4 ml per cm2. For this ME cells were pipetted in 0-2 ml of medium onto round 1-3 cm2 coverslips. This volume does not overflow the edge and allows cellsto settle over the whole surface. For confluent cultures 2-4 x io6 cells were added percoverslip (1-5-3-0 x io5 per cm2), and for sparse cultures io4 cells (8 x io3 per cm2).Some experiments were done using overcrowded cultures with I -6XIO 6 cells percoverslip (1-2 xio6 per cm2). Confluent and overcrowded cultures produced con-tinuous sheets of cells with no bare surface visible. In sparse cultures most of thesurface was bare. As with whole Petri dishes, between half and two-thirds of the cellsadded attached to the glass. After 5 h the coverslips were placed in 5 ml of medium in50-ml dishes. At successive time intervals, 1-4 x io5 Py cells were added to the mediumallowing a calculated I-I x io4 cells per coverslip (8-5 x io3 per cm2). Five hours later(when previous studies had shown Py cells to be firmly attached to the ME cells orbare surface), the coverslips were transferred singly or in pairs (confluent or crowdedand sparse) to new dishes containing 5 ml of fresh medium. Control coverslips weresimilarly manipulated but were not exposed to Py cells.

Growth inhibition of transformed cells 299

For estimates of the number of Py cells in the mixed cultures, the carbon-markingtechnique used previously was not satisfactory because of carbon loss in successivegenerations, and two other methods were used. In the total count method the totalnumber of cells per mixed culture was determined in duplicate cultures after suspen-sion and counting in a haemocytometer chamber. (Coverslip cultures were transferredto small bottles for suspension.) More than 150 cells were counted per culture exceptfor some sparse cultures on coverslips where fewer cells were available in the countingchambers. Similar counts were made on equivalent duplicate pairs of control culturesof ME cells without Py cells, and the number of Py cells in the mixed cultures wasthen estimated by subtraction of the mean counts.

In the colony count method advantage was taken of the ability of Py cells but notME cells to grow in agar suspension culture (Sanders &Burford, 1964; Macpherson &Montagnier, 1964). Cells from duplicate mixed cultures were suspended and the totalcells from each culture, or an appropriate proportion, were then mixed with 1-5 ml of0-34% agar medium at 44 °C and plated again in duplicate on a preformed base of°"5 % a 8 a r medium. To provide a uniform feeder effect and to mask differences due tounequal numbers of ME cells from the mixed cultures, a large excess (io6) of freshME cells was added to each agar culture. After 6 or 7 days of incubation at 37 °C, 80 ormore colonies of Py cells were counted per plate.

The total count method has the disadvantage that it is somewhat inaccurate and itassumes that the number of ME cells in the controls is the same as in the mixed cul-tures. The selective colony count method is more accurate and gives a direct estimateof the number of Py cells. It still depends on an assumption that the plating efficiencyof Py cells is unaffected by conditions in the mixed cultures. However, it will be seenfrom the experimental findings (Figs. 2, 4) that the plating efficiency of Py cells inagar was not markedly affected, at least by the initial number of ME cells.

RESULTS

Petri dish cultures

Py cells were added to 18-h confluent and sparse Petri dish cultures of ME cells, andtotal cells were then counted after suspension of duplicate cultures on successive days(total count method). Table 1 shows the results, including counts on duplicate platesof ME cells alone. Fig. 1 gives the calculated numbers of Py cells alone, in comparisonwith the numbers of Py cells cultured in parallel on bare surface, i.e. in sparse MEcultures. It will be seen that there was a rise in the estimated number of Py cells inboth types of culture for 2 days. Py-ceti growth on ME cells was then inhibited whilegrowth on bare surface continued. This suggested that there was a delay before con-fluent ME cell cultures became inhibitory to Py cells. Additional confirmation that themain early increase in mixed cultures was due to Py cells, and not to a stimulation ofME cells, was obtained in another experiment by identifying the hamster karyotypein 16 out of 18 cells in mitosis (kindly performed by Dr I. A. Macpherson).

A similar type of experiment was then carried out, in which the confluent ME cellswere left in culture for 4 days before Py cells were added in fresh medium. The results

30° M. G. P. Stoker, M. Shearer and C. O'Neill

Table i. Growth of ME cells and Py cells in mixed cultures

Days inA

(

ME cells

i

2

3

4

S

4*

5

6

culture

AddedPy cells

o

i

2

3

4

o

I

2

Total no. of cells

Confluent MEA

Py cellsabsent

5-i4-66 i6-36-66-75-i6-25'54-8

3-53 74 95'O4 657

O"2 X IO

Py cellsadded

——6-76 9

10-210-9

7-68-58-78-i

——

4"34 95H5'5

per plate, x

SparseA

Py cellsabsent

o-i

o-o6o- i

O-2

O-2

o- i

o-3

o-3O 2

0 - 2

OO7

OO80 - 2

O-2

O'2

0 ' 2

I O 8

ME

0*2 X IO

Py cellsadded

——0 7

°'54-83'39-89 6

12-612-5

—

—0 9

0 9

3-35'O

Sparse and confluent cultures of ME cells set up in Petri dishes at time o andPy cells added after 1 or 4 days. Total counts determined in haemocytometer after sus-pension on successive days. Medium/substrate, 0-3 ml/cm*.• For confluent ME only: control (sparse) cultures seeded previous day.

6 -

s-o6

Py cells

4

Py cells

Days Age ofME culture

Fig. 1. Growth curves of Py cells derived from data in Table 1. Numbers of Py cellswere estimated by differences of mean counts of control cultures (ME cells alone) andmixed cultures (ME cells and Py cells). Medium growth surface ratio 0-3 ml/cm1.• — • , Py cells on confluent cultures of ME cells; O — O, Py cells on sparse

cultures of ME cells.

Growth inhibition of transformed cells 301

of this delayed addition of Py cells, also given in Table 1 and Fig. 1, show that by4 days the cultures were immediately inhibitory to Py cells.

Some difficulty was experienced in maintaining an equal pH in the confluent andsparse ME cultures in Petri dishes. When the pH was controlled by alteration of CO^concentration or use of medium with higher bicarbonate content, however, a similardelay followed by inhibition was observed for Py cells on confluent ME cells.

Coverslip cultures in excess medium

Previous studies and additional findings reported later in this paper make it unlikelythat the inhibition of Py cells is due to overall changes in the medium in confluentME cultures. However, to minimize differences in the medium over confluent andsparse cultures, further studies were carried out on coverslips in 5 ml of medium,thus giving a 13-fold increase in the relative amount of medium.

A feature of these coverslip cultures which soon became apparent was the highersaturation density of the ME cells compared to that found in whole Petri dish cultures.Maximum ME-cell density in whole Petri dish cultures, or on coverslips included insuch cultures, varied between 2-0 x io6 and 4-0 x 10s cells per cm2, while growth oncoverslips alone in excess medium reached densities of 4-0 x 10s to 6-o x ioB cellsper cm2. This higher density is presumably due to the higher relative volume ofmedium diminishing the effect of diffusible substances which are added or removed,and which perhaps operate in addition to inhibition by contact. It may be noted thatthe ME cells on glass or plastic form a contiguous sheet at about 2*0 x io6 cells per cm2

and are then oriented in parallel indicating contact inhibition of movement. Oncoverslips in excess medium a further one or two divisions are therefore possible aftermost of the cells are in continuous contact. Finally, however, cell division ceases evenin repeatedly changed excess medium, and numbers remain static until the cell sheetstrips, usually about 48 h later (see, for example, Fig. 3).

Several experiments, using the total count method to study growth of Py cells overME cells on coverslips, showed the same initial multiplication of Py cells as in Petridish cultures. The smaller total numbers of cells made the haemocytometer countsunsatisfactory, however, so this method was discarded and Py cells were assayed by thecolony count method. Py cells were allowed to attach to confluent and sparse MZ?-cellcoverslip cultures which had been grown for various times. The coverslips were trans-ferred to fresh medium and the cells from 2 confluent and 2 sparse coverslips weresuspended and plated in agar, after 5 h or 24 h of further incubation. In this way theinhibitory effect of ME cultures was tested by a 19-h 'pulse' of Py cells. Fig. 2 showsthe results of 3 separate experiments with the number of colony-forming Py cellsrecovered after addition to sparse and confluent ME cultures of various ages. In 2 ofthe experiments, the initial recovery of colony-forming cells was slightly less (70 %)from the sparse than from the confluent cultures, but this does not affect theconclusions. On barely confluent but growing ME cultures the growth is similar tothat on bare substrate. The denser ME cell cultures, grown for 3 or more dayshowever, inhibit the growth of Py cells, thus confirming the results based on totalcell counts.

302 M. G. P. Stoker, M. Shearer and C. O'Neill

A more detailed examination of the interrelationship between M£-cell growth andinhibition of added Py cells is shown in Fig. 3. In this experiment Py cells were addedon successive days to pairs of pre-existing cultures of a single batch of ME cells. Theinhibition is expressed as the number of Py cells present after 24 h growth on confluentME cells, as a percentage of the controls grown for the same period on sparse MEcells (i.e. on bare surface). A percentage greater than 100 shows stimulation of Pyby confluent ME cells, while less than 100 shows inhibition. The initial number of

1 2 3 4 4ME cells, days since plating

50S • 5-48 5-54 5 24 5 63 • 5-65No. per cm3 log,,,

Fig. 2. Growth of Py cells between 5 and 24 h after addition to confluent andsparse ME-cell coverslip cultures on different days (3 separate experiments), I-I xio4 Py cells (8-5 x io3 per cm1) were added per coverslip culture of ME cells. After5 and 24 h, numbers of colony-forming Py cells were determined by agar suspensionculture. Numbers of ME cells were determined by haemocytometer counts on controlcultures without Py cells. Medium/growth surface ratio 4 ml/cm1. •—•, Py cells onME cells as indicated; O — O, Py cells on sparse ME cells.

colony-forming Py cells was not determined in this experiment, but from other ex-periments it may be assumed that the control Py cells would have increased 4-fold in24 h; a value of 25% in Fig. 3 would therefore represent absence of growth of Pycells in the confluent ME cultures. Examination of both stained and unstained cover-slips showed that the ME cells were confluent on day 1 but that they continued tomultiply until a density of 4 x io5 per cm2 was reached on the third day.

Compared to controls on bare surface it will be seen that Py-cell growth was stimu-lated when in contact with dividing ME cells. When ME-ceW growth stopped, however,added Py cells were also inhibited. There may have been a delay between cessation ofME cell growth, and transmission of the effect to Py cells, but the long pulse period of24 h necessary to detect Py cell inhibition makes precise timing difficult.

Growth inhibition of transformed cells 3°3

Pycells

84-1

o

120

100

80

60

40

20

-« Confluent •— *-

-

-

-

/

/

>

*

s*

-

-

MEcells

- 60

o

- 5 5

50

Days

Fig. 3. Relationship between ME cell numbers on coverslips and inhibition of Py cellsadded on successive days (single experiment). Number of Py cells determined after 24 hgrowth by agar suspension culture. Inhibition of Py cells shown in histogram as percen-tage : Py cells on confluent ME compared to Py cells on sparse ME. Number of MEcells (O — O) determined by haemocytometer counts in controls without Py cells.Medium/growth surface ratio 4 ml/cm1.

Overcrowded cultures

There were two possible reasons to explain the delay in inhibition of Py cells byconfluent ME cells. The first was that the growth of Py cells was permitted so long asthere was any growth of the ME cells, even if this was barely detectable, as shown inTable 1. The second was that Py cells were inhibited by any contact with ME cells,even with young confluent cultures of ME cells, but that the transfer of inhibitoryeffect took several days to develop. To investigate this, overcrowded ME cultures oncoverslips were set up, giving densities of about 9 x io6 attached and spread cells percm2, that is higher than the maximum number obtained by growth. The results givenin Figs. 4 and 5 show that, despite some growth of Py cells added on day 2, there wasrelative inhibition from day 1, so that a long delay was not essential for inhibition tooccur.

The first point on Fig. 5, however, shows the results of a separate experiment inwhich the Py cells were added to the coverslips simultaneously with the high densityof ME cells. Under these circumstances there was little inhibition. Py cells added after

20 CellSci.i

304 M. G. P. Stoker, M. Shearer and C. O'Neill

one day in this experiment were, as usual, strongly inhibited (not shown). It is there-fore necessary for ME cells to stick and spread or go through some other change beforethe culture becomes inhibitory.

Attempts to detect changes in medium

A simple explanation of the observation that Py cells were inhibited in mixedcultures when the ME cells were also inhibited would be a depletion of some limitingsubstance in the medium. Py cells alone grow to a higher final density than ME cells,

59

2 3ME cells, days since plating

5-92 5-92 5-96 5 96No. per cm1 log10

594

Fig. 4. Growth of Py cells for between 5 and 24 h after addition to overcrowdedand sparse ME cultures. Single experiment, otherwise as for Fig. 2.

or even ME cells with the added Py cells, but it was possible that ME cells selectivelydestroyed or removed an essential factor for Py cells. Evidence against this explanationwas produced previously by showing that colony formation by Py cells was not in-hibited if the Py cells were situated a short distance from the edge of a confluentME-cell sheet in the same culture medium (Stoker, 1964), and this has been confirmedeven in cultures where the medium was continuously mixed. However, the effect ofincreasing the relative volume of medium on the saturation density of ME cellsprompted a re-examination of the possibility of medium depletion as a cause ofPj>-cell inhibition.

Medium was pooled after incubation for 4 days over confluent ME cultures onPetri dishes. Such cultures are known to be highly inhibitory to added Py cells. Mediumwas also collected and pooled from sparse ME cultures when still not confluent after4 days. The pH of the confluent culture medium was adjusted to 7-4, equal to sparse

Growth inhibition of transformed cells 305

culture medium and to fresh medium, by addition of sodium bicarbonate. The poolsof confluent and sparse culture medium, and also fresh medium, were then re-distributed in duplicate in fresh Petri dishes. To each dish was then added a coverslipto which 2 x io5 Py cells had been added 24 h previously. Counts of Py cells on indi-vidual coverslips in the different media were made in haemocytometer chambers aftersuspension on successive days. The results given in Fig. 6 show that Py cells grew aswell in medium from confluent cultures as in medium from sparse cultures or freshmedium.

Pycells

120

_23 100

8*S 80

•5£ 60

!

" 40

20

-Confluent-MEcells

6 0

60o

"3

5 0

Days

Fig. 5. Relationship between AfE-cell numbers and inhibition of added Py cells. Asfor Fig. 3, but with overcrowded ME cells. Data for day o to 1 from separate experi-ment in which ME cells and Py cells were mixed before addition to coverslips. Remain-der from experiment shown in Fig. 4.

Secondly, some of the experiments using coverslips already reported were carriedout with confluent and sparse coverslip cultures side by side in one dish in the samemedium. The presence of an inhibitory confluent coverslip had no effect on the growthof Py cells on bare substrate in the same medium. Moreover, replenishment of mediumor a change from the standard medium to a highly enriched medium (Vogt & Dulbecco,1963) had no effect on the inhibitory action of confluent ME cell cultures.

Though late development of medium depletion could be demonstrated in agarcultures of ME cells held for 5 days (see below), the results made it unlikely that theP^-cell inhibition in fluid medium was due to depletion caused by the ME cells. It also

306 M. G. P. Stoker, M. Shearer and C. O'Neill

ruled out inhibition due to addition of a stable diffusible inhibitor released into themedium.

There remained the possibility of an unstable, and/or poorly diffusible inhibitor,acting only a short distance from the ME cells. Various attempts have been made todetect such a short range inhibitor, so far without success. These may be summarizedas follows:

(i) Very small volumes of medium (0-3 ml) placed on established confluentMZ?-cell layers for 24 h to give a depth of 300 (i had no subsequent effect on cell growth,or uptake of pHJthymidine by Py cells.

6 5 -

6 0 -

I 5-5-1

5-0-

4-5-

2Days

3

Fig. 6. Growth of Py cells in various media (4 ml/an1). After initial seeding of 2 x 10'Py cells on coverslips 24 h previously, the coverslips were placed in the media shownand haemocytometer counts were made after suspension at intervals. ( x — x , Freshmedium; O—O, medium after 4 days on sparse ME-cell culture 0-3 ml/cm1).•— • , medium after 4 days on confluent AfE-cell culture (o'3 ml/cm*).

(2) When confluent ME cells and Py cells were grown on opposite sides of the samemillipore filter, the ME cells exerted a feeder effect on the Py cells but no inhibitioncould be detected.

(3) Bottles with alternate strips of confluent ME cells and bare areas in differentrelative sizes were incubated while rotating at about 1 rev/min to ensure mixing ofmedium. Varying volumes of medium were tested. In all experiments added Py cellsgrew to form colonies only on the bare areas, and there was no inhibitory zone adjacentto the trailing or leading edges of the confluent strips.

(4) It was previously reported that there was some reduction in Py-cell colony sizein agar when added in a separate layer of agar over confluent ME cells. Further ex-

Growth inhibition of transformed cells 307

periments showed inhibition of Py-colony formation when the cells were added to thesurface of agar medium previously held for 5 days over confluent ME cells. This in-hibition was removed when the Py cells were added in fresh medium but not bufferedsalt solution, and it is therefore concluded that medium depletion can occur after longculture in agar, and that a diffusible inhibitor was not responsible for the inhibition ofthe Py cells under these conditions.

DISCUSSION

The results reported here confirm the earlier report that growth of polyoma-trans-formed cells is inhibited in mixed cultures with freshly isolated normal fibroblasts.It was previously suggested, however, that the inhibition occurred simply by contactbetween cells, either ME cells and ME cells, or ME cells and Py cells, but notbetween Py cells and Py cells. The experiments reported here show that contact withME cells per se is not sufficient to inhibit Py cells.

This conclusion arises from the observation that already confluent, parallel-orientedcultures of ME cells will continue to grow for one or two more divisions if incubatedon coverslips in large volumes of medium. This is in agreement with results givenin a recent paper by Kruse & Miedema (1965), which appeared after our experimentshad been completed. These authors have shown that cell layers in continuouslyperfused medium continue to multiply after confluent monolayers have formed. Thedensity generally achieved varied with the cell type, but the Wi-38 human diploidcells, which can be compared to the mouse embryo cells used in this laboratory, grewto about the same densities (a maximum of 5-6-5 x ioB cells per cm2, or in their usefulterminology, 3-6—5 monolayer equivalents). In their perfused cultures, growth did notstop completely in the time shown but there was a change from maximal growth tovery slow growth at a density of 3-2 x io8 cells per cm2. The authors suggest that ashift in cell regulation occurs at the critical cell density where maximal proliferationceases, and they show a change in production and uptake of certain amino acids. Theother cells used by Kruse & Miedema were tumour cells or stable cell lines which inperfused medium reached even higher densities, comparable to those found in thislaboratory with Py cells.

When Py cells were added to confluent but growing ME cells, they also continuedto grow optimally. However, once the ME cells had stopped growing, even in excessmedium, the cultures became inhibitory to Py cells which were in contact. It musttherefore be concluded that the essential requirement for growth inhibition of Pycells is contact with or close proximity to a non-growing ME cell. In contrast, contact orproximity of Py cells to other Py cells does not result in growth inhibition, as judgedby the high densities achieved, and by the presence of mitoses in multilayered colonies(House & Stoker, 1966).

To explain the inhibition of Py cell growth in ME cell cultures the followingexplanations might be put forward:

(1) Depletion of medium by ME cells leading to starvation of both ME cells andPy cells.No evidence of general medium depletion could be found under conditions where

308 M. G. P. Stoker, M. Shearer and C. O'Neill

Py cells were inhibited, except in long-continued cultures under agar. Moreover,medium over inhibiting layers of ME cells does not affect growth of Py cells in the samevessel, even in continuously agitated medium, unless they are in direct contact orclose proximity to the ME cells. Depletion if it is operative in Py-cell inhibition musttherefore be limited to competition at the surface of the ME cells, and must involvesubstances which are not limiting in pure P_y-cell cultures of much higher total density.This might occur if the change in regulation of ME cells at critical density resulted in avery large increase in demand for a critical substance which was essential for Py cellsin only small concentrations, and which was therefore not limiting in pure Py cellcultures.

(2) Action of an inlribitor from static cultures of ME cells. It is reasonable to supposethat the diminution in macromolecular synthesis shown in static cultures by Levineet al. (1965) is associated with feed-back inhibition and perhaps repressor mechanisms,and that inhibitory compounds may be present intracellularly, and possibly may alsobe released into the intracellular environment. The lack of general effect on the mediumand the need for contact or close proximity to ME cells for inhibition of Py cells wouldmean that, to be effective, any supposed inhibitors must be acting on Py cells at a veryshort range through low diffusibility or rapid inactivation. Evidence that inhibition isdue to release of such inhibitors is at present lacking but cannot be excluded becausetheir presumed properties would make them very difficult to detect. As an extension ofthis hypothesis, inhibition might result from transfer of cytoplasm between ME cellsand Py cells, either by intracellular connexions as in nerve cells, or by phagocytosis ofcytoplasm-containing vesicles released by budding from neighbouring cells. There isat present no direct evidence to support this type of mechanism, but recent findings bySubak-Sharpe, Biirk & Pitts (1966) in this laboratory may be highly relevant. Theyhave used a variant of Py cells which lacks inosinic pyrophosphorylase and will notincorporate hypoxanthine (Subak-Sharpe, 1965). In mixed cultures with normal cellswhich possess the enzyme, there is evidence from autoradiography that ability to in-corporate labelled hypoxanthine is acquired by the variant cells, but only if they are inapparent contact with normal, enzyme-containing cells. This suggests that either thelabelled nucleotide or RNA, or the enzyme or a substance required for its synthesis, istransferred between the neighbouring cells but not through the medium to distantcells. Similar experiments using Py-cell variants are now in progress to investigatetransfer from ME cells to Py cells, under conditions where growth inhibition is knownto occur.

(3) Inhibition by direct contact between Py cells and ME cells, through a mechanisminitiated in the opposing plasma membranes. Contact between membranes is known toaffect motility of cells by the well-known experiments of Abercrombie & Heaysman(1954). It has been shown here, however, that simple cell-to-cell contact is notsufficient since no inhibition of growth is transferred by addition of Py cells to con-fluent ME cells which show the orientation due to inhibition of movement, but whichare still growing. It is still possible that the surface of an ME cell may be further changedwhen cell growth stops, and at that stage acquire the property of transmitting the growthinhibition to Py cells.

Growth inhibition of transformed cells 309

The phenomena described in this paper do not in any way explain the growth in-hibition in static cultures of ME cells. Such inhibition may be partly determined bythe rate of medium change, but the ultimate stasis or slowing of growth in crowdedcultures is probably due to other unknown factors. Whatever these factors are theyoperate less effectively or not at all in crowded Py-cell cultures. Once the inhibition hasbeen established in the ME cells, however, Py cells in the same culture are affected.

Thus we may suppose two separable and consecutive events. First, initiation ofinhibition due to unknown factors in crowded cultures. Secondly, inhibition itself dueto the operation of controlling substances inside the cell, perhaps also associated withchanges in the cell surface. Normal fibroblasts are subject to both initiating and con-trolling factors. Polyoma-transformed cells are insensitive or less sensitive to theinitiating factors but are quite sensitive to the factors operating in the controlled stage.This may make the Py cell a useful tool for the investigation of growth regulation innormal cells.

Finally it should be made clear that the findings reported apply at present only topolyoma-transformed BHK21 cells, and normal mouse and hamster fibroblasts. Onthe contrary it appears that SV40 virus-transformed 3 T3 cells are not inhibited by theuntransformed 3 T3 cells (Todaro, Green & Goldberg, 1964). The 3 T3 cell, despiteits strong contact inhibition is aneuploid, and may invoke a form of growth regulationwhich is different from that of normal fibroblasts. Alternatively cell-to-cell transfer maybe more efficient between normal and transformed fibroblasts than between normaland transformed 3 Tj cells. Further study will be needed with other model systemsbefore any generalizations can safely be made.

REFERENCES

ABERCROMBIE, M. & HEAYSMAN, J. E. M. (1954). Social behaviour of cells in tissue culture. II .Monolayering of fibroblasts. Expl Cell Res. 6, 293-306.

BARSKI, G. & BELEHRADEK Jr, J. (1965). fitude microcinematographique du mecanisme d'inva-sion canc6reuse en cultures de tissu normal associ6 aux cellules malignes. Expl Cell Res. 37,464-480.

HOUSE, W. & STOKER, M. G. P. (1966). Structure of normal and polyoma virus-transformedhamster cell cultures. J. Cell Sci. 1, 169-173.

KRUSE Jr., P. F. & MIEDEMA, E. (1965). Production and characterization of multiple-layeredpopulations of animal cells. J. Cell Biol. 27, 273-279.

LEVINE, M., BECKER, Y., BOONB, C. W. & EAGLE, H. (1965). Contact inhibition, macro-molecular synthesis, and polyribosomes in cultured human diploid fibroblasts. Proc. natn.Acad. Sci. U.S.A. 53, 350-356.

MACPHERSON, I. & MONTAGNIER, L. (1964). Agar suspension culture for the selective assay ofcells transformed by polyoma virus. Virology 23, 291-294.

SANDERS, F. K. & BURFORD, B. O. (1964). Ascites tumours from BHK21 cells transformedin vitro by polyoma virus. Nature, Lond. 201, 786-789.

STOKER, M. (1964). Regulation of growth and orientation in hamster cells transformed bypolyoma virus. Virology 24, 165-174.

SUBAK-SHARPE, H. (1965). Biochemically marked variants of the Syrian hamster fibroblast lineBHK21 and its derivatives. Expl Cell. Res. 38, 106-119.

SUBAK-SHARPE, H., BORK, R. R. & PITTS, J. D. (1966). Metabolic co-operation by cell to celltransfer between genetically different mammalian cells in tissue culture. Heredity, Lond.(in the press).

310 M. G. P. Stoker, M. Shearer and C. O'Neill

TODARO, G. J. & GREEN, H. J. (1963). Quantitative studies of the growth of mouse embryocells in culture and their development into established lines. J. Cell Biol. 17, 299-313.

TODARO, G. J., GREEN, H. & GOLDBERG, B. D. (1964). Transformation of properties of anestablished cell line by SV40 and polyoma virus. Proc. natn. Acad. Sci. U.S.A. 51, 66-73.

VOGT, M. & DULBECCO, R. (1963). Steps in the neoplastic transformation of hamster embryocells by polyoma virus. Proc. natn. Acad. Sci. U.S.A. 49, 171-179.

(Received 12 March 1966)