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Regulation of growth and orientation in hamster cells transformed by polyoma virus
24, 165-174 (1964)
VIROLOGY
Regulation
of Growth
and
Transformed
Orientation
by Polyoma
MICHAEL Institute
of Virology,
in Hamster
Cells
Virus’
STOKER
University
of Glasgow, Scotland
Accepted June 10, 1964 After neoplastic transformation by polyoma virus, hamster cells multiply and move when in contact with one another, resulting in a piled up and random arrangement of cells. Contact with normal fibroblasts, however, inhibits multiplication of the transformed cells and leads to their regular orientation. Contact with irradiated transformed cells also inhibits multiplication. It is suggested that contact inhibition involves two functions, emission of and response to a contact-promoted signal, and that polyoma transformed cells still retain the ability to respond. I!VTRODUCTION
Freshly cultured mammalian cells from normal tissue continue to move on solid substrates until the cell density is such that the cells are all in close contact. Movement then ceases with the cells finally oriented to give maximum contact with minimal superimposition, resulting in parallel orientation of elongated, fibroblastic, cells. This is the phenomenon of contact inhibition described by Abercrombie and his colleagues (see =Ibercrombie and Ambrose, 1962). When freshly cultured cells reach this confluent state cell division also ceases. Contact inhibition of division, however, is probably distinct
from
contact
inhibition
of movement
and can be distinguished in certain types of cell. The BHK21 line of hamster fibroblasts for example grows with parallel orientation like freshly isolated cells, but cell division continues even when cells are in close contact. hIany types of tumour cell in culture continue to move when in close contact so that the cells cross each other at random and pile up, rather than preserve a single oriented layer. They may also continue to multiply until the cell density on the substrate is 1 Work Council.
supported
by
the
Medical
much higher than the maximum achieved by normal cells in culture. This loss of contact inhibition of movement and growth shown by tumour cells in culture is in accord with their apparently unrestricted growth and in vim, though as tendency to metastasize Defendi et al. (1963) have pointed out,, the correlation is not complete. Hamster fibroblasts transformed by polyoma vir?ls show the typical random orientation and unrestricted growth of tumour cells in culture when they are in contact with one another, but preliminary observations suggested that the transformed cells were not entirely devoid of contact inhibition when in contact with other types of cell. It was first noticed by an inhibitory action of irradiated cells used in feeder layers (Stoker, 1962). This paper repot% the results of a study on the effect of contact between polyoma-transformed hamster cells and various other cells. METHODS
Cells. The polyoma-transformed cells (Py cells) were derived from clone 13 of the BHK21 line of hamster fibroblasts after transformation by polyoma virus using standard methods (Stoker and Abel, 1962). The Py cells were recloned, grown to about log cells, and stored at -70”. They were
Research 165
166
STOKER
used after less than 2 months growth since deep, with mitotic cells at all levels (W. C. storage. Two Py cell clones were used with House and M. Stoker, unpublished). However, if the colony lies over an irradiated similar results. Both were predominantly diploid and contained no detectable infec- giant cell used in a feeder layer there may be tive polyoma virus, and both were easily a hole in the colony with Py cells reaching transplantable. the edge of the giant cell but none lying on top. The same effect may be seen with irFreshly isolated mouse and hamster embryo fibroblasts were used after one or radiated giant cells from fresh hamster or two subcultures. mouse embryo cultures, untransformed For X-irradiation cells were exposed to BHK21 cells, or even Py cells. Giant cells 1700 r to give a survival of less than 10e5. produced by the action of bromodeoxyuriCell counts, based on about 200 cells, were dine, and spontaneous giant cells appearing made in haemocytometer chambers. In in untreated cultures, also inhibit cells in Py comparative growth curves the counts were colonies. This suggested that Py cells were done without knowledge of the identity of in some way sensitive to contact with nondividing cells and would not migrate over the sample. A cell-marking technique (Stoker, 1964) them. based on phagocytosis of carbon was used to Failure of Colony Formation by Py Cells on identify cells in mixtures. Confluent Cell Sheets Media and solutions. The standard medium was 10 ‘33uninactivated calf serum and Since Py cells multiply well on top of one 10% tryptose phosphate broth in Eagle’s another it was to be expected that they medium modified to contain twice the standwould grow and form colonies on preexisting ard concentration of vitamins and amino monolayers of other cells. Known numbers acids. Cells were suspended in 0.0.5% trypsin of Py cells were therefore added to cultures (Difco) and 0.0002% Versene in Tris saline of various types of cells prepared the preafter two rapid washes in the same solution. vious day. These included homologous Py Cultures. Falcon plastic 5ti-mm petri cells and untransformed BHK21 cells, both dishes or glass 60.mm petri dishes were used irradiated so that developing Py colonies throughout. Confluent cultures were made would not be obscured, and irradiated and by inoculating dishes with 4 million normal unirradiated mouse and hamster embryo cells or 6 million Py cells. Sparse cultures cells. were made with 2 to ci hundred thousand To minimize differences in medium over cells. cultures of different density these preexisting For certain experiments monolayer cul- cultures were prepared on one side of each tures were made on one side of the dish only. petri dish (see Methods) while the other Half the normal cell number was inoculated side remained bare except for a few disinto dishes which were incubated in a sloped lodged cells. The viable Py cells were added position so that the cell suspension covered evenly over the whole dish and after incuonly about half the surface. When the cells bation numbers of colonies were compared had attached the medium was changed and on the two sides. The results in Table 1 the dish was laid flat. show the actual numbers of colonies on the The agar suspension method of culture two sides, and also the density of colonies (Macpherson and Montagnier, 1964) was since the two sides were not always equal in used to select for Py cells. area. The cultures on hamster embryo cells were not suitable for counting but showed RESULTS the same type of effect as the mouse embryo Contact Inhibition by Giant Cells in Colonies cells. The following points may be noted. of Transformed Cells 1. A sparse preexisting cell layer enhanced colony formation by Py cells compared to Py cells in colonies continue to divide the bare substrate, presumably due to the without migrating away from each other until at 7 days the colony may be 10 cells feeder effect. For unknown reasons this was
REGI’L.ATIOII;
IX
TRANSFORMED TABLE
COLONIES
OF
PY CELLS
ON
HAMSTER
167
CELLS
1
PREEXISTING
CELL BEETS
IN HALF PLATES
Py cells added Preexisting cell sheet
Confiuent side Bare side
Py cells irradiated
cells
irradi-
Mouse embryo not irradiated
Mouse embryo irradiated a Colony
cells
cells
density
Per sq. cm.
Colony density= per sq. cm.
Colonies per culture
200 200
5.3 8.3
3, 3, 4 58, 92, 67
0.45 4.3
200 200 _______~
8.3 8.3
57, 54, 23, 43 11, 22, 0, 14
5.4 0.89
Confluent side Bare side
200 200
8.3 a.3
0, 0, 0, 0 10, 17, 10, 9
0 0.72
Sparse side Bare side
200 200
8.3 8.3
12, 6, 23, 12 22, 14, 23, 21
0.9 1.7
Conflue~it side Bare side
400 400
16.6 lG.G
0, 0 87, 82
0 5.3
Sparse side Bare side .-.~-~
400 400
16.6 16.6
72, 73 98, 78
9.1 5.5
Confluent side Bare side
400 400
16.6 16.6
0, 0 77, 65
Sparse side Bare side BHK21 ated
Per culture
-
shown to allow for inequality
not shown with the sparse irradiated BHK21 culture, despite the fact that these cells usually make efficient feeder layers. 2. A confluent preexisting cell layer on the other hand, inhibited colony formation by Py cells almost completely. 3. Irradiated Py cells, BHK21 cells, mouse embryo cells, and freshly cultured hamster embryo cells all inhibited Py colonies, and uni~radiated mouse and hamster embryo cells were equally effective. Figure 1 shows a typical example of a culture with inhibition of Py colonies over a confluent area of irradiated mouse embryo cells. 4. There was a sharp del~~a~cation at the edge of the preexisting confluent culture, and colonies of Py cells were found right up to the edge of this line. At the boundary the parts of the Py colonies which were in direct contact with the preexisting cell sheet were distorted, and when in contact with oriented cells the edge of the Py colony also oriented in parallel. Figure 2 shows a colony of Py cells distorted by contact with an advancing
0 4.4
of the two sides.
Frc;. 1. Failure of Py cells to form colonies on a confluent sheet of mouse cells. A petri dish was prepared with a preformed confluent monolayer of X-irradiated mouse embryo cells covering only part of the surface (lower part); Py cells were added to the whole surface, and formed colonies only on the bare part. Stained Jenner-Giemsa. Magnification : X 1.0.
168
STOKER
edge of unirradiated hamster embryo cells. This all suggested that the inhibiting effect of the confluent cell sheets required very close proximity or actual contact. Mouse and hamster embryo fibrobIasts seemed to be both effective in inhibiting the appearance of Py colonies (originating from hamster cells) and since the former were generally available in the laboratory they were used in subsequent experiments.
To test further the effect of cell separation on colony inhibition, use was made of the ability of Py cells to grow suspended in agar (Macpherson and Montagnier, 1964). Preexisting cultures in half petri dishes were set up as before, but were covered with 0.5% agar medium to a depth of 0.G mm. When this had set, Py celfs in 0.33 7%agar medium were added on top and after incubatioK~ were examined for colony formation. It will be seen from Table 2 that Py cells separated by agar from confluent mouse cells do form colonies. The number of colonies is less than than that over sparse cells, but this may be due to medium depletion localized by the agar. The lowest and most variable numbers of colonies were found over bare substrate, presl~nlably due to lack of a feeder effect. Attachmennt of Py Cells on Other Cells
FIG. 2. Colony of Py cells distorted with advancing sheet of hamster Stained Jenner-Giemsa. Magnification:
Failure of Py cells to form colonies on preexisting cell sheets might be due to lack of stable attachment or failure to spread. To investigate this, Py cells marked with carbon (E%oker, 1964) were added to half cultures of irradiated mouse embryo fibroblasts and of irradiated Py cells. The number of marked cells which adhered and spread on confluent cell layers was compared with the number adhering to the bare surface on the other half of the same culture. The results expressed as cells per field are shown on Table 3. There is no significant difference in number of cells attached to the confluent cell sheets which could account for the failure to form colonies. In another experiment dishes with confluent, sparse, or no preexisting cell sheet were inoculated with black Py cells. After
by contact fibrobh&s. X 40. TABLE
OF PY CELLS SEPARATED
COLONIES
BY
AGAB
2
FROM PREEXISTIM
CELL
SHEETS
IN HALF PLATES
Py cells added Preexisting cell sheet .Mouse embryo irradiated
cells
Confluent side Bare side Sparse side Bare side
Per culture
Per sq. cm.
Colonies per culture
Colony density per sq. cm.
-- .._.--
~.
500 500
21 21
21, 15, 14, 16 20, G, 3, 7
1.8 0.6
500 500
21 21
38, 38, 36 3, 16, 4
3.5 0.6
REGULATION
IN
TRANSFORMED
Orientation of Py Cells on Normal Cells Though Py cells attached and spread on preexisting cells, there was a marked difference in orientation of the cells falling on glass compared to those falling on confluent mouse cells which were already oriented in a regular parallel fashion. The Py cells on glass took up the usual random arrangement (Fig. 3a), but those attaching to mouse fibroblasts rapidly oriented in regular parallel fashion, aligned with the mouse cells (Fig. 3b). This arrangement of cells was still
illultiplication of Py Cells in Contact with Other Cells When 5 X lo5 Py cells were added to a %-mm petri dish the number rose to about 10 million cells in 4-6 days (Fig. 4). In eontrast total cell counts after the same number of Py cells were added to confluent monolayers of irradiated cells, showed only a small increase (on irradiated mouse cells) or a drop (on irradiated Py cells) over the initial cell number and did not reach the same density as Py cells growing alone. This suggested that Py cells grew less well or not at all in contact with irradiated cells. However the actual number of Py cells was not known and was obscured by the large excess of cells from the underlying monolayer. Direct counts of marked Py cells in situ
3
ATTACHMENT OF MARKED PY CELLS ON PREEXISTING CONFLUENT CELL SHEETSCOUNTED in Situ
Preexisting cell sheet
l-2 Hours
Mouse embryo cells irradiated Nil Py cells irradiated Nil a Mean numbers standard error.
20 Hours
17.0%
1.5
21.6 f
1.4
17.4 f 15.4 f 20.5 f
1.6 1.0 2.8
24.4 f
1.G
of marked
cells per field with
TABLE A~~A~~~~~~ Preexisting Mouse embryo Confluent Sparse Nil
4
OF ~L~ARKED PY CELLS ON PREEXISTING Marked Py cells added per culture
cell sheet
169
CELLS
found after 20 hours in culture (Fig. 3c), at a time when the Py cells on the bare surface were already increasing in number and showing the typical piled-up disorientation. The observations and the distortion of orien~tion of Py colonies in contact with advancing normal cells (see previous seetion) suggested that the Py cells might be sensitive to contact with normal cells. This alone might account for the failure of Py cells to form recognisable piled-up colonies on confluent cell sheets of normal cells, without any inhibition in cell multiplication. The nlultipli~ation of Py cells in contact with other cells was therefore investigated next.
4 hours the cell sheets were washed and the number of attached Py cells determined after suspension by counts of black cells in haemocytometer chambers. Table 4 shows the results on duplicate cultures and also indicates that Py cells attach firmly to irradiated mouse cells or Py cells.
TABLE
HAMSTER
CELL SHEETS Marked Py cells per culture adherent 4 hoursa
cells irradiated
Py cells irradiated Confluent Sparse Nil Q Duplicate (or quadruplicate) and suspension of cells.
cultures
5.7 5.7 5.7
5.55 5.73 5.6
5.66 5.71 5.68
6.0 6.0 6.0
5.68 5.78 5.79 5.87
5.82 5.76 5.95 5.92
were counted
in haemocytometer
chamber,
after
washing
STOKE I<.
FIG. 3. Py cells I&belled with carbon attached to different parts of the same petri dish aft,er 2 hours. (a) On bare surface; (b) on preformed monolayer of irradiated mouseembryo cells. Note oriented arrangement on mouse embryo cells. (c) Samea8 (b) after 20hours. Stained Je~~ner-Giemsa. Magnification: X 200.
on confiuent monolayers or on bare glass were unsatisfactory because the reduction in the amount of carbon per cell following division nlade it difficult to distinguish the marked cells from unmarked cells containing lipid granules. When the cells in such cultures were suspended, however, the black cells could be distinguish~ from unmarked (white) cells in a haemocytometer chamber even after cell division (Stoker, 1964). 5 X 10” black Py cells were added to confluent or sparse cultures of irradiated mouse cells and both black and white cells counted in pairs of cultures after suspension at successive time intervals. Table 5 shows the results. It will be seen that black cells faIIing on glass (sparse mouse ceIIsf increased in number whereas black cells on confluent mouse cells showed no increase. The increase in black cells growing on glass
FIG. 3(b)
FIG. 3(c)
REGULATION
IN
TRANSFORMED
4
6
HAMSTER
)
171
CELLS
4
DAYS
FIG. 4. Growth of Py cells on bare substrate, or on confluent cell sheet. 105,7cells were added to dishes containing sparse (circles) or confluent (squares) preexisting monolayers of irradiated mouse embryo cells (left-hand figure), or irradiated Py cells (right-hand figure). Total counts were made after suspension of cells at 4 hours, and on the 4th and 6th day.
suggests at most one cell division, but it represents only a part of the total increase (shown by the total counts). This is because the loss of carbon with cell division leads to increasing numbers of white cells (Stoker, 1964). This result indicated that Py cells grew poorly or not at all on confluent irradiated mouse cells, but the method limited observation to a few divisions. To follow growth of Py cells for longer periods the numbers of viable cells were counted by suspending and replating under conditions where identifiable colonies would arise from Py cells with high efficiency. For this the cells were plated on glass or in agar. The
agar method was necessary when Py cells were plated with an excess of unirradiated BHK21 cells, which also grow well on glass but fail to grow in agar. The results in Table 5 show that there is a small increase in numbers of Py cells on confluent mouse cells, but a much larger increase when the same number of cells fall on glass in the sparse cultures. Sparse unirradiated cultures of BHK21 cells rapidly became confluent during the experiment, and this probably accounts for the small increment in numbers of Py cells in these cultures. Confluent BHK21 cells appear to kill the Py cells.
172
STOKER TABLE 5 GROWTH OF PY CELLS ON PREEXISTING CELL SEIEETS Preexisting culture (24 hours)
PWY;
Colony forming Py cells (loglo) per culture* after:
~log10) Mouse embryo cells
Not irradiated Irradiated
Mouse embryo cells
BHK21 cells
_--. Confluent Sparse Confluent sparse
Not irradiated
Confluent Sparse
Irradiated
Confluent Sparse
Not irradiated
5.3
Confluent Sparse
4.0
4 hours
96 hours
Inc;;txn;nt 96 hours
5.47
2.2
6.2
14.6
5.61
5.53
6.34
6.43
1.7 14.6
5.11" 5.18
5.11 5.04
5.42 6.34
5.42 5.18
5.28 5.23
3.42b 3.23
3.77
2.2
4.34
12.6
3.46
3.11
4.28 4.50
6.6 25.0
3.39
2.78
3.11
3.96
.-..-.-
0.15 6.7
* Figures based on counts of about 200 colonies after plating on glass” or in agarb. DISCUSSION
The experiments described in this paper show that the unrestricted growth and disoriented arrangement of polyoma-transformed hamster fibroblasts can be affected by contact with or close proximity to untra~forr~~ed cells of at least two species. X-irradiation of the normal cells does not prevent this effect, and even transformed cells after irradiation will prevent the multiplication of unirradiated transformed cells. The orientation of transfornled cells falling on a monolayer of normal cells, could be due to the nonspecific phenomenon of contact guidanee (Weiss, 1962), but the distortion of a transformed colony meeting an advancing sheet of oriented cells, and the failure initially to overgrow these cells, suggests that some more active inhibition of movement, and avoidance of contact is taking place. It is to be hoped that this point will be elucidated by time lapse cinematography with marked cells.2 z Since this paper was submitted the author has seen a report by G. Barski and J. Belehradek (&‘zptE. cell Res., in press), which is in close agreement with findings reported here. By time lapse ei~ematogra~hy the movement of two types of
Because the marker system availabIe is not adequate, it has not been possible to use the same quantitative techniques to investigate the interaction of viable transformed cells in contact with one another. It is clear however that transforI~led cells in colonies or congruent monolayer cultures continue to divide when in contact. Vertical sections of transformed cell colonies show mitotic figures at all levels including the centre of the colony, where cells may be separated from the substrate below or the medium above by a layer four or five cells thick. It has also been shown by time lapse cine~tography that Py cells, but not normal embryo cells or untransformed BHK21 cells, move actively when in contact with each other {AI~lbrose, personal col~~~~lu~~ation). The effect on growth and movement (i.e., orientation) following contact therefore appears to be as follows for the cells used in these experiments : 1. Normal cells inhibit normal cells. 2. Normal cells inhibit transformed cehs. 3. Transforn~ed cells do not inhibit transformed cells. -.-mouse tumor cells is shown to be arrested by contact with compact sheets of normal mouse embryo cells.
REGULATION
IN
TRANSFORMED
7.0 _ 0 ,‘O
6.0
n n n
n 1
20
40
60
so
I
HOURS
FIG. 5. Growth of marked Py cells on bare substrate or on confluent cell sheet. 105.7 marked Py cells were added to sparse (circles) or confluent (squares) monolayer cultures of irradiated mouse cells. Counts of total cells (open circles and squares) and marked cells (filled circles and squares) were made after suspension at successive times.
4. Transformed nondividing cells (after X-ray, etc.) inhibit transformed cells. These findings could be explained by postulating that in inhibiting each other normal cells display two functions. First, we suppose that the normal cell surface or some other part contains active factors or signals which inhibit other cells by contact. Second, the same normal cells respond to these inhibiting factors, or signals, from other cells. In other words each normal cell must be able both to emit and receive the contact signals. Polyoma-transformed cells seem to be sensitive to normal contact signals, but they do not inhibit each other. It is therefore suggested that they have lost the ability to emit the contact signal without losing their ability to respond.
HAMSTER
CELLS
173
This loss of emitting function is apparently dependent on cell viability because it is regained by transformed cells which are not dividing after irradiation, BUDR treatment, or spontaneous loss of ability to divide. Loss of the emitting function in the viable transformed cells could be due to the presence of surface material which masks active groups, providing this does not interfere with ability to receive the contact signal. It is of interest that Defendi and Gasic (1963) have reported a large amount of acid mucopolysaccharide detected histochemically on the surface of polyoma transformed cells, compared to untransformed cells. Sensitivity of polyoma transformed cells to contact with normal cells explains why transformation is not detected if virus-infected cells are initially cultured under crowded conditions. The majority of cells are untransformed, so the few transformed cells will be isolated from each other but touching normal cells, which presumably restrict division and movement. If on the other hand the transformed cells can start with free space around them, they may then reach an adequate clone size before coming in contact with normal cells so that some transformed cells in the centre of the colony are isolated from normal cell contacts, and continue to grow. Such clones may finally “overflow” and grow on top of the normal cell sheet if they become thick enough for a lower insulating layer of transformed cells to mask the normal contacts. In other words, the clone of transformed cells needs to reach a critical size, with enough cell to cell contacts, to escape from control by normal cells. Extension of hypotheses based on in vitro systems to explain the behaviour of tumours in animals is hazardous. Absence of contact inhibition of movement is believed to be related to the ability of tumour cells to infiltrate and metastasize, but spontaneous variants of BHIi21 cells (Defendi et al., 1963; Stoker and Macphcrson, 1964) may be isolated which are highly malignant though they grow in oriented fashion and pile up only slightly more than nontransplantable fibroblasts. Loss of contact inhibition of division may be more important in
174
STOKER
tumour cells, but there probably are other unknown factors involved as well. nievertheless the sensitivity of transformed cells to contact with normal cells in vi&v described above may help to explain some phenomena in animals. For example, polyoma tumours look highly malignant and rapidly growing, but like rodent ulcers they have a sharply defined edge and metastasize poorly if at all. This may be due to the inability of single tumour cells which are released from the main mass to escape from the inhibiting contacts with normal cells. When large numbers of polyoma-transformed cells are transplanted into an animal, a tumour appears rapidly; with lower cell numbers however, the latent period before a tumour reaches the same size may be disproportio~~ately long, and of course very small cell numbers may result in 110 tumour at all. It is now suggested that the tumour cells can grow fast and unrest,rictedly only when they reach a critical concentration which allows enough contacts between tumour cells alone. The extent to which these factors apply to cells transformed by other tumour viruses or to tumour cells in general, is not known. S~r40-t~nsforln~ tu~~~our cells are sensitive to contact inhibition of movement by giant cells (Black, personal ~ol~lmunication), and the expression of Rous-transformed cells as visible foci is inhibited by overcrowding, as with polyoma transformation. There are insu~cient data, however, to warrant generalization at this stage. There is oue striking phenomenon in clinical medicine which might possibly be explained by contact inhibition of n~ultiplication of single tumour cells by normal cells. With certain types of localized malignant tumours, individual tumour cells can be recognized in the circulating blood, but judging by the number of secondary tumours found at postn~orte~~~,the majority of these cells must fail to metastasize. There could obviously be several explanations of this effect, but it may be due to the inability of single t~~n~o~~rcells to escape from normal cell contacts. In the studies by Abercrombie et al. (1937)
tumour cells showed no contact inhibition of movement when in contact with normal cells, but this is not necessarily in conflict with the findings reported here. They used different techlli~ues, and the mouse sarcoma cells tested were highly anaplastic. If normal cells do indeed have two functions concerned in contact inhibition, we may expect that tumours might also arise from loss of the second function, ability to receive the contact signal. This defect would lead to a highly l~~aligna~~t cell which could metastasize from single cells, and it should show no contact inhibition by normal cells in culture. ACKNOWLEDGMENT I am grateful to Miss Anne Smith for her skilled technical assistance. REFERENCES ABERCROMBIE, M., and AMBROSE, E. J. (1962).
The surface properties of cancer cells-a review. Cancer Res. 22, 525-545. ABERCRO~BIE, PI., REAYSMAN, J. E. M., and KARTHAUSER, X. M. (1957). Social behaviour of cells in tissue culture III. Mutual influence of sarcoma celIs and fibroblasts. Expll. Cell Res. 13, 276-291. DEFENDI, V., and GASIC, G. (1963). Surface mucopotysaccharides of polyoma virus transformed cells. J. ~e~Z,ula~Camp. Physiol. 62, 23-26. DEFENDI, V., LEHMAN, J., and KRAEMEH, P. (1963). “Morphologically normal” hamster cells with malignant properties. Virology 19, 592598. MACPHERSON, I., and MONTAGNIER, L. (1964). Agar suspension culture for the seIective aksay of cells transformed by polyoma virus. ~~~~010~~ 23, 291-294. STOKER, M. (1962). Studies on transformat8ion by polyoma virus in vitro. C&a Found. S#mp. Il’unaour Viruses Murine Origin, pp. 365-376. STOKER, M. (1964). A simple marker technique for cells in culture. Exptl. Cell Res. in press. STOKER, M., and ABEL, P. (1962). Conditions affecting t~ransformation by polyoma virus. Cold Spring Harbor Sy,np. &z&ant. Biol. 27, 375-386. STOKER, RI., and MACPHERSON, I. (1964). The Syrian hamster fihroblast cell line BHKZI and its derivatives. Suture in press. WEISS, P. (1962). “Attraction fields” between growing tissue cultures. Science 115, 293-296.
VIROLOGY
Regulation
of Growth
and
Transformed
Orientation
by Polyoma
MICHAEL Institute
of Virology,
in Hamster
Cells
Virus’
STOKER
University
of Glasgow, Scotland
Accepted June 10, 1964 After neoplastic transformation by polyoma virus, hamster cells multiply and move when in contact with one another, resulting in a piled up and random arrangement of cells. Contact with normal fibroblasts, however, inhibits multiplication of the transformed cells and leads to their regular orientation. Contact with irradiated transformed cells also inhibits multiplication. It is suggested that contact inhibition involves two functions, emission of and response to a contact-promoted signal, and that polyoma transformed cells still retain the ability to respond. I!VTRODUCTION
Freshly cultured mammalian cells from normal tissue continue to move on solid substrates until the cell density is such that the cells are all in close contact. Movement then ceases with the cells finally oriented to give maximum contact with minimal superimposition, resulting in parallel orientation of elongated, fibroblastic, cells. This is the phenomenon of contact inhibition described by Abercrombie and his colleagues (see =Ibercrombie and Ambrose, 1962). When freshly cultured cells reach this confluent state cell division also ceases. Contact inhibition of division, however, is probably distinct
from
contact
inhibition
of movement
and can be distinguished in certain types of cell. The BHK21 line of hamster fibroblasts for example grows with parallel orientation like freshly isolated cells, but cell division continues even when cells are in close contact. hIany types of tumour cell in culture continue to move when in close contact so that the cells cross each other at random and pile up, rather than preserve a single oriented layer. They may also continue to multiply until the cell density on the substrate is 1 Work Council.
supported
by
the
Medical
much higher than the maximum achieved by normal cells in culture. This loss of contact inhibition of movement and growth shown by tumour cells in culture is in accord with their apparently unrestricted growth and in vim, though as tendency to metastasize Defendi et al. (1963) have pointed out,, the correlation is not complete. Hamster fibroblasts transformed by polyoma vir?ls show the typical random orientation and unrestricted growth of tumour cells in culture when they are in contact with one another, but preliminary observations suggested that the transformed cells were not entirely devoid of contact inhibition when in contact with other types of cell. It was first noticed by an inhibitory action of irradiated cells used in feeder layers (Stoker, 1962). This paper repot% the results of a study on the effect of contact between polyoma-transformed hamster cells and various other cells. METHODS
Cells. The polyoma-transformed cells (Py cells) were derived from clone 13 of the BHK21 line of hamster fibroblasts after transformation by polyoma virus using standard methods (Stoker and Abel, 1962). The Py cells were recloned, grown to about log cells, and stored at -70”. They were
Research 165
166
STOKER
used after less than 2 months growth since deep, with mitotic cells at all levels (W. C. storage. Two Py cell clones were used with House and M. Stoker, unpublished). However, if the colony lies over an irradiated similar results. Both were predominantly diploid and contained no detectable infec- giant cell used in a feeder layer there may be tive polyoma virus, and both were easily a hole in the colony with Py cells reaching transplantable. the edge of the giant cell but none lying on top. The same effect may be seen with irFreshly isolated mouse and hamster embryo fibroblasts were used after one or radiated giant cells from fresh hamster or two subcultures. mouse embryo cultures, untransformed For X-irradiation cells were exposed to BHK21 cells, or even Py cells. Giant cells 1700 r to give a survival of less than 10e5. produced by the action of bromodeoxyuriCell counts, based on about 200 cells, were dine, and spontaneous giant cells appearing made in haemocytometer chambers. In in untreated cultures, also inhibit cells in Py comparative growth curves the counts were colonies. This suggested that Py cells were done without knowledge of the identity of in some way sensitive to contact with nondividing cells and would not migrate over the sample. A cell-marking technique (Stoker, 1964) them. based on phagocytosis of carbon was used to Failure of Colony Formation by Py Cells on identify cells in mixtures. Confluent Cell Sheets Media and solutions. The standard medium was 10 ‘33uninactivated calf serum and Since Py cells multiply well on top of one 10% tryptose phosphate broth in Eagle’s another it was to be expected that they medium modified to contain twice the standwould grow and form colonies on preexisting ard concentration of vitamins and amino monolayers of other cells. Known numbers acids. Cells were suspended in 0.0.5% trypsin of Py cells were therefore added to cultures (Difco) and 0.0002% Versene in Tris saline of various types of cells prepared the preafter two rapid washes in the same solution. vious day. These included homologous Py Cultures. Falcon plastic 5ti-mm petri cells and untransformed BHK21 cells, both dishes or glass 60.mm petri dishes were used irradiated so that developing Py colonies throughout. Confluent cultures were made would not be obscured, and irradiated and by inoculating dishes with 4 million normal unirradiated mouse and hamster embryo cells or 6 million Py cells. Sparse cultures cells. were made with 2 to ci hundred thousand To minimize differences in medium over cells. cultures of different density these preexisting For certain experiments monolayer cul- cultures were prepared on one side of each tures were made on one side of the dish only. petri dish (see Methods) while the other Half the normal cell number was inoculated side remained bare except for a few disinto dishes which were incubated in a sloped lodged cells. The viable Py cells were added position so that the cell suspension covered evenly over the whole dish and after incuonly about half the surface. When the cells bation numbers of colonies were compared had attached the medium was changed and on the two sides. The results in Table 1 the dish was laid flat. show the actual numbers of colonies on the The agar suspension method of culture two sides, and also the density of colonies (Macpherson and Montagnier, 1964) was since the two sides were not always equal in used to select for Py cells. area. The cultures on hamster embryo cells were not suitable for counting but showed RESULTS the same type of effect as the mouse embryo Contact Inhibition by Giant Cells in Colonies cells. The following points may be noted. of Transformed Cells 1. A sparse preexisting cell layer enhanced colony formation by Py cells compared to Py cells in colonies continue to divide the bare substrate, presumably due to the without migrating away from each other until at 7 days the colony may be 10 cells feeder effect. For unknown reasons this was
REGI’L.ATIOII;
IX
TRANSFORMED TABLE
COLONIES
OF
PY CELLS
ON
HAMSTER
167
CELLS
1
PREEXISTING
CELL BEETS
IN HALF PLATES
Py cells added Preexisting cell sheet
Confiuent side Bare side
Py cells irradiated
cells
irradi-
Mouse embryo not irradiated
Mouse embryo irradiated a Colony
cells
cells
density
Per sq. cm.
Colony density= per sq. cm.
Colonies per culture
200 200
5.3 8.3
3, 3, 4 58, 92, 67
0.45 4.3
200 200 _______~
8.3 8.3
57, 54, 23, 43 11, 22, 0, 14
5.4 0.89
Confluent side Bare side
200 200
8.3 a.3
0, 0, 0, 0 10, 17, 10, 9
0 0.72
Sparse side Bare side
200 200
8.3 8.3
12, 6, 23, 12 22, 14, 23, 21
0.9 1.7
Conflue~it side Bare side
400 400
16.6 lG.G
0, 0 87, 82
0 5.3
Sparse side Bare side .-.~-~
400 400
16.6 16.6
72, 73 98, 78
9.1 5.5
Confluent side Bare side
400 400
16.6 16.6
0, 0 77, 65
Sparse side Bare side BHK21 ated
Per culture
-
shown to allow for inequality
not shown with the sparse irradiated BHK21 culture, despite the fact that these cells usually make efficient feeder layers. 2. A confluent preexisting cell layer on the other hand, inhibited colony formation by Py cells almost completely. 3. Irradiated Py cells, BHK21 cells, mouse embryo cells, and freshly cultured hamster embryo cells all inhibited Py colonies, and uni~radiated mouse and hamster embryo cells were equally effective. Figure 1 shows a typical example of a culture with inhibition of Py colonies over a confluent area of irradiated mouse embryo cells. 4. There was a sharp del~~a~cation at the edge of the preexisting confluent culture, and colonies of Py cells were found right up to the edge of this line. At the boundary the parts of the Py colonies which were in direct contact with the preexisting cell sheet were distorted, and when in contact with oriented cells the edge of the Py colony also oriented in parallel. Figure 2 shows a colony of Py cells distorted by contact with an advancing
0 4.4
of the two sides.
Frc;. 1. Failure of Py cells to form colonies on a confluent sheet of mouse cells. A petri dish was prepared with a preformed confluent monolayer of X-irradiated mouse embryo cells covering only part of the surface (lower part); Py cells were added to the whole surface, and formed colonies only on the bare part. Stained Jenner-Giemsa. Magnification : X 1.0.
168
STOKER
edge of unirradiated hamster embryo cells. This all suggested that the inhibiting effect of the confluent cell sheets required very close proximity or actual contact. Mouse and hamster embryo fibrobIasts seemed to be both effective in inhibiting the appearance of Py colonies (originating from hamster cells) and since the former were generally available in the laboratory they were used in subsequent experiments.
To test further the effect of cell separation on colony inhibition, use was made of the ability of Py cells to grow suspended in agar (Macpherson and Montagnier, 1964). Preexisting cultures in half petri dishes were set up as before, but were covered with 0.5% agar medium to a depth of 0.G mm. When this had set, Py celfs in 0.33 7%agar medium were added on top and after incubatioK~ were examined for colony formation. It will be seen from Table 2 that Py cells separated by agar from confluent mouse cells do form colonies. The number of colonies is less than than that over sparse cells, but this may be due to medium depletion localized by the agar. The lowest and most variable numbers of colonies were found over bare substrate, presl~nlably due to lack of a feeder effect. Attachmennt of Py Cells on Other Cells
FIG. 2. Colony of Py cells distorted with advancing sheet of hamster Stained Jenner-Giemsa. Magnification:
Failure of Py cells to form colonies on preexisting cell sheets might be due to lack of stable attachment or failure to spread. To investigate this, Py cells marked with carbon (E%oker, 1964) were added to half cultures of irradiated mouse embryo fibroblasts and of irradiated Py cells. The number of marked cells which adhered and spread on confluent cell layers was compared with the number adhering to the bare surface on the other half of the same culture. The results expressed as cells per field are shown on Table 3. There is no significant difference in number of cells attached to the confluent cell sheets which could account for the failure to form colonies. In another experiment dishes with confluent, sparse, or no preexisting cell sheet were inoculated with black Py cells. After
by contact fibrobh&s. X 40. TABLE
OF PY CELLS SEPARATED
COLONIES
BY
AGAB
2
FROM PREEXISTIM
CELL
SHEETS
IN HALF PLATES
Py cells added Preexisting cell sheet .Mouse embryo irradiated
cells
Confluent side Bare side Sparse side Bare side
Per culture
Per sq. cm.
Colonies per culture
Colony density per sq. cm.
-- .._.--
~.
500 500
21 21
21, 15, 14, 16 20, G, 3, 7
1.8 0.6
500 500
21 21
38, 38, 36 3, 16, 4
3.5 0.6
REGULATION
IN
TRANSFORMED
Orientation of Py Cells on Normal Cells Though Py cells attached and spread on preexisting cells, there was a marked difference in orientation of the cells falling on glass compared to those falling on confluent mouse cells which were already oriented in a regular parallel fashion. The Py cells on glass took up the usual random arrangement (Fig. 3a), but those attaching to mouse fibroblasts rapidly oriented in regular parallel fashion, aligned with the mouse cells (Fig. 3b). This arrangement of cells was still
illultiplication of Py Cells in Contact with Other Cells When 5 X lo5 Py cells were added to a %-mm petri dish the number rose to about 10 million cells in 4-6 days (Fig. 4). In eontrast total cell counts after the same number of Py cells were added to confluent monolayers of irradiated cells, showed only a small increase (on irradiated mouse cells) or a drop (on irradiated Py cells) over the initial cell number and did not reach the same density as Py cells growing alone. This suggested that Py cells grew less well or not at all in contact with irradiated cells. However the actual number of Py cells was not known and was obscured by the large excess of cells from the underlying monolayer. Direct counts of marked Py cells in situ
3
ATTACHMENT OF MARKED PY CELLS ON PREEXISTING CONFLUENT CELL SHEETSCOUNTED in Situ
Preexisting cell sheet
l-2 Hours
Mouse embryo cells irradiated Nil Py cells irradiated Nil a Mean numbers standard error.
20 Hours
17.0%
1.5
21.6 f
1.4
17.4 f 15.4 f 20.5 f
1.6 1.0 2.8
24.4 f
1.G
of marked
cells per field with
TABLE A~~A~~~~~~ Preexisting Mouse embryo Confluent Sparse Nil
4
OF ~L~ARKED PY CELLS ON PREEXISTING Marked Py cells added per culture
cell sheet
169
CELLS
found after 20 hours in culture (Fig. 3c), at a time when the Py cells on the bare surface were already increasing in number and showing the typical piled-up disorientation. The observations and the distortion of orien~tion of Py colonies in contact with advancing normal cells (see previous seetion) suggested that the Py cells might be sensitive to contact with normal cells. This alone might account for the failure of Py cells to form recognisable piled-up colonies on confluent cell sheets of normal cells, without any inhibition in cell multiplication. The nlultipli~ation of Py cells in contact with other cells was therefore investigated next.
4 hours the cell sheets were washed and the number of attached Py cells determined after suspension by counts of black cells in haemocytometer chambers. Table 4 shows the results on duplicate cultures and also indicates that Py cells attach firmly to irradiated mouse cells or Py cells.
TABLE
HAMSTER
CELL SHEETS Marked Py cells per culture adherent 4 hoursa
cells irradiated
Py cells irradiated Confluent Sparse Nil Q Duplicate (or quadruplicate) and suspension of cells.
cultures
5.7 5.7 5.7
5.55 5.73 5.6
5.66 5.71 5.68
6.0 6.0 6.0
5.68 5.78 5.79 5.87
5.82 5.76 5.95 5.92
were counted
in haemocytometer
chamber,
after
washing
STOKE I<.
FIG. 3. Py cells I&belled with carbon attached to different parts of the same petri dish aft,er 2 hours. (a) On bare surface; (b) on preformed monolayer of irradiated mouseembryo cells. Note oriented arrangement on mouse embryo cells. (c) Samea8 (b) after 20hours. Stained Je~~ner-Giemsa. Magnification: X 200.
on confiuent monolayers or on bare glass were unsatisfactory because the reduction in the amount of carbon per cell following division nlade it difficult to distinguish the marked cells from unmarked cells containing lipid granules. When the cells in such cultures were suspended, however, the black cells could be distinguish~ from unmarked (white) cells in a haemocytometer chamber even after cell division (Stoker, 1964). 5 X 10” black Py cells were added to confluent or sparse cultures of irradiated mouse cells and both black and white cells counted in pairs of cultures after suspension at successive time intervals. Table 5 shows the results. It will be seen that black cells faIIing on glass (sparse mouse ceIIsf increased in number whereas black cells on confluent mouse cells showed no increase. The increase in black cells growing on glass
FIG. 3(b)
FIG. 3(c)
REGULATION
IN
TRANSFORMED
4
6
HAMSTER
)
171
CELLS
4
DAYS
FIG. 4. Growth of Py cells on bare substrate, or on confluent cell sheet. 105,7cells were added to dishes containing sparse (circles) or confluent (squares) preexisting monolayers of irradiated mouse embryo cells (left-hand figure), or irradiated Py cells (right-hand figure). Total counts were made after suspension of cells at 4 hours, and on the 4th and 6th day.
suggests at most one cell division, but it represents only a part of the total increase (shown by the total counts). This is because the loss of carbon with cell division leads to increasing numbers of white cells (Stoker, 1964). This result indicated that Py cells grew poorly or not at all on confluent irradiated mouse cells, but the method limited observation to a few divisions. To follow growth of Py cells for longer periods the numbers of viable cells were counted by suspending and replating under conditions where identifiable colonies would arise from Py cells with high efficiency. For this the cells were plated on glass or in agar. The
agar method was necessary when Py cells were plated with an excess of unirradiated BHK21 cells, which also grow well on glass but fail to grow in agar. The results in Table 5 show that there is a small increase in numbers of Py cells on confluent mouse cells, but a much larger increase when the same number of cells fall on glass in the sparse cultures. Sparse unirradiated cultures of BHK21 cells rapidly became confluent during the experiment, and this probably accounts for the small increment in numbers of Py cells in these cultures. Confluent BHK21 cells appear to kill the Py cells.
172
STOKER TABLE 5 GROWTH OF PY CELLS ON PREEXISTING CELL SEIEETS Preexisting culture (24 hours)
PWY;
Colony forming Py cells (loglo) per culture* after:
~log10) Mouse embryo cells
Not irradiated Irradiated
Mouse embryo cells
BHK21 cells
_--. Confluent Sparse Confluent sparse
Not irradiated
Confluent Sparse
Irradiated
Confluent Sparse
Not irradiated
5.3
Confluent Sparse
4.0
4 hours
96 hours
Inc;;txn;nt 96 hours
5.47
2.2
6.2
14.6
5.61
5.53
6.34
6.43
1.7 14.6
5.11" 5.18
5.11 5.04
5.42 6.34
5.42 5.18
5.28 5.23
3.42b 3.23
3.77
2.2
4.34
12.6
3.46
3.11
4.28 4.50
6.6 25.0
3.39
2.78
3.11
3.96
.-..-.-
0.15 6.7
* Figures based on counts of about 200 colonies after plating on glass” or in agarb. DISCUSSION
The experiments described in this paper show that the unrestricted growth and disoriented arrangement of polyoma-transformed hamster fibroblasts can be affected by contact with or close proximity to untra~forr~~ed cells of at least two species. X-irradiation of the normal cells does not prevent this effect, and even transformed cells after irradiation will prevent the multiplication of unirradiated transformed cells. The orientation of transfornled cells falling on a monolayer of normal cells, could be due to the nonspecific phenomenon of contact guidanee (Weiss, 1962), but the distortion of a transformed colony meeting an advancing sheet of oriented cells, and the failure initially to overgrow these cells, suggests that some more active inhibition of movement, and avoidance of contact is taking place. It is to be hoped that this point will be elucidated by time lapse cinematography with marked cells.2 z Since this paper was submitted the author has seen a report by G. Barski and J. Belehradek (&‘zptE. cell Res., in press), which is in close agreement with findings reported here. By time lapse ei~ematogra~hy the movement of two types of
Because the marker system availabIe is not adequate, it has not been possible to use the same quantitative techniques to investigate the interaction of viable transformed cells in contact with one another. It is clear however that transforI~led cells in colonies or congruent monolayer cultures continue to divide when in contact. Vertical sections of transformed cell colonies show mitotic figures at all levels including the centre of the colony, where cells may be separated from the substrate below or the medium above by a layer four or five cells thick. It has also been shown by time lapse cine~tography that Py cells, but not normal embryo cells or untransformed BHK21 cells, move actively when in contact with each other {AI~lbrose, personal col~~~~lu~~ation). The effect on growth and movement (i.e., orientation) following contact therefore appears to be as follows for the cells used in these experiments : 1. Normal cells inhibit normal cells. 2. Normal cells inhibit transformed cehs. 3. Transforn~ed cells do not inhibit transformed cells. -.-mouse tumor cells is shown to be arrested by contact with compact sheets of normal mouse embryo cells.
REGULATION
IN
TRANSFORMED
7.0 _ 0 ,‘O
6.0
n n n
n 1
20
40
60
so
I
HOURS
FIG. 5. Growth of marked Py cells on bare substrate or on confluent cell sheet. 105.7 marked Py cells were added to sparse (circles) or confluent (squares) monolayer cultures of irradiated mouse cells. Counts of total cells (open circles and squares) and marked cells (filled circles and squares) were made after suspension at successive times.
4. Transformed nondividing cells (after X-ray, etc.) inhibit transformed cells. These findings could be explained by postulating that in inhibiting each other normal cells display two functions. First, we suppose that the normal cell surface or some other part contains active factors or signals which inhibit other cells by contact. Second, the same normal cells respond to these inhibiting factors, or signals, from other cells. In other words each normal cell must be able both to emit and receive the contact signals. Polyoma-transformed cells seem to be sensitive to normal contact signals, but they do not inhibit each other. It is therefore suggested that they have lost the ability to emit the contact signal without losing their ability to respond.
HAMSTER
CELLS
173
This loss of emitting function is apparently dependent on cell viability because it is regained by transformed cells which are not dividing after irradiation, BUDR treatment, or spontaneous loss of ability to divide. Loss of the emitting function in the viable transformed cells could be due to the presence of surface material which masks active groups, providing this does not interfere with ability to receive the contact signal. It is of interest that Defendi and Gasic (1963) have reported a large amount of acid mucopolysaccharide detected histochemically on the surface of polyoma transformed cells, compared to untransformed cells. Sensitivity of polyoma transformed cells to contact with normal cells explains why transformation is not detected if virus-infected cells are initially cultured under crowded conditions. The majority of cells are untransformed, so the few transformed cells will be isolated from each other but touching normal cells, which presumably restrict division and movement. If on the other hand the transformed cells can start with free space around them, they may then reach an adequate clone size before coming in contact with normal cells so that some transformed cells in the centre of the colony are isolated from normal cell contacts, and continue to grow. Such clones may finally “overflow” and grow on top of the normal cell sheet if they become thick enough for a lower insulating layer of transformed cells to mask the normal contacts. In other words, the clone of transformed cells needs to reach a critical size, with enough cell to cell contacts, to escape from control by normal cells. Extension of hypotheses based on in vitro systems to explain the behaviour of tumours in animals is hazardous. Absence of contact inhibition of movement is believed to be related to the ability of tumour cells to infiltrate and metastasize, but spontaneous variants of BHIi21 cells (Defendi et al., 1963; Stoker and Macphcrson, 1964) may be isolated which are highly malignant though they grow in oriented fashion and pile up only slightly more than nontransplantable fibroblasts. Loss of contact inhibition of division may be more important in
174
STOKER
tumour cells, but there probably are other unknown factors involved as well. nievertheless the sensitivity of transformed cells to contact with normal cells in vi&v described above may help to explain some phenomena in animals. For example, polyoma tumours look highly malignant and rapidly growing, but like rodent ulcers they have a sharply defined edge and metastasize poorly if at all. This may be due to the inability of single tumour cells which are released from the main mass to escape from the inhibiting contacts with normal cells. When large numbers of polyoma-transformed cells are transplanted into an animal, a tumour appears rapidly; with lower cell numbers however, the latent period before a tumour reaches the same size may be disproportio~~ately long, and of course very small cell numbers may result in 110 tumour at all. It is now suggested that the tumour cells can grow fast and unrest,rictedly only when they reach a critical concentration which allows enough contacts between tumour cells alone. The extent to which these factors apply to cells transformed by other tumour viruses or to tumour cells in general, is not known. S~r40-t~nsforln~ tu~~~our cells are sensitive to contact inhibition of movement by giant cells (Black, personal ~ol~lmunication), and the expression of Rous-transformed cells as visible foci is inhibited by overcrowding, as with polyoma transformation. There are insu~cient data, however, to warrant generalization at this stage. There is oue striking phenomenon in clinical medicine which might possibly be explained by contact inhibition of n~ultiplication of single tumour cells by normal cells. With certain types of localized malignant tumours, individual tumour cells can be recognized in the circulating blood, but judging by the number of secondary tumours found at postn~orte~~~,the majority of these cells must fail to metastasize. There could obviously be several explanations of this effect, but it may be due to the inability of single t~~n~o~~rcells to escape from normal cell contacts. In the studies by Abercrombie et al. (1937)
tumour cells showed no contact inhibition of movement when in contact with normal cells, but this is not necessarily in conflict with the findings reported here. They used different techlli~ues, and the mouse sarcoma cells tested were highly anaplastic. If normal cells do indeed have two functions concerned in contact inhibition, we may expect that tumours might also arise from loss of the second function, ability to receive the contact signal. This defect would lead to a highly l~~aligna~~t cell which could metastasize from single cells, and it should show no contact inhibition by normal cells in culture. ACKNOWLEDGMENT I am grateful to Miss Anne Smith for her skilled technical assistance. REFERENCES ABERCROMBIE, M., and AMBROSE, E. J. (1962).
The surface properties of cancer cells-a review. Cancer Res. 22, 525-545. ABERCRO~BIE, PI., REAYSMAN, J. E. M., and KARTHAUSER, X. M. (1957). Social behaviour of cells in tissue culture III. Mutual influence of sarcoma celIs and fibroblasts. Expll. Cell Res. 13, 276-291. DEFENDI, V., and GASIC, G. (1963). Surface mucopotysaccharides of polyoma virus transformed cells. J. ~e~Z,ula~Camp. Physiol. 62, 23-26. DEFENDI, V., LEHMAN, J., and KRAEMEH, P. (1963). “Morphologically normal” hamster cells with malignant properties. Virology 19, 592598. MACPHERSON, I., and MONTAGNIER, L. (1964). Agar suspension culture for the seIective aksay of cells transformed by polyoma virus. ~~~~010~~ 23, 291-294. STOKER, M. (1962). Studies on transformat8ion by polyoma virus in vitro. C&a Found. S#mp. Il’unaour Viruses Murine Origin, pp. 365-376. STOKER, M. (1964). A simple marker technique for cells in culture. Exptl. Cell Res. in press. STOKER, M., and ABEL, P. (1962). Conditions affecting t~ransformation by polyoma virus. Cold Spring Harbor Sy,np. &z&ant. Biol. 27, 375-386. STOKER, RI., and MACPHERSON, I. (1964). The Syrian hamster fihroblast cell line BHKZI and its derivatives. Suture in press. WEISS, P. (1962). “Attraction fields” between growing tissue cultures. Science 115, 293-296.