J. Cell Sci. i, 169-173 (1966) 169

Printed in Great Britain

STRUCTURE OF NORMAL AND POLYOMA

VIRUS-TRANSFORMED HAMSTER

CELL CULTURES

W. HOUSE AND M. G. P. STOKERMedical Research Council Unit for Experimental Virus Research,Institute of Virology, University of Glasgow

SUMMARY

Vertical sections of colonies and confluent sheets of normal and polyoma virus-transformedBHK21 cells, and of a spontaneous malignant variant of BHK21 cells, and also of freshlyisolated hamster fibroblasts, were examined to determine the structural interrelationship of thecells in relation to their varying transplantability.

All cells except the freshly isolated fibroblasts formed multilayers, but the virus-transformedcells were arranged in the form of a loose network, in which cell division took place. The untrans-formed BHK21 cells were very tightly packed and mitosis was rare. A spontaneous malignantvariant which superficially resembled the normal BHK21 cells was shown by section to havefeatures in common with virus-transformed cells.

INTRODUCTION

The BHK21 line of hamster fibroblasts differs from freshly isolated cells in severalrespects but it retains the parallel-oriented arrangement of cells which is charac-teristic of normal fibroblasts in colonies and in confluent cultures (Macpherson &Stoker, 1962; Stoker & Macpherson, 1964). Polyoma virus transformation of thesecells results in a greatly increased transplantability and in a random arrangement ofcells, in marked contrast to the regular orientation of untransformed cells (Mac-pherson, 1963). Despite the fact that untransformed and transformed BHK21 cellsgrow to about the same cell density per unit area of substrate (2 x io6 cells/cm2),isolated colonies and sheets of transformed cells appear to be thicker or more piled-upthan untransformed cells, when viewed from above or below, i.e. at right angles to thesupporting substrate. However, Defendi, Lehman & Kraemer (1963) and others haveobserved that BHK21 cells may sometimes become highly transplantable withoutknown virus infection and that these variant cells retain the parallel orientation ofnormal fibroblasts in culture, closely resembling the original BHK21 cells, whichhave a very low transplantability. For more detailed observations of the morphologyand interrelationships of these various cell types in culture, colonies and cell layerswere studied in vertical section.

170 W. House and M. G. P. Stoker

MATERIALS AND METHODS

The cell lines used and their designation have been described by Stoker & Mac-pherson (1964). BHK 21/13 (abbreviated C13) is a cloned line of diploid hamsterfibroblasts, and BHK2iji3/PyY (abbreviated Py Y) is a subclone of these cells trans-formed by polyoma virus (Stoker & Macpherson, 1964). Both lines were used after40-50 generations since cloning. BHK2i/Is3 (abbreviated Is3) is a spontaneousvariant, isolated from BHK21 and kindly sent by Dr Gottlieb-Stematsky. It was usedafter an unknown but certainly large number of generations. Extensive tests failed toreveal mycoplasmas in the C13 or Py Y clones used, but Is3 cells were found to containmycoplasmas after the completion of the investigation. Freshly isolated kidney fibro-blasts (HKF) were also examined; these were prepared by standard methods fromkidneys of 3-day-old hamsters and used after one subculture. Calf serum 10% andtryptose phosphate broth 10% in modified Eagle's medium was used throughout.

Sections of cell colonies of C13, PyY and Is3 were obtained by plating 500 singlecells on to 60-mm plastic tissue-culture dishes. After 7 days of incubation at 37 °Cthe growth medium was removed, and after washing with Eagle's medium the colonieswere treated in situ with standard histological reagents as follows: Bouin's fixative,30 min; 90 % ethanol, 20 min; ethanol, 20 min; and ethanol/chloroform (equalparts, v/v), 20 min. Chloroform was then added to the dishes to dissolve a thin layerof plastic so that after 3-5 min gentle agitation the colonies detached from the surfaceof the dish. The colonies were then transferred using a wide-bore pipette to a glasscontainer of fresh chloroform and left for 20 min. Subsequently, the colonies weretreated as follows, using a wide-bore pipette for all transfers: chloroform/paraffin wax(equal parts, v/v), 20 min; paraffin wax (two changes) 60 min. The colonies wereembedded vertically in fresh paraffin wax. After the paraffin wax had set, the blockswere trimmed to present the colony vertically to the knife, and sections were cut at8-10 fi, mounted on slides and stained with haematoxylin and eosin.

Sections of confluent cell layers of C13, PyY and Is3 were obtained by plating2 x io4 cells in 60-mm plastic dishes with the medium as previously described andincubating at 37 °C. To obtain a monolayer of HKF cells, 2 x io6 cells were plated.After 7 days' incubation (with changes of medium on the 3rd and 5th days) cell layerswere treated in the same way as the colonies. After 3-5 min of treatment with chloro-form the cell layers came off the dishes as complete sheets and at the embedding stageonly parts of the sheets were used. Sections were cut at 8-10 ju,, mounted and stainedwith haematoxylin and eosin.

The mean number of cells per colony was obtained from seven replicate cultures,by dividing the number of cells obtained from each dish after trypsinization by thenumber of colonies observed.

Numbers of cells in confluent sheets were determined as the mean of seven replicatecultures.

Normal and transformed hamster cells 171

RESULTS

Structure of colonies

Table 1 shows that the average number of cells per colony was approximately the samefor C13, Py Y and Is 3 cells. Typical colonies of the three cell types are seen in Figs. 1-3,respectively. The similarity of C13 and Is3 cells is shown with the cells arranged inparallel bundles, and in contrast the criss-cross random arrangement of cells in thecolony of Py Y cells. Figs. 4-6 show typical sections of colonies. The untransformedC13 colonies (Fig. 4) are not monolayers, and the centres of the colonies are up tofour cells thick. The proximity of the nuclei to each other and their elongated shape iscompatible with close packing of the cells, with very little intercellular space. Mitoticfigures are rarely seen in fully developed colonies at 7 days.

It will be seen that the colonies of polyoma-transformed BHK21 cells are quitedistinct (Fig. 5). They consist of cells piled up at the colony centre to a depth of tencells. The nuclei are separated and rounded, and there are spaces between the cells,suggesting a loose network. Fig. 7 shows that cells may be seen in mitosis at variousdepths, and at the surface and bottom layer.

Despite the superficial similarity to C13 colonies, the sections of colonies of Is 3cells (Fig. 6) show clear distinctions. The Is3 colonies are not much thicker than C13colonies in terms of cell numbers but the physical distance from top to bottom isgreater. The cells are larger and the nuclei more rounded and there is some unstainedintercellular space. Though the extent of piling up is much less than in colonies ofPyY cells, in general appearance Is3 cells are more like PyY cells than C13 cells.

Many other colonies of the three types of cell have been examined, and though thereis variability in size and depth the differences described above are consistent.

Structure of cell sheets

Sections were also made of cell sheets grown to confluent cultures on plastic dishes.It will be seen from Table 1 that C13, Py Y and Is3 all show approximately the samedensity of cells after 7 days' growth. Fresh hamster-fibroblast sheets on the other handare of different density, and it is known that they do not increase on further incubation.As in the colonies, the C13 and Is3 cells were oriented in parallel and appeared to beidentical when viewed vertically while Py Y cells were randomly arranged.

Sections of these cell layers showed little variation from one part of the layer toanother, and Figs. 8-11 show typical regions of this type of culture. As in colonies the

Table 1. Average number of cells in colonies and cell sheets ofCi3,PyY,Is3andHKF

Cell line C13 PyY Is3 HKF

0-30 x io7

Average number ofcells/colony

Average number ofcells/confluent culture

0

1

•94 x

•00 x

I O 4

I O 7

1

0

•02 x

•90 x

I O 4

I O 7

0-87

o-95

X

X

I O 4

I O 7

172 W. House and M. G. P. Stoker

C13 cells are multilayered, about 3 cells thick, and closely packed, while the Py Y cellsare 4-6 cells thick, but with rounded, well-spaced nuclei and intercellular space,indicating much looser packing. The Is 3 cell layers are like the C13 layers, about3 cells thick, but the nuclei are separated more than in C13 layers and are not sotightly packed.

DISCUSSION

Untransformed BHK21J13 cells with a low transplantability show parallel orienta-tion, and E. J. Ambrose and K. Shepley (unpublished observation) have demonstratedby time-lapse cinematography that they are subject to contact inhibition of movementin confluent cultures. It had also been assumed that the cells ceased to grow and re-mained as monolayers when cultures became confluent. The sections, however, showthat C13 cells do continue to multiply for one or two divisions while in close contactwith each other and form multilayers several cells thick. This explains the ability of thecells to reach high densities on the substrate, compared to freshly isolated fibroblasts.

The sections of PyY cells show a much thicker layer than C13 cells, due partly tolooser packing and partly to the shape of the cells, which are more rounded and do nottake up the extremely elongated shape of the C13 cells. This may be due to activemovement but it may also be connected with the loss of anchorage-dependence whichaccompanies transformation. C13 cells resemble other normal fibroblasts in that theywill only multiply if they can attach to and elongate on a substrate; even in the multi-layered C13 cultures it is possible that each cell has an attachment to the substrate.After transformation the cells become independent of this requirement and willmultiply in suspension, for example in soft agar or in fluid.media. The section ofcolonies and monolayers of Py Y cells and the occurrence of mitosis in all layers suggeststhat the cells are in effect growing in suspension, in a loose matrix which is functionallyindependent of the substrate surface.

The highly malignant Is 3 cells in culture have previously appeared to be in-distinguishable from the relatively poorly transplantable C13 cells. However, thesections of the Is 3 cultures show that they are different from the C13 cells and inter-mediate between these and PyY cells. The Is3 cells examined contained a myco-plasma and the effect of this organism on the cells is unknown, but it may be noted thatthere is no similar change in morphology and structure of C13 and PyY cells whenthese lines have been deliberately infected with the same mycoplasma.

This investigation shows that vertical sections of cultured cell layers may revealfeatures which are not obvious in the usual view through the thickness of the layer.Further studies are needed to determine whether the features correlated with hightransplantability are shown by other types of tumour cells.

We are grateful to Miss A. McCaffery and Mr H. Young for their assistance with the histo-logy and to Mr A. Mcllroy for the photography.

Journal of Cell Science, Vol. i, No. z

Figs. 1-3. Typical colonies showing the regular arrangement of C13 (Fig. 1) and Is3(Fig. 3) cells, and in contrast a colony of PyY cells (Fig. 2). Leishman stain, x 25.Figs. 4-6. Sections of colonies of C13 cells (Fig. 4), PyY cells (Fig. 5), and Is3cells (Fig. 6). Plastic substrate at bottom. Stained with haematoxylin and eosin. x 720.Fig. 7. A section of a colony of PyY cells to show cells in mitosis at different levels.Plastic substrate at bottom. Stained with haematoxylin and eosin. x 720.

W. HOUSE AND M. G. P. STOKER {Facing p. 172)

Journal of Cell Science, Vol. i, No. 2

8

!T*K

10

11

Figs. 8-11. Sections of confluent cell layers of HKF cells (Fig. 8), C13 cells (Fig. 9),PyY cells (Fig. 10), and Is3 cells (Fig. 11). Plastic substrate at bottom. Stained withhaematoxylin and eosin. x 720.

W. HOUSE AND M. G. P. STOKER

Normal and transformed hamster cells 173

REFERENCES

DEFENDI, V., LEHMAN, J. & KRAEMER, P. (1963). 'Morphologically normal' hamster cells withmalignant properties. Virology 19, 592-598.

MACPHERSON, I. (1963). Characteristics of polyoma-transformed cells, jf. natn. Cancer Inst.30, 795-815-

MACPHERSON, I. & STOKER, M. (1962). Polyoma transformation of hamster cell clones. Virology16, 147-151.

STOKER, M. & MACPHERSON, I. (1964). Syrian hamster fibroblast cell line BHK21 and itsderivatives. Nature, Lond. 203, 1355-1357.

{Received 24 November 1965)