CYTOLOGY AND GROWTH CHARACTER- ISTICS OF HUMAN TUMOUR ASTROCYTES TRANSFORMED BY ROUS SARCOMA VIRUS

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1 J. Cell Sci. 5, (1969) 583 Printed in Great Britain CYTOLOGY AND GROWTH CHARACTER- ISTICS OF HUMAN TUMOUR ASTROCYTES TRANSFORMED BY ROUS SARCOMA VIRUS E. H. MACINTYRE, R. A. GRIMES AND A. E. VATTER Department of Pathology, University of Colorado Medical Center, and Webb-Waring Institute for Medical Research Denver, Colorado 80220, U.S.A. SUMMARY Human tumour astrocytes (118MG) were exposed in vitro to the Engelbreth-Holm strain of Rous sarcoma virus at a multiplicity of infection of one. Morphological transformation of the cells in 60-mm plates was complete in 9-11 weeks after viral exposure. The transformed cells {EH-i 18MG) grew as a monolayer, upon which nests of rounded cells developed. From these nests, rounded cells shed into the medium and in turn on seeding formed a monolayer and repeated the cycle. The transformed cells contained the viral group-specific antigen in the cytoplasm, and produced Rous sarcomas in chickens. No complete virus was demonstrated in the mammalian cells. Electron micrographs of the parent 118MG and transformed EH-118MG cells gave further details of the structure of cytoplasmic protrusions which were formed in some of the free EH-i 18MG but not in the 118MG cells. Their nature is completely unknown. INTRODUCTION It has been known for many years that Rous sarcoma virus (RSV) produces a characteristic cancer, the fibrosarcoma, in a wide vaiiety of animals (Rous, 1911; Ahlstrom & Forsby, 1962; Munroe & Windle, 1963; Svet-Moldavsky, Trubcheninova & Ravkina, 1967). Recently, investigators have studied the effect of RSV on cells of the mammalian central nervous system. RSV injected into this system produces two tumours sarcoma of the leptomeninges (which cover the brain) (Rabotti, Grove, Sellers & Anderson, 1966), and a pleomorphic cancer of the supporting glial tissue glioblastoma multiforme (Rabotti & Raine, 1964; Rabotti et al. 1966). These are readily distinguishable from the classical Rous fibrosarcoma. Fibroblasts (Halberstaedter, Doljanski & Tenenbaum, 1941), epithelial cells (Ephrussi & Temin, i960) and certain human astrocytes (Macintyre, 1968; Ponten & Macintyre, 1968 a) show morphological transformation and a variable loss of contact inhibition (Abercrombie & Heaysman, 1954; Hayflick & Moorhead, 1961; Eagle, 1965) on exposure to RSV in tissue culture. The Rous-transformed human astrocytes exhibit additional distinctive growth characteristics. The cells develop in a continuous cycle, which includes monolayer, multilayer, and free-growing states. It is the purpose of this communication to describe in detail this in vitro behaviour, and the comparative cytology of these states.

2 584 E. H. Macintyre, R. A. Grimes and A. E. Valter MATERIALS AND METHODS The human tumour astrocyte strain, 118MG, was grown from primary culture of a malignant cerebral tumour, a glioblastoma multiforme (Ponten & Macintyre, 1968a). The Roustransformed human glial cells, designated EH-118MG (Macintyre, 1968; Ponten & Macintyre, 1968a), were developed through the action of the Engelbreth-Holm strain of Rous sarcoma virus (EH-RSV) on 118MG cells in tissue culture. All cells were maintained in Eagle's minimum essential medium (obtained from Schwarz Biochemical Co.) supplemented by 10% foetal calf serum (FCS) (Hyland Laboratories), by penicillin at 150 units and by streptomycin at 50 fig per ml. Calf serum (CS) (Hyland Laboratories) replaced foetal calf serum for viral studies (Rubin, 1960a). Falcon plastic Petri dishes and Belico roller bottles were used for routine cultures, and medium was changed biweekly. Eagle's modified spinner medium (Schwarz Biochemical Co.) with 20 % FCS was employed in Belico spinners. Trypsin (Hyland Laboratories) at 025 % served to remove attached cells for transfer. Ethylenediaminetetraacetate (EDTA) (Sigma Biochemical Co.) at 0-05 % in phosphate-buffered saline (PBS) was substituted for trypsin when preparing cells for growth curves, after comparative counts had shown no essential difference between the resultant values. EDTA did not lyse the cells and separated clumped cells. All counts for growth curves were performed in triplicate. Primary fibroblast cultures were prepared from 10-day-old fertilized Hyline eggs, after the method of Rubin (19606). Interference studies for virus production used published methods (Rubin, 1961), as did direct (Vogt & Rubin, 1961) and indirect (Kelloff & Vogt, 1966) fluorescence procedures for detecting species-specific and group-specific viral antigens respectively. Hamster antisera directed against Rous sarcoma virus tumour were purchased from Flow Laboratories, Inglewood, California. Fluorescein-labelled goat antisera against hamster serum globulins were obtained from Antibodies Incorporated, Davis, California. Normal hamster sera used in control experiments were purchased from Colorado Serum Company, Denver, Colorado. Chicken antisera against avian tumour viruses were prepared and their globulin fractions conjugated to fluorescein isothiocyanate by published methods (Vogt & Rubin, 1961). Quail cells were supplied through the courtesy of Dr C. Moscovici. The oncogenicity of the human astrocytes was tested by subcutaneous injection of 1-2 x io 3 cells into the wing web of 4-week-old Hyline chickens. Both 118MG cells and the Rous-transformed EH-118MG cells were tested for their ability to produce tumours. The birds were sacrificed 2 weeks after tumour development, and specimens were taken for electron microscopy and tissue culture. Chromosome preparations were made following the procedure of Saksela & Moorhead (1962). Autoradiographs of EH-118MG cells labelled with o-oi /tci/ml tritiated thymidine ( 3 H-TdR) for 24 h were prepared by a technique already reported (Ponten & Macintyre, 19686) and stained with toluidine blue. Wright's or May-GrOnwald Giemsa stains were used for all other preparations. The following procedure was used to prepare cells for electron microscopy. All steps until infiltration with plastic were performed at o C C. A freshly prepared mixture of 2-5 % glutaraldehyde and 1 % osmium tetroxide (1:2) in 01 M cacodylate buffer, ph 74 (Hirsch & Fedorko, 1968) was added in equal quantity to medium containing living cells. After 2 min, the cells were harvested, pelletted at slow speed, and resuspended in fresh fixative for min. The cells were then washed several times with cacodylate buffer, by repeated sedimentation and resuspension, and the pellet was post-fixed in 0-25 % uranyl acetate in 001 M acetate buffer, ph 63 for 20 min. Several rinses with buffer were followed by dehydration of the cell pellet in 70, 80, and 97 % hydroxypropyl methacrylate, allowing 5 min for each concentration. The cells were infiltrated with a series of hydroxypropyl methacrylate and Epon mixtures and embedded in Epon. Areas were selected for electron microscopy by optical-microscopic study of sections fim thick. Sections were stained with lead actate for 2 min, and examined in a Philips EM 300 electron microscope. In certain preparations in which mucopolysaccharide preservation was desired, specimens were prepared according to Luft (1964) with ruthenium red which binds to acid mucopolysaccharides.

3 Human Rom-transformed tumour astrocytes 585 RESULTS The human malignant astrocytes (118MG), which were subsequently exposed to RSV, grew in multilayered loosely packed bundles of fusiform cells (Figs. 2, 3), criss-crossed by bipolar spongioblasts (Ponten & Macintyre, ICJCSC). The astrocyte cytoplasm was deeply basophilic and contained numerous r.cidophilic granules Free EH-J18MG (roller) o ^ 50 Attached EH-U8MG Free EH-N8MG (seeded on plates) MG (plates) Days Fig. 1. Comparative growth curves. Free EH-118MG cells (roller): Plates for this growth curve were seeded from free cells growing in a Bellco roller bottle. EH- 118MG cells to be seeded were rinsed briefly with 005 % EDTA in PBS on plates, and several times with PBS, before trypsinization. This served to remove all free cells and all attached round cells. EH-118MG: The plates for this growth curve were seeded from free round EH-118MG cells. 118MG: Plates for this growth curve were seeded from trypsinized cell sheets. Neurofibrils were demonstrable (Shein, 1965). The nucleus of the majority of the astrocytes was single, irregular and contained two or three large nucleoli. Approximately 5 % of the astrocytes were giant forms. Their nucleus was usually large and single, but multilobed variants (Fig. 2) were occasionally found. Exposure of 118MG cells 2 days after cell seeding to EH-118 RSV at a multiplicity of infection of 1 resulted in the following sequence of events. Four weeks after addition of virus,

4 586 E. H. Macintyre, R. A. Grimes and A. E. Valter scattered islets of mitosing, refractile, oval cells appeared within the now densely overgrown 118MG cells. One week later, nests of round cells (3-6 cells in each), some in mitosis, appeared studding the cytoplasmic border of the other transformed cells (Fig. 3), which by now had a stellate or undulant outline. Scattered giant cells were observed in the base monolayer at this time. Free round cells, similar in appearance to those in the nests, were observed in the medium within a few days. The areas of transformation expanded centrifugally as a monolayer of cells carrying multilayered cell nests. Giant cells increased in number (Fig. 4). The transformed cells replaced the parent cells in 60-mm Petri dishes completely within a further 4-6 wjeks, that is, 9 11 weeks after addition of the virus (Fig. 5). The nests of round cells increased in population to each within this same period (Fig. 5). Free round cells (Fig. 5) increased in number steadily. In the fully transformed cell complex, the vessel surface was covered by a monolayer of undulant or stellate cells, interspersed by pleomorphic giant cells (Figs. 6 8). The latter were much more numerous at 20% of the population than the 118MG giant cells. The basal monolayer carried numerous multilayered colonies of round cells in mitosis (Figs. 5-7), and similar free cells lay in the medium. Some of the free EH-118MG cells carried several well-defined cytoplasmic protrusions (Fig. 9). These outpouchings were usually of a different consistency and staining quality than the cytoplasm from which they arose. Similar structures were seen (Fig. 10) in giant cells, and at the non-contiguous borders of round cell pairs. An example of cytoplasmic fusion is shown (Fig. 11). The multinuclear cell contained two distinct types of nucleus. The relationship among the morphological types seen in the fully transformed plate was clarified by further studies. It was observed that the free round EH-118MG cells could attach to a fresh surface. They sent out cytoplasmic processes and within a few hours formed a monolayer whose morphology was identical to that of the undulant- to stellate-bordered EH-118MG cells described above. Giant cells were few at this time, but increased in number progressively from a level of 10 % to 30 % at 3 months, always as part of the basal layer. The stellate and giant cell base layer completely covered the 60-mm plate within 5-10 days (depending upon the number of cells seeded). At this time few mitoses could be found in the base layer, although DNA synthesis continued (Figs. 6-8). Attached round cells first appeared 2-4 days after the initial cell seeding. They lay as groups of 1-5 cells associated with the cytoplasmic border of the base layer (of both mononuclear and giant) cells (Figs. 3, 4, 6, 7). The nests increased in number and shed free cells as already described. Mitoses were also present among the free cells in the medium. The numbers of free round EH-118MG cells seemed limited only by the amount of nutrient available. These cells could be seen by the naked eye as a distinct granularity in the medium. Bipolar spongioblasts, identical in morphology to those seen in the parent 118MG cells, appeared 1-2 days after cell seeding, and increased in number with time. The cell complex formed by seeding the round EH-118MG cells was identical in architecture to that of the original Rous-transformed 118MG cells. The substitution of stellate EH-118MG for round EH-118MG cells as initial seed gave the same final results. These findings indicated a clear relationship between the attached stellate,

5 Human Rous-transformed tumour astrocytes 587 attached round, and free round EH-118MG cells. These three types represented different morphological states of the same cell. Initial results from cloning further supported this concept. The cells have been in continuous culture for 2 years, and still maintain the same growth characteristics. EH-118MG cells may therefore be considered a cell line (Hayflick & Moorhead, 1961). Comparative growth curves were prepared using (i) attached stellate and (ii) free round EH-118MG cells for initial seeding (Fig. 1). The plates were rinsed gently several times with PBS after cell attachment. Inspection had shown no attached round colonies at this time and multiple rinses served to remove any free round cells which might subsequently have attached or have multiplied in free state and vitiated resultant counts. With both types of original seed (Fig. 1), the cells attached in equivalent numbers, multiplied at comparable rates, and attained a final total of 8 x io 6 per 60-mm plate in 17 days. The final architecture of the cell complexes was in all ways comparable to that already described. The free cells were all viable as judged by eosin and trypan blue exclusion. Parent 118MG cells (Fig. 1) had a slower growth rate than the virustransformed cells. The question arose whether this system could continue to produce free cells indefinitely and continuously and if such cells would remain viable. In order to find out, the free EH-118MG cells were seeded in Bellco roller jars. Cell attachment was complete within 14 h and the medium with its free round cells was. poured off and replaced without rinsing. Attached cells grew as a monolayer; round-cell colony and free cell formation proceeded subsequently; giant cells and bipolar spongioblasts developed as before. The growth curve of free EH-118MG cells in such a system (Fig. 1) indicated that on day 1 a significant number (5-6 x io 6 ) of EH-118MG cells remained free in the medium, although at this time no attached round-cell colonies were seen. These free cells were considered to represent the residuum of unattached cells left after pouring off the medium at 14 h. Comparable free cells had been removed from the previous growth curves by multiple rinsing shortly after cell attachment. The total free cell count continued to increase after 24 h although no attached round colonies were found at this time. This suggested that the free cells underwent mitosis, and that attached round-cell nests contributed to the increase in round cells, but were not the sole agent. The free cell population rose continuously throughout the 13-day period of this study. Parent 118MG cells did not grow well in roller jars. The plating ability, and thus the viability, of the round EH-118MG cells removed at 14 h from the previous experiment, reported in Fig. 1, were then tested as follows. The free cells were seeded at 0-18 x io 6 on 60-mm plates. Within 14 h, 0-15 x io 6 cells had attached per plate. It was concluded that the cells which had not attached to the Bellco jar were inherently capable of so doing, but had lacked available surface area on the vessel. Autoradiographic studies of EH-118MG cells (Figs. 6 8) demonstrated that both the stellate and the round types synthesized DNA and underwent mitosis. The different morphological types of giant cells also participated in this process, and some showed a gradient of 3 H-TdR uptake (Figs. 6, 8). These investigations are being pursued further.

6 58S E. H. Macintyre, R. A. Grimes and A. E. Vatter The presence of complete Rous sarcoma virus in the medium from EH-118MG cells was sought repeatedly using plaque assay and interference studies. The EH 118MG cells were examined by direct fluorescence and electron microscopy for the virus. They were co-cultivated with chicken and quail cells, and both cells and medium subsequently investigated for virus. Quail cells were used to check the possibility that RSV (O) (Vogt, 1967) was the infective agent. All such studies failed to show virus. The interference and fluorescence studies were repeated by Dr C. Moscovici (personal communication), with the same negative results. Injection of 1-2 x io 6 EH-118MG cells subcutaneously into the wing web of chickens was followed within 3-6 weeks by the development of typical Rous sarcomas of chicken karyotype in 7 out of 25. No tumours were found in chickens injected similarly with 118MG cells not transformed by Rous sarcoma virus. The chickens were sacrificed 2 weeks after tumour development, and specimens were taken for electron microscopy and tissue culture. Again, no virus was detected. Indirect fluorescence microscopy of the EH-118MG cells showed that the groupspecific antigen (Kelloff & Vogt, 1966) was present within the cytoplasm of all the transformed cells. The specific staining was much brighter in the round cells, probably because the cytoplasm of the attached stellate cells was spread out more and was thinner while that of the round cells was more concentrated and thicker. The degree of fluorescence was therefore considered equivalent after taking cytoplasmic volume into consideration. The 118MG cells (not transformed by RSV) gave no specific fluorescence, nor did EH-118MG cells when normal hamster serum was substituted for hamster antiserum directed against the Rous sarcoma tumour antigen (Kelloff & Vogt, 1966). Electron microscopy permitted the comparison of parent 118MG cells and the two main types of Rous-transformed offspring. Nuclear pleomorphism and nucleolar hyperplasia were present and equivalent in all. Ribosomes were abundant, usually as polysomes. Rough endoplasmic reticulum varied in amount and was occasionally dilated. All cells were highly phagocytic of carbon (Fig. 12) and engulfed dead cells. Mitochondria were plentiful (Figs. 13, 14) and occasionally abnormal in shape and cristal pattern (Figs. 13, 15). Surface coatings of mucopolysaccharide as observed after ruthenium red fixation (Fig. 15) appeared comparable in all cultures. Lysosomes of all stages were much more common and the Golgi system was better developed in the parent 118MG cells than in the transformed cells (Fig 13). Some transformed cells had multiple protrusions of the cell border (Figs. 15, 16). These had a variable content of organelles. Usually they were rich in ribosomes but poor in endoplasmic reticulum and mitochondria. Occasionally the two latter organelles were numerous (Fig. 15). This was in contrast to the remainder of the cytoplasm, whose organelle content was comparable to that of the other transformed cells. Free structures whose content was analogous to that of the protrusions were observed both by light and electron microscopy. Only transformed cells had numerous large (1-2/mi) cytoplasmic vacuoles (Fig. 14). These were without characteristic features. The phagocytosed carbon was found within similar sized vacuoles. In addition to these structures, autophagic vacuoles were present. Glycogen granules were identified in

7 Human Rous-transformed tumour astrocytes 589 cells of all types. No membrane-bound glycogen granules, annular lamellae, complete or incomplete virus particles (Di Stefano & Dougherty, 1965; Courington & Vogt, 1967) were seen in any of the multiple sections examined. Comparison of the filament distribution and content of all tumour cell types was disappointing. Filaments were plentiful, usually randomly distributed (Figs ), and rarely in parallel bundles (Fig. 16). They were absent from the cytoplasmic border of the round EH-118MG cells (Figs. 12 and 14-16) (Courington & Vogt, 1967). DISCUSSION The present investigation studied the effect of Rous sarcoma virus on human malignant astrocytes. This is in contrast to the studies of previous workers who have concentrated exclusively upon the response of normal tissue to this virus (Rous, 1911; Halberstaedter et al. 1941; Ephrussi & Temin, i960; Ahlstrom & Forsby, 1962; Munroe & Windle, 1963; Rabotti & Raine, 1964; Rabotti et al. 1966; Svet-Moldavsky et al. 1967). It is therefore not surprising that a new morphological and behavioural pattern should emerge. This does not necessarily imply that only malignant cells would manifest this particular response to RSV, or even that all malignant astrocytes would so react. It has long been considered that obedience to the laws of contact inhibition (Abercrombie & Heaysman, 1954; Hayflick & Moorhead, 1961; Eagle, 1965) is a hallmark of normalcy in tissue culture, while the reverse holds for malignant cells which, to a variable degree, disobey such laws (Abercrombie, Heaysman & Karthauser, 1957; Stoker, 1964). Exceptions to this generalization are well documented (Defendi, Lehman & Kraemer, 1963; Todaro & Green, 1966; Macintyre & Ponte'n, 1967), but the cycle of EH-118MG growth described above presents a further modification. This cycle encompasses both distinct change in morphology and a radical alteration in cell-to-cell relationship. These cells change from monolayer to multilayer to free state and back to monolayer, and metamorphose from stellate to round at the same time. Earlier workers (Doljanski & Tenenbaum, 1942) studying Rous-transformed fibroblasts in plasma clot cultures suggested that the 2 cell types seen spindle and round were interchangeable, depending on physical conditions. Time-lapse cinematography will be necessary to follow relationships among EH-118MG cells further. The manifestation of Rous-induced changes in fibroblast behaviour in vitro may be suppressed by substituting foetal calf serum for calf serum in the growth medium (Rubin, 1960a). This does not hold for EH-118MG cells which have been supplemented with foetal calf serum since their inception. Unhealthy cells often shed into the medium. The free, round EH-118MG cells were in excellent state, as evidenced by their cell-plating ability, organelle preservation and dye exclusion. DNA synthesis and mitosis took place in both stellate cells and round-cell colonies and was found among free cells. A substantial increase in the number of free cells was found regularly in roller jars before any contribution could have been made from attached round-cell nests. One 2-fold increase in cell count was regularly found in spinners, although this was not maintained. Information seems sufficient to support a tentative suggestion that

8 590 E. H. Macintyre, R. A. Grimes and A. E. Vatter round-cell proliferation proceeds both in cell colonies and in the free state. The observation that DNA synthesis continued among monlayers of stellate EH-118MG cells, although mitosis had ceased, and that there was a gradation of DNA uptake present within the same nucleus of certain giant cells is under further study. The tumour from which the original parent cells 118MG were grown, a glioblastoma multiforme, was characterized histologically by pleomorphism. Differentiation within this tumour was variably expressed, and showed as areas of primitive spongioblasts, of bipolar spongioblasts (pilar astrocytes), of multinucleated cells, and of fibrillary astrocytes. The parent 118MG and the Rous transformed EH-118MG cells retained some features of tumour pleomorphism and cell differentiation in tissue culture. The latter was manifested by the development of bipolar spongioblasts (pilar astrocytes) and giant cells in vitro. Rous sarcoma induces cell fusion (Moses & Kohn, 1963). Rous sarcoma contributed to the comparative increase in numbers of giant cells and in the development of their bizarre morphology in EH-118MG cultures. Figure 11 clearly demonstrates 2 sets of nuclei with different chromatin patterns within the same cell evidence of cytoplasmic fusion. The fact that no complete virus was detectable suggests that fusion is a property of the virus genotype, and does not depend upon the production of complete virus. The general pattern of growth in the EH-118MG cells, was somewhat reminiscent of the cell-feeder relationship existing between fibroblasts and colonies of lymphoid cells (Benyesh-Melnick, Fernbach & Lewis, 1963; Ponten & Macintyre, 1968 a). The lymphoid cells developed in close association with fibroblasts as colonies from which free round cells detached into the medium. The resemblance to EH-118MG growth ended there. The lymphoid system had an absolute requirement for a continuing replenishment of feeder cells, and the lymphoid cells were unable to plate out on a free surface. The failure to demonstrate complete virus in the Rous-transformed mammalian cells is not novel (Svoboda, Chyle, Simkovic & Hilgert, 1963; Simkovi, Svoboda & Valentova, 1963; Jensen, Girardi, Gilden & Koprowski, 1964; Huebner et al. 1964). The apparent absence of complete virus in chickens bearing sarcomas induced by EH-118MG cells is more difficult to explain. The addition of helper virus (Hanafusa, Hanafusa & Rubin, 1963) may be necessary to permit the production of complete Rous virus. This will be subject to further study. The presence of at least part of the Rous sarcoma virus genome in the EH-118MG cells is shown by the induction of chicken sarcomata by the EH-118MG cells, and by the demonstration of the avian leukosis and sarcoma group specific antigen in the EH-118MG cells by indirect fluorescence. Electron-microscopic findings confirmed previous reports on the rich ribosomal content of malignant cells, their nuclear pleomorphism and nucleolar hyperplasia (Bernhard, 1958; Haguenau, Febvre & Arnoult, 1962). The apparent peripheral accumulation of mucopolysaccharide on the surface of both EH-118MG and 118MG cells suggests that it may be a more general finding than previously shown (Todaro & Green, 1966; Courington & Vogt, 1967). Further studies are required to confirm the absolute specificity of ruthenium red for acid mucopolysaccharides in this system. Decreased lysosomal content has been noted in normal cells growing in logarithmic

9 Human Rous-transformed tumour astrocytes 591 phase compared with the same cells in 'stationary' phase (Gordon, Miller & Bensch, 1965), and an analogous situation may hold for Rous-transformed compared with parent 118MG cells. The cytoplasmic protrusions seen in some free EH-118MG cells are a feature not previously described in Rous-transformed cells. The work of two sets of investigators (Di Stefano & Dougherty, 1965; Courington & Vogt, 1967) is particularly apposite for comparison here. They studied the fine structure of chicken cells which transformed and contained the Rous sarcoma genome, but which did not produce sufficient complete virus to permit its detection by the then standard techniques. Neither group described such large cytoplasmic expansions although small pseudopodia were shown (Courington & Vogt, 1967). The protrusions may serve as cell-to-cell fusion contacts, as the ruffled border is never found between contiguous cells. They may separate from the cell as a form of excretion or secretion. They may even be a form of pseudopodium. The authors would like to express their sincere appreciation to Dr P. K. Vogt, Dr R. M. Mulligan and Dr C. Moscovici for helpful suggestions made during the course of this work.the authors are also deeply appreciative of the help and encouragement given by Dr J. Ponton in the early stages. Mrs D. Gillespie, Mrs D. Gaskin, Miss M-B. Wall, Miss K. Lindquist and Mrs L. King rendered skilful technical assistance. This study was supported by PHS grants NBO 7983 and CAO 5164 and by a Milheim Foundation grant. REFERENCES ABERCROMBIE, M. & HEAYSMAN, J. E. M. (1954). Observations on the social behaviour of cells in tissue culture. II. Monolayering of fibroblasts. Expl Cell Res. 6, ABERCROMBIE, M., HEAYSMAN, J. E. M. & KARTHAUSER, M. M. (1957). Social behaviour of cells in tissue culture. III. Mutual influence of sarcoma cells and fibroblasts. Expl Cell Res. 13, AHLSTROM, C. G. & FORSBY, N. (1962). Sarcomas in hamsters after injection with Rous chicken tumour material. J. exp. Med. 115, BENYESH-MELNICK, M., FERNBACH, D. J. & LEWIS, R. T. (1963). Studies on human leukemia. I. Spontaneous lymphoblastoid transformation of fibroblastic bone marrow cultures derived from leukemic and non-leukemic children. J. natn. Cancer Inst. 31, BERNHARD, W. (1958). Electron microscopy of tumor cells and rumor viruses. A review. Cancer Res. 18, COURINGTON, D. & VOGT, P. K. (1967). Electron microscopy of chick fibroblasts infected by defective Rous sarcoma virus and its helper. J. Virol. 1, DEFENDI, V., LEHMAN, J. & KRAEMER, P. (1963). 'Morphologically normal' hamster cells with malignant properties. Virology 19, Di STEFANO, H. S. & DOUGHERTY, R. M. (1965). Cytological observations of 'nonproducer' Rous sarcoma cells. Virology 27, DOLJANSKI, L. & TENENBAUM, E. (1942). Studies on Rous sarcoma cells cultivated in vitro. I. Cellular composition of pure cultures of Rous sarcoma cells. Cancer Res. 2, EAGLE, H. (1965). Metabolic controls in cultured mammalian cells. Science, N.Y 148, EPHRUSSI, B. & TEMIN, H. (i960). Infection of chick iris epithelium with the Rous sarcoma virus in vitro. Virology 11, GORDON, G. B., MILLER, L. R. & BENSCH, K. G. (1965). Studies on the intracellular digestive process in mammalian tissue culture cells. J. Cell Biol. 25, HAGUENAU, F., FEBVRE, H. & ARNOULT, J. (1962). Mode de formation intra-cellulaire du virus du sarcome de Rous: Etude ultrastructurale. J. Microscopie i, HALBERSTAEDTER, L., DOLJANSKI, L. & TENENBAUM, E. (1941). Experiments on the cancerization of cells in vitro by means of Rous sarcoma agent. Br. J. exp. Path. 22,

10 592 E. H. Macintyre, R. A. Grimes and A. E. Vatter HANAFUSA, H., HANAFUSA, T. & RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. natn. Acad. Sci. U.S.A. 49, HAYFLICK, L. & MOORHEAD, P. S. (1961). The serial cultivation of human diploid cell strains. Expl Cell Res. 25, HIRSCH, J. G. & FEDORKO, E. (1968). Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and 'post fixation' in uranyl acetate. J. CellBiol. 38, HUEBNER, R. J., ARMSTRONG, D., OKUYAN, M., SARMA, P. S. & TURNER, H. C. (1964). Specific complement-fixing viral antigens in hamster and guinea pig tumors induced by the Schmidt-Ruppin strain of avian sarcoma. Proc. natn. Acad. Sci. U.S.A. 51, JENSEN, F. C, GIRARDI, A. J., GILDEN, R. V. & KOPROWSKI, H. K. (1964). Infection of human and simian tissue cultures with Rous sarcoma virus. Proc. natn. Acad. Sci. U.S.A. 52, KELLOFF, G. & VOGT, P. K. (1966). Localization of avian tumor group-specific antigen in cell and virus. Virology 29, LUFT, J. H. (1964). Electron microscopy of cell extraneous coats as revealed by ruthenium red staining. J. Cell Biol. 23, 54A-55A. MACINTYRE, E. H. (1968). Transformation of glioblastoma cells in tissue culture by Rous sarcoma virus. Bad. Proc. p. 175, Abstract V 181. MACINTYRE, E. H. & PONTEN, J. (1967). Interaction between normal and transformed bovine fibroblasts in culture. I. Cells transformed by Rous sarcoma virus. J. Cell Sci. 2, MOSES, E. & KOHN, A. (1963). Polykaryocytosis induced by Rous sarcoma virus in chick fibroblasts. Expl Cell Res. 32, MUNROE, J. S. & WINDLE, W. F. (1963). Tumor induced in primates by chicken sarcoma virus. Science, N.Y. 140, PONTEN, J. & MACINTYRE, E. H. (1968a). Long term culture of normal and neoplastic human glial tissue. Ada path, microbiol. scand. 74, PONTEN, J. & MACINTYRE, E. H. (19686). Interaction between normal and transformed bovine fibroblasts in culture. II. Cells transformed by polyoma virus, jf. Cell Sci. 3, RABOTTI, G. F., GROVE, A. S. JR., SELLERS, R. L. & ANDERSON, W. R. (1966). Induction of multiple brain tumours (gliomata and leptomeningeal sarcomata) in dogs by Rous sarcoma virus. Nature, Lond. 209, RABOTTI, G. F. & RAINE, W. A. (1964). Brain tumours induced in hamsters inoculated intracerebrally at birth with Rous sarcoma virus. Nature, Lond. 204, Rous, P. (1911). A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J. exp. Med. 13, RUBIN, H. (1960a). The suppression of morphological alterations in cells infected with Rous sarcoma virus. Virology 12, RUBIN, H. (19606). A virus in chick embryos which induces resistance in vitro to infection with Rous sarcoma virus. Proc. natn. Acad. Sci. U.S.A. 46, RUBIN, H. (1961). The nature of a virus-induced cellular resistance to Rous sarcoma virus. Virology 13, SAKSELA, E. & MOORHEAD, P. S. (1962). Enhancement of secondary constrictions and the heterochromatic X in human cells. Cytogenetics 1, SHEIN, H. M. (1965). Propagation of human fetal spongioblasts and astrocytes in dispersed cell cultures. Expl Cell Res. 40, SIMKOVIC, D., SVOBODA, J. & VALENTOVA, N. (1963). Clonal analysis of line XCt,. rat tumour cells (derived from tumour XC) grown in vitro. Folia biol., Praha 9, STOKER, M. (1964). Regulation of growth and orientation in hamster cells transformed by polyoma virus. Virology 24, SVET-MOLDAVSKY, G. J., TRUBCHENINOVA, L. & RAVKINA, L. I. (1967). Pathogenicity of the chicken sarcoma virus (Schmidt-Ruppin) for amphibians and reptiles. Nature, Lond. 214, SVOBODA, J., CHYLE, P., SIMKOVIC, D. & HILGERT, I. (1963). Demonstration of the absence of infectious Rous virus in rat tumour XC, whose structurally intact cells produce Rous sarcoma when transferred to chicks. Folia biol., Praha 9,

11 Human Rous-transformed tumour astrocytes 593 TODARO, G. J. & GREEN, H. (1966). Cell growth and the initiation of transformation by SV40. Proc. natn. Acad. Sci. U.S.A. 55, VOGT, P. K. (1967). A virus produced by 'nonproducing' Rous sarcoma cells. Proc. natn. Acad. Sci. U.S.A. 58, VOGT, P. K. & RUBIN, H. (1961). Localization of infectious virus and viral antigen in chick fibroblasts during successive stages of infection with Rous sarcoma virus. Virology 13, (Received 13 February 1969) 38 Cell Sci. 5

12 594 E- H. Macintyre, R. A. Grimes and A. E. Vatter Fig. 2. The 118MG tumour astrocytes are generally fusiform, and lie in multilayered, loosely packed, irregular bundles. A few giant cells (single arrow) and bipolar spongioblasts (double arrow) are found. Acidophilic granules are seen in the cytoplasm of some astrocytes. May-Griinwald Giemsa, x 165. Fig. 3. Seven days after overt transformation, this Rous-transformed focus of tumour astrocytes includes several round-cell colonies (single arrow) superimposed on a basal layer of stellate or undulant cells. The colonies lie at the cytoplasmic border of the basal cells. One giant cell (double arrow) and two bipolar astrocytes (triple arrow) also lie within the focus. The left upper border of the illustration is occupied by a tangled mat of parent tumour astrocytes (118MG). Live tissue culture, x 100. Fig. 4. Three weeks after overt transformation, the round-cell colonies have increased in size and cell content. Giant cells (single arrow) are more frequent. This focus is bordered (double arrow) by 118MG cells not transformed by RSV. Live tissue culture, x 100. Fig. 5. This picture was taken through 5 ml of tissue-culture fluid in a Petri dish to demonstrate the numerous free EH-i 18MG forms. Giant cells, some immense (single arrow), stellate basal cells (double arrow), and round-cell nests (triple arrow) are also well represented. Live tissue culture, x 50.

13 Human Rous-transformed tumour astrocytes

14 596 E. H. Macintyre, R. A. Grimes and A. E. Vatter Fig. 6. This autoradiogram was made from a fully transformed cell complex. The base layer consists of stellate or undulant cells, interspersed with bizarre giant cells. Roundcell nests (single arrow) lie at the cytoplasmic border of contiguous base layer cells. Toluidine blue, x 165. Fig. 7. Stellate and giant cells as well as round-cell colonies show diffuse nuclear labelling by tritiated thymidine. Toluidine blue, x 500. Fig. 8. This EH-118MG giant cell is multinucleated. Its nuclei contain different amounts of tritiated thymidine. Toluidine blue, x 500. Fig. 9. Many cytoplasmic protrusions extend from the surface of the EH-118MG cell. These are highly vacuolated and less basophilic than the rest of the cytoplasm. The cell has phagocytosed carbon. Wright Giemsa, x 2000.

15 8 Human Rous-transformed tumour astrocytes 597

16 598 E. H. Macintyre, R. A. Grimes and A. E. Vatter Fig. 10. This multinucleated EH 118MG cell is studded with multiple cytoplasmic outpouchings, which are absent from its border with the other giant cell. Wright Giemsa, x 600. Fig. 11. This giant EH-118MG cell has 3 nuclei. Two resemble each other closely in general morphology and degree of chromaticity, but are quite different in these respects from the third nucleus. Wright Giemsa, x Fig. 12. The culture was exposed to carbon for 2 h before fixation. This EH-118MG cell contains irregular clumps of material, considered to be carbon, within a singlemembraned cytoplasmic organelle. x Fig. 13. This area of //<?MG-cell cytoplasm has a well-developed Golgi system (single arrows), numerous lysosomes of all stages, free ribosomes, focal collections of nonoriented membranes and tubules, and non-dilated rough endoplasmic reticulum. x

17 Human Rous-transformed tumour astrocytes 599

18 6oo E. H. Macintyre, R. A. Grimes and A. E. Valter Fig. 14. This free round EH-11SMG cell carries small microvillar projections on its surface. The cytoplasm has numerous vacuoles, nests of mitochondria, free ribsosomes and an unusually well-developed Golgi apparatus (single arrows). The unique nuclear shape gives evidence of its pleomorphism. Few filaments are seen, x Fig. 15. This EH-118MG cell came from the medium of a roller bottle. Its surface carries broad-based protrusions. One such includes mitochondria and rough endoplasmic reticulum in addition to the more normal abundant ribosomes. There is a suggestion that some demarcation of a small projection from the cell surface is beginning (single arrow). A large, abnormally shaped mitochondrion is seen (double arrow). Loosely arranged filaments do not extent into the protrusions. The flocculent material external to the surface may be mucopolysaccharide. Ruthenium red fixative, x

19 Human Rons-transformed tumour astrocytes 601

20 602 E. H. Macintyre, R. A. Grimes and A. E. Votter Fig. 16. This micrograph shows one of many cytoplasmic protrusions of this free EH 118MG cell. These are rich in ribosomes. The well-defined collections of filaments in parallel association (single arrow) with the cytoplasm are" an unusual finding. Such filaments do not extend into the protrusions, x