ELECTRON-MICROSCOPIC OBSERVATIONS OF POLYOMA VIRUS-TRANSFORMED MOUSE CELLS TREATED WITH SPECIFIC IMMUNE SERUM

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1 J. Cell Sci. 7, (1970) 7II Printed in Great Britain ELECTRON-MICROSCOPIC OBSERVATIONS OF POLYOMA VIRUS-TRANSFORMED MOUSE CELLS TREATED WITH SPECIFIC IMMUNE SERUM G.NEGRONI AND RITA TILLY Department of Environmental Carcinogenesis, Imperial Cancer Research Fund, Mill Hill, London, N.W.y, England SUMMARY Transformed, non-malignant cells from a polyoma virus-induced mouse fibrosarcoma were treated with immune serum raised in isologous mice, and fresh guinea-pig serum (complement). Electron microscopy showed that reduction in cell viability in vitro was associated with damage to cell membranes. The extravasated cell organelles showed only minimal changes. INTRODUCTION Walls & Negroni (1966) reported the isolation in tissue culture of a transformed but non-malignant clone of cells from a polyoma virus-induced fibrosarcoma of C 3 H Bi mice. These cells, when inoculated into C. A H Bi mice, did not grow as tumours but immunized the animals against malignant cells derived from the same fibrosarcoma. Furthermore the serum from mice thus immunized prevents the growth in vitro of both malignant and non-malignant polyoma virus-transformed cells (the colonyinhibition test, Hellstrom & Sjogren, 1965). We describe in this note the morphological changes observed by electron microscopy in these cells treated with such immune serum in the presence of fresh guinea-pig complement. These changes provide an explanation for cell death and growth failure in vitro. MATERIALS AND METHODS Cells Transformed non-malignant cells were grown in Falcon plastic Petri dishes with Eagle's minimal essential medium (MEM) containing 10% calf serum and antibiotics. The cultures were treated with 0-25 % trypsin solution in phosphate-buffered saline (PBS) ph 7-2 at 37 C for 5 min. The single cell suspension in MEM was counted in a haemocytometer and the cell concentration adjusted to 1 x io 5 cells in 0'2 ml of MEM. A suspension of control mouse embryo cells (secondary cultures) was prepared in a similar way. Immunological procedures Fresh guinea-pig serum stored at 20 C was used as complement. Control serum came from untreated C 3 H Bi mice. The antiserum against the transformed cells was prepared in C 3 H Bi mice by 6 intraperitoneal inoculations, each of 1 x io 6 non-malignant cells, twice weekly. The mice were bled under

2 712 G. Negroni and R. Tilly anaesthesia from the subclavian veins and the serum separated from the clotted blood by centrifugation. A volume of o-2 ml of serum diluted with an equal volume of PBS (ph 7-2) was mixed with 0-2 ml of MEM containing 1 x io 5 non-malignant cells. The mixture was incubated in a water bath at 37 C for 1 h and shaken at frequent intervals. Then o-6 ml of fresh guinea-pig serum was added. The preparation was shaken at intervals and left at 37 C for different periods of time. Viability tests Suspensions of 1 x io 5 cells treated either with immune or control serum for 30 min followed by complement for 45 min were seeded in 100-mm plastic Petri dishes to test cell viability. The cultured cells were later stained using the May Griinwald and Giemsa techniques. Electron microscopy For electron microscopy the cells were pelleted by centrifugation at g for 10 min in polyethylene Beem capsules (as supplied by Polaron Ltd., London, N.3) so that fixation and embedding could be performed without further disturbance of the cells. Pellets were fixed with 1 % osmium tetroxide buffered with veronal acetate, dehydrated with graded ethanols, embedded in Araldite and sectioned with a Huxley ultramicrotome. Sections were stained with uranyl acetate and lead citrate before being examined with a Siemens Elmiskop I electron microscope. EXPERIMENTS AND RESULTS Two separate experiments were carried out. In the first the non-malignant cells were treated either with immune serum and complement or with control serum and complement and prepared for electron microscopy after 45 min. Untreated cells were also used as controls. Electron microscopy showed that no morphological changes had occurred in the cells treated with control serum and complement, or in the untreated cells, whereas complete disruption of the cells was observed after treatment with immune serum and complement. In order that this disruption might be studied, in a second experiment the mixture of immune serum and complement was left in contact with non-malignant transformed cells for different times (10, 20 or 30 min) so that early damage could be observed. Controls comprised (a) non-malignant transformed cells, control serum and complement; (b) non-malignant transformed cells and immune serum (no complement); and (c) 1 x io 5 normal mouse embryo cells, control (or immune) serum and complement. Samples of transformed non-malignant cells were tested for viability after treatment with immune or control serum for 30 min followed by complement for 45 min. The results can be summarized as follows. The viability tests in the plastic dishes showed that the cells treated with control serum and complement produced a confluent sheet of cells in 3 days, whilst the cells treated with immune serum and complement produced only 4 colonies after 7 days incubation at 37 C. In the electron-microscopic studies no morphological changes were observed in (a) the non-malignant transformed cells treated with control serum and complement (Fig. 1); (b) nonmalignant transformed cells treated with immune serum but no complement; and (c) normal mouse embryo cells treated with control (or immune) serum and complement. Morphological changes were observed in the non-malignant transformed cells

3 Transformed cells treated with immune serum 713 treated with immune serum and complement. These cells showed discontinuity of the cell membranes; the sizes of the lesions varied (Fig. 2). The number of cells showing interruption of the cell membranes increased with the time of contact with complement and immune serum, the majority of cells being affected after 30 min of contact with complement and immune serum. Cell organelles were seen in the gaps of the cell membranes and in the intercellular spaces (Fig. 2). When the discontinuity of the cell membranes was not seen as the plane of section did not pass through the lesion, it was still possible to distinguish damaged cells as they showed a peripheral emptiness (Fig. 3) due to loss of organelles through the lesion which increased with the time of contact with complement and immune serum until finally only the nucleus remained (Fig. 4). Ruptured cells discharged their contents, which appeared relatively unchanged until very late (immune serum 30 min plus complement 30 min), except for the granulated endoplasmic reticulum which appeared as discrete circles (Fig. 2). DISCUSSION The non-malignant transformed cells exposed to specific immune serum die only if complement is present. Death is associated with a lesion of the cell membrane. These findings are similar to those of Borsos, Dourmashkin & Humphrey (1964), Humphrey & Dourmashkin (1965) and Dourmashkin & Rosse (1966), who studied negatively stained preparations in a system involving specific haemolysin, red cells and fresh complement. However, our results, which are based on observations of sectioned cells, do not allow any quantitative assay. In experiments carried out with Gross' lymphoma cells, immune serum and complement, Jakobsson & Wahren (1965) also described a discontinuity in cell membranes. As far as one can judge from their published pictures, the lesions in their system are less extensive than those described here. This may be a result either of a difference in the organization of the two types of cells or of a difference in the potency of the immune serum. Although it is difficult to assess the specificity of an antiserum directed against mammalian cell antigens, we have demonstrated that the antiserum used in our experiments reacts with the surface of cells transformed by polyoma virus; haemagglutination inhibition tests showed no reaction with polyoma virus itself. Cocultivation of the polyoma virus-transformed cells with normal cells (Walls & Negroni, 1966) did not show the presence of cytopathic infectious organisms; the possible contamination with Mycoplasma or bacteria was excluded by bacteriological tests. Furthermore it was shown by the colony inhibition tests that the antiserum produced against the transformed non-malignant cells inhibited the growth of malignant cells from other polyoma virus-induced tumours, but not the growth of normal mouse embryo, mouse kidney and non-malignant L-cells (unpublished results). It seems, therefore, that the 'new' surface antigens are associated with the transformation induced by polyoma virus. We should like to thank Mr Peter E. Robins and Miss Joan Calvert for their careful, intelligent work and assistance.

4 714 G. Negroni and R. Tilly REFERENCES BORSOS, T., DOURMASHKIN, R. R. & HUMPHREY, J. H. (1964). Lesions in erythrocyte membranes caused by immune haemolysis. Nature, Lond. 202, DOURMASHKIN, R. R. & ROSSE, W. F. (1966). Morphological changes in the membranes of red blood cells undergoing haemolysis. Am. J. Med. 41, HELLSTROM, I. & SJOGREN, H. O. (1965). Demonstration of H-2 isoantigens and polyoma specific tumor antigens by measuring colony formation in vitro. Expl Cell Res. 40, HUMPHREY, J. H. & DOURMASHKIN, R. R. (1965). Electron microscopy studies of immune cell types. In Ciba Fdn Symp. on Complement (ed. G. E. W. Wolstenholme & J. Knight), PP London: Churchill. JAKOBSSON, S. V. & WAHREN, B. (1965). Electron microscopy of Gross' lymphoma cells treated with a tumour specific antiserum. Expl Cell Res. 37, WALLS, E. J. & NEGRONI, G. (1966). The properties of cell clones derived from a polyoma induced mouse tumour. Eur. jf. Cancer 2, (Received 15 April 1970)

5 Transformed cells treated with immune serum 715 >:%.^*j:xm*$-j* Fig. 1. Transformed non-malignant cell after treatment with control serum for 60 min and complement for 10 min. x CEL 7

6 G. Negroni and R. Tilly Fig. 2. Transformed non-malignant cell after treatment with immune serum for 60 min and complement for 30 min. Lesions are more extensive and this cell is losing its nucleus in addition to other organelles. The endoplasmic reticulum appears as discrete circles, x 7500.

7 Transformed cells treated with immune serum 717 Fig. 3. Transformed non-malignant cell after treatment with immune serum for 60 min and complement for 10 min, showing peripheral emptiness and swollen endoplasmic reticulum. x

8 7i8 G. Negroni and R. Tilly Fig. 4. Completely disrupted transformed non-malignant cells after treatment with immune serum for 60 min and complement for 20 min. x 9000.