J. Cell Sci. 10, 769-787 (1972) 769 Printed in Great Britain STUDIES ON CHEMICALLY INDUCED CELL FUSION Q. F. AHKONG, F. C. CRAMP, D. FISHER, J. I. HOWELL AND J. A. LUCY Department of Biochemistry, Royal Free Hospital School of Medicine, University of London, 8 Hunter St., London WCiN iBP, England SUMMARY Hen erythrocytes that were fixed after treatment with lysolecithin in aqueous solution for 30 s at 37 °C showed evidence of bridge formation between adjacent lysed cells. Generally, the homokaryons that were produced using lysolecithin in this way contained large numbers of nuclei. These giant syncytia had damaged nuclear membranes and unstable plasma mem- branes; complete disintegration of the syncytia occurred within 1 min of adding lysolecithin to the erythrocytes. In order to localize the action of lysolecithin, the fusing agent was incorporated into micro- droplets of lipid. Cell fusion following the addition of lysolecithin in an aqueous glyceride- lecithin emulsion was slower than with lysolecithin in aqueous solution, taking 10-30 min, and it was accompanied by considerably less damage to the plasma and nuclear membranes. The fused erythrocytes, which usually contained only two or three nuclei, lysed slowly during the 45 min following fusion, and lysis could be arrested by cooling the fused cells. The plasma membranes of lysed, multinucleated cells remained intact at 37 °C for at least 90 h. Mouse fibroblast-hen erythrocyte heterokaryons formed with the aid of the emulsion were more stable than those produced with lysolecithin in solution, but the hybrid cells nevertheless had damaged subcellular organelles. Viable clones of hybrid mouse-hamster fibroblast cells were obtained using the emulsion although, possibly owing to reduced viability of the lysole- cithin-treated cells, only at twice the frequency of spontaneously produced hybrids. INTRODUCTION In addition to its well known haemolytic action, lysolecithin has been reported to induce phagocytosis (Burdzy, Munder, Fischer & Westphal, 1964), to cause elongation of surface microvilli (Wilkinson & Cater, 1969), to increase the permeability of cell membranes (Mehrishi & Butterworth, 1969), and to cause pre-lytic sphering of erythrocytes (Klibansky & DeVries, 1963). Lysolecithin has been implicated in various processes involving membrane fusion in vivo such as the release of catecholamines (Blaschko, Firemark, Smith & Winkler, 1967; Winlder, 1971) and histamine (Hogberg & Uvnas, 1957) from membrane-bound compartments within cells, and also in membrane fusion occurring in the lysosomal vacuolar system (Lucy, 1969). The calcium-dependent ATPase of fragmented sarcoplasmic reticulum requires small quantities of unsaturated acids or lysolecithin for its activity (Fiehn & Hasselbach, 1970), and lysolecithin-treatment of this membrane system gives rise to elongated, irregularly shaped, branching tubular structures which may arise, in part, from rearrangement of the vesicles with membrane fusion (Agostini & Hasselbach, 1971). 770 Q. F. Ahkong and others It has been found in this laboratory that lysolecithin can induce cells to fuse in vitro with the formation of both multinucleated homokaryons (Howell & Lucy, 1969) and heterokaryons (Poole, Howell & Lucy, 1970). Cell fusion was, however, accom- panied by extensive degradation of the treated cells. In the present paper, interac- tions between fusing cells, and the degradative changes observed when hen erythro- cytes are treated with a solution of lysolecithin are reported in more detail. One aim of the studies described here was to see if cells could be fused by lysoleci- thin without extensive membrane damage, since this would provide circumstantial evidence for the possibility that lysolecithin may be concerned in some of the processes of membrane fusion occurring in living cells. In addition, in view of pos- sible future applications of lysolecithin-induced cell fusion in investigations employing cell hybridization, it is important to know whether or not viable cell hybrids can be obtained with the aid of lysolecithin. If the effects of lysolecithin on the plasma mem- branes of cells can be localized, cell fusion might occur in the absence of widespread damage to membranes. One way of localizing lysolecithin is to include it in the lipid phase of an aqueous emulsion of fat. Since about 10% of the phospholipid of the nascent fat droplets of cow's milk is lysolecithin and the secretion of milk fat has been suggested to depend on exocytosis (Keenan, Morre, Olson, Yunghans & Patron, 1970), it seems possible that the presence of lysolecithin in the nascent fat droplets of milk may be at least partially responsible for membrane fusion involved in the secretion of milk fat by exocytosis. With this in mind, emulsions of fat containing lysolecithin have been prepared and used in experiments on cell fusion. These emulsions have been found to induce the formation of relatively stable polykaryocytes and heterokaryons. MATERIALS AND METHODS Blood (approximately 2 ml) was removed from the brachial vein of an adult hen, and taken into a syringe already containing 1 ml of citrate anticoagulant solution (DeGowin, Hardin & Alsever, 1949). It was then transferred to a large volume of this solution and well mixed. The cells were washed twice in a large volume (approximately 20 ml) of the anticoagulant solution, and then twice in isotonic saline. The packed cell volume was measured and 1 ml of packed cells diluted to 85 ml with isotonic saline. After dilution, cells were counted in a haemocyto- meter: the population was generally 7 — 8 x io8 cells/ml. Lysolecithin used in the initial experiments was a gift from Professor G. R. Webster. It was prepared from ovolecithin with snake venom (Agkistrodcm p. piscivorus) and was chromato- graphically pure. Comparable preparations were obtained from Lipid Products, Epsom, and were also prepared in our laboratory. All preparations of lysolecithin behaved similarly in our experiments. Ovolecithin was prepared according to the method of Papahadjopoulos & Miller (1967). It was stored in chloroform, under nitrogen, in small, sealed aliquots containing known quantities of phospholipid P (Perry, Tampion & Lucy, 1971). Glyceryl trioleate was obtained from B.D.H. and glyceryl dioleate from K. & K. Laboratories Inc.; thin-layer chromatography (Cater & Hallinan, 1971) revealed the presence of mono-, di- and tri-glycerides in both of these preparations. No significant differences were observed between the 2 preparations in our experiments with lysolecithin. Chemically induced cell fusion 771 Lysolecithin in aqueous solution The techniques used have been described in a previous paper (Howell & Lucy, 1969): the final incubation mixture (1-7 ml) consisted of 4X108 avian erythrocytes, and 70-560/tg of lysolecithin in 106 mM NaCl and 44 mM sodium acetate at a final pH of 57. In early experi- ments, acetate buffer was occasionally used at pH 50. Cells were incubated at 37 °C. Lysolecithin in lipid emulsions A solution in chloroform of lysolecithin, lecithin and either glyceryl dioleate or glyceryl trioleate was prepared in a siliconized flask. All traces of chloroform were then removed by evaporation to dryness from an atmosphere of nitrogen in a rotary evaporator. The lipids were allowed to swell for 15-30 min in a 3:5 ratio mixture (v/v) of 09 % NaCl and 015 M sodium acetate buffer at pH 56 or in Dulbecco's medium (pH 72), and then sonicated under a stream of nitrogen at about 4 °C with a 60-W ultrasonic disintegrator tuned to maximum intensity (using a i-9-cm diameter titanium probe, M.S.E. Ltd., London) for 1-2 min or until the bulk of the lipids was dispersed. All emulsions were used within 10 min of preparation, and con- tained 30 mg of glyceryl dioleate (or glyceryl trioleate), 270 /ig lecithin and 30 /tg lysolecithin per ml. Washed hen erythrocytes were suspended in 09 % NaCl, and 08 ml of the lipid emulsion in the buffered saline (pH 56) was added to 05 ml of red cell suspension containing approxi- mately 8 x io8 cells/ml. Mouse LS fibroblasts were derived from a culture kindly provided by Professor S. J. Pirt. The cells were grown as a monolayer in Eagle's medium, supplemented with 10% foetal calf serum; they were harvested by shaking, washed 3 times with Hanks's saline and suspended in 09% NaCl just before use. 16 ml of lipid emulsion in the buffered saline (pH 56) was added at 4 °C to 10 ml of a mixed cell suspension in 09% NaCl (containing 73 x io7 hen erythrocytes and 8-5 x io6 fibroblasts). The treated cells were centrifuged into a pellet to facili- tate fusion. After 5 min at 4 °C the preparation was incubated at 37 °C for 15 min. Cells were fixed for electron microscopy by gently resuspending them in the glutaraldehyde fixative. A control experiment without the lipid emulsion was undertaken to determine the effect of the low pH on the ultrastructural appearance of the fibroblasts and on their ability to exclude trypan blue. Electron microscopy Cells were fixed in suspension immediately following incubation with lysolecithin by the addition of ice-cold cacodylate-buffered glutaraldehyde (Glauert, 1965). They were postfixed in osmium tetroxide containing ruthenium red (Pate & Ordal, 1967), stained in block with uranyl acetate (Farquhar & Palade, 1965) and embedded in Araldite (Glauert, Rogers & Glauert, 1956). Sections were cut on an LKB Ultratome III and stained with lead citrate (Venable & Coggeshall, 1965) when necessary. An AEI EM 6 B electron microscope was used at an accelerating voltage of 80 kV and micrographs were taken at an initial magnification of x 5000—15000. Cell hybridization Cells of the mouse fibroblast line, 3T3 TK~, and of the hamster fibroblast line, wg 3 IMP", were kindly provided by Professor G. Pontecorvo. Both cell lines received 2 passages in Dulbecco's medium supplemented with 10% foetal calf serum before experimental use. The cells were grown to confluent monolayers and were seeded in 90-mm Petri dishes 20 h prior to hybridization.