176 BioEssays Vol. 2, NO. 4

Cell Fusion Henry Harris

There is a tendency for living things to join up, establish linkages, live inside each other, return to earlier arrangements, get along, whenever possible, This is the way of the world.

The new phenomenon of cell fusion, a laboratory trick on which much of today's science of molecular genetics relies for its data, is the simplest and most spectacular symbol of the tendency. In a way, it is the most unbiologic of all phenomena, violating the most fundamental myths of the last century, for it denies the importance of specificity, integrity, and separateness in living things. Any cell - man, animal,Jish, fowl, or insect - given the chance and under the right conditions, brought into contact with any other cell, however foreign, will fuse with it. Cytoplasm willflow easily from one to the other, the nuclei will combine, and it will become, for a time anyway, a single cell with two complete, alien genomes, ready to dance, ready to multiply. It is a Chimera, a Griffon, a Sphinx, a Ganesha, a Peruvian God, a Ch'i-lin, an omen of good fortune, a wish for the world.

Lewis Thomas in The Lives of a Cell'

It is very difficult for anyone to give an accurate account of how he came to make a particular discovery, especially many years after the event. Memory is all too fallible, and even when labora- tory notes or other documentation have survived, they rarely plot the tortuous network of thought processes that led to the experiment being tried. On looking back, one can hardly avoid seeing oneself as the mover of events, whereas, of course, more often than not one is moved by them. If I have any confidence in what I have to say, it is because, in my own case, the events leading up to the discovery are punctuated by a series of coincidences, all fortunately docu- mented, that place some restraints on my imagination.

A key event was my decision in August 1959 to accept an offer to become the head of a newly constructed Department of Cell Biology at the John Innes Institute, then at Bayfordbury, Hertfordshire. From 1952 to 1959 I had worked at the Sir William Dunn School of Pathology in Oxford, entirely on animal cells, and my decision to move to

a botanical institute met with some surprise, and even misgiving, on the part of my colleagues in Oxford, including Florey who had been my supervisor and mentor. Indeed, the decision was taken while he was away. Between Oxford and Bayfordbury, I spent nine months at the National Cancer Institute in Bethesda as a guest scientist, and arrived at the John Innes in June 1960. For the first time I was exposed to the plant world, not only as a subject of scientific study, but also as a source of pleasure, for I was a city boy and at that time barely knew a carrot from a turnip. I remain permanently grateful to Bayfordbury for opening my eyes. It was, however, agreed that I should continue to pursue my own experiments on animal cells, and I think the Department of Cell Biology was the first in which work on animal and plant cells was done pari passu in the same laboratory.

In the summer of 1961, John Fin- cham, who had joined the staff of the John Innes as head of the Department of Genetics, returned to the Institute from the USA. He was then exploring the mechanism of complementation at the glutamic dehydrogenase locus in the bread mould, Neurospora crassa, which was, since the work of Beadle and Tatum, a celebrated object of genetical research.l It was the daily contact with Fincham's work that concentrated my mind on the sexual and parasexual processes of mycelial fungi in which fusion of hyphae brings together within a single cytoplasm nuclei of different genotype (heterokaryosis). It was prob- ably during the first half of 1962 that the idea of producing heterokaryons of somatic animal cells first took shape in my mind. The prospect of inducing parasexual processes in mammalian cells was, ofcourse, immensely attractive, for it was clear to anyone interested in the study of such cells that progress was in large part frustrated by the inapplica- bility of the genetic techniques that had proved so immensely fruitful in the study of bacteria and fungi. But I could not at that time see how it was to be done. In this1 wasnot alone. Pontecorvo, who had discovered the parasexual cycle in fungi,3 had been exploring the possibility of applying parasexual gen-

etics to mammalian cells for some time, but so far had nothing to report. So there, for me, matters stood until, towards the end of 1962, an issue of Experimental Cell Research arrived that contained three consecutive papers, two by Okada and one by Okada and T a d ~ k o r o . ~ - ~ These papers described the fusion of Ehrlich ascites cells induced by high concentrations of a parainfluenza virus, the haemagglutina- ting virus of Japan (HVJ). As I read them, the penny dropped. Okada was not, of course, the first to describe virus-induced cell fusion. The earliest descriptions of this phenomenon go back to the nineteenth c e n t ~ r y ; ~ and Enders and Peebless had already shown, as early as 1954, that cells growing in vitro could be induced to fuse together if viruses were added to the cultures. I was, in general, aware of the phenome- non of virus-induced cell fusion before I read Okada's papers, but those papers were determinative in two respects. First, Okada showed that, with HVJ virus, fusion could be induced very rapidly and on a large scale; and second, Okada and Tadokoro showed that the infectivity of the virus could be destroyed by doses of ultraviolet light that left intact its ability to fuse cells together. It became clear to me on the day I read those papers that, if one wanted to induce parasexual processes in somatic animal cells, the thing to do was to use inactivated viruses and, in particular, the virus that Okada had described. I wrote to Okada for reprints and received them in due course. But I did nothing more about it.

There were two reasons for this. The first was that there was no virologist at the John Innes, no one who was familiar with the standard techniques of virus culture, isolation and titration. This was not an insuperable difficulty, for the John Innes was less than an hour's drive from the National Institute of Medical Research at Mill Hill, and I could certainly have obtained preparations of the virus from Mill Hill had I made the effort. The second reason was more fundamental : I was too busily involved in something else. That something else was the metabolism of nuclear RNA. In 1959 the experiments I was doing on the

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accept each other. Some years later Pontecorvo, in a review of my Dunham Lect~res ,~ gave it as his view that this could not have been predicted. Perhaps not, but it is difficult to see why I should have tried the experiment at all, if I hadn’t though there was a chance that it would come off.

Well, one day in the middle of October 1964, I gave the two kinds of cells to John Watkins, who treated them with the inactivated Sendai virus, seeded the treated cells onto coverslips and incubated them at 37 “C overnight. The following morning he brought me the stained preparations of the coverslips to look at. The cultures clearly contained many multinucleate cells in which nuclei of two different morphological types were present in the one cytoplasm. But morphology, of course, was not enough. So I labelled the HeLa cells with tritiated thymidine and asked John to treat the labelled HeLa cells and unlabelled Ehrlich cells with the inactivated virus. Autoradiographs of the resulting multi- nucleate cells showed labelled and un- labelled nuclei within the one cell (Fig. 1). There was no doubt that we had made heterokaryons from cells of different species. But what could they do? This was a vital question, for although Okada had been studying virus-induced cell fusion since 1958,12 none of his papers contained any infor-

kinetics of turnover of nuclear RNAS had led me to the view that much of the RNA synthesized in the cell nucleus was broken down there and not transferred to the cytoplasm of the cell. This idea received a more than cool reception: it was either ignored or treated with derision. In the long run this was no bad thing, for it brought out in me a streak of obstinacy that I knew I had but that I had never before had to use product- ively in my science. In 1962 I was accumulating, and ramming home, the evidence that much of the rapidly labelled nuclear RNA underwent some form of degradation within the cell nucleus,1° and I was groping towards an understanding of what the biological significance of intranuclear RNA turn- over might be. My ideas on this question were summarized in a review that I wrote in 1964 in which I proposed that intranuclear RNA turnover was the reflection of an evolutionary mechanism that permitted eukaryotic cells to try out new kinds of nucleic acids with impunity.I1 It was about a decade before my experimental data on the kinetics of RNA turnover were accepted, and almost two decades before my hazy evolutionary ideas began to find an echo. This long and wintry period was no doubt excellent for my moral fibre, but it induced too strong an emotional engagement in the problem of RNA turnover for me to be deflected from it by a new idea, however promising it might be.

What deflected me in the end was an event in which many people had a hand - my return to Oxford. Florey had resigned from the Chair of Pathology in 1962 and in 1963 I was appointed to succeed him. There had been no virologist at the Dunn School at the time I left it in 1959, but between then and my return in October 1963, John Watkins had joined the staff. He was an expert virologist and was, in fact, at the time of my arrival actually working with one of the herpes viruses that can, under certain circumstances, induce cell fusion. Not many months elapsed before I spoke to John about my ideas and showed him the reprints I had received from Okada. I asked him whether he might be able to obtain the HVJ virus so that we could try it out. He wrote to H. G. Pereira at Mill Hill, from whom I could no doubt have had the virus in 1962, had I asked. Pereira replied that he didn’t exactly have HVJ, but had something that he called ‘Sendai’ virus and that he thought was the same thing. When the virus reached

terms of haemagglutinating units and inactivated it with ultraviolet light. We were ready to go.

The two cell types that I chose for our first experiments were HeLa (from man) and Ehrlich ascites cells (from mouse). There were two reasons for this, one technical but trivial, the other quite fundamental. The trivial reason was that the two kinds of nuclei were easily distinguishable on morphological grounds alone, so that if we succeeded in forming heterokaryons, we would know at once. The second reason was that I regarded it as essential to find out whether viable heterokaryons could be formed from cells derived from differ- ent species. This was crucial because genetic analysis required markers and, at that time, there were very few geneti- cally stable biochemical markers that permitted different tissue culture cells from the one species to be distinguished. If one could cross the species barrier, a plenitude of markers would become available. I actually believed that, at the level of the cell, the species barrier could becrossed. I argued that themechanisms responsible for histo-or cyto-incompat- ibility in nature resided in the cell surface, whereas the intracellular metab- olism of all animal cells was remarkably similar. I thought that if cells from different animal species could be amal- gamated into a single unit, they would

- Oxford, John grew it up, titrated it in Fig. 1. A heterokaryon showing one labelled HeLa nucleus and one unlabelled Ehrlich nucleus.

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mation about the viability of his fused cells: no indication of how long they survived, or even whether they retained any interesting metabolic functions. Viewed retrospectively, this is perhaps surprising, but Okada both before and after 1965 was primarily interested in the mechanism of cell fusion itself, so that the absence of any attempt to explore the subsequent fate of the fused cells is perhaps understandable. In any case, for what I wanted to do with the heterokaryons, the information that Okada had not provided was indis- pensable.

It wasn’t long before I knew that the man-mouse heterokaryons would do virtually everything I wanted them to do. The genes of both kinds of nuclei were transcribed, translated and replica- ted in the fused cells; and, at mitosis, binucleate heterokaryons would com- monly form only a single spindle and hence generate two mononucleate daughters that contained within a single nucleus the chromosomes of both parent cells. With the help of Charles Ford and his colleagues, we made a cytogenetic analysis of the hybrid mitoses in our cu1t~res.l~ We did not, however, succeed in isolating a hybrid cell line from our HeLa-Ehrlich cross, even though we could identify hybrid clones in the cultures, essentially because, in the absence of any selective system, the hybrids were rapidly over grown by the HeLa cells. The first hybrid cell line to be derived from Sendai-virus- induced cell fusion was a cross between Armenian and Chinese hamster cells reported by Yerganian and Nell in 1966.14 By the beginning of February 1965 I had succeeded in making hetero- karyons with highly differentiated cells, such as lymphocytes, monocytes and even nucleated erythrocytes, and was busy studying their behavio~r.’~ It was clear that it was possible to fuse essen- tially any animal cell with any other and that, for cell biologists, Pandora’s box had been opened.

A few words are in order about the relationship between my experiments in 1964 and 1965 and the earlier experi- ments of Barski and of Ephrussi dealing with hybrid cells that arose spon- taneously in mixed cultures of two different mouse cell lines. I was not aware of the existence of the latter ex- periments during my John Innes period. The work was not at that time well known, except perhaps to cytogeneti- cists, who did not seem to know what to make of it; and I did not myself become interested in cytogenetics until much later. As far as I can remember, I first

Fig. 2 A cartoon from the London Daily Mirror of I5 February 1965.

learnt about Barski’s discovery and Eplrussi’s development of it from a volume entitled Cytogenetics of Cells in Culture which I received towards the end of 1964. In it there was a review article by BarskP and another by Ephrussi and his colleague^.^' I was not at that time especially impressed by the experimental possibilities offered by the work reviewed in those two articles, mainly because it was limited to intraspecific hybrids derived from similar established mouse cell lines, but also because the formation of the hybrids was rare, uncontrolled and obscure in origin (Barski and Belehradek had earlier produced cine- matographic evidence that the hybrid cells arose by a process of nuclear trans- fer betweencellsl8). I later learnt that this pessimistic view of the usefulness of the spontaneous mouse hybrids described by Barski and Ephrussi was shared by others. I did, indeed, wonder whether these hybrids might have arisen through the activity of stray viruses contamina- ting the cultures, but the relationship between the hybrids discovered by Barski and the virus-induced hetero- karyons that Watkins and I had made did not become clear until the fate of the latter had been elucidated and their ability to generate mononucleate hybrid cells demonstrated. The line of thought that prompted my experiments derived, as I have already explained, from Fincham’s work with Neurospora, via Okada and not via Barski and Ephrussi.

When, on the 13 February 1965, the paper by John Watkins and myself appeared in Nature,lB it evoked an astonishing reaction in the world’s

press. What caught the imagination of journalists was the fact that the species barrier could be crossed. This seems to have shaken some deeply cherished assumptions about the uniqueness of man, and many of the newspaper reports showed striking similarities with the paintings of Hieronymus Bosch. Fig. 2, from the London Daily Mirror of 15 February, is an early example. Looking back on it now, I must confess that, in one respect, the journalists were right. I now think the most important consequence of our experiment was that it broke down conceptual limitations that had prevented scientists from attempting experiments that already lay within their technical competence. Once it was clear that virtually any cell from any animal species could be fused with any other and that the fused cells had a good chance of surviving, a torrent of novel experiments was unleashed, which, within a few years, transformed our approach to some of the central questions in cell biology. Four years after the appearance of our paper, Nature produced an editorial entitled ‘Cell fusion: a new gift in biology’.20 The opening paragraph contains a dramatic prophecy ( a not uncommon feature of editorials in Nature): ‘No-one would have predicted the quiet revolu- tion that chromatography would bring about in biology and chemistry. Equally, it would be rash to predict that cell fusion will be the midwife to several of the most significant biological dis- coveries in the 1970s. Rash perhaps, but tempting because. . . .’ Well, how has it, in fact, gone? It is well nigh impossible

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ROOTS to avoid overestimating the importance of one’s own experiments, but I like to think ;hat the work done in Oxford in 1964 and 1965 opened up possibilities, both conceptual and methodological, that, with the passage of time and with contributions by many other hands, permitted the development for mamma- lian cells of formal methods of gene mapping (which, among other things, led to the construction of a detailed map of the human genome); deepened our understanding of some inherited dis- eases; threw new light on the mechan- isms that regulate gene expression and those that determine malignancy; and eventually, a decade later, produced monoclonal antibodies. I also like to think that the harvest is not yet all in.

REFERENCES 1 THOMAS, L. (1974). The Lives o f a Cell pp. 147-148. Bantam Books, Toronto, New York, London. 2 FINCHAM, J. R. S. (1962). Genetically determined multiple forms of glutamic dehydrogenase in Neurospora crassa. J. Mol. Biol. 4, 251-214. 3 PONTECORVO, G. (1956). The parasexual cycle in fungi. Annu. Rev. Microbiol. 10, 393-400. 4 OKADA, Y. (1962). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. 1. Microscopic observation ofgiant polynuclear cell formation. Exp. Cell Res. 26, 98-107.

5 OKADA, Y. & TADOKORO, J. (1962). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. 11. Quantitative analysis of giant polynuclear cell formation. Exp. Cell Res.

6 OKADA, Y. (1962). Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich’s ascites tumor cells. 111. Relationship between cell condition and fusion reaction or cell degeneration reaction. Exp. Cell Res. 26, 119-128. 7 HARRIS, H. (1970). Cell Fusion, pp. 1-3. The Dunham Lectures. Oxford University Press. 8 ENDERS, J. F. & PEEBLES, T. C. (1954). Propagation in tissue cultures of cytopatho- genic agents from patients with measles. Proc. SOC. Exp. Biol. Med. 86,277-286. 9 HARRIS, H. (1959). Turnover of nuclear and cytoplasmic ribonucleic acid in two types of animal cell, with some further observations on the nucleolus. Biochem. J.

10 HARRIS, H. (1962). The labile nuclear ribonucleic acid of animal cells and its relevance to the messenger-ribonucleic acid hypothesis. Biochem. J. 84,6OP-61P. 11 HARRIS, H. (1965). The short-lived RNA in the cell nucleus and its possible role in evolution. In Evolving Genes and Proteins (ed. V. Bryson & H. J. Vogel) pp. 469-477. Academic Press, New York. 12 OKADA, Y. (1958). The fusion of Ehrlich’s tumor cells caused by HVJ virus in vitro. Biken’s J. 1, 103-110. 13 HARRIS, H., WATKINS, J. F., CAMPBELL, G. LEM., EVANS, E. P. & FORD, C. E. (1965). Mitosis in hybrid cells derived from

26, 108-1 18.

73, 362-369.

mouse and man. Nature (Lond.) 207, 606-608. 14 YERGANIAN, G. & NELL, M. B. (1966). Hybridization of dwarf hamster cells by UV-inactivated Sendai virus. Proc. Nut1 Acad. Sci. USA 55, 1066-1073. 15 HARRIS, H. (1965). Behaviour of differ- entiated nuclei in heterokaryons of animal cells from different species. Nature (Lond.)

16 BARSKI, G. (1964). Cytogenetic altera- tions in mixed cultures of mammalian somatic cells in vitro. In Cytogenetics of Cells in Culture (ed. R. J. C. Harris), pp. 1-11. Academic Press, New York & London. 17 EPHRUSSI, B., SCALETTA, L. J., STEN- CHEVER, M. A. & YOGHIDA, M. C. (1964). Hybridization of somatic cells in vitro. In Cytogenetics of Cells in Culture (ed. R. J. C. Harris), pp. 13-25. Academic Press, New York & London. 18 BARSKI, G. & BELEHRADEK, J. (1963). Transfert nuclkaire intercellulaire en cultures mixtes in vitro. Exp. Cell Res. 29, 102-1 1 1. 19 HARRIS, H. & WATKINS, J. F. (1965). Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species. Nature (Lond.) 205, 640-646. 20 ANON. (1969) Cell fusion: a new gift to biology. Nature (Lond.) 223, 1039-1041.

206, 583-588.

Medicine at the Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.

International Union of Biochemistry 13th International Congress

25-30 August 1985 To be held at: International Congress Centrum RAI, Amsterdam, the Netherlands

There will be 40 symposia in 12 different areas of biochemical research: the genome -structural organization and replication; gene expression and transcriptional controls; nucleic acid-protein interactions, including protein biosynthesis; protein structure and function, including enzymology; metabolic regulation; membrane structure and functions; bioenergetics; cellular growth, differentiation, and transformation; hormones; molecular and cellular immunology; molecular mechanisms of disease; applied biochemistry.

Forms and information may be obtained from: In addition, colloquia will be held in each of these subject areas.

13th International Congress of Biochemistry c/o Organisatie Bureau Amsterdam bv Europaplein 1078 GZ Amsterdam The Netherlands

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