The Croonian Lecture, 1971: Cell Fusion and the Analysis of Malignancy

The Croonian Lecture, 1971: Cell Fusion and the Analysis of MalignancyAuthor(s): Henry HarrisSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 179, No.1054 (Oct. 12, 1971), pp. 1-20Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/75945 .

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Proc. B. Soc. Lond. B. 179, 1-20 (1971) Printed in Great Britain

THE CROONIAN LECTURE, 1971

Cell fusion and the analysis of malignancy

BY HENRY HARRIS, F.R.S

Sir William Dunn School of Pathology, University of Oxford

(Delivered 17 June 1971-Received 17 June 1971)

The cells of the body do not normally engage in sex. Nor is it easy to see that sexual activity would greatly benefit them. For sex is ultimately merely a device to facilitate the accumulation in a single individual of favourable mutations occurring separately in different individuals; and since the cells in the body are, at least in

large part, genetically identical, the advantages to be gained by genetic exchange are obviously limited. In recent years, however, a technique has been devised that

imposes a form of artificial sexuality on somatic cells, and it has been found that somatic cells of widely different genetic constitutions can be induced to undergo genetic amalgamation and exchange. A few years ago, Professor Hayes, in a Leeuwenhoek Lecture (Hayes I966) described how sex in bacteria is mediated by an infectious particle which produces a change in the cell wall of the 'male' bacterium that enables it to make intimate contact with the 'female' bacterium. A connexion is then established between the cytoplasms of the two bacteria and

through this connexion transfer of genetic material may take place. The imposition of sexuality on somatic cells is achieved by a mechanism which, viewed superficially, is reminiscent of bacterial conjugation. An animal virus, whose normal mode of

entry into the cell appears to involve fusion between the viral membrane and the cell membrane, is used to facilitate fusion between the cell membranes of con-

tiguous cells (Okada 1958; Harris & Watkins I965). Cytoplasmic bridges are thus established which eventually determine the complete coalescence of the cytoplasms of adjacent cells (Schneeberger & Harris 1966). In this way multinucleated cells are formed which contain various numbers of nuclei, and different kinds of nuclei if cells of different kinds are brought together (Harris, Watkins, Ford & Schoefl I966). The virus now commonly used to produce cell fusion is the Sendai virus, a member of the parainfluenza group of myxoviruses, although many other viruses can achieve the same effect. Unlike the sex particle in bacteria, however, Sendai virus will produce fusion of somatic cells even after its nucleic acid has been destroyed (Okada & Tadokoro 1962; Neff & Enders 1968); the viral envelope is all that is required for this effect. The standard reagent for inducing cell fusion is Sendai virus inactivated by large doses of ultraviolet light or by appropriate treat- ment with ,-propriolactone.

Vol. I79. B. (12 October I97i) [ I ]



Most cells types from a very wide range of animal species are susceptible to the action of Sendai virus, so that fusion can be achieved between cells from different animal species and cells showing very different forms of specialization. I have

already reviewed before this Society the general properties of such fused cells

(Harris i966). These cells are, as I have said, initially multinucleate; and many experiments have now been done on the interactions of different nuclei and cytoplasms brought together in this way (Harris I970). But for the purposes of the

present lecture I shall be concerned with the mononucleate hybrid cells that are

produced when the nuclei of a binucleate (and occasionally trinucleate) cell fuse

together. This nuclear fusion takes place when the binucleate cell enters mitosis. The two nuclei commonly enter mitosis together; and, when this occurs, a single spindle may be formed and all the chromosomes may become alined along one

metaphase plate. The cell then divides to give rise to two mononucleate daughter cells each of which contains within a single nucleus the chromosomes of both parent cells. Many of these composite mononucleate cells, even when they are derived from parents of widely different animal species, are capable of indefinite multipli- cation.

Genetic analysis of somatic cells requires, however, not only that different chromosomal complements be brought together; the chromosomes must also segregate. Hybrid somatic cells do not appear to undergo the regular systematic segregation normally seen at meiosis; but for the purposes of genetic analysis a useful alter- native is provided by chromosome loss (Ephrussi, Scaletta, Stenchever & Yoshida

I964). The mechanisms responsible for loss of chromosomes in hybrid cells remain rather unclear, despite a good deal of intensive study. Virtually all such cells under-

go a slow progressive loss of chromosomes on continued cultivation, and this may be determined in some cases by non-disjunction (Handmaker I97I). In certain cell combinations large numbers of chromosomes are eliminated during the early cell

divisions, possibly at the very first division (Weiss & Green 1967; Nabholz, Mig- giano & Bodmer 1969). Some evidence suggests that these drastic early losses may be produced by spindle abnormalities of one kind or another. Whatever the mechanism of chromosome loss, the end result is the production of cells which, from the genetic point of view, may be regarded as segregants of the original hybrid cell

population. The experiments I now wish to describe had a very simple objective: to deter-

mine whether the daughter cells resulting from the fusion of a malignant cell and a

non-malignant one were malignant or not. (Malignancy is here defined as the

ability of tumour cells to grow progressively and kill their host.) One might at first glance suppose that the solution of so simple a problem would not pose any great difficulty; but when one considers that the injection of cells into an animal selects for malignancy and that hybrid cells continually generate segregants from which certain parental chromosomes are eliminated, the complexities of the investi-

gation become apparent. The work began as a collaboration between Professor

George Klein of the Department of Tumour Biology at the Karolinska Institute

2 Henry Harris



The Croonian Lecture, 1971 3

and myself. At various stages we were joined by Dr O. J. Miller and Miss D. Quinn at Oxford and Drs P. Worst, T. Tachibana, U. Bregula and F. Wiener at Stock- holm. The enterprise would long ago have foundered but for the skill and enthusiasm of these colleagues.

TABLE 1. EXPERIMENTAL TUMOURS

histocom- genotype patibility

tumour type origin of host class Ehrlich mammary spontaneous ? ?

carcinoma SEWA sarcoma polyoma virus A.SW H-2S

(osteogenic) MSWBS sarcoma methylchol- A.SW H-28

anthrene YAC lymphoma Moloney virus A H-2a YACIR lymphoma Moloney virus A H-2a TA3 mammary spontaneous A H-2a

carcinoma

If we were to have any hope of obtaining an unequivocal answer to our question it was clear that, to begin with, we should have to use tumour cells whose malignancy was not in doubt. We therefore chose for our experiments six transplantable mouse tumours, which grew as well in the solid as in the ascitic form, and which

produced progressive fatal neoplasms in 100 % of genetically compatible mice with inocula of 100 cells or less., More lethal cell populations can hardly be obtained. Table I lists the six tumours and some of their properties. The Ehrlich tumour

appears originally to have been a spontaneous mammary carcinoma. It arose in Ehrlich's laboratory in a mouse of unknown genetic constitution and has been passaged continuously in many different strains of mice for more than seventy years. The cells of this tumour possess remarkably efficient mechanisms for masking histocompatibility antigens and hence grow freely in any strain of mouse. SEWA and MSWBS are both sarcomas, the first induced by polyoma virus, the second by the chemical carcinogen methylcholanthrene. Both tumours bear the H-2s group of histocompatibility antigens and their growth is limited to mice of the A.SW strain. SEWA also bears the specific transplantation antigen characteristic of polyoma virus infection. YAC is a lymphoma induced by Moloney virus. It bears the H-2a group of histocompatibility antigens and a specific surface antigen characteristic of Moloney virus infection. The tumour grows only in A strain mice. YACIR is a subline of YAC selected for diminished expression of the Moloney virus-induced surface antigen by continuous passage in mice immunized against this antigen. TA3 is a spontaneous mammary carcinoma which arose originally in an A strain mouse in T. S. Hauschka's laboratory. It bears the H-2a group of histocompatibility antigens but readily transgresses histocompatibility barriers.



For technical reasons these tumours were first fused with two derivatives of the L cell line, an established line of mouse fibroblasts that has been maintained in culture for more than fifteen years. One of these derivatives, the A9 cell, was selected for resistance to 8-azaguanine and lacks the enzyme inosinic acid pyrophosphorylase; the other, the B 82 cell, was selected for resistance to 5-bromodeoxy- uridine and lacks the enzyme thymidine kinase (Littlefield 1964). These enzymic defects in the L cell derivatives facilitate the selection of the hybrid cells, for culture media can be employed that inhibit the growth of cells lacking these enzymes. However, since it was possible that the growth of the hybrid cells in vivo might be influenced by the presence of a severe enzymic defect in one of the

TABLE 2. GROWTH OF L CELL DERIVATIVES IN VIVO

no. takes (total no. animals with progressive

tumours/total no. percentage cell type inoculated) takes A9 3/25 12 B 82 3/48 6 A9RI 22/66 33

All animals were X-irradiated newborn C3H mice. Inocula varied between 5 x 104 and 2 x 106 cells injected subcutaneously.

parent cells, hybrids were also constructed between some of the tumour cells and a

presumptive revertant of the A9 cell in which inosinic acid pyrophosphorylase activity had been restored. This cell line, known as A 9 RI, arose spontaneously in cultures of A 9 cells maintained in a selective medium thatinhibits thegrowth of cells

lacking inosinic acid pyrophosphorylase. In the case of crosses between the A 9 RI cells and the tumour cells, a selective medium could not be used to facilitate the isolation of the hybrid cells; and these were obtained by procedures that exploited differences in surface properties between the hybrids and the parental cell lines. Differences in surface properties formed the basis of successful selective procedures for several other hybrid lines that could not be isolated by means of selective media. Where the cultural characteristics of the hybrids closely resembled those of the parental cells, and no selective medium was appropriate, the fused cell population was cloned, and clones having morphological features suggesting hybridity were isolated. Karyological investigation of these clonal populations eventually permitted the identification, and hence isolation, of the desired hybrids.

The L cell line was originally derived from C3Hl mice and thus bore the H-2k

histocompatibility antigen complex. This complex was also present on the surface of the three L cell derivatives, A 9, B 82 and A 9 RI. As shown in table 2, these three cell lines had a very low level of tumorigenicity in syngeneic C3H mice: even when

assayed in X-irradiated (4 J kg-l) newborn mice, which provide optimal conditions for cell growth in vivo, these cells produced tumours in only 6 to 33 % of

4 Henry Harris




the recipients with inocula between 5 x 104 and 2 x 106 cells. The 50 % tumour dose

(the inoculum required to produce tumours in 50% of the recipient animals) exceeded that of the tumour cells by a factor of at least 105.

TABLE 3. CHROMOSOMAL CONSTITUTION OF PARENTAL CELL LINES

total chromosome no. r-

Ak

range mode

50-69 53-70 37-52

71-94 42-44 28-30 39-41 40-42 40-41

56 57 49

76 43 29 40 40 40

no. bi-armed chromosomes C A

range

22-27 23-29 14-26

0-2 0-1 8-11 0-1

0 0

mode

25 27 23

1 1

10 1 0 0 O

O

TABLE 4. CHROMOSOMAL CONSTITUTION OF HYBRID CELL LINES

cell line

Ehrlich/A 9 Ehrlich/A 9 Ehrlich/A 9 SEWA/A 9 SEWA/A 9 MSWBS/A 9 MSWBS/A 9 Ehrlich/B 82 Ehrlich/A9RI Ehrlich/A9RI YAC/A 9 YACIR/A 9

date of examination

15. x. 68 12. ix. 69 25. v. 70 10. ii. 69 27. x. 69

3. iii. 69 5. ix. 69 9. ix. 69

26. vi. 69 5. ix. 69

23. iii. 70 2. iv. 70

total chromosome no. A?

range 104-150 71-87 65-108 81-113 71-95 65-109 72-85 86-156 74-136 85-105 83-103 78-94

mode 128 83

(73-77) 94

(84-87) 86

(80-82) 112

(118-122) (96-98)

88 92

no. bi-armed chromosomes AX

range mode 11-30 24 11-26 16 13-23 16 17-29 21 20-30 23 29-40 36 27-38 32 13-30 (22) 12-26 (20) 17-24 20 24-28 26 26-28 27

Parentheses denote weak mode.

Hybrid cells are usually identified by their karyotype: they contain in varying proportions the chromosomes of both parent cells. In table 3 the chromosomal constitutions of the parental cells are shown, and in table 4 the chromosomal constitutions of the hybrid cells derived from them. It will be seen that, in general, the hybrid cells initially have chromosome numbers approximating to the sum of the two parental chromosome sets. In some cases the modal chromosome number of the hybrid cells is exactly what one would expect from the fusion of two modal parent cells; in other cases the hybrid cells have a slightly lower chromosome

cell line

in vitro A9 B 82 A9RI

tumour Ehrlich SEWA MSWBS YAC YACIR TA 3



number than that to be expected from the sum of the two parental modes. The

hybrid nature of the cells was confirmed in all cases by the presence of parental marker chromosomes, or by the presence of the two parental H-2 antigen complexes, or both. As shown in table 4, the hybrid cells lose chromosomes on progressive cultivation in vitro; but the loss is very gradual and little change in chromosome number is usually seen in the first few weeks. These hybrids between the tumour cells and the established cell lines thus resemble many other intraspecific hybrid cells that have been described in having relatively stable chromosomal constitutions initially composed of something pretty close to the sum of the two parental chromosome sets (Ephrussi et al. I964; Littlefield I966; Engel, McGee & Harris

I969). In testing hybrid cells for their ability to grow progressively in vivo it is, of

course, obvious that histocompatible test animals must be used. This means, in

practice, that where the two parent cells are derived from mice of known genotypes the hybrid cells are tested in the F1 progeny of the parental mouse strains. All the tumour cell lines grew as well in the F1 hybrid animals as in the parental strains. However, it was possible that prolonged cultivation in vitro might have

generated minor degrees ofhistoincompatibility between the cell lines and the animals from which they were initially derived; and it was also possible that new antigenic combinations might have been generated by the cell fusion itself. For this reason our tests for malignancy were carried out on genetically compatible newborn animals that had received 4 J kg-l of whole body irradiation before test. Since these X-irradiated newborn mice have little immunological reactivity and permit the growth of tumours bearing foreign histocompatibility antigens, we felt con- fident that our tests would not be complicated in any important way by histo-

incompatibility reactions between cells and host. I shall present evidence later that our confidence was not misplaced.

The first hybrid cells we tested were those resulting from the fusion of Ehrlich cells with A 9 cells; and it was at once obvious that the tumorigenicity of these

hybrids was in no way comparable to that of the Ehrlich cells. With inocula between 3 x 104 and 3.5 x 106 cells, tumours developed in only about a third of the X-irradiated newborn mice. This take incidence was of the same order as that obtained with A9 cells. In statistical terms, these results suggested that in the

hybrid cell population only one cell in about 105 or 106 was able to produce a

tumour, whereas virtually every Ehrlich cell can do so. The question, of course, at once arose whether the very small minority of hybrid cells that did give rise to tumours differed in their chromosomal constitution from the hybrid cell population as a whole. Karyotypes of the cells in the tumours arising from Ehrlich/A 9

hybrids were therefore examined. It was found that in all cases the tumours had chromosome numbers much lower than that of the hybrid cells injected. Whereas the modal chromosome number of the latter was about 128, the tumour cell

populations had modes in the region of 80. The tumours were none the less derived from hybrid cells, for their karyotypes contained marker chomosomes from both

6 Henry Harris




parent cells. These results thus indicated that the hybrid cells with chromosome

complements approximating to the sum of the two parental chromosome sets had

very little, if any, ability to grow progressively in vivo; the tumours were all com-

posed of cells from which certain chromosomes had been eliminated.

cell type

Ehrlich/A 9

TABLE 5. GROWTH OF HYBRID CELLS IN VIVO

no. takes (total no.

animals with progressive tumours/

genotype total no. of host inoculated)

C3IH 4/12 (Jan. to June 1969)

Ehrlich/A 9 (July to Dec. 1969)

Ehrlich/A 9 (Dec. 1969 to June 1970)

SEWA/A9 MSWBS/A9 Ehrlich/B 82 Ehrlich/A 9 RI YAC/A9 YACIR/A9

C3H

C311

2/32

2/38

C3HxA.SWF1 C3H xA.SWF1 C3H1 C3H C3HxAF1 C3HxAF1

8/25 13/30 3/41

25/96 1/27 6/52

percentage takes

33

6

5

32 43

7 26

4 12

Inocula varied between 3 x 104 and 3.5 x 106 cells.

This result raised several obvious questions. (1) Is the suppression of malignancy by cell fusion limited to the Ehrlich ascites tumour? (2) Is the effect peculiar to the A 9 cell? (3) Does the metabolic defect in the A 9 cell (inosinic acid pyrophosphorylase deficiency) play an important role in the suppression of malignancy? (4) Can the suppressive effect be exercised by normal diploid cells? (5) Is it in general the case that when a malignant cell and a non-malignant cell are fused together, the

hybrids with unreduced chromosome complements are not malignant? (6) Is a loss of chromosomes essential for the production of malignant variants from a non-

malignant hybrid cell population? (7) If loss of chromosomes is essential, is it

necessary to eliminate certain specific chromosomes, or is it enough simply to achieve some overall reduction in chromosome number?

Tests on SEWA/A9, MSWBS/A9, YAC/A9 and YACIR/A9 hybrids at once revealed that the suppressive effect was not limited to the Ehrlich cell; the malignancy of all these tumours was dramatically reduced by fusion with the A 9 cell. As shown in table 5, the take incidences for all these hybrids were not very different from those obtained with A9 cells. Tests on Ehrlich/B82 and Ehrlich/A9RI hybrids gave similar results. It was thus not only the A 9 cell that had the ability to suppress malignancy, and not only L cell derivatives with hereditary metabolic defects. The A 9 RI cells did not have the metabolic defect of A 9 cells (inosinic acid



pyrophosphorylase deficiency), but the take incidence for the Ehrlich/A9RI hybrids, although a little higher than that obtained with the Ehrlich/A9 hybrids, was actually lower than that obtained with the A 9RI cells themselves. The 50 % tumour doses for all these hybrids were about 10000 times higher than those for the parental tumours. Examination of the chromosomal constitutions of the tumours

arising from the injection of the hybrid cells revealed in all cases a situation essen-

tially similar to that found with the Ehrlich/A9 hybrids. The tumours were not

produced by progressive growth of the hybrid cells injected but by selective over-

growth of a minority cell population with a lower modal chromosome number.

TABLE 6. GROWTH OF HYBRID TUMOUR CELLS IN VIVO

AFTER TWO MONTHS CULTIVATION IN VITRO


tumours*/total no. cell type inoculated)

Ehrlih/A 9 (1) 4/4 Ehrlich/A9 (2) 4/4 Ehrlich/A9 (3) 4/4 Ehrlich/B 82 8/8 Ehrlich/A9 itI 4/4

* All animals were dead within 3 to 4 weeks of inoculation.

Several tumours derived from Ehrlich/A9, Ehrlich/B82 and Ehrlich/A9RI

hybrids were explanted, grown continuously in vitro for 2 months, and then

tested again for growth in vivo. As shown in table 6, 100 % take incidences were

obtained in all cases. This result confirms the karyological observations in demon-

strating that the hybrid tumours were indeed produced by cell selection; and it

further shows that continued cultivation of the cells in vitro does not in itself

suppress malignancy. It thus appeared that when any of these highly malignant tumour cells were fused with L cell derivatives of low tumorigenicity, the latter

contributed to the hybrid cell some factor or factors that suppressed the malignant

phenotype of the tumour cell. The hybrid cells resulting from such fusions had little

or no ability to grow progressively in the animal so long as they retained the com-

plete chromosome sets of both parent cells; but when certain chromosomes were

eliminated, the malignant phenotype reappeared and the segregant cells were again able to produce tumours.

The question remains whether restoration of the malignant phenotype requires the elimination of certain specific chromosomes or whether a non-specific reduc-

tion in chromosome number will suffice. An analysis of the tumorigenicity of

Ehrlich/A9 hybrids after prolonged cultivation in vitro provides an answer to this

question. Although chromosome loss in this hybrid takes place very slowly in

vitro, after a period of eighteen months' cultivation the modal chromosome number

of the hybrid cell population was reduced from an initial 128 to about 80. This

8 Henry Harris



The Croonian Lecture, 1971

reduction was thus comparable to that seen in the tumours arising from these

hybrids where modal chromosome numbers in the region of 80 were usually found.

But, as shown in table 5, the take incidences for these hybrids with chromosome numbers reduced by prolonged cultivation in vitro were, if anything, even lower than those obtained with the original unreduced hybrid cell population. It is therefore clear that to generate malignant segregants from the non-malignant hybrid cells, it is not enough simply to reduce the overall number of chromosomes in the

cell; certain specific chromosomes have to be eliminated. These results also imply that injection of the hybrid cells into the animal must select for the loss of a different set of chromosomes from those eliminated by growth in vitro. Analysis of the

patterns of elimination of parental marker chromosomes in the two situations has confirmed that this is indeed the case.

TABLE 7. GROWTH OF EHRLICH/FIBROBLAST HYBRIDS IN VIVO


cell line tumours/total no. percentage or clone inoculated takes

Ehrlich/T 6 6 123/123 100 (wild type)

clonal series clone 1 12/12 100 clone 2 27/28 97 clone 3 20/20 100 clone 4: 36/45 80 clone 5 11/11 100 clone 7 7/8 88 clone 7b 11/12 92 clone 8 26/28 93 clone 9 10/13 77 clone 11 6/10 60

Inocula varied between 104 and 2 x 106 cells.

Can a normal diploid cell suppress the malignant phenotype? Hybrids were made between Ehrlich cells and diploid fibroblasts isolated directly from CBA mice homozygous for the T 6 chromosome translocation, which provides an easily identifiable marker for the diploid fibroblast component in the hybrid cell. Three wild type hybrid populations were tested first, each derived from a separate fusion experiment. With inocula comparable to those used in testing the Ehrlich/A 9

hybrids, take incidences close to 100 % were obtained for all three populations. But wild type populations are populations grown under conditions that select for the most rapidly growing cells. It therefore seemed worth while to examine a number of clonal populations, each derived from a separate primary fusion, for it was possible that the tumorigenicity of some of the clones might differ from that of the wild type populations. The take incidences for a number of clonal populations of Ehrlich/fibroblast hybrids are shown in table 7. Although all of these populations

9



Henry Harris

gave high take incidences, it will be seen that some of the clones, even with inocula

up to 2 x 106 cells, did not give the 100 % take incidence that would be expected for Ehrlich cells; and measurement of the growth rate of some of the tumours

produced from these hybrids also showed some variation from clone to clone. It

:0 - wild type

10 - I clone 1

0? ~~ ~ ~~I ILL I// . I I I - I LO 0- 2

~0- I I I I I I 31 I

LO -

0- 5

30- 8

20 -

LO -

0 I J 30 - 5h

10 -

.0 -

~~L~JIII I ImImlu i ibda I 1fi

po - .0 -

o~~~~~~~~~~~~~~~~~~~~ .0 - Ci I i00i

.O - 4

I?~L I 10 I/I

80 90 100 110 140 150 160 170 180 190 chromosome number

200 210 220 230 240 >245

FIGURE 1. Distribution of chromosomes in clonal populations of Ehrlich/fibroblast hybrids. The expected chromosome number for a hybrid cell resulting from the fusion of one modal Ehrlich cell and one diploid fibroblast would be 116. Except for grossly polyploid clones, all the hybrid populations have chromosome numbers well below that to be expected for the sum of the two parental chromosome sets.

10

2 1

3 2 0

0

a)

at 1-

0 0

0 a) I

0 m

0 3

02

z I

1 L 1E 19

u It 2

to -4r

00 m

.11




thus appeared that fusion with diploid fibroblasts had produced some changes in the capacity of the Ehrlich cells to grow progressively in vivo, but these changes were not very impressive.

One might be tempted to conclude that diploid fibroblasts were essentially ineffective, or at least much less effective than the L cell derivatives, in suppressing the malignancy of the Ehrlich tumour cell. But analysis of the chromosomal constitutions of the Ehrlich/fibroblast hybrids again revealed a situation of greater complexity. The expected chromosome number for a hybrid cell produced by the fusion of one modal Ehrlich cell and one diploid fibroblast would be 116. However, as shown in figure 1, all populations of Ehrlich/fibroblast hybrids, except some grossly polyploid clones, showed modal chromosome numbers between 91 and 102. Such reduced modal chromosome numbers were seen as soon as enough cells could be generated to permit chromosome analysis; and it has, in fact, not proved possible to isolate a population of Ehrlich/fibroblast hybrids with a chromosomal complement corresponding to the sum of the two parental modes. Chromosome losses apparently occur very rapidly in these hybrids, and karyological analyses of populations grown continuously in vitro indicate a high degree of chromosomal instability compared with the hybrids made by fusing the Ehrlich cells with L cell derivatives. It is possible that this instability may be related to the fact that the L cell derivatives have long been adapted to growth in vitro, whereas the freshly isolated fibroblasts have not; but, whatever the cause of the instability, it pre- cludes any firm conclusion about the capacity of diploid fibroblasts to suppress malignancy. For rapid and extensive elimination of chromosomes will obviously greatly increase the chances of producing malignant segregants in the hybrid cell population and will thus tend to generate high take incidences. The tumours produced from Ehrlich/fibroblast hybrids showed substantial variation in chromosome number; but no tumour was found with a chromosomal constitution corresponding to that expected for the complete sum of the two parental chromosome sets.

The Ehrlich tumour is highly aneuploid and its cells have a wide range of chromosome numbers. It seemed possible that if the diploid fibroblasts were fused with tumour cells showing a less variable chromosome number, some hybrid lines might be produced containing essentially complete parental chromosome sets. It was reasonable to hope that such hybrid lines, if they could be obtained, might provide a less equivocal test of the ability of the diploid cell to suppress malignancy. The fibroblasts were therefore fused with SEWA cells, which show a narrow range of 42 to 44 chromosomes (with a mode of 43), and with TA 3 cells, which are virtually euploid. For the experiments with the SEWA cells, CBA fibroblasts homozygous for the T6 translocation were again used; but, in order to eliminate the possibility that some complication might be introduced by the presence of this translocation, the TA3 cells were fused with karyologically normal fibroblasts isolated from ACA mice. (ACA mice have a private histocompatibility system which permits ready identification of the hybrid cells by immunological methods.)

11.



12 Henry Harris

Figure 2 shows the chromosomal constitutions of fifteen clones of SEWA/fibroblast hybrids, each derived from a different primary fusion. The expected chromosome number for a hybrid cell containing one modal chromosome set from the SEWA cell and one diploid chromosome set would be 83; but it will be seen that, as soon as they could be analysed, the majority of the clonal populations already

clone I

0 20- 2

40- 20-

0 40- ' 20-

1 0 .. .......,.

u 40- 20a -

o 0

w 401- 7 202

60 70 80 90

60 S- 8 11

modal SEWA cell and one diploid fibroblast would be 83. Most of the clones have chromo-

40- 20- 9

some numbers well below to be expected for the sum of the two parental

chromosome sets, but clones 2, 3, 5, 13 and20- 15 have modes close to this figure.

o 20- 20-

40 1 0-

0 70 .. 9.0

modal SEWA cell and one diploid fibroblast would be 83. ~Most of the clones have .hrom-

chromosome sets, but clones 2, 3, 5, 13 and 15 have modes close to this figure.




had chromosome numbers a good deal short of this. However, five clones did initially show modes in the region of 80 and some of these retained this high chromosome number for several weeks of subcultivation in vitro, long enough, in any case, to

permit the tumorigenicity of the clones to be tested. The results of the in vivo tests are shown in table 8. It will be seen that all the clones with reduced chromosome numbers gave take incidences not far short of 100 %; but in three of the clones with initially unreduced chromosome numbers the take incidences were greatly reduced. Compared with the parental SEWA tumour cells, these reduced take incidences represent increases in the 50 % tumour doses of at least 105. It thus

TABLE 8. GROWTH OF SEWA/FIBROBLAST HYBRIDS IN VIVO


tumours/total no. percentage cell line inoculated) takes

SEWA/T6T6 15/16 94 (wild type)

clonal series clone 1 4/4 100 clone 2 11/19 58 clone 3 13/27 48 clone 4 7/7 100 clone 5 6/6 100 clone 6 3/3 100 clone 7 7/7 100 clone 10 12/12 100 clone 13 16/31 51 clone 14 15/15 100 clone 15 3/3 100 clone 16 18/20 90

Inocula varied between 1.2 x 104 and 3.6 x 106 cells.

appears that the diploid fibroblast can suppress the malignant phenotype, but only in hybrid cells in which something close to the complete chromosome sets of both parent cells in retained. This conclusion is supported by chromosomal analysis of some forty tumours produced by the injection of the SEWA/fibroblast hybrid cells. The cells in these tumours showed a large spread of chromosome numbers, as might be expected from the chromosomal instability of the hybrid cells in vitro; but, even so, the modes, where clear modes could be established, indicated that the tumours were dominated by cells from which substantial numbers of chromosomes had been eliminated. No tumour was found composed of cells with chromosomal constitutions corresponding to the complete sum of the two parental chromosome sets.

Since our results showed that the tumorigenicity of Ehrlich/fibroblast and SEWA/ fibroblast hybrids could be profoundly influenced by chromosome loss, a special

13



study was made of TA 3/ACA fibroblast hybrids that were selected for high chromosome numbers. Populations of cells were isolated with chromosome numbers

exceeding the sum of the two parental chromosome sets (80). These cells, with modes between 110 and 120 must at one stage have contained at least three parental chromosome sets. It was hoped that such hybrids might retain the chromosomes

responsible for suppression of malignancy over a longer period of cultivation and thus provide a more decisive demonstration of this suppression. Table 9 shows the results of the in vivo tests on three such populations, one wild type and two clonal.

TABLE 9. GROWTH OF TA3/FIBROBLAST HYBRIDS IN VIVO

no. takes (total no. animals with

chromosome number progressive ,r - A. A tumours/total no. percentage

cell line mode range inoculated) takes

wild type 117 100-117 19/54 35 clone 5 112 110-121 6/19 32 clone 7 114 66-121 2/10 20

Inocula varied between 1.2 x 104 and 3.6 x 106 cells.

It will be seen that the take incidences, despite the high inocula, are very low

comparable, in fact, to those obtained with hybrids between tumour cells and L cell derivatives. Chromosomal analysis of the tumours arising from the TA 3/fibroblast hybrids showed once again that the hybrid tumours were not produced by the

progressive growth of the whole cell population injected, but by the selective

overgrowth of cells from which chromosomes had been eliminated. Whereas the modes of the three cell populations injected were 117, 112 and 114, no tumour had a mode greater than 103, and the great majority had modes in the region between 80 and 90. These findings thus indicate that the differences in overall take incidence between the hybrids produced by fusing the tumour cells with L cell derivatives and those produced by fusing the tumour cells with diploid fibroblasts are not to be explained by the greater capacity of the L cell derivatives to suppress malignancy. The higher take incidences seen with tumour/fibroblast hybrids are due to their chromosomal instability which generates malignant segregants at a much higher frequency. That this is the correct interpretation of the data is

supported by an examination of the take incidences of tumour/fibroblast hybrids as a function of the length of time that the cells have been grown in vitro. In table 10 the take incidences of individual batches of TA3/fibroblast hybrids are set out in a chronological order that reflects the period of cultivation in vitro. It will be seen that these hybrids initially have virtually no capacity for progressive growth in

vivo; but, as the period of cultivation in vitro is extended and chromosome losses

supervene, a marked increase in the take incidence occurs. In using X-irradiated newborn syngeneic mice for our assays of growth in vivo,

we chose, from the immunological point of view, the least reactive system available.

14 Henry Harris




If these animals do not provide immunologically neutral conditions for testing the tumorigenicity of the hybrid cells, it is difficult to see how this property can be tested at all. I now wish to present the evidence for the conclusion that these animals did provide a satisfactory test system and that our assays for tumorigenicity were not complicated to any important degree of histoincompatibility between the inoculum and the host. As I have mentioned previously, these X- irradiated newborn mice permit the growth of tumours bearing foreign histo-

compatibility antigens; and it was therefore not surprising that where populations

TABLE 10. PROGRESSIVE INCREASE IN TAKE INCIDENCE OF TA 3

FIBROBLAST HYBRIDS ON CONTINUED PASSAGE IN VITRO

date of no. cells final date genotype no. cell type inoculation inoculated of examination of host takes

TA3/fibro- 11. vi. 70 4.1 x104 24. vii. 70 A 0/3 blast hybrid 11. vi. 70 4.1 x10 10. ix. 70 A 0/3 (clone 6) 2. vii. 70 4.4 x 104 18. ix. 70 A x ACA 0/1

A 0/1 5. vii. 70 1.6 x 104 3. ix. 70 A 0/17

20. vii. 70 1.6x 104 12. x. 70 Ax ACA 0/1 4. viii. 70 3.2 x 104 23. x. 70 A 1/4

10. viii. 70 2.4 x 104 23. x. 70 A 0/2 13. viii. 70 5.7 x104 3. xii. 70 A 1/1 20. viii. 70 3.3 x104 18. ix. 70 A x ACA 1/1 20. viii. 70 3.3x 104 25. ix. 70 Ax ACA 1/1 24. viii. 70 8.2 x 104 23. x. 70 A x ACA 2/3

A 3/3 17. ix. 70 6.3 x104 3. xii. 70 A 1/4 28. ix. 70 1.8x 104 23. x. 70 A 3/3 22. iii. 71 9.0 x 104 19. v. 71 A x ACA 2/3

of hybrid cells showed high or low tumorigenicity when tested in X-irradiated newborn syngeneic mice, these differences in tumorigenicity were also apparent when the hybrid cells were tested in X-irradiated newborn allogeneic mice. This was the case even for different clonal populations of the one hybrid cell type. Dif- ferent clones of SEWA/fibroblast hybrids showed marked differences in tumorigenicity when assayed in X-irradiated newborn A.SW x CBA F1 hybrid mice (syngeneic); but the differences in tumorigenicity between these clones could also be revealed by assays in X-irradiated newborn A.SW or CBA mice (allogeneic). Table 10 shows that the rising take incidence of TA3/fibroblast hybrids on continued cultivation in vitro could be demonstrated not only in the syngeneic A x ACA F1 mice but also in the parental A strain. Immunological tests showed that in these SEWA/fibroblast and TA3/fibroblast hybrids both groups of parental histocompatibility antigens were fully expressed. These findings thus eliminate the trivial objection that the suppression of malignancy seen in our experiments might simply have been due to mis-matching between the hybrid cells and the animals in which they were tested; but they do not eliminate a more sophisticated



Henry Harris

argument, which runs as follows. Since the progressive growth of the hybrid tumours in vivo obviously involves cell selection, might it not be possible that those cells that do grow progressively in vivo are cells that have lost the genetic determinants for the histocompatibility antigens of one parent or the other, or at least cells that express these antigens less strongly. An analysis of the segregation patterns of surface antigens in tumours derived from Ehrlich/A 9 hybrid cells eliminates this possibility also.

The cells of the Ehrlich tumour, having been grown for several decades in mice of a wide variety of foreign genotypes, possess mechanisms that suppress or mask the histocompatibility antigens normally found on the cell surface (Hauschka & Amos I957). When a cell in which the histocompatibility antigen complex is fully expressed is fused with the Ehrlich cell, the suppressive mechanisms of the latter continue to operate and the new histocompatibility antigens introduced into the hybrid cell are also suppressed. When A 9 cells bearing the H-2k antigen complex are fused with the Ehrlich cell, this antigen complex can be detected in the hybrid cells only in very low concentrations or not at all (Harris et al. I969). One might therefore

suppose that if antigenic suppression were related to malignancy, these hybrid cells would grow readily in the test animal; but they are among the least tumori- genie hybrids that we have tested. The tumours arising from the injection of these

hybrids are produced, as I have already described, by progressive overgrowth of

segregants from which variable numbers of chromosomes have been eliminated.

Analysis of these segregant tumours for the presence of the H-2k antigens donated

by the A 9 parent cell reveals that in some of them these antigens remain suppressed; but in others the H-2k antigens are fully restored (Klein, Gars & Harris

I970). These findings demonstrate that the absence of the H-2k antigens in the

original Ehrlich/A9 hybrids was due to antigenic suppression and not to loss of the genetic determinants for these antigens; but they also demonstrate that the

ability of the hybrid cells to grow progressively in the animal is not linked to the

expression or suppression of the histocompatibility antigens. The factors governing the expression of these antigens and those determining progressive growth in vivo

segregate independently. The Ehrlich cell can suppress other antigens introduced into the hybrid cell. The surface of the A 9 cell bears antigens determined by resi- dent viruses. These too can only be detected in low concentrations, or not at all, in Ehrlich/A 9 hybrids; but, like the H-2 antigens, these virus-induced antigens may also reappear in some of the segregant tumours (Grundner et al. I97i). It is thus clear that malignancy, as defined by the ability of the hybrid cells to grow progressively in X-irradiated newborn syngeneic mice, is determined by mechanisms

independent of those that determine the expression of either the histocompatibility antigens or the virus-induced surface antigens. The suppression of malignancy observed in our experiments and the differences in tumorigenicity between one

hybrid cell and another are not explicable in immunological terms. The general conclusions to be drawn from our investigations, despite the great

variety of the experimental material, are clear. When a malignant cell is fused with

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a non-malignant one, or one of greatly reduced malignancy, the resulting hybrid cell, provided that it retains something close to the complete chromosome sets of both parent cells, has lit-le capacity for growth in vivo. In our material the tumours that derive from the injection of such hybrid cells are produced not by progressive growth of the whole cell population injected, but by selective overgrowth of cells from which some chromosomes have been eliminated; and the evidence indicates that the generation of malignant variants in the hybrid cell population requires not merely an overall reduction in chromosome number, but the elimination of some specific chromosomes donated by the non-malignant parent cell. It is therefore difficult to escape the conclusion that in the hybrid cell the non-malignant partner contributes some factor or factors, linked to specific chromosomes, that can suppress the malignancy of the parental tumour cell or hold it in check. What then is the nature of this contribution?

It could be argued that normal diploid cells, or other non-malignant cells, contain specific suppressors of malignancy; but a less fanciful explanation becomes apparent if the possibility is considered that malignancy might ultimately be due to some lesion or lesions involving genetic (or stable epigenetic) loss. If the malignant phenotype were due to some form of genetic loss, or the synthesis of some non- functional gene product, then one would expect that this defect would be made good by a normal cell in which the relevant gene or genes are unimpaired. Comple- mentation between the malignant and the normal cell would then be expected to restore the normal phenotype. Moreover, if more than one type of genetic loss, or impaired gene function, could determine a malignant phenotype, it should be possible in some cases to show complementation between different kinds of malignant cell: hybrid cells of non-malignant phenotype might then be produced from two malignant parent cells. We have therefore studied the properties of hybrids produced by fusing cells from two different sarcomas with lymphoma cells. The sarcomas (MSWBS and MBA) were induced by methylcholanthrene; the lymphoma (YACIR) by Moloney virus. The MSWBS and YACIR tumours have already been described. MBA is a solid transplantable sarcoma carried in CBA mice and bearing the H-2k histocompatibility antigen complex. In order to obtain cell populations suitable for fusion, the solid tumour was explanted and the cells grown for several days in vitro. The MBA/YACIR and MSWBS/YACIR hybrids showed chromosomal constitutions corresponding closely to the sum of the two parental chromosome sets. (For the MSWBS/YACIR hybrids an almost exact correspondence could be established because the YACIR cells were essentially euploid and the MSWBS cells had a very tight mode and a number of marker chromosomes.) Individual clones of both hybrids were tested in the usual way in X-irradiated newborn syngeneic animals. The MBA/YACIR hybrids gave virtually 100% take incidences, comparable to those given by the two parent cells. In this case, complementation clearly had not occurred. Indeed, these hybrids provide the most decisive evidence that malignancy is not suppressed in some non- specific way by cell fusion itself. However, the MSWBS/YACIR hybrids gave

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substantially lower take incidences than the two parent cells, and the tumorigenicity of some of the clones was comparable to that of hybrids between the two parental tumour cells and A 9 cells. This finding indicates that the malignant phenotype can, in some cases be suppressed by fusing one malignant tumour cell with another. While this experiment does not prove that the genetic lesions responsible for

malignancy involve losses of function, the results so closely mimic classical genetic complementation, that the idea, it seems to me, must be taken seriously. The difference in tumorigenicity between the MBA/YACIR hybrids and the MSWBS/ YACIR hybrids might be due to differences in the stability of the two chromosome sets, or to different patterns of chromosome elimination under the selective pres- sures of growth in vitro or in vivo. But such a clear cut difference also suggests the

possibility that the basic lesion responsible for malignancy might not be identical in the two sarcomas, even though both were induced by methylcholanthrene.

Whatever the precise nature of the genetic lesions responsible for malignancy, the present findings clearly delineate the operation of two processes in the formation of a malignant tumour: the generation of genetic variation in the cell population as a whole and selection by the body of certain specific variants that are capable of

progressive growth. All our results can be summarized very simply in such Dar- winian terms. The tumour cells used in our experiments are the end products of extreme selection; they have been selected for the ability to grow progressively in vivo. When such cells are fused with cells that have not been selected for this

property (non-malignant cells), the malignant character of the tumour cells is not

expressed in the hybrids, or is expresed much less strongly. In order to obtain a sub-

population of malignant cells from these essentially non-malignant hybrids, one must select again; and the frequency with which malignant subpopulations can be selected is a function of the degree of genetic variation in the hybrid cell population on which the selection operates. This view of the genesis of a malignant tumour does not require that an oncogenic agent should be highly specific in the genetic lesions that it induces. It would be enough if such an agent raised the level of genetic variation in the exposed cell population to a point where the specific lesions re-

sponsible for malignancy, although random events, had a high probability of occur-

ring. Oncogenic agents such as chemical carcinogens or radiation would then act not by converting normal cells to malignant cells, but by generating enough genetic variation to permit malignant variants to occur with predictable frequency. This selectionist interpretation of malignancy implies, however, that the vast

majority of the variants produced, and hence the vast majority of the chromosomal aberrations seen in pre-cancerous conditions, are essentially irrelevant to the production of the malignant state.

But what of oncogenic viruses ? They obviously represent a genetic increment to the cell, not a genetic loss. And is it not reasonable to suppose that their role in the

production of malignancy is a direct and specific one? The results that we have obtained with virus-induced tumours lead me to think differently. The SEWA tumour was induced by polyoma virus. The cells carry the virus and show the

18 lHenry Harris




characteristic polyoma transplantation antigen. The YAC tumour was induced by Moloney virus. The cells also carry the virus and show the Moloney-specific surface antigen. When hybrids are made between these tumours and A9 cells, the hybrid cells continue to carry the oncogenic viruses of the parental tumour cells; they continue to show the specific surface antigens associated with the presence of these viruses; and they have, at least in some degree, the morphological features and growth patterns characteristic of virus-transformed cells. Yet SEWA/A 9 and YAC/A 9 hybrid cells, so long as they retain something like the complete parental chromosome sets, have little or no ability to grow progressively in vivo: the take incidences obtained with these hybrids are of the same order as those obtained with A9 cells. This, in my view, makes it unlikely that malignancy, as defined by progressive growth in vivo, is determined directly by some diffusible product of the viral genes. If it were so determined, one would expect malignancy to be dominant in these hybrid cells and not recessive, as is found to be the case. There is no doubt, of course, that some oncogenic viruses do produce direct effects on cellular morpho- logy and behaviour: it has been shown, for example, that viral gene products induce changes in the surface properties of the cell (Benjamin & Burger I970) and in the mechanisms that control the replication of the cellular DNA (Dulbecco & Eckhart 1970). But my interpretation of the significance of these effects in relation to the genesis of malignancy is that they generate the genetic instability, the genetic variation, on which selection for malignancy can operate. Some oncogenic viruses may well be very efficient in generating the right kind of genetic instability; but the genetic lesion or lesions that actually determine progressive growth in vivo might none the less involve functional losses even in tumours induced by viruses.

In conclusion, I should like to say something about the generality of our findings. If the fundamental lesions responsible for malignancy are indeed some form of genetic or epigenetic loss, then one would expect that in hybrids between malignant and non-malignant cells recessive inheritance of malignancy would be the rule. But our evidence on the nature of the genetic lesions is at best suggestive; and it may well be that a detailed examination of a wider range of experimental material will reveal cases in which malignancy is inherited as a dominant character. I do not believe that any such cases have yet been established; but their existence would not detract from the interest of the observations that I have described. That malignancy can be suppressed, and suppressed by the activity of a normal body cell is, it seems to me, no small thing; and perhaps I may be forgiven for hoping that the further exploration of this phenomenon may contribute in some small way to our understanding of what remains one of the most distressing of human maladies.

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REFERENCES

Benjamin, T. L. & Burger, M. M. 1970 Proc. natn. Acad. Sci. U.S.A. 67, 929-934. Dulbeeco, R. & Eekhart, W. 1970 Proc. natn. Acad. Sci. U.S.A. 67, 1775-1781. Engel, E., McGee, B. J. & Harris, H. 1969 J. Cell Sci. 5, 93-120. Ephrussi, B., Sealetta, L. J., Stenchever, M. A. & Yoshida, M. C. 1964 Cytogenetics of cells in

culture (ed. R. J. C. Harris), pp. 13-25. New York and London: Academic Press. Grundner, G., Fenyo, E. M., Klein, G., Klein, E., Bregula, U. & Harris, H. 1971 Expl Cell

Res. (in the Press). Handmaker, S. D. I97I Nature, Lond. (in the Press). Harris, H. I966 Proc. R. Soc. Lond. B 166, 358-368. Harris, H, 1970 Cell fusion. Oxford: Clarendon Press. Harris, H., Miller, O. J., Klein, G., Worst, P. & Tachibana, T. 1969 Nature, Lond. 223,

363-368. Harris, H. & Watkins, J. F. I965 Nature, Lond. 205, 640-646. Harris, H., Watkins, J. F., Ford, C. E. & Schoefl, G. I. I966 J. Cell Sci. 1, 1-30. Hauschka, T. S. & Amos, D. B. 1957 Ann. N.Y. Acad. Sci. 69, 561-579. Hayes, W. I966 Proc. R. Soc. Lond. B 165, 1-19. Klein, G., Gars, U. & Harris, H. 1970 Expl Cell Res. 62, 149-160. Littlefield, J. W. 1964 Science, N.Y. 145, 709-710. Littlefield, J. W. I966 Expl Cell Res. 41, 190-196. Nabholz, M., Miggiano, V. & Bodmer, W. I969 Nature, Lond. 223, 358-363. Neff, J. M. & Enders, J. F. I968 Proc. Soc. exp. Biol. Med. 127, 260-267. Okada, Y. I958 Biken's J. 1, 103-110. Okada, Y. & Tadokoro, J. I962 Expl Cell Res. 26, 108-118. Schneeberger, E. E. & Harris, H. I966 J. Cell Sci. 1, 401-406. Weiss, M. C. & Green, H. I967 Proc. natn. Acad. Sci. U.S.A. 58, 1104-1111.



The Croonian Lecture, 1971: Cell Fusion and the Analysis of Malignancy

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Transcript of The Croonian Lecture, 1971: Cell Fusion and the Analysis of Malignancy