DNA SYNTHESIS AND MITOSIS IN FUSED CELLS · cells, even though the single cells whic tho for fusm...

J. Cell Sci. 5, 603-624 (1969) 603

Printed in Great Britain

DNA SYNTHESIS AND MITOSIS IN FUSED

CELLS

I. HeLa HOMOKARYONS

R. T. JOHNSON AND H. HARRISSir William Dunn School of Pathology, University of Oxford, England

SUMMARY

When HeLa cells growing in asynchronous culture are fused together by inactivated Sendaivirus, synchrony of DNA synthesis and mitosis is rapidly imposed in the resultant multinucleatecells, even though the single cells which fuse to form these multinucleate cells are at differentstages of the cell cycle. Some measure of nuclear co-ordination can already be observed withina few hours of cell fusion, and maximal synchrony is achieved within 2 days. By the end of thesecond day after fusion, asynchronous DNA synthesis or mitosis is rare. In binucleate cellsthis high order of synchrony is maintained for at least 5 days, but in cells containing greaternumbers of nuclei some loss of synchrony begins to appear after the third day. The resultsindicate that the ability of the multinucleate cell to co-ordinate nuclear events is not impairedby the use of virus to facilitate cell fusion.

INTRODUCTION

The technique of inducing cell fusion by the use of Sendai virus (HaemagglutinatingVirus of Japan) was introduced by Okada and his colleagues (Okada, Suzuki & Hosaka,1957; Okada, 1958, 1962a, b; Okada & Tadokoro, 1962). Harris & Watkins (1965),Harris (1965) and Harris et al. (1965) showed that both differentiated and undifferen-tiated cells from different species of animal could be successfully fused together to formviable heterokaryons. The development of this work has been reviewed by Harris,Watkins, Ford & Schoefl (1966) and Harris (1968).

When cells of different types are fused together, co-ordination of DNA synthesisamong the nuclei in the heterokaryons is variable. In some cases, a high order ofsynchrony is imposed on these nuclei; in other cases there is little or no synchrony(Harris & Watkins, 1965; Harris, 1965, 1966; Harris et al. 1966). The workdescribed in the present papers deals with the measurement of synchrony of DNAsynthesis and mitosis in various kinds of fused cells. The first paper describes theresults obtained with homokaryons produced by fusing HeLa cells together. Theobservations made on these cells, which contain genetically similar nuclei, provide abase-line against which the results obtained with different kinds of heterokaryon canbe assessed. The second paper deals with heterokaryons produced by fusing HeLacells with chick embryo erythrocytes; and the third with heterokaryons produced byfusing HeLa cells with Ehrlich ascites cells.

604 R. T. Johnson and H. Harris

MATERIALS AND METHODS

Techniques of cell culture and cell fusion

The HeLa cells were fused together by means of inactivated Sendai virus. After fusion, thehomokaryons were grown on coverslips. All the relevant techniques have been described pre-viously (Harris et al. 1966).

Cytological examination of cover slip cultures

For some hours after cell fusion the multinucleate cells were not flat enough for satisfactoryanalysis. To flatten these large cells without damage, a modification of the apparatus of Gaillard& Schaberg (1964) was constructed. This is essentially a suction apparatus, and consists of ateflon block resting on a microscope slide. Interposed between the two is a filter paper. Runningthrough the block is a circular hole, the diameter of which corresponds exactly to a hole madein the filter paper. The slide rests on a stainless steel base-plate, and the pressure in the systemcan be regulated by a lever with adjustable weights. An 11-mm circular coverslip bearing thecells is placed at the bottom of the hole in the block, and phosphate-buffered saline is pipettedon to it. When the levers are placed in position, the filter paper exerts a suction pressure whichdrags the fluid level down over the cells, producing gentle, but firm, flattening. Multinucleatecells were collected from suspension by means of a Cytocentrifuge (Shandon Scientific Instru-ments, London). Similar machines have been described in detail by Watson (1966).

The preparations of flattened cells on slides or coverslips were fixed and stained with May-Grunwald-Giemsa as described by Harris et al. (1966). When mitotic figures were to be exam-ined, Weigert'8 iron haematoxylin stain was used (Drury & Wallington, 1967). This gave goodnuclear definition with little cytoplasmic staining.

Autoradiography

Thymidine-6-[3H], at a specific activity of 5 Ci/mM was used to label nuclei which weresynthesizing DNA. The radioactive material was obtained from The Radiochemical Centre,Amersham, Bucks, and was added to the culture medium at a final concentration of 5-10 /tCi/ml.The labelled cells were fixed and prepared for autoradiography as described by Harris et al.(1966). Autoradiographs were exposed for periods ranging from 7 days to 2 months as required.

Procedure for determining whether cells at any particular stage of the cell cycle wereselected during fusion

In order to determine whether HeLa cells at any particular stage of the cell cycle fused pre-ferentially with each other, a 50-ml suspension culture was prepared and labelled for 40 minwith 200 /tCi of tritiated thymidine. The cells were removed and collected by centrifugation,and the supernatant was discarded. One sample of the cells was thoroughly washed 3 times inphosphate-buffered saline containing unlabelled thymidine at a concentration of IO~3M. Smearswere prepared from both the washed and unwashed samples. Some of the cells which had beenwashed in the thymidine solution were then fused together. Immediately after fusion, the cellswere flattened on to slides by means of the Cytocentrifuge. The smears and the preparationsof flattened cells were then subjected to autoradiography.

Measurement of nuclear synchrony

Nuclear synchrony is the term used in these experiments to express the extent to which thenuclei in multinucleate cells are co-ordinated in the replication of their DNA. It is essentiallya measure of the synchrony of the phase of DNA synthesis (5 phase) in the individual nuclei;it does not necessarily imply synchronization of the passage of these nuclei into or out of thephase of DNA synthesis (from Gx into S, or from S into Gj).

The populations of fused cells were labelled with tritiated thymidine for 40 min and thensubjected to autoradiography. In each multinucleate cell the numbers of labelled and

DNA synthesis and mitosis in fused cells. I 605

unlabelled nuclei were scored. For homokaryons containing genetically similar nuclei, a singlebinomial term can be used to calculate the distributions of nuclear labelling which would beexpected to occur if there were no co-ordination between nuclei. If p equals the proportion ofnuclei which are labelled and q the proportion which are not, then, since p + q = i, the prob-ability of the random occurrence of multinucleate cells containing no labelled nuclei, nucleiwhich are all labelled, and nuclei some of which are labelled and some not labelled is given byexpansion of the binomial term (p + q)n, where n = the total number of nuclei present. Forexample, a binucleate cell can have three patterns of labelling: no nuclei labelled, one nucleuslabelled, both nuclei labelled. These are represented by the binomial expansion p% + ipq + g3.This method of arriving at the distribution of labelling patterns to be expected for nucleisynthesizing DNA at random has been applied independently by Okada & Murayama (1965)and Yamanaka & Okada (1966).

Similar calculations can be used to test for co-operation between genetically unlike nuclei inheterokaryons. For such cells the distribution of labelling to be expected in the absence ofinteraction between the nuclei can be derived as follows. In a heterokaryon containing, say,nl HeLa nuclei and n2 Ehrlich nuclei, let px be the proportion of HeLa nuclei not labelled,and qx the proportion of HeLa nuclei labelled. Similarly, let p. be the proportion of Ehrlichnuclei not labelled and q* the proportion labelled. The over-all distribution of labelling will thenbe given by expanding the binomial expression

and multiplying the terms, one from each bracket, appropriately in pairs. For example, ifnL = 3 and vt — 2, then the probability, under random conditions, of heterokaryons showing2 out of 3 HeLa nuclei not labelled and 1 out of 2 Ehrlich nuclei labelled would be given as:

3 P'igi x 2/>2?2-

Having thus determined the patterns of labelling to be expected in homokaryons or hetero-karyons if DNA synthesis in their nuclei were not co-ordinated in any way, it is possible tomeasure the degree of synchronization, that is, the degree of deviation from randomness, bycomparing the observed patterns of labelling with those calculated from the binomial equations.The degree of deviation from randomness can be expressed as the x1 value adjusted to thecorrect degree of freedom for each class of multinucleate cell as determined by the totalnumber of nuclei which it contains, x1 can thus be used as a measure of nuclear synchrony; ingeneral, the higher the x1 value, the higher the level of synchrony.

OBSERVATIONS

Randomness of fusion with respect to the cell cycle

Between 30% and 40% of HeLa cells growing in suspension culture are in thephase of DNA synthesis at any one time. It is possible to test whether this proportionof .S-phase cells is found in the multinucleate cells immediately after fusion. If theproportion of 5-phase nuclei in homokaryons immediately after cell fusion was thesame as the proportion of S-phase cells in the culture from which the homokaryonswere made, this would indicate that the fusion process did not select cells from anyparticular stage of the cell cycle. If selection for S- or G-phase cells did occur duringfusion, then the proportion of 5-phase nuclei in the multinucleate cells would bedifferent from the proportion of 5-phase cells in the original culture.

Table 4 shows the results of one such experiment. The proportion of labelled nucleiin the original cell culture was not changed by washing the cells in unlabelled thymi-dine solution. This indicates that little radioactivity derived from tritiated thymidineis incorporated after the cells are washed, and that little or no quenching of the labelled


intracellular pools is achieved by the wash with unlabelled thymidine. The proportionof labelled cells in the smears taken from the suspension culture is the same as theproportion of labelled nuclei in the homokaryons made from this culture. This indi-cates that little or no preferential selection of S- or G-phase cells occurred during theprocess of fusion. This result is in agreement with the work of Yamanaka & Okada(1966), who studied virus-induced fusion of KB cells.

Increase in nuclear synchrony during t/ie first day after fusion

In order to assess whether DNA synthesis in the individual nuclei within homo-karyons became co-ordinated with time, the following experiment was carried out. Onehour after cell fusion, some of the fused cells were exposed briefly to tritiated thymi-dine, spun down in the Cytocentrifuge and then prepared for autoradiography. Therest of the cells were grown on coverslips for 24 h and then exposed to the tritiatedthymidine. Examination of the autoradiographs prepared from the two sets of cellspermitted an analysis of the changes in labelling pattern which occurred in the multi-nucleate cells during the first 24 h.

Table 1 shows the pattern of labelling of the nuclei in homokaryons in 1- and 24-hsamples taken from two such experiments. The major change in the pattern of labellingin all classes of homokaryon over the first 24 h is an increase in the frequency of cellsshowing synchronously labelled or synchronously unlabelled nuclei.

Table 2 shows the proportion of labelled nuclei in all classes of homokaryon takentogether compared with the proportions of labelled nuclei in each separate class ofhomokaryon from bi- to pentanucleate cells. In both experiments, in both i-h and24-h samples, there is a good correlation between the proportion of labelled nuclei inall classes of homokaryon taken together and the proportion of labelled nuclei in eachindividual class. This suggests that, within these limits, the number of nuclei in thecell does not influence the number of nuclei which synthesize DNA.

In Fig. 1 the observed patterns of nuclear labelling in homokaryons containing2, 3 and 4 nuclei are compared with the random patterns calculated from the appro-priate binomial expansions. The following points emerge from these histograms.(1) The frequency of asynchronous nuclear labelling was far higher in the i-h thanin the 24-h samples. (2) At 24 h, the great majority of multinucleate cells showedhighly synchronous nuclear labelling. At 1 h, completely unlabelled cells were presentin excess, as might be expected from the low over-all incidence of labelling. (3) Therewas a greater similarity between the observed patterns and the calculated randompatterns in the i-h than in the 24-h samples.

Table 3 shows the x2 values for the comparisons between these observed and cal-culated labelling patterns. The summated ^2 value for each class of multinucleate cellin the 24-h sample was much greater than this value in the i-h sample. The summatedX2 values increased with the number of nuclei per cell, so that for tetranucleate cellsthe x2 value had a much higher level of significance than for binucleate or trinucleatecells. This effect is seen clearly only in the 24-h samples, when nuclear synchrony hasbeen firmly imposed; a slight effect is sometimes seen in the i-h samples. Theseresults indicate that during the first day after fusion the nuclei in the homokaryons


become highly synchronized with respect to DNA synthesis. This is reflected in thepatterns of nuclear labelling 24 h after cell fusion, and in the high levels of significanceof the x2 values for comparisons between the observed patterns of labelling and the

Expt 224 h

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

Number of nuclei labelled per cell0 1 2 3 4

Fig. i. The observed patterns of nuclear labelling in homokaryons containing 2, 3 and4 nuclei, compared with the random patterns calculated from the appropriate binomialexpansions. Samples were taken 1 h and 24 h after cell fusion. Data from two separateexperiments are given. Black columns are observed values; white columns calculatedrandom values.


random patterns calculated from the appropriate binomial expansions. However, eveni h after cell fusion, there were substantial differences between the observed and thecalculated random patterns. Since cell fusion does not select cells in any particularstage of the cell cycle, it appears that some nuclear co-ordination is established evenduring the first hour after fusion.

It might be argued that the progressive increase in nuclear synchrony observed inthese homokaryons is not due to increased co-ordination between initially asynchronousnuclei, but to selective death of those multinucleate cells which show asynchronouslabelling. This possibility can be dismissed. There is very little cell death during thefirst 24 h after fusion, and the total number of multinucleate cells in the populationremains virtually constant during this period (Fig. 2). The formal alternative, namely,that asynchronous multinucleate cells die, while synchronous multinucleate cellsmultiply to keep the total number of multinucleate cells constant, can also be dis-missed. At mitosis, the multinucleate cells do not generate multinucleate daughtercells, but undergo nuclear fusion to produce increasing numbers of mononucleatedaughters (Harris et al. 1966).

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Time (h) after cell fusion

Fig. 2. The relative numbers of mononucleate and multinucleate cells in the fused cellpopulation during the first day after fusion. (Each number represents the total numberof cells counted in 15 random high-power fields.) —, mononucleate cells; , multi-nucleate cells.

Nuclear synchrony during the first 5 days after cell fusion

In the following experiments cells were briefly exposed to tritiated thymidine atintervals during the first 5 days after fusion, and the resulting autoradiographs wereanalysed in the usual way.

Experiment 1. This experiment covers the period from 5 to 27 h after fusion.Figure 3 A shows the percentage of mononucleate and multinucleate cells with nucleiin the phase of DNA synthesis during this period. A high dose of virus (4000 h.a.u.)was used in this fusion, and the number of cells synthesizing DNA was initially verylow. During the subsequent 12 h, however, a sharp rise in the percentage of labelledcells took place. This artificial form of synchronization is seen clearly when high dosesof virus are used to induce the cell fusion; it appears to be due to the fact that highconcentrations of virus delay the entry of many cells into the S-phase. The effect isseen in both mononucleate and multinucleate cells. This type of induced synchrony is


much less marked when lower doses of virus (in the region of 500 h.a.u.) are used(Fig. 3B). The level of nuclear labelling in the various classes of homokaryon did notdiffer much from the over-all level of labelling in the multinucleate population as awhole, indicating again that the number of nuclei in the homokaryon does not greatlyinfluence the incidence of DNA synthesis in the cell. Figures 4, 5 and 6 show theobserved patterns of labelling in bi-, tri- and tetranucleate homokaryons during thefirst day after cell fusion. The observed and the calculated random patterns for bi-nucleate cells are very different (Fig. 4). The levels of the synchronous labelling pat-terns (both nuclei either labelled or unlabelled) were always higher than those of theasynchronous labelling pattern (one of the nuclei labelled); and the levels of the twosynchronous patterns showed a reciprocal relationship—that is, as the level of cells

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Fig. 3. A, Percentage of mononucleate and multinucleate cells synthesizing DNAduring the first day after cell fusion; 4000 h.a.u. of virus were used in this case to inducefusion. B, Similar data from another cell fusion in which 500 h.a.u. of virus were used.• , mononucleate cells; x , multinucleate cells.

with no nuclei labelled decreased, so the level of cells with both nuclei labelled in-creased. During the first day after cell fusion, the proportion of binucleate cells showingasynchronous labelling fluctuated from below 10% to above 25%.

The labelling patterns in tri- and tetranucleate cells (Figs. 5, 6) are essentially similarto those seen in binucleate cells. Again, synchronously labelled and unlabelled cellspredominate and tend to show a reciprocal relationship. The various types of asyn-chronous labelling patterns also show reciprocal relationships.

Figure 7 shows the summated x2 values for the comparisons between the observedand calculated random labelling patterns in the bi-, tri- and tetranucleate cells in thisexperiment. The x2 values were high for all classes of cell 5 h after fusion, fell between12 to 18 h and then rose again. This indicates that a high level of co-ordination be-tween the nuclei in these homokaryons was already established within 5 h. The levelof co-ordination fluctuated during the first day after fusion, but then increasedprogressively.

Experiment 2. An intermediate dose of virus (2000 h.a.u.) was used in this experi-ment, and the results obtained during the second day after fusion are presented. In

3 Q Cell Sci. 5

6io R. T. Johnson and H. Harris

both bi- and trinucleate cells, to a lesser degree in tetranucleate cells, and even in cellswith higher numbers of nuclei, synchronous labelling patterns predominated. Evenin very large cells having io or more nuclei the most common labelling patterns werethose showing either all or none of the nuclei labelled. Figures 8-10 show the observedand calculated random patterns for bi- tri- and tetranucleate cells. For all classes of

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Fig. 4. Nuclear labelling patterns in binucleate cells during the first day after cellfusion. The observed patterns are shown above and the calculated random patternsbelow. O, neither nucleus labelled; • , 1 nucleus labelled; x , both nuclei labelled.

cell, synchronous nuclear labelling was more common and asynchronous labelling lesscommon than in the calculated random labelling patterns. For example, the observedpatterns in binucleate cells (Fig. 8) show a reciprocal relationship between syn-chronously labelled and completely unlabelled cells and a nearly constant low level ofcells with only one nucleus labelled. The calculated random patterns show a nearlyconstant 50% level of cells with asynchronous nuclear labelling and about a 25 % levelof cells with synchronous labelling. Similar relationships are seen in the tri- and


tetranucleate cells (Figs. 9, 10). These results indicate that nuclear synchrony withinthese cells, imposed very early during the first day after cell fusion, was maintained andincreased on the second day. During this period the relationships between the indi-vidual patterns of labelling in each class of homokaryon become more clearly defined.The summated x2 values for the comparisons between the observed and calculatedrandom patterns of labelling in this experiment are shown in Fig. 11. A comparisonbetween Figs. 7 and 11 indicates that nuclear synchrony was more precise in all threeclasses of cell on the second day after fusion.

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Fig. 5. Nuclear labelling patterns in trinucleate cells during the first day after cellfusion. The observed patterns are shown above and the calculated random patternsbelow. O, no nuclei labelled; • , 1 nucleus labelled; x , 2 nuclei labelled; D, 3 nucleilabelled.

Experiment 3. The results were obtained during the 4th day after cell fusion. Thepatterns of labelling in bi-, tri- and tetranucleate cells on the 4th day after fusion wereessentially similar to those found on the 2nd day. Figure 12 shows the summated x2

values for the comparisons between the observed and calculated random patterns oflabelling. The x2 values for binucleate cells remained approximately constant during

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the 4th day after fusion at about the level found at the end of the 2nd day. The %2

values for trinucleate cells at the beginning of the 4th day were at about the level foundat the end of the 2nd day, but during the 4th day these values declined. The x2 valuesfor tetranucleate cells were scattered, but the levels were, in general, below those found

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Fig. 6. Nuclear labelling patterns in tetranucleate cells during the first day after cellfusion. The observed patterns are shown above and the calculated random patternsbelow. O, no nuclei labelled; • , 1 nucleus labelled; x , 2 nuclei labelled; D, 3 nucleilabelled; A, 4 nuclei labelled.


on the 2nd day. These results indicate that, in binucleate cells, nuclear synchrony istightly maintained for 4 days after cell fusion. In tri- and tetranucleate cells synchronybegins to break down towards the end of the 4th day. The level of synchrony in thesecells was found, in other experiments, to decline still further during the 5th day aftercell fusion.

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Fig. 7. x2 values for the comparisons between the observed patterns of nuclear labellingin bi-, tri- and tetranucleate cells and the random patterns calculated from the appro-priate binomial expansions. O, binucleate cells; • , trinucleate cells; x , tetranucleatecells.

Synchrony of mitosis in homokaryons

Synchrony of mitosis in multinucleate cells, although not always the rule, is a com-mon event in a large range of plant and animal cells (Flemming, 1880; Fell & Hughes,1949; Gonzalez, 1967). Mitoses in homokaryons were examined from 2 h to 50 h aftercell fusion, and the incidence of mitotic figures (from prophase onwards) was recorded.For comparison, mitotic counts were also made on mononucleate cells growing in thesame cultures.

Table 5 shows the results of one such experiment. Two hours after cell fusion, over70% of all multinucleate cells containing mitotic figures showed asynchronous mitosis,that is, some of the nuclei in the cell had not entered mitosis. Twenty-three hours and33 hours after cell fusion, only about 20% of the homokaryons containing mitoticfigures showed asynchronous mitosis. Fifty hours after fusion, only 16-5% of themitoses in homokaryons were asynchronous. Aberrant spindle formations were fre-quently seen. Of these the commonest were tri-polar spindles. Occasionally, one ormore chromosomes failed to attach properly to the main metaphase plate and sub-sequently gave rise to micronuclei. These were very common at later stages in verylarge homokaryons.


Except in the 23-h sample, the percentage of nuclei in mitosis in the homokaryonsscored in Table 5 was higher than in the single cells in the same preparations. Thismay reflect the greater complexity, and hence duration, of the act of mitosis in multi-nucleate cells, especially when a common spindle is formed for 2 or more sets ofchromosomes. Oftebro & Wolf (1967), in an analysis of mitosis in naturally occurring

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Fig. 8. Nuclear labelling patterns in binucleate cells during the second day after cellfusion. The observed patterns are shown above and the calculated random patternsbelow. O, neither nucleus labelled; • , 1 nucleus labelled; x , both nuclei labelled.

HeLa multinucleate cells, described the formation of various forms of aberrant spindleand noted that, in these cells, the duration of metaphase was markedly prolonged. Inthe present experiments, the majority of mitotic figures seen were, in fact, meta-phases. Frequently, asyncrirony of mitosis was very slight, one nucleus just precedingthe other into prophase. Not a single case was found of a homokaryon in which onenucleus was in metaphase and one in prophase. The rarity of such cells conforms withthe observations of Oftebro & Wolf (1967), who showed that, in their preparations,

DNA synthesis and mitosis infused cells. 1 615

all nuclei were synchronized by metaphase even though the entry of the nuclei intoprophase could be slightly out of phase.

Table 6 shows the incidence of synchronous and asynchronous mitoses in all classesof homokaryon. The major difference between the 2-h and the later samples is the

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Fig. 9. Nuclear labelling patterns in trinucleate cells during the second day after cellfusion. The observed patterns are shown above and the calculated random patternsbelow. O, no nuclei labelled; • , 1 nucleus labelled; x , 2 nuclei labelled; D, 3 nucleilabelled.

decrease in the number of asynchronous mitoses in the latter. This effect is even moreclearly seen in Table 7, which shows a marked increase in the incidence of synchronousmitosis in bi-, tri- and tetranucleate cells with time. The increased co-ordination ofmitosis over the 2-day period following cell fusion essentially parallels the increasedco-ordination of DNA synthesis.

DISCUSSIONMost multinucleate cells occurring spontaneously in tissue culture exhibit syn-

chronized DNA synthesis and mitosis in their nuclei (Fell & Hughes, 1949; Nakanishi


& Nakahora, 1956; Mitsuhashi, 1967; Church, 1967; Oftebro & Wolf, 1967; Pera &Wolf, 1967; Pera & Schwarzacher, 1968; Fernandez-G6mez, 1968). In a few cases,asynchrony has been observed in such multinucleate cells (Stubblefield, 1964; Sand-berg, Sofuni, Takagi & Moore, 1966; Kato & Sandberg, 1968), but always under theinfluence of drugs such as colchicine. The present experiments indicate clearly thatwhen cells of similar genetic constitution are fused together by inactivated virus,synchrony of DNA synthesis and mitosis is rapidly imposed in the resultant multi-nucleate cells, even though the single cells which fuse to form these multinucleate

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Fig. 10. Nuclear labelling patterns in tetranucleate cells during the second day aftercell fusion. The observed patterns are shown above and the calculated random patternsbelow. O, no nuclei labelled; • , 1 nucleus labelled; x , 2 nuclei labelled; D, 3 nucleilabelled; A, 4 nuclei labelled.

cells are at different stages of the cell cycle. A similar imposition of nuclear synchronyin virus-induced homokaryons has been found by Yamanaka & Okada (1966) and byWesterveld (1969). In the present experiments, the homokaryons were formed fromasynchronous cell populations; in the experiments of Westerveld, the cell populationswere artificially synchronized before fusion. None the less, the final levels of nuclearsynchrony achieved in the two sets of experiments were comparable. This observation


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70 72 74 76 78 80 82 84 86 88 90 92Time (h) after cell fusion

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Fig. 11. Xs values for the comparisons between the observed patterns of nuclear label-ling in bi-, tri- and tetranucleate cells and the random patterns calculated from theappropriate binomial expansions. O, binucleate cells; 9 , trinucleate cells; x , tetra-nucleate cells.Fig. 12. x' values for the comparisons between the observed patterns of nuclearlabelling in bi-, tri- and tetranucleate cells and the random patterns calculated fromthe appropriate binomial expansions. O, binucleate cells; 0 , trinucleate cells; x,tetranucleate cells.

emphasizes the remarkable ability of the fused cell to co-ordinate nuclear events anddemonstrates further that the use of virus to facilitate cell fusion does not impair thisability.

Dr R. T. Johnson was in receipt of a Medical Research Council Scholarship for training inresearch methods.

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OFTEBRO, R. & WOLF, I. (1967). Mitosis of bi- and multinucleate HeLa cells. Expl Cell Res. 48,39-52.

OKADA, Y. (1958). The fusion of Ehrlich's tumor cells caused by HVJ virus. Biken's J. 1, 103-110.

OKADA, Y. (1962a). Analysis of giant polynuclear cell formation caused by HVJ virus fromEhrlich's ascites tumor cells. I. Microscopic observation of giant polynuclear cell formation.Expl Cell Res. 26, 98-107.

OKADA, Y. (19626). Analysis of giant polynuclear cell formation caused by HVJ virus fromEhrlich's ascites tumor cells. III. Relationship between cell condition and fusion reaction orcell degeneration reaction. Expl Cell Res. 26, 119—128.

OKADA, Y. & MURAYAMA, F. (1965). Multinucleated giant cell formation by fusion betweencells of two different strains. Expl Cell Res. 40, 154-158.

OKADA, Y., SUZUKI, T. & HOSAKA, Y. (1957). Interaction between influenza virus and Ehrlich'stumor cells. III . Fusion phenomenon of Ehrlich's tumor cells by the action of HVJ Z strain.Med.J. Osaka Univ. 7, 709-717.

OKADA, Y. & TADOKORO, J. (1962). Analysis of the giant polynuclear cell formation caused byHVJ virus from Ehrlich's ascites tumor cells. II. Quantitative analysis of giant polynuclearcell formation. Expl Cell Res. 26, 108-118.

PERA, F. & SCHWARZACHER, H. G. (1968). Formation and division of binucleated cells in kidneycell cultures of Microtus agrestis. Humangenetik 6, 158-162.

PERA, F. & WOLF, U. (1967). DNS-Replikation und Morphologie dcr X-Chromosomenwahrend der Synthese Periode bei Microtus agrestis. Chromosoma 22, 378—389.

SANDBERC, A. A., SOFUNI, T., TAKAGI, N. & MOORE, G. E. (1966). Chronology and pattern ofhuman chromosome replication. IV. Autoradiographic studies of binucleate cells. Proc. natn.Acad. Sci. U.S.A. 56, 105-110.

STUBBLEFIELD, E. (1964). DNA synthesis and chromosomal morphology of Chinese hamstercells cultured in media containing JV-deacetyl-Ar-methylcolchicine (colcemid). In Cyto-genetics of Cells in Culture (ed. R. J. C. Harris), pp. 223-248. New York and London:Academic Press.

WATSON, P. (1966). A slide centrifuge: an apparatus for concentrating cells in suspension onto amicroscope slide. J. Lab. din. Med. 68, 494-501.

WESTERVELD, A. (1969). Expl Cell Res. (in the Press).YAMANAKA, T. & OKADA, Y. (1966). Cultivation of fused cells resulting from treatment of cells

with HVJ. I. Synchronization of the stages of DNA synthesis of nuclei involved in fusedmultinucleated cells. Biken's J. 9, 159—175.

{Received 24 February 1969)

Tab

le i

. D

NA

sy

nthe

sis

in n

ucle

i in

hom

okar

yons

one

hou

r an

d tw

enty

-fou

r ho

urs

afte

r ce

ll f

usio

n

Cla

ss 2

Cla

ss 3

Cla

ss 4

Cla

ss 5

Cla

ss 6

Cla

ss 8

Tim

e (h

) ,

* ,

afte

r ce

ll N

o. n

ucle

i N

o. n

ucle

i N

o. n

ucle

i N

o. n

ucle

i N

o. n

ucle

i N

o.

nucl

eifu

sion

la

bell

ed

No.

cel

ls

labe

lled

N

o.

cells

la

bell

ed

No

. ce

lls

labe

lled

N

o. c

ells

la

bell

ed

No.

cel

ls

labe

lled

N

o. c

ells

24

o i 2

121 69

66

185

116

349

100

4546

15322

73

o 1 2 3 o 1 2 3

2928 15 10 3921 20 46 2220 15 9

58

11 13 14

o 1 2 3 4 o 1 2 3 4 o 1 2 3 4 o 1 2 3 4

Experiment 1

11 S 9 17 10 7 4 12

Exp

erim

ent

2o 7 6 3 o

22

1 3 5

o 1 3 5

CLA

SS :

Th

e ho

mok

aryo

ns a

re d

ivid

ed i

nto

clas

ses

in a

ccor

danc

e w

ith

the

tota

l nu

mbe

r of

nuc

lei

whi

ch t

hey

cont

ain.

C

lass

2ar

e di

kary

ons,

cla

ss 3

con

tain

3 n

ucle

i, et

c.N

o.

nucl

ei l

abel

led:

Th

e fi

gure

s in

dica

te t

he n

umbe

r of

lab

elle

d nu

clei

in

the

mul

tinu

clea

te c

ell.

No

. ce

lls:

Th

e fi

gure

s in

dica

te t

he t

otal

num

ber

of c

ells

sho

win

g th

e pa

rtic

ular

pat

tern

of

nucl

ear

labe

llin

g in

dica

ted

in

the

adja

cent

co

lum

n.

Tab

le 2

. T

he p

ropo

rtio

n of

nuc

lei

synt

hesi

zing

D

NA

in

all

cla

sses

of

hom

okar

yon

take

n

tog

ethe

r, c

om

pa

red

wit

h

the

pro

po

rtio

ns

of s

uch

nucl

ei i

n th

e in

divi

dual

cla

sses

Tim

e (h

)af

ter

cell

fusi

on i

24 1

24

All

hom

okar

yons

take

n to

geth

er

Tot

aln

o.

nucl

ei

905

1978

66

010

00

No.

N

ucle

inu

clei

la

bell

edla

bell

ed

%

34°

37-6

ii3

3 5

73

24

8 3

76

32

0 3

20

Tot

alno

.nu

clei

51

2

1300 38

249

6

Cla

ss 2

No

.nu

clei

labe

lled

20

1

81

4

137

168

Nuc

lei

labe

lled

/o

39

36

26

35

93

39

Tot

aln

o.

nucl

ei

Cla

ss 3

No

.nu

clei

labe

lled

Exp

erim

ent

12

46

378

88 199

Exp

erim

ent

219

82

88

77

79

Nuc

lei

labe

lled

%

35

852

-6

38

927

-4

, Tot

aln

o.

nucl

ei

13

22

00 64 156

Cla

ss 4

A

No

.nu

clei

labe

lled

47 84

28

54

Nuc

lei

labe

lled

%

35-6

42

0

43'7

34'6

To

tal

no

.nu

clei

15 55 10

35

Cla

ss 5

A

No

.nu

clei

labe

lled

4 23 2

10

Nuc

lei

labe

lled

(%)

26-7

41-8

20

0

28

6

0 sS01 » Si.


Table 3. x2 values for the comparison between the observed patterns of nuclear labellingin HeLa homokaryons and the random patterns calculated from the appropriate binomialexpansions

(Data from 2 separate experiments are given. In all cases P = 0001.)

Time (h)after cell

fusion

i

2 4

1

2 4

f

Class 2

Individual Summated

;v2

2 99 0

7-0

—

—

15-0

18-o5-4——

3 11 0 9

97-——

7-428-827-9—

—

A'2

——18-9————

38-4——

——

23-7——

——

64-1—

—

ClassA

3

Individual SummatedA'2

Experiment 13-02-3

i -71 2 6

—

3 9 39-9

14-0

3 2 9—

Experiment 24-84 - i0-9

1 0 0

—

1 2 9

23-4o-5

75-6—

A'2

———

1 9 6

—

—

—

—

96-1

—

—

—

—

19-8

—

—

—

—

112-4

—

Class 4A

Individual Summated•\/2 -i/2A A

7-7 —4-0 —

6-5 —239 —

I-I 432

45'6 —5-o —

131 —

4-9 —1409 209-5

io-o —

5-2 —0-04 —

0 —

3'7 18-9793 —33-7 —17-2 —0-4 —

260-6 391-2

Tab

le 4

. R

ando

mne

ss o

f fus

ion

wit

h re

spec

t to

the

cel

l cyc

leO

Sto to

Pre

-was

h sm

ears

*P

ost-

was

h sm

ears

fP

ost-

was

h fu

sion

%

No.

nuc

lei

Per

cent

age

of

No

. nu

clei

P

erce

ntag

e of

N

o.

nucl

ei

Per

cent

age

ofT

ota

l no

. nuc

lei

labe

lled

nucl

ei l

abel

led

To

tal

no. n

ucle

i la

bell

ed

nucl

ei l

abel

led

Tot

al n

o. n

ucle

i la

bell

ed

nucl

ei l

abel

led

2000

73°

36-5

718

35

977

338

-6

* S

mea

rs o

f ce

lls f

rom

a

susp

ensi

on

cult

ure

expo

sed

to t

riti

ated

f

Th

e sa

me

prep

arat

ions

, b

ut

was

hed

wit

h un

labe

lled

th

ymid

ine

thym

idin

e fo

r 40

min

. T

hese

sm

ears

wer

e no

t w

ashe

d in

unl

abel

led

solu

tion

bef

ore

auto

radi

ogra

phy.

thym

idin

e so

luti

on b

efor

e be

ing

prep

ared

for

aut

orad

iogr

aphy

. %

Pre

para

tion

s of

hom

okar

yons

pro

duce

d fr

om t

he

labe

lled

cul

ture

afte

r th

e ce

lls

had

been

was

hed

wit

h un

labe

lled

thy

mid

ine.

Tab

le 5

. Mit

osis

in

hom

okar

yons

Tim

e (h

) af

ter

cell

fus

ion

To

tal

num

ber

of s

ingl

e H

eLa

cells

Per

cent

age

of s

ingl

e H

eLa

cells

in

mit

osis

Tot

al n

umbe

r of

hom

okar

yons

Per

cent

age

of h

omok

aryo

ns i

n m

itos

isT

otal

num

ber

of n

ucle

i in

hom

okar

yons

Per

cent

age

of n

ucle

i in

mit

osis

in

hom

okar

yons

Per

cent

age

of h

omok

aryo

ns s

how

ing

asyn

chro

nous

mit

osis

Per

cent

age

of h

omok

aryo

ns s

how

ing

sync

hron

ous

mit

osis

Asy

nchr

onou

s m

itos

es a

s a

perc

enta

geof

all

mit

oses

in

hom

okar

yons

Per

cent

age

of n

ucle

i st

ill

in i

nter

phas

ein

hom

okar

yons

whi

ch h

ad e

nter

edm

itos

is

2

IOOO 3-2

IOOO

"•5

2586 63

23

1022 4-i

IOOO 5-4

2371 4'2

33

IOOO 3-4

IOOO130

2452 106

50

IOOO 7-0

IOOO 16-4

2331 14-4

8-5

30

73'9

47-2

I-

I

4-3

20-4

14-5

99

23-8

14-5

2-7

16-5 9-2

a

Tab

le 6

. Sy

nchr

onou

s an

d as

ynch

rono

us m

itos

is i

n ho

mok

aryo

ns

Tim

e (h

) af

ter

cell

fusi

on

2

23 33 5°

Cla

ss A

No.

nuc

lei

in m

itosi

s

0 1 2

— — —

0 1 2

— — —

0 1 2

— — —

0 1 2

— — —

2

\

No.

cel

ls

567

41

28

— — —

71

0 64

1— — —

60

7 16 78 — — —

61

9 16

us — — —

Cla

ss1

No.

nuc

lei

in m

itosi

s

0 1 2 3— —

0 1 2 3— —

0 1 2 3— —

0 1 2 3— —

3 No.

cel

ls

19

2 18 4 1

— — 15

7 3 0 2

— — 17

1 2 61

6

— — 15

7 4 3 17 — —

Cla

ssI N

o. n

ucle

iin

mito

sis

0 1 2 3 4—

0 1 2 3 4—

0 1 2 3 4—

0 1 2 3 4—

4 No.

cel

ls

785 8 1 0

— 52 1 1 0 0

— 593 0 0 2

— 42 2 0 1 6

Cla

ss

( No.

nuc

lei

in m

itosi

s

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

5s.

No.

cel

ls

25 3 0 1 0 0 14 0 0 0 0 0 15 0 0 0 1 0 6 0 0 0 0 0

a PS.

fN §. <*>C} t5 a. 3 ft.*

*•

*. a O

S<s

>

Tab

le 7

. In

crea

sing s

ynch

rony

of m

itosi

s in

hom

oka

ryons

wit

h

tim

e

Tim

e (h

)af

ter

cell

fusi

on 2

23

33 5°

No.

cel

ls

63

6

757

70

1

75°

Bin

ucle

ate

cells

No.

nuc

lei

in m

itos

is

0 1 2

— —

0 1 2

— —

0 1 2

— —

0 1 2

— —

Cel

ls s

how

ing

indi

cate

dpa

tter

n of

mit

osis

(%

)

89

26

44

'4 — —

93-8

o-8

64

— —

86-6

2-3

II

-I

— —

82-s

21

I5-3 — —

No.

cel

ls

21

5

16

2

195

18

1

Tri

nucl

eate

cel

ls

No

. nu

clei

in m

itos

is

0 1 2 3—

0 1 2 3—

0 1 2 3—

0 1 2 3—

i Cel

ls s

how

ing

indi

cate

dpa

tter

n of

mit

osis

(%

)

89-3 8-4

19

0-4 —

96

91-

9

0 1-2

87-7

I-O

3-1 8-2

—

86-7

2'2

1-7

9'4

Tet

ranu

clea

te c

ells

1

No

. ce

lls

92 54 64 5°

No.

nuc

le:

in m

itos

is

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

Cel

ls s

how

ing

indi

cate

di

patt

ern

ofm

itos

is (

%)

84-8 5'4

8-7

11

0

96

3i-

8

i-8

0 0

92

2

4'7

0 0 3'i

84-0

40

0 2-O

I2"O

hnson a A. tq L 2.

DNA SYNTHESIS AND MITOSIS IN FUSED CELLS · cells, even though the single cells whic tho for fusm...

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