dna synthesis and mitosis in fused cellskaryon formed the starting point of the present...

jr. Cell Sci. 5, 645-697 (1969) 645Printed in Great Britain

DNA SYNTHESIS AND MITOSIS IN FUSED

CELLS

III. HeLa-EHRLICH HETEROKARYONS

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

SUMMARY

Unlike HeLa homokaryons and HeLa-erythrocyte heterokaryons, heterokaryons made byfusing HeLa cells with Ehrlich ascites cells do not, in general, succeed in synchronizing DNAsynthesis or mitosis. Indeed, a form of antisynchrony is observed in which most of the Ehrlichnuclei synthesize DNA and most of the HeLa nuclei do not. This anomalous situation appearsto be the consequence of competition between the Ehrlich nuclei and the HeLa nuclei for somefactor or factors essential for DNA synthesis. In this competition the Ehrlich nuclei are over-whelmingly successful, so that synthesis of DNA in the HeLa nuclei is largely inhibited. Thepatterns of DNA synthesis in these heterokaryons are thus essentially determined by the pro-portions of the two kinds of nuclei in the cell.

INTRODUCTION

While the two preceding papers (Johnson & Harris, 1969a, b) show that synchronyof DNA synthesis can be achieved both in homokaryons produced by the fusion oflike cells and in heterokaryons produced by the fusion of unlike cells, the originalobservations of Harris & Watkins (1965) on heterokaryons produced by the fusion ofHeLa cells and Ehrlich ascites cells showed that such synchrony was not alwaysimposed on the nuclei in the multinucleate cell. Harris & Watkins found that, duringthe first 7 days after cell fusion, the proportion of heterokaryons showing any formof nuclear labelling after a brief exposure to tritiated thymidine declined from about70% to 30%, but, throughout this period, the proportion of Ehrlich nuclei labelledremained about 80%, whereas the proportion of HeLa nuclei labelled was 30% orless. This disparity between the behaviour of the two sorts of nuclei in the hetero-karyon formed the starting point of the present investigation.

MATERIALS AND METHODS

Measurement of growth and DNA synthesis in Ehrlich ascites tumour cells in the peritonealcavity of the mouse.

The Ehrlich ascites tumour used in the present experiments was a tetraploid line (modalchromosome number 76), maintained in this laboratory for 4 years by serial passage in theperitoneal cavity of non-inbred Swiss mice. In order to determine the approximate rate ofgrowth of the tumour, the method of Klein & Revesz (1953) and Edwards etal. (i960) was used.An inoculum of 2 x 10' Ehrlich cells was injected into the peritoneal cavity of a number ofSwiss mice. Each day a mouse was killed, and as many cells as possible were removed by

646 R. T. Johnson and H. Harris

repeated washing of the peritoneal cavity with i-ml volumes of physiological saline solution.When the saline was free of cells, as viewed under the microscope, the washings were pooled,and the total number of cells in the suspension estimated. The various cell types and theirproportions in the suspension were determined from flattened preparations deposited ontoslides by the Cytocentrifuge.

In order to assess the proportion of cells synthesizing DNA at any one time, 50 /iCi of tritiatedthymidine in 1 ml of sterile saline were injected into the peritoneal cavity of replicate pairs ofmice. One hour later the mice were killed, and smears made of the cells in the peritoneal cavity.These smears were subjected to autoradiography in the usual way. Lennartz & Maurer (1964)have shown that tritiated thymidine is available for about 30 min.

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

The procedure here was essentially similar to that described for HeLa homokaryons (Johnson& Harris, 1969a). On the one hand, HeLa cells in suspension culture were exposed briefly totritiated thymidine and then fused with unlabelled Ehrlich cells. The proportion of labelledHeLa nuclei in the heterokaryons was then compared with the proportion of labelled cells inthe original suspension culture. On the other hand, Ehrlich cells were labelled with tritiatedthymidine in the peritoneal cavity and then fused with unlabelled HeLa cells. The proportionof labelled Ehrlich nuclei in the heterokaryons was then compared with the proportion oflabelled cells in the ascites tumour. In this way it was possible to determine whether the fusionprocedure selected either for or against Ehrlich or HeLa cells in the phase of DNA synthesis.The identification of HeLa and Ehrlich nuclei in the heterokaryon presented no difficulty,because the two types of nucleus are easily distinguishable on morphological grounds.

Cytological and autoradiographic techniquesThese were a3 described in the paper dealing with HeLa homokaryons (Johnson & Harris,

1969a).

OBSERVATIONS

Randomness of fusion with respect to the cell cycle

Ehrlich cells. In Table 1 the proportion of Ehrlich cells labelled by a brief exposureto tritiated thymidine in the peritoneal cavity is compared with the proportion oflabelled Ehrlich nuclei in heterokaryons produced by fusing these labelled Ehrlichcells with unlabelled HeLa cells. As in the previous experiments with HeLa homo-karyons, Table 1 shows that little radioactivity derived from tritiated thymidine isincorporated after the cells are washed in unlabelled thymidine solution, and thatlittle or no quenching of the labelled intracellular pools is achieved by this washingprocedure.

Comparison of the washed smears of labelled Ehrlich cells with the preparations ofheterokaryons showed that the numbers of labelled Ehrlich nuclei were slightly greaterin the heterokaryons, particularly in the samples taken 3-5 h after cell fusion. Thedisparity, which reached a maximum of approximately 10%, was greatest with 3- and4-day-old tumours. It is possible that the higher proportion of labelled nuclei in theheterokaryons was due to the exclusion from the fusion process of cells in mitosis,which were very numerous in young tumours. At later stages, when the cells weredividing much less rapidly, this disparity was much smaller. In any case, this effectis much too small to account for the preferential labelling of Ehrlich nuclei observedin these heterokaryons by Harris & Watkins (1965).

DNA synthesis and mitosis in fused cells. HI 647

HeLa cells. Table 2 shows the results of a similar experiment in which labelled HeLacells were fused with each other and with unlabelled Ehrlich cells. The data indicatethat fusion did not select either for or against HeLa cells in the phase of DNAsynthesis.

Growth and DNA synthesis in populations of Ehrlich cells in vivo

There are at least two distinct phases in the growth of Ehrlich ascites tumours in theperitoneal cavity of the mouse. During the first 5 or 6 days after inoculation, theincrease in cell number is generally exponential, but, after this, there is a progressive

2000

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Fig. 1. Growth curve of the Ehrlich ascites tumour in the peritoneal cavity of themouse. An inoculum of 2 x io8 cells was injected at the point shown by the arrow, andcell numbers were corrected for contamination with other cell types.

fall in the rate of cell multiplication. The data of Klein & Revesz (1953), Baserga (1963)and Lala & Patt (1966) all show essentially similar growth curves. Usually by the 7thday after inoculation Ehrlich cells begin to penetrate the mesothelium and basementmembrane of the peritoneum (Birbeck & Wheatley, 1965). This renders the analysisof the kinetics of tumour growth, after this point, very hazardous (Edwards et al.i960).

As shown in Fig. i, the growth of the Ehrlich cells used in the present experimentswas essentially similar to that described by other workers.

Figure 2 shows the percentage of cells in S phase on successive days. From the


3rd to the 7th day after inoculation there was a steady decrease in the percentage ofcells labelled by a brief exposure to tritiated thymidine. The percentage of cellslabelled fell from about 70% to 35% during this period, and from the 7th to the13th day remained constant at about the 35% level. This pattern of DNA synthesisis similar in many respects to that described by Baserga (1963).

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Fig. 2. The percentage of cells in the phase of DNA synthesis during the growth of thetumour. An inoculum of 2 x io' cells was injected at the point shown by the arrow.

Fusion of HeLa cells tvith Ehrlich cells derived from progressively older tumours

These experiments were undertaken to see whether the age of the Ehrlich tumouraffected the extremely high incidence of DNA synthesis in Ehrlich nuclei in hetero-karyons. The Ehrlich cells were removed from the mice 3, 7, 11, 13 and 15 days after theinitial inoculation of 2 x io6 cells. The labelling pattern of the Ehrlich nuclei was, ingeneral, examined 1 h and 24 h after cell fusion. In Table 3 the data from a selectionof such experiments are shown.

One hour after cell fusion about 40% of the residual single HeLa cells were in Sphase, a value very similar to that found in these cells growing asynchronously in sus-pension. Twenty-four hours after fusion, the percentage of residual single HeLa cellsin S phase was higher in some experiments, but much the same in others. The labellingof HeLa nuclei in heterokaryons was more variable than in homokaryons, but therewas a substantial fall in the level of labelling of the HeLa nuclei in heterokaryons 24 hafter fusion. In almost all cases, the percentage of HeLa nuclei labelled fell to about20%. One hour after fusion, this value was about 40%, that is, about the same as thevalue found in single HeLa cells in the same culture. We may therefore conclude thatfusion of the HeLa cells with Ehrlich cells does not immediately change the incidenceof DNA synthesis in the HeLa nuclei, but that 24 h later the incidence of DNA syn-thesis is markedly reduced. This appears to be true whether the Ehrlich cells weretaken from a young tumour growing exponentially or from one where the growth ratewas extremely slow.

In all samples 1 h after fusion, the multinucleate Ehrlich cells and the residual singleEhrlich cells showed an increase in the incidence of DNA synthesis compared withthe level found in the tumour in vivo. The effect was most marked in the cells takenfrom the 7-day tumour, where the incidence of DNA synthesis in the Ehrlich nuclei

DNA synthesis and mitosis in fused cells. Ill 649

doubled after fusion. This enhancement of DNA synthesis was more marked inyounger tumours than in older ones. It is possible that incubation of the Ehrlich cellsand Ehrlich homokaryons in vitro initially mimics the reinoculation of the tumourinto a fresh mouse. This procedure is known to produce an immediate stimulation ofDNA synthesis in the inoculated cells (Baserga & Gold, 1963; Lala & Patt, 1968;Wiebel & Baserga, 1968).

Twenty-four hours after cell fusion there was a marked reduction in the labellingof the HeLa nuclei in heterokaryons and a marked increase in the labelling of Ehrlichnuclei. On average, about 75 % of all Ehrlich nuclei in the heterokaryons were labelled24 h after fusion, and the level of labelling at this time was essentially independent ofthe age of the tumour at the time of fusion. This indicates that, in the heterokaryon,the incidence of DNA synthesis in the Ehrlich nuclei is restored within 24 h to thelevel found in young tumours in exponential growth.

Analysis of nuclear labelling in HeLa-Ehrlich heterokaryons

Tables 4 and 5 show the labelling of HeLa and Ehrlich nuclei in heterokaryons 1and 24 h after cell fusion. The percentage of HeLa nuclei labelled in all classes ofheterokaryon 1 h after fusion is very similar to the percentage of nuclei labelled in theHeLa homokaryons from the same population (Table 6). This indicates that, duringthe first hour after formation of the heterokaryons, DNA synthesis in the HeLa nucleiis not greatly reduced, as it is 24 h later. Twenty-four hours after fusion, the HeLanuclei in the heterokaryons show a low incidence of DNA synthesis and the Ehrlichnuclei a high incidence irrespective of the total number of nuclei in the cell. This istrue even for cells containing up to 20 nuclei.

Tables 7 and 8 show the patterns of labelling of HeLa and Ehrlich nuclei in hetero-karyons of different nuclear constitutions 1 h and 24 h after cell fusion. The Ehrlichcells were taken from an 11-day-old tumour, and this particular experiment wasanalysed in detail because the sample counted 24 h after fusion contained 10000heterokaryons and was therefore thought to be large enough to give an unbiasedestimate for the total population. Essentially similar patterns of labelling were foundwith tumours of different ages.

One hour after fusion the level of labelling of the HeLa nuclei in heterokaryonsreflected, in general, that found in an asynchronous culture of HeLa cells; the levelof labelling of the Ehrlich nuclei was slightly higher than that found in the tumourin vivo. Tetranucleate cells were an exception to this general statement. Whereas theEhrlich nuclei in all three subgroups of tetranucleate heterokaryons showed a high levelof labelling in comparison with the Ehrlich nuclei in binucleate heterokaryons, thelevel of labelling of the HeLa nuclei fell progressively as the number of Ehrlich nucleiin the heterokaryon increased.

The significance of this effect becomes clearer in the labelling patterns 24 h afterfusion. By this time it is clear that the relative proportions of HeLa and Ehrlich nucleiin the heterokaryons largely determine the labelling patterns of the two types of nuclei.

The original observations of Harris & Watkins (1965) on HeLa-Ehrlich hetero-karyons posed two major questions about the regulation of DNA synthesis. Why


should DNA synthesis in these heterokaryons be largely asynchronous, when, ingeneral, other multinucleate cells exhibit synchronous DNA synthesis? And whyshould the Ehrlich nuclei show such high levels of labelling? These questions can belargely resolved by analysis of the data in Table 8, from which it will be seen that boththese effects are due to the interactions between different ratios of the two types ofnuclei.

When the two types of nuclei are present in the heterokaryon in equal proportionsthe level of labelling in the HeLa nuclei falls to between 15 and 20%, while the levelof labelling in the Ehrlich nuclei reaches 70 to 75%. Heterokaryons of the 1H1E,2H2E and 3H3E type all show very similar patterns of labelling. As the relativenumber of Ehrlich nuclei increases, there is a progressive fall in the level of HeLalabelling, but also a fall in the level of Ehrlich labelling. (See, for example, the series1H1E, 1H2E, 1H3E, and the series 2H2E, 2H3E.) As the relative number ofHeLa nuclei increases, the level of HeLa labelling rises and the level of Ehrlichlabelling remains high or even rises further. (See, for example, the series 1H1E,2H1E, 3H1E, 4H1E, and the series 2H2E, 3H2E, 4H2E, 5H2E.)

When the 24-h samples are compared with the i-h samples or with the cells fromwhich the heterokaryons were made, it is clear that in the heterokaryon a new regimeis imposed: DNA synthesis in the HeLa nuclei is suppressed, while that in the Ehrlichnuclei is enhanced.

The relationship between the labelling of the Ehrlich nuclei and that of the HeLa nucleiTable 9 shows the relationship between the incidence of DNA synthesis in the

Ehrlich nuclei in heterokaryons and the incidence of DNA synthesis in the HeLanuclei in the same cells. In all classes of heterokaryon a similar pattern of labelling isseen, but this is clearest in binucleate heterokaryons. In these, when the Ehrlichnucleus was labelled, only about 20% of the HeLa nuclei in the same cell were alsolabelled; but this figure was very much higher than that found when the Ehrlichnuclei were not labelled. In the latter case, 5 % or less of the HeLa nuclei were foundto be labelled. In cells containing higher numbers of nuclei, the number of HeLanuclei labelled decreased as the number of Ehrlich nuclei labelled increased. On theother hand, as the number of HeLa nuclei in the cell increased, so the proportionof them which were labelled also increased. All these observations point to the sameconclusion. In a heterokaryon DNA synthesis in the Ehrlich nucleus is enhanced atthe expense of the HeLa nucleus and the intensity of the effect depends upon theratio of the two types of nuclei in the cell. It is as if the Ehrlich nucleus acted as aparasite in the heterokaryon, arrogating to itself the materials required for DNAsynthesis and permitting DNA synthesis in the HeLa nucleus only when its own re-quirements had been met.

In Tables 10 and 11 the observed patterns of labelling are compared with the randompatterns calculated from the appropriate binomial expansions. The results indicatethat, 1 h after fusion (Table 10), the deviation from randomness was not as markedas it was 24 h after fusion (Table 11), when the enhancement of Ehrlich labelling andthe suppression of HeLa labelling became more obvious.

DNA synthesis and mitosis infused cells. Ill 651

Tables 12 and 13 show the x2 values for the comparisons between the observed andcalculated random patterns of labelling for bi-, tri- and tetranucleate heterokaryons.A comparison between the summated x2 values of the i-h and the 24-h samples againreveals the increase in disparity between observed and calculated random values at24 h. This is apparent not only in the binucleate cells, but also in all types of tri-tetra-, penta- and hexanucleate cells. It is clear that DNA synthesis in the hetero-karyons becomes increasingly non-random during the first day after fusion as a resultof the dominance exercised by the Ehrlich component. The high x2 values in theseexperiments, despite the high level of labelling of the Ehrlich nuclei and the low levelof labelling of the HeLa nuclei, indicate that, within each of the 2 groups of nuclei,a large measure of synchronization is achieved. The Ehrlich nuclei are synchronizedin S phase, the HeLa nuclei in G phase.

Synchrony of DNA synthesis in the subpopulations of HeLa and Ehrlich nuclei inheterokaryons

Synchrony within the 2 subpopulations of nuclei was assessed by comparison be-tween the observed and the calculated random patterns of labelling in heterokaryonscontaining 2, 3 and 4 nuclei of one or other type. The results are shown as histogramsin Figs. 3-6. From the results obtained for pairs of HeLa nuclei (Fig. 3), the followingconclusions can be drawn: (i) The labelling patterns 1 h and 24 h after fusion differmainly in the frequency of synchronously unlabelled pairs of nuclei. At 24 h, theseoccur with high frequency, indicating a high order of synchrony in G phase, (ii) Thecalculated random patterns of labelling were closer to the observed patterns in the i-hsamples than in the 24-h samples. This is due to the much greater frequency of HeLanuclei in S phase 1 h after fusion than 24 h later, (iii) The frequency of unlabelledpairs of HeLa nuclei increased as the number of Ehrlich nuclei in the cell increased.This relationship was quite clear in the 24-h samples, but was perceptible even in thei-h samples.

The effect of increasing numbers of Ehrlich nuclei on the labelling of triplets ofHeLa nuclei (Fig. 4) was, in general, similar to that seen in pairs of HeLa nuclei.However, the synchrony in triplets of HeLa nuclei was less rigid than in pairs. Thissuggests that the influence of the Ehrlich nuclei may be countered to some extent byincreasing the number of HeLa nuclei in the cell. This view is supported by theobservations on quadruplets of HeLa nuclei, in which synchronous G phase was lesscommon than in triplets or pairs.

The labelling patterns in pairs and triplets of Ehrlich nuclei (Figs. 5 and 6) are, insome ways, the reverse of the HeLa patterns. The following conclusions can be drawnfrom the Ehrlich patterns: (i) The i-h samples show a slightly enhanced level oflabelling of the Ehrlich nuclei compared with that seen in the tumour in vivo.This effect, as measured by the increased frequency of synchronously labelled pairsand triplets of Ehrlich nuclei, was more obvious in younger than in older tumours,(ii) The 24-h samples in all experiments showed a high incidence of Ehrlich nuclei inS phase. In both pairs and triplets of nuclei there was a high level of synchrony,(iii) An increase in the number of HeLa nuclei per cell from 1 to 2 increased the


frequency of synchronously labelled pairs of Ehrlich nuclei; but a further increasein the number of HeLa nuclei produced no further enhancement of Ehrlich labelling.The optimum enhancement of Ehrlich labelling in these cells appears to be achievedby 2 HeLa nuclei per cell.

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Fig. 3. Patterns of labelling in pairs of HeLa nuclei in heterokaryons containingdifferent numbers of Ehrlich nuclei. The observed patterns of labelling are shown inthe left of each pair of histograms, and the random patterns, calculated from theappropriate binomial expansions, are shown in the right histogram of each pair.

Tables 14-18 show the ^2 values for the comparisons between the observed and thecalculated random patterns of labelling for the HeLa and Ehrlich nuclei in thesevarious types of heterokaryon.


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Patterns of nuclear labelling on the second day after cell fusion

Figure 7 shows the percentage of single HeLa cells, HeLa homokaryons and HeLa-Ehrlich heterokaryons, from the same culture, showing some form of nuclear labelling18 to 45 h after cell fusion. After an initial decrease, the percentage of single cellslabelled remained more or less constant throughout this period, but the percentage ofhomokaryons and heterokaryons showing some form of labelling decreased. Table 19shows the proportions of HeLa and Ehrlich nuclei labelled in this experiment. (TheEhrlich cells were derived from a 4-day-old tumour.) It will be seen that the incidenceof DNA synthesis in the HeLa nuclei in heterokaryons fell from the 18th to the 30thhour, and then began to rise. Forty-five hours after cell fusion the level of labellingof the HeLa nuclei in heterokaryons approached that seen in homokaryons and single


HeLa cells. It thus appears that the suppression of HeLa DNA synthesis exerted bythe Ehrlich component in the heterokaryons becomes more pronounced during thecourse of the first day after fusion, but is relaxed to some extent towards the end of thesecond day. The level of labelling of the Ehrlich nuclei also fell during the second day,from 94% at 18 h to 33% at 45 h. During a large part of the second day there wasthus a marked reduction in the labelling of both HeLa and Ehrlich nuclei. As on the

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first day after fusion, there was no correlation between the level of labelling of eitherHeLa or Ehrlich nuclei and the total number of nuclei in the heterokaryon.

Tables 20-23 show the patterns of DNA synthesis in bi-, tri- and tetranucleateheterokaryons during the second day after fusion. These patterns are essentially similarto those seen on the first day, even though the level of labelling of the Ehrlich nucleiwas much reduced. The labelling of the HeLa and the Ehrlich nuclei was alwaysrelated to the relative proportions of the two types of nuclei in the cell.

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Figure 8 shows the proportions of unlabelled HeLa and Ehrlich nuclei in bi-, tri-and tetranucleate heterokaryons during the second day. There are certain generalfeatures in the curves for all types of heterokaryon. The proportion of HeLa nucleinot synthesizing DNA remained high in most heterokaryons, with the greatest varia-tion occurring in cells containing many HeLa and few Ehrlich nuclei. The proportionof Ehrlich nuclei not synthesizing DNA was initially low but increased with time.There is a marked similarity between the curves for the HeLa nuclei in heterokaryonscontaining equal numbers of HeLa and Ehrlich nuclei and those for the HeLa nuclei


in heterokaryons containing a high HeLa to Ehrlich ratio. The curves for the Ehrlichnuclei in these two sorts of heterokaryon were also very similar. It is clear that theproportions of HeLa nuclei not synthesizing DNA were greatest when the HeLa toEhrlich ratio was low. More HeLa nuclei synthesized DNA when equal numbers ofHeLa and Ehrlich nuclei were present, and still more when the number of HeLanuclei in the-eell exceeded the number of Ehrlich nuclei. These results again supportthe view that more HeLa nuclei synthesize DNA when the number of HeLa nucleiin the cell is high.

18 21 27 30 33 36Time (h) after cell fusion

39 •42 45

Fig. 7. Percentage of single HeLa cells, HeLa homokaryons and HeLa-Ehrlichheterokaryons, from the same culture, showing some form of nuclear labelling 18 to45 h after cell fusion. O, single HeLa cells; • , HeLa homokaryons; x , HeLa-Ehrlich heterokaryons.

The relationship between labelling of Ehrlich nuclei and labelling of HeLa nucleiin heterokaryons during the second day after cell fusion is shown in Table 24. It willbe seen that the proportion of cells containing both HeLa and Ehrlich nuclei labelleddecreased between 18 and 27 h after fusion, and then increased. There was an evenmore marked reduction from 21 h onward in the proportion of cells containing alabelled HeLa nucleus and an unlabelled Ehrlich nucleus, and the incidence of suchcells remained uniformly low. On the other hand, the incidence of cells showinglabelled Ehrlich nuclei but unlabelled HeLa nuclei remained high. It thus appears,once again, that the Ehrlich nuclei dominate the resources for DNA synthesis at theexpense of the HeLa nuclei, but this domination is relaxed towards the end of thesecond day after fusion. The reason for the increased incidence of DNA synthesis in


the HeLa nuclei towards the end of the second day is not clear. This increase mayrepresent a belated co-ordination of DNA synthesis between the two sorts of nuclei,or a reduced demand on the part of the Ehrlich nuclei for the materials required forDNA synthesis. A reduction in demand could result from the progressive retardationof DNA synthesis in the Ehrlich nuclei.

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-X

...a

- * —

..o-

•• .• • • • •

"~x—""

...


As in the experiments analysed on the first day after fusion, the two subpopulationsof HeLa and Ehrlich nuclei generally show opposite forms of synchrony, that is, theHeLa nuclei are largely unlabelled, while the Ehrlich nuclei are largely labelled. Each

1716151413121110

5 7« 6

54321

1 . A

0001

001

002

005 I0-1 o-

0-2

0508

18 21 24 27 30 33 36Time (h) after cell fusion

39 42

Fig. 9. x1 values for the comparisons between the observed patterns of labelling and thecalculated random patterns in HeLa-Ehrlich heterokaryons containing 1 HeLanucleus and 1 Ehrlich nucleus. (Second day after cell fusion.)

110 r-

90

70

S 60

•5 50

* 4 0

30

20

10

o

0001001

18 21 24 27 30 33 36Time (h) after cell fusion

39 42

Fig. 10. x* values for the comparisons between the observed patterns of labelling andthe calculated random patterns in trinucleate HeLa-Ehrlich heterokaryons. (Secondday after cell fusion.) • , 1H2E cells; O, 2H1E cells.

of the subpopulations of nuclei within the heterokaryon has a high order of synchrony,but this tends to decrease as the numbers of nuclei in the subpopulations increase. Thefall in the level of Ehrlich labelling during the second day is not associated with aperceptible loss of synchrony. As the frequency of pairs of labelled Ehrlich nuclei fell,


so the frequency of pairs of unlabelled nuclei rose. The reason for the fall in the level ofEhrlich labelling is not clear. It may be due to the passage of these nuclei from S phaseinto G2 phase, or to a decline in the general level of metabolic activity of the cell, or both.

0 1 2 0 1 2 0 1 2 0 1 2 0 1 2

Number of nuclei labelled per cell0 1 2

Fig. 11. Patterns of labelling in pairs of HeLa nuclei in heterokaryons containing2 HeLa nuclei and 1 Ehrlich nucleus. The observed patterns of labelling are shownin the left of each pair of histograms, and the random patterns, calculated from theappropriate binomial expansions, are shown in the right histogram of each pair. Thenumber over each pair of histograms represents the time after cell fusion in hours.

Mitosis in HeLa-Ehrlich heterokaryons

Whereas mitosis in HeLa homokaryons was very common and usually synchronous,little mitotic activity, by comparison, was seen in HeLa-Ehrlich heterokaryons; andmany of the mitoses observed appeared to involve only Ehrlich nuclei. This depressionof mitosis might be a consequence of the inhibition of DNA synthesis in the HeLanuclei in these heterokaryons; the HeLa nuclei might not yet have duplicated their

42-2

66o R. T. Johnson and H. Harris

DNA when the Ehrlich nuclei were ready to enter mitosis, and this could result in themitosis being held in abeyance. Mitosis of the Ehrlich nuclei, while the HeLa nucleiin the cell remained in interphase, would then represent a partial escape from thisform of restraint. Nonetheless, synchronous mitosis of the HeLa and Ehrlich nucleidoes occur in some cells, and this event may result in nuclear fusion, as previouslydescribed (Harris, Watkins, Ford & Schoefl, 1966). The relative infrequency of mitosisand the uncertainty in the identification of the nuclei involved make a quantitativeexamination of mitotic events in these heterokaryons impracticable.

24

0 1 2 3 t i l ) 0 1 2 3 0 1 2 3

0 1 2 3 0 1 2 . 3 0 1 2 3 0 1 2 3 0 1 2 3

Number of nuclei labelled per cell0 1 2 3

Fig. 12. Patterns of labelling in triplets of HeLa nuclei in heterokaryons containing3 HeLa nuclei and i Ehrlich nucleus. The observed patterns of labelling are shown inthe left of each pair of histograms, and the random patterns, calculated from the appro-priate binomial expansions, are shown in the right histogram of each pair. The numberover each pair of histograms represents the time after cell fusion in hours.

DISCUSSION

The observations on HeLa homokaryons and on HeLa-erythrocyte heterokaryonsmake it clear that cells fused together by virus can achieve synchrony of nuclear eventseven when the cells are derived from widely different species, when they show differentforms of specialization and different levels of metabolic activity, and when they are atdifferent stages of the cell cycle at the time of fusion. But the observations on HeLa-


Ehrlich heterokaryons show that this co-ordination is not inevitable. In these hetero-karyons a new phenomenon is revealed: competition between the two types of nucleiin the composite cell for some factor or factors which are essential for the synthesisof DNA. The Ehrlich nuclei arrogate these limiting factors to themselves and thusinhibit the synthesis of DNA in the HeLa nuclei. The end result is that, in these

1 2 0 1 1 0 1 2 0 1 2 0 1 2

Number of nuclei labelled per cell0 1 2

Fig. 13. Patterns of labelling in pairs of Ehrlich nuclei in heterokaryons containing1 HeLa nucleus and 2 Ehrlich nuclei. The observed patterns of labelling are shown inthe left of each pair of histograms, and the random patterns, calculated from theappropriate binomial expansions, are shown in the right histogram of each pair. Thenumber over each pair of histograms represents the time after cell fusion in hours.

heterokaryons, a form of what is virtually antisynchrony is established: the greatmajority of the Ehrlich nuclei synthesize DNA, while the great majority of the HeLanuclei do not. The Ehrlich nuclei thus behave essentially as parasites in the multi-nucleate cell.


The basis for this successful parasitism is obscure. It is unlikely to be due to quali-tative differences in the mechanisms of DNA synthesis, since the relevant metabolicpathways appear to be the same in HeLa and Ehrlich cells (Keir & Smellie, 1959;Smellie, Keir & Davidson, 1959; Smellie, 1962; McAuslan & Joklik, 1962; Brent,

0 1 2 0 1 2 0 1 2 0 1 2


Fig. 14. Patterns of labelling in pairs of Ehrlich nuclei in heterokaryons containing2 HeLa and 2 Ehrlich nuclei. The observed patterns of labelling are shown in the leftof each pair of histograms, and the random patterns, calculated from the appropriatebinomial expansions, are shown in the right histogram of each pair. The number overeach pair of histograms represents the time after cell fusion in hours.

Butler & Crathorn, 1965). Bianchi (1961) has shown that the level of thymidine kinasein human tumours is very much lower than in normal or malignant mouse tissues.But this difference can hardly explain the present findings. Thymidine kinase isunlikely to be a limiting factor in the synthesis of DNA; and experiments in progresson the behaviour of heterokaryons produced by fusing Ehrlich cells with normal


mouse fibroblasts indicate that the Ehrlich nucleus competes favourably against thenormal mouse nucleus also. Certain peculiarities in the organization of the cell cyclein Ehrlich cells may be relevant. It has been shown that, during the early period ofexponential growth, the Ehrlich cells have little or no Gx phase and synthesize DNAcontinuously throughout the major part of interphase (Baserga, 1963; Lala & Patt,1966). On the other hand, HeLa cells, like other cells in culture, have a substantialGx phase (Painter & Drew, 1959; Puck & Steffen, 1963). This means that DNA syn-thesis in the Ehrlich nucleus can be initiated at stages of the cell cycle which normallydo not support DNA synthesis. If this property of the Ehrlich cell resides, at leastpartially, in the organization of the Ehrlich nucleus, it could provide a basis for theparasitism exhibited by this nucleus in the heterokaryon. Whether this ability tocompete favourably for the prerequisites of DNA synthesis is simply a peculiarity ofthe Ehrlich nucleus, or whether this property is also present, in varying degrees, inthe nuclei of other malignant cells, this question, although obscured by the vaguenessof our definitions of malignancy, is not without interest.

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

REFERENCES

BASERGA, R. (1963). Mitotic cycle of ascites tumour cells. Archs Path. 75, 156-161.BASERGA, R. & GOLD, R. (1963). The uptake of tritiated thymidine by newly transplanted

Ehrlich ascites tumour cells. Expl Cell Res. 31, 576—585.BIANCHI, P. A. (1961). Thymidine kinases in human tumours. Biochcm.J. 81, 21P-22P.BIRBECK, M. S. C. & WHEATLEY, D. N. (1965). An electron microscope study of the invasion

of ascites tumour cells into the abdominal wall. Cancer Res. 25, 490-498.BRENT, T. P., BUTLER, J. A. V. & CRATHORN, A. R. (1965). Variations in phosphokinase acti-

vities during the cell cycle in synchronous populations of HeLa cells. Nature, Lond. 207,176-177.

EDWARDS, J. L., KOCH, A. L., YOUNIS, P., FREESE, H. L., LAITE, M. B. & DONALSON, J. T.

(i960). Some characteristics of DNA synthesis and the mitotic cycle in Ehrlich ascitestumour cells. J. biophys. biochem. Cytol. 7, 273-282.

HARRIS, H. & WATKINS, J. F. (1965). Hybrid cells derived from mouse and man: artificialheterokaryons of mammalian cells from different species. Nature, Lond. 205, 640—646.

HARRIS, H., WATKINS, J. F., FORD, C. E. & SCHOEFL, G. I. (1966). Artificial heterokaryons ofanimal cells from different species. J. Cell Sci. 1, 1—30.

JOHNSON, R. T. & HARRIS, H. (1969a). DNA synthesis and mitosis in fused cells. I. HeLahomokaryons. J. Cell Sci. 5, 603-624.

JOHNSON, R. T. & HARRIS, H. (19696). DNA synthesis and mitosis in fused cells. II. HeLa-chick erythrocyte heterokaryons. J. Cell Sci. 5, 625-643.

KEIR, H. M. & SMELLIE, R. M. S. (1959). Studies on the biosynthesis of DNA in extracts ofmammalian cells. II. The enzymic formation of deoxyribonucleoside triphosphate. Biochim.biophys. Ada 35, 405-412.

KLEIN, G. & REVESZ, L. (1953). Quantitative studies on the multiplication of neoplastic cellsin vivo. I. Growth curves of Ehrlich and MCIM ascites tumours. J. natn. Cancer Inst. 14,229-277.

LALA, P. K. & PATT, H. M. (1966). Cytokinetic analysis of tumour growth. Proc. natn. Acad.Sci. U.S.A. 56, 1735-1742.

LALA, P. K. & PATT, H. M. (1968). A characterization of the boundary between the cycling andresting stages in ascites tumour cells. Cell & Tissue Kinetics 1, 137-146.


LENNARTZ, K. J. & MAURER, W. (1964). Autoradiographische Bestimmung der Dauer der DNS-Verdopplung und der Generationszeit beim Ehrlich-Ascites Tumor der Maus durch Doppel-Markierung mit "C-und 3H-Thymidin. Z. Zellforsch. mikrosk. Anat. 63, 478-495.

MCAUSLAN, B. R. & JOKLIK, W. K. (1962). Stimulation of the thymidine phosphorylatingsystem in HeLa cells infected with poxvirus. Biochem. biophys. Res. Commun. 8, 486-491.

PAINTER, R. B. & DREW, R. M. (1959). Studies on deoxyribonucleic acid metabolism in humancancer cell cultures (HeLa). I. Temporal relationships of DNA synthesis to mitosis and turn-over time. Lab. Invest. 8, 278-285.

PUCK, T. T. & STEFFEN, J. (1963). Life cycle analysis of mammalian cells. I. A method forlocalizing metabolic events within the life cycle, and its application to the action of colcemideand sublethal doses of X-irradiation. Biophys. J. 3, 379-397.

SMELLIE, R. M. S. (1962). Studies on the biosynthesis of deoxyribonucleic acid. Proc. Ilth Ann.Reunion Soc. Chem. Phys. (1961), pp. 89-95.

SMELLIE, R. M. S., KEIR, H. M. & DAVIDSON, J. N. (1959). Studies on the biosynthesis of DNAby extracts of mammalian cells. I. Incorporation of 3H-thymidine. Biochim. biophys. Ada 35,389-404.

WIEBEL, F. & BASERGA, R. (1968). Cell proliferation in newly transplanted Ehrlich ascitestumour cells. Cell fif Tissue Kinetics 1, 273-280.

(Received 24 February 1969)

Tab

le i

. R

ando

mne

ss o

f fus

ion

wit

h re

spec

t to

the

cel

l cyc

le:

Ehr

lich

cel

ls

Age

of

tum

our

(day

s)

3 4 5 6 7 8 9 IO 11 12 13

Pre-

was

hsm

ears

, to

tal

no.

nucl

ei

995

—

1824

—

1268

70

9

438

354

562

467

427

476

525

447

482

439

487

275

75i

481

943

672

Pre-

was

hsm

ears

, no

.nu

clei

lab

elle

d

738

-

1147

—

798

4°7

185

158

191

149

183

178

186

186

158

139

170

69

100

187

367

169

Pre-

was

hsm

ears

, %

nuc

-le

i la

belle

d

74-2

—

62

9 —

62

9 5

74

42

2 4

46

34

0 3

19

42-9

37

'43

5 4

44'i

32-8

31

-7

34'9

25

-1

I3'3

38

-9

38

9 2

51

Post

-was

hsm

ears

, to

tal

no.

644

52

5

1057 47

5

589

654

536

435

64

2

44

0

"7

nucl

ei

— — 121

8

29

7

10

0

48

05

10

526

557

516

92

0

Post

-was

hsm

ears

no.

nucl

eila

belle

d

45i

—

3°9

—

652

646

198

115

194

19

238

172

199

189

157

194

220

136

114

203

224

3°7

Post

-was

hsm

ears

, %

nucl

ei l

abel

led

700

—

58-9

—

61

7 5

30

41-7

38

-7

329

190

36-4

35-

83

71

37

1

36

1 3

69

34'3

24

-4

25-9

39

'3

38-8

33-

4

Post

-was

hfu

sion

, to

tal

no.

nucl

ei (

time

afte

r fu

sion

)

3-5

h 8

5 h

255

238

85 h

353

35 h

55

h11

0 50

7

35 h

gh

129

340

9h

42

3— 4h

465

7h

28

3

45

h 8

h14

7 3

4'

95

h2

41

7h

386

Post

-was

hfu

sion

, no

.nu

clei

lab

elle

d

Post

-was

hfu

sion

, %

of

nucl

ei l

abel

led

(tim

e af

ter

fusi

on)

(tim

e af

ter

fusi

on)

35

h 8

5h

204

198

85

h2

52

35 h

5

5 h

68

263

35 h

9 h

68

174

9h

14

1

4h

187

7h

"3

4-5

h 8

h61

11

7

95

h7

i7

h•7

5

35

h8

5h

80

0 8

32

85 h

71-4

35 h

55

h6

18

51

9

35 h

9h

52-7

51

-29

h33

'3

4h

40-2

7h

39

94

5 h

8 h

4i-

5 34

'39

5 h

29

57

h45

-3

• 1

synt s. a ^- s 3- 1 a.

Pre-

was

h sm

ears

: sm

ears

of

Ehr

lich

asci

tes

cells

pre

pare

d 1

h af

ter

the

intr

aper

itone

al i

njec

tion

of t

ritia

ted

thym

idin

e.

The

se s

mea

rs w

ere

not

was

hed

befo

re b

eing

pre

pare

d fo

r au

tora

diog

raph

y.

Post

-was

h sm

ears

:

the

sam

e pr

epar

atio

ns b

ut w

ashe

d w

ith u

nlab

elle

d th

ymid

ine

solu

tion

befo

reau

tora

diog

raph

y. P

ost-

was

h fu

sion

: pr

epar

atio

ns o

f he

tero

kary

ons

prod

uced

by f

usin

g th

e w

ashe

d la

belle

d E

hrlic

h ce

lls w

ith u

nlab

elle

d H

eLa

cells

.

ON

ON


Table 2. Randomness of fusion with respect to the cell cycle: HeLa cells

Total no. nucleiNo. nuclei

labelled% of nuclei

labelled

Pre-wash smearsHeLa homokaryonsHeLa-Ehrlich heterokaryons

1140

1085

2 0 8

S ' 4497

88

45-i45-842-3

Pre-wash smears: smears of HeLa cells prepared after a brief exposure to tritiated thymidine.These smears were not washed before being prepared for autoradiography. The labelled HeLacells were, however, washed in unlabelled thymidine solution before fusion.

Table 3. Fusion of HeLa cells with Ehrlich cells derived from progressively older tumours

Ehrlich cells synthesizing DNA in vivo (%)

Age of tumour (days)Time after cell fusion (h)Total no. single HeLacells

Single HeLa cellslabelled (%)

Total no. HeLa nuclei inHeLa homokaryons

% of HeLa nucleilabelled in HeLahomokaryons

Total no. HeLa nuclei inheterokaryons

% of HeLa nucleilabelled inheterokaryons

Total no. single Ehrlichcells

% of single Ehrlichcells labelled

Total no. Ehrlich nucleiin Ehrlich homokaryons*

% of Ehrlich nucleilabelled in Ehrlichhomokaryons

Total no. Ehrlich nucleiin heterokaryons

% of Ehrlich nucleilabelled in heterokaryons

Total no. HeLahomokaryons

% of HeLa homokaryonslabelled

Total no. Ehrlichhomokaryons*

% of Ehrlichhomokaryons labelled

Total no. of heterokaryons% of heterokaryons labelled

3i

319

40-4

291

43-6

407

37-8

117S

S5-6

3 3 2

75-6

380

80-3

1 2 2

5 9 °

142

92-3

306. 88-2

73

\

32 4

1000

6i-s

2454

49'9

1299

25-9

—

—

—

—

i i 5 5

73-2

1022

57-7

—

—

1000

92-4

71

1000

41-0

9°5

37-6

8 0 2

40-3

1520

78-0

4 4 2

70-8

723

77-5

376

57-2

196

88-2

607

87-5

S9' >

72 4

1019

70'6

1979

57-2

1318

9 6

—

—

—

—

1206

83-

855

70-

—

—

1 1 1 1

86-

* Ehrlich homokaryons were studied from preparationsi-h samples could be examined since these Ehrlich cells (

-

f

11

1

5 0 0

42-6

660

37'3

996

42-8

1267

51-2

1039

60-3

1027

3 655

277

2 55-6

461

8I - I

6857 81-3

made with

!5* v

1 1

24469

3 9 9

991

32-4

14967

22-6

—

—

—

—

13397

70-8

398

40-2

—

1000087-0

28

151

238

41-2

114

33'3

1 2 0

39-2

535

47'i

2 4 6

42-5

126

44-4

49

4 6 9

I O 5

66-7

99636

33

13

2 41000

4cv 1

1881

2 9 2

8002

18-5

—

—

—

—

4458

72-1

687

43-8

—

2007

88-2

1 the Cytocentrifuge. Onk:annot be grown in vitro.

Tab

le 4

. DN

A

synt

hesi

s in

the

HeL

a nu

clei

in

HeL

a-E

hrli

ch h

eter

okar

yons

Age

of

tum

our

(day

s) 3 3 7 71

1

11 15

13

Tim

e (h

)af

ter

cell

fusi

on i24

i

24 I

24 I

24

All

clas

ses

A

Tot

al n

o.of

nuc

lei

40

71:

199

80

2

1318 99

614

967

12

0

8002

%o

fnu

clei

labe

lled

37-8

25'9

40-3 9-6

42-8

22-6

39-2 i8-5

Cla

ss

Tot

al n

o.of

nuc

lei

168

67

433

279

729

557

35 57 275

2 nucl

eila

bell

ed

39

92

09

41

59

44

10

16-4

31

617

-1

Cla

ss 3

Tot

al n

o.of

nuc

lei

12

2

337

28

92

84

325

3487 38 52

8

%o

fnu

clei

labe

lled

36-9

29-1

42-6

n-6

43-1

27-4

44

72

1-0

Cla

ss 4

A1

Tot

al n

o.of

nuc

lei

861

90

15

214

82

22

2546 18 71

3

%o

fnu

clei

labe

lled

4°7

30-0

33-6 8-

i

42-8

27-5

38

915

-0

Cla

ss 5

Tot

al n

o.of

nuc

lei

19

77 29 30 93

1301

382

4

%o

fnu

clei

labe

lled

23'5

48-1

37-9

0 50-5

24-1

66-7

15-4

Cla

sses

Tot

al n

o.of

nuc

lei

14

21

— 57 6118

25 430

63

6-10 %

of

nucl

eila

bell

ed

21-4

14-3

— io-s

37

724

-375

'0i8

- 3

1 Us. a C6 » s R..

3

Age

of

tum

our

(day

s)

3 3 7 71

1 11 15

13

Tim

e (h

)af

ter

cell

fusi

on 12

4 1'

24 1

24 1

24

All

Cla

sses

r Tot

al n

o.of

nuc

lei

41

1

1155

74

912

0810

14

1339

71

22

4458

^1

nucl

eila

bell

ed

8i-8

73-2

77'4

83-2

65-1

70-8

43'4

72-1

Cla

ss

Tot

al n

o.of

nuc

lei

168

674

332

79

729

556

66 572

75

2

%"o

fnu

clei

labe

lled

78

673

-97

77

86-2

62

7

74'4

45'6

70

9

Cla

ss

r Tot

al n

o.of

nuc

lei

133

293

263

22

3

30

330

18 37

336

3

%o

fnu

clei

labe

lled

82-7

70-0

79-5

81-2

64-4

67-8

43'2

73-8

Cla

ss,

A_

1 Tot

al n

o.of

nuc

lei

86 134

12

8

11

2

23

1

2122 1

537

5

4N

nucl

eila

bell

ed

87-2

72-4

72-7

76-8

67

16

96

33-3

72-3

Cla

ss,

A_

_

r Tot

al n

o.of

nuc

lei

18

44 26 27

10

1

1084

54

16

5

% o

fnu

clei

labe

lled

77-8

8i-8

76-9

70-4

66

37

00

40-0

75-o

Cla

sses

Tot

al n

o.of

nuc

lei

61

0

— 40 84

1433 8

1610

6-10 %

of

nucl

eila

bell

ed

83-3

1000 — 70

0

69-0

71-0

50-0

69

8

fused 1

1

ON

Tab

le 6

. D

NA

syn

thes

is i

n H

eLa

hom

okar

yons

OS

ON 00

All

Cla

sses

Cla

ss 2

Cla

ss 3

Cla

ss 4

Cla

ss 5

C

lass

es 6

-10

Age

of

Tim

e (h

)tu

mou

r af

ter

cell

(day

s)

fusi

onT

otal

no.

of n

ucle

i

% o

fnu

clei

labe

lled

Tot

al n

o.of

nuc

lei

%o

fnu

clei

labe

lled

Tot

al n

o.of

nuc

lei

%o

fnu

clei

labe

lled

% o

f %

of

% o

fT

otal

no.

nu

clei

T

otal

no.

nu

clei

T

otal

no.

nu

clei

of n

ucle

i la

bell

ed

of n

ucle

i la

bell

ed

of n

ucle

i la

bell

edSr

- s S 17 7

11

11

13

241

241

241

24

291

2449 90

519

78 660

114

1881

45'7

49

837

-657

-33

76

32-0

33-3

29-2

172

1508 51

213

00 882

496 74 768

45-3

39'3

62

635

-93

39

29

735

'0

84 246

378

198

288 27

47i

38-1

45-7

35-8

52-6

38-9

27-4

44-5

28

7

2426

813

220

0 64 156 8

272

50-0

49

635

-642

-0

43"7

34-6

25-0

25-0

90 IS 55 10 35 5 175

53'3

26

741

-82O

-O28

640

-024

-6

73 44 185

ioo-

o42

-5

27-3

19-0

The

se f

igur

es w

ere

obta

ined

fro

m h

omok

aryo

ns p

rese

nt i

n th

e sa

me

cell

popu

lati

ons

as t

he h

eter

okar

yons

ana

lyse

d in

Tab

les

4 an

d 5.


Class

Table 7. Labelling of HeLa and Ehrlich nuclei in heterokaryons

having different ratios of the two types of nuclei

(1 h after cell' fusion.)

Nuclearconstitution

Total no.cells

Total no.HeLanuclei

% HeLanuclei

labelled

Total no.Ehrlichnuclei

% Ehrlichnuclei

labelled

2

3

4

1H1E2H1E1H2E

3H1E2H2E1H3E

295

in

96

255831

Nuclear constitution: H = HeLataken from an 11-day-old tumour.

295

22296

75116

3i

nucleus; E =

41-0

37-4S94

45-345-725-8

Ehrlich nucleus.

295

in

192

25116

93

The

62-767662-588-o61270-0

Ehrlich cells were

Table 8. Labelling of HeLa and Ehrlich nuclei in heterokaryons

having different ratios of the two types of nuclei

(24 h after cell fusion.)

Class

2

3

4

5

5

6

6

Nuclearconstitution

1H1E

2H1E1H2E

3H1E2H2E1H3E

4H1E3H2E

2H3E1H4E

5H1E4H2E

3H3E2H4Ei H S E

Total no.cells

5666

1352

783

345686141

68229

162

18

1681

120

383

Total no.HeLanuclei

5666

2704783

i°351372

141

272

687

32418

80324360

763

% HeLanuclei

labelled

1 6 6

305163

34'3181

I 2 - I

42-323'410-5

22-2

33-828-1

l 7-83'9

33-3

Total no.Ehrlichnuclei

5666

13521566

3451372423

68458486

7216

162

360152

15

% Ehrlichnuclei

labelled

74'4

73'761-2

7i-375-o5i-8

79'474-566358-3

81-374-7

74'452-633'3

Tab

le 9

. T

he r

elat

ions

hip

betw

een

labe

llin

g of

Ehr

lich

nuc

lei

and

labe

llin

g of

HeL

a nu

clei

in

hete

roka

ryon

s

(24

h af

ter

cell

fus

ion.

)

Nuc

lear

cons

titu

tion

1H1E

1H2E

1H3E

2H1E

3H1E

Pat

tern

of

nucl

ear

labe

llin

g

iH l

abel

led

whe

n iE

lab

elle

diH

lab

elle

d w

hen

iE u

nlab

elle

diH

unl

abel

led

whe

n iE

lab

elle

diH

unl

abel

led

whe

n iE

unl

abel

led

iH l

abel

led

whe

n 2E

lab

elle

diH

lab

elle

d w

hen

iE l

abel

led

and

iE u

nlab

elle

diH

lab

elle

d w

hen

2E u

nlab

elle

diH

lab

elle

d w

hen

3E l

abel

led

iH l

abel

led

whe

n 2E

lab

elle

dan

d iE

unl

abel

led

iH l

abel

led

whe

n iE

lab

elle

dan

d 2E

unl

abel

led

iH l

abel

led

whe

n 3E

unl

abel

led

2H l

abel

led

whe

n iE

lab

elle

diH

lab

elle

d an

d iH

unl

abel

led

whe

n iE

lab

elle

d2H

unl

abel

led

whe

n iE

lab

elle

d2H

lab

elle

d w

hen

iE u

nlab

elle

diH

lab

elle

d an

d iH

unl

abel

led

whe

n iE

unl

abel

led

2H u

nlab

elle

d w

hen

iE u

nlab

elle

d3H

lab

elle

d w

hen

iE l

abel

led

2H l

abel

led

and

iH u

nlab

elle

dw

hen

iE l

abel

led

iH l

abel

led

and

2H u

nlab

elle

dw

hen

iE l

abel

led

( Tot

alno

. ce

lls

49

817

649

817

6

53

10

19

— — — — 88 88 88 39 39 39 23 23 23

3 —

^%

of

cell

ssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

26-5 5-1

73'5

94

94i-

5

IO

O 0

— — — — 33'°

13

6

53'4

2'6 7'7

8 9-7

30

4

4'3

21-7

( Tot

alno

. ce

lls

687

no

687

no 41 4

9— — — — 9

5 95 95 20

20

20

22

22

22

Age

of

tum

our

(day

s)

7 % o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

io-8 0

9

89-2

99-1

I2'2

25-O O 6-3

I2'6

8I-

I

5'°

5'°

90-0

91 0

13

6

Tot

alno

. ce

lls

4217

1449

4217

1449 42

4n

o

24

9

43 36 18

44

997

997

997

355

355

355

246

24

6

24

6

11 %

of

cells

show

ing

indi

cate

dpa

tter

n of

labe

llin

g

19

5

4'8

79-3

95'2

26-2

12-7 1-

2

3O

'2 8'3 0

2-3

19-8

26-6

53'7

13-8

19-4

66-8

II'O

25-6

26-4

To

tal

no.

cell

s

19

58

0

19

58

0

25 12

11 3 3

— — 186

186

186 54 54 54 128

12

8

12

8

13 % o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

22-1 5-

0

77-9

95'°

2O-O O O O O

12

4

22'6

65'I

II

'I

II

-I

77-8 5'5

5'5

24

2

to • a § a1 2.

Tab

le 9

(co

nt.)

Age

of

tum

our

(day

s)

Nuc

lear

cons

titu

tion

Pat

tern

of

nucl

ear

labe

llin

gT

otal

no.

cells

% o

f ce

lls

show

ing

indi

cate

dpa

tter

n of

labe

llin

g

% o

f ce

lls

show

ing

% o

f ce

lls

show

ing

% o

f ce

lls

show

ing

—

—„

indi

cate

d in

dica

ted

indi

cate

dT

otal

pa

tter

n of

T

otal

pa

tter

n of

T

otal

pa

tter

n of

no.

cells

la

bell

ing

no.

cells

la

bell

ing

no.

cells

la

bell

ing

3H u

nlab

elle

d w

hen

iE l

abel

led

3H l

abel

led

whe

n iE

unl

abel

led

2H l

abel

led

and

iH

unla

bell

edw

hen

iE u

nlab

elle

diH

lab

elle

d an

d 2H

unl

abel

led

whe

n iE

unl

abel

led

3H u

nlab

elle

d w

hen

iE u

nlab

elle

d4H

1E

4H

la

bell

ed w

hen

1E l

abel

led

3H l

abel

led

and

iH

unla

bell

edw

hen

iE l

abel

led

2H l

abel

led

and

2H u

nlab

elle

dw

hen

iE l

abel

led

iH l

abel

led

and

3H u

nlab

elle

dw

hen

iE l

abel

led

4H u

nlab

elle

d w

hen

iE l

abel

led

4H l

abel

led

whe

n iE

un

labe

lled

3H l

abel

led

and

iH

unla

bell

edw

hen

iE u

nlab

elle

d2H

lab

elle

d an

d 2H

unl

abel

led

whe

n iE

unl

abel

led

iH l

abel

led

and

3H u

nlab

elle

dw

hen

iE

unla

bell

ed4H

unl

abel

led

whe

n iE

un

labe

lled

5H1E

5H

lab

elle

d w

hen

iE l

abel

led

4H l

abel

led

and

iH

unla

bell

edw

hen

iE l

abel

led

3H l

abel

led

and

2H u

nlab

elle

dw

hen

iE l

abel

led

2H l

abel

led

and

3H u

nlab

elle

dw

hen

iE l

abel

led

23 16 16 16 16

43

5 oI2

'S 6-3

81-3

22

3 3 •3 3

77'3 o o o

ioo-

o

246 99 99 99 99

37-0 61

23-2

17-2

53'5

128 49 49 49 49 90 90 90 90 90 27 27 27 27 27 37 37 37 37

64-8 o 8-2

79-6 2'2 5-6

89

200

63

3 o1

I-I

7-4

22-2

59-3 o

io-8 S'4

io-8

b 1 O o

Tab

le 9

(co

nt.)

Age

of

tum

our

(day

s)

Nuc

lear

cons

titut

ion

Patte

rn o

f nu

clea

r la

belli

ngT

otal

no.

cells

% o

f ce

llssh

owin

gin

dica

ted!

patte

rn o

£la

belli

ngT

otal

% o

f ce

llssh

owin

gin

dica

ted

patte

rn o

fno

. ce

lls

labe

lling

Tot

alno

. ce

lls

% o

f ce

llssh

owin

gin

dica

ted

patte

rn o

fT

otal

% o

f ce

llssh

owin

gin

dica

ted

patte

rn o

fla

belli

ng

no.

cells

la

belli

ng

iH l

abel

led

and

4H u

nlab

elle

dw

hen

iE l

abel

led

5H u

nlab

elle

d w

hen

iE l

abel

led

5H l

abel

led

whe

n iE

unl

abel

led

4H l

abel

led

and

iH u

nlab

elle

dw

hen

iE u

nlab

elle

d3H

lab

elle

d an

d 2H

unl

abel

led

whe

n iE

unl

abel

led

2H l

abel

led

and

3H u

nlab

elle

dw

hen

iE u

nlab

elle

diH

lab

elle

d an

d 4H

unl

abel

led

whe

n iE

unl

abel

led

5H u

nlab

elle

d w

hen

iE u

nlab

elle

d2H

2E

2H l

abel

led

whe

n 2E

lab

elle

diH

lab

elle

d an

d iH

unl

abel

led

whe

n 2E

lab

elle

d2H

unl

abel

led

whe

n 2E

lab

elle

d2H

lab

elle

d w

hen

iE l

abel

led

and

iE u

nlab

elle

diH

lab

elle

d an

d iH

unl

abel

led

whe

n iE

lab

elle

d an

d iE

unla

belle

d2H

unl

abel

led

whe

n iE

lab

elle

dan

d iE

unl

abel

led

2H l

abel

led

whe

n 2E

unl

abel

led

iH l

abel

led

and

iH u

nlab

elle

dw

hen

2E u

nlab

elle

d2H

unl

abel

led

whe

n 2E

unl

abel

led

22 22 222

22-7

409 O

22 22 22 6

4'5

4'5

91-0 o

445

445 44

513

9

139

139

102

102

102

112

18-4

70-3 8-6

23

7

67-6 39 i-o

95'i

37 37 17 17 17 17 17 17 55 55 55 18 18 18 14

29-7

43-2 o o 59 23-5

706

25-5

72-7 56

38

9

55-6 7-1 o

929

a a a-

Tab

le 9

(co

nt.)

Age

of

tum

our

(day

s)

Nuc

lear

cons

titu

tion

Pat

tern

of

nucl

ear

labe

llin

gT

otal

no.

cell

s

% o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

% o

f ce

llssh

owin

gi

di

d

% o

f ce

llssh

owin

gin

dica

ted

indi

cate

dT

otal

pa

tter

n of

T

otal

pa

tter

n of

^

r „.

no.

cells

la

bell

ing

no.

cells

la

bell

ing

no.

cell

s la

bell

ing

% o

f ce

llssh

owin

gin

dica

ted

Tot

al

patt

ern

of

Co !. t

3H2E

3H

lab

elle

d w

hen

2E l

abel

led

2H l

abel

led

and

iH u

nlab

elle

dw

hen

2E l

abel

led

iH l

abel

led

and

2H u

nlab

elle

dw

hen

2E l

abel

led

3H u

nlab

elle

d w

hen

2E l

abel

led

3H l

abel

led

whe

n iE

lab

elle

dan

d iE

unl

abel

led

2H l

abel

led

and

iH u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

iH l

abel

led

and

2H u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

3H u

nlab

elle

d w

hen

iE l

abel

led

and

iE u

nlab

elle

d3H

lab

elle

d w

hen

2E u

nlab

elle

d2H

lab

elle

d an

d iH

unl

abel

led

whe

n 2E

unl

abel

led

iH l

abel

led

and

2H u

nlab

elle

dw

hen

2E u

nlab

elle

d3H

unl

abel

led

whe

n 2E

unl

abel

led

£ 4H

2E

4H l

abel

led

whe

n 2E

lab

elle

d' w

3H

lab

elle

d an

d iH

unl

abel

led

?•

whe

n 2E

lab

elle

d2H

lab

elle

d an

d 2H

unl

abel

led

whe

n 2E

lab

elle

diH

lab

elle

d an

d 3H

unl

abel

led

whe

n 2E

lab

elle

d

126

126

126

126 69 69 69

8-7

198

17-5

61-9 1 "4

I4-S

26-1

59 59 59 59 22 22

11

9

16-9

66

14-

5

9-1

09 24 24 24 24 —

5<vo 0

i6- 7

29

2

54-2

—

22 14

14

14 58 58 58 58

77-3 0 0 0

ioo-o 1-7 8-6

24-1

13-8

5= § OS OJ

Tab

le 9

(co

nt.)

Age

of

tum

our

(day

s)O

S

Nuc

lear

cons

titu

tion

Pat

tern

of

nucl

ear

labe

llin

gT

otal

no.

cells

% o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

Tot

alno

. ce

lls

% o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

bell

ing

Tot

alno

. ce

lls

% o

f ce

lls

show

ing

indi

cate

dpa

tter

n of

labe

llin

g

13 .A % o

f ce

lls

show

ing

indi

cate

dT

otal

pa

tter

n of

no.

cell

s la

bell

ing

5H2E

4H

unl

abel

led

whe

n 2E

lab

elle

d4H

lab

elle

d w

hen

iE l

abel

led

and

iE

unla

bell

ed3H

lab

elle

d an

d iH

un

labe

lled

whe

n iE

lab

elle

d an

d iE

unla

bell

ed2H

lab

elle

d an

d 2H

unl

abel

led

whe

n iE

lab

elle

d an

d iE

unla

bell

ediH

lab

elle

d an

d 3H

unl

abel

led

whe

n iE

lab

elle

d an

d iE

unla

bell

ed4H

unl

abel

led

whe

n iE

lab

elle

dan

d iE

unl

abel

led

4H l

abel

led

whe

n 2E

unl

abel

led

3H l

abel

led

and

iH

unla

bell

edw

hen

2E u

nlab

elle

d2H

lab

elle

d an

d 2H

unl

abel

led

whe

n 2E

unl

abel

led

iH l

abel

led

and

3H u

nlab

elle

dw

hen

2E u

nlab

elle

d4H

unl

abel

led

whe

n 2E

unl

abel

led

5H l

abel

led

whe

n 2E

lab

elle

d4H

lab

elle

d an

d iH

un

labe

lled

whe

n 2E

lab

elle

d3H

lab

elle

d an

d 2H

unl

abel

led

whe

n 2E

lab

elle

d2H

lab

elle

d an

d 3H

unl

abel

led

whe

n 2E

lab

elle

diH

lab

elle

d an

d 4H

unl

abel

led

whe

n 2E

lab

elle

d

58 24 24 24 24 24

4'2

12-5

41-7

41-7

a

14 14 14

14

14

32 32 32

32

32

o 0

7-i

7-i

85

79'

463 9'

4

28-1 94

arris

Tab

le 9

(co

nt.)

Nuc

lear

cons

titut

ion

Patte

rn o

f nu

clea

r la

belli

ng

5H u

nlab

elle

d w

hen

2E l

abel

led

5H l

abel

led

whe

n iE

lab

elle

dan

d iE

unl

abel

led

4H l

abel

led

and

iH u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

3H l

abel

led

and

2H u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

2H l

abel

led

and

3H u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

iH l

abel

led

and

4H u

nlab

elle

dw

hen

iE l

abel

led

and

iEun

labe

lled

5H u

nlab

elle

d w

hen

iE l

abel

led

and

iE u

nlab

elle

d5H

lab

elle

d w

hen

2E u

nlab

elle

d4H

lab

elle

d an

d iH

unl

abel

led

whe

n 2E

unl

abel

led

3H l

abel

led

and

2H u

nlab

elle

dw

hen

2E u

nlab

elle

d2H

lab

elle

d an

d 3H

unl

abel

led

whe

n 2E

unl

abel

led

iH l

abel

led

and

4H u

nlab

elle

dw

hen

2E u

nlab

elle

dSH

unl

abel

led

whe

n 2E

unl

abel

led

t

3t

\

% o

f ce

llssh

owin

gin

dica

ted

Tot

al

patt

ern

ofno

. ce

lls

labe

lling

—

——

—

—

—

—

—

—

—

—

—

—

—

—

——

—

—

—

—

—

—

—

—

—

Age

of

tum

our

(day

s)A

7 % o

f ce

llssh

owin

gin

dica

ted

Tot

al

patte

rn o

fno

. ce

lls

labe

lling

—

——

—

—

—

—

—

—

—

—

—

—

—

—

——

—

—

—

—

—

—

—

—

—

< Tot

alno

. cel

ls— — — — — — — — — — — — —

11 %

of

cells

show

ing

indi

cate

dpa

ttern

of

labe

lling

— — — — — — — — — — — — —

Tot

alno

. ce

lls

32 18 18 18 18 18 18 7 7 7 7 7 7

A % o

f ce

llssh

owin

gin

dica

ted

patte

rn o

fla

belli

ng

37'5 0 5'6

5'6

22-2

22-2

44'4 0 0 0

14

3 0

8s'7

!

Tab

le 9

(co

nt.)

Age

of

tum

our

(day

s)A

13

Nuc

lear

cons

titut

ion

Patt

ern

of n

ucle

ar l

abel

ling

Tot

alno

. ce

lls

% o

f ce

llssh

owin

gin

dica

ted

patt

ern

ofla

belli

ng

% o

f ce

llssh

owin

gin

dica

ted

% o

f ce

llssh

owin

g%

of

cells

show

ing

indi

cate

din

dica

ted

indi

cate

dT

otal

pa

tter

n of

T

otal

pa

tter

n of

T

otal

pa

tter

n of

no.

cells

la

belli

ng

no.

cells

la

belli

ng

no.

cells

la

belli

ng

2H3E

2H

lab

elle

d w

hen

3E l

abel

led

iH l

abel

led

and

iH u

nlab

elle

dw

hen

3E l

abel

led

2H u

nlab

elle

d w

hen

3E l

abel

led

2H l

abel

led

whe

n 2E

lab

elle

dan

d iE

unl

abel

led

iH l

abel

led

and

iH u

nlab

elle

dw

hen

2E l

abel

led

and

iEun

labe

lled

2H u

nlab

elle

d w

hen

2E l

abel

led

and

iE u

nlab

elle

d2H

lab

elle

d w

hen

iE l

abel

led

and

2E u

nlab

elle

diH

lab

elle

d an

d iH

unl

abel

led

whe

n iE

lab

elle

d an

d 2E

unla

belle

d2H

unl

abel

led

whe

n iE

lab

elle

dan

d 2E

unl

abel

led

2H l

abel

led

whe

n 3E

unl

abel

led

iH l

abel

led

and

iH u

nlab

elle

dw

hen

3E u

nlab

elle

d2H

unl

abel

led

whe

n 3E

unl

abel

led

64 64 64 57 57

14-1

17-2

68-8 o 7-0

57 16 16

93

0 0

6-3

16 25 25 25

937 o o

1000

20 20 20

7 7 4 4 4 4

5'0

2O'0

286

71-4 o

ioo-

o

a s &3

DNA synthesis and mitosis infused cells. Ill

Table 10. Relationship between observed and calculated random

patterns of nuclear labelling in HeLa-Ehrlich heterokaryons

(i h after cell fusion.)

677

Pattern of labelling

1HU1EU1HU1EL1HL1EU1HL1EL

2HU1EU1HL1HU1EU2HU1EL2HL1EL1HL1HU1EL2HL1EL

1HU2EU1HL2EU1HU1EL1EU1HL1EL1EU1HU2EL1HL2EL

3HU1EU1HL2HU1EU3HU1EL2HL1HU1EU1HL2HU1EL3HL1EU2HL1HU1EL3HL1EL

2HU2EU1HL1HU2EU2HU1EL1EU2HL2EU2HU2EL1HL1HU1EL1EU2HL1EL1EU1HL1HU2EL2HL2EL

1HU3EU1HL3EU1HU1EL2EU1HL1EL2EU1HU2EL1EU1HL2EL1EU1HU3EL1HL3EL

No. cellsshowing '% of cellsindicated showing indicatedpattern

Class 2 (1H1E)

73I O I

3784

Class 3 (2H1E)

131 2

4 i11

19

15

Class 3 (1H2E)11

91418

1430

Class 4 (3H1E)1

1

51

90

86

Class 4 (2H2E)

7351

81 0

81 0

6

Class 4 (1H3E)

30

3334

1 0

5

pattern

2 4 734'21 2 5

28-5

1 1 7

io-83 6 9

9 917-113-5

ii-59 4

14-61 8 714-63i-3

4-0

4-0

2 0 0

4-0

36-00

32-024-0

I 2 - I

5-28-61 7

13-817-213-817-210-3

9 70

9 79 79 7

1 2 9

32'31 6 1

Calculatedrandom values

for percentage ofcells showing

indicated pattern

22-O

3 7-o15-32 5 7

1 2 7

15-226-5

4-53 1 7

9 5

5 78-4

19-027-8i5-923-2

3-68-9

14-47'4

35-82 0

2 9 6

8-2

4'47-5

14-03 ' i

I I ' O

2 3 6

9-918-67-8

2 0

0 7

14-04-9

3 2 711-425-58-8


Table 11. Relationship between observed and calculated randompatterns of nuclear labelling in HeLa-Ehrlich heterokaryons

(24 h after cell fusion.)

Pattern of labelling

1HU1EU1HU1EL1HL1EU1HL1EL

2HU1EU1HL1HU1EU2HU1EL2HL1EU1HL1HU1EL2HL1EL

1HU2EU1HL

dna synthesis and mitosis in fused cellskaryon formed the starting point of the present...

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