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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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Days after inoculation
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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
648 R. T. Johnson and H. Harris
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).
« DA
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^ 40"oo3 20
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0 1 2 3 4 5 6 7 8 9 10 11 12 13t Days after inoculation
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
650 R. T. Johnson and H. Harris
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
652 R. T. Johnson and H. Harris
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.
DNA synthesis and mitosis in fused cells. Ill 653
Triplets of HeLanuclei in
3H1E cells
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Fig. 4. Patterns of labelling in triplets 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 the appro-priate binomial expansions, are shown in the right histogram of each pair.
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
654 R. T. Johnson and H. Harris
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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Number of nuclei labelled per cellFig. 5. Patterns of labelling in pairs of Ehrlich nuclei in heterokaryons containingdifferent numbers of HeLa 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.
DNA synthesis and mitosis infused cells. Ill 655
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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Fig. 6. Patterns of labelling in triplets of Ehrlich nuclei in heterokaryons containingdifferent numbers of HeLa nuclei. The observed patterns of labelling are shown inthe left of each pair of histograms, arid the random patterns, calculated from theappropriate binomial expansions, are shown in the right histogram of each pair.
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
656 R. T. Johnson and H. Harris
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
DNA synthesis and mitosis infused cells. Ill 657
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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0-4
0-2
0-8 '
06
0-4
0-2
0-8
0-6
0-4
0-2
08
0-6 '
0-4
0-2
0-8 '
06
0-4
0-2
x
-
-
-
i ©x—
-
-
~ o—••
; X—"
-
, o
-
-i. a-''
- "~—X—""
-
-
-
n.
— X _ ^
. - O - "
•••o
-X
...a
- * —
..o-
•• .• • • • •
"~x—""
...
658 R. T. Johnson and H. Harris
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,
DNA synthesis and mitosis infused cells. Ill 659
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-
DNA synthesis and mitosis infused cells. Ill 661
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.
662 R. T. Johnson and H. Harris
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
Number of nuclei labelled per cell
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
DNA synthesis and mitosis in fused cells. Ill 663
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.
664 R. T. Johnson and H. Harris
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
666 R. T. Johnson and H. Harris
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.
DNA synthesis and mitosis in fused cells. Ill 669
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
678 R. T. Johnson and H. Harris
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