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Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells
Experimentul
119
Cell Research 26, 119-128 (19G2)
ANALYSIS
OF GIANT
CAUSED
POLYNUCLEAR
BY HVJ ASCITES
VIRUS TUMOR
CELL FORMATION
FROM
EHRLICH’S
CELLS
III. RELATIONSHIP BETVVEEN CELL CONDITION AND FUSIOS REACTIOn’ OR CELL DEGENERATION REACTION YOSHIO Department
OKADA
of Preventive Medicine, Research Institute for Microbial Osaka University, Osaka, Japan
Diseases,
Received May 29, 1961
As
described in a previous report [j 1, the cell fusion reaction is initiated in z~itro by the activity of HVJ when the cells are incubated with shaking at 37°C. Without shaking, the efficiency of the fusion reaction is very low and marked degeneration of the cells occurs. The necessity of shaking and some other features of the fusion reaction are discussed in this report. MATERIALS
AND
METHODS
Virus.-Egg adapted Z strain of HVJ was purified and concentrated as described in the previous report by differential centrifugation. Purified HVJ was resuspended in glucose and bicarbonate-free Hanks’ solution buffered with M/i00 phosphate (Solution A), or in Hanks’ solution with 0.003 M bicarbonate without glucose (Solution B). Cell.--Washed Ehrlich’s tumor cells (ETC) were resuspended in both types of Hank’s solution given above. Cell count.-Immediately after adding and mixing one drop of 2 per cent trypan blue the cell sample was introduced into a Fuchs-Rosenthal cytometer and photographed with a phase contrast microscope at low magnification. The numbers of degenerated cells stained and viable cells remaining colorless were counted from the photographs. The fusion index [S] was calculated with the formula. Cell number in non-virus control tube Fusion index = ___ __ - 1.0. Cell number in test tube The rate of the degenerated cells was expressed as fractions in per cent of the original cell number, because (1) cell number decreases during fusion reaction, (2) under suitable conditions there is no increase of cell degeneration during the reaction, and (3) almost all degenerated cells are mononucleated cells. Experimental
Cell Research 26
120
Yoshio Okada EXPERIMENTALS
Oxygen containing Warburg HAU/ml placed in the tumor
uptrrke during the fusion reaction.--One ml of tumor cell suspension 2.2 X 107 cells/ml was introduced into the main chamber of a flask. Then 0.5 ml sample of HVJ at a concentration of 16,000 was placed in the side arm, and 0.2 ml of 20 per cent KOH was the center well containing a piece of filter paper. In this experiment cells and H\‘J were suspended in Solution A.
4.c
0.0
Fig. 1. Fig. l.-Oxygen no-virus control
uptake sample.
Fig. 2. during
the fusion
reaction.
0 -0,
virus
added test sample;
A-
A
Fig. 2.-Shaking effect on the fusion reaction. 0 - 0, fusion index of samples with shaking; 0 - - 0, fusion index of samples without shaking; A - A , % of degenerated cells of samples with shaking; A - - A , % of degenerated cells of samples without shaking.
After preincubation of 3i”C for 15 min, the HVJ was transferred into the main chamber and then the oxygen uptake was determined manometrically for 60 min. As controls, 0.5 ml of the suspending medium in place of HVJ, or 1 ml of the same medium in place of the tumor cell suspension were used. As indicated in Fig. 1, the oxygen uptake of the cell-virus mixture n-as slightly higher than that of the tumor cell control; the HV.J control sample Experimental
Cell Research 26
121
Analysis of fused cell formation caused by HVJ. II1
showed no oxygen uptake. This elevation in the rate of oxygen uptake is rather more marked in the first 5 min than that in the later stage. Though the difference between the test and control samples is slight, in repeated experiments the difference \vas consistent. As described previously, after addition of HVJ the cells promptly agglutinate and within 5 min opposed cells in the aggregates start fusing and the reaction is completed in 30 min. At this stage of the reaction the cell number of the test sample decreased to about 23 per cent of the control, as the fusion index of this sample was 3.3, and as the result the total cell surface area of the test sample becomes smaller than that of the control. Despite this reduction, oxygen uptake is slightly elevated especially in the early stage of the fusion reaction. It seems the fusion reaction requires some extra energy. Seressity of shnkiny for the fusion reaction.-As reported previously, the shaking of ETC-H\‘.J mixture is essential for the formation of typical fused cells in vitro [s]. Without shaking, the appearance of fused cells was greatl) decreased. When H\‘.J treated with sonic vibration or freeze-thawing was used, more significant differences appeared between cells which were shaken and those which were not. The hemolytic activity of H\‘.J is increased with these treatments [3], as that of mumps virus [l] or NDV [‘LI, but the fusion activity decreases [(;I. In Fig. 2, a case of the fusion reaction using sonically treated HVJ is shown. The fusion activity of this virus preparation gave a fusion index of 4.3 while the original native virus showed a fusion index of 7.9 under the same conditions. In this experiment the effect of shaking is prominent and by the incubation at 37°C for 60 min with shaking the fusion reaction occurred in proportion to the virus titer and no specifically degenerated cell appeared. On the other hand, using the same preparation of the virus, when the cell-rirus mixture was incubated without shaking the fusion reaction did not occur, while cell degeneration took place roughly proportional to the virus tiiers. As sholvn in the preceding paper, the fusion activity of H\‘J is different from the other characteristics of the virus and it seems also that the fusion reaction which occurs with shaking is different from the cell degeneration reaction which develops without shaking. Cell degeneration occurred typically \vhen a high concentration of ISTC and sonically treated virus were used. \Yhen native H\‘.J \vas used, cell degeneration was only slight in the absence of shaking. Effect of rrerntion 0~1 the fusion rerrction-In these experiments, ETC and HVJ samples \vere used suspended in Solution 13. Warburg flasks with 1 ml of 20 per cent ETC (about 2 x 107 cells/ml) in the main chamber and 0.5 ml Erperimenful
Cell Research
26
122
Yoshio Okada
of 16,000 HAU/ml of HVJ in the side arm were flowed at 4°C with 1000 ml of the desired gas mixtures containing 1 per cent CO,, O-99 per cent air and the remainder iYz. After gas exchange, HVJ samples were tipped into the main chamber of the flasks, kept for 20 min at 4°C and then incubated at 37°C for 60 min with shaking. 5.0 4.0 g 2 3.0
60 e
5 'Z .: 2.0
40 &
1.0
20 &
0 : ’
0
0.0 3
5
10 Air content.
20
Fig. 3.-Aeration effect on the fusion reaction and the cell degeneration reaction. 0 -0, fusion index when native HVJ was used; 0 ~- 0 , fusion index when sonicated HVJ was used; *-., % of degenerated cells when native HVJ was used; A -- a , % of degenerated cells when sonicated HVJ was used.
99
per cent
As shown in Fig. 3, using O-l per cent air, no fused cells appeared and a slight cell degeneration occurred. With an increased amount of air, a strong fusion reaction occurred and the development of the reaction reached a plateau with more than 20 per cent air. Cell degeneration did not take place when the air content was 5 per cent or more. It seems that the fusion activity of HVJ causes the fusion of the cells under aerobic conditions but under anaerobic conditions the cell fusion reaction does not occur and degeneration of the cells results. When sonically treated HVJ was used in place of native HVJ the air effect was similar to that of native HVJ, but the maximum fusion index (at air content of 20 per cent or more) was very low, compared to that of the native HVJ. In this case, a marked cell degeneration occurred in low air content, and 92 per cent of the cells were degenerated under anaerobic conditions. This fact also indicates the independence of the fusion activity of HVJ from the cell degenerating activity of the virus which can proceed under anaerobic conditions and may be closely related with the hemolytic activity. The above data would indicate that oxygen is essential for the fusion reaction in glucose free medium. Fused cell formation under anaerobic conditions in a medium containing glucose has not yet succeeded because the pH of the medium dropped markedly during incubation at 37°C. Thus the statement in the previous report [S] that aeration was not important for the fusion reaction must be discarded. Experimental
Cell Research 26
Analysis
of fused cell formation
caused by HVJ.
123
III
The effect of’ 2,kiinitrophenol (DLX’P) on the fusion reaction.-From the above observations it may be concluded that the fusion reaction is a process which requires a specific expenditure of energy. When the effect of DNP is studied, the results seem to support this conclusion.
-100
0
0.5 1.1 2.1 4.2 8.5 Final concentration of DNP (X 10-h M)
6.0
17
0
7.0 PH
Fig. 4.
Fig. 5.
Fig. 4.-Effect of 2,4-dinitrophenol on the fusion reaction. O-O, fusion index of test sample; A - A , degenerated cells in test sample; a - - a , degenerated cells in no-virus control sample. Fig. 5.-pH
dependence
of the fusion reaction.
0-
0, fusion index;
A-
A , degenerated
cells.
In the experiment, ETC or HVJ were suspended in Solution A at pH 7.4. DNP dissolved in the same solution was adjusted to pH 7.4 with sodium hydroxide. One ml of 10 per cent ETC, 0.5 ml of 16,000 HAU/ml of native HVJ and 0.3 ml of various concentrations of D&I- were mixed in test tubes at 4°C. After 20 min incubation at 4X, the test tubes were incubated further at 37°C for 60 min with shaking. L\s indicated in Fig. 4, the fusion index of the non-DNP control was 6.3 and with the increase in DNP concentration the fusion reaction was inhibited and at a final DNP concentration of 2.1 X 1O-4 M or more the fusion reaction scarcely occurred. At 1.1 x 1W4 JI or less of DNP concentration there was no increase in cell degeneration as compared to the non-virus controls, but at the 2.1 x lo-” M or higher concentration of DNP a significant cell degeneration, up to 100 per cent, occurred with the increase in DSP concentration in the test samples compared to the controls. Compared with the data in the previous section, the 1 .l x 1O-4 M DNP sample appears to provide conditions similar to those of l-3 per cent air content shown in Fig. 3 as regards the fusion reaction. Experimental
Cell Research 26
124
Yoshio Okada
In the gas exchange experiments, the cell degeneration ratio was 92 per cent in the case of sonically treated virus, and that of native virus was 19 per cent under anaerobic conditions. It must be noted 100 per cent cell degeneration could be attained using native virus in DXP experiments. It might be that such a high degree of blocking in the energy supply of the cells by 2.1 x 10~~ 3/ or more DNP concentration could never be established by gas exchange. Generally speaking, the cell surface or cell membrane substances \\-ould have a quick turn-over rate under optimal conditions and some energy supplied from the cell would be required to maintain its several functions. By the reaction \vith the H\‘.J virus, some changes \vould be caused in the cell, on the surface or inside. The energy supplying system would then be intact even after the reaction xvith the virus, some of the changes may be repaired, the cell surface would be maintained normally and cell fusion would occur. But when the system is blocked the changes may initiate disorders in the semi-permeability or some other property of the cell surface and lead to cell degeneration. The sonically treated or freeze-thawed HVJ are more potent in this cytolytic activity. pH dependence of the fusion reclction.--ETC and HVJ were suspended in Solution n. One ml of HVJ and 1 ml of ETC suspensions were mixed in test tubes at 4°C and 1000 ml of air, containing CO, at concentrations of 30, 9, ti, 3, 1, and 0 per cent, were introduced into the tubes separately at 4°C. Then the test tubes were incubated at 37°C for 60 min with shaking. The starting pH of each tubes was 6.0, 6.45, 6.7, 7.0, 7.4 and 7.8 at 37°C respectively and scarcely changed during the incubation. As indicated in Fig. 5, the fusion reaction shows a remarkable pH dependence and the optimal pH range is 7.3 to 7.6. The pH range permitting the fusion reaction is very narrow compared to the case of the hemolysis in the fowl red cell-H\‘.1 system [3]. Outside the optimal pH range, the fusion efficiency decreased markedly. At pH 6.0 fused cells do not appear and some single, mononuclear cells in the samples are degenerated and become stainable with trypan blue. At this pH there was no increase in degenerated cell number in the virus-free control. It is very suggestive that the optimal pH range for the fusion reaction and the optimal pH for usual mammalian cells are common. Tempernture dependence of the fkion reaction.--ETC and H\‘.J \vere suspended in Solution A. One ml of 10 per cent suspension of ETC (about 107 cells/ml) and an equal volume of 8000 HAU/ml of HVJ were mixed in two series of test tubes and incubated at 4°C for 20 min. One series was then placed at 23°C and the other at 37°C and both were incubated \vith shaking. Experimenlcd
Cell Hesearch 26
Analysis
of fused cell formafion
caused by HVJ.
III
Findings during the course of incubation in the first and second cases are quite different. In the case of incubation at 23°C the efficiency of the fusion is very low compared to that in the 37°C incubation, even after 1.50 min incubation (Table I). But this fact does not necessarily mean the delay in the reaction rate at lowered temperature because no agglutinated cells are found in the sample after the incubation, but suggests that the virus particles adsorbed ‘I’.-\RI,P: I. Incubation temperature,
“C
Temperature Incubation time, min
dependence
Sample
of f&ion Fusion index
reaction. ?A of degenerated cells
23
150
control test
0.0 2.0
5.2 6.3
37
60
control test
0.0 6.3
2.6 2.5
onto the cells during the incubation at 4°C had already disappeared from the cell surface in 150 min incubation at 23°C and the major part of the cell aggregates formed at 4°C had dispersed again into single cells and did not produce the fused cells at 23°C. The cell metabolism may be suppressed at 23°C compared to that at 37°C but the enzymatic activity of the virus should develop at this temperature, though it is suppressed to a certain extent owing to the temperature constant. This consideration leads the author to the conclus?on that the fusion reaction does not consist only of the enzymatic activity of the virus, but that some unknown activities of the cells also play a part in the reaction.
DISCUSSION
Surveying all findings described in the reports of this series, the author is inclined to the conclusion that the fusion reaction is initiated not only by the activity of the virus, but that the metabolic activity of the participant cells is also essential. Oxygen uptake rate is elevated during the fusion reaction, especially during the first .5 min, and this fact should be compared with the morphological observation in electron micrographs reported in the first paper of this series, that the virus particles once adsorbed onto the cells disappear from the cell surfaces within 5 min from the onset of the incubation Experimental
Cell Research 26
Yoshio Okada at 37°C. Moreover, the supplementary experiments, in which the cell-virus mixtures were shaken at 37°C for the desired period of time and then kept stationary, indicated that the first 5 min shaking determines the cells to the development of giant cell formation. These findings suggest that the most important determination is established in just the early stage of the 37°C incubation, within 5 min from the onset. The necessity of shaking at 37°C for the fusion reaction, and the effects of aerobic and anaerobic conditions on the reaction, clearly indicate the requirement of oxygen for the fusion reaction. Further, the difference in the fusion efficiency in the presence or absence of 2,4-dinitrophenol also shows the necessity of energy for the development of the fusion reaction. The question whether the energy from glycolysis can be used for the reaction cannot yet be tested because of technical difficulties in preventing a lowering of the pH of the surrounding medium due to glycolysis. While these are the factors which have rather direct effects on the reaction, the fact that the optimal pH range for the reaction is narrow but is the same as that of mammalian cells in general, and the fact that the fusion efficiency at 23°C is far lower than that at 37X, support the existence of positive correlation of the metabolic state of the cells in the fusion reaction. The author considers that the oxygen consumed excessively could be used as energy for the fusion reaction. Moore et rrl. [4] and Prince and Ginsberg [7j reported that if a ETCNDV mixture is incubated at 37°C in uifro and then implanted in the abdominal cavity of a mouse, the ETC degenerates in 24 hr owing to the lethal activity of ND\‘. But n-hen a ETC-NDV mixture is incubated at 21 “C for several hours and then implanted in the abdominal cavity of a mouse, the ETC stays intact and shows no trace of the lethal activity of the virus. It is interesting that the direction of the reaction caused by the lethal activity of NDV is determined in vitro. These authors only by the preliminary incubation temperature suggest that the killing of the cells by the virus develops after the completion of a kind of stable union between Ehrlich’s tumor cell and NDV and this stable union is established only at 37°C and not at 21°C. The phenomenon, however, may be interpreted as follows. The cell degeneration is initiated only by the ND\’ infection in this case and the virus particles adsorbed onto the cell surface must be introduced into the cell by so-called viropecsis for the establishment of the infection. The cell surface activity is high at 37°C but low at 21 “C. Therefore the virus particles cannot be introduced into the cells even after a prolonged incubation time at 2 1 “C, and in this case the virus particles once adsorbed onto the cells are eventually released from the cell surface by the receptor-destroying enzyme or by changes in the physical Experimental
Cell Research 26
Analysis of fused cell lormation caused by HVJ. III condition of the cell surface. This virus liberation frees the cells from the lethal activity of the virus. Analogous to this consideration, the number of H\‘J virus particles introduced into ETC during the incubation period may be less at 23°C than at 37°C. As described in the preceding report, the fusion reaction seems to be due to a direct action between adsorbed virus particle and the cell surfaces. There would, however, still remain a possibility that only virus particles taken into a cell or infecting particles initiate the fusion reaction. It must be mentioned that the cell degeneration through killing by ND\’ reported by Moore et crl. and Prince and Ginsberg is different from the cell degeneration by HVJ described in this report. Whereas, in the former case, cell degeneration occurs in 24 hr in vivo and only one virus particle is required for one cell, in the latter case cell degeneration is completed within ii0 min at 37°C in vitro and requires the same order of high concentration virus as the face of the fusion reaction. Fusion activity of the native virus is stronger than that of sonically treated H\‘,J but the cell degenerating activity of the former under certain conditions is weaker than that of the latter. It seems that fusion activity of HVJ is different from cell degenerating activity. Hemolytic activity of H\‘J is strongl? stimulated by sonic vibration or freeze-thawing treatments. Hemolysis in the H1’dFfo\\-l red cell system is completed at 37°C within 60 min and the degree of hemolysis is determined by the virus concentration used. Metabolic systems or enzyme systems in the red cell are very limited compared with other cells. E’I’C is degenerated at 37°C within 60 min by H\‘.J, especially sonically treated H\‘J, under certain special conditions, such as anaerobiosis, absence of shaking or presence of DNP. The cell metabolism ma? be limited under these conditions. Cell degeneration by H1’,J is similar to hemolysis by the virus. The degeneration of the cell by HVJ is recovered n-hen suitable conditions are available. Under conditions medial to cell fusion and cell degeneration, cells neither fuse nor degenerate. It may be that the cells can protect themselves against the cell degenerating activity of HV.J but cannot have the system for fusion. The assumption that cell degeneration could be recovered only by the cell fusion and if the cell fusion does not occur the cells must degenerate, is not correct. Cell fusion reaction is independent of cell degeneration. Cell degeneration by HVJ could be rather compared with the lpsis from without in bacteriophage-bacteria systems.
Experimenfal
Cell Research 26
128
Yoshio Okada SUMMARY
Aerobic conditions are essential for polynuclear giant cell formation of ETC by HVJ virus. Optimum pH range of the reaction is 7.3 to 7.6 and at low temperatures, such as 23”C, the fusion efficiency decreases. When an HVJ-ETC mixture is incubated without shaking or under anaerobic conditions or in the presence of DNP at 37°C for 60 min, cells agglutinated by HVJ disaggregate freely and cell degeneration appears. From these results, it seems that the fusion reaction is initiated not only by the activity of the virus, but the metabolic activity of the participant cells is also essential. Cell degenerating activity of HVJ is independent from its fusion activity, and it may have a close relationship with its hemolytic activity. There is, however, a difference between the hemolytic action and the cell degenerating action; under suitable conditions, cell degeneration is repaired by the cell itself and only under conditions of metabolic inhibition does the reaction occur. In contrast, hemolysis is completed under all conditions studied. The author wishes to express his thanks to Professor Konosuke Fukai for his interest. Thanks are also expressed to Dr. S. Kano for supplying some pure chemical agents for this study, and to Dr. J. Tadokoro and Miss K. Kawai for generous assistance in carrying out the experiments. Dr. E. D. Hanson kindly revised the English. The work was supported by a Grant in Aid for the Waksman Foundation of Japan INC. REFERENCES CHU, L. W. and MORGAN, H. R., J. Exptl. Med. 91, 393 (1950). GRANOFF, A. and HENLE, W., J. Immunol. 72, 318 (1954). HOSAKA, Y., Biken’s J. 1, 70 (1958). MOORE, A. E., DIAMOND, C., MACKEY, H. H. and SAB~CHEWSKY, L., Proc. 81, 498 (1952). 5. OKADA, Y., Biken’s .J. 1, 103 (1958). 6. OKADA, Y. and TADOKORO, J., Erptl. Cell Research 26, 108 (1962). 7. PRINCE, A. M. and GINSBERG, H. S., -7. Exptl. Med. 79, 107 (1957).
1. 2. 3. 4.
Experimental
Cell Research 26
Sot. Ezpfl.
Viol.
Med.
119
Cell Research 26, 119-128 (19G2)
ANALYSIS
OF GIANT
CAUSED
POLYNUCLEAR
BY HVJ ASCITES
VIRUS TUMOR
CELL FORMATION
FROM
EHRLICH’S
CELLS
III. RELATIONSHIP BETVVEEN CELL CONDITION AND FUSIOS REACTIOn’ OR CELL DEGENERATION REACTION YOSHIO Department
OKADA
of Preventive Medicine, Research Institute for Microbial Osaka University, Osaka, Japan
Diseases,
Received May 29, 1961
As
described in a previous report [j 1, the cell fusion reaction is initiated in z~itro by the activity of HVJ when the cells are incubated with shaking at 37°C. Without shaking, the efficiency of the fusion reaction is very low and marked degeneration of the cells occurs. The necessity of shaking and some other features of the fusion reaction are discussed in this report. MATERIALS
AND
METHODS
Virus.-Egg adapted Z strain of HVJ was purified and concentrated as described in the previous report by differential centrifugation. Purified HVJ was resuspended in glucose and bicarbonate-free Hanks’ solution buffered with M/i00 phosphate (Solution A), or in Hanks’ solution with 0.003 M bicarbonate without glucose (Solution B). Cell.--Washed Ehrlich’s tumor cells (ETC) were resuspended in both types of Hank’s solution given above. Cell count.-Immediately after adding and mixing one drop of 2 per cent trypan blue the cell sample was introduced into a Fuchs-Rosenthal cytometer and photographed with a phase contrast microscope at low magnification. The numbers of degenerated cells stained and viable cells remaining colorless were counted from the photographs. The fusion index [S] was calculated with the formula. Cell number in non-virus control tube Fusion index = ___ __ - 1.0. Cell number in test tube The rate of the degenerated cells was expressed as fractions in per cent of the original cell number, because (1) cell number decreases during fusion reaction, (2) under suitable conditions there is no increase of cell degeneration during the reaction, and (3) almost all degenerated cells are mononucleated cells. Experimental
Cell Research 26
120
Yoshio Okada EXPERIMENTALS
Oxygen containing Warburg HAU/ml placed in the tumor
uptrrke during the fusion reaction.--One ml of tumor cell suspension 2.2 X 107 cells/ml was introduced into the main chamber of a flask. Then 0.5 ml sample of HVJ at a concentration of 16,000 was placed in the side arm, and 0.2 ml of 20 per cent KOH was the center well containing a piece of filter paper. In this experiment cells and H\‘J were suspended in Solution A.
4.c
0.0
Fig. 1. Fig. l.-Oxygen no-virus control
uptake sample.
Fig. 2. during
the fusion
reaction.
0 -0,
virus
added test sample;
A-
A
Fig. 2.-Shaking effect on the fusion reaction. 0 - 0, fusion index of samples with shaking; 0 - - 0, fusion index of samples without shaking; A - A , % of degenerated cells of samples with shaking; A - - A , % of degenerated cells of samples without shaking.
After preincubation of 3i”C for 15 min, the HVJ was transferred into the main chamber and then the oxygen uptake was determined manometrically for 60 min. As controls, 0.5 ml of the suspending medium in place of HVJ, or 1 ml of the same medium in place of the tumor cell suspension were used. As indicated in Fig. 1, the oxygen uptake of the cell-virus mixture n-as slightly higher than that of the tumor cell control; the HV.J control sample Experimental
Cell Research 26
121
Analysis of fused cell formation caused by HVJ. II1
showed no oxygen uptake. This elevation in the rate of oxygen uptake is rather more marked in the first 5 min than that in the later stage. Though the difference between the test and control samples is slight, in repeated experiments the difference \vas consistent. As described previously, after addition of HVJ the cells promptly agglutinate and within 5 min opposed cells in the aggregates start fusing and the reaction is completed in 30 min. At this stage of the reaction the cell number of the test sample decreased to about 23 per cent of the control, as the fusion index of this sample was 3.3, and as the result the total cell surface area of the test sample becomes smaller than that of the control. Despite this reduction, oxygen uptake is slightly elevated especially in the early stage of the fusion reaction. It seems the fusion reaction requires some extra energy. Seressity of shnkiny for the fusion reaction.-As reported previously, the shaking of ETC-H\‘.J mixture is essential for the formation of typical fused cells in vitro [s]. Without shaking, the appearance of fused cells was greatl) decreased. When H\‘.J treated with sonic vibration or freeze-thawing was used, more significant differences appeared between cells which were shaken and those which were not. The hemolytic activity of H\‘.J is increased with these treatments [3], as that of mumps virus [l] or NDV [‘LI, but the fusion activity decreases [(;I. In Fig. 2, a case of the fusion reaction using sonically treated HVJ is shown. The fusion activity of this virus preparation gave a fusion index of 4.3 while the original native virus showed a fusion index of 7.9 under the same conditions. In this experiment the effect of shaking is prominent and by the incubation at 37°C for 60 min with shaking the fusion reaction occurred in proportion to the virus titer and no specifically degenerated cell appeared. On the other hand, using the same preparation of the virus, when the cell-rirus mixture was incubated without shaking the fusion reaction did not occur, while cell degeneration took place roughly proportional to the virus tiiers. As sholvn in the preceding paper, the fusion activity of H\‘J is different from the other characteristics of the virus and it seems also that the fusion reaction which occurs with shaking is different from the cell degeneration reaction which develops without shaking. Cell degeneration occurred typically \vhen a high concentration of ISTC and sonically treated virus were used. \Yhen native H\‘.J \vas used, cell degeneration was only slight in the absence of shaking. Effect of rrerntion 0~1 the fusion rerrction-In these experiments, ETC and HVJ samples \vere used suspended in Solution 13. Warburg flasks with 1 ml of 20 per cent ETC (about 2 x 107 cells/ml) in the main chamber and 0.5 ml Erperimenful
Cell Research
26
122
Yoshio Okada
of 16,000 HAU/ml of HVJ in the side arm were flowed at 4°C with 1000 ml of the desired gas mixtures containing 1 per cent CO,, O-99 per cent air and the remainder iYz. After gas exchange, HVJ samples were tipped into the main chamber of the flasks, kept for 20 min at 4°C and then incubated at 37°C for 60 min with shaking. 5.0 4.0 g 2 3.0
60 e
5 'Z .: 2.0
40 &
1.0
20 &
0 : ’
0
0.0 3
5
10 Air content.
20
Fig. 3.-Aeration effect on the fusion reaction and the cell degeneration reaction. 0 -0, fusion index when native HVJ was used; 0 ~- 0 , fusion index when sonicated HVJ was used; *-., % of degenerated cells when native HVJ was used; A -- a , % of degenerated cells when sonicated HVJ was used.
99
per cent
As shown in Fig. 3, using O-l per cent air, no fused cells appeared and a slight cell degeneration occurred. With an increased amount of air, a strong fusion reaction occurred and the development of the reaction reached a plateau with more than 20 per cent air. Cell degeneration did not take place when the air content was 5 per cent or more. It seems that the fusion activity of HVJ causes the fusion of the cells under aerobic conditions but under anaerobic conditions the cell fusion reaction does not occur and degeneration of the cells results. When sonically treated HVJ was used in place of native HVJ the air effect was similar to that of native HVJ, but the maximum fusion index (at air content of 20 per cent or more) was very low, compared to that of the native HVJ. In this case, a marked cell degeneration occurred in low air content, and 92 per cent of the cells were degenerated under anaerobic conditions. This fact also indicates the independence of the fusion activity of HVJ from the cell degenerating activity of the virus which can proceed under anaerobic conditions and may be closely related with the hemolytic activity. The above data would indicate that oxygen is essential for the fusion reaction in glucose free medium. Fused cell formation under anaerobic conditions in a medium containing glucose has not yet succeeded because the pH of the medium dropped markedly during incubation at 37°C. Thus the statement in the previous report [S] that aeration was not important for the fusion reaction must be discarded. Experimental
Cell Research 26
Analysis
of fused cell formation
caused by HVJ.
123
III
The effect of’ 2,kiinitrophenol (DLX’P) on the fusion reaction.-From the above observations it may be concluded that the fusion reaction is a process which requires a specific expenditure of energy. When the effect of DNP is studied, the results seem to support this conclusion.
-100
0
0.5 1.1 2.1 4.2 8.5 Final concentration of DNP (X 10-h M)
6.0
17
0
7.0 PH
Fig. 4.
Fig. 5.
Fig. 4.-Effect of 2,4-dinitrophenol on the fusion reaction. O-O, fusion index of test sample; A - A , degenerated cells in test sample; a - - a , degenerated cells in no-virus control sample. Fig. 5.-pH
dependence
of the fusion reaction.
0-
0, fusion index;
A-
A , degenerated
cells.
In the experiment, ETC or HVJ were suspended in Solution A at pH 7.4. DNP dissolved in the same solution was adjusted to pH 7.4 with sodium hydroxide. One ml of 10 per cent ETC, 0.5 ml of 16,000 HAU/ml of native HVJ and 0.3 ml of various concentrations of D&I- were mixed in test tubes at 4°C. After 20 min incubation at 4X, the test tubes were incubated further at 37°C for 60 min with shaking. L\s indicated in Fig. 4, the fusion index of the non-DNP control was 6.3 and with the increase in DNP concentration the fusion reaction was inhibited and at a final DNP concentration of 2.1 X 1O-4 M or more the fusion reaction scarcely occurred. At 1.1 x 1W4 JI or less of DNP concentration there was no increase in cell degeneration as compared to the non-virus controls, but at the 2.1 x lo-” M or higher concentration of DNP a significant cell degeneration, up to 100 per cent, occurred with the increase in DSP concentration in the test samples compared to the controls. Compared with the data in the previous section, the 1 .l x 1O-4 M DNP sample appears to provide conditions similar to those of l-3 per cent air content shown in Fig. 3 as regards the fusion reaction. Experimental
Cell Research 26
124
Yoshio Okada
In the gas exchange experiments, the cell degeneration ratio was 92 per cent in the case of sonically treated virus, and that of native virus was 19 per cent under anaerobic conditions. It must be noted 100 per cent cell degeneration could be attained using native virus in DXP experiments. It might be that such a high degree of blocking in the energy supply of the cells by 2.1 x 10~~ 3/ or more DNP concentration could never be established by gas exchange. Generally speaking, the cell surface or cell membrane substances \\-ould have a quick turn-over rate under optimal conditions and some energy supplied from the cell would be required to maintain its several functions. By the reaction \vith the H\‘.J virus, some changes \vould be caused in the cell, on the surface or inside. The energy supplying system would then be intact even after the reaction xvith the virus, some of the changes may be repaired, the cell surface would be maintained normally and cell fusion would occur. But when the system is blocked the changes may initiate disorders in the semi-permeability or some other property of the cell surface and lead to cell degeneration. The sonically treated or freeze-thawed HVJ are more potent in this cytolytic activity. pH dependence of the fusion reclction.--ETC and HVJ were suspended in Solution n. One ml of HVJ and 1 ml of ETC suspensions were mixed in test tubes at 4°C and 1000 ml of air, containing CO, at concentrations of 30, 9, ti, 3, 1, and 0 per cent, were introduced into the tubes separately at 4°C. Then the test tubes were incubated at 37°C for 60 min with shaking. The starting pH of each tubes was 6.0, 6.45, 6.7, 7.0, 7.4 and 7.8 at 37°C respectively and scarcely changed during the incubation. As indicated in Fig. 5, the fusion reaction shows a remarkable pH dependence and the optimal pH range is 7.3 to 7.6. The pH range permitting the fusion reaction is very narrow compared to the case of the hemolysis in the fowl red cell-H\‘.1 system [3]. Outside the optimal pH range, the fusion efficiency decreased markedly. At pH 6.0 fused cells do not appear and some single, mononuclear cells in the samples are degenerated and become stainable with trypan blue. At this pH there was no increase in degenerated cell number in the virus-free control. It is very suggestive that the optimal pH range for the fusion reaction and the optimal pH for usual mammalian cells are common. Tempernture dependence of the fkion reaction.--ETC and H\‘.J \vere suspended in Solution A. One ml of 10 per cent suspension of ETC (about 107 cells/ml) and an equal volume of 8000 HAU/ml of HVJ were mixed in two series of test tubes and incubated at 4°C for 20 min. One series was then placed at 23°C and the other at 37°C and both were incubated \vith shaking. Experimenlcd
Cell Hesearch 26
Analysis
of fused cell formafion
caused by HVJ.
III
Findings during the course of incubation in the first and second cases are quite different. In the case of incubation at 23°C the efficiency of the fusion is very low compared to that in the 37°C incubation, even after 1.50 min incubation (Table I). But this fact does not necessarily mean the delay in the reaction rate at lowered temperature because no agglutinated cells are found in the sample after the incubation, but suggests that the virus particles adsorbed ‘I’.-\RI,P: I. Incubation temperature,
“C
Temperature Incubation time, min
dependence
Sample
of f&ion Fusion index
reaction. ?A of degenerated cells
23
150
control test
0.0 2.0
5.2 6.3
37
60
control test
0.0 6.3
2.6 2.5
onto the cells during the incubation at 4°C had already disappeared from the cell surface in 150 min incubation at 23°C and the major part of the cell aggregates formed at 4°C had dispersed again into single cells and did not produce the fused cells at 23°C. The cell metabolism may be suppressed at 23°C compared to that at 37°C but the enzymatic activity of the virus should develop at this temperature, though it is suppressed to a certain extent owing to the temperature constant. This consideration leads the author to the conclus?on that the fusion reaction does not consist only of the enzymatic activity of the virus, but that some unknown activities of the cells also play a part in the reaction.
DISCUSSION
Surveying all findings described in the reports of this series, the author is inclined to the conclusion that the fusion reaction is initiated not only by the activity of the virus, but that the metabolic activity of the participant cells is also essential. Oxygen uptake rate is elevated during the fusion reaction, especially during the first .5 min, and this fact should be compared with the morphological observation in electron micrographs reported in the first paper of this series, that the virus particles once adsorbed onto the cells disappear from the cell surfaces within 5 min from the onset of the incubation Experimental
Cell Research 26
Yoshio Okada at 37°C. Moreover, the supplementary experiments, in which the cell-virus mixtures were shaken at 37°C for the desired period of time and then kept stationary, indicated that the first 5 min shaking determines the cells to the development of giant cell formation. These findings suggest that the most important determination is established in just the early stage of the 37°C incubation, within 5 min from the onset. The necessity of shaking at 37°C for the fusion reaction, and the effects of aerobic and anaerobic conditions on the reaction, clearly indicate the requirement of oxygen for the fusion reaction. Further, the difference in the fusion efficiency in the presence or absence of 2,4-dinitrophenol also shows the necessity of energy for the development of the fusion reaction. The question whether the energy from glycolysis can be used for the reaction cannot yet be tested because of technical difficulties in preventing a lowering of the pH of the surrounding medium due to glycolysis. While these are the factors which have rather direct effects on the reaction, the fact that the optimal pH range for the reaction is narrow but is the same as that of mammalian cells in general, and the fact that the fusion efficiency at 23°C is far lower than that at 37X, support the existence of positive correlation of the metabolic state of the cells in the fusion reaction. The author considers that the oxygen consumed excessively could be used as energy for the fusion reaction. Moore et rrl. [4] and Prince and Ginsberg [7j reported that if a ETCNDV mixture is incubated at 37°C in uifro and then implanted in the abdominal cavity of a mouse, the ETC degenerates in 24 hr owing to the lethal activity of ND\‘. But n-hen a ETC-NDV mixture is incubated at 21 “C for several hours and then implanted in the abdominal cavity of a mouse, the ETC stays intact and shows no trace of the lethal activity of the virus. It is interesting that the direction of the reaction caused by the lethal activity of NDV is determined in vitro. These authors only by the preliminary incubation temperature suggest that the killing of the cells by the virus develops after the completion of a kind of stable union between Ehrlich’s tumor cell and NDV and this stable union is established only at 37°C and not at 21°C. The phenomenon, however, may be interpreted as follows. The cell degeneration is initiated only by the ND\’ infection in this case and the virus particles adsorbed onto the cell surface must be introduced into the cell by so-called viropecsis for the establishment of the infection. The cell surface activity is high at 37°C but low at 21 “C. Therefore the virus particles cannot be introduced into the cells even after a prolonged incubation time at 2 1 “C, and in this case the virus particles once adsorbed onto the cells are eventually released from the cell surface by the receptor-destroying enzyme or by changes in the physical Experimental
Cell Research 26
Analysis of fused cell lormation caused by HVJ. III condition of the cell surface. This virus liberation frees the cells from the lethal activity of the virus. Analogous to this consideration, the number of H\‘J virus particles introduced into ETC during the incubation period may be less at 23°C than at 37°C. As described in the preceding report, the fusion reaction seems to be due to a direct action between adsorbed virus particle and the cell surfaces. There would, however, still remain a possibility that only virus particles taken into a cell or infecting particles initiate the fusion reaction. It must be mentioned that the cell degeneration through killing by ND\’ reported by Moore et crl. and Prince and Ginsberg is different from the cell degeneration by HVJ described in this report. Whereas, in the former case, cell degeneration occurs in 24 hr in vivo and only one virus particle is required for one cell, in the latter case cell degeneration is completed within ii0 min at 37°C in vitro and requires the same order of high concentration virus as the face of the fusion reaction. Fusion activity of the native virus is stronger than that of sonically treated H\‘,J but the cell degenerating activity of the former under certain conditions is weaker than that of the latter. It seems that fusion activity of HVJ is different from cell degenerating activity. Hemolytic activity of H\‘J is strongl? stimulated by sonic vibration or freeze-thawing treatments. Hemolysis in the H1’dFfo\\-l red cell system is completed at 37°C within 60 min and the degree of hemolysis is determined by the virus concentration used. Metabolic systems or enzyme systems in the red cell are very limited compared with other cells. E’I’C is degenerated at 37°C within 60 min by H\‘.J, especially sonically treated H\‘J, under certain special conditions, such as anaerobiosis, absence of shaking or presence of DNP. The cell metabolism ma? be limited under these conditions. Cell degeneration by H1’,J is similar to hemolysis by the virus. The degeneration of the cell by HVJ is recovered n-hen suitable conditions are available. Under conditions medial to cell fusion and cell degeneration, cells neither fuse nor degenerate. It may be that the cells can protect themselves against the cell degenerating activity of HV.J but cannot have the system for fusion. The assumption that cell degeneration could be recovered only by the cell fusion and if the cell fusion does not occur the cells must degenerate, is not correct. Cell fusion reaction is independent of cell degeneration. Cell degeneration by HVJ could be rather compared with the lpsis from without in bacteriophage-bacteria systems.
Experimenfal
Cell Research 26
128
Yoshio Okada SUMMARY
Aerobic conditions are essential for polynuclear giant cell formation of ETC by HVJ virus. Optimum pH range of the reaction is 7.3 to 7.6 and at low temperatures, such as 23”C, the fusion efficiency decreases. When an HVJ-ETC mixture is incubated without shaking or under anaerobic conditions or in the presence of DNP at 37°C for 60 min, cells agglutinated by HVJ disaggregate freely and cell degeneration appears. From these results, it seems that the fusion reaction is initiated not only by the activity of the virus, but the metabolic activity of the participant cells is also essential. Cell degenerating activity of HVJ is independent from its fusion activity, and it may have a close relationship with its hemolytic activity. There is, however, a difference between the hemolytic action and the cell degenerating action; under suitable conditions, cell degeneration is repaired by the cell itself and only under conditions of metabolic inhibition does the reaction occur. In contrast, hemolysis is completed under all conditions studied. The author wishes to express his thanks to Professor Konosuke Fukai for his interest. Thanks are also expressed to Dr. S. Kano for supplying some pure chemical agents for this study, and to Dr. J. Tadokoro and Miss K. Kawai for generous assistance in carrying out the experiments. Dr. E. D. Hanson kindly revised the English. The work was supported by a Grant in Aid for the Waksman Foundation of Japan INC. REFERENCES CHU, L. W. and MORGAN, H. R., J. Exptl. Med. 91, 393 (1950). GRANOFF, A. and HENLE, W., J. Immunol. 72, 318 (1954). HOSAKA, Y., Biken’s J. 1, 70 (1958). MOORE, A. E., DIAMOND, C., MACKEY, H. H. and SAB~CHEWSKY, L., Proc. 81, 498 (1952). 5. OKADA, Y., Biken’s .J. 1, 103 (1958). 6. OKADA, Y. and TADOKORO, J., Erptl. Cell Research 26, 108 (1962). 7. PRINCE, A. M. and GINSBERG, H. S., -7. Exptl. Med. 79, 107 (1957).
1. 2. 3. 4.
Experimental
Cell Research 26
Sot. Ezpfl.
Viol.
Med.