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Multinucleated giant cell formation by fusion between cells of two different strains
131 MULTINUCLEATED
GIANT
BETWEEN
CELLS
FORMATION
OF TWO DIFFERENT
Y. OKADA Department
CELL
BY FUSION STRAINS
and F. MURAYAMA
of Preventive Medicine,
Research Institute for Microbial Osaka University, Osaka, Japan
Diseases,
Received April 6, 1965
M.a,lc+NANT tumor cells
and cells of established tissue culture strains are indicated to have high cell fusion capacities by HVJ [4, 51. The reaction steps in the fusion are (I) adsorption of HVJ onto the cell surface and mutual attachment of the participant cells at 4”C, (II) loosening or disconnecting of the virus adsorption sites of the cell surface structure at 37”C, (III) reconnection of the disconnected sites or fusion of the sites with those of the neighbouring cell surface at 37”C, and (IV) mixing of the inner components of the participant cells and spherical giant cell formation at 37°C [4]. It is very easy to mix two different kinds of cells in any ratio for the cell fusion reaction, since the cells are utilized in the form of suspensions. Thus, giant cell formation by fusion between different cells is considered to be possible when cells of two different strains in a mixed cell sample agglutinate together at random after the addition of HVJ at 4°C. Moreover, it was observed microscopically that red blood cells from man, mouse and fowl agglutinated together randomly with Ehrlich ascites tumor cells, or KB cells on addition of HVJ. This stimulated us to carry out experiments on giant cell formation between cells of different strains induced by HVJ. Previously, multinucleated giant cell formation between mouse ascites tumor cells of different strains was described [9]. In this paper, the distribution of demonstrable fusion of cells of different strains to form multinucleated giant cells was examined. Method.-For the differentiation of the cells of two strains, the nuclei of the cells of one strain were heavily labeled with 3H-thymidine during monolayer-culturing (incubated for 72 hr in bovine serum-YLH medium containing 1 to 5 &/ml of 3Hthymidine at 37°C). Monolayer-cultured cells were trypsinized and suspended in modified Hanks’ solution after washing, as described previously [5]. HVJ was purified by differential centrifugation and resuspended in modified Hanks’ solution [5]. Cells of two strains were mixed (I *: 107 cells/ml), and one ml of the cell sample and 0.5 ml of HVJ sample (100 to 3200 HAU/ml) were put into a test tube. After keeping the mixture for 10 min at 4”C, the tube was incubated with shaking at 37°C for 60 min. After completion of the cell fusion reaction, the sample was smeared on a slideglass, dried and fixed with methanol. It was then treated with 5 per cent PCA (perchloric acid) for 16 hr at 10°C to elute out the RNA; DNA was not liberated b> this treatment. In some cases RNAase treatment was used instead of PCA treatment. \Vhen RNA was not eluted, the B-rays from 3H were absorbed with RNA. The treated sample was dipped into autoradiographic emulsion (Kodak NTB-2 or KonishiRoku NR-Ml) and exposed for 7 days in a dark cold room. Then, after developing it 1 The present work was reported at the lith
istry,
on the 26th November,
Experimenial
Cell Research 40
1964.
meeting of the .Japan Society for Cellular
Chcm-
Cell fusion of two different
15.5
strains by HV.J
was stained with giemsa. 500 to 1000 giant cells with between two and seven nuclei were counted on the plate, determining the number of labeled and non-labeled nuclei in each giant cell. The frequency distribution of labeled nuclei in the giant cells was calculated and compared with the theoretical value for (i) cells of two strains fusing
iJ 2N
Ki
tielo
=2,
1
he’
ETC=I
14
Fig. 2.
Fig. 1.
Fig. l.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of KB cells and HeLa cells by HVJ. The starting cell sample contained KB cells and HeLa cells in the ratio of 2.1: 1, and 85.6 per cent of the KB cells had been heavily labeled with 3H-thymidine. The effect of addition of 320 HAD/ml of HVJ is shown. Theoretical value (i) indicates the case, when KB cells and HcLa cells fuse independently; theoretical value (ii) indcates the case when these two types fuse together as readily as homologous cells. I;ig. S.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of KB cells and Ehrlich ascites tumor cells. The ratio of KB cells to Ehrlich ascites tumor cells in the starting sample was 1:1.4 and 96.0 per cent of the KB cells had been labeled with 3H-thymidine. The effect of addition of 1600 HAD/ml of HVJ is shown.
independently, and for (ii) the two strains fusing together as readily cells. The theoretical value was calculated by the following formula, Frequency
distribution
_ .C,Pr(l
as homologous
--P)“-‘,
where n is the total number of nuclei in a giant cell, r the number of labeled nuclei in a giant cell, and P the ratio of number of labeled cells to total cells in the initial mixture. Experimenfal
Cell Hesearch 40
156
Y. Okada and F. Murayama
ResuZfs.-(A) As a control, labeled KB cells were mixed with non-labeled KB cells and fused by HVJ. The observed frequency distribution of labeled nucleus members in the giant cells agreed well with the theoretical value for (ii) above. This shows that this method is useful for analysis of cell fusion between cells of different strains.
Fig. 3.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of Ehrlich ascites tumor cells and mouse embryo fibroblasts. The ratio of MEF cells to ETC cells was 1.2: 1 in the starting sample, and 73.2 per cent of the MEF cells had been labeled with sH-thymidine. The effect of addition of 2560 HAU/ml of HVJ is shown.
(B) The giant cell formation between cells of different strains of human origin was determined. As indicated in Fig. 1, KB cells fused as readily with HeLa cells as homologous cells. The value observed for giant cells with two and three nuclei varied from the theoretical values for (ii), because the starting material was contaminated with cells with two and three nuclei. KB cells also fused readily with FL cells. (C) The possibility of giant cell formation between cells of human and animal origin was tested. 3H-labeled KB cells and non-labeled L cells (from mouse) were mixed and fused by HVJ. The distribution of labeled nucleus members in the giant cells agreed well with the theoretical distribution for (ii). KB cells also fused with PS cells (from pig). (D) These results show that the cells of established tissue culture strains have a kind of homologous surface structure. It was considered that the loss of specificity was due to long term culturing of these cells in vitro. They suggest the further test of whether tissue culture line cells fuse with mouse ascites tumor cells which have been Experimental
Cell Research 40
Cell fusion of two different
strains by HVJ
137
serially passaged in vivo. 3H-labeled KB cells were mixed with non-labeled Ehrlich ascites tumor cells and fused by HVJ. In this case, giant cells formed by fusion between KB cells and Ehrlich ascites tumor cells also appeared. However, when a low titer of HVJ (e.g. 400 HAU/ml) was used, labeled nuclei were dominant in all giant
Fig. 4.-Autoradiographs of giant cells formed by fusion of two different kinds of cells. (A) A giant cell from a cell sample containing 3H-thymidine labeled KB cells and non-labeled Ehrlich ascites tumor cells. Three labeled nuclei and two non-labeled nuclei are found. (B) A giant cell from a cell sample containing 3H-thymidine labeled KB cells and non-labeled PS cells. Two labeled nuclei and four non-labeled nuclei are observed.
cells. On increasing the titer, fused giant cells formed from KB and Ehrlich ascites tumor cells were more efficiently formed, as in the case with a mixture of two lines of tissue culture cells, as shown in Fig. 2. This may be because the Ehrlich ascites tumor cells require more HVJ than KB cells for cell fusion [5]. (E) To study the fusion reaction between cells with high and low fusion capacities from the same animal or different animals, Ehrlich ascites tumor cells or KB cells and mouse embryo fibroblasts were used. Mouse embryos aged 18 days were trypsinized and cultured for one week as described in the previous report [5], and labeled with 3H-thymidine during the culturing. About 73 per cent of the cells were heavily labeled. These mouse embryo fibroblasts (MEF) were mixed with nonlabeled Ehrlich ascites tumor cells (ETC) and fused by HVJ. The result is shown in Fig. 3. MEF scarcely fused with ETC and most giant cells were found to have been formed entirely from ETC, while almost all the MEF cells remained as separate cells. However, some giant cells containing many ETC-nuclei and one or two MEF-nuclei were observed in fusion. When HVJ was added to a mixture of MEF and ETC at 4X, much cell agglutination occurred and no free cells remained, and MEF formed cell clumps with ETC. However, almost all the MEF cells were excluded from the Experimental
Cell Research 40
cell fusion process during the cell fusion reaction at 37°C. MEF did not fuse with KB cells as readily as with ETC cells. Discussion.PFrom the above results, it is concluded that giant cell formation by fusion between different kinds of cells take place well between cell strains with high cell fusion capacities [5], and the giant cell formation was observed even between cells of different species of animals. As reported by Coombs et al. [I], cells of established tissue cultured strains maintain the species specificity of the original animal and this specificity is detected immunologically in the cell surface. Nevertheless, our results indicate that the cell membrane of one strain can become connected, in a form maintaining functional membrane structure, with that of a strain derived from another kind of animal as well as with that of a homologous strain. It seems that the cell surface structure of these cells lose a kind of specificity. The cells having a high cell fusion capacity may be malignant cells or long term cultured undifferentiated cells, while short term cultured cells or mature somatic cells have a very low or no cell fusion capacity [5]. Recently the authors observed that in the MEF-HVJ system, reaction steps (I), (II) and (III) of fusion are demonstrable; the cell surface is disconnected by HVJ and then becomes reconnected during incubation at 37°C but only at its own disconnecting sites. In the case of ETC or KB cells, the reconnection occurs with disconnecting sites of neighbouring cells as well as with its own disconnecting sites [3]. Hence, it is considered that some kind of specificity of the cell surface structure must exist in cells showing a very low or no cell fusion capacity, but not in malignant or undifferentiated tissue culture cells.
REFERENCES 1. COOMSS, R. Y., 3. ORADA, Y. 4. OKADA, Y.
2. OKADA, 5. ~
R. A., DANIEL, M. R., GURNER, B. W. and KELUS, A., Immunology Biken’s J. 4, 145 (1961). and MURAYAMA, li., Unpublished data. and TADOKORO, J., Ezptl Cell Res. 26, 98, 108 and 119 (1962).
ibid. 32, 417 (1963).
Experimental
Cell Research 40
4, 55 (1961).
GIANT
BETWEEN
CELLS
FORMATION
OF TWO DIFFERENT
Y. OKADA Department
CELL
BY FUSION STRAINS
and F. MURAYAMA
of Preventive Medicine,
Research Institute for Microbial Osaka University, Osaka, Japan
Diseases,
Received April 6, 1965
M.a,lc+NANT tumor cells
and cells of established tissue culture strains are indicated to have high cell fusion capacities by HVJ [4, 51. The reaction steps in the fusion are (I) adsorption of HVJ onto the cell surface and mutual attachment of the participant cells at 4”C, (II) loosening or disconnecting of the virus adsorption sites of the cell surface structure at 37”C, (III) reconnection of the disconnected sites or fusion of the sites with those of the neighbouring cell surface at 37”C, and (IV) mixing of the inner components of the participant cells and spherical giant cell formation at 37°C [4]. It is very easy to mix two different kinds of cells in any ratio for the cell fusion reaction, since the cells are utilized in the form of suspensions. Thus, giant cell formation by fusion between different cells is considered to be possible when cells of two different strains in a mixed cell sample agglutinate together at random after the addition of HVJ at 4°C. Moreover, it was observed microscopically that red blood cells from man, mouse and fowl agglutinated together randomly with Ehrlich ascites tumor cells, or KB cells on addition of HVJ. This stimulated us to carry out experiments on giant cell formation between cells of different strains induced by HVJ. Previously, multinucleated giant cell formation between mouse ascites tumor cells of different strains was described [9]. In this paper, the distribution of demonstrable fusion of cells of different strains to form multinucleated giant cells was examined. Method.-For the differentiation of the cells of two strains, the nuclei of the cells of one strain were heavily labeled with 3H-thymidine during monolayer-culturing (incubated for 72 hr in bovine serum-YLH medium containing 1 to 5 &/ml of 3Hthymidine at 37°C). Monolayer-cultured cells were trypsinized and suspended in modified Hanks’ solution after washing, as described previously [5]. HVJ was purified by differential centrifugation and resuspended in modified Hanks’ solution [5]. Cells of two strains were mixed (I *: 107 cells/ml), and one ml of the cell sample and 0.5 ml of HVJ sample (100 to 3200 HAU/ml) were put into a test tube. After keeping the mixture for 10 min at 4”C, the tube was incubated with shaking at 37°C for 60 min. After completion of the cell fusion reaction, the sample was smeared on a slideglass, dried and fixed with methanol. It was then treated with 5 per cent PCA (perchloric acid) for 16 hr at 10°C to elute out the RNA; DNA was not liberated b> this treatment. In some cases RNAase treatment was used instead of PCA treatment. \Vhen RNA was not eluted, the B-rays from 3H were absorbed with RNA. The treated sample was dipped into autoradiographic emulsion (Kodak NTB-2 or KonishiRoku NR-Ml) and exposed for 7 days in a dark cold room. Then, after developing it 1 The present work was reported at the lith
istry,
on the 26th November,
Experimenial
Cell Research 40
1964.
meeting of the .Japan Society for Cellular
Chcm-
Cell fusion of two different
15.5
strains by HV.J
was stained with giemsa. 500 to 1000 giant cells with between two and seven nuclei were counted on the plate, determining the number of labeled and non-labeled nuclei in each giant cell. The frequency distribution of labeled nuclei in the giant cells was calculated and compared with the theoretical value for (i) cells of two strains fusing
iJ 2N
Ki
tielo
=2,
1
he’
ETC=I
14
Fig. 2.
Fig. 1.
Fig. l.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of KB cells and HeLa cells by HVJ. The starting cell sample contained KB cells and HeLa cells in the ratio of 2.1: 1, and 85.6 per cent of the KB cells had been heavily labeled with 3H-thymidine. The effect of addition of 320 HAD/ml of HVJ is shown. Theoretical value (i) indicates the case, when KB cells and HcLa cells fuse independently; theoretical value (ii) indcates the case when these two types fuse together as readily as homologous cells. I;ig. S.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of KB cells and Ehrlich ascites tumor cells. The ratio of KB cells to Ehrlich ascites tumor cells in the starting sample was 1:1.4 and 96.0 per cent of the KB cells had been labeled with 3H-thymidine. The effect of addition of 1600 HAD/ml of HVJ is shown.
independently, and for (ii) the two strains fusing together as readily cells. The theoretical value was calculated by the following formula, Frequency
distribution
_ .C,Pr(l
as homologous
--P)“-‘,
where n is the total number of nuclei in a giant cell, r the number of labeled nuclei in a giant cell, and P the ratio of number of labeled cells to total cells in the initial mixture. Experimenfal
Cell Hesearch 40
156
Y. Okada and F. Murayama
ResuZfs.-(A) As a control, labeled KB cells were mixed with non-labeled KB cells and fused by HVJ. The observed frequency distribution of labeled nucleus members in the giant cells agreed well with the theoretical value for (ii) above. This shows that this method is useful for analysis of cell fusion between cells of different strains.
Fig. 3.-Distribution of giant cells having labeled and non-labeled nucleus members appearing after fusion of Ehrlich ascites tumor cells and mouse embryo fibroblasts. The ratio of MEF cells to ETC cells was 1.2: 1 in the starting sample, and 73.2 per cent of the MEF cells had been labeled with sH-thymidine. The effect of addition of 2560 HAU/ml of HVJ is shown.
(B) The giant cell formation between cells of different strains of human origin was determined. As indicated in Fig. 1, KB cells fused as readily with HeLa cells as homologous cells. The value observed for giant cells with two and three nuclei varied from the theoretical values for (ii), because the starting material was contaminated with cells with two and three nuclei. KB cells also fused readily with FL cells. (C) The possibility of giant cell formation between cells of human and animal origin was tested. 3H-labeled KB cells and non-labeled L cells (from mouse) were mixed and fused by HVJ. The distribution of labeled nucleus members in the giant cells agreed well with the theoretical distribution for (ii). KB cells also fused with PS cells (from pig). (D) These results show that the cells of established tissue culture strains have a kind of homologous surface structure. It was considered that the loss of specificity was due to long term culturing of these cells in vitro. They suggest the further test of whether tissue culture line cells fuse with mouse ascites tumor cells which have been Experimental
Cell Research 40
Cell fusion of two different
strains by HVJ
137
serially passaged in vivo. 3H-labeled KB cells were mixed with non-labeled Ehrlich ascites tumor cells and fused by HVJ. In this case, giant cells formed by fusion between KB cells and Ehrlich ascites tumor cells also appeared. However, when a low titer of HVJ (e.g. 400 HAU/ml) was used, labeled nuclei were dominant in all giant
Fig. 4.-Autoradiographs of giant cells formed by fusion of two different kinds of cells. (A) A giant cell from a cell sample containing 3H-thymidine labeled KB cells and non-labeled Ehrlich ascites tumor cells. Three labeled nuclei and two non-labeled nuclei are found. (B) A giant cell from a cell sample containing 3H-thymidine labeled KB cells and non-labeled PS cells. Two labeled nuclei and four non-labeled nuclei are observed.
cells. On increasing the titer, fused giant cells formed from KB and Ehrlich ascites tumor cells were more efficiently formed, as in the case with a mixture of two lines of tissue culture cells, as shown in Fig. 2. This may be because the Ehrlich ascites tumor cells require more HVJ than KB cells for cell fusion [5]. (E) To study the fusion reaction between cells with high and low fusion capacities from the same animal or different animals, Ehrlich ascites tumor cells or KB cells and mouse embryo fibroblasts were used. Mouse embryos aged 18 days were trypsinized and cultured for one week as described in the previous report [5], and labeled with 3H-thymidine during the culturing. About 73 per cent of the cells were heavily labeled. These mouse embryo fibroblasts (MEF) were mixed with nonlabeled Ehrlich ascites tumor cells (ETC) and fused by HVJ. The result is shown in Fig. 3. MEF scarcely fused with ETC and most giant cells were found to have been formed entirely from ETC, while almost all the MEF cells remained as separate cells. However, some giant cells containing many ETC-nuclei and one or two MEF-nuclei were observed in fusion. When HVJ was added to a mixture of MEF and ETC at 4X, much cell agglutination occurred and no free cells remained, and MEF formed cell clumps with ETC. However, almost all the MEF cells were excluded from the Experimental
Cell Research 40
cell fusion process during the cell fusion reaction at 37°C. MEF did not fuse with KB cells as readily as with ETC cells. Discussion.PFrom the above results, it is concluded that giant cell formation by fusion between different kinds of cells take place well between cell strains with high cell fusion capacities [5], and the giant cell formation was observed even between cells of different species of animals. As reported by Coombs et al. [I], cells of established tissue cultured strains maintain the species specificity of the original animal and this specificity is detected immunologically in the cell surface. Nevertheless, our results indicate that the cell membrane of one strain can become connected, in a form maintaining functional membrane structure, with that of a strain derived from another kind of animal as well as with that of a homologous strain. It seems that the cell surface structure of these cells lose a kind of specificity. The cells having a high cell fusion capacity may be malignant cells or long term cultured undifferentiated cells, while short term cultured cells or mature somatic cells have a very low or no cell fusion capacity [5]. Recently the authors observed that in the MEF-HVJ system, reaction steps (I), (II) and (III) of fusion are demonstrable; the cell surface is disconnected by HVJ and then becomes reconnected during incubation at 37°C but only at its own disconnecting sites. In the case of ETC or KB cells, the reconnection occurs with disconnecting sites of neighbouring cells as well as with its own disconnecting sites [3]. Hence, it is considered that some kind of specificity of the cell surface structure must exist in cells showing a very low or no cell fusion capacity, but not in malignant or undifferentiated tissue culture cells.
REFERENCES 1. COOMSS, R. Y., 3. ORADA, Y. 4. OKADA, Y.
2. OKADA, 5. ~
R. A., DANIEL, M. R., GURNER, B. W. and KELUS, A., Immunology Biken’s J. 4, 145 (1961). and MURAYAMA, li., Unpublished data. and TADOKORO, J., Ezptl Cell Res. 26, 98, 108 and 119 (1962).
ibid. 32, 417 (1963).
Experimental
Cell Research 40
4, 55 (1961).