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Inhibition of virus-induced fusion of ehrlich ascites tumor cells by cytochalasin B and D
Life Sciences Vol . 20, PP- 117-122, 1977 . Printed in the U .S .A .
Pergamon Press
INHIBITION OF VIRUS-INDUCED FUSION OF EHRLICH ASCITES TUMOR CELLS BY CYTOCHALASIN B AND D Akira Asano* and Yoshio Okada** * Institute for Prot@4n Research and Research Institute for Microbial Diseases , Osaka University, Osaka 565, Japan (Received in final form November 15, 1976)
SUMMARY: Cytochalasin B and D were found to inhibit HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells . Nearly complete inhibition was attained by 4 uM (2 pg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor . The inhibition was largely reversible . Since the transport of 2-deoxyglucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sug1kr transport . Instead, it was suggested that the inhibition was due to the action of the drugs on microfilm-ants . The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process . Various mammalian cells undergo fusion by the action of paramyxoviruses such as HVJ (Sendai virus) (1,2) and ATP is required for this and eome other membrane fusion phenomena (2-4) . However, the precise roles of ATP in these processes are not yet well defined . In a previous paper (5), we reported that HVJ-induced fusion of Ehrlich ascites tumor cells is stimulated by treatments that increase the intracellular level of 3',5'-cyclic AMP and suggested that ATP is required for this cyclic AMP-dependent step -of the fusion process . In this communication, we present circumstantial evidence that microfilaments are involved in the virusinduced cell fusion . Since the function of microfilaments depends on ATP (6), this intracellular structure seems to be another site of ATP action in the fusion process . Materials and Methods Cytochalasin B was-obtained from Imperial Chemical Industries, Ltd., and cytochalasin D was a generous gift from Shionogi Co ., Osaka. Ehrlich ascites tumor cells were grown in ddo mice, har vested, and washed as described (5) . The washed cells were suspended in a balanced salt solution (BSS) consisting of 0 .14 M NaCl, 54 mM RC1, 0.34 mM Na 2HPO , 0 .44 mM RH PO , and 10 mm Trio-HC1 buffer (pH 7 .6) . HVJ, Z train, was cglltfvated in embryonated eggs, purified, and suspended in BSS as described previously (5) .
11 8
Cytochalasin Inhibition of Cell Fusion
vol . 20, No . 1, 1977
The dose of the virus was expressed in terms of its hemagglutination units (HAU), which was determined by Salk's pattern method (7) . Cell fusion was measured as follows . A desired dose of HVJ was mixed with Ehrlich ascites cells suspended in BSS containing 2 mM CaCl (BSS-Ca2+) at 0° to make the final volume of 1 ml . The mixture wis incubated at 0° for 15 min to complete the virusmediated aggregation of the cells . The fusion reaction was then started by raising the temperature to 37° and stopped after 15 min incubation by cooling the mixture rapidly in ice . The number of cells was counted before and after the experiment by the method of Tadokoro and Okada (8) . Since the decrease in cell number is equivalent to the frequency of cell fusion, this was taken as a The effects of cytochalasin B measure of the fusion frequency . and D on fusion frequency were studied by including the drugs dissolved in dimethyl sulfoxide (DMSO) in the reaction mixture . Control experiments in which the same amount of DMSO was added were also performed . For determination of intracellular ATP, a cell suspension was The mixture was neumixed with an equal volume of 10 % HC104 . tralized with 3 M K2CO3 and then analyzed for ATP by the-firefly luciferase method (9) as described by Asano et al . (10) . Cyclic AMP was determined by the competitive binding method as described previously (5) . For measurement of 2-deoxyglucose transport, 0 .2 ml of a 5 8 suspension of the tumor cells in BSS-Ca2+ was preincubated at 0° for 10 min with the inhibitor or the same volume of the solvent (DMSO or ethanol) . The system was brought to 30° and 0 .2 pmole of [1- 3H)2-deoxyglucose (0 .2 juCi, Radiochemical Centre, Amersham, England) was added . The reaction was stopped 0, 2, 4, and 6 min after the addition of deoxyglucose by adding 20 vol . of the cold medium . The cells were collected on a Toyo GA100 glass-fiber filter overlaid with a DP-70 prefilter and washed two times with 5 ml each of the cold medium= the filtration and washing were completed within 1 min . The cells on the filter were then counted in a Beckman LS-250 scintillation spectrometer by using POP-to&QLene-Triton X-100 (4 g/1,000m1/500 ml) . Results and Discussion The requirement of intracellular ATP for HVJ-induced fusion of Ehrlich ascites tumor cells was further confirmed by using reagents that decrease intracellular ATP by different mechanisms . Thus, in addition to anaerobiosis and 2,4-dinitrophenol reported in a previous paper (3), g-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, an uncoupler of oxidative phosphorylation), valinomysin (an ionophore), and 2-deoxyglucose (a substrate of hexokinase leading to accumulation of a non-metabolizable sugar phosphate) were all effective in depleting the ATP level and in inhibiting the fusion reaction almost completely . Rutamycin (an energy transfer inhibitor) and rotenone (an electron transfer inhibitor) were partially effective in both respects . It is now established that microfilaments are involved in dynamic membrane reactions such as phagocytosis (11,12) and cap formation (13) and that ATP is required for the function of micro filaments (6) . It was, therefore, of interest to examine a possible role of microfilaments in the cell fusion reaction . As shown in Table I, the virus-induced fusion of Ehrlich ascites cells was inhibited by cytochalasin B, a drug which has been
Vol. 20, No . 1, 1977
Cytochalasin Inhibition of Call Fusion
119
TABLE I Inhibition of HVJ-Induced Cell Fusion by Cytochalasin B HVJ
Additions
_
Concentration
Cell Number in 1/80 mm 3
___
264± 7
<0 .999
__-
__-
160
<0 .99
129 -
(100)
135±13 209±19
<0 .999
_--
+
DMSO + Cytochalasin B
20 pl/ml 1 pg/ml
+
2 pg/ml
+
4 pg/ml
104±10
166±13
Decrease Fusion in Cell (% of Number Control)
P
---
0 .999
98 55
81 61 34
Fusion of Ehrlich ascites tumor cells was determined as described in "Materials and Methods", except that 2 .1 x 10 7 of the tumor cells and 500 HAU of the virus were used and DMSO which was used as solvent for cytochalasin B .was included to a final concentration of 2 8 in all experiments . Means t standard deviations were shown in 4th column . Probability of difference of the means (P) was caluculated for each set of experiments, i .e . between cell number in control experiment (line 2) ârnd either those with the indicated amount of the inhibitors (lines 3,4 and 5) or that without addition of HVJ (line 1), based on the t-test (28) . TABLE II Effects of Cytochalasin B and-D and Phlorizin on 2-Deoxyglucose Transport into Ehrlich Ascites Tumor Cells Expt .
Additions None
Concentration ---
698 cpm
1.
Cytochalasin B
Cytochalasin D
100 Pg/ml
658
2.
None Cytochalasin D
--20 pg/ml
2,500 3,205-
Phlorizin
25 pg/ml
2-Deoxyglucose Transported
2 mM
27
154
Reaction was performed as described in "Materials and Methods" except that 4 .4 x 10 6 cells in Expt . 1 and 5 .0 x 106 cells in Expt . 2 were used . DMSO was included to a final concentration of 2 .5 8 (v/v) as solvent in Expt . 1 and ethanol used as solvent was added to a final concentration of 1 .0 8 (v/v) in Expt . 2 . shown to be an inhibitor of microfilaments (14,15) . The sensitivity of the fusion reaction to cytochalasin B was comparable to that observed for the other membrane reactions (12-14) . However,
120
Cytochalasin Inhibition of Call Fusion
Vol. 20, No . 1, 1977
since cytochalasin B has also been shown to inhibit facilitated diffusion of sugars through plasma membrane (16,17), it was not possible to conclude unequivocally the involvement of microfilaments in cell fusion solely based on these results . we therefore tested the effect of cytochalasin D, which is also an inhibitor of microfilament function (18-20) and has been shown to cause no effect on sugar transport in Vero, MDBK and HEp2 cells (except for partial inhibition of the transport in HeLa cells) (19) . In this study, we confirmed that the transport of 2-deoxyglucose into Ehrlich ascites cells was actually not inhibited by this drug (Table II) . As shown in Fig . 1, on the other hand, cytochalasin D was much more inhibitory than cytochalasin B to the fusion of Ehrlich ascites cells, as is the case for the other membrane reactions (12,14) . 100
0
100
pXF
0 .01
1. 0
0 .1
10
CYTOCHALASIN (}ig/m1 ) FIG. 1 Effects of cytochalasin B and D on cell fusion and intracellular ATP level . Cell fusion was performed as descr bed in the legend of Table I, except that 1 .9 x 10 cells and 1,000 HAU of the virus were used in case of cytochalasin D treatment . After the cessation of fusion reaction by cooling, 0 .5 ml aliquot of the reaction mixture of cytochalasin D series was taken from each tube and ATP content was determined as described in "Materials and Methods" . (x), cytochalasin D, fusion ; (0 ), cytochalasin D, ATP level ; (t7), cytochalasin B, fusion . Fig. 1 also shows that cytochalasin D did not affect the ATP level in the cells, indicating that the inhibition observed was not due to depletion of intracellular ATP . As reported by Warner and Perdue (21), cytochalasin B has no effect on ATP level of cultured fibroblasts . Although cyclic AMP is stimulatory to the fusion reaction (5), the addition of cytochalasin D (10 Xg/ml) did not alter the intracellular level of the cyclic nucleotide (K . Ohki, unpublished observation) . Furthermore, phlorizin, which inhibited the 2-deoxyglucose transport (Table II), did not inhibit the cell fusion reaction (data not shown) . These observations,
Vol . 20, No . 1, 1977
Cytochalasin Inhibition of Cell Fusion
12 1
therefore, indicated that the inhibition of cell fusion by cytochalasin B and D was conceivably due to their action on microfilaments . The inhibition of cell fusion by cytochalasin D was reversible at least partially (Table III) . At a higher concentration of the drug (2 pg/ml), the recovery of the fusion activity was greater when bovine serum albumin was included in the washing medium,probably because the protein could bind cytochalasin D, as it is known to bind cytochalasin B (22) . The cell fusion reaction was usually performed after preincubation of the cells with the drug at 0° for 10 min in the presence of HVJ . However, cytochalasin D (0 .4 pg/ml) caused 48 % inhibition of the fusion even without the preincubation as compared with 60 8 inhibition attained after the preincubation, indicating that the drug could reach its target rather rapidly . TABLE III Reversibility of Inhibition by Cytochalasin D Concentration
Wash
0 .2 pg/ml
None
"
BSS+Albumin
19
2 .0 Kg/ml
None
99
"
BSS+Albumin
35
"
"
BSS
BSS
Inhibition 78 8 17
75
Preincubation of the tumor cells with cytochalasin D was performed as in the case of fusion reaction except that HVJ was omitted, then the. mixture was cooled by placing in ice . After addition of 3 ml of ice-cold BSS-Ca 2 + either not containing or containing 10 mg/ml of bovine serum albumin, cells were centrifuged down at 2,000 rpm for 10 min. The pellets were resuspended with 3 ml of the medium as indicated and recentrifuged . Centrifugation and suspending cycles were repeated twice more . Then, the cells were washed again with 6 ml of BSS-Ca 2 + to remove bovine serum albumin . The cells were finally resuspended in the original volume of BSS-Ca2+ and cell fusion was performed as described in "Materials and Methods", except that 1 .8 10 7 cells and 4,000 HAU of HVJ were used .
x
Although microfilaments have been shown to be involved in a variety of ATP-requiring membrane phenomena, their precise roles in these processes are still obscure . It is known that bare lipid bilayers can fuse each other under appropriate conditions (23-25) . The role of microfilaments in the cell fusion reaction may, therefore, be visualized .a s a regulator of the movement of cell surface glycoproteins which appear to prevent the direct contact of the lipid bilayers . Association of microfilaments (or actin) with certain membrane components such as O(-actinin (26) or actin-
122
Cytochalasin Inhibition of Cell Fusion
Vol . 20, No . 1, 1977
binding protein (27) may play a role in this connection . Further studies on the mode of action of cytochalasins and the mechanism of interaction of membrane components with cytoplasmic factors are, however, required for complete understanding of the mechanism of cell fusion . ACKNOWLEDGMENTS : We would like to thank Prof . Ryo Sato of our laboratory for his helpful discussions and critical reading of the manuscript . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 .
Y . Okada, Biken's J . 1 103-110 (1958) . G . Poste and A .G . Allison, Biochim . Biophys . Acta 300 421-465 (1973) . Y . Okada, F . Murayama and Y . Yamada, Virol . 2i_1115-: _ 30 (1966) . H . Rusmuesen, Science 170 404-412 (197057 . K . Ohki, S . Nakama, A .Asano and Y . Okada, Biochem . Biophys . Res . Commune . 67 331-337 (1975) . J. C . W . Chan and N . Kamiya, Cell Struct . Function 1 1-9 (1975) . Y . Okada and. F . Muraysma, Eja . Cell Res . 52 34-42 (1968) . Y . Okada and J . Tadokoro, Ea . Cell Res . 26 108-118 (1962) . P . E . Stanley and S . G . Williams, Anal. Biochem . 29 381-392 (1969) . A . Asano, N . S . Cohen, R . F . Baker and A . F . Brodie, _J . Biol . Chem . 248 3386-3397 (1973) . G . G . Klaus, Eue. Cell Res . 79 73-78 (1973) . N . Mimura and A. Asano,Nature 261 319-321 (1976) . S . de Ptreis, Nature 250 54-56 1974) . S . B . Carter, Nature 213 261-264 (1967) . J . A . Spudich and S . Lin, Proc . Nat . Aced . Sci . 6 9 442-446 (1972) . S . B . Mizel and L . Wilson, J . Biol. Chem . 2474102-4105 (1972) . R . D . Estensen and P . G . W .Plagemann, Proc .Nat . Acad . Sci . 69 1430-1434 (1972) . E . Puszkin, S . Puszkin, L . W . Lo and S . W . Tanenbaum, _J . Biol . Chem . 248 7754-7761 (1973) . A . F . Miranda, G . C . Godman, A . D . Dietch and S . W . Tanenbaum, _J . Cell Biol . 61 481-500 (1974) . ' A . F . Miranda, G . C . Godman and S . W . Tanenbaum, J . Cell Biol . 62 406-423 (1974) . _ D . A . Warner, and J . F . Perdue, _J . Cell Biol . _55 242-244 (1972) . S . Lin, D . V . Santi and J . A . Spudich, _J . Biol . Chem . 249 2268-2274 (1974) . C . Taupin and H . M . McConnell, "Mitochondria Biomembrane?", North-Holland, Amsterdam (1972) . D . Papahadjopoulos, G . Poste, B . E . Schaeffer and W . J . Vail, Biochim . Biophys . Acts, 352 10-28 (1974) . R . Lawaczieck, M . Kainisho, J . L . Girardet and S . I . Chan, Nature 256 584586 (1975) . E . Lazarides and K . Burridge, Cell 6 289-298 (1975) . T . P . Stossel and J . H . Hartwig, _J . Cell Biol . 68 602-619 (1976) . L . Saunders and R . Flaming, "Mathematics and Statistics", The Pharmaceutical Press, London (1971) .
Pergamon Press
INHIBITION OF VIRUS-INDUCED FUSION OF EHRLICH ASCITES TUMOR CELLS BY CYTOCHALASIN B AND D Akira Asano* and Yoshio Okada** * Institute for Prot@4n Research and Research Institute for Microbial Diseases , Osaka University, Osaka 565, Japan (Received in final form November 15, 1976)
SUMMARY: Cytochalasin B and D were found to inhibit HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells . Nearly complete inhibition was attained by 4 uM (2 pg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor . The inhibition was largely reversible . Since the transport of 2-deoxyglucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sug1kr transport . Instead, it was suggested that the inhibition was due to the action of the drugs on microfilm-ants . The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process . Various mammalian cells undergo fusion by the action of paramyxoviruses such as HVJ (Sendai virus) (1,2) and ATP is required for this and eome other membrane fusion phenomena (2-4) . However, the precise roles of ATP in these processes are not yet well defined . In a previous paper (5), we reported that HVJ-induced fusion of Ehrlich ascites tumor cells is stimulated by treatments that increase the intracellular level of 3',5'-cyclic AMP and suggested that ATP is required for this cyclic AMP-dependent step -of the fusion process . In this communication, we present circumstantial evidence that microfilaments are involved in the virusinduced cell fusion . Since the function of microfilaments depends on ATP (6), this intracellular structure seems to be another site of ATP action in the fusion process . Materials and Methods Cytochalasin B was-obtained from Imperial Chemical Industries, Ltd., and cytochalasin D was a generous gift from Shionogi Co ., Osaka. Ehrlich ascites tumor cells were grown in ddo mice, har vested, and washed as described (5) . The washed cells were suspended in a balanced salt solution (BSS) consisting of 0 .14 M NaCl, 54 mM RC1, 0.34 mM Na 2HPO , 0 .44 mM RH PO , and 10 mm Trio-HC1 buffer (pH 7 .6) . HVJ, Z train, was cglltfvated in embryonated eggs, purified, and suspended in BSS as described previously (5) .
11 8
Cytochalasin Inhibition of Cell Fusion
vol . 20, No . 1, 1977
The dose of the virus was expressed in terms of its hemagglutination units (HAU), which was determined by Salk's pattern method (7) . Cell fusion was measured as follows . A desired dose of HVJ was mixed with Ehrlich ascites cells suspended in BSS containing 2 mM CaCl (BSS-Ca2+) at 0° to make the final volume of 1 ml . The mixture wis incubated at 0° for 15 min to complete the virusmediated aggregation of the cells . The fusion reaction was then started by raising the temperature to 37° and stopped after 15 min incubation by cooling the mixture rapidly in ice . The number of cells was counted before and after the experiment by the method of Tadokoro and Okada (8) . Since the decrease in cell number is equivalent to the frequency of cell fusion, this was taken as a The effects of cytochalasin B measure of the fusion frequency . and D on fusion frequency were studied by including the drugs dissolved in dimethyl sulfoxide (DMSO) in the reaction mixture . Control experiments in which the same amount of DMSO was added were also performed . For determination of intracellular ATP, a cell suspension was The mixture was neumixed with an equal volume of 10 % HC104 . tralized with 3 M K2CO3 and then analyzed for ATP by the-firefly luciferase method (9) as described by Asano et al . (10) . Cyclic AMP was determined by the competitive binding method as described previously (5) . For measurement of 2-deoxyglucose transport, 0 .2 ml of a 5 8 suspension of the tumor cells in BSS-Ca2+ was preincubated at 0° for 10 min with the inhibitor or the same volume of the solvent (DMSO or ethanol) . The system was brought to 30° and 0 .2 pmole of [1- 3H)2-deoxyglucose (0 .2 juCi, Radiochemical Centre, Amersham, England) was added . The reaction was stopped 0, 2, 4, and 6 min after the addition of deoxyglucose by adding 20 vol . of the cold medium . The cells were collected on a Toyo GA100 glass-fiber filter overlaid with a DP-70 prefilter and washed two times with 5 ml each of the cold medium= the filtration and washing were completed within 1 min . The cells on the filter were then counted in a Beckman LS-250 scintillation spectrometer by using POP-to&QLene-Triton X-100 (4 g/1,000m1/500 ml) . Results and Discussion The requirement of intracellular ATP for HVJ-induced fusion of Ehrlich ascites tumor cells was further confirmed by using reagents that decrease intracellular ATP by different mechanisms . Thus, in addition to anaerobiosis and 2,4-dinitrophenol reported in a previous paper (3), g-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, an uncoupler of oxidative phosphorylation), valinomysin (an ionophore), and 2-deoxyglucose (a substrate of hexokinase leading to accumulation of a non-metabolizable sugar phosphate) were all effective in depleting the ATP level and in inhibiting the fusion reaction almost completely . Rutamycin (an energy transfer inhibitor) and rotenone (an electron transfer inhibitor) were partially effective in both respects . It is now established that microfilaments are involved in dynamic membrane reactions such as phagocytosis (11,12) and cap formation (13) and that ATP is required for the function of micro filaments (6) . It was, therefore, of interest to examine a possible role of microfilaments in the cell fusion reaction . As shown in Table I, the virus-induced fusion of Ehrlich ascites cells was inhibited by cytochalasin B, a drug which has been
Vol. 20, No . 1, 1977
Cytochalasin Inhibition of Call Fusion
119
TABLE I Inhibition of HVJ-Induced Cell Fusion by Cytochalasin B HVJ
Additions
_
Concentration
Cell Number in 1/80 mm 3
___
264± 7
<0 .999
__-
__-
160
<0 .99
129 -
(100)
135±13 209±19
<0 .999
_--
+
DMSO + Cytochalasin B
20 pl/ml 1 pg/ml
+
2 pg/ml
+
4 pg/ml
104±10
166±13
Decrease Fusion in Cell (% of Number Control)
P
---
0 .999
98 55
81 61 34
Fusion of Ehrlich ascites tumor cells was determined as described in "Materials and Methods", except that 2 .1 x 10 7 of the tumor cells and 500 HAU of the virus were used and DMSO which was used as solvent for cytochalasin B .was included to a final concentration of 2 8 in all experiments . Means t standard deviations were shown in 4th column . Probability of difference of the means (P) was caluculated for each set of experiments, i .e . between cell number in control experiment (line 2) ârnd either those with the indicated amount of the inhibitors (lines 3,4 and 5) or that without addition of HVJ (line 1), based on the t-test (28) . TABLE II Effects of Cytochalasin B and-D and Phlorizin on 2-Deoxyglucose Transport into Ehrlich Ascites Tumor Cells Expt .
Additions None
Concentration ---
698 cpm
1.
Cytochalasin B
Cytochalasin D
100 Pg/ml
658
2.
None Cytochalasin D
--20 pg/ml
2,500 3,205-
Phlorizin
25 pg/ml
2-Deoxyglucose Transported
2 mM
27
154
Reaction was performed as described in "Materials and Methods" except that 4 .4 x 10 6 cells in Expt . 1 and 5 .0 x 106 cells in Expt . 2 were used . DMSO was included to a final concentration of 2 .5 8 (v/v) as solvent in Expt . 1 and ethanol used as solvent was added to a final concentration of 1 .0 8 (v/v) in Expt . 2 . shown to be an inhibitor of microfilaments (14,15) . The sensitivity of the fusion reaction to cytochalasin B was comparable to that observed for the other membrane reactions (12-14) . However,
120
Cytochalasin Inhibition of Call Fusion
Vol. 20, No . 1, 1977
since cytochalasin B has also been shown to inhibit facilitated diffusion of sugars through plasma membrane (16,17), it was not possible to conclude unequivocally the involvement of microfilaments in cell fusion solely based on these results . we therefore tested the effect of cytochalasin D, which is also an inhibitor of microfilament function (18-20) and has been shown to cause no effect on sugar transport in Vero, MDBK and HEp2 cells (except for partial inhibition of the transport in HeLa cells) (19) . In this study, we confirmed that the transport of 2-deoxyglucose into Ehrlich ascites cells was actually not inhibited by this drug (Table II) . As shown in Fig . 1, on the other hand, cytochalasin D was much more inhibitory than cytochalasin B to the fusion of Ehrlich ascites cells, as is the case for the other membrane reactions (12,14) . 100
0
100
pXF
0 .01
1. 0
0 .1
10
CYTOCHALASIN (}ig/m1 ) FIG. 1 Effects of cytochalasin B and D on cell fusion and intracellular ATP level . Cell fusion was performed as descr bed in the legend of Table I, except that 1 .9 x 10 cells and 1,000 HAU of the virus were used in case of cytochalasin D treatment . After the cessation of fusion reaction by cooling, 0 .5 ml aliquot of the reaction mixture of cytochalasin D series was taken from each tube and ATP content was determined as described in "Materials and Methods" . (x), cytochalasin D, fusion ; (0 ), cytochalasin D, ATP level ; (t7), cytochalasin B, fusion . Fig. 1 also shows that cytochalasin D did not affect the ATP level in the cells, indicating that the inhibition observed was not due to depletion of intracellular ATP . As reported by Warner and Perdue (21), cytochalasin B has no effect on ATP level of cultured fibroblasts . Although cyclic AMP is stimulatory to the fusion reaction (5), the addition of cytochalasin D (10 Xg/ml) did not alter the intracellular level of the cyclic nucleotide (K . Ohki, unpublished observation) . Furthermore, phlorizin, which inhibited the 2-deoxyglucose transport (Table II), did not inhibit the cell fusion reaction (data not shown) . These observations,
Vol . 20, No . 1, 1977
Cytochalasin Inhibition of Cell Fusion
12 1
therefore, indicated that the inhibition of cell fusion by cytochalasin B and D was conceivably due to their action on microfilaments . The inhibition of cell fusion by cytochalasin D was reversible at least partially (Table III) . At a higher concentration of the drug (2 pg/ml), the recovery of the fusion activity was greater when bovine serum albumin was included in the washing medium,probably because the protein could bind cytochalasin D, as it is known to bind cytochalasin B (22) . The cell fusion reaction was usually performed after preincubation of the cells with the drug at 0° for 10 min in the presence of HVJ . However, cytochalasin D (0 .4 pg/ml) caused 48 % inhibition of the fusion even without the preincubation as compared with 60 8 inhibition attained after the preincubation, indicating that the drug could reach its target rather rapidly . TABLE III Reversibility of Inhibition by Cytochalasin D Concentration
Wash
0 .2 pg/ml
None
"
BSS+Albumin
19
2 .0 Kg/ml
None
99
"
BSS+Albumin
35
"
"
BSS
BSS
Inhibition 78 8 17
75
Preincubation of the tumor cells with cytochalasin D was performed as in the case of fusion reaction except that HVJ was omitted, then the. mixture was cooled by placing in ice . After addition of 3 ml of ice-cold BSS-Ca 2 + either not containing or containing 10 mg/ml of bovine serum albumin, cells were centrifuged down at 2,000 rpm for 10 min. The pellets were resuspended with 3 ml of the medium as indicated and recentrifuged . Centrifugation and suspending cycles were repeated twice more . Then, the cells were washed again with 6 ml of BSS-Ca 2 + to remove bovine serum albumin . The cells were finally resuspended in the original volume of BSS-Ca2+ and cell fusion was performed as described in "Materials and Methods", except that 1 .8 10 7 cells and 4,000 HAU of HVJ were used .
x
Although microfilaments have been shown to be involved in a variety of ATP-requiring membrane phenomena, their precise roles in these processes are still obscure . It is known that bare lipid bilayers can fuse each other under appropriate conditions (23-25) . The role of microfilaments in the cell fusion reaction may, therefore, be visualized .a s a regulator of the movement of cell surface glycoproteins which appear to prevent the direct contact of the lipid bilayers . Association of microfilaments (or actin) with certain membrane components such as O(-actinin (26) or actin-
122
Cytochalasin Inhibition of Cell Fusion
Vol . 20, No . 1, 1977
binding protein (27) may play a role in this connection . Further studies on the mode of action of cytochalasins and the mechanism of interaction of membrane components with cytoplasmic factors are, however, required for complete understanding of the mechanism of cell fusion . ACKNOWLEDGMENTS : We would like to thank Prof . Ryo Sato of our laboratory for his helpful discussions and critical reading of the manuscript . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 .
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