Life Sciences vol . 18, pp . Printed in the U.S .A .
1383-1390,
1976 .
Pergamoa Praee
EFFECT OF COLCHICINE ON vIRIIS-INDUCED FIISIO~i OF HUMAN ERYTHROCYTES &iyotoshi 3ekiguchi and Akira Asano Institute for Protein Research, Osaka University, Suite, Osaka, Japan (Received in final form May 4, 1976) Simamary : Colchicine was found to stimulate the virus-induced fusion of human erythrocytes . Colchicine also stimulated the rata of hemolysis, but had no effect on its final ertent, suggesting that the enhanced rate of envelope fusion, _i ._e . virus to cell, caused by colchicine resulted in the stimulation of cell to cell fusion . The fact that affective doses of colchicine were at mülimolar concentrations, together with the absence of mièrotubules in human erythrocytes, indicates that the target of colchicine action ie not this aubcellular tubular system . Instead, the peripéeral meabrane protein, spectrin, may be a likely candidate for the site of colchicine action . Colchicine, a well known antimitotic agent, has been shown to affect several cellular processes which accompany topographical changes of cell membranes, such an cell agglutination by plant lectins (1,2), phagocytosie (3,4), cap for mation (5) and secretion phenomena (6,7,8) . In these processes, the effective doses of colchicine are nicromolar concentrations, and therefore, it is believed that this type of effect of colchicine should be manifested through the disruption of nicrotubulea . On the other hand, there seems to aziet another kind of membrane phenomena which is also influenced by colchicine . The temperature dependent topographical changes of Tetrahyneaa pyriformie plasma membrane (9) and sugar transport in fat cell ghosts (10) are the latter ezanples . In these cases the effective doses of wlchicine are millimolar concentrations, suggest- . ing that another type of action mechanism rather than microtubule disruption may be involved .In the present study, we ezamined the effect of colchicine on the HvJ(Sendai virus)-induced fusion reaction of human erythrocytes which provides a suitable model system for studying the mechanise of membrane fusion and the mode of action of colchicine . MATERIALS AND METHODS HvJ, Z strain, was grown in chicken eggs, harvested, and partially purified by differental centrifugation e~deacribed previously (11), except that the purified virus was suspended in Ca -free Tricine-buffered saline (TBS ; 140 mM NaCl, 5.4 mTf RC1 sad 40 mM Trieine-NaOH, pH 7 .6) ; this medium was used throughout the fusion ezperiments unless otherwise stated . The virus dose was e~rnesed in terms of its hemagglutination unite determined by Salk'e pattern method (12) . Human red blood cells were obtained from a blood bank and used within 4 weeks after drawing . ABBREVIATIONS : HVJ, Heeagglutinating virus of Japan; Tricine, N-Trie(hydro:yeethyl)methyl glycine; TBS, Triciae-buffered saline . 1383
1384
Colchicine Stimulation of Cell Fusion
Vol. 18, No . 12
Cell fusion was assayed as follows . A desired dose of HVJ in 0 .5 ml of the medium was added~to 0 .5 ml of a cell suspension kept in an ice bath and the mixture was allowed to stand for 15 min in the cold to complete cell agglutina tion . The fusion reaction was then started by raising the temperature to 37 ° and terminated after 30 min by the addition of 1 ml of cold medium, and rapid cooling is ân ice bath . A 0.2 ml portion of mutate was diluted to 50 .0 ml with 0 .9 X NaCl solution and subjected to cell number counting is a Cooper counter (Model A, Cooper Electronics) with as aperture of 100 pm . The efficiencq of cell fusion was eapressed either as percent decrease is cell number or ae fusion index (13) . We could confirm that this new method can be satisfactorily applied to quantitative studies of erythrocyte fusion under various conditions . Possible errors, which could be introduced by insufficient dissociation of cell aggregates, were checked every time by phase contrast microscopy and found to be insignificant. If cell aggregates are not thoroughly resolved after completion of fusion reaction, degree of fusion estimated by this method should be higher than that measured by the microscopic method (13) . Contrary to expectation, fusion frequency estimated by the Cooper counter method was always slightly lower than that obtained by the orthodox microscopic method . This discrepancy may be attributed to eatenaive hemolysis caused by the virus, in other words, resultant ghosts which are very difficult to settle oa hemocytometer may not be counted by the microscopic method . Fusion of Ehrlich ascites tumor cells was measured as described previously (14) .
100
..
80
..
c~
2 W
r
60
4 3 X 2 W O Z 2
a 40 W Z 20 _O N 0 0
2
4
6
COLCH ICINE
8
10
0
(mM
FIGURE 1 Effect of colchicine on the fusion reaction of humas erythrocytes . Erythrocytée suspended in TBS containing different concentrations of colchicine were agglutinated by HVJ(400 HAU/ml) at 0 ° sad then incubated for 30 min at 37 ° . Fusion frequency was expressed either as percent decrease is original cell number or fusion index.
Vol. 18, No . 12
Colchicine Stimulation of Cell Fusion
1385
Hemolyeis was determined by measuring hemoglobin content of the supernatant of the fusion reaction at 540 nm . Oxidation of hemoglobin was checked spectrophotometrically, and all the hemoglobin content measurment was completed before spectrum change was observed . Cell agglutination was estimated as described for fusion, except that incubation at 37 ° was omitted and particle number counting in the Coulter counter was performed below 10 ° . Iodination of HVJ was performed at 4° by a chlo~~ine T method(15) . Cell agglutination, hemolysis and cell fusion activitea of I-HVJ thus prepared were not appreciably decreased from the original preparation . RESULTS When an erythrocyte suspension was incubated with the virus at 0° to 4 ° for 15 min and then at 37 ° for 30 min, the cells fused with each other producing "polyerythrocytes" . Addition of colchicine to the reaction medium resulted
100 .. 80 cA 80 N } 40 O
W
TIME(min) FIGURE 2 Effect of colchicine on hemolyeis at different temperatures . Erythrocytes were agglutinated by HVJ(960 HAII/ml) at 0° is the presence ( " ) or absence (0) of 10 mM colchicine . Resultant cell aggregates were then transferred to specified temperatures and incubated for different periods as indicated . Following the addition of 4 volumes of cold TBS, samples were iame diately centrifuged at 4° and the hemoglobin content is the supernatant was determined as described under "~TERTATR AND I~THODS" . No detectable heaalysie was observed with controls containing 10 uM colchicine but not containing HVJ under any conditions employed . is a significant stimulation of the fusion reaction, accompanied by the formation of huge "polyerythrocytes" . As shown in Fig.l, the fusion frequency was enhanced by colchicine is a concentration-dependent way; about a three fold stimulation was obtained by the addition of 10 mM colchicine . The effect of colchicine on the fusion reaction was found to be reversible, because stimulation of the reaction was undetectable when cells, which were preincubated with
1386
Colchicine Stimulation of Cell Pueion
pol. 18, No . 12
10 mM colchicine at 37 ° for 15 aia, were washed with cold TBS (unpublished data) . Similar or greater stimulation by the drug was also observed in HpJinduced fusion of Ehrlich aecitea tumor celle.(data not shown) Thus this stimulation may have general importance in the virus-induced fusion reaction . It ie well known that interaction of HpJ with erythrocytes leads to eatensive hemolytic (16) and there is evidence that the hemolytic activity of the vitae is closely correlated to its fusion-inducing activity (17) . Therefore,
25°
37°
3d°
S
:Q.~~;~~ ~ .~
r ~~ i
~, ,.~4 ~~
i
FIGIIRE 3 Effect of colchicine on the fusion reaction of human erythrocytes at different temperatures .( la and lb, 25 °C ; 2a and 2b, 30 °C ; 3a and 3b, 37°C) . Ezperimeatal conditions were the same se in Fig.2 ezcept that the incubation time was fazed for 30 mis and 10 ~ colchicine was included in experiments lb, 26 and 3b . Phase contrast microscopy . the affect of colchicine on the rate of hemolytic was studied (Fig . 2) . When cells were fused by HpJ is the praeeace of lO .mM colchicine at 37 ° , the rate of heanlysis was significantly accelerated and the initial lag phase was diminished, whereas the maximal level of hemolytic attained after 30 min incubation was almost uachaaged. This tendency was more clearly shown at lower temperatures . When the fusion was carried out without colchicine at 23 ° , the early lag phase continued for sore than 10 sin and it took more than one hour to reach the mar i~l level . The addition of colchicine markedly diminished the lag phase and enhanced the rate of hemolytie . In accordance with the stimulation of hemolytic, colchicine enhanced the formation of large fused cells . Moreover, it could
Vol . 18, No . 12
Colchicine Stimulation of Cell Fusion
1387
bring about eztenaivn call fusion even at 25 ° whereas is the absence of colchicine the cells remained is the aggregated state and no fused cells were detectable (Fig . 3) . Since the rate of henolysis might be affected by the amount of HVJ absorbed on the surface of erythrocytes and the eztent of the subsequent virus-induced cell agglutination, the effect of colchicine on these preliminary pro cesses for cell fusion were also e=a,°~npd . Making use of I-labeled HVJ, we observed that colchicine did not c~~ge the amount of HVJ absorbed to erythrocytes ( for ezample, 4,314 cpm of I-HVJ was absorbed on the cells in the presence of 10 mM colchicine, whereas 4,275 cpm of those was bound to the cells in the absence of colchicine) . The virus-induced agglutination was also unaffected by colchicinn . As can be sees in Fig . 4, the agglutination of cells was not appreciably affected by 10 mM colchicine .
Z O
a a 0 a W V Q _2 C_9 O W O 0
6.4
12.8
19~2
25.6 32.0
H V J (I 02 HAU/ml
FIGURE 4 Dose responce of HVJ-induced agglutination of human erythrocytes . The erythrocytes suspension was agglutinated by different doses of the virus for 30 min at 0 ° . The resultant call aggrngates warn diepersad by shaking with a Vortez Mizer, and then apparent cell number in the sample was measured by a Cooper counter below 10 ° . The eztent of cell agglutination was ezpressed as the relative decrease in original call auaber . ( " ) with 10 mM colchicine, (Q) without colchicine .
1388
Colchicine Stimulation of Cell Fusion
Vol . 18, No . 12
The other poseibilitq that the stimulation ~fas due to the inhibition of elution rate of HVJ from the cells with a consequent enhancement of hemolysisl ~d fusion frequency was also ezperimeatally checked by measuring release of I-HVJ from cell aggregates . Hunan erythrocytes was, however, found to be unsuitable for this purpose, because virus-bearing membrane fragments produced from hemolysed erythrocyte membrane may not precipitate under the centrifugal conditions employed sad thus obscure the elution of HVJ . Since stimulation of fusion of Ehrlich ascites tumor cells by the addition of colchicine was very similar, if not identical, with that of human erythrocytes as described above, the tumor cg~~s were used instead of human erythrocytes in this study. Rates of elution of I-HVJ during fusion reaction (2 nia to 15 min) from the virus-cell aggregates were identical within the experimental errors either by the abaeact or the presence of 5 mM colchicine . DISCUSSION Virus-induced cell fusion proceeds by the way of at least three different steps, namely, cell agglutination, envelope fusion (fusion between virus envelope and cell membrane), and subsequent cell-cell fusion . From electron microscopic observation sad also from the change in hemolytic activity of freeze thawed or oemotically shocked virus (18), it has been suggested that the virusinduced hemolytic results from envelope fusion with hemoglobin being released through a "hole" in the fused virus envelope (18,19) . If this hypothesis is too sympliatic, at least we could state that hemolytic is caused by interaction (or fusion) of the virus envelope with the cell membrane . Thus we can utilize hemolysis as a kind of measure of envelope fusion . Significant stimulation of hemolyais caused by colchicine, therefore, suggested that colchicine stimulated envelope fusion, resoling in enhancement of the subsequent cell-cell fusion . This effect of colchicine on envelope fusion was confirmed by another method, a spin-labelling technique which we have recently developed for the quantitative assay of envelope fusion sad cell-cell fusion (20) . Although it is well known that colchicine affects various cell surface phenomena in which the involvement of microtubulea is implicated (1-8), it is unlikely that the colchicine-induced stimulation of cell fusion ie due to the dissociation of microtubules . Firstly, erythrocytes are thought to lack microtubules, and secondly, the concentration of colchicine required for the stimulation is several thousand tines higher than that needed for the disruption of microtubules . However, the reversibility of the colchicine action, together with the existence of several membrane phenomena which were also affected by millimolar concentrations of colchicine, suggest that colchicine binds to cer~ tain membrane component(s) . Since colchicine with its low molecular weight and lack of charged group could easily penetrate through biological membrane, and since ESB spectra of erythrocyte membrane labeled with lecithin or phosphatidylserine containing spin-labeled fatty acid (20) were not affected by colchicine (T . Maeda, unpublished observation), a likely candidate for the site of colchicine interaction is the peripheral membrane protein, apectrin . Colchicine has recently been reported to bind to this protein (21) . Taking into consideration the idea that apectrin plays an important role in regulating the topological distribution of membrane glycoproteins(22) and the fact that cluster formation of membrane glgcoproteins was observed upon interaction of HVJ with erythrocytes (23), it is tempting to speculate that colchicina stimulates membrane fusion by binding to apectrin which in turn causes redistribution of membrane glycoproteina. Studies along this line are now progress in our laboratory . ACKNOWLEDGEMENT: We are indebted to Prof . Ryo Sato of our laboratory for his kind help, stimulating discussions, and for reading this manuscript . Also ve would like to thank to Dr . Y.Okada of Research Institute for Microbial Deseases for helpful discussions and for donating the $VJ preparations .
Vol . 18, No . 12
Colchicine Stimulation of Cell Fusion
1389
BEFEBENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .
B.D .Berlia and T .E .iJkena, Natura,238, 120-122(1972) T.E .IIkena,J.Z .Borysenko,M .J .Rarnovsky and R.D .Berlin, J.Cell Bio1 .,61, 7082 (1974) H.D .Berlin, Nature, 235, 44-45 (1972) J.M .Oliver, T .E .IIkena and R.D .Berlin, Proc . Nat . Acad . Sci. II .S .A ., 71, 394398 (1974) G.M .Edelman, LYahara and J .L .Wang, Proc .Nat .Acad.Sci .II.S .A ., 70, 1442-1446 (1973) E .Gillespie and L.M .Lichitenstein, J .C1in.Invest., _51, 2941-2947 (1972) J.A .Williams and J.Wolff, J.Cell Biol . 54, 157-165 (1972) N.B .Thoa, G.F .Wooten, J .Aaelrod and I .J .Ropin, Proc .Nat .Acad.Sc1 .II.S .A .,69, 520-522 (1972) F .Wunderlich,R.Muller and V .Speth, Science, 182, 1136-1138 (1973) R .Cheng and G.Ratsoyaanis, Biochem.Biophys .Res .Commua.,64, 1069-1075(1975) Y. Okada; Bikea's J ., 1, 103-110(1958) J.E .Salk, J.Inmuno1 .,49, 87-97(1944) Y .Okada and J .Tadokoro, Ezptl.Cell Bes .,26, 119-128(1963) R.Ohki,S .Nakama,A.Asano and Y .Okada, Blochem.Biophys .Res .Commun .,67, 331337(1975) W.H .Hunter, in "Handbook of Experimental Immunology", D.M.Weir,ed .(1973), Blackwell Scientific Publications, pp . 17 .3-17.4 . Y .Hosaka, Biken's J . 1, 70-89(1958) C.Howe and C.Morgan, J.Virol ., 3, 70-81(1969) R.Apostolov and V .Damjaaovic, Microbios, 8, 257-266(1973) R .Apostolov and J.D .Almeida, J.Gen .Virol ., 15, 227-234(1972) T.Maeda, A.Asano,R .Ohki,Y .Okada and S .Ohaishi, Biochem.,14, 3736-3741(1975) G.M .Fuller, J .M .Boughter and M.Morazzaai, Abstract 9th Internat .Congr .Bioch . p .287(1973) G.L .Nicolson and R.G .Painter,J .Cell Biol ., 59, 395-406(1973) T .BHchi, M.Agnet and C .Howe, J.Virol .,ll, 1004-1014(1973)
1383-1390,
1976 .
Pergamoa Praee
EFFECT OF COLCHICINE ON vIRIIS-INDUCED FIISIO~i OF HUMAN ERYTHROCYTES &iyotoshi 3ekiguchi and Akira Asano Institute for Protein Research, Osaka University, Suite, Osaka, Japan (Received in final form May 4, 1976) Simamary : Colchicine was found to stimulate the virus-induced fusion of human erythrocytes . Colchicine also stimulated the rata of hemolysis, but had no effect on its final ertent, suggesting that the enhanced rate of envelope fusion, _i ._e . virus to cell, caused by colchicine resulted in the stimulation of cell to cell fusion . The fact that affective doses of colchicine were at mülimolar concentrations, together with the absence of mièrotubules in human erythrocytes, indicates that the target of colchicine action ie not this aubcellular tubular system . Instead, the peripéeral meabrane protein, spectrin, may be a likely candidate for the site of colchicine action . Colchicine, a well known antimitotic agent, has been shown to affect several cellular processes which accompany topographical changes of cell membranes, such an cell agglutination by plant lectins (1,2), phagocytosie (3,4), cap for mation (5) and secretion phenomena (6,7,8) . In these processes, the effective doses of colchicine are nicromolar concentrations, and therefore, it is believed that this type of effect of colchicine should be manifested through the disruption of nicrotubulea . On the other hand, there seems to aziet another kind of membrane phenomena which is also influenced by colchicine . The temperature dependent topographical changes of Tetrahyneaa pyriformie plasma membrane (9) and sugar transport in fat cell ghosts (10) are the latter ezanples . In these cases the effective doses of wlchicine are millimolar concentrations, suggest- . ing that another type of action mechanism rather than microtubule disruption may be involved .In the present study, we ezamined the effect of colchicine on the HvJ(Sendai virus)-induced fusion reaction of human erythrocytes which provides a suitable model system for studying the mechanise of membrane fusion and the mode of action of colchicine . MATERIALS AND METHODS HvJ, Z strain, was grown in chicken eggs, harvested, and partially purified by differental centrifugation e~deacribed previously (11), except that the purified virus was suspended in Ca -free Tricine-buffered saline (TBS ; 140 mM NaCl, 5.4 mTf RC1 sad 40 mM Trieine-NaOH, pH 7 .6) ; this medium was used throughout the fusion ezperiments unless otherwise stated . The virus dose was e~rnesed in terms of its hemagglutination unite determined by Salk'e pattern method (12) . Human red blood cells were obtained from a blood bank and used within 4 weeks after drawing . ABBREVIATIONS : HVJ, Heeagglutinating virus of Japan; Tricine, N-Trie(hydro:yeethyl)methyl glycine; TBS, Triciae-buffered saline . 1383
1384
Colchicine Stimulation of Cell Fusion
Vol. 18, No . 12
Cell fusion was assayed as follows . A desired dose of HVJ in 0 .5 ml of the medium was added~to 0 .5 ml of a cell suspension kept in an ice bath and the mixture was allowed to stand for 15 min in the cold to complete cell agglutina tion . The fusion reaction was then started by raising the temperature to 37 ° and terminated after 30 min by the addition of 1 ml of cold medium, and rapid cooling is ân ice bath . A 0.2 ml portion of mutate was diluted to 50 .0 ml with 0 .9 X NaCl solution and subjected to cell number counting is a Cooper counter (Model A, Cooper Electronics) with as aperture of 100 pm . The efficiencq of cell fusion was eapressed either as percent decrease is cell number or ae fusion index (13) . We could confirm that this new method can be satisfactorily applied to quantitative studies of erythrocyte fusion under various conditions . Possible errors, which could be introduced by insufficient dissociation of cell aggregates, were checked every time by phase contrast microscopy and found to be insignificant. If cell aggregates are not thoroughly resolved after completion of fusion reaction, degree of fusion estimated by this method should be higher than that measured by the microscopic method (13) . Contrary to expectation, fusion frequency estimated by the Cooper counter method was always slightly lower than that obtained by the orthodox microscopic method . This discrepancy may be attributed to eatenaive hemolysis caused by the virus, in other words, resultant ghosts which are very difficult to settle oa hemocytometer may not be counted by the microscopic method . Fusion of Ehrlich ascites tumor cells was measured as described previously (14) .
100
..
80
..
c~
2 W
r
60
4 3 X 2 W O Z 2
a 40 W Z 20 _O N 0 0
2
4
6
COLCH ICINE
8
10
0
(mM
FIGURE 1 Effect of colchicine on the fusion reaction of humas erythrocytes . Erythrocytée suspended in TBS containing different concentrations of colchicine were agglutinated by HVJ(400 HAU/ml) at 0 ° sad then incubated for 30 min at 37 ° . Fusion frequency was expressed either as percent decrease is original cell number or fusion index.
Vol. 18, No . 12
Colchicine Stimulation of Cell Fusion
1385
Hemolyeis was determined by measuring hemoglobin content of the supernatant of the fusion reaction at 540 nm . Oxidation of hemoglobin was checked spectrophotometrically, and all the hemoglobin content measurment was completed before spectrum change was observed . Cell agglutination was estimated as described for fusion, except that incubation at 37 ° was omitted and particle number counting in the Coulter counter was performed below 10 ° . Iodination of HVJ was performed at 4° by a chlo~~ine T method(15) . Cell agglutination, hemolysis and cell fusion activitea of I-HVJ thus prepared were not appreciably decreased from the original preparation . RESULTS When an erythrocyte suspension was incubated with the virus at 0° to 4 ° for 15 min and then at 37 ° for 30 min, the cells fused with each other producing "polyerythrocytes" . Addition of colchicine to the reaction medium resulted
100 .. 80 cA 80 N } 40 O
W
TIME(min) FIGURE 2 Effect of colchicine on hemolyeis at different temperatures . Erythrocytes were agglutinated by HVJ(960 HAII/ml) at 0° is the presence ( " ) or absence (0) of 10 mM colchicine . Resultant cell aggregates were then transferred to specified temperatures and incubated for different periods as indicated . Following the addition of 4 volumes of cold TBS, samples were iame diately centrifuged at 4° and the hemoglobin content is the supernatant was determined as described under "~TERTATR AND I~THODS" . No detectable heaalysie was observed with controls containing 10 uM colchicine but not containing HVJ under any conditions employed . is a significant stimulation of the fusion reaction, accompanied by the formation of huge "polyerythrocytes" . As shown in Fig.l, the fusion frequency was enhanced by colchicine is a concentration-dependent way; about a three fold stimulation was obtained by the addition of 10 mM colchicine . The effect of colchicine on the fusion reaction was found to be reversible, because stimulation of the reaction was undetectable when cells, which were preincubated with
1386
Colchicine Stimulation of Cell Pueion
pol. 18, No . 12
10 mM colchicine at 37 ° for 15 aia, were washed with cold TBS (unpublished data) . Similar or greater stimulation by the drug was also observed in HpJinduced fusion of Ehrlich aecitea tumor celle.(data not shown) Thus this stimulation may have general importance in the virus-induced fusion reaction . It ie well known that interaction of HpJ with erythrocytes leads to eatensive hemolytic (16) and there is evidence that the hemolytic activity of the vitae is closely correlated to its fusion-inducing activity (17) . Therefore,
25°
37°
3d°
S
:Q.~~;~~ ~ .~
r ~~ i
~, ,.~4 ~~
i
FIGIIRE 3 Effect of colchicine on the fusion reaction of human erythrocytes at different temperatures .( la and lb, 25 °C ; 2a and 2b, 30 °C ; 3a and 3b, 37°C) . Ezperimeatal conditions were the same se in Fig.2 ezcept that the incubation time was fazed for 30 mis and 10 ~ colchicine was included in experiments lb, 26 and 3b . Phase contrast microscopy . the affect of colchicine on the rate of hemolytic was studied (Fig . 2) . When cells were fused by HpJ is the praeeace of lO .mM colchicine at 37 ° , the rate of heanlysis was significantly accelerated and the initial lag phase was diminished, whereas the maximal level of hemolytic attained after 30 min incubation was almost uachaaged. This tendency was more clearly shown at lower temperatures . When the fusion was carried out without colchicine at 23 ° , the early lag phase continued for sore than 10 sin and it took more than one hour to reach the mar i~l level . The addition of colchicine markedly diminished the lag phase and enhanced the rate of hemolytie . In accordance with the stimulation of hemolytic, colchicine enhanced the formation of large fused cells . Moreover, it could
Vol . 18, No . 12
Colchicine Stimulation of Cell Fusion
1387
bring about eztenaivn call fusion even at 25 ° whereas is the absence of colchicine the cells remained is the aggregated state and no fused cells were detectable (Fig . 3) . Since the rate of henolysis might be affected by the amount of HVJ absorbed on the surface of erythrocytes and the eztent of the subsequent virus-induced cell agglutination, the effect of colchicine on these preliminary pro cesses for cell fusion were also e=a,°~npd . Making use of I-labeled HVJ, we observed that colchicine did not c~~ge the amount of HVJ absorbed to erythrocytes ( for ezample, 4,314 cpm of I-HVJ was absorbed on the cells in the presence of 10 mM colchicine, whereas 4,275 cpm of those was bound to the cells in the absence of colchicine) . The virus-induced agglutination was also unaffected by colchicinn . As can be sees in Fig . 4, the agglutination of cells was not appreciably affected by 10 mM colchicine .
Z O
a a 0 a W V Q _2 C_9 O W O 0
6.4
12.8
19~2
25.6 32.0
H V J (I 02 HAU/ml
FIGURE 4 Dose responce of HVJ-induced agglutination of human erythrocytes . The erythrocytes suspension was agglutinated by different doses of the virus for 30 min at 0 ° . The resultant call aggrngates warn diepersad by shaking with a Vortez Mizer, and then apparent cell number in the sample was measured by a Cooper counter below 10 ° . The eztent of cell agglutination was ezpressed as the relative decrease in original call auaber . ( " ) with 10 mM colchicine, (Q) without colchicine .
1388
Colchicine Stimulation of Cell Fusion
Vol . 18, No . 12
The other poseibilitq that the stimulation ~fas due to the inhibition of elution rate of HVJ from the cells with a consequent enhancement of hemolysisl ~d fusion frequency was also ezperimeatally checked by measuring release of I-HVJ from cell aggregates . Hunan erythrocytes was, however, found to be unsuitable for this purpose, because virus-bearing membrane fragments produced from hemolysed erythrocyte membrane may not precipitate under the centrifugal conditions employed sad thus obscure the elution of HVJ . Since stimulation of fusion of Ehrlich ascites tumor cells by the addition of colchicine was very similar, if not identical, with that of human erythrocytes as described above, the tumor cg~~s were used instead of human erythrocytes in this study. Rates of elution of I-HVJ during fusion reaction (2 nia to 15 min) from the virus-cell aggregates were identical within the experimental errors either by the abaeact or the presence of 5 mM colchicine . DISCUSSION Virus-induced cell fusion proceeds by the way of at least three different steps, namely, cell agglutination, envelope fusion (fusion between virus envelope and cell membrane), and subsequent cell-cell fusion . From electron microscopic observation sad also from the change in hemolytic activity of freeze thawed or oemotically shocked virus (18), it has been suggested that the virusinduced hemolytic results from envelope fusion with hemoglobin being released through a "hole" in the fused virus envelope (18,19) . If this hypothesis is too sympliatic, at least we could state that hemolytic is caused by interaction (or fusion) of the virus envelope with the cell membrane . Thus we can utilize hemolysis as a kind of measure of envelope fusion . Significant stimulation of hemolyais caused by colchicine, therefore, suggested that colchicine stimulated envelope fusion, resoling in enhancement of the subsequent cell-cell fusion . This effect of colchicine on envelope fusion was confirmed by another method, a spin-labelling technique which we have recently developed for the quantitative assay of envelope fusion sad cell-cell fusion (20) . Although it is well known that colchicine affects various cell surface phenomena in which the involvement of microtubulea is implicated (1-8), it is unlikely that the colchicine-induced stimulation of cell fusion ie due to the dissociation of microtubules . Firstly, erythrocytes are thought to lack microtubules, and secondly, the concentration of colchicine required for the stimulation is several thousand tines higher than that needed for the disruption of microtubules . However, the reversibility of the colchicine action, together with the existence of several membrane phenomena which were also affected by millimolar concentrations of colchicine, suggest that colchicine binds to cer~ tain membrane component(s) . Since colchicine with its low molecular weight and lack of charged group could easily penetrate through biological membrane, and since ESB spectra of erythrocyte membrane labeled with lecithin or phosphatidylserine containing spin-labeled fatty acid (20) were not affected by colchicine (T . Maeda, unpublished observation), a likely candidate for the site of colchicine interaction is the peripheral membrane protein, apectrin . Colchicine has recently been reported to bind to this protein (21) . Taking into consideration the idea that apectrin plays an important role in regulating the topological distribution of membrane glycoproteins(22) and the fact that cluster formation of membrane glgcoproteins was observed upon interaction of HVJ with erythrocytes (23), it is tempting to speculate that colchicina stimulates membrane fusion by binding to apectrin which in turn causes redistribution of membrane glycoproteina. Studies along this line are now progress in our laboratory . ACKNOWLEDGEMENT: We are indebted to Prof . Ryo Sato of our laboratory for his kind help, stimulating discussions, and for reading this manuscript . Also ve would like to thank to Dr . Y.Okada of Research Institute for Microbial Deseases for helpful discussions and for donating the $VJ preparations .
Vol . 18, No . 12
Colchicine Stimulation of Cell Fusion
1389
BEFEBENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .
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