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The effects of cytochalasin B and dimethyl sulfoxide on the movement of mouse kidney cells
DEVELOPMENTAL
BIOLOGY
35, 176-179 (1973)
The Effects of Cytochalasin B and Dimethyl Sulfoxide Movement of Mouse Kidney Cells I. Effect on the Outgrowth
on the
of Cells’
F. J. DYE Department Deportment
of Biology, Western of Cytogenetics,
Connecticut State College, The New England Institute, Accepted
June
Danbury, Ridgefield,
Connecticut Connecticut
06810, and 06877
4, 1973
The following experiments were undertaken to determine the effect of cytochalasin B (CB) on the movement of mouse kidney epitheliocytes in primary culture. Fragments of mouse kidney were cultured in Sykes-Moore chambers with control medium (McCoy’s 5a, modified), CB in dimethyl sulfoxide (DMSO), and DMSO. CB, 10 pg/ml, in 1% DMSO completely inhibited the outgrowth of cells from the kidney fragments. These effects were reversible. Unexpectedly, 1% DMSO suppressed the outgrowth of the kidney cells. A myriad of phenomenological effects of CB on mammalian cells have been reported in the recent literature. Most investigators report no effect of DMSO on the cells. These results suggest that DMSO is affecting the movement of mouse kidney cells. INTRODUCTION
The movement of sheets of cells is involved in such diverse phenomena as wound healing and morphogenetic movements. The mechanism of movement by sheets of mammalian epitheliocytes is obscure (Abercrombie and Middleton, 1968; Dye, 1971; Carter, 1972). This study was undertaken to determine what the effects of cytochalasin B, a fungal metabolite, would reveal about the mechanism of such movement. Dimethyl sulfoxide (DMSO) is a commonly employed solvent for chemicals, including cytochalasin B (CB), that resist dissolving in aqueous media. Examination of the controls set up for these experiments revealed that DMSO inhibits cell movement. The effects of CB and DMSO on the outgrowth of cells from fragments of mouse kidney in vitro are reported here. MATERIALS
AND
METHODS
Cell cultures. Primary cultures of neonatal mouse kidney were set up as follows. ‘This work was supported in part by a Grant-inAid of Research from the Society of the Sigma Xi. 176
Mice of the CFl-S strain (Carworth Farms), with one exception (see Fig. l), were sacrificed by cervical dislocation, and the kidneys were fragmented with scalpels in Hank’s balanced salt solution (GIBCO). Five fragments of less than 1 mm3 were placed on the glass coverslip of a partially assembled Sykes-Moore culture chamber. Fragments were matched as to size, so that the size of the fragments would not be variable; i.e., the fragments in control and experimental cultures were of the same size. After the culture chamber was assembled, approximately 0.8 ml of medium was added by injection. Generally, at least four of the five fragments in each culture attached so that they could be observed microscopically. The short-term cultures were maintained at 37°C. Media and chemicals. The basic medium used and the control medium throughout was McCoy’s 5% modified medium with 30% fetal calf serum (GIBCO) to which penicillin, streptomycin, and neomycin were added. Cytochalasin B (CB) was purchased from Imperial Chemical Industries, United Kingdom and dissolved in dimethyl sulfox-
177
BRIEF NOTES
ide (DMSO; Sigma Chemical Co.) to a concentration of 1 mg CB/l ml DMSO. A working solution was obtained by diluting the CB-DMSO in the basic medium (see above) to a concentration of 10 pg CB/ml of medium (containing 1% DMSO). Further dilutions of this working solution were made to obtain various concentrations. Data collection. The cultures were observed by phase contrast microscopy, and the cells surrounding each kidney fragment were manually counted. Outgrowth is here defined as the number of cells that leave the fragment and move out onto the glass coverslip as a monolayer of cells. Replacement of medium. Whenever medium containing CB (CB-medium) was replaced with control medium (see above), the following procedure was followed. The upper ring and coverslip of the SykesMoore chamber were removed, leaving behind a pond of medium (CB-medium) trapped by the gasket. The CB-medium was removed with a syringe and the kidney fragments were washed (3x) with control medium. Finally, the chamber was reassembled and fresh medium was added by injection. RESULTS
during the first 2 days in vitro (Fig. 2). With 1% DMSO alone there is always some outgrowth by 48 hrs, and by 72 hr the average number of cells/fragment with outgrowth may be at control levels. Inhibition of Cell Outgrowth CBIml of Medium
by 10 /Ig
Since no outgrowth of cells occurs in the presence of 10 pg CB/ml of media with 1% DMSO (Fig. l), whereas some outgrowth does occur in 1% DMSO alone by either 24 hr or 48 hr, it was not determined whether CB alone inhibits cell outgrowth or whether CB in the presence of 1% DMSO has its action potentiated by the DMSO; or whether the CB potentiates the effect of the DMSO. Effect of DMSO Outgrowth
Concentration
on Cell
The effects of concentrations of DMSO of 0.1% and 0.01% are difficult to interpret. Generally, these concentrations cause some suppression of outgrowth, whether measured as the number of fragments with cellular outgrowth or as the average number of cells per fragment with cellular outgrowth.
Inhibition of Cell Outgrowth by 10 pg CBIml of Medium with DMSO
Effect of CB Outgrowth
The effect of 10 pg CB/ml of medium with 1% DMSO is the complete inhibition of the outgrowth of cells from the kidney fragments. If the CB medium is replaced by control medium within 48 or 72 hr (Fig. l), the inhibition of outgrowth is completely removed. On the other hand, replacement of the CB medium during the sixth day does not completely reverse the inhibition, nor is the effect of the removal of the CB in DMSO as rapid (Fig. 1). The control media used did not contain DMSO.
The concentrations of CB used of less than 10 pg/ml do not completely inhibit cell outgrowth after 24 hr. Since 1 pg of CB is in 0.1% DMSO and 0.1 pg of CB is in 0.01% DMSO and the effects of these concentrations of DMSO are difficult to interpret, the noteworthy effect of 1 pg and 0.1 pg of CB is that they do not cause total inhibition of cell outgrowth, in the concentrations of DMSO used.
Suppression of Cell Outgrowth DMSO
by 1%
The concentration of DMSO (1%) often used suppresses the outgrowth of cells
Concentration
on Cell
DISCUSSION
Recent reports indicate that the initial effect of CB may be on the plasma membrane of the cell (Beck et al., 1972; Cohn et al., 1972; Estensen and Plagemann, 1972; Krishan, 1972; Plagemann and Estensen,
178
DEVELOPMENTAL 400
r
Bro~ocu
VOLUME
34.1973
.a
FIG. 1. The effect of 10 pg CB/ml of medium with 1% DMSO on the outgrowth of cells from mouse kidney fragments. Mice approximately 1 week (-1 and 24 hr (- - - -) old were used. The father of one mouse (-1 was a “brown” mouse of unknown origin, whereas the other parents were CFl-S. The a curves show outgrowth of cells in control medium, and the b curves show outgrowth of cells after CB medium with DMSO was replaced by control medium. Letters X, y, and z show the days on which the CB medium was removed. Each curve represents two cultures. All cultures were set up on day 0.
FIG. 2. The effect of 1% DMSO on the outgrowth of cells from mouse kidney fragments. Mice approximately 5 days (- - - -) and 24 hr (-1 old were used. The a curves show outgrowth of cells in control medium, and the b curves show outgrowth of cells in 1% DMSO. On each day for the given mouse there is a significant difference in the amount of outgrowth between the control and 1% DMSO cultures. Student’s t test: p =
1972; Zigmond and Hirsch, 1972; Kletzien and Perdue, 1973). Related to this, it is interesting to note that DMSO appears to be extremely effective in altering the configuration of proteins (Rammler and Zaf-
faroni, 1967) and is able to modify the stereoisomery of fatty acids (Muset and Martin-Esteve, 1965), both types of molecules being components of plasma membranes. If two compounds (e.g., DMSO and CB) have the same target organelle (e.g., the plasma membrane), it would not be surprising to find that their simultaneous application elicits effects different from those resulting from their individual application. Furmanski and Lubin (1972) reported that 2% DMSO inhibited neurite extension by neuroblastoma cells and suggested the possibility that in some systems an agent under study acts synergistically with DMSO. Recently, Landauer and Salam (1972) reported that the effects of some teratogens were reduced in incidence and expressivity when given in DMSO rather than in water, whereas malformations induced by sulfanilamide occurred with higher frequency and expression in DMSO than in aqueous solution. REFERENCES M., and MIDDLETON, C. A. (1968). Epithelial-mesenchymal interactions affecting locomotion of cells in culture. In “Epitbelialmesenchymal Interactions” (R. Fleischmajor and R. E. Billingham, eds.), pp. 56-63. Williams & Wilkins, Baltimore, Maryland.
ABERCROMBIE,
BRIEF NOTES BECK, J. S., JAY, A. W. L., and SAARI, J. T. (1972). Effects of cytochalasin B on osmotic fragility and deformability of human erythrocytes. Can. J. Whysiol. Pharmacol. 50, 684-688. CARTER, S. B. (1972). Tools in cytology. Endeaoour 31, 77-82. COHN, R. H., BANERJEE, S. D., SHELTON, E. R., and BERNFIELD, M. R. (1972). Cytochalasin B: lack of effect on mucopolysaccharide synthesis and selective alterations in precursor uptake. F’roc. Nat. Acad. Sci. U.S. 69, 2865-2869. DYE, F. J. (1971). The in vitro movement of mammalian epithelial cells. Oncology 25. 79-82. ESTENSEN, R. D., and PLACEMANN, P. G. W. (1972). Cytochalasin B: inhibition of glucose and glucosamine transport. Proc. Nat. Acad. Sci. U.S. 69, 1430-1434. FURMANSKI, P., and LLJBIN, M. (1972). Effects of dimethylsulfoxide on expression of differentiated functions in mouse neuroblastoma. J. Nat. Cancer ht. 48, 1355-1361. KLETZIEN, R. F., and PERDUE. J. F. (1973). The inhibition of sugar transport in chick embryo fibro-
179
blasts by cytochalasin B. J. Biol. Chem. 248, 711-719. KRISHAN, A. (1972). Cytochalasin-B: time-lapse cinematographic studies on its effects on cytokinesis. J. Cell. Viol. 54, 657-664. LANDAUER. W., and SALAM, N. (1972). Aspects of dimethyl sulfoxide as solvent for teratogens. Develop. Biol. 28,35-46. MUSET, P. P. and MARTIN-ESTE~E, J. (1965). Physiological cell permeability and pharmacological action of DMSO. Experientia 21, 649-651. PLACEMANN, P. G. W., and ESTENSEN, R. D. (1972). Cytochalasin B VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells. J. Cell Biol. 55, 179-185. RAMMLER, D. H., and ZAFFARONI, A. (1967). Biological implications of DMSO based on a review of its chemical properties. Ann. N. Y. Acad. Sci. 141, 13-23. ZICMOND, S. H., and HIRSCH, J. G. (1972). Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp. Cell Res. 73, 383-393.
BIOLOGY
35, 176-179 (1973)
The Effects of Cytochalasin B and Dimethyl Sulfoxide Movement of Mouse Kidney Cells I. Effect on the Outgrowth
on the
of Cells’
F. J. DYE Department Deportment
of Biology, Western of Cytogenetics,
Connecticut State College, The New England Institute, Accepted
June
Danbury, Ridgefield,
Connecticut Connecticut
06810, and 06877
4, 1973
The following experiments were undertaken to determine the effect of cytochalasin B (CB) on the movement of mouse kidney epitheliocytes in primary culture. Fragments of mouse kidney were cultured in Sykes-Moore chambers with control medium (McCoy’s 5a, modified), CB in dimethyl sulfoxide (DMSO), and DMSO. CB, 10 pg/ml, in 1% DMSO completely inhibited the outgrowth of cells from the kidney fragments. These effects were reversible. Unexpectedly, 1% DMSO suppressed the outgrowth of the kidney cells. A myriad of phenomenological effects of CB on mammalian cells have been reported in the recent literature. Most investigators report no effect of DMSO on the cells. These results suggest that DMSO is affecting the movement of mouse kidney cells. INTRODUCTION
The movement of sheets of cells is involved in such diverse phenomena as wound healing and morphogenetic movements. The mechanism of movement by sheets of mammalian epitheliocytes is obscure (Abercrombie and Middleton, 1968; Dye, 1971; Carter, 1972). This study was undertaken to determine what the effects of cytochalasin B, a fungal metabolite, would reveal about the mechanism of such movement. Dimethyl sulfoxide (DMSO) is a commonly employed solvent for chemicals, including cytochalasin B (CB), that resist dissolving in aqueous media. Examination of the controls set up for these experiments revealed that DMSO inhibits cell movement. The effects of CB and DMSO on the outgrowth of cells from fragments of mouse kidney in vitro are reported here. MATERIALS
AND
METHODS
Cell cultures. Primary cultures of neonatal mouse kidney were set up as follows. ‘This work was supported in part by a Grant-inAid of Research from the Society of the Sigma Xi. 176
Mice of the CFl-S strain (Carworth Farms), with one exception (see Fig. l), were sacrificed by cervical dislocation, and the kidneys were fragmented with scalpels in Hank’s balanced salt solution (GIBCO). Five fragments of less than 1 mm3 were placed on the glass coverslip of a partially assembled Sykes-Moore culture chamber. Fragments were matched as to size, so that the size of the fragments would not be variable; i.e., the fragments in control and experimental cultures were of the same size. After the culture chamber was assembled, approximately 0.8 ml of medium was added by injection. Generally, at least four of the five fragments in each culture attached so that they could be observed microscopically. The short-term cultures were maintained at 37°C. Media and chemicals. The basic medium used and the control medium throughout was McCoy’s 5% modified medium with 30% fetal calf serum (GIBCO) to which penicillin, streptomycin, and neomycin were added. Cytochalasin B (CB) was purchased from Imperial Chemical Industries, United Kingdom and dissolved in dimethyl sulfox-
177
BRIEF NOTES
ide (DMSO; Sigma Chemical Co.) to a concentration of 1 mg CB/l ml DMSO. A working solution was obtained by diluting the CB-DMSO in the basic medium (see above) to a concentration of 10 pg CB/ml of medium (containing 1% DMSO). Further dilutions of this working solution were made to obtain various concentrations. Data collection. The cultures were observed by phase contrast microscopy, and the cells surrounding each kidney fragment were manually counted. Outgrowth is here defined as the number of cells that leave the fragment and move out onto the glass coverslip as a monolayer of cells. Replacement of medium. Whenever medium containing CB (CB-medium) was replaced with control medium (see above), the following procedure was followed. The upper ring and coverslip of the SykesMoore chamber were removed, leaving behind a pond of medium (CB-medium) trapped by the gasket. The CB-medium was removed with a syringe and the kidney fragments were washed (3x) with control medium. Finally, the chamber was reassembled and fresh medium was added by injection. RESULTS
during the first 2 days in vitro (Fig. 2). With 1% DMSO alone there is always some outgrowth by 48 hrs, and by 72 hr the average number of cells/fragment with outgrowth may be at control levels. Inhibition of Cell Outgrowth CBIml of Medium
by 10 /Ig
Since no outgrowth of cells occurs in the presence of 10 pg CB/ml of media with 1% DMSO (Fig. l), whereas some outgrowth does occur in 1% DMSO alone by either 24 hr or 48 hr, it was not determined whether CB alone inhibits cell outgrowth or whether CB in the presence of 1% DMSO has its action potentiated by the DMSO; or whether the CB potentiates the effect of the DMSO. Effect of DMSO Outgrowth
Concentration
on Cell
The effects of concentrations of DMSO of 0.1% and 0.01% are difficult to interpret. Generally, these concentrations cause some suppression of outgrowth, whether measured as the number of fragments with cellular outgrowth or as the average number of cells per fragment with cellular outgrowth.
Inhibition of Cell Outgrowth by 10 pg CBIml of Medium with DMSO
Effect of CB Outgrowth
The effect of 10 pg CB/ml of medium with 1% DMSO is the complete inhibition of the outgrowth of cells from the kidney fragments. If the CB medium is replaced by control medium within 48 or 72 hr (Fig. l), the inhibition of outgrowth is completely removed. On the other hand, replacement of the CB medium during the sixth day does not completely reverse the inhibition, nor is the effect of the removal of the CB in DMSO as rapid (Fig. 1). The control media used did not contain DMSO.
The concentrations of CB used of less than 10 pg/ml do not completely inhibit cell outgrowth after 24 hr. Since 1 pg of CB is in 0.1% DMSO and 0.1 pg of CB is in 0.01% DMSO and the effects of these concentrations of DMSO are difficult to interpret, the noteworthy effect of 1 pg and 0.1 pg of CB is that they do not cause total inhibition of cell outgrowth, in the concentrations of DMSO used.
Suppression of Cell Outgrowth DMSO
by 1%
The concentration of DMSO (1%) often used suppresses the outgrowth of cells
Concentration
on Cell
DISCUSSION
Recent reports indicate that the initial effect of CB may be on the plasma membrane of the cell (Beck et al., 1972; Cohn et al., 1972; Estensen and Plagemann, 1972; Krishan, 1972; Plagemann and Estensen,
178
DEVELOPMENTAL 400
r
Bro~ocu
VOLUME
34.1973
.a
FIG. 1. The effect of 10 pg CB/ml of medium with 1% DMSO on the outgrowth of cells from mouse kidney fragments. Mice approximately 1 week (-1 and 24 hr (- - - -) old were used. The father of one mouse (-1 was a “brown” mouse of unknown origin, whereas the other parents were CFl-S. The a curves show outgrowth of cells in control medium, and the b curves show outgrowth of cells after CB medium with DMSO was replaced by control medium. Letters X, y, and z show the days on which the CB medium was removed. Each curve represents two cultures. All cultures were set up on day 0.
FIG. 2. The effect of 1% DMSO on the outgrowth of cells from mouse kidney fragments. Mice approximately 5 days (- - - -) and 24 hr (-1 old were used. The a curves show outgrowth of cells in control medium, and the b curves show outgrowth of cells in 1% DMSO. On each day for the given mouse there is a significant difference in the amount of outgrowth between the control and 1% DMSO cultures. Student’s t test: p =
1972; Zigmond and Hirsch, 1972; Kletzien and Perdue, 1973). Related to this, it is interesting to note that DMSO appears to be extremely effective in altering the configuration of proteins (Rammler and Zaf-
faroni, 1967) and is able to modify the stereoisomery of fatty acids (Muset and Martin-Esteve, 1965), both types of molecules being components of plasma membranes. If two compounds (e.g., DMSO and CB) have the same target organelle (e.g., the plasma membrane), it would not be surprising to find that their simultaneous application elicits effects different from those resulting from their individual application. Furmanski and Lubin (1972) reported that 2% DMSO inhibited neurite extension by neuroblastoma cells and suggested the possibility that in some systems an agent under study acts synergistically with DMSO. Recently, Landauer and Salam (1972) reported that the effects of some teratogens were reduced in incidence and expressivity when given in DMSO rather than in water, whereas malformations induced by sulfanilamide occurred with higher frequency and expression in DMSO than in aqueous solution. REFERENCES M., and MIDDLETON, C. A. (1968). Epithelial-mesenchymal interactions affecting locomotion of cells in culture. In “Epitbelialmesenchymal Interactions” (R. Fleischmajor and R. E. Billingham, eds.), pp. 56-63. Williams & Wilkins, Baltimore, Maryland.
ABERCROMBIE,
BRIEF NOTES BECK, J. S., JAY, A. W. L., and SAARI, J. T. (1972). Effects of cytochalasin B on osmotic fragility and deformability of human erythrocytes. Can. J. Whysiol. Pharmacol. 50, 684-688. CARTER, S. B. (1972). Tools in cytology. Endeaoour 31, 77-82. COHN, R. H., BANERJEE, S. D., SHELTON, E. R., and BERNFIELD, M. R. (1972). Cytochalasin B: lack of effect on mucopolysaccharide synthesis and selective alterations in precursor uptake. F’roc. Nat. Acad. Sci. U.S. 69, 2865-2869. DYE, F. J. (1971). The in vitro movement of mammalian epithelial cells. Oncology 25. 79-82. ESTENSEN, R. D., and PLACEMANN, P. G. W. (1972). Cytochalasin B: inhibition of glucose and glucosamine transport. Proc. Nat. Acad. Sci. U.S. 69, 1430-1434. FURMANSKI, P., and LLJBIN, M. (1972). Effects of dimethylsulfoxide on expression of differentiated functions in mouse neuroblastoma. J. Nat. Cancer ht. 48, 1355-1361. KLETZIEN, R. F., and PERDUE. J. F. (1973). The inhibition of sugar transport in chick embryo fibro-
179
blasts by cytochalasin B. J. Biol. Chem. 248, 711-719. KRISHAN, A. (1972). Cytochalasin-B: time-lapse cinematographic studies on its effects on cytokinesis. J. Cell. Viol. 54, 657-664. LANDAUER. W., and SALAM, N. (1972). Aspects of dimethyl sulfoxide as solvent for teratogens. Develop. Biol. 28,35-46. MUSET, P. P. and MARTIN-ESTE~E, J. (1965). Physiological cell permeability and pharmacological action of DMSO. Experientia 21, 649-651. PLACEMANN, P. G. W., and ESTENSEN, R. D. (1972). Cytochalasin B VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells. J. Cell Biol. 55, 179-185. RAMMLER, D. H., and ZAFFARONI, A. (1967). Biological implications of DMSO based on a review of its chemical properties. Ann. N. Y. Acad. Sci. 141, 13-23. ZICMOND, S. H., and HIRSCH, J. G. (1972). Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp. Cell Res. 73, 383-393.