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Studies on serum stimulation of mouse fibroblast migration
Preliminary notes 499
Studies on serum stimulation of mouse fibroblast migration
serum. After centrifugation, the cells were washed twice in medium and plated at the appropriate density in 55-mm (Falcon) plastic dishes.
L. WOLF and A. LIPTON, Section of Oncology, Department of Medicine, The Milton S. Hershey Medical Center, Hershey, Pa 17033, USA
Migration
Summary. Evidence is presented that the factor present
in rat serum that uromotes the migration of mouse fi~i~sblas.s+(3T3) is a high mol. wtmolecule. Amino and Ca2+ are essential for full expression of aciivity. Once exposed to serum there is an 8-10 h lag period before cell migration can be detected. Cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B, all inhibit cellular migration from a wound edge.
The growth of fibroblasts in culture ceases when the population reaches a characteristic cell density. If a wound is then made by scratching such a confluent cell monolayer, cells migrate into the denuded area, synthesize DNA and divide [l, 21. A serum factor which stimulates the movement of cells and is thus responsible for ‘wound healing’ has been demonstrated [3]. The migration-promoting factor has been shown to be distinct from the growth and survival factors [3-51. In the present communication we describe some of the characteristics of the migration of mouse fibroblasts under the direction of the serum migration-promoting factor.
assay. Migration-promoting activity was assayed by the use of wounds in confluent 3T3 cells. The cultures were grown from lo5 cells cultured in Dulbecco & Vogt’s modification of Eagle’s medium with 10 % calf serum, in a CO2 incubator at 37°C for 5 days. The cultures were washed 3 times with serumfree medium, incubated for 15 min, and then 2 linear wounds (about 5 mm wide) were made in the monolayer with a wedge-tipped Teflon rod. The cultures were then washed twice more with serum-free medium, incubated in serum-free medium for 30 min, and washed again before the addition of 5 ml of serum-free medium plus the samples to be tested. After incubation for -the times inhicated, cultures were washed once with Tris-saline (pH 7.4), fixed for 5 min with 98 % acetic acid, 95 % ethanol 1 : 3, and stained for IO min with 0.5 % crystal violet in 95 % ethanol. The stained cultures were washed twice with distilled water and dried at room temperature. The number of cells migrating from the wound edge was determined with an ocular net micrometer, 1 x 1 cm (Unitron N3M), in a wide-field x 12.5 eyepiece, and-a x 6.3 obiective. Ten different fields (each 1.6 x 1.6 mm contiguous with the wound edge) were counted for each sample assayed, and the migrating cells per field were averaged.
of serum. All the described operations were performed at 0-4°C. All sera were stored at - 20°C and were tested without heating. Sephadex (Pharmacis) gels susnended in solutions at the appropriate pH and-salt co&. were equilibrated for 334 days prior to use. Columns were packed and serum (at the appropriate pH) was applied to the column. Rat serum fractionation with ammonium sulfate was performed with undiluted serum and with serum diluted I : IO with water. Solutions were stirred vigorously during the addition of the salt.
Fractionation
Materials and Methods Blood of rats (Sprague Dawley) was obtained by heart puncture. Serum was obtained by allowing the blood to clot at room temperature for 60 min. Cohn fractions were purchased from Nutritional Biochemicals Corp. Pituitary hormones were kindly supplied by Dr James Lewis, Scripps Clinic and Research Foundation, La Jolla, Calif. Other hormones were purchased from commercial sources. Actinomycin D was purchased from Sigma; colchitine and cycloheximide from Nutritional Biochemicals Corp.; velban from Lilly and cytochalasin B from Imperial Chemical Industry, Ltd. Cells and culture media. The 3T3 cells were originally obtained from Dr Dennis Cunningham, Department of Microbiology, University of California, Irvine, Calif. Cells were cultured in Dulbecco & Vogt’s modification of Eagle’s medium with 10 % calf serum. The cells were transformed using 0.05 % trypsin in Ca2+and Mg2+-free Tris-saline solution, pH 7.4; the action of the trypsin was terminated by the addition of
Results Serum and urine have been shown to be a ready source of migration-promoting activity for 3T3 cells in all species tested [3]. Gel filtration at pH 7.4 reveals that the migration-promoting activity in human serum is due to the presence of a high mol. wt molecule (fig. 1). (Indeed this activity is retained by Amicon UM 10 and PM 30 filters.) Serum migration promoting activity is not depleted by growing or resting 3T3 or SV3T3 cells. The serum migration activity is stable for up to 6 months if stored at 4°C. Serum from a hepatectomized rat (6-12 h Exptl Cell Res 80 (1973)
500 Preliminary notes
MW 700.000
MW 160,000
MW67,OOO
MW 45,000
MW 12,384
1.60I .40I .20-
-240
l.OO.80-
-200
.60.40-
- 1.60
.20-1.20
-
2
20
22
24
26
28
30
32
34
36
38
40
42
44
46
46
50
52
54
56
58
60 62
64
66
68
so
70 60
Fig. I. Abscissa: fraction no.; ordinate: (left) absorbance at 280 nm and 300 nm; (right) migration index. n-m, A,,, and Azeo; O-O, 3T3 migration-promoting activity. Gel filtration of 8 ml rat serum on Sephadex G-200 column (80 x 3 cm) at pH 7.4, 0.01 M Tris buffer. 5 ml fractions were collected. 0.4 ml of each fraction was assayed. The following markers were used: cytochrome c (12 384); ovalbumen (45 000); bovine albumin (67 000); human y globulin (160 000); thyroglobulin (700 000).
after surgery) contains the same amount of migration-promoting activity as does serum from normal adult rats. Ammonium sulfate fractionation of rat serum at pH 8.0 spreads the migration-promoting activity for 3T3 cells throughout all fractions. This pattern resembled that obtained for growth-promoting activity [5]. Table 1. Migration-promoting activity dialysed rat serum in deficient media
Media
Migrationpromoting activity (X)
Complete Amino acids Calcium Magnesium Calcium and magnesium
100 40 2 12 Cells come off dish
of
0.05 ml of the appropriate medium was added to the standard wound assay. After 24 h incubation, cultures were fixed and counted as described under Materials and Methods. Exptl CeN Res 80 (1973)
The following materials were assayed and were found to be inactive in promoting migration of 3T3 cells-AMP, CAMP, dibutyryl-CAMP, l-thyroxine, bradykinin, testosterone, 17-P-estradiol, FSH, LH, ACTH, TSH, somatotropin, prolactin, prostaglandin E, melantonin, insulin, epinephrine and hydrocortisone. Combining each of these compounds with 3T3 or SV3T3 growth factor [5] also failed to cause migration of 3T3 cells. The serum migration-promoting activity was also not affected by the presence of dexamethasone (0.02-2.0 pg/ml) in the medium. Steroids are known to retard woundhealing in man. 3T3 cells will not migrate into a denuded area in the absence of serum. Serum alone is, however, not sufficient to achieve migration of mouse fibroblasts. The migration of 3T3 cells is dependent upon the presence of amino acids, calcium, and magnesium in the medium. In the absence of any of these, the migration-
Preliminary notes promoting capacity of that particular serum is not fully expressed (table 1). When complete medium is replaced, the migrationpromoting activity is restored. The time course of cell migration under the influence of serum was next studied. There is a lag period of 8-10 h after addition of serum to a wound edge before significant migration can be detected (table 2). If serum is present for a stated time interval (e.g. O-4, O-8, or O-12 h) and then replaced with serum-free medium, further migration is not seen. There would thus appear to be an 8-10 h lag period after addition of serum before any cell migration is seen. In addition, serum must constantly be present to have continued (maximal) cell movement. The availability of chemicals which inhibit a variety of cell functions allows us to study the role of these functions in fibroblast migration under the influence of serum. Calf serum plus varying concentrations of each inhibitory substance were added to the standard wounded monolayer. Serum-induced migration of fibroblasts is inhibited by cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B (table 3). After 24-h exposure in each case this inhibiTable 2. Relationship between cell migration and time after exposure to serum in a wounded 3T3 culture
Hours
Average number of cells migrating from 1.6 mm wound edge
0 2 4 7 II 24
0.07 0.05 0.22 0.35 2.43 6.10
0.05 ml calf serum was added to wounded confluent 3T3 cultures at time zero. At the appropriate interval cultures were fixed and counted as described under Materials and Methods.
501
Table 3. Inhibition of serum-induced jibroblast migration Average
Sample
number of cells migrating from I .6 mm wound edge
E0.05 ml calf serum (CS) CS -i-cycloheximide (0.5-5 pg/ml) CS + velban (0.005-l pg/ml) CS + colchicine (0.03-0.5 pg/ml) CS + act D (0.05-l pg/ml) CS + cytochalasin B (0.05-10 ,ug/ml)
0.07 5.60 0.00 0.00 0.00 0.00 0.00
3T3 cells were prepared as described in the standard wound assay. 0.05 ml calf serum plus various concentrations of each inhibitor were added at time zero. At 24 h of incubation cultures were fixed and counted. All cultures were still viable at 24 h as replacement with fresh medium containing calf serum would result in the expected migration.
tion is reversible if the cultures are washed and replaced with fresh serum-containing medium. These same cells then migrate in normal fashion once free of inhibitory substances. If inhibitors are added at 0, 4, 7, 10 or 13 h after addition of calf serum, further migration is prevented. Discussion The migration of 3T3 mouse fibroblasts into a wound depends upon the presence of serum [3]. This is contrary to an earlier conclusion of other authors [7]. The serum migrationpromoting activity is a large molecule (greater than 200 000 D) that is pepsin labile [3]. The serum migration-promoting activity has been physically separated from other factors present in serum that promote cell growth and survival. Serum growth factors are depleted by resting or growing 3T3 cells [8, 91. Neither the migration-promoting factor nor the survival factor [6] is depleted under these conditions. The migration-promoting Exptl Cell Res 80 (1973)
502 Preliminary
notes
activity and growth factors are present in human urine. The migration of a cell into a denuded area is a complex phenomenon. The serum migration-promoting activity must be present. Amino acids, calcium and magnesium must also be present for the full expression of the migration potential. Absence of any of these components will reduce the amount of migration of fibroblasts. A complex series of intracellular functions must also be available in order to permit the motility of fibroblasts. Actinomycin D, cycloheximide, velban, colchicine and cytochalasin B reversibly inhibit the serum migration-promoting potential. The presence of contractile microfilaments has previously been shown to be necessary for cell motility [IO, 111. The 8-10 h delay after addition of serum before migration is detected is further indirect evidence for a cascade of intracellular interactions initiated by the serum migration-promoting factor that result in cell motility. Numerous cellular functions are intimately involved in the intermediate reactions that result in fibroblast migration.
Exptl Cell Res 80 (1973)
We wish to thank Dr R. W. Holley and Miss lngrid Klinger for their suggestion to pursue this study and for many stimulating discussions. This investigation was supported by a research grant from ACS (no. VC-78) (William Rankin and Elizabeth Forbes Rankin Memorial grant for Cancer Research).
References 1. Todaro, G J, Lazar, G K & Green, H, J cell camp physiol 66 (1965) 325. 2. Todaro, G J, Matsuya, G, Bloom, A, Robbins, A & Green, H, Growth regulating substances for animal cells in culture (ed V Defendi & M Stoker). Wistar inst symp monogr 7 (1967) 87. 3. Lipton, A, Klinger, I, Paul, D & Holley, R W, Proc natl acad sci US 68 (1971) 2799. 4. Holley, R W & Kiernan, J A, Proc natl acad sci US 60 (1968) 300. 5. Paul, D, Lipton, A & Klinger, I, Proc natl acad sci US 68 (1971) 645. 6. Lipton, A, Paul, D, Henahan, M, Klinger, I & Holley, R W, Exptl cell res 74 (1972) 466. 7. Raff, E C & Houck, J C, J cell physiol 74 (1969) 235. 8. Jainchill, J L & Todaro, G J, Exptl cell res 59 (1970) 137. 9. Holley, R W & Kiernan, J A, Growth control in cell cultures. CIBA Foundation symposium (ed J Knight & G E Wolstenholme) p. 3. Churchill, London (1971). 10. Carter, S‘B, kature 213 (1967) 261. 11. Gail. M H & Boone. C W. Exntl cell res 68 (1971)
Received May 15, 1973
Studies on serum stimulation of mouse fibroblast migration
serum. After centrifugation, the cells were washed twice in medium and plated at the appropriate density in 55-mm (Falcon) plastic dishes.
L. WOLF and A. LIPTON, Section of Oncology, Department of Medicine, The Milton S. Hershey Medical Center, Hershey, Pa 17033, USA
Migration
Summary. Evidence is presented that the factor present
in rat serum that uromotes the migration of mouse fi~i~sblas.s+(3T3) is a high mol. wtmolecule. Amino and Ca2+ are essential for full expression of aciivity. Once exposed to serum there is an 8-10 h lag period before cell migration can be detected. Cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B, all inhibit cellular migration from a wound edge.
The growth of fibroblasts in culture ceases when the population reaches a characteristic cell density. If a wound is then made by scratching such a confluent cell monolayer, cells migrate into the denuded area, synthesize DNA and divide [l, 21. A serum factor which stimulates the movement of cells and is thus responsible for ‘wound healing’ has been demonstrated [3]. The migration-promoting factor has been shown to be distinct from the growth and survival factors [3-51. In the present communication we describe some of the characteristics of the migration of mouse fibroblasts under the direction of the serum migration-promoting factor.
assay. Migration-promoting activity was assayed by the use of wounds in confluent 3T3 cells. The cultures were grown from lo5 cells cultured in Dulbecco & Vogt’s modification of Eagle’s medium with 10 % calf serum, in a CO2 incubator at 37°C for 5 days. The cultures were washed 3 times with serumfree medium, incubated for 15 min, and then 2 linear wounds (about 5 mm wide) were made in the monolayer with a wedge-tipped Teflon rod. The cultures were then washed twice more with serum-free medium, incubated in serum-free medium for 30 min, and washed again before the addition of 5 ml of serum-free medium plus the samples to be tested. After incubation for -the times inhicated, cultures were washed once with Tris-saline (pH 7.4), fixed for 5 min with 98 % acetic acid, 95 % ethanol 1 : 3, and stained for IO min with 0.5 % crystal violet in 95 % ethanol. The stained cultures were washed twice with distilled water and dried at room temperature. The number of cells migrating from the wound edge was determined with an ocular net micrometer, 1 x 1 cm (Unitron N3M), in a wide-field x 12.5 eyepiece, and-a x 6.3 obiective. Ten different fields (each 1.6 x 1.6 mm contiguous with the wound edge) were counted for each sample assayed, and the migrating cells per field were averaged.
of serum. All the described operations were performed at 0-4°C. All sera were stored at - 20°C and were tested without heating. Sephadex (Pharmacis) gels susnended in solutions at the appropriate pH and-salt co&. were equilibrated for 334 days prior to use. Columns were packed and serum (at the appropriate pH) was applied to the column. Rat serum fractionation with ammonium sulfate was performed with undiluted serum and with serum diluted I : IO with water. Solutions were stirred vigorously during the addition of the salt.
Fractionation
Materials and Methods Blood of rats (Sprague Dawley) was obtained by heart puncture. Serum was obtained by allowing the blood to clot at room temperature for 60 min. Cohn fractions were purchased from Nutritional Biochemicals Corp. Pituitary hormones were kindly supplied by Dr James Lewis, Scripps Clinic and Research Foundation, La Jolla, Calif. Other hormones were purchased from commercial sources. Actinomycin D was purchased from Sigma; colchitine and cycloheximide from Nutritional Biochemicals Corp.; velban from Lilly and cytochalasin B from Imperial Chemical Industry, Ltd. Cells and culture media. The 3T3 cells were originally obtained from Dr Dennis Cunningham, Department of Microbiology, University of California, Irvine, Calif. Cells were cultured in Dulbecco & Vogt’s modification of Eagle’s medium with 10 % calf serum. The cells were transformed using 0.05 % trypsin in Ca2+and Mg2+-free Tris-saline solution, pH 7.4; the action of the trypsin was terminated by the addition of
Results Serum and urine have been shown to be a ready source of migration-promoting activity for 3T3 cells in all species tested [3]. Gel filtration at pH 7.4 reveals that the migration-promoting activity in human serum is due to the presence of a high mol. wt molecule (fig. 1). (Indeed this activity is retained by Amicon UM 10 and PM 30 filters.) Serum migration promoting activity is not depleted by growing or resting 3T3 or SV3T3 cells. The serum migration activity is stable for up to 6 months if stored at 4°C. Serum from a hepatectomized rat (6-12 h Exptl Cell Res 80 (1973)
500 Preliminary notes
MW 700.000
MW 160,000
MW67,OOO
MW 45,000
MW 12,384
1.60I .40I .20-
-240
l.OO.80-
-200
.60.40-
- 1.60
.20-1.20
-
2
20
22
24
26
28
30
32
34
36
38
40
42
44
46
46
50
52
54
56
58
60 62
64
66
68
so
70 60
Fig. I. Abscissa: fraction no.; ordinate: (left) absorbance at 280 nm and 300 nm; (right) migration index. n-m, A,,, and Azeo; O-O, 3T3 migration-promoting activity. Gel filtration of 8 ml rat serum on Sephadex G-200 column (80 x 3 cm) at pH 7.4, 0.01 M Tris buffer. 5 ml fractions were collected. 0.4 ml of each fraction was assayed. The following markers were used: cytochrome c (12 384); ovalbumen (45 000); bovine albumin (67 000); human y globulin (160 000); thyroglobulin (700 000).
after surgery) contains the same amount of migration-promoting activity as does serum from normal adult rats. Ammonium sulfate fractionation of rat serum at pH 8.0 spreads the migration-promoting activity for 3T3 cells throughout all fractions. This pattern resembled that obtained for growth-promoting activity [5]. Table 1. Migration-promoting activity dialysed rat serum in deficient media
Media
Migrationpromoting activity (X)
Complete Amino acids Calcium Magnesium Calcium and magnesium
100 40 2 12 Cells come off dish
of
0.05 ml of the appropriate medium was added to the standard wound assay. After 24 h incubation, cultures were fixed and counted as described under Materials and Methods. Exptl CeN Res 80 (1973)
The following materials were assayed and were found to be inactive in promoting migration of 3T3 cells-AMP, CAMP, dibutyryl-CAMP, l-thyroxine, bradykinin, testosterone, 17-P-estradiol, FSH, LH, ACTH, TSH, somatotropin, prolactin, prostaglandin E, melantonin, insulin, epinephrine and hydrocortisone. Combining each of these compounds with 3T3 or SV3T3 growth factor [5] also failed to cause migration of 3T3 cells. The serum migration-promoting activity was also not affected by the presence of dexamethasone (0.02-2.0 pg/ml) in the medium. Steroids are known to retard woundhealing in man. 3T3 cells will not migrate into a denuded area in the absence of serum. Serum alone is, however, not sufficient to achieve migration of mouse fibroblasts. The migration of 3T3 cells is dependent upon the presence of amino acids, calcium, and magnesium in the medium. In the absence of any of these, the migration-
Preliminary notes promoting capacity of that particular serum is not fully expressed (table 1). When complete medium is replaced, the migrationpromoting activity is restored. The time course of cell migration under the influence of serum was next studied. There is a lag period of 8-10 h after addition of serum to a wound edge before significant migration can be detected (table 2). If serum is present for a stated time interval (e.g. O-4, O-8, or O-12 h) and then replaced with serum-free medium, further migration is not seen. There would thus appear to be an 8-10 h lag period after addition of serum before any cell migration is seen. In addition, serum must constantly be present to have continued (maximal) cell movement. The availability of chemicals which inhibit a variety of cell functions allows us to study the role of these functions in fibroblast migration under the influence of serum. Calf serum plus varying concentrations of each inhibitory substance were added to the standard wounded monolayer. Serum-induced migration of fibroblasts is inhibited by cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B (table 3). After 24-h exposure in each case this inhibiTable 2. Relationship between cell migration and time after exposure to serum in a wounded 3T3 culture
Hours
Average number of cells migrating from 1.6 mm wound edge
0 2 4 7 II 24
0.07 0.05 0.22 0.35 2.43 6.10
0.05 ml calf serum was added to wounded confluent 3T3 cultures at time zero. At the appropriate interval cultures were fixed and counted as described under Materials and Methods.
501
Table 3. Inhibition of serum-induced jibroblast migration Average
Sample
number of cells migrating from I .6 mm wound edge
E0.05 ml calf serum (CS) CS -i-cycloheximide (0.5-5 pg/ml) CS + velban (0.005-l pg/ml) CS + colchicine (0.03-0.5 pg/ml) CS + act D (0.05-l pg/ml) CS + cytochalasin B (0.05-10 ,ug/ml)
0.07 5.60 0.00 0.00 0.00 0.00 0.00
3T3 cells were prepared as described in the standard wound assay. 0.05 ml calf serum plus various concentrations of each inhibitor were added at time zero. At 24 h of incubation cultures were fixed and counted. All cultures were still viable at 24 h as replacement with fresh medium containing calf serum would result in the expected migration.
tion is reversible if the cultures are washed and replaced with fresh serum-containing medium. These same cells then migrate in normal fashion once free of inhibitory substances. If inhibitors are added at 0, 4, 7, 10 or 13 h after addition of calf serum, further migration is prevented. Discussion The migration of 3T3 mouse fibroblasts into a wound depends upon the presence of serum [3]. This is contrary to an earlier conclusion of other authors [7]. The serum migrationpromoting activity is a large molecule (greater than 200 000 D) that is pepsin labile [3]. The serum migration-promoting activity has been physically separated from other factors present in serum that promote cell growth and survival. Serum growth factors are depleted by resting or growing 3T3 cells [8, 91. Neither the migration-promoting factor nor the survival factor [6] is depleted under these conditions. The migration-promoting Exptl Cell Res 80 (1973)
502 Preliminary
notes
activity and growth factors are present in human urine. The migration of a cell into a denuded area is a complex phenomenon. The serum migration-promoting activity must be present. Amino acids, calcium and magnesium must also be present for the full expression of the migration potential. Absence of any of these components will reduce the amount of migration of fibroblasts. A complex series of intracellular functions must also be available in order to permit the motility of fibroblasts. Actinomycin D, cycloheximide, velban, colchicine and cytochalasin B reversibly inhibit the serum migration-promoting potential. The presence of contractile microfilaments has previously been shown to be necessary for cell motility [IO, 111. The 8-10 h delay after addition of serum before migration is detected is further indirect evidence for a cascade of intracellular interactions initiated by the serum migration-promoting factor that result in cell motility. Numerous cellular functions are intimately involved in the intermediate reactions that result in fibroblast migration.
Exptl Cell Res 80 (1973)
We wish to thank Dr R. W. Holley and Miss lngrid Klinger for their suggestion to pursue this study and for many stimulating discussions. This investigation was supported by a research grant from ACS (no. VC-78) (William Rankin and Elizabeth Forbes Rankin Memorial grant for Cancer Research).
References 1. Todaro, G J, Lazar, G K & Green, H, J cell camp physiol 66 (1965) 325. 2. Todaro, G J, Matsuya, G, Bloom, A, Robbins, A & Green, H, Growth regulating substances for animal cells in culture (ed V Defendi & M Stoker). Wistar inst symp monogr 7 (1967) 87. 3. Lipton, A, Klinger, I, Paul, D & Holley, R W, Proc natl acad sci US 68 (1971) 2799. 4. Holley, R W & Kiernan, J A, Proc natl acad sci US 60 (1968) 300. 5. Paul, D, Lipton, A & Klinger, I, Proc natl acad sci US 68 (1971) 645. 6. Lipton, A, Paul, D, Henahan, M, Klinger, I & Holley, R W, Exptl cell res 74 (1972) 466. 7. Raff, E C & Houck, J C, J cell physiol 74 (1969) 235. 8. Jainchill, J L & Todaro, G J, Exptl cell res 59 (1970) 137. 9. Holley, R W & Kiernan, J A, Growth control in cell cultures. CIBA Foundation symposium (ed J Knight & G E Wolstenholme) p. 3. Churchill, London (1971). 10. Carter, S‘B, kature 213 (1967) 261. 11. Gail. M H & Boone. C W. Exntl cell res 68 (1971)
Received May 15, 1973