Proc. Nati. Acad. Sci. USA Vol. 74, No. 4, pp. 1619-1621, April 1977 Cell Biology
Local starvation for epidermal growth factor cannot explain density-dependent inhibition of normal human glial cells (growth control/cell density/epidermal growth factor -receptors)
BENGT WESTERMARK The Wallenberg Laboratory, University of Uppsala, S-751 22 Uppsala, Sweden
Communicated by George Klein, January 24, 1977
ABSTRACT Mouse epidermal growth factor (mEGF) is a potent growth promoter of human glial cells in sparse cultures, whereas very little stimulation of growth in dense cultures is induced by the factor. In the present communication, the possibility that the density-dependent inhibition is caused by a reduced binding/uptake of the factor was scrutinized. It was found that the number of mEGF binding sites was 20,000 and 35,000 per cell in sparse and dense cultures, respectively. The dissociation constant of the binding reaction was not influenced by the cell density. It was concluded that crowded cells are not starved for the factor add that a decrease in number or affinity of the EGF receptors can be excluded as a cause of the inhibition. Two basically different hypotheses have been proposed to explain why normal surface-attached cells in culture cease to multiply under crowded conditions. The original one supposed that contact between cells elicited signals (1) that were inhibitory for the cell cycle. The hypothesis derived its main support from the intimate correlation between local density and cell multiplication and the fact that inhibition does not seem to start until cells begin to touch each other (2, 3). During the last few years the "contact inhibition" theory has been contradicted by a number of experimental results recently reviewed by Holley (4). Many center around the importance of the serum concentration in controlling the terminal cell density (5) and in stimulating mitoses in density-inhibited cultures (6). In an extension of such studies, Stoker (7) demonstrated that local starvation of crowded cells for a growth factor may be caused by a juxtacellular "diffusion boundary layer." Alternatively, a reduced uptake of a growth factor may be due to a diminished cell surface area of crowded cells (8-10). So far the evidence for local starvation as a cause of density-dependent growth inhibition has been entirely circumstantial because decisive experiments on the uptake and metabolism of growth factors have been hampered by lack of pure and potent molecules. Recently, the purified and well-characterized polypeptide, mouse epidermal growth factor (mEGF) (11, 12), has been found to stimulate growth of cultured fibroblastic (13-15) and glial (16) cells. Human glial cells (16) could be stimulated in the absence of serum proteins other than albumin. Using iodinated EGF, Cohen and collaborators (15, 17) have shown that the factor binds to a membrane receptor and is then transferred within and degraded. Using basically the same technology in this investigation, I have scrutinized the possibility that the density inhibition of the mEGF response of glial cells (16) is due to a lowered binding/uptake of the molecule.
MATERIAL AND METHODS Cell Line and Culture Conditions. The normal human glial line U-787 CG was initiated from nonneoplastic brain tissue as Abbreviation: mEGF, mouse epidermal growth factor.
1619
described (18). The growth characteristics of this cell line (19) as well as of other human normal glial lines (18) have been published. The cells were routinely grown in Nunc 50 mm petri dishes in Ham's nutrient medium F-10 with 10% fetal calf serum (Grand Island Biochemical Co.), 50 ,g/ml of streptomycin, and 100 units/ml of penicillin. Medium was renewed twice a week. Subcultivation was performed after trypsinization (0.25% trypsin in phosphate-buffered saline). The cultures were kept at 370 in a humidified atmosphere containing 5% CO2. Dense cultures were established by seeding trypsinized glial cells at a 1:3 split ratio from confluent cultures into 50 mm Nunc petri dishes 14 days before the actual experiment. Medium was renewed twice a week. After 14 days, stationary dense monolayers had formed (18). Sparse cultures were initiated 2 days before the experiment at a 1:6 split ratio. lodination of mEGF. mEGF was kindly provided by Stanley Cohen, Vanderbilt University, Nashville, TN and was labeled with 125I (carrier free, obtained from Radiochemical Centre, Amersham, U.K.) by the chloramine-T method (20). Specific activity is given in each experiment. Assay of 12SI-Labeled mEGF Binding to Glial Cells. Each culture was washed twice with F-10 medium containing 1 mg/ml of human serum albumin and then given 1.5 ml of such medium. 1251-labeled mEGF was added at indicated concentrations. After incubation at 37° for the given time, the cultures were washed nine times with ice-cold phosphate-buffered saline (Dulbecco's A solution, supplemented with 0.1 mg/ml of MgSO4-7H20 and CaC12, respectively), containing 1% calf serum. The cells were then lysed in 0.3 M NaOH and the radioactivity was determined in a Beckman gamma counter. Nonspecific Binding. This binding was determined in the presence of 10 ,g of unlabeled mEGF, and values of 1-3% were subtracted from experimental values. Growth of Glial Cells in mEGF Containing Medium. Glial cells from confluent cultures were treated with trypsin, pooled, and seeded at a 1:6 split ratio into 50 mm Nunc dishes using 5 ml per dish of F-10 medium with 1% fetal calf serum. The low concentration of serum was required for long-term survival (16). mEGF was added at 1, 5, and 20 ng/ml final concentration, respectively. Controls were incubated without mEGF. The respective medium was renewed three times a week. At intervals, cell numbers were determined on duplicates from each set of cultures. Cells were counted in an electronic cell counter (Celloscope, AB Lars Ljungberg, Stockholm, Sweden). RESULTS AND DISCUSSION Fig. 1 shows that the binding of '25I-labeled mEGF reached saturable levels with saturation at a lower degree of binding per cell in the sparse than in the dense cultures. Scatchard plots (21) (Fig. 2) of the binding data indicated that, at saturation, 20,000 molecules were bound per cell in sparse cultures and 35,000 molecules per cell in dense cultures. Recently it has been shown
~2
Proc. Natl. Acad. Sci. USA 74 (1977)
Cell Biology: Westermark
1620
a
Q
a)
4L-
Co 10
CLL 11
., 0
-
0X
E CO 0
'lo-
-0
-
U.0
0)0 (0~~~~~~~~~~~~~~~~~1
15
10 1251-labeled mEGF, nM
5
0
5
10
Time, hr
FIG. 1. Concentration dependence of the binding of 12WI-labeled mEGF to sparse (O--- -0; 14 X 103 cells per cm2) and dense (0-0; 83 X 103 cells per cm2) cultures of glial cells. Each culture received 1.5 ml of F-10 medium with 1 mg of human serum albumin/ml and 1251-labeled mEGF (14,230 cpm/ng) was added at different concentrations. After 60 min of incubation at 37°, the cultures were harvested and the radioactivity was determined as described in the
FIG. 3. Kinetics of the binding of 125I-labeled mEGF to sparse (O--- -0; 15 X 103 cells per CM2) and dense (0-0; 77 X 103 cells per cm2) cultures of glial cells during continuous exposure to the factor. Cultures were given 1.5 ml of F-10 medium with 1 mg of human serum albumin/ml and 6.4 ng of 1251-labeled mEGF/ml (1.1 nm; 156,360 cpm/ng) and incubated at 37'. At intervals, duplicates from each set were harvested and the radioactivity was determined as described in
text.
the text.
that also the amount of insulin receptors on BALB 3T3 cells was increased in dense compared to sparse cultures (22). The apparent dissociation constants of the 125I-labeled mEGF binding reactions were 0.50 nM and 0.54 nM, respectively; these values are similar to that for human fibroblasts (14, 15). For the initiation of DNA synthesis, a maximal response was obtained with 1-2 ng/ml (0.17-0.33 nM) of mEGF in both sparse and dense glial cultures (16) and thus well below saturation of binding, which indicates spare receptors (23) as on fibroblasts (14, 15).
0.5o
X a)
m
10
20
0
40
60
'251-labeled mEGF bound, pM
FIG. 2. Scatchard plots of the data of Fig. 1. The upper panel represents sparse cultures and the lower panel dense cultures.
Stationary glial cells have to be continuously exposed to mEGF for at least 12 hr before any cells are committed to DNA synthesis (24). The kinetics of mEGF binding to sparse and
dense cultures during this lag period should therefore be of interest (Fig. 3). In both cultures, there was initially a rapid increase in cell associated radioactivity which then leveled off until a slow decrease finally occurred. Control experiments showed that, the decrease was not due to a depletion of the labeled compound. The phenomenon is supposed to represent a decrease in amount of binding sites (17, 25) due to internal uptake of the ligand-receptor complex. Though a minor difference in slope of the respective curves can be noted, the kinetics of the binding to sparse and dense cultures were very similar. However, as in Fig. 1, sparse cells bound less mEGF than did dense cells. This difference was also found when
binding was assayed at 00. The rate of disappearance of cell-bound 125I-labeled mEGF was also determined. Cultures were labeled for 60 min, extensively washed, and reincubated at 370; the remaining cellassociated radioactivity was determined at intervals. Only a small difference in radioactivity could be noted between sparse (half-life, 30 min) and dense (half-life, 40 min) cultures. There is therefore no evidence for an increased degradation of the factor in crowded cultures as has been suggested (4). The binding data on sparse and dense glial cell cultures exclude the possibility that the difference in response to mEGF (16) is related to cellular starvation for the factor as postulated by the diffusion boundary layer theory (7). A decrease in number and/or affinity of the receptors can likewise be excluded. Because cell crowding does not lead to a diminished access to mEGF, increasing the factor concentration above the optimal dose level of 1-2 ng/ml (16) should not lead to any further increase in terminal cell density. Growth curves (Fig. 4) demonstrate the lack of correspondence between mEGF concentration and terminal cell density. In this respect, mEGF differs from whole serum (5, 26). The fact that terminal cell density
increases with the serum concentration may be due to the presence of several growth factors in serum. In fact, the cell may have a whole set of receptors with individual specificity for
Cell Biology: Westermark
Proc. Natl. Acad. Sci. USA 74 (1977)
so/
50
E) .-
X10
.*1
5
// I.,/
/
t//
0
3
7
13
20
Days
FIG. 4. Multiplication of glial cells at different concentrations of mEGF. mEGF was added at 1 (....), 5 (--- -), and 20 (-) ng/ml, respectively. Controls were incubated without mEGF (----.). All media contained 1% fetal calf serum. Cell numbers were determined at indicated time intervals.
different growth factors and the maximal, terminal cell density is not obtained unless all of these factors are present at optimal concentrations. Other candidates for such glial cell factors may be somatomedin B (27, 28), fibroblast growth factor (28, 29), and platelet factors (30-32). The concentrations of these factors in serum may be so low that an optimal concentration can never be obtained if unprocessed serum is used as the source. The author is indebted to Dr. Stanley Cohen, Vanderbilt University, Nashville, TN, U.S.A. for advice and a supply of pure mEGF. Dr. Carl-Henrik Heldin, Institute of Medical and Physiological Chemistry, Uppsala University, Uppsala, Sweden, kindly iodinated the growth factor. I thank Miss Annika Magnusson and Miss Marianne Pettersson for their skillful assistance. Supported by grants from the Swedish Cancer Society (Project No. 55-B75-11XA). 1. Stoker, M. G. P. (1964) Virology 24,164-174. 2. Siminovitch, J. & Axelrad, X. (1963) in Fifth Canadian Cancer
Conference, Honey Harbor (Academic Press, New York), pp. 149-165.
1621
3. Todaro, G. J., Green, H. & Goldberg, B. (1964) Proc. Natl. Acad. Sci. USA 51, 66-73. 4. Holley, R. W. (1975) Nature 258, 487-490. 5. Holley, R. W. & Kiernan, J. A. (1965) Proc. Natl. Acad. Sci. USA 53,12-19. 6. Todaro, G. J., Lazar, G. K. & Green, H. (1965) J. Cell. Comp. Physiol. 66,325-333. 7. Stoker, M. G. P. (1973) Nature 246,200-203. 8. Castor, L. N. (1968) J. Cell. Physiol. 72,161-172. 9. Zetterberg, A. & Auer, G. (1970) Exp. Cell Res. 62, 262-270. 10. Dulbecco, R. & Elkington, J. (1973) Nature 246,197-199. 11. Cohen, S. & Savage, C. R., Jr. (1974) Recent Prog. Horm. Res. 30,551-572. 12. Cohen, S. & Taylor, J. M. (1974) Recent Prog. Horm. Res. 30, 533-550. 13. Armelin, H. A. (1973) Proc. Natl. Acad. Sci. USA 70, 27022706. 14. Hollenberg, M. D. & Cuatrecasas, P. (1973) Proc. Natl. Acad. Sci. USA 70,2964-2968. 15. Carpenter, H., Lembach, K. J., Morrison, M. M. & Cohen, S. (1975) J. Biol. Chem. 250, 4297-4304. 16. Westermark, B. (1976) Biochem. Biophys. Res. Commun. 69, 304-310. 17. Carpenter, G. & Cohen, S. (1977) J. Cell. Biol. 71, 159-171. 18. Ponten, J., Westermark, B. & Hugosson, R. (1969) Exp. Cell Res. 58,393-400. 19. Lindgren, A., Westermark, B. & Ponten, J. (1975) Exp. Cell Res. 95,311-319. 20. Hunter, W. M. & Greenwood, F. C. (1962) Nature 194, 495497. 21. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 22. Thomopoulus, P., Roth, J., Lovelace, E. & Pastan, I. (1976) Cell 8,417-423. 23. Cuatrecasas, P. (1975) in Adv. in Cyclic Nucleotide Research, eds. Drummond, G. I., Greengard, P. & Robinson, G. A. (Raven Press, New York), Vol. 5, pp. 79-104. 24. Lindgren, A. & Westermark, B. (1976) Exp. Cell Res. 99, 357362. 25. Gavin, J. R., III, Roth, J., Neville, D. M., de Meyts, P. & Buell, D. N. (1974) Proc. Natl. Acad. Sci. USA 71,84-88. 26. Westermark, B. (1971) Exp. Cell Res. 69, 259-264. 27. Fryklund, L., Uthne, K., Sievertsson, H. & Westermark, B. (1975) Biochem. Biophys. Res. Commun. 61, 950-956. 28. Westermark, B. & Wasteson, A. (1975) in Adv. Metabol. Disorders, eds. Luft, R. & Hall, K. (Academic Press, New York), Vol. 8, pp. 85-100. 29. Gospodarowicz, D. (1974) Nature 249, 123-127. 30. Balk, S. D. (1971) Proc. Natl. Acad. Sci. USA 68,271-275. 31. Ross, R., Glomset, J., Kariya, B. & Harker, L. (1974) Proc. Natl. Acad. Sci. USA 71,1207-1210. 32. Westermark, B. & Wasteson, A. (1976) Exp. Cell Res. 98, 170-174.
Local starvation for epidermal growth factor cannot explain density-dependent inhibition of normal human glial cells (growth control/cell density/epidermal growth factor -receptors)
BENGT WESTERMARK The Wallenberg Laboratory, University of Uppsala, S-751 22 Uppsala, Sweden
Communicated by George Klein, January 24, 1977
ABSTRACT Mouse epidermal growth factor (mEGF) is a potent growth promoter of human glial cells in sparse cultures, whereas very little stimulation of growth in dense cultures is induced by the factor. In the present communication, the possibility that the density-dependent inhibition is caused by a reduced binding/uptake of the factor was scrutinized. It was found that the number of mEGF binding sites was 20,000 and 35,000 per cell in sparse and dense cultures, respectively. The dissociation constant of the binding reaction was not influenced by the cell density. It was concluded that crowded cells are not starved for the factor add that a decrease in number or affinity of the EGF receptors can be excluded as a cause of the inhibition. Two basically different hypotheses have been proposed to explain why normal surface-attached cells in culture cease to multiply under crowded conditions. The original one supposed that contact between cells elicited signals (1) that were inhibitory for the cell cycle. The hypothesis derived its main support from the intimate correlation between local density and cell multiplication and the fact that inhibition does not seem to start until cells begin to touch each other (2, 3). During the last few years the "contact inhibition" theory has been contradicted by a number of experimental results recently reviewed by Holley (4). Many center around the importance of the serum concentration in controlling the terminal cell density (5) and in stimulating mitoses in density-inhibited cultures (6). In an extension of such studies, Stoker (7) demonstrated that local starvation of crowded cells for a growth factor may be caused by a juxtacellular "diffusion boundary layer." Alternatively, a reduced uptake of a growth factor may be due to a diminished cell surface area of crowded cells (8-10). So far the evidence for local starvation as a cause of density-dependent growth inhibition has been entirely circumstantial because decisive experiments on the uptake and metabolism of growth factors have been hampered by lack of pure and potent molecules. Recently, the purified and well-characterized polypeptide, mouse epidermal growth factor (mEGF) (11, 12), has been found to stimulate growth of cultured fibroblastic (13-15) and glial (16) cells. Human glial cells (16) could be stimulated in the absence of serum proteins other than albumin. Using iodinated EGF, Cohen and collaborators (15, 17) have shown that the factor binds to a membrane receptor and is then transferred within and degraded. Using basically the same technology in this investigation, I have scrutinized the possibility that the density inhibition of the mEGF response of glial cells (16) is due to a lowered binding/uptake of the molecule.
MATERIAL AND METHODS Cell Line and Culture Conditions. The normal human glial line U-787 CG was initiated from nonneoplastic brain tissue as Abbreviation: mEGF, mouse epidermal growth factor.
1619
described (18). The growth characteristics of this cell line (19) as well as of other human normal glial lines (18) have been published. The cells were routinely grown in Nunc 50 mm petri dishes in Ham's nutrient medium F-10 with 10% fetal calf serum (Grand Island Biochemical Co.), 50 ,g/ml of streptomycin, and 100 units/ml of penicillin. Medium was renewed twice a week. Subcultivation was performed after trypsinization (0.25% trypsin in phosphate-buffered saline). The cultures were kept at 370 in a humidified atmosphere containing 5% CO2. Dense cultures were established by seeding trypsinized glial cells at a 1:3 split ratio from confluent cultures into 50 mm Nunc petri dishes 14 days before the actual experiment. Medium was renewed twice a week. After 14 days, stationary dense monolayers had formed (18). Sparse cultures were initiated 2 days before the experiment at a 1:6 split ratio. lodination of mEGF. mEGF was kindly provided by Stanley Cohen, Vanderbilt University, Nashville, TN and was labeled with 125I (carrier free, obtained from Radiochemical Centre, Amersham, U.K.) by the chloramine-T method (20). Specific activity is given in each experiment. Assay of 12SI-Labeled mEGF Binding to Glial Cells. Each culture was washed twice with F-10 medium containing 1 mg/ml of human serum albumin and then given 1.5 ml of such medium. 1251-labeled mEGF was added at indicated concentrations. After incubation at 37° for the given time, the cultures were washed nine times with ice-cold phosphate-buffered saline (Dulbecco's A solution, supplemented with 0.1 mg/ml of MgSO4-7H20 and CaC12, respectively), containing 1% calf serum. The cells were then lysed in 0.3 M NaOH and the radioactivity was determined in a Beckman gamma counter. Nonspecific Binding. This binding was determined in the presence of 10 ,g of unlabeled mEGF, and values of 1-3% were subtracted from experimental values. Growth of Glial Cells in mEGF Containing Medium. Glial cells from confluent cultures were treated with trypsin, pooled, and seeded at a 1:6 split ratio into 50 mm Nunc dishes using 5 ml per dish of F-10 medium with 1% fetal calf serum. The low concentration of serum was required for long-term survival (16). mEGF was added at 1, 5, and 20 ng/ml final concentration, respectively. Controls were incubated without mEGF. The respective medium was renewed three times a week. At intervals, cell numbers were determined on duplicates from each set of cultures. Cells were counted in an electronic cell counter (Celloscope, AB Lars Ljungberg, Stockholm, Sweden). RESULTS AND DISCUSSION Fig. 1 shows that the binding of '25I-labeled mEGF reached saturable levels with saturation at a lower degree of binding per cell in the sparse than in the dense cultures. Scatchard plots (21) (Fig. 2) of the binding data indicated that, at saturation, 20,000 molecules were bound per cell in sparse cultures and 35,000 molecules per cell in dense cultures. Recently it has been shown
~2
Proc. Natl. Acad. Sci. USA 74 (1977)
Cell Biology: Westermark
1620
a
Q
a)
4L-
Co 10
CLL 11
., 0
-
0X
E CO 0
'lo-
-0
-
U.0
0)0 (0~~~~~~~~~~~~~~~~~1
15
10 1251-labeled mEGF, nM
5
0
5
10
Time, hr
FIG. 1. Concentration dependence of the binding of 12WI-labeled mEGF to sparse (O--- -0; 14 X 103 cells per cm2) and dense (0-0; 83 X 103 cells per cm2) cultures of glial cells. Each culture received 1.5 ml of F-10 medium with 1 mg of human serum albumin/ml and 1251-labeled mEGF (14,230 cpm/ng) was added at different concentrations. After 60 min of incubation at 37°, the cultures were harvested and the radioactivity was determined as described in the
FIG. 3. Kinetics of the binding of 125I-labeled mEGF to sparse (O--- -0; 15 X 103 cells per CM2) and dense (0-0; 77 X 103 cells per cm2) cultures of glial cells during continuous exposure to the factor. Cultures were given 1.5 ml of F-10 medium with 1 mg of human serum albumin/ml and 6.4 ng of 1251-labeled mEGF/ml (1.1 nm; 156,360 cpm/ng) and incubated at 37'. At intervals, duplicates from each set were harvested and the radioactivity was determined as described in
text.
the text.
that also the amount of insulin receptors on BALB 3T3 cells was increased in dense compared to sparse cultures (22). The apparent dissociation constants of the 125I-labeled mEGF binding reactions were 0.50 nM and 0.54 nM, respectively; these values are similar to that for human fibroblasts (14, 15). For the initiation of DNA synthesis, a maximal response was obtained with 1-2 ng/ml (0.17-0.33 nM) of mEGF in both sparse and dense glial cultures (16) and thus well below saturation of binding, which indicates spare receptors (23) as on fibroblasts (14, 15).
0.5o
X a)
m
10
20
0
40
60
'251-labeled mEGF bound, pM
FIG. 2. Scatchard plots of the data of Fig. 1. The upper panel represents sparse cultures and the lower panel dense cultures.
Stationary glial cells have to be continuously exposed to mEGF for at least 12 hr before any cells are committed to DNA synthesis (24). The kinetics of mEGF binding to sparse and
dense cultures during this lag period should therefore be of interest (Fig. 3). In both cultures, there was initially a rapid increase in cell associated radioactivity which then leveled off until a slow decrease finally occurred. Control experiments showed that, the decrease was not due to a depletion of the labeled compound. The phenomenon is supposed to represent a decrease in amount of binding sites (17, 25) due to internal uptake of the ligand-receptor complex. Though a minor difference in slope of the respective curves can be noted, the kinetics of the binding to sparse and dense cultures were very similar. However, as in Fig. 1, sparse cells bound less mEGF than did dense cells. This difference was also found when
binding was assayed at 00. The rate of disappearance of cell-bound 125I-labeled mEGF was also determined. Cultures were labeled for 60 min, extensively washed, and reincubated at 370; the remaining cellassociated radioactivity was determined at intervals. Only a small difference in radioactivity could be noted between sparse (half-life, 30 min) and dense (half-life, 40 min) cultures. There is therefore no evidence for an increased degradation of the factor in crowded cultures as has been suggested (4). The binding data on sparse and dense glial cell cultures exclude the possibility that the difference in response to mEGF (16) is related to cellular starvation for the factor as postulated by the diffusion boundary layer theory (7). A decrease in number and/or affinity of the receptors can likewise be excluded. Because cell crowding does not lead to a diminished access to mEGF, increasing the factor concentration above the optimal dose level of 1-2 ng/ml (16) should not lead to any further increase in terminal cell density. Growth curves (Fig. 4) demonstrate the lack of correspondence between mEGF concentration and terminal cell density. In this respect, mEGF differs from whole serum (5, 26). The fact that terminal cell density
increases with the serum concentration may be due to the presence of several growth factors in serum. In fact, the cell may have a whole set of receptors with individual specificity for
Cell Biology: Westermark
Proc. Natl. Acad. Sci. USA 74 (1977)
so/
50
E) .-
X10
.*1
5
// I.,/
/
t//
0
3
7
13
20
Days
FIG. 4. Multiplication of glial cells at different concentrations of mEGF. mEGF was added at 1 (....), 5 (--- -), and 20 (-) ng/ml, respectively. Controls were incubated without mEGF (----.). All media contained 1% fetal calf serum. Cell numbers were determined at indicated time intervals.
different growth factors and the maximal, terminal cell density is not obtained unless all of these factors are present at optimal concentrations. Other candidates for such glial cell factors may be somatomedin B (27, 28), fibroblast growth factor (28, 29), and platelet factors (30-32). The concentrations of these factors in serum may be so low that an optimal concentration can never be obtained if unprocessed serum is used as the source. The author is indebted to Dr. Stanley Cohen, Vanderbilt University, Nashville, TN, U.S.A. for advice and a supply of pure mEGF. Dr. Carl-Henrik Heldin, Institute of Medical and Physiological Chemistry, Uppsala University, Uppsala, Sweden, kindly iodinated the growth factor. I thank Miss Annika Magnusson and Miss Marianne Pettersson for their skillful assistance. Supported by grants from the Swedish Cancer Society (Project No. 55-B75-11XA). 1. Stoker, M. G. P. (1964) Virology 24,164-174. 2. Siminovitch, J. & Axelrad, X. (1963) in Fifth Canadian Cancer
Conference, Honey Harbor (Academic Press, New York), pp. 149-165.
1621
3. Todaro, G. J., Green, H. & Goldberg, B. (1964) Proc. Natl. Acad. Sci. USA 51, 66-73. 4. Holley, R. W. (1975) Nature 258, 487-490. 5. Holley, R. W. & Kiernan, J. A. (1965) Proc. Natl. Acad. Sci. USA 53,12-19. 6. Todaro, G. J., Lazar, G. K. & Green, H. (1965) J. Cell. Comp. Physiol. 66,325-333. 7. Stoker, M. G. P. (1973) Nature 246,200-203. 8. Castor, L. N. (1968) J. Cell. Physiol. 72,161-172. 9. Zetterberg, A. & Auer, G. (1970) Exp. Cell Res. 62, 262-270. 10. Dulbecco, R. & Elkington, J. (1973) Nature 246,197-199. 11. Cohen, S. & Savage, C. R., Jr. (1974) Recent Prog. Horm. Res. 30,551-572. 12. Cohen, S. & Taylor, J. M. (1974) Recent Prog. Horm. Res. 30, 533-550. 13. Armelin, H. A. (1973) Proc. Natl. Acad. Sci. USA 70, 27022706. 14. Hollenberg, M. D. & Cuatrecasas, P. (1973) Proc. Natl. Acad. Sci. USA 70,2964-2968. 15. Carpenter, H., Lembach, K. J., Morrison, M. M. & Cohen, S. (1975) J. Biol. Chem. 250, 4297-4304. 16. Westermark, B. (1976) Biochem. Biophys. Res. Commun. 69, 304-310. 17. Carpenter, G. & Cohen, S. (1977) J. Cell. Biol. 71, 159-171. 18. Ponten, J., Westermark, B. & Hugosson, R. (1969) Exp. Cell Res. 58,393-400. 19. Lindgren, A., Westermark, B. & Ponten, J. (1975) Exp. Cell Res. 95,311-319. 20. Hunter, W. M. & Greenwood, F. C. (1962) Nature 194, 495497. 21. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 22. Thomopoulus, P., Roth, J., Lovelace, E. & Pastan, I. (1976) Cell 8,417-423. 23. Cuatrecasas, P. (1975) in Adv. in Cyclic Nucleotide Research, eds. Drummond, G. I., Greengard, P. & Robinson, G. A. (Raven Press, New York), Vol. 5, pp. 79-104. 24. Lindgren, A. & Westermark, B. (1976) Exp. Cell Res. 99, 357362. 25. Gavin, J. R., III, Roth, J., Neville, D. M., de Meyts, P. & Buell, D. N. (1974) Proc. Natl. Acad. Sci. USA 71,84-88. 26. Westermark, B. (1971) Exp. Cell Res. 69, 259-264. 27. Fryklund, L., Uthne, K., Sievertsson, H. & Westermark, B. (1975) Biochem. Biophys. Res. Commun. 61, 950-956. 28. Westermark, B. & Wasteson, A. (1975) in Adv. Metabol. Disorders, eds. Luft, R. & Hall, K. (Academic Press, New York), Vol. 8, pp. 85-100. 29. Gospodarowicz, D. (1974) Nature 249, 123-127. 30. Balk, S. D. (1971) Proc. Natl. Acad. Sci. USA 68,271-275. 31. Ross, R., Glomset, J., Kariya, B. & Harker, L. (1974) Proc. Natl. Acad. Sci. USA 71,1207-1210. 32. Westermark, B. & Wasteson, A. (1976) Exp. Cell Res. 98, 170-174.
