Initiation of 3T3 Fibroblast Cell Division by Epidermal Growth Factor STEVEN P. ROSE, REBECCA M. PRUSS AND HARVEY R. HERSCHMAN Department o f Biological Chemistry, UCLA School of Medicine and Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles, California WOO24
ABSTRACT Epidermal Growth Factor (EGF) at concentrations of lo-$ to lo-'' M initiates cell division in both confluent and low density non-dividing 3T3 cells. Four days after addition of EGF to confluent or low density non-dividing 3T3 cells there is a 2- and 5-fold increase, respectively, in cell number.
Macromolecular components present in serum are required for division of 3T3 murine fibroblasts in culture. The saturation density of these cells has been shown to be directly proportional to the concentration of serum in the growth medium (Holley and Kiernan, '71). Studies with confluent or low density cultures of 3T3 cells indicate that a number of factors are able to stimulate proliferation of non-dividing populations. Agents as diverse as serum (Bombik and Burger, '73), proteolytic enzymes (Burger, '70), phorbol myristate acetate (Sivak, '72), insulin (Bombik and Burger, '73) glucocorticoids (Thrash and Cunningham, '73), and the recently described Fibroblast Growth Factor (FGF) isolated from pituitary extracts (Gospodarowicz, '74; Rudland et al., '74), will initiate 3T3 fibroblast proliferation in cell culture. Epidermal Growth Factor (EGF) is a low molecular weight (6,000) polypeptide isolated from the male mouse submaxillary gland. Injection of purified EGF into neonatal mice causes precocious eye opening and tooth eruption (Cohen, '62), and stimulates keratinization of epidermal tissue (Cohen and Elliott, '63). In addition, it is a potent co-carcinogen for epidermal carcinogenesis (Reynolds et al., '65; Rose et al., in preparation). EGF is extremely stable, can easily be isotopically labeled and retain biological activity (Covelli et al., '72a), is simply purified in relatively large quantities (Savage and Cohen, '72), and has been completely sequenced (Savage et al., '72). Stimulation of incorporaJ. CELL. PHYSIOL., 86: 593-598.
tion of labeled precursors into DNA, RNA and protein of HeLa and KB cells (Covelli et al., '72b), and into DNA of cultured human fibroblasts (Hollenberg and Cuatrecasas, '73) by EGF has been reported, and Hollenberg and Cuatrecasas ('73) have recently suggested that "the polypeptide may serve as a model compound for those substances in serum which promote cell growth." We present here a study of the ability of EGF to initiate cell division in non-dividing confluent and low-density cultures of 3T3 cells. MATERIALS AND METHODS
EGF purification The initial steps in our EGF purification were identical to those described by Bocchini and Angeletti ('69) for Nerve Growth Factor (NGF). The submaxillary glands from 300 male Swiss-Webster mice (3540 g body weight) were homogenized for two minutes in cold water (3 ml/g glands) using a Waring blender, followed by homogenization in a Potter homogenizer for one minute. After centrifugation for 30 minutes at 23,3000 x g the supernatant was diluted with a volume of cold water equal to 50% of its volume. To nine volumes of the supernatant was slowly added one volume of 0.2 M streptomycin sulfate in 0.1 M Tris-HC1 buffer of pH 7.5. After 30 minutes on ice, the mixture was centrifuged at 23,300 X g for 30 minutes and the supernatant was lyophilized. One milliliter of water/gram of Received Jan. 7, '75. Accepted Apr. 1, '75.
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glands was added to the lyophilized supernatant and the material which did not go into solution was removed by centrifugation at 16,300 X g for 20 minutes. The supernatant was applied to an upwardflow Sephadex G-100 column ( 5 x 90 c m ) equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5) containing 5 x M EDTA. The column was run at 25 ml/hr, and 13 ml fractions were collected. NGF and EGF eluted from the column i n well separated peaks. Fractions from the G 1 0 0 column were assayed for EGF by double immunodiffusion on a microscope slide (Ouchterlony and Nilsson, '73), using an antiserum kindly provided by Dr. Stanley Cohen. The EGF containing fractions from two G-100 column runs (derived from the submaxillary gland extract of 600 mice) were combined, diluted with a n equal volume of water and loaded onto a DEAE-cellulose column ( 1.7 X 16.8 c m ) which had been equilibrated with 0.02 M Tris-HC1 buffer, pH 7.5. After a 50 ml wash with equilibrating buffer, the EGF was eluted with a salt gradient prepared by pumping 0.1 M NaCl in equilibrating buffer into a 200 ml constant volume mixing chamber containing equilibrating buffer. Fractions of 15 ml were collected. Two peaks with EGF activity, as judged by reactivity in the immunodiffusion assay and ability to elicit early eyelid opening and tooth eruption in neonatal mice (Cohen, '62), were eluted from the column. The EGF was concentrated using an Arnicon ultrafiltration unit with a UM-2 membrane. The recovery of EGF i n the two peaks ranged between 30 and 48 mg in various EGF preparations. Polyacrylumide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was carried out as described by Weber and Osborn ('69). Polyacrylamide gel electrophoresis at pH 9.5 was done by the method of Davis ('64) with the exception that the gels were pre-run at 2 majtube for 15 minutes before the application of the protein samples and no spacer gel was used. Polyacrylamide gel electrophoresis at pH 9.5 in the presence of p-mercaptoethanol, was a modification
of the Davis ('64) system in which 10 mM p-mercaptoethanol was present in the polyacrylamide gels and in the reservoir buffer solutions. Before electrophoresis in this system, the purified EGF solution was incubated for two hours at 37" in the presence of 10 mM p-mercaptoethanol. Gels were stained with 0.25% Coomassie blue in 45% methanol and 9% acetic acid and destained in 20% ethanol and 7.5% acetic acid. Cell growth
Swiss 3T3 cells (clone 42) were obtained from Dr. C. Fred Fox. Cells were grown at 37" in a 10% CO, atmosphere in 60 m m tissue culture plates (Falcon) in 5 ml of Dulbecco's Modified Eagle's medium (DME, Gibco) containing 5% (except where indicated) fetal calf serum (FCS, Reheis), 100 units/ml penicillin and 100 yg/ml streptomycin. Cell counts Cells were harvested with 1 ml of a 0.05% solution of trypsin i n Saline A (Puck et al., '51) containing 0.02% EDTA. One ml of DME containing 10% FCS was immediately added to prevent cell clumping. Cell numbers represent the average of eight haemocytometer counts of the cells from a single plate. Protein determinations
The concentration of the purified EGF was determined using the value for EiTm at 280 of 30.9 (Taylor et. al., '72). RESULTS
EGF purification. In our EGF purification procedure, two peaks with EGF activity were eluted from the DEAE-cellulose column by the salt gradient. Savage and Cohen ('72) using an ammonium acetate gradient elution of EGF from DEAEkellulose also observe two peaks with EGF activity. EGF eluting in the first peak, used for the experiments described subsequently, was analyzed by pH 9.5, SDS and pH 9.5-p-mercaptoethanol polyacrylamide gel electrophoresis (fig. 1 ) . In both the SDS and pH 9.5-p-mercaptoethanol systems only a single protein band with rapid
EGF STIMULATION OF CELL DIVISION
595
Fig. 1 Polyacrylamide gel electrophoresis of the DEAE peak I EGF fractions in ( a ) the pH 9.5, ( b ) sodium dodecyl sulfate a n d ( c ) p H 9.5-p-mercaptoethanol systems. The amount of protein loaded onto t h e gels (left to right) w a s 52 pg, 87 p g , 46 p g , 92 pg, 33 pg and 66 fig.
mobility is seen suggesting that the protein bands in the pH 9.5 system (fig. l a , right gel) are aggregate forms of the EGF molecule. Initiation of cell division by EGF. Addition of 13 ng/ml EGF to confluent quiescent 3T3 cultures results i n a marked stimulation in the rate of incorporation of "-thymidine into TCA precipitable material (data not shown). Maximal incorporation occurs approximately 24 hours after EGF addition. Similar results have previously been reported with human fibroblasts by Hollenberg and Cuatrecasas ('73). In order to determine whether EGF could initiate cell division (as opposed to stimulation of thymidine incorporation)
we counted cell number daily following addition of EGF to non-dividing confluent 3T3 cultures (fig. 2 top). A doubling in total cell number occurs four days after addition of EGF to the confluent cultures. 3T3 cells can be maintained at low cell density well below confluency, in a concentration of serum which does not allow cell division, but maintains cell viability. A fetal calf serum concentration of 0.125% in DME is able to maintain viability of 3T3 cells at a density of 5 x lo4 cells per 60 m m plate for at least six days. If additional serum is added to cells maintained at this density, growth curves identical to those obtained directly in 10% serum are observed (data not shown). EGF is also able to stimulate cell division
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Fig. 2 ( T o p ) EGF induced initiation of cell division of confluent non-dividing 3T3 cells. Cells were plated at 5 x lo4 cells per plate in 5 m l of medium. Five days after plating the cultures were fed with fresh medium. On day 8, control plates (0) received 0.1 ml of conditioned medium (obtained from additional day 8 plates ), while experimental plates ( 0 )received 0.1 ml of conditioned medium containing 32.5 ng of EGF. Final concentration of EGF was thus 6.5 ng/ml. Each point represents 8 haemocytometer counts from a single plate. ( B o t t o m ) EGF induced initiation of cell division of low density 3T3 cell. Cells were plated at 5 x lo4 cells per 60 nim plate in medium containing 10% FCS. This medium was removed eight hours later, and replaced with DME containing 0.125% FCS. Control plates received 0.1 ml of conditioned medium on day 1 ( 3 )while experimental plates received 0.1 ml of conditioned medium containing 32.5 ng of EGF either on day 1 alone ( 0 ) or on day 1 and again on day 3 ( 0 ) .Cell counts were determined as described in figure 2 ( t o p ) .
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EGF STIMULATION OF CELL DIVISION
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I I 1 0.1 1.o 10.0 EGF (ngiml medium) Fig. 3 Concentration dependence of EGF induced initiation of cell division in confluent 3T3 cultures. Cultures were grown as described in figure 2 ( t o p ) . O n day 8, varying concentrations of EGF were added. Cell number was determined four days later as described in figure 2. I
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in cultures of non-dividing 3T3 cells which are at a low density, and are not in contact with one another. Addition of EGF at a concentration of 6.5 ng/ml to low-density non-dividing 3T3 cultures results in an approximately 5-fold increase in cell number after four days (fig 2 bottom). Concentration of EGF required for initiation of cell division. EGF dependent initiation of cell division in confluent quiescent 3T3 cultures occurs at very low concentrations. A detectable increase in cell number can be observed at concentrations in the 1 ng/ml range. A maximal effect is observed at 6.5 ng/ml (fig. 3). This factor is thus effective at concentrations on the order of 10- to lo-'' M. DISCUSSION
These studies demonstrate that EGF is a potent initiator of 3T3 cell division at concentrations ( 1 0 - ~ - 1 0 - ~ M~) which are in the range of the circulating levels of EGF in mouse serum, and well below (by nearly 2 orders of magnitude) that of EGF in mouse milk (Byyny et al., '74). Thus, there is good reason to believe that its ability to stimulate incorporation of 3H-thy-
midine and initiate cell division in nondividing 3T3 cells reflects relevant biological activity for the protein in developing animals. Insulin and Fibroblast Growth Factor are the only other purified polypeptide growth factors reported to initiate cell division in 3T3 cultures. Only Fibroblast Growth Factor and EGF are active at concentrations in the physiological range for polypeptide hormones. Insulin is apparently required in much higher concentrations when used alone to stimulate DNA synthesis (Bombik and Burger, '73; Holley and Kiernan, '74). Gospodarowicz ('74) reports that purified Fibroblast Growth Factor yields from bovine pituitary glands average about 1 mg/kg. FGF activity is 5-fold greater in whole brain, but i t is apparently more difficult to purify FGF from brain than pituitary (Gospodarowicz, '74). In contrast, the purification procedure of Savage and Cohen ('72) yields 500-700 mg of EGF per kilogram of mouse salivary tissue, while our purification procedure yields 250-400 mg/kg. The ready accessibility, small size, stability and known primary structure of EGF make it an ideal subject for chemical modification
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S. P. ROSE, R. M. PRUSS AND H. R. HERSCHMAN
studies to determine a “minimal requirement” for polypeptide initiation of DNA synthesis and/or cell division in nondividing cells. Recent studies with Fibroblast Growth Factor, glucocorticoids, insulin, and serum fractions have demonstrated that various combinations of these agents act synergistically to initiate DNA synthesis and cell division in different clones of Swiss 3T3 and Balb 3T3 cells (Rudsland et al., ’74; Holley and Kiernan, ’74). Experiments are now in progress’ to study the requirements and interactions with glucocoriticoids, serum, and insulin in the EGF syFtem. ACKNOWLEDGMENTS
This work was supported by Grant CA 15276-01 from the National Cancer Institute and Contract AT (04-1) GEN-12 between the Atomic Energy Commission and the University of California RMP was supported by NIH Training Grant GM 00364. LITERATURE CITED Bocchini, V., and P. U. Angeletti 1969 The nerve growth factor: Purification as a 30,000molecular-weight protein. Proc. Nat. Acad. Sci. (U.S.A.), 64: 787-794. Bombik, B. M., and M. M. Burger 1973 c-AMP and the cell cycle: Inhibition of growth stimulation. Exptl. Cell Res., 80: 88-94. Burger, M. M. 1970 Proteolytic enzymes initiating cell division and escape from contact inhibition of growth. Nature, 227: 17C-171. Byyny, R. L., D. N. Orth, S. Cohen and E. Doyne 1974 Epidermal growth factor: Effects of androgens and adrenergic agents. Endocrinology, 95: 776-782. Cohen, S. 1962 Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening i n the new-born animal. J. Biol. Chem., 237: 1555-1562. Cohen, S., and G.A. Elliott 1963 The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. Invest. Dermat., 40: 1-5. Covelli, I., R. Rossi, R. Mozzi and L. Frati 1972a Synthesis of bioactive 13lI-labeled epidermal growth factor and its distribution i n rat tissues. Eur. J. Biochem., 27: 225-230. Covelli, I., R. Mozzi, R. Rossi and L. Frati 197213 The mechanism of action of epidermal growth factor. Hormones, 3: 183-191.
Davis, B. J. 1964 Disc electrophoresis-I1 method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121: 404-427. Gospodarowicz, D. 1974 Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone o n 3T3 cell growth. Nature, 249: 123-127. Hollenberg, M. D., and P. Cuatrecasas 1973 Epidermal growth factor: Receptors in human fibroblasts and modulation of action by cholera toxin. Proc. Nat. Acad. Sci., (U.S.A.), 70: 2964-2968. Holley, R. W., and J. A. Kiernan 1971 Studies of serum factors required by 3T3 and SV 3T3 cells. In: Ciba Foundation Symposium o n Growth Control i n Cell Cultures. G. E. W. Wolstenholme and J. Knight, eds. Churchill Livingstone (London), pp. 3-10. Holley, R. W., and J. A. Kiernan 1974 Control of the initiation of DNA synthesis in 3T3 cells: Serum factors. Proc. Nat. Acad. Sci. (U.S.A.), 71 : 2908-291 1 . Ouchterlony, O., and L. A. Nilsson 1973 Immunodiffusion and immunoelectrophoresis. In: Handbook of Experimental Immunology. D. M. Weir, ed. Blackwell Scientific Publications, pp. 19, 1-19, 39. Puck, T. T., S. J. Cieciura and H. W. Fisher 1951 Clonal growth in &To of human cells with fibroblastic morphology. J. Exptl. Med., 106: 145-157. Reynolds, V. H., F. H. Boehm and S. Cohen 1965 Enhancement of chemical carcinogenesis by a n epidermal growth factor. Surgical Forum, 16: 108-109. Rudland, P. S., W. Seifert and D. Gospodarowicz 1974 Growth control i n cultured mouse fibroblasts: Induction of the pleiotypic and mitogenic responses by a purified growth factor. Proc. Nat. Acad. Sci. (U.S.A.), 71: 2600-2604. Savage, C. R., and S. Cohen 1972 Epidermal growth factor and a new derivative, rapid isolation procedures and biological and chemical characterization, J. Biol. Chem., 247: 76097611. Savage, C. R., T. Inagami and S. Cohen 1972 The primary structure of epidermal growth factor. J. Biol. Chem., 247: 7612-7621. Sivak, A. 1972 Induction of cell division: Role of cell membrane sites. J .Cell. Physiol., 80: 167-174. Taylor, J. M., W. M. Mitchell and S. Cohen 1972 Epidermal Growth Factor. Physical and Chemical Properties. J. Biol. Chem., 247: 5928-5934. Thrash, C. R., and D. D. Cunningham 1973 Stimulation of division of density inhibited fibroblasts by glucocorticoids. Nature, 242: 399401. Weber, K., and M. Osborn 1969 The reliability of molecular weight determinations by dodecyl sulfate-aolvacrvlamide gel electrophoresis. J. Biol. Chem., 244: 4406-4412.
ABSTRACT Epidermal Growth Factor (EGF) at concentrations of lo-$ to lo-'' M initiates cell division in both confluent and low density non-dividing 3T3 cells. Four days after addition of EGF to confluent or low density non-dividing 3T3 cells there is a 2- and 5-fold increase, respectively, in cell number.
Macromolecular components present in serum are required for division of 3T3 murine fibroblasts in culture. The saturation density of these cells has been shown to be directly proportional to the concentration of serum in the growth medium (Holley and Kiernan, '71). Studies with confluent or low density cultures of 3T3 cells indicate that a number of factors are able to stimulate proliferation of non-dividing populations. Agents as diverse as serum (Bombik and Burger, '73), proteolytic enzymes (Burger, '70), phorbol myristate acetate (Sivak, '72), insulin (Bombik and Burger, '73) glucocorticoids (Thrash and Cunningham, '73), and the recently described Fibroblast Growth Factor (FGF) isolated from pituitary extracts (Gospodarowicz, '74; Rudland et al., '74), will initiate 3T3 fibroblast proliferation in cell culture. Epidermal Growth Factor (EGF) is a low molecular weight (6,000) polypeptide isolated from the male mouse submaxillary gland. Injection of purified EGF into neonatal mice causes precocious eye opening and tooth eruption (Cohen, '62), and stimulates keratinization of epidermal tissue (Cohen and Elliott, '63). In addition, it is a potent co-carcinogen for epidermal carcinogenesis (Reynolds et al., '65; Rose et al., in preparation). EGF is extremely stable, can easily be isotopically labeled and retain biological activity (Covelli et al., '72a), is simply purified in relatively large quantities (Savage and Cohen, '72), and has been completely sequenced (Savage et al., '72). Stimulation of incorporaJ. CELL. PHYSIOL., 86: 593-598.
tion of labeled precursors into DNA, RNA and protein of HeLa and KB cells (Covelli et al., '72b), and into DNA of cultured human fibroblasts (Hollenberg and Cuatrecasas, '73) by EGF has been reported, and Hollenberg and Cuatrecasas ('73) have recently suggested that "the polypeptide may serve as a model compound for those substances in serum which promote cell growth." We present here a study of the ability of EGF to initiate cell division in non-dividing confluent and low-density cultures of 3T3 cells. MATERIALS AND METHODS
EGF purification The initial steps in our EGF purification were identical to those described by Bocchini and Angeletti ('69) for Nerve Growth Factor (NGF). The submaxillary glands from 300 male Swiss-Webster mice (3540 g body weight) were homogenized for two minutes in cold water (3 ml/g glands) using a Waring blender, followed by homogenization in a Potter homogenizer for one minute. After centrifugation for 30 minutes at 23,3000 x g the supernatant was diluted with a volume of cold water equal to 50% of its volume. To nine volumes of the supernatant was slowly added one volume of 0.2 M streptomycin sulfate in 0.1 M Tris-HC1 buffer of pH 7.5. After 30 minutes on ice, the mixture was centrifuged at 23,300 X g for 30 minutes and the supernatant was lyophilized. One milliliter of water/gram of Received Jan. 7, '75. Accepted Apr. 1, '75.
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glands was added to the lyophilized supernatant and the material which did not go into solution was removed by centrifugation at 16,300 X g for 20 minutes. The supernatant was applied to an upwardflow Sephadex G-100 column ( 5 x 90 c m ) equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5) containing 5 x M EDTA. The column was run at 25 ml/hr, and 13 ml fractions were collected. NGF and EGF eluted from the column i n well separated peaks. Fractions from the G 1 0 0 column were assayed for EGF by double immunodiffusion on a microscope slide (Ouchterlony and Nilsson, '73), using an antiserum kindly provided by Dr. Stanley Cohen. The EGF containing fractions from two G-100 column runs (derived from the submaxillary gland extract of 600 mice) were combined, diluted with a n equal volume of water and loaded onto a DEAE-cellulose column ( 1.7 X 16.8 c m ) which had been equilibrated with 0.02 M Tris-HC1 buffer, pH 7.5. After a 50 ml wash with equilibrating buffer, the EGF was eluted with a salt gradient prepared by pumping 0.1 M NaCl in equilibrating buffer into a 200 ml constant volume mixing chamber containing equilibrating buffer. Fractions of 15 ml were collected. Two peaks with EGF activity, as judged by reactivity in the immunodiffusion assay and ability to elicit early eyelid opening and tooth eruption in neonatal mice (Cohen, '62), were eluted from the column. The EGF was concentrated using an Arnicon ultrafiltration unit with a UM-2 membrane. The recovery of EGF i n the two peaks ranged between 30 and 48 mg in various EGF preparations. Polyacrylumide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was carried out as described by Weber and Osborn ('69). Polyacrylamide gel electrophoresis at pH 9.5 was done by the method of Davis ('64) with the exception that the gels were pre-run at 2 majtube for 15 minutes before the application of the protein samples and no spacer gel was used. Polyacrylamide gel electrophoresis at pH 9.5 in the presence of p-mercaptoethanol, was a modification
of the Davis ('64) system in which 10 mM p-mercaptoethanol was present in the polyacrylamide gels and in the reservoir buffer solutions. Before electrophoresis in this system, the purified EGF solution was incubated for two hours at 37" in the presence of 10 mM p-mercaptoethanol. Gels were stained with 0.25% Coomassie blue in 45% methanol and 9% acetic acid and destained in 20% ethanol and 7.5% acetic acid. Cell growth
Swiss 3T3 cells (clone 42) were obtained from Dr. C. Fred Fox. Cells were grown at 37" in a 10% CO, atmosphere in 60 m m tissue culture plates (Falcon) in 5 ml of Dulbecco's Modified Eagle's medium (DME, Gibco) containing 5% (except where indicated) fetal calf serum (FCS, Reheis), 100 units/ml penicillin and 100 yg/ml streptomycin. Cell counts Cells were harvested with 1 ml of a 0.05% solution of trypsin i n Saline A (Puck et al., '51) containing 0.02% EDTA. One ml of DME containing 10% FCS was immediately added to prevent cell clumping. Cell numbers represent the average of eight haemocytometer counts of the cells from a single plate. Protein determinations
The concentration of the purified EGF was determined using the value for EiTm at 280 of 30.9 (Taylor et. al., '72). RESULTS
EGF purification. In our EGF purification procedure, two peaks with EGF activity were eluted from the DEAE-cellulose column by the salt gradient. Savage and Cohen ('72) using an ammonium acetate gradient elution of EGF from DEAEkellulose also observe two peaks with EGF activity. EGF eluting in the first peak, used for the experiments described subsequently, was analyzed by pH 9.5, SDS and pH 9.5-p-mercaptoethanol polyacrylamide gel electrophoresis (fig. 1 ) . In both the SDS and pH 9.5-p-mercaptoethanol systems only a single protein band with rapid
EGF STIMULATION OF CELL DIVISION
595
Fig. 1 Polyacrylamide gel electrophoresis of the DEAE peak I EGF fractions in ( a ) the pH 9.5, ( b ) sodium dodecyl sulfate a n d ( c ) p H 9.5-p-mercaptoethanol systems. The amount of protein loaded onto t h e gels (left to right) w a s 52 pg, 87 p g , 46 p g , 92 pg, 33 pg and 66 fig.
mobility is seen suggesting that the protein bands in the pH 9.5 system (fig. l a , right gel) are aggregate forms of the EGF molecule. Initiation of cell division by EGF. Addition of 13 ng/ml EGF to confluent quiescent 3T3 cultures results i n a marked stimulation in the rate of incorporation of "-thymidine into TCA precipitable material (data not shown). Maximal incorporation occurs approximately 24 hours after EGF addition. Similar results have previously been reported with human fibroblasts by Hollenberg and Cuatrecasas ('73). In order to determine whether EGF could initiate cell division (as opposed to stimulation of thymidine incorporation)
we counted cell number daily following addition of EGF to non-dividing confluent 3T3 cultures (fig. 2 top). A doubling in total cell number occurs four days after addition of EGF to the confluent cultures. 3T3 cells can be maintained at low cell density well below confluency, in a concentration of serum which does not allow cell division, but maintains cell viability. A fetal calf serum concentration of 0.125% in DME is able to maintain viability of 3T3 cells at a density of 5 x lo4 cells per 60 m m plate for at least six days. If additional serum is added to cells maintained at this density, growth curves identical to those obtained directly in 10% serum are observed (data not shown). EGF is also able to stimulate cell division
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Fig. 2 ( T o p ) EGF induced initiation of cell division of confluent non-dividing 3T3 cells. Cells were plated at 5 x lo4 cells per plate in 5 m l of medium. Five days after plating the cultures were fed with fresh medium. On day 8, control plates (0) received 0.1 ml of conditioned medium (obtained from additional day 8 plates ), while experimental plates ( 0 )received 0.1 ml of conditioned medium containing 32.5 ng of EGF. Final concentration of EGF was thus 6.5 ng/ml. Each point represents 8 haemocytometer counts from a single plate. ( B o t t o m ) EGF induced initiation of cell division of low density 3T3 cell. Cells were plated at 5 x lo4 cells per 60 nim plate in medium containing 10% FCS. This medium was removed eight hours later, and replaced with DME containing 0.125% FCS. Control plates received 0.1 ml of conditioned medium on day 1 ( 3 )while experimental plates received 0.1 ml of conditioned medium containing 32.5 ng of EGF either on day 1 alone ( 0 ) or on day 1 and again on day 3 ( 0 ) .Cell counts were determined as described in figure 2 ( t o p ) .
597
EGF STIMULATION OF CELL DIVISION
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I I 1 0.1 1.o 10.0 EGF (ngiml medium) Fig. 3 Concentration dependence of EGF induced initiation of cell division in confluent 3T3 cultures. Cultures were grown as described in figure 2 ( t o p ) . O n day 8, varying concentrations of EGF were added. Cell number was determined four days later as described in figure 2. I
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in cultures of non-dividing 3T3 cells which are at a low density, and are not in contact with one another. Addition of EGF at a concentration of 6.5 ng/ml to low-density non-dividing 3T3 cultures results in an approximately 5-fold increase in cell number after four days (fig 2 bottom). Concentration of EGF required for initiation of cell division. EGF dependent initiation of cell division in confluent quiescent 3T3 cultures occurs at very low concentrations. A detectable increase in cell number can be observed at concentrations in the 1 ng/ml range. A maximal effect is observed at 6.5 ng/ml (fig. 3). This factor is thus effective at concentrations on the order of 10- to lo-'' M. DISCUSSION
These studies demonstrate that EGF is a potent initiator of 3T3 cell division at concentrations ( 1 0 - ~ - 1 0 - ~ M~) which are in the range of the circulating levels of EGF in mouse serum, and well below (by nearly 2 orders of magnitude) that of EGF in mouse milk (Byyny et al., '74). Thus, there is good reason to believe that its ability to stimulate incorporation of 3H-thy-
midine and initiate cell division in nondividing 3T3 cells reflects relevant biological activity for the protein in developing animals. Insulin and Fibroblast Growth Factor are the only other purified polypeptide growth factors reported to initiate cell division in 3T3 cultures. Only Fibroblast Growth Factor and EGF are active at concentrations in the physiological range for polypeptide hormones. Insulin is apparently required in much higher concentrations when used alone to stimulate DNA synthesis (Bombik and Burger, '73; Holley and Kiernan, '74). Gospodarowicz ('74) reports that purified Fibroblast Growth Factor yields from bovine pituitary glands average about 1 mg/kg. FGF activity is 5-fold greater in whole brain, but i t is apparently more difficult to purify FGF from brain than pituitary (Gospodarowicz, '74). In contrast, the purification procedure of Savage and Cohen ('72) yields 500-700 mg of EGF per kilogram of mouse salivary tissue, while our purification procedure yields 250-400 mg/kg. The ready accessibility, small size, stability and known primary structure of EGF make it an ideal subject for chemical modification
598
S. P. ROSE, R. M. PRUSS AND H. R. HERSCHMAN
studies to determine a “minimal requirement” for polypeptide initiation of DNA synthesis and/or cell division in nondividing cells. Recent studies with Fibroblast Growth Factor, glucocorticoids, insulin, and serum fractions have demonstrated that various combinations of these agents act synergistically to initiate DNA synthesis and cell division in different clones of Swiss 3T3 and Balb 3T3 cells (Rudsland et al., ’74; Holley and Kiernan, ’74). Experiments are now in progress’ to study the requirements and interactions with glucocoriticoids, serum, and insulin in the EGF syFtem. ACKNOWLEDGMENTS
This work was supported by Grant CA 15276-01 from the National Cancer Institute and Contract AT (04-1) GEN-12 between the Atomic Energy Commission and the University of California RMP was supported by NIH Training Grant GM 00364. LITERATURE CITED Bocchini, V., and P. U. Angeletti 1969 The nerve growth factor: Purification as a 30,000molecular-weight protein. Proc. Nat. Acad. Sci. (U.S.A.), 64: 787-794. Bombik, B. M., and M. M. Burger 1973 c-AMP and the cell cycle: Inhibition of growth stimulation. Exptl. Cell Res., 80: 88-94. Burger, M. M. 1970 Proteolytic enzymes initiating cell division and escape from contact inhibition of growth. Nature, 227: 17C-171. Byyny, R. L., D. N. Orth, S. Cohen and E. Doyne 1974 Epidermal growth factor: Effects of androgens and adrenergic agents. Endocrinology, 95: 776-782. Cohen, S. 1962 Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening i n the new-born animal. J. Biol. Chem., 237: 1555-1562. Cohen, S., and G.A. Elliott 1963 The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J. Invest. Dermat., 40: 1-5. Covelli, I., R. Rossi, R. Mozzi and L. Frati 1972a Synthesis of bioactive 13lI-labeled epidermal growth factor and its distribution i n rat tissues. Eur. J. Biochem., 27: 225-230. Covelli, I., R. Mozzi, R. Rossi and L. Frati 197213 The mechanism of action of epidermal growth factor. Hormones, 3: 183-191.
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