X)21-972X/78/0046-0001$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 1 Printed in U.S.A.
EPIDERMAL GROWTH FACTOR STIMULATES SECRETION OF HUMAN CHORIONIC GONADOTROPIN BY CULTURED HUMAN CHORIOCARCINOMA CELLS R. Benveniste, K.V.. Speeg, Jr., G. Carpenter., S. Cohen, J. Lindner and D. Rabinowitz Departments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232 ABSTRACT. Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCGalpha. Epidermal growth factor is a polypeptide with a molecular weight of about 6,000, which has been isolated from the submaxillary gland of the male mouse (1) and from human urine (2). The chemical and biological characteristics of EGF have been reviewed (3). It has been reported that JEG-3, a human choriocarcinoma cell line, secretes biologically active hCG (4) and free hCG-alpha subunit (5). We now report that EGF binds to JEG-3 cells, stimulates the secretion of hCG and to a lesser extent free hCG-alpha. This observation suggests for the first time a role for EGF as a trophic modulator of hormonal secretion.
The medium of treated cultures was collected daily, centrifuged to remove cell debris, and then stored at -20°C until hCG or hCG-alpha was assayed. After removal of culture medium, the cell sheet was collected by scraping, washed with 0.9% NaCl, disrupted by sonication, and assayed for protein by the method of Lowry et al. (6) using bovine serum albumin (BSA) as a standard. EGF was isolated from human urine (2) or mouse submaxillary gland (1) by methods previously reported. The preparation of 125i_E3f ancj t n e procedure for measuring specific binding of EGF directly to monolayers of cultured cells has been described elsewhere (1, 2). The concentrations of immunoreactive hCG and hCG-alpha were determined by homologous radioimmunoassays (RIA); there was no significant crossreactivity of hCG-alpha in the hCG RIA, or of hCG in the hCG-alpha RIA (7).
MATERIALS AND METHODS The JEG-3 cells were generously provided by Dr. P. 0. Kohler (Baylor Univ. Sch. of Med. ). Cells were cultured in plastic flasks in Dulbecco's Modified Eagle Medium (DMEM) enriched with 10% fetal calf serum (FCS) at 37°C after gassing with 90% air-10% C02- For each experiment, cells were collected with trypsin-EDTA, pooled, aliquoted to replicate plastic flasks, and fed DMEM containing 10% FCS. Specific experimental manipulation is described in the Figure legends.
RESULTS Removal of JEG-3 cells from serumcontaining medium resulted in a sharp decrease in the secretion of hCG and hCG-alpha into the culture medium (Figure 1A & IB). The decrease of hCGalpha secretion slightly preceded the decline of hCG secretion. Addition for 24, 48, or 72 hours of EGF effected a 2-to 3-fold increase in the amount of hCG secreted into the culture medium (Figure 1A). Maximal stimulation was reached after 24 hours and extending this period of exposure failed to cause a further rise in hCG but main-
Submitted September 25, 1977 Supported by grants from the National Institute of Health: 1-R01HD-10128-01; the American Cancer Society: 1N-25P-4; HD00700, and HD05797. 169
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RAPID COMMUNICATIONS
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JCE & M • 1978 Vol 46 • No 1
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Fig. 1. Effect of EGF upon hCG and hCG-alpha secretion in JEG-3 cells grown in serum-free medium. Cells were subcultured.and fed in DMEM+10% FCS. 48 hours later, the cells were refed with serum-free DMEM (Day 0 ) . On Day 4, mEGF (20 ng/ml) was added for the indicated times. The EGF solution contained BSA such that 100 yg BSA was dispensed to each flask. 100 yg BSA without EGF was given to control flasks during the period EGF was present. All cells were refed at 24 hour intervals with serum-free medium with or without EGF. At the indicated times, the medium was assayed for hCG (A) and hCG-alpha (B) and cell sheet for protein content. Each point represents the average +/SEM for three flasks. tained a plateau level of stimulation. Removal of EGF was followed by the disappearance of the stimulatory effect with hCG levels decreasing toward those of control flasks with a half-time of about two days. The effect of EGF on free hCG-alpha secretion (Figure IB) was smaller than that observed for hCG, resulting in only a modest increase (about 20%) of free alpha subunit secretion relative to the levels found in controls. The influence of the composition of the cell medium on EGF-stimulated hCG secretion is shown in Figure 2A. In all media investigated, EGF induced about a two-fold increase in hCG secretion relative to control flasks. The hCG levels found in control flasks varied with the proteins added to DMEM, and maximal secretion was ob-
tained in medium fortified with 10% calf serum (CS). Figure 2B indicates that in all media investigated, a smaller effect of EGF on hCG-alpha secretion was observed. The binding of iodinated hEGF (5yci/yg) was assessed in dishes containing subconfluent JEG-3 cells. The hormone (72,000 cpm in 1.5 ml of DMEM-0.1% BSA) was added to dishes containing 6.5x10$ attached cells, with or without an excess (20 yg) of unlabeled mEGF. After 1 hour of incubation at 37°C, dishes with 125ihEGF alone showed a mean binding of 10,500 * 300 cpm, while only 410 cpm ± 40 cpm were found in dishes to which 20 yg of unlabeled mEGF had been added. DISCUSSION Secretions of hCG and hCG-alpha by
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 16:49 For personal use only. No other uses without permission. . All rights reserved.
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D CONTROL • EGF
Fig. 2. Effect of EGF upon hCG and hCG-alpha secretion in JEG-3 cells in different media. Cells were subcultured and fed DMEM + 10% FCS. On Day 0, medium was changed and replaced each 24 hours with DMEM alone, DMEM + 0.1% BSA, DMEM + 10% gamma-Globulin-free calf serum, DMEM + 1% calf serum, and DMEM + 10% calf serum. Medium was added with or without 10 ng/ml of mEGF. Medium was assayed for hCG (A), and hCG-alpha (B). Results are expressed in ng of hCG or hCG-alpha per ml of the medium content of flasks collected 48 hours after DayO. the human choriocarcinoma JEG-3 cells were markedly influenced by the serum proteins present in the culture medium, and maximum secretion was attained in medium fortified with 10% CS (Figure 2A and 2B). This is consistent with the rapid decline of both hCG and hCG-alpha following transfer of JEG-3 cells from 10% FCS medium to serum-free medium (Figure 1A and IB). A similar decline in hCG secretion has been observed in BeWo choriocarcinoma cells (8). In all media investigated EGF elicited an enhanced secretion of hCG. Only a modest effect was observed on hCG-alpha secretion. From the results shown in Figure 1A, it appears that the effect of EGF on hCG secretion requires the continuous presence of the growth factor since withdrawal of EGF from the medium is followed by a decline of hCG to control levels. Also, the decay of the stimulatory effect following removal of EGF seems independent of the length of cell exposure to EGF. As shown in Figure IB, the EGF effect on hCGalpha is more modest, slightly different from control values. However, it is possible that the overall production of alpha subunit was stimulated by EGF with a large part of the newly synthesized subunit used in the production of the intact hormone. We have observed in JEG-3 cells, dibutyryl cAMP stimulates hCG and
hCG-alpha to about the same extent (Benveniste and Speeg, in preparation) whereas EGF results in little increase in free alpha subunit. This difference in the effects of EGF and of dibutyryl cAMP may reflect the experimental conditions since dibutyryl cAMP induces a much greater stimulation than does EGF, or may be due to a difference in the mechanism of action between EGF and dibutyryl cAMP. The pattern of secretion of hCG during pregnancy is well defined with peak levels attained at the end of first trimester. Little is known about the control of placental hCG synthesis and secretion. The JEG-3 cells have been shown to retain several characteristics of the normal syncytiotrophoblastic cell (4). The data presented in this report demonstrate the binding of EGF and suggest the presence on JEG-3 cells of high affinity receptors. Interestingly, high levels of EGF receptor have been detected in term placenta (9). Our data provide no evidence that EGF exerts a physiological effect on hCG secretion, but this notion is worthy of exploration. REFERENCES 1. Carpenter, G., K.J. Lembach, M.M. Morrison, and S. Cohen, Characterization of the binding of 1251-
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labeled EGF to human fibroblasts, J Biol Chem, 250, 4297, 1975. 2 5 Carpenter, G., and S. Cohen, ' I labeled EGF: Binding, internalization, and degradation in human fibroblasts, J Cell Biol, 7T_f 159 1976. Carpenter, G., and S. Cohen, In Litwack, G., (ed.), Biochemical actions of hormones, Vol. 5, Academic Press, New York, In press. Kohler, P.O., and W.E. Bridson, Isolation of hormone-producing clonal lines of human choriocarcinoma, J Clin Endocrinol Metab, 32, 683, 1971. Benveniste, R., M. Conway, S. Cohen, D. Puett, and D. Rabinowitz, Secretion of choriogonadotropin and of a large form of alpha subunit by cultured human choriocarcinoma cells, Clin Research, 25, 48A, 1977.
JCK& M • 197* Vol 4
Vol. 46, No. 1 Printed in U.S.A.
EPIDERMAL GROWTH FACTOR STIMULATES SECRETION OF HUMAN CHORIONIC GONADOTROPIN BY CULTURED HUMAN CHORIOCARCINOMA CELLS R. Benveniste, K.V.. Speeg, Jr., G. Carpenter., S. Cohen, J. Lindner and D. Rabinowitz Departments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232 ABSTRACT. Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCGalpha. Epidermal growth factor is a polypeptide with a molecular weight of about 6,000, which has been isolated from the submaxillary gland of the male mouse (1) and from human urine (2). The chemical and biological characteristics of EGF have been reviewed (3). It has been reported that JEG-3, a human choriocarcinoma cell line, secretes biologically active hCG (4) and free hCG-alpha subunit (5). We now report that EGF binds to JEG-3 cells, stimulates the secretion of hCG and to a lesser extent free hCG-alpha. This observation suggests for the first time a role for EGF as a trophic modulator of hormonal secretion.
The medium of treated cultures was collected daily, centrifuged to remove cell debris, and then stored at -20°C until hCG or hCG-alpha was assayed. After removal of culture medium, the cell sheet was collected by scraping, washed with 0.9% NaCl, disrupted by sonication, and assayed for protein by the method of Lowry et al. (6) using bovine serum albumin (BSA) as a standard. EGF was isolated from human urine (2) or mouse submaxillary gland (1) by methods previously reported. The preparation of 125i_E3f ancj t n e procedure for measuring specific binding of EGF directly to monolayers of cultured cells has been described elsewhere (1, 2). The concentrations of immunoreactive hCG and hCG-alpha were determined by homologous radioimmunoassays (RIA); there was no significant crossreactivity of hCG-alpha in the hCG RIA, or of hCG in the hCG-alpha RIA (7).
MATERIALS AND METHODS The JEG-3 cells were generously provided by Dr. P. 0. Kohler (Baylor Univ. Sch. of Med. ). Cells were cultured in plastic flasks in Dulbecco's Modified Eagle Medium (DMEM) enriched with 10% fetal calf serum (FCS) at 37°C after gassing with 90% air-10% C02- For each experiment, cells were collected with trypsin-EDTA, pooled, aliquoted to replicate plastic flasks, and fed DMEM containing 10% FCS. Specific experimental manipulation is described in the Figure legends.
RESULTS Removal of JEG-3 cells from serumcontaining medium resulted in a sharp decrease in the secretion of hCG and hCG-alpha into the culture medium (Figure 1A & IB). The decrease of hCGalpha secretion slightly preceded the decline of hCG secretion. Addition for 24, 48, or 72 hours of EGF effected a 2-to 3-fold increase in the amount of hCG secreted into the culture medium (Figure 1A). Maximal stimulation was reached after 24 hours and extending this period of exposure failed to cause a further rise in hCG but main-
Submitted September 25, 1977 Supported by grants from the National Institute of Health: 1-R01HD-10128-01; the American Cancer Society: 1N-25P-4; HD00700, and HD05797. 169
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 16:49 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
170
JCE & M • 1978 Vol 46 • No 1
A.
2500-
EGF •—• o—a
j
2000-
B.
EGF A—1\ •—«
1500 -
O—D
500
1\
1000
1000-
500 -
500 -
n 2
3
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9
DAYS IN SERUM-FREE MEDIUM
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Fig. 1. Effect of EGF upon hCG and hCG-alpha secretion in JEG-3 cells grown in serum-free medium. Cells were subcultured.and fed in DMEM+10% FCS. 48 hours later, the cells were refed with serum-free DMEM (Day 0 ) . On Day 4, mEGF (20 ng/ml) was added for the indicated times. The EGF solution contained BSA such that 100 yg BSA was dispensed to each flask. 100 yg BSA without EGF was given to control flasks during the period EGF was present. All cells were refed at 24 hour intervals with serum-free medium with or without EGF. At the indicated times, the medium was assayed for hCG (A) and hCG-alpha (B) and cell sheet for protein content. Each point represents the average +/SEM for three flasks. tained a plateau level of stimulation. Removal of EGF was followed by the disappearance of the stimulatory effect with hCG levels decreasing toward those of control flasks with a half-time of about two days. The effect of EGF on free hCG-alpha secretion (Figure IB) was smaller than that observed for hCG, resulting in only a modest increase (about 20%) of free alpha subunit secretion relative to the levels found in controls. The influence of the composition of the cell medium on EGF-stimulated hCG secretion is shown in Figure 2A. In all media investigated, EGF induced about a two-fold increase in hCG secretion relative to control flasks. The hCG levels found in control flasks varied with the proteins added to DMEM, and maximal secretion was ob-
tained in medium fortified with 10% calf serum (CS). Figure 2B indicates that in all media investigated, a smaller effect of EGF on hCG-alpha secretion was observed. The binding of iodinated hEGF (5yci/yg) was assessed in dishes containing subconfluent JEG-3 cells. The hormone (72,000 cpm in 1.5 ml of DMEM-0.1% BSA) was added to dishes containing 6.5x10$ attached cells, with or without an excess (20 yg) of unlabeled mEGF. After 1 hour of incubation at 37°C, dishes with 125ihEGF alone showed a mean binding of 10,500 * 300 cpm, while only 410 cpm ± 40 cpm were found in dishes to which 20 yg of unlabeled mEGF had been added. DISCUSSION Secretions of hCG and hCG-alpha by
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171
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D CONTROL • EGF
Fig. 2. Effect of EGF upon hCG and hCG-alpha secretion in JEG-3 cells in different media. Cells were subcultured and fed DMEM + 10% FCS. On Day 0, medium was changed and replaced each 24 hours with DMEM alone, DMEM + 0.1% BSA, DMEM + 10% gamma-Globulin-free calf serum, DMEM + 1% calf serum, and DMEM + 10% calf serum. Medium was added with or without 10 ng/ml of mEGF. Medium was assayed for hCG (A), and hCG-alpha (B). Results are expressed in ng of hCG or hCG-alpha per ml of the medium content of flasks collected 48 hours after DayO. the human choriocarcinoma JEG-3 cells were markedly influenced by the serum proteins present in the culture medium, and maximum secretion was attained in medium fortified with 10% CS (Figure 2A and 2B). This is consistent with the rapid decline of both hCG and hCG-alpha following transfer of JEG-3 cells from 10% FCS medium to serum-free medium (Figure 1A and IB). A similar decline in hCG secretion has been observed in BeWo choriocarcinoma cells (8). In all media investigated EGF elicited an enhanced secretion of hCG. Only a modest effect was observed on hCG-alpha secretion. From the results shown in Figure 1A, it appears that the effect of EGF on hCG secretion requires the continuous presence of the growth factor since withdrawal of EGF from the medium is followed by a decline of hCG to control levels. Also, the decay of the stimulatory effect following removal of EGF seems independent of the length of cell exposure to EGF. As shown in Figure IB, the EGF effect on hCGalpha is more modest, slightly different from control values. However, it is possible that the overall production of alpha subunit was stimulated by EGF with a large part of the newly synthesized subunit used in the production of the intact hormone. We have observed in JEG-3 cells, dibutyryl cAMP stimulates hCG and
hCG-alpha to about the same extent (Benveniste and Speeg, in preparation) whereas EGF results in little increase in free alpha subunit. This difference in the effects of EGF and of dibutyryl cAMP may reflect the experimental conditions since dibutyryl cAMP induces a much greater stimulation than does EGF, or may be due to a difference in the mechanism of action between EGF and dibutyryl cAMP. The pattern of secretion of hCG during pregnancy is well defined with peak levels attained at the end of first trimester. Little is known about the control of placental hCG synthesis and secretion. The JEG-3 cells have been shown to retain several characteristics of the normal syncytiotrophoblastic cell (4). The data presented in this report demonstrate the binding of EGF and suggest the presence on JEG-3 cells of high affinity receptors. Interestingly, high levels of EGF receptor have been detected in term placenta (9). Our data provide no evidence that EGF exerts a physiological effect on hCG secretion, but this notion is worthy of exploration. REFERENCES 1. Carpenter, G., K.J. Lembach, M.M. Morrison, and S. Cohen, Characterization of the binding of 1251-
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172
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labeled EGF to human fibroblasts, J Biol Chem, 250, 4297, 1975. 2 5 Carpenter, G., and S. Cohen, ' I labeled EGF: Binding, internalization, and degradation in human fibroblasts, J Cell Biol, 7T_f 159 1976. Carpenter, G., and S. Cohen, In Litwack, G., (ed.), Biochemical actions of hormones, Vol. 5, Academic Press, New York, In press. Kohler, P.O., and W.E. Bridson, Isolation of hormone-producing clonal lines of human choriocarcinoma, J Clin Endocrinol Metab, 32, 683, 1971. Benveniste, R., M. Conway, S. Cohen, D. Puett, and D. Rabinowitz, Secretion of choriogonadotropin and of a large form of alpha subunit by cultured human choriocarcinoma cells, Clin Research, 25, 48A, 1977.
JCK& M • 197* Vol 4
