0021-972X/90/7102-0329$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 2 Printed in U.S.A.
Epidermal Growth Factor (EGF) Receptors in Human Chorionic Gonadotropin-Producing Tumor: Transplantation in Nude Mice and the Effect of EGF on Tumor Growth YUKITAKA MIYACHI,* TAKASHI TERAZONO, NORIKO NAGAO, TORU SHOJI, AND MINORU IRIE First Department of Internal Medicine, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ota-ku, Tokyo 143, Japan
EGF caused an increase in the rate of tumor growth, while 50 fig EGF strongly inhibited tumor growth. The concentration of [126I]EGF binding in the tumor treated with low doses of EGF was high, and that in the tumor treated with high doses of EGF was low. These changes in EGF binding were attributable to the changes in the number of high affinity EGF receptors with no significant alteration in binding affinity. In conclusion, the existence of high concentrations of EGF receptors with high affinity and specificity to EGF was demonstrated in an hCGproducing tumor transplanted in nude mice and appeared to be correlated with tumor growth. (J Clin Endocrinol Metab 7 1 : 329-334, 1990)
ABSTRACT. We examined the presence and characteristics of epidermal growth factor (EGF) receptors in hCG producing tumors (CC-2-JCK) transplanted in female nude mice. We also examined the in vivo effects of EGF on tumor growth. Specific receptors with apparent dissociation constants of 3.89 x 10~10 and 1.0 x 10"9 M and binding capacities of 5.96 x 10~10 and 1.52 X 10"9 M/mg protein for EGF have been identified in the hCGproducing tumor. [125I]EGF binding to the tumor tissues was time, temperature, and tissue weight dependent and specific. EGF and transforming growth factor-a (TGFa) competed for [ m I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGFa. TGF/3 competed for [126I]EGF binding slightly. Five micrograms of
E
Materials and Methods
PIDERMAL growth factor (EGF), a 6040-dalton polypeptide, is a potent mitogen and induces a number of biological responses in various tissues; these responses are thought to be mediated by interaction with specific plasma membrane receptors. Specific binding sites for EGF have been documented to exist in a number of human tumor tissues, such as squamous cell carcinomas of the lung (1), gliomas (2), meningiomas (2), and A431 human epidermoid carcinomas (3-5). Human placental cells (6-8) and choriocarcinoma cells (9-11) also contain EGF receptors, suggesting that EGF plays important roles in normal as well as abnormal cultured placental cell growth. These studies have mainly been concerned with cultured cells in vitro; studies of EGF receptors and the effects of EGF on hCG-producing tumors are limited. In this study we examined the presence and characterization of EGF receptors in hCGproducing tumors transplanted in female nude mice. In addition, we studied the effect of EGF on tumor growth in vivo.
Animals Six-week-old female nude mice were obtained from Sankyo Laboratory Co. (Tokyo, Japan). Human choriocarcinoma tissues, originally excised from human choriocarcinoma of the uterus and transplanted in nude mice (CC-2-JCK; Central Institute for Experimental Animals, Kawasaki, Japan) (12) were used in the following experiments. The serially transplanted tumors in nude mice restored the morphology and functions of the original tumor after more than 100 passages (12). The fragments of this human choriocarcinoma tissue were transplanted sc in the infrascapular region of nude mice with the use of a 12-gauge trocar needle. Four weeks after the transplantation, tumor tissues were excised and stored at —70 C until analysis of EGF receptors. P25IJEGF binding to hCG-producing tumor tissues The frozen tumor tissues were homogenized in 20 vol icecold 0.01 M phosphate-buffered saline (PBS) using a glass homogenizer, and the homogenate was filtered through stainless steel mesh no. 200. The filtrate then was centrifuged at 1500 X g for 30 min. In the supernatant no specific binding of [125I]EGF was found; therefore, we used the precipitate as the
Received October 30,1989. * To whom requests for reprints should be addressed.
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crude membrane fraction in the following studies. The precipitate was resuspended in PBS, and aliquots of the filtrate were incubated with [125I]EGF in the presence or absence of unlabeled EGF (Wakunaga Pharmaceutical Co., Hiroshima, Japan) in 0.5 mL PBS containing 0.1% BSA. [125I]EGF used in binding assays was produced by the lactoperoxidase method (13) using Na125I (Amersham/Searle Corp., Arlington Heights, IL) to a specific activity of approximately 100 nCi/iig. After incubation, 1 mL ice-cold PBS was added, and the tubes were centrifuged at 1500 X g for 15 min. The precipitate was washed with an additional 1 mL PBS. The radioactivity of the precipitate was measured with an autogamma counter. Tissue from six different tumors was used for the following studies, except for the experiment in which EGF was administered. The tumor tissue obtained from one nude mouse was used for each experiment. Examination of the time course of binding of [125I]EGF to hCG-producing tumor tissues at 4, 24, and 37 C was performed using the method described above. Specific binding was calculated by subtracting nonspecific binding from total binding. Binding of [125I]EGF as a function of tissue weight was examined by incubating [125I]EGF with aliquots of tumor tissue homogenates (2.1-32.7 fig protein/tube) for 3 h at 37 C in the presence or absence of unlabeled EGF. The specificity of [125I]EGF binding to hCG-producing tumor tissues was evaluated using competition binding of unlabeled peptides added to the incubation tube containing [125I]EGF, and binding was conducted, as described above, at 37 C for 3 h. The effects of anti-EGF receptor antibody (Oncogene Science, Inc., Manhasset, NY) on the binding of [125I]EGF to hCGproducing tumor tissues were evaluated. Aliquots of tumor tissue homogenates were preincubated with different concentrations of anti-EGF receptor antibody (final concentrations 1:1,500,1:17,000, and 1:180,000) at 37 C for 60 min and centrifuged at 1,500 X g for 30 min. The precipitates were washed, and [125I]EGF binding was performed as described above. Binding of [125I]EGF to A431 epidermoid carcinoma cells and HeLa cells was conducted as follows. A431 (1.0 x 106 cells or 25.3 ng protein/tube) and HeLa cells (1.7 x 106 cells or 42.7 Hg protein/tube) were incubated with [125I]EGF in the presence or absence of unlabeled EGF at 37 C in 0.5 mL PBS containing 0.1% BSA. After 3 h the cells were rinsed with PBS, and the radioactivity in the cells was counted with an autogamma counter. Specific [125I]EGF binding was analyzed employing the method of Scatchard (14). Protein values of the solutions were determined using the method of Lowry et al (15), with BSA as the standard. Student's t test was used to evaluate the statistical significance of the difference.
JCE & M • 1990 Vol71«No2
EGF at doses of 5 and 50 ng in 100 fiL PBS was started on the day of tumor transplantation. The animals were inspected daily, and tumor size was determined with a caliper along two dimensions. At the end of the 4-week experimental period, the animals were killed, and the tumors were excised and weighed. EGF receptors and hCG contents in the tissues were determined as described above. Results Effect of temperature on the time course of [125IJEGF binding The time course of specific binding of [125I]EGF (66 nM) to hCG-producing tumor tissues was examined at 4, 24, and 37 C. As shown in Fig. 1, specific binding of [125I] EGF reached a plateau after 2 h at 4 C. At 24 and 37 C, specific binding of [125I]EGF approached a near plateau after 2 h, but slight increases were observed until 5 h. The amounts of specific binding of [125I]EGF after 5 h at 4, 24, and 37 C were 3.2%, 8.9%, and 12.7%, respectively. On the basis of these results, analysis of [125I]EGF binding to tumor tissues was performed at 37 C for 3 h. Binding of P^IJEGF as a function of tissue weight Specific binding of [125I]EGF (44 nM) to tumor tissues was proportional to tissue weight from 2.1-16.4 /ig tissue protein in a nearly linear fashion. Nineteen and 27.1%
37°
24°
Measurement of EGF and hCG Amounts of EGF and hCG in the supernatant of the tissue homogenate were determined using the specific EGF and hCG RIAs, as described previously (16, 17). Effects of EGF on the growth of hCG-producing tumor transplanted in nude mice hCG-producing tumor fragments (50 mg) were transplanted sc in nude mice, as described above. Daily sc administration of
0 15 30
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FIG. 1. Effects of time and temperature on specific binding of [125I] EGF to hCG-producing tumor tissues. Aliquots of the tissue homogenate (32.7 ng protein/tube) were incubated with [125I]EGF (66 nM) in the presence or absence of unlabeled EGF (33.3 nM) at the appropriate temperature (37, 24, and 4 C). At the indicated times specific binding was calculated by subtracting nonspecific binding from total binding. Each point represents the mean of duplicate assays.
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EGF RECEPTOR IN hCG-PRODUCING TUMOR of [125I]EGF were bound specifically to 16.4 and 32.7 fig tissue protein, respectively. Specificity of f125IJEGF binding (Fig. 2) The specificity of [125I]EGF binding to tumor tissues was examined by comparing the ability of other peptides to prevent binding of [125I]EGF (66 nM). High concentrations of insulin-like growth factor-I (IGF-I, Fujisawa Pharmaceutical Co., Osaka, Japan), IGF-II (Sumitomo Pharmaceutical Co., Osaka, Japan), fibroblast growth factor (FGF; Toyobo Co., Osaka, Japan) and multiplication-stimulating activity (MSA; Collaborative Research, Inc., Waltham, MA) failed to compete effectively with binding of [125I]EGF. Only a slight decrease in the binding of [125I]EGF was observed at high concentrations of transforming growth factor-/? (TGF-/3; supplied by Dr. T. Tsushima) and 7s nerve growth factor (7sNGF; Toyobo Co., Osaka, Japan). Unlabeled EGF and TGFa (supplied by Dr. T. Tsushima) competed for [125I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGFa. 125
Effects ofanti-EGF receptor antibody on [ IJEGF binding (Fig. 3) Representative displacement curves for labeled EGF are shown in Fig. 3. Specific binding of [125I]EGF (22 nM) to tumor tissue decreased from 6.5% to 5.9% in the presence of a 1:17,000 dilution of antibody and to 2.7% in the presence of a 1:1,500 dilution of antibody. 100r*—A
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Scatchard analysis of P25I]EGF binding to tumor tissues Scatchard analysis of [125I]EGF binding to tumor tissues generated a curvilinear line, suggesting the presence of both high and low affinity binding sites with apparent dissociation constants of 3.89 X 10~10 and 1.0 X 10~9 M and binding capacities of 5.96 X 10"10 and 1.52 X 10"9 M/mg protein, respectively. EGF levels in hCG-producing tumors transplanted in nude mice EGF concentrations in the supernatant of tumor tissue homogenates were less than 10 pg/mg protein, and thus essentially not detectable. Binding of f125IJEGF to hCG-producing tumors, A431 cells, and HeLa cells Specific bindings of [125I]EGF (33 nM) to hCG-producing tumor and HeLa cells were 67.7% and 4% of specific binding to A431 cells in terms of protein concentration. Effect of EGF on the growth of hCG-producing tumors transplanted in nude mice (Fig. 4a) After transplantation of the tumor in nude mice, the size of the tumors progressively increased. During first 14 days of the treatment, EGF (5 and 50 fig) caused an increased rate of growth compared to that with PBS treatment. After 20 days of treatment, however, the higher amount of EGF (50 tig) strongly inhibited tumor growth; indeed, the size of the transplanted tumor after 1 month of daily administrations of 50 fig EGF was close to the initial size. Weight and EGF receptor concentrations of excised tumor tissues (Fig. 4b)
^ 50 EGF
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FIG. 2. Specificity of [125I]EGF binding to tumor tissues. Aliquots of tissue homogenates (32.7 ng protein/tube) were incubated with [125I] EGF (66 nM) and the indicated concentrations of the unlabeled peptides (EGF, TGFa, TGF0, IGF-I, IGF-II, FGF, MSA, and 7sNGF) for 3 h at 37 C. Each point represents the mean of duplicate [125I]EGF specific binding values.
The weights of excised tumor tissues (mean ± SE) after 1 month of treatment with PBS and 5 and 50 fig EGF were 325.0 ± 56.1, 862.5 ± 104.3, and 133.3 ± 48.8 mg (Fig. 4b), respectively. The average weight of the tumors treated with 5 fig EGF was 2.7 times larger than that of those treated with PBS. Proliferative responses of the tumors to EGF were biphasic, with a stimulation of growth at 5 fig EGF and an inhibition of growth at 50 fig EGF. Specific bindings of [125I]EGF (mean ± SE) to tumor tissues treated with PBS and 5 and 50 fig EGF were 3.75 ± 0.33%, 6.18 ± 0.51%, and 2.40 ± 0.71% (Fig. 5), respectively. The capacities of the high affinity binding sites in these tumor tissues were 2.01 x 10~10, 2.58 X 10"10, and 1.60 X 10"10 M/mg protein, and their association constants were similar (3.33 x 10~10, 3.50 X 10~10 and 3.33 x 10~10 M, respectively), suggesting that the changes in capacities of high affinity EGF-binding sites were induced by EGF treatment.
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control anti EGF receptor Ab 1 : 180.000 anti EGF receptor Ab 1 : 17.000
FlG. 3. Effects of anti-EGF receptor antibody on [125I]EGF binding. Aliquots of tissue homogenates (8.2 ^gprotein/tube) were preincubated with different concentrations of anti-EGF receptor antibody (Ab; final concentrations, 1:1,500, 1:17,000 and 1:180,000) for 60 min at 37 C, washed, and incubated with [126I]EGF (22 nM) for 3 h at 37 C in the presence or absence of different concentrations of EGF. Each point represents the mean of duplicate [128I]EGF specific binding values. B/T, Bound to total ratio.
anti EGF receptor Ab 1 : 1500
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FlG. 4. Effect of EGF on the growth of hCG-producing tumor transplanted in nude mice. hCG-producing tumor was transplanted into normal 6-week-old female nude mice, as described in Materials and Methods. Daily administration of EGF (5 and 50 Mg/100 /xh PBS) or PBS (100 nL) was started on the day of operation (day 0). a, Tumor size was determined daily. Tumor size (square millimeters) is given as the mean ± SE. Each group consisted of four mice. (O--O, PBS treated; • • 5 ng EGF-treated; X X, 50 Mg EGF-treated. b, On the 29th day of the treatment, mice were killed, and the tumor tissues were weighed. Each value represents the mean ± SE of four tumor tissues.
u. O UJ
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FlG. 5. [125I]EGF binding to hCG-producing tumors treated with PBS and 5 and 50 ng EGF for 1 month. Aliquots of tissue homogenates (8.2 ng protein/tube) obtained from PBS-treated and 5 and 50 ng EGFtreated mice were incubated with [125I]EGF (22 nM) in the presence of different amounts of unlabeled EGF. [125I]EGF binding (mean ± SE) was calculated by subtracting the mean of duplicate nonspecific binding tubes from the mean of quadruplicate binding tubes. B/T, Bound to total ratio.
Discussion hCG concentration in excised tumor tissues (Fig. 6) Concentrations of hCG (mean ± SE) in tumor tissues treated with PBS and 5 and 50 Mg EGF were 9.78 ± 1.32, 9.88 ± 1.82, and 7.95 ± 1.27 nmol/mg protein, respectively. A significantly lower level of hCG (P < 0.01) was observed in tumor tissue treated with high doses of EGF.
The presence of EGF receptors has been demonstrated in A431 human epidermoid carcinoma cells (3-5), several human squamous cell carcinomas (1), gliomas (2), and meningiomas (2) as well as in the human placental tissues (6-8) and choriocarcinoma cells (9-11). Our data obtained regarding the presence of EGF receptors in
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EGF RECEPTOR IN hCG-PRODUCING TUMOR
10
c "5 *•> o a a) 8
o E O
o
6-
5-
T
T77fx vffx control
EGF
rfm EGF
5/yg 50/ig FIG. 6. hCG concentrations in hCG-producing tumor tissues treated with PBS and 5 and 50 ng EGF for 1 month. Each value represents the mean ± SE of four tumor tissues.
hCG-producing tumors transplanted in nude mice are in accordance with the presence of EGF receptors in choriocarcinoma cells. Specific receptors for EGF were identified in the hCGproducing tumors (CC-2-JCK) transplanted in nude mice; moreover, specific [125I]EGF binding to the tumor tissues is time, temperature, and tissue weight dependent. [125I]EGF specific binding is also saturable, specific, and of high affinity. Furthermore, the concentration of EGF receptors appeared to be correlated with the growth of hCG-producing tumors. The high concentration of EGF receptors in these hCG-producing tumors (CC-2-JCK) was comparable to that in A431 human epidermoid carcinoma cells in terms of protein concentration. A431 cells are thought to be the richest cells in terms of EGF receptor content. The Kd of the high affinity EGF receptor in our hCG-producing tumors was 3.89 X 10~10 M, which is comparable to those in A431 human epidermoid carcinoma cells (Kd, 1.4 X 10~10 M) (2, 3, 5) and choriocarcinoma cells (Kd, 1 x 1(T10 M) (11). [125I]EGF binding to the hCG-producing tumor tissues exhibited a high degree of specificity. TGFa, one of the
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TGFs synthesized and secreted by some human tumor lines (18, 19), competed for [125I]EGF binding to the receptor, while TGF/3 and 7sNGF competed for [125I] EGF binding only slightly. TGFa, although antigenically different from EGF, has enough structural similarity to EGF to compete for [125I]EGF binding (20). At present, it is not known if a receptor exists in hCG-producing tumors (CC-2-JCK) that is specific for TGFa and does not recognize EGF. As EGF was not detectable in hCGproducing tumor extracts, it is possible that TGFa, although we did not measure it in these tumor tissues, may react with EGF receptors. The effect of EGF on the growth of hCG-producing tumors transplanted in nude mice was biphasic, with stimulation at low and inhibition at high concentrations of EGF. These data are similar to those found in A431 human epidermoid carcinoma cells, the growth of which was inhibited by high amounts of EGF (21, 22). This phenomenon seems to be common in tissues with high concentrations of EGF receptors, such as A431 human epidermoid carcinomas cells and hCG-producing tumor (CC-2-JCK), since variant clonal A431 cells with reduced EGF receptors show resistance to EGF-mediated growth inhibition (21). In our experiment the EGF receptor concentration in growth-inhibited tumor tissues treated with high doses of EGF and in growth-promoted tumor tissues treated with low doses of EGF was lower or higher, respectively, than that in PBS-treated tumor tissues. The decrease or increase in EGF binding was attributable, by Scatchard analysis, to a decrease or increase in the number of high affinity EGF receptors, with no significant alteration in binding affinity. These results suggest that the concentration of high affinity EGF receptor appears to be correlated with the growth of the tumor. This is in agreement with the report of Santon et al (23), who showed that the concentration of EGF receptor is the major factor in tumorigenicity of A431 cells in nude mice. A high concentration of EGF receptors appears to facilitate the growth of tumor cells in vivo as well as in vitro. Interactions between implanted cells and the host animal are complex, and the concentration of EGF receptors may be one factor determining tumor growth; higher concentrations of EGF receptors, however, appear to contribute significantly to the growth of hCG-producing tumors in nude mice. In conclusion, the presence of high concentrations of EGF receptor with high affinity and specificity to EGF was demonstrated in hCG-producing tumors transplanted in nude mice and appeared to be correlated with tumor growth. hCG-producing tumors transplanted in nude mice in continuous passages provide a useful in vivo model for the study of the actions of EGF in relation to tumor growth and hormone production.
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Acknowledgments The authors thank Miss Kieko Kashiwabara for typing the manuscript. We also wish to acknowledge Dr. Y. Ueyama of Tokai University for providing CC-2-JCK, Dr. T. Tsushima of Tokyo Women's Medical College for supplying TGFa and TGF/3, and Dr. Y. Yamaguchi of Tokyo University for providing the HeLa cells and A431 cells.
11.
References
13.
1. Filmus J, Pollak MN, Cailleau R, Buick RN. MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem Biophys Res Commun. 1985; 128:898-905. 2. Liebermann TA, Nusbaum HR, Razon N, et al. Amplification enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature (Lond). 1985;313:144-7. 3. Lin CR, Chijen WS, Kruijer W, et al. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984;224:843-8. 4. Ullrich A, Coussens L, Hayflick JS, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature (Lond). 1984;309:418-24. 5. Xu Y-H, Ishii S, Clark AJL, et al. Human epidermal growth factor receptor cDNA is homologous to a variety of RNAs overproduced in A431 carcinoma cells. Nature (Lond). 1984;309:806-10. 6. Lai WH, Guyda JH. Characterization and regulation of epidermal growth factor receptors in human placental cell cultures. J Clin Endocrinol Metab. 1984;58:344-52. 7. Rao CV, Ramani M, Chegini N, et al. Topography of human placental receptors for epidermal growth factor. J Biol Chem. 1985;260:1705-10. 8. Hout RI, Foidart JM, Nardone RM, Stromberg K. Differential modulation of human chorionic gonadotropin secretion by epidermal growth factor in normal and malignant placental cultures. J Clin Endocrinol Metab. 1981;53:1059-63. 9. Benveniste R, Speeg Jr KV, Carpenter G, Cohen S, Linder J, Rabinowitz D. Epidermal growth factor stimulates secretion of human chorionic gonadotropin by cultured human choriocarcinoma cells. J Clin Endocrinol Metab. 1978;46:169-72. 10. Bahn RS, Speeg Jr KV, Ascoli M, Rabin D. Epidermal growth
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factor stimulates production of progesterone in cultured human choriocarcinoma cells. Endocrinology. 1980; 107:2121-3. Hirata Y, Sueoka S, Uchibashi M, et al. Specific binding sites for epidermal growth factor and its effect on human chorionic gonadotropin secretion by cultured tumor cell lines: comparison between trophoblastic and non-trophoblastic cells. Acta Endocrinol (Copenh). 1982;101:281-6. Hayashi H. A study of tumor growth, cell differentiation and hormone production in two transplantable human choriocarcinoma in nude mice. Keio Igaku. 1977;54:465-81. Miyachi Y, Vaitukaitis JL, Nieschlag E, Lipsett MB. Enzymatic radioiodination of gonadotropins. J Clin Endocrinol Metab. 1972;34:23-8. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci. 1949;51:600-72. Lowry OH, Rosenbrough NJ, Fau AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:26575. Ihara T, Miyachi Y. Study in distribution of epidermal growth factor (EGF) in rat tissues. Folia Endocrinol. 1988;64:593-605. Mizuchi A, Kitagawa N, Miycahi Y, Iio M. Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay. J Immunol Methods. 1984; 74:36974. Sherwin SA, Minna JD, Gazdar AF, Todaro GJ. Expression of epidermal and nerve growth factor receptors and soft agar growth factor production by human lung cancer cells. Cancer Res. 1981;41:3538-42. Todaro GJ, FDryling C, DeLarco JE. Transformong growth factors production by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci USA. 1980;77:5258-62. Massaque J, Czech MP, Iwata K, DeLarco JE, Todaro GJ. Affinity labelling of a transforming growth factor receptor that does not interact with epidermal growth factor. Proc Natl Acad Sci USA. 1982;79:6822-6. Kawamoto T, Mendelsohn J, Le A, Sato GH, Laazar CS, Gill GH. Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells. J Biol Chem. 1984;259:7761-6. Barnes DW. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum free cell culture. J Cell Biol. 1982;93:l-4. Santon BJ, Cronin MR, Macleod CL, Mendelsohn M, Masui H, Gill GN. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res. 1986;46:4701-5.
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Vol. 71, No. 2 Printed in U.S.A.
Epidermal Growth Factor (EGF) Receptors in Human Chorionic Gonadotropin-Producing Tumor: Transplantation in Nude Mice and the Effect of EGF on Tumor Growth YUKITAKA MIYACHI,* TAKASHI TERAZONO, NORIKO NAGAO, TORU SHOJI, AND MINORU IRIE First Department of Internal Medicine, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ota-ku, Tokyo 143, Japan
EGF caused an increase in the rate of tumor growth, while 50 fig EGF strongly inhibited tumor growth. The concentration of [126I]EGF binding in the tumor treated with low doses of EGF was high, and that in the tumor treated with high doses of EGF was low. These changes in EGF binding were attributable to the changes in the number of high affinity EGF receptors with no significant alteration in binding affinity. In conclusion, the existence of high concentrations of EGF receptors with high affinity and specificity to EGF was demonstrated in an hCGproducing tumor transplanted in nude mice and appeared to be correlated with tumor growth. (J Clin Endocrinol Metab 7 1 : 329-334, 1990)
ABSTRACT. We examined the presence and characteristics of epidermal growth factor (EGF) receptors in hCG producing tumors (CC-2-JCK) transplanted in female nude mice. We also examined the in vivo effects of EGF on tumor growth. Specific receptors with apparent dissociation constants of 3.89 x 10~10 and 1.0 x 10"9 M and binding capacities of 5.96 x 10~10 and 1.52 X 10"9 M/mg protein for EGF have been identified in the hCGproducing tumor. [125I]EGF binding to the tumor tissues was time, temperature, and tissue weight dependent and specific. EGF and transforming growth factor-a (TGFa) competed for [ m I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGFa. TGF/3 competed for [126I]EGF binding slightly. Five micrograms of
E
Materials and Methods
PIDERMAL growth factor (EGF), a 6040-dalton polypeptide, is a potent mitogen and induces a number of biological responses in various tissues; these responses are thought to be mediated by interaction with specific plasma membrane receptors. Specific binding sites for EGF have been documented to exist in a number of human tumor tissues, such as squamous cell carcinomas of the lung (1), gliomas (2), meningiomas (2), and A431 human epidermoid carcinomas (3-5). Human placental cells (6-8) and choriocarcinoma cells (9-11) also contain EGF receptors, suggesting that EGF plays important roles in normal as well as abnormal cultured placental cell growth. These studies have mainly been concerned with cultured cells in vitro; studies of EGF receptors and the effects of EGF on hCG-producing tumors are limited. In this study we examined the presence and characterization of EGF receptors in hCGproducing tumors transplanted in female nude mice. In addition, we studied the effect of EGF on tumor growth in vivo.
Animals Six-week-old female nude mice were obtained from Sankyo Laboratory Co. (Tokyo, Japan). Human choriocarcinoma tissues, originally excised from human choriocarcinoma of the uterus and transplanted in nude mice (CC-2-JCK; Central Institute for Experimental Animals, Kawasaki, Japan) (12) were used in the following experiments. The serially transplanted tumors in nude mice restored the morphology and functions of the original tumor after more than 100 passages (12). The fragments of this human choriocarcinoma tissue were transplanted sc in the infrascapular region of nude mice with the use of a 12-gauge trocar needle. Four weeks after the transplantation, tumor tissues were excised and stored at —70 C until analysis of EGF receptors. P25IJEGF binding to hCG-producing tumor tissues The frozen tumor tissues were homogenized in 20 vol icecold 0.01 M phosphate-buffered saline (PBS) using a glass homogenizer, and the homogenate was filtered through stainless steel mesh no. 200. The filtrate then was centrifuged at 1500 X g for 30 min. In the supernatant no specific binding of [125I]EGF was found; therefore, we used the precipitate as the
Received October 30,1989. * To whom requests for reprints should be addressed.
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crude membrane fraction in the following studies. The precipitate was resuspended in PBS, and aliquots of the filtrate were incubated with [125I]EGF in the presence or absence of unlabeled EGF (Wakunaga Pharmaceutical Co., Hiroshima, Japan) in 0.5 mL PBS containing 0.1% BSA. [125I]EGF used in binding assays was produced by the lactoperoxidase method (13) using Na125I (Amersham/Searle Corp., Arlington Heights, IL) to a specific activity of approximately 100 nCi/iig. After incubation, 1 mL ice-cold PBS was added, and the tubes were centrifuged at 1500 X g for 15 min. The precipitate was washed with an additional 1 mL PBS. The radioactivity of the precipitate was measured with an autogamma counter. Tissue from six different tumors was used for the following studies, except for the experiment in which EGF was administered. The tumor tissue obtained from one nude mouse was used for each experiment. Examination of the time course of binding of [125I]EGF to hCG-producing tumor tissues at 4, 24, and 37 C was performed using the method described above. Specific binding was calculated by subtracting nonspecific binding from total binding. Binding of [125I]EGF as a function of tissue weight was examined by incubating [125I]EGF with aliquots of tumor tissue homogenates (2.1-32.7 fig protein/tube) for 3 h at 37 C in the presence or absence of unlabeled EGF. The specificity of [125I]EGF binding to hCG-producing tumor tissues was evaluated using competition binding of unlabeled peptides added to the incubation tube containing [125I]EGF, and binding was conducted, as described above, at 37 C for 3 h. The effects of anti-EGF receptor antibody (Oncogene Science, Inc., Manhasset, NY) on the binding of [125I]EGF to hCGproducing tumor tissues were evaluated. Aliquots of tumor tissue homogenates were preincubated with different concentrations of anti-EGF receptor antibody (final concentrations 1:1,500,1:17,000, and 1:180,000) at 37 C for 60 min and centrifuged at 1,500 X g for 30 min. The precipitates were washed, and [125I]EGF binding was performed as described above. Binding of [125I]EGF to A431 epidermoid carcinoma cells and HeLa cells was conducted as follows. A431 (1.0 x 106 cells or 25.3 ng protein/tube) and HeLa cells (1.7 x 106 cells or 42.7 Hg protein/tube) were incubated with [125I]EGF in the presence or absence of unlabeled EGF at 37 C in 0.5 mL PBS containing 0.1% BSA. After 3 h the cells were rinsed with PBS, and the radioactivity in the cells was counted with an autogamma counter. Specific [125I]EGF binding was analyzed employing the method of Scatchard (14). Protein values of the solutions were determined using the method of Lowry et al (15), with BSA as the standard. Student's t test was used to evaluate the statistical significance of the difference.
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EGF at doses of 5 and 50 ng in 100 fiL PBS was started on the day of tumor transplantation. The animals were inspected daily, and tumor size was determined with a caliper along two dimensions. At the end of the 4-week experimental period, the animals were killed, and the tumors were excised and weighed. EGF receptors and hCG contents in the tissues were determined as described above. Results Effect of temperature on the time course of [125IJEGF binding The time course of specific binding of [125I]EGF (66 nM) to hCG-producing tumor tissues was examined at 4, 24, and 37 C. As shown in Fig. 1, specific binding of [125I] EGF reached a plateau after 2 h at 4 C. At 24 and 37 C, specific binding of [125I]EGF approached a near plateau after 2 h, but slight increases were observed until 5 h. The amounts of specific binding of [125I]EGF after 5 h at 4, 24, and 37 C were 3.2%, 8.9%, and 12.7%, respectively. On the basis of these results, analysis of [125I]EGF binding to tumor tissues was performed at 37 C for 3 h. Binding of P^IJEGF as a function of tissue weight Specific binding of [125I]EGF (44 nM) to tumor tissues was proportional to tissue weight from 2.1-16.4 /ig tissue protein in a nearly linear fashion. Nineteen and 27.1%
37°
24°
Measurement of EGF and hCG Amounts of EGF and hCG in the supernatant of the tissue homogenate were determined using the specific EGF and hCG RIAs, as described previously (16, 17). Effects of EGF on the growth of hCG-producing tumor transplanted in nude mice hCG-producing tumor fragments (50 mg) were transplanted sc in nude mice, as described above. Daily sc administration of
0 15 30
60
120
180 T I M E (MIN)
300
FIG. 1. Effects of time and temperature on specific binding of [125I] EGF to hCG-producing tumor tissues. Aliquots of the tissue homogenate (32.7 ng protein/tube) were incubated with [125I]EGF (66 nM) in the presence or absence of unlabeled EGF (33.3 nM) at the appropriate temperature (37, 24, and 4 C). At the indicated times specific binding was calculated by subtracting nonspecific binding from total binding. Each point represents the mean of duplicate assays.
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EGF RECEPTOR IN hCG-PRODUCING TUMOR of [125I]EGF were bound specifically to 16.4 and 32.7 fig tissue protein, respectively. Specificity of f125IJEGF binding (Fig. 2) The specificity of [125I]EGF binding to tumor tissues was examined by comparing the ability of other peptides to prevent binding of [125I]EGF (66 nM). High concentrations of insulin-like growth factor-I (IGF-I, Fujisawa Pharmaceutical Co., Osaka, Japan), IGF-II (Sumitomo Pharmaceutical Co., Osaka, Japan), fibroblast growth factor (FGF; Toyobo Co., Osaka, Japan) and multiplication-stimulating activity (MSA; Collaborative Research, Inc., Waltham, MA) failed to compete effectively with binding of [125I]EGF. Only a slight decrease in the binding of [125I]EGF was observed at high concentrations of transforming growth factor-/? (TGF-/3; supplied by Dr. T. Tsushima) and 7s nerve growth factor (7sNGF; Toyobo Co., Osaka, Japan). Unlabeled EGF and TGFa (supplied by Dr. T. Tsushima) competed for [125I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGFa. 125
Effects ofanti-EGF receptor antibody on [ IJEGF binding (Fig. 3) Representative displacement curves for labeled EGF are shown in Fig. 3. Specific binding of [125I]EGF (22 nM) to tumor tissue decreased from 6.5% to 5.9% in the presence of a 1:17,000 dilution of antibody and to 2.7% in the presence of a 1:1,500 dilution of antibody. 100r*—A
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Scatchard analysis of P25I]EGF binding to tumor tissues Scatchard analysis of [125I]EGF binding to tumor tissues generated a curvilinear line, suggesting the presence of both high and low affinity binding sites with apparent dissociation constants of 3.89 X 10~10 and 1.0 X 10~9 M and binding capacities of 5.96 X 10"10 and 1.52 X 10"9 M/mg protein, respectively. EGF levels in hCG-producing tumors transplanted in nude mice EGF concentrations in the supernatant of tumor tissue homogenates were less than 10 pg/mg protein, and thus essentially not detectable. Binding of f125IJEGF to hCG-producing tumors, A431 cells, and HeLa cells Specific bindings of [125I]EGF (33 nM) to hCG-producing tumor and HeLa cells were 67.7% and 4% of specific binding to A431 cells in terms of protein concentration. Effect of EGF on the growth of hCG-producing tumors transplanted in nude mice (Fig. 4a) After transplantation of the tumor in nude mice, the size of the tumors progressively increased. During first 14 days of the treatment, EGF (5 and 50 fig) caused an increased rate of growth compared to that with PBS treatment. After 20 days of treatment, however, the higher amount of EGF (50 tig) strongly inhibited tumor growth; indeed, the size of the transplanted tumor after 1 month of daily administrations of 50 fig EGF was close to the initial size. Weight and EGF receptor concentrations of excised tumor tissues (Fig. 4b)
^ 50 EGF
0 O.OO1 0.01
0.1
1
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Peptides (nM)
FIG. 2. Specificity of [125I]EGF binding to tumor tissues. Aliquots of tissue homogenates (32.7 ng protein/tube) were incubated with [125I] EGF (66 nM) and the indicated concentrations of the unlabeled peptides (EGF, TGFa, TGF0, IGF-I, IGF-II, FGF, MSA, and 7sNGF) for 3 h at 37 C. Each point represents the mean of duplicate [125I]EGF specific binding values.
The weights of excised tumor tissues (mean ± SE) after 1 month of treatment with PBS and 5 and 50 fig EGF were 325.0 ± 56.1, 862.5 ± 104.3, and 133.3 ± 48.8 mg (Fig. 4b), respectively. The average weight of the tumors treated with 5 fig EGF was 2.7 times larger than that of those treated with PBS. Proliferative responses of the tumors to EGF were biphasic, with a stimulation of growth at 5 fig EGF and an inhibition of growth at 50 fig EGF. Specific bindings of [125I]EGF (mean ± SE) to tumor tissues treated with PBS and 5 and 50 fig EGF were 3.75 ± 0.33%, 6.18 ± 0.51%, and 2.40 ± 0.71% (Fig. 5), respectively. The capacities of the high affinity binding sites in these tumor tissues were 2.01 x 10~10, 2.58 X 10"10, and 1.60 X 10"10 M/mg protein, and their association constants were similar (3.33 x 10~10, 3.50 X 10~10 and 3.33 x 10~10 M, respectively), suggesting that the changes in capacities of high affinity EGF-binding sites were induced by EGF treatment.
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control anti EGF receptor Ab 1 : 180.000 anti EGF receptor Ab 1 : 17.000
FlG. 3. Effects of anti-EGF receptor antibody on [125I]EGF binding. Aliquots of tissue homogenates (8.2 ^gprotein/tube) were preincubated with different concentrations of anti-EGF receptor antibody (Ab; final concentrations, 1:1,500, 1:17,000 and 1:180,000) for 60 min at 37 C, washed, and incubated with [126I]EGF (22 nM) for 3 h at 37 C in the presence or absence of different concentrations of EGF. Each point represents the mean of duplicate [128I]EGF specific binding values. B/T, Bound to total ratio.
anti EGF receptor Ab 1 : 1500
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0.1 EGF (nM)
(b) 1.000
(a) 300 r
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m 2 500
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c
fi 3 100
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I
control EGF EGF (5«) (50w)
FlG. 4. Effect of EGF on the growth of hCG-producing tumor transplanted in nude mice. hCG-producing tumor was transplanted into normal 6-week-old female nude mice, as described in Materials and Methods. Daily administration of EGF (5 and 50 Mg/100 /xh PBS) or PBS (100 nL) was started on the day of operation (day 0). a, Tumor size was determined daily. Tumor size (square millimeters) is given as the mean ± SE. Each group consisted of four mice. (O--O, PBS treated; • • 5 ng EGF-treated; X X, 50 Mg EGF-treated. b, On the 29th day of the treatment, mice were killed, and the tumor tissues were weighed. Each value represents the mean ± SE of four tumor tissues.
u. O UJ
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FlG. 5. [125I]EGF binding to hCG-producing tumors treated with PBS and 5 and 50 ng EGF for 1 month. Aliquots of tissue homogenates (8.2 ng protein/tube) obtained from PBS-treated and 5 and 50 ng EGFtreated mice were incubated with [125I]EGF (22 nM) in the presence of different amounts of unlabeled EGF. [125I]EGF binding (mean ± SE) was calculated by subtracting the mean of duplicate nonspecific binding tubes from the mean of quadruplicate binding tubes. B/T, Bound to total ratio.
Discussion hCG concentration in excised tumor tissues (Fig. 6) Concentrations of hCG (mean ± SE) in tumor tissues treated with PBS and 5 and 50 Mg EGF were 9.78 ± 1.32, 9.88 ± 1.82, and 7.95 ± 1.27 nmol/mg protein, respectively. A significantly lower level of hCG (P < 0.01) was observed in tumor tissue treated with high doses of EGF.
The presence of EGF receptors has been demonstrated in A431 human epidermoid carcinoma cells (3-5), several human squamous cell carcinomas (1), gliomas (2), and meningiomas (2) as well as in the human placental tissues (6-8) and choriocarcinoma cells (9-11). Our data obtained regarding the presence of EGF receptors in
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EGF RECEPTOR IN hCG-PRODUCING TUMOR
10
c "5 *•> o a a) 8
o E O
o
6-
5-
T
T77fx vffx control
EGF
rfm EGF
5/yg 50/ig FIG. 6. hCG concentrations in hCG-producing tumor tissues treated with PBS and 5 and 50 ng EGF for 1 month. Each value represents the mean ± SE of four tumor tissues.
hCG-producing tumors transplanted in nude mice are in accordance with the presence of EGF receptors in choriocarcinoma cells. Specific receptors for EGF were identified in the hCGproducing tumors (CC-2-JCK) transplanted in nude mice; moreover, specific [125I]EGF binding to the tumor tissues is time, temperature, and tissue weight dependent. [125I]EGF specific binding is also saturable, specific, and of high affinity. Furthermore, the concentration of EGF receptors appeared to be correlated with the growth of hCG-producing tumors. The high concentration of EGF receptors in these hCG-producing tumors (CC-2-JCK) was comparable to that in A431 human epidermoid carcinoma cells in terms of protein concentration. A431 cells are thought to be the richest cells in terms of EGF receptor content. The Kd of the high affinity EGF receptor in our hCG-producing tumors was 3.89 X 10~10 M, which is comparable to those in A431 human epidermoid carcinoma cells (Kd, 1.4 X 10~10 M) (2, 3, 5) and choriocarcinoma cells (Kd, 1 x 1(T10 M) (11). [125I]EGF binding to the hCG-producing tumor tissues exhibited a high degree of specificity. TGFa, one of the
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TGFs synthesized and secreted by some human tumor lines (18, 19), competed for [125I]EGF binding to the receptor, while TGF/3 and 7sNGF competed for [125I] EGF binding only slightly. TGFa, although antigenically different from EGF, has enough structural similarity to EGF to compete for [125I]EGF binding (20). At present, it is not known if a receptor exists in hCG-producing tumors (CC-2-JCK) that is specific for TGFa and does not recognize EGF. As EGF was not detectable in hCGproducing tumor extracts, it is possible that TGFa, although we did not measure it in these tumor tissues, may react with EGF receptors. The effect of EGF on the growth of hCG-producing tumors transplanted in nude mice was biphasic, with stimulation at low and inhibition at high concentrations of EGF. These data are similar to those found in A431 human epidermoid carcinoma cells, the growth of which was inhibited by high amounts of EGF (21, 22). This phenomenon seems to be common in tissues with high concentrations of EGF receptors, such as A431 human epidermoid carcinomas cells and hCG-producing tumor (CC-2-JCK), since variant clonal A431 cells with reduced EGF receptors show resistance to EGF-mediated growth inhibition (21). In our experiment the EGF receptor concentration in growth-inhibited tumor tissues treated with high doses of EGF and in growth-promoted tumor tissues treated with low doses of EGF was lower or higher, respectively, than that in PBS-treated tumor tissues. The decrease or increase in EGF binding was attributable, by Scatchard analysis, to a decrease or increase in the number of high affinity EGF receptors, with no significant alteration in binding affinity. These results suggest that the concentration of high affinity EGF receptor appears to be correlated with the growth of the tumor. This is in agreement with the report of Santon et al (23), who showed that the concentration of EGF receptor is the major factor in tumorigenicity of A431 cells in nude mice. A high concentration of EGF receptors appears to facilitate the growth of tumor cells in vivo as well as in vitro. Interactions between implanted cells and the host animal are complex, and the concentration of EGF receptors may be one factor determining tumor growth; higher concentrations of EGF receptors, however, appear to contribute significantly to the growth of hCG-producing tumors in nude mice. In conclusion, the presence of high concentrations of EGF receptor with high affinity and specificity to EGF was demonstrated in hCG-producing tumors transplanted in nude mice and appeared to be correlated with tumor growth. hCG-producing tumors transplanted in nude mice in continuous passages provide a useful in vivo model for the study of the actions of EGF in relation to tumor growth and hormone production.
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Acknowledgments The authors thank Miss Kieko Kashiwabara for typing the manuscript. We also wish to acknowledge Dr. Y. Ueyama of Tokai University for providing CC-2-JCK, Dr. T. Tsushima of Tokyo Women's Medical College for supplying TGFa and TGF/3, and Dr. Y. Yamaguchi of Tokyo University for providing the HeLa cells and A431 cells.
11.
References
13.
1. Filmus J, Pollak MN, Cailleau R, Buick RN. MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem Biophys Res Commun. 1985; 128:898-905. 2. Liebermann TA, Nusbaum HR, Razon N, et al. Amplification enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature (Lond). 1985;313:144-7. 3. Lin CR, Chijen WS, Kruijer W, et al. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984;224:843-8. 4. Ullrich A, Coussens L, Hayflick JS, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature (Lond). 1984;309:418-24. 5. Xu Y-H, Ishii S, Clark AJL, et al. Human epidermal growth factor receptor cDNA is homologous to a variety of RNAs overproduced in A431 carcinoma cells. Nature (Lond). 1984;309:806-10. 6. Lai WH, Guyda JH. Characterization and regulation of epidermal growth factor receptors in human placental cell cultures. J Clin Endocrinol Metab. 1984;58:344-52. 7. Rao CV, Ramani M, Chegini N, et al. Topography of human placental receptors for epidermal growth factor. J Biol Chem. 1985;260:1705-10. 8. Hout RI, Foidart JM, Nardone RM, Stromberg K. Differential modulation of human chorionic gonadotropin secretion by epidermal growth factor in normal and malignant placental cultures. J Clin Endocrinol Metab. 1981;53:1059-63. 9. Benveniste R, Speeg Jr KV, Carpenter G, Cohen S, Linder J, Rabinowitz D. Epidermal growth factor stimulates secretion of human chorionic gonadotropin by cultured human choriocarcinoma cells. J Clin Endocrinol Metab. 1978;46:169-72. 10. Bahn RS, Speeg Jr KV, Ascoli M, Rabin D. Epidermal growth
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factor stimulates production of progesterone in cultured human choriocarcinoma cells. Endocrinology. 1980; 107:2121-3. Hirata Y, Sueoka S, Uchibashi M, et al. Specific binding sites for epidermal growth factor and its effect on human chorionic gonadotropin secretion by cultured tumor cell lines: comparison between trophoblastic and non-trophoblastic cells. Acta Endocrinol (Copenh). 1982;101:281-6. Hayashi H. A study of tumor growth, cell differentiation and hormone production in two transplantable human choriocarcinoma in nude mice. Keio Igaku. 1977;54:465-81. Miyachi Y, Vaitukaitis JL, Nieschlag E, Lipsett MB. Enzymatic radioiodination of gonadotropins. J Clin Endocrinol Metab. 1972;34:23-8. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci. 1949;51:600-72. Lowry OH, Rosenbrough NJ, Fau AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:26575. Ihara T, Miyachi Y. Study in distribution of epidermal growth factor (EGF) in rat tissues. Folia Endocrinol. 1988;64:593-605. Mizuchi A, Kitagawa N, Miycahi Y, Iio M. Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay. J Immunol Methods. 1984; 74:36974. Sherwin SA, Minna JD, Gazdar AF, Todaro GJ. Expression of epidermal and nerve growth factor receptors and soft agar growth factor production by human lung cancer cells. Cancer Res. 1981;41:3538-42. Todaro GJ, FDryling C, DeLarco JE. Transformong growth factors production by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci USA. 1980;77:5258-62. Massaque J, Czech MP, Iwata K, DeLarco JE, Todaro GJ. Affinity labelling of a transforming growth factor receptor that does not interact with epidermal growth factor. Proc Natl Acad Sci USA. 1982;79:6822-6. Kawamoto T, Mendelsohn J, Le A, Sato GH, Laazar CS, Gill GH. Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells. J Biol Chem. 1984;259:7761-6. Barnes DW. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum free cell culture. J Cell Biol. 1982;93:l-4. Santon BJ, Cronin MR, Macleod CL, Mendelsohn M, Masui H, Gill GN. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res. 1986;46:4701-5.
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