HUMAN CHORIONIC GONADOTROPIN-LIKE PROTEINS: SECRETION IN NONPREGNANT HUMANS AND PRODUCTION BY BACTERIA* WILLIAM D. ODELLt, and (by invitation) JEANINE GRIFFIN, SANJEEV GROVER and DOUGLAS T. CARRELL SALT LAKE CITY
Human Chorionic Gonadotropin Secretion by Normal Humans: Human chorionic gonadotropin (hCG) and the other human glycoprotein hormones (luteinizing hormone [hLH], follicle stimulating hormone [hFSH], and thyroid stimulating hormone [hTSH]) are biochemically very similar and are composed of two subunits, alpha and beta, which are not covalently bound. The amino acid sequence of the alpha subunit is identical for all four glycoprotein hormones and this protein is encoded by a single gene (1, 2). The beta subunit of hLH is also encoded by a single gene, whereas the beta subunit for hCG is encoded by up to eight genes or pseudogenes (3). The beta subunit of hLH differs from that of hCG in two ways: a) hCG has a carboxyl-terminal extension or prolongation of 37 amino acids, not present on hLH, b) the remainder of the hCG beta has 82% homology to that of hLH (4, 5). In mammalian systems, LH and hCG bind to the same receptor and possess similar biological functions. Human CG has usually been considered a hormone of pregnancy, acting to prolong corpus luteum function or of neoplasms derived from trophoblastic tissue (6). However, in the years 1966-1972, several case reports appeared, describing patients with various types of nontrophoblastic neoplasms that produced hCG (7-9). The first radioimmunoassay for hCG, reported from our laboratory in 1967 (6, 10), reacted equally with hLH and hCG. This assay, and also the bioassays then used to quantify hCG, therefore depended predominantly on quantitative differences in the amount of hCG present to distinguish it from hLH; in normal pregnancy, for example, we estimate the serum concentrations of hCG to be 10-8 M, whereas hLH circulates in nonpregnant humans at 10-10 M. In 1972, Vaitukaitis et al. (11), using polyclonal antisera developed against the beta subunit of hCG, developed an assay with increased specificity for hCG. Human LH still reacted in this assay, * Department of Internal Medicine, University of Utah Medical Center, Salt Lake City, Utah. t Address correspondence and reprint requests to: William D. Odell, M.D., Ph.D., Department of Internal Medicine, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132.
238
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CHORIONIC GONADOTROPIN
but the dose response line for hLH differed from that of hCG, and significant cross-reaction was seen only when hCG and hLH were present in about equal molar concentrations-a phenomenon that was not then known to occur physiologically. However, employing this improved hCG assay, surprising findings were reported. Braunstein et al. (12) reported that 7% of sera from 828 nonpregnant patients with a variety of carcinomas, including stomach, colon, lung, and pancreas, contained an hCGlike material. Furthermore, since testicular teratocarcinomas produce hCG, these workers studied extracts of normal human testes and reported that human testes contained an hCG-like material (13). Braunstein et al. (13) employed a concanavalin A (Con-A) purification technique, and using this technique, they reported that extracts of other tissues did not contain hCG. Con-A is a plant lectin that binds carbohydrate-rich placental hCG avidly. In 1977, however, a series of studies from our laboratory, employing a glacial acetic acid extraction technique that does not depend on carbohydrate presence on hCG, revealed, surprisingly, that extracts of many normal human tissues (e.g., liver, kidney, lung) contained an hCG-like material (14, 15). Further studies showed that this "normal tissue hCG" showed very little binding to Con-A and that on gel chromatography, it had a molecular volume compatible with carbohydrate-free hCG (16-18). In addition, hCG could be extracted from all carcinomas studied, irregardless of histological type (14, 17). The hCG from carcinomas showed highly variable binding to Con-A-ranging from 4% to 86% (Table 1). Braunstein et al. (19) confirmed that extracts of most of all normal tissues contained an hCG-like substance. These studies of hCG, and additional similar studies of ACTH-like materials, vasopressin and the free alpha subunit of hCG, led us to the hypothesis that so-called "ectopic hormone syndromes" were caused by increased production by a cancer, of protein hormone-like substances, usually produced in smaller amounts by normal tissues (14, 16); that is, so-called ectopic hormone production is not ectopic. TABLE 1 hCG Binding to Concanavalin A % Bound ± SEM
Range
Normal Tissue (10) 6.1 ± 1.6 (0.0-14.6) Cancer Tissue (9) 31.2 ± 9.1 (4.0-86.0) Placenta (4) 92.5 ± 0.9 (90.1-94.0) Pregnant Serum (3) 100.0 Cancer Serum (8) 54.7 ± 11.9 (3.1-92.5) Reproduced from Odell WD and Saito E: Protein hormone-like materials from normal and cancer cells-"ectopic" hormone production. In: Mirand EA, Hutchinson WB, Mihich E, eds. Cancer Management, The 13th International Cancer Congress, Part E. New York: Alan R. Liss, Inc.; 1983:247.
240
WILLIAM D. ODELL
In 1978, Matsuura et al. (20) developed an entirely specific assay for hCG. This assay used polyclonal antibodies against the unique carboxyltail portion of the beta subunit of hCG. This assay, while very specific, lacked sensitivity because of the low affinity of these antisera. Nevertheless, the great specificity of this assay permitted Matsuura et al. (21) and Chen et al. (22) to show that urine and pituitary from nonpregnant humans contained an hCG-like material. Borkowski et al. (23, 24) subsequently extracted large volumes of plasma from nonpregnant subjects and demonstrated the presence of an hCG-like material. These findings of production of hCG and its circulation in blood of normal humans depended on three generations of progressively more specific assay systems. The fourth generation employed immunoradiometric (IRMA) or so-called sandwich assays. These assays employed monoclonal antibodies and were capable of greatly increased specificity as well as specificity. Employing such an assay, Armstrong et al. (25) confirmed that hCG was detectable in urine of nonpregnant humans. Further studies from our laboratory were directed to determining the tissue source of the hCG present in blood and urine of nonpregnant humans. To assist in answering this question we developed an extremely sensitive (.04 mIU/ml) and entirely specific assay for the intact hCG molecule (holo-hCG) (26). A second assay based on similar principles was developed for hLH (27). The hCG assay showed no reaction with the free alpha or beta subunits of hCG and no reaction with hTSH or hFSH. Immunochemical grade hLH showed .15% reaction in the assay, which interestingly, we were later able to show, was caused by contamination of this pituitary hLH with small amounts of hCG from pituitary (28). When we further purified this hLH, we also showed hLH had no cross-reaction in the hCG assay (Fig. 1). Using these assay systems, we found that if repeated serum samples were obtained, small amounts of hCG were detectable in every normal eugonadal or postmenopausal subject (29, 30). Furthermore, we were able to show that this hCG was secreted in a pulsatile fashion, in parallel with hCG in postmenopausal women (29), and in women during the normal menstrual cycle (29) (Figs. 2-4). These studies from our laboratory, as well as quite independent studies by Stenman et al. (31), also showed that hCG was stimulated by gonadotropin releasing hormone (GnRH) and suppressed by GnRH agonists and estrogens (29, 32) (Fig. 5). Human CG concentrations in sera from hypopituitary patients or from men with prostate cancer, treated with GnRH agonists, were very low or undetectable. All these findings suggested that the major source of hCG in blood of normal humans was the pituitary gland, and not other peripheral tissues. In further studies (reported in 1990) employing human fetal pituitaries
241
CHORIONIC GONADOTROPIN 50,000-
40,000 z
hCG
30,000-
m 0~ X
20,000-
A/
10,000-
0 ~~~~u~~7-%~
100
102
104
eta hCGA
1hFSH hTSH 106
HORMONE CONC. (pg)
a z
hCG
M
0
hLH
0.
hLH affinity purified
U
1
10
100
1000
10000
100000
HORMONE (pg/tube) B FIG. 1A. Immunoradiometric assay for hCG. The free alpha subunit and beta subunit, as well as purified hFSH and hTSH showed no reaction (
Human Chorionic Gonadotropin Secretion by Normal Humans: Human chorionic gonadotropin (hCG) and the other human glycoprotein hormones (luteinizing hormone [hLH], follicle stimulating hormone [hFSH], and thyroid stimulating hormone [hTSH]) are biochemically very similar and are composed of two subunits, alpha and beta, which are not covalently bound. The amino acid sequence of the alpha subunit is identical for all four glycoprotein hormones and this protein is encoded by a single gene (1, 2). The beta subunit of hLH is also encoded by a single gene, whereas the beta subunit for hCG is encoded by up to eight genes or pseudogenes (3). The beta subunit of hLH differs from that of hCG in two ways: a) hCG has a carboxyl-terminal extension or prolongation of 37 amino acids, not present on hLH, b) the remainder of the hCG beta has 82% homology to that of hLH (4, 5). In mammalian systems, LH and hCG bind to the same receptor and possess similar biological functions. Human CG has usually been considered a hormone of pregnancy, acting to prolong corpus luteum function or of neoplasms derived from trophoblastic tissue (6). However, in the years 1966-1972, several case reports appeared, describing patients with various types of nontrophoblastic neoplasms that produced hCG (7-9). The first radioimmunoassay for hCG, reported from our laboratory in 1967 (6, 10), reacted equally with hLH and hCG. This assay, and also the bioassays then used to quantify hCG, therefore depended predominantly on quantitative differences in the amount of hCG present to distinguish it from hLH; in normal pregnancy, for example, we estimate the serum concentrations of hCG to be 10-8 M, whereas hLH circulates in nonpregnant humans at 10-10 M. In 1972, Vaitukaitis et al. (11), using polyclonal antisera developed against the beta subunit of hCG, developed an assay with increased specificity for hCG. Human LH still reacted in this assay, * Department of Internal Medicine, University of Utah Medical Center, Salt Lake City, Utah. t Address correspondence and reprint requests to: William D. Odell, M.D., Ph.D., Department of Internal Medicine, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132.
238
239
CHORIONIC GONADOTROPIN
but the dose response line for hLH differed from that of hCG, and significant cross-reaction was seen only when hCG and hLH were present in about equal molar concentrations-a phenomenon that was not then known to occur physiologically. However, employing this improved hCG assay, surprising findings were reported. Braunstein et al. (12) reported that 7% of sera from 828 nonpregnant patients with a variety of carcinomas, including stomach, colon, lung, and pancreas, contained an hCGlike material. Furthermore, since testicular teratocarcinomas produce hCG, these workers studied extracts of normal human testes and reported that human testes contained an hCG-like material (13). Braunstein et al. (13) employed a concanavalin A (Con-A) purification technique, and using this technique, they reported that extracts of other tissues did not contain hCG. Con-A is a plant lectin that binds carbohydrate-rich placental hCG avidly. In 1977, however, a series of studies from our laboratory, employing a glacial acetic acid extraction technique that does not depend on carbohydrate presence on hCG, revealed, surprisingly, that extracts of many normal human tissues (e.g., liver, kidney, lung) contained an hCG-like material (14, 15). Further studies showed that this "normal tissue hCG" showed very little binding to Con-A and that on gel chromatography, it had a molecular volume compatible with carbohydrate-free hCG (16-18). In addition, hCG could be extracted from all carcinomas studied, irregardless of histological type (14, 17). The hCG from carcinomas showed highly variable binding to Con-A-ranging from 4% to 86% (Table 1). Braunstein et al. (19) confirmed that extracts of most of all normal tissues contained an hCG-like substance. These studies of hCG, and additional similar studies of ACTH-like materials, vasopressin and the free alpha subunit of hCG, led us to the hypothesis that so-called "ectopic hormone syndromes" were caused by increased production by a cancer, of protein hormone-like substances, usually produced in smaller amounts by normal tissues (14, 16); that is, so-called ectopic hormone production is not ectopic. TABLE 1 hCG Binding to Concanavalin A % Bound ± SEM
Range
Normal Tissue (10) 6.1 ± 1.6 (0.0-14.6) Cancer Tissue (9) 31.2 ± 9.1 (4.0-86.0) Placenta (4) 92.5 ± 0.9 (90.1-94.0) Pregnant Serum (3) 100.0 Cancer Serum (8) 54.7 ± 11.9 (3.1-92.5) Reproduced from Odell WD and Saito E: Protein hormone-like materials from normal and cancer cells-"ectopic" hormone production. In: Mirand EA, Hutchinson WB, Mihich E, eds. Cancer Management, The 13th International Cancer Congress, Part E. New York: Alan R. Liss, Inc.; 1983:247.
240
WILLIAM D. ODELL
In 1978, Matsuura et al. (20) developed an entirely specific assay for hCG. This assay used polyclonal antibodies against the unique carboxyltail portion of the beta subunit of hCG. This assay, while very specific, lacked sensitivity because of the low affinity of these antisera. Nevertheless, the great specificity of this assay permitted Matsuura et al. (21) and Chen et al. (22) to show that urine and pituitary from nonpregnant humans contained an hCG-like material. Borkowski et al. (23, 24) subsequently extracted large volumes of plasma from nonpregnant subjects and demonstrated the presence of an hCG-like material. These findings of production of hCG and its circulation in blood of normal humans depended on three generations of progressively more specific assay systems. The fourth generation employed immunoradiometric (IRMA) or so-called sandwich assays. These assays employed monoclonal antibodies and were capable of greatly increased specificity as well as specificity. Employing such an assay, Armstrong et al. (25) confirmed that hCG was detectable in urine of nonpregnant humans. Further studies from our laboratory were directed to determining the tissue source of the hCG present in blood and urine of nonpregnant humans. To assist in answering this question we developed an extremely sensitive (.04 mIU/ml) and entirely specific assay for the intact hCG molecule (holo-hCG) (26). A second assay based on similar principles was developed for hLH (27). The hCG assay showed no reaction with the free alpha or beta subunits of hCG and no reaction with hTSH or hFSH. Immunochemical grade hLH showed .15% reaction in the assay, which interestingly, we were later able to show, was caused by contamination of this pituitary hLH with small amounts of hCG from pituitary (28). When we further purified this hLH, we also showed hLH had no cross-reaction in the hCG assay (Fig. 1). Using these assay systems, we found that if repeated serum samples were obtained, small amounts of hCG were detectable in every normal eugonadal or postmenopausal subject (29, 30). Furthermore, we were able to show that this hCG was secreted in a pulsatile fashion, in parallel with hCG in postmenopausal women (29), and in women during the normal menstrual cycle (29) (Figs. 2-4). These studies from our laboratory, as well as quite independent studies by Stenman et al. (31), also showed that hCG was stimulated by gonadotropin releasing hormone (GnRH) and suppressed by GnRH agonists and estrogens (29, 32) (Fig. 5). Human CG concentrations in sera from hypopituitary patients or from men with prostate cancer, treated with GnRH agonists, were very low or undetectable. All these findings suggested that the major source of hCG in blood of normal humans was the pituitary gland, and not other peripheral tissues. In further studies (reported in 1990) employing human fetal pituitaries
241
CHORIONIC GONADOTROPIN 50,000-
40,000 z
hCG
30,000-
m 0~ X
20,000-
A/
10,000-
0 ~~~~u~~7-%~
100
102
104
eta hCGA
1hFSH hTSH 106
HORMONE CONC. (pg)
a z
hCG
M
0
hLH
0.
hLH affinity purified
U
1
10
100
1000
10000
100000
HORMONE (pg/tube) B FIG. 1A. Immunoradiometric assay for hCG. The free alpha subunit and beta subunit, as well as purified hFSH and hTSH showed no reaction (
