EP0517902A1

EP0517902A1 - Protein with human choriogonadotropin inhibitory effect

Info

Publication number: EP0517902A1
Application number: EP92903686A
Authority: EP
Inventors: Glenn David Braunstein; Song-Guang Ren
Original assignee: Cedars Sinai Medical Center
Current assignee: Cedars Sinai Medical Center
Priority date: 1990-12-28
Filing date: 1991-12-19
Publication date: 1992-12-16
Also published as: AU9164891A; AU641835B2; PT99953A; EP0517902A4; WO1992012178A1; CA2075981A1

Abstract

Substance ayant un effet inhibiteur sur la production de choriogonadotropine humaine (hCG), comprenant une protéine isolée à partir de cellules déciduales humaines ayant une analyse d'acide aminé préliminaire ainsi qu'une masse moléculaire comprise entre 7000 et environ 10000 daltons, et utilisée pour inhiber la production de hCG in vivo ou in vitro, dont la mesure fournit une indication d'une cause d'inhibition de hCG, ou pouvant être utilisée dans le traitement de tumeurs secrétant de la hCG.Substance having an inhibitory effect on the production of human choriogonadotropin (hCG), comprising a protein isolated from human decidual cells having a preliminary amino acid analysis as well as a molecular mass of between 7,000 and approximately 10,000 daltons, and used for inhibit the production of hCG in vivo or in vitro, the measurement of which provides an indication of a cause of inhibition of hCG, or which can be used in the treatment of tumors secreting hCG.

Description

DESCRIPTION

*-. Protein With Human Chorioσonadotropin

Inhibitory Effect

This invention relates to a decidual inhibitory protein (DIP) having an inhibitory effect on the produc¬ tion of human choriogonadotropin (hCG) . More particu¬ larly, this invention relates to a protein isolated from 5 human decidual cells with a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons (as measured by ultrafiltration and gel chromatography) which may be used to inhibit hCG produc¬ tion in vivo (to induce abortion) or _in vitro. measured 10 .in vivo or in vitro to diagnose the cause of hCG inhibi¬ tion as an indication of potential miscarriage, or used in the treatment of hCG-secreting tumors.

Background

The fetoplacental unit plays an essential role in the

15 complicated hormonal interplay that is necessary for a successful pregnancy. Human choriogonadotropin (hCG) , a hormone secreted by the trophoblast cells of the placenta, is present in the blood and urine during pregnancy. The primitive trophoblast begins producing hCG soon after

20 fertilization and concentrations of hCG rise to peak values at 8-12 weeks of pregnancy, then decrease to a plateau level that is maintained during the remainder of the pregnancy. hCG may also be produced by certain trophoblastic or ectopic neoplasms.

25 Following fertilization, hCG stimulates the sustained secretion of progesterone by the corpus luteum. which is necessary for the growth of the uterine endometriu . hCG also is a stimulus for the production of fetal testicular androgens.

30 Decreased production of hCG can result in spontaneous abortion. Many abnormal pregnancies are associated with decreased levels of hCG as compared to normal pregnancies of the same duration. Most of these pregnancies end in spontaneous abortions. (B. Saxena, in Endocrinology of Pregnancy, Fuchs et al. Eds, Harper & Row, Philadelphia (1983) pp. 50-72) .

The decidualized endometrium of the uterus also pro¬ duces substances necessary for gestation. The decidual¬ ized endometrium and the placental trophoblast are con¬ tiguous tissues, and prior studies have investigated the possibility that each may influence the function of the other. Studies performed over a decade ago showed that hCG secretion from placental explants rose over the initial 72 hour period, leading researchers to suggest that loss of an endogenous hCG inhibitory factor (lost as it was removed from culture during changes of growth media) was responsible. (A. Golander et al., Endo¬ crinology 102:597-605 (1978)). Yuen and coworkers have suggested that prolactin released by the decidua inhibits hCG production by trophoblasts. (B.H. Yuen, et al. Am. J. Obstet. Gynecol. 154:336-340 (1986)). Conversely, tropho¬ blast hCG secretion has been found to be stimulated by interleukin-1 produced by placental phagocytic cells and decidual cells. (A. Flynn, et al.. Science, 218:475-476 (1982) and S. Yagel et al., J. Clin. Endocrinol. Metab.. 68:992-995 (1989)).

Recently, Vicovac and Genvacev provided evidence, through co-incubation of trophoblasts and decidual cells, that the decidua has an inhibitory effect on the synthesis of hCG and total protein by trophoblast cells. (L.J. Vicovac et al., Placenta 9:109-115, (1988)). They con¬ cluded that some endometrial secretory product or products act to inhibit total protein synthesis and hCG secretion and/or synthesis by the trophoblast tissue.

Characterization of an endometrial hCG inhibitor was heretofore unknown. Potential uses for such a substance include as an agent to induce therapeutic abortions, as a basis to develop methods of evaluating hCG inhibition in vivo, or as a treatment of hCG-secreting tumors. The present inventors noted the inhibitory effect of a sub¬ stance produced by decidual cells, and searched for the active material. As a result, the present inventors have found that a protein of a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons (as measured by ultrafiltration and gel chromatog¬ raphy) is produced by the decidua and has an inhibitory effect on hCG production by the trophoblast and by JEG-3 choriocarcinoma cells.

Summary of the Invention

The present invention is directed to a composition of matter which inhibits hCG production by human trophoblast cells and JEG-3 choriocarcinoma cells. The present inven- tion is also directed to a method for inhibiting hCG release comprising contacting hCG producing tissue with an effective amount of the composition of the invention. The present invention is also directed to methods of evaluat¬ ing hCG inhibition .in vivo and .in vitro, comprising guan- tifying the amount of the composition of the invention produced in vivo or in vitro.

The protein of the present invention may also be of use in the treatment of hCG-secreting tumors. Since the placental trophoblast is the normal site of synthesis of hCG, it is understandable that both gestational and non- gestational trophoblastic tumors synthesize and secrete hCG. Indeed, hCG measurements have been quite useful for the diagnosis of these tumors, staging the tumors, and for monitoring the effects of therapy. In addition, some nontrophoblastic tumors may produce hCG ectopically. hCG may act as a growth factor for some tumors (Melmed S. and BraunaLeiπ GD: Human chorionic gonadotropin stimulates proliferation of Nb 2 rat lymphoma cells. J. Clin. Endocrinol. Metab 56:1068-1070, (1983)). Therefore, the use of a material which suppresses the production of hCG may suppress the growth of an hCG-secreting tumor. This has been previously shown with the use of anti-hCG anti¬ bodies in animal tumor models.

The present composition comprises a substantially- pure polypeptide of a preliminary amino acid analysis and a molecular weight of about 7,000 to about 10,000 daltons (as measured by ultrafiltration and gel chromatography) , wherein said polypeptide inhibits the production of hCG by human trophoblast and JEG-3 choriocarcinoma cells. This polypeptide can be isolated from cultures of human deci- dual cells by cultivation and centrifugation of the cul¬ ture medium. As used herein, "substantially pure" is intended to mean that DIP has been isolated from the decidual cells.

Brief Description of the Drawings Figure 1 is a graph showing the relationship between hCG release by trophoblast cells and initial decidual cell concentration in conculture of trophoblast cells (lxlO⁶/ well) and decidual cells during 120 hours of incubation. Figure 2 is a graph showing the time course of inhibition of hCG release from primary trophoblast cell cultures by DCM. Trophoblast cells were exposed to control medium or 50% DCM. The medium was removed and replaced with control medium or DCM each 24 hours during the 96 hour incubation period. Each point presents the mean of 20 wells in three different placental experiments. DCM -= Decidual conditioned medium.

Figure 3 is a graph showing DCM inhibition of the stimulatory effect of cAMP on hCG secretion by JEG-3 cells. JEG-3 cells were exposed to control media, 25% DCM, 0.5 mM 8-bromo-cAMP plus different doses of DCM, or 0.5 mM 8-bromo-cAMP plus 25% RPMI-1788 cell conditioned media for 72 hours. DCM = Decidual conditioned medium.

Figure 4 is a graph showing the dose-response rela¬ tionship between DCM and hCG release. JEG-3 cells and trophoblast cells were exposed to different concentrations of DCM for 72 hours. Each bar represents mean + SEM of hCG in six wells. DCM - Decidual conditioned medium.

Figure 5 is a graph showing the inhibitory activity of different fractions of DCM fractionated by ultrafiltra- tion and chromatography. JEG-3 cells were exposed for 72 hours to control medium, 25% DCM, ultrafiltrated (through YM-10) or chromatographed (through Sephadex G-50) fractions of DCM. Each bar represents the mean+SEM of hCG in six wells from at least two experiments. DCM = Deci- dual conditioned medium.

Detailed Description of the Invention

The isolation and characterization of DIP will be further described by reference to the following detailed examples.

A. Materials and Methods

1. Preparation of Cells

Term placenta and associated membranes were obtained from 10 healthy pregnant women undergoing Cesarean sec¬ tion. Cytotrophoblasts were isolated from villus tissue using the method of H.J. Kliman et al., (Endocrinology 118:1567-1582 (1986)). Briefly, washed villus tissue was minced and then digested with 0.125% trypsin (Sigma, St. Louis, MO) and 0.2 g/L DNase (Sigma) at 37°C for 30 in. Dispersed tissue was applied to a 5-70% percoll (Sigma) gradient. After centrifugation at 1200 X g, the middle layer containing cytotrophoblasts was collected and washed. Decidual cells were isolated from decidual tissue dissected from placental membranes by the method of M.B. Braverman et al., (J. Clin. Endocrinol. Metab. 58:521-525 (1984)). This procedure involved the digestion of deci¬ dual tissue with 0.25% coliagenase A (Mannhem, Indiana¬ polis, IN) at 37"C for 2 hours, followed by centrifugation through a 20-60% percoll gradient. Cell viability was greater than 90% using trypan blue exclusion. Human choriocarcinoma cell line JEG-3 was purchased from American Type Culture Collection (Rockville, MD) .

2. Cell Incubation

In co-culture studies, the trophoblast (lxlO⁶ cells/ ml/well) or decidual cells (0.83-7.5xl0⁵ cells/ml/well) , or trophoblast plus decidual cells (sum of both cell numbers/ 2ml/well) were cultured in 24-multiwell culture plates with 16 mm well diameter (Costar, Cambridge, MA) contain¬ ing CMRL-1066 medium (GIBCO, Grand Island, NY) supple- mented with 10% fetal bovine serum (GIBCO) , 25 mM HEPES (Sigma) , 1 U/ml insulin (Nordisk, Gentofte, Denmark) , 10 mM glutamine (Irving, Santa Ana, CA) , 100 U/ml penicil¬ lin and 100 ug/ l streptomycin (GIBCO) in an atmosphere of 5% C0₂-95% air at 37°C for 120 h. The medium was removed and replaced each 24 h.

In other studies, the trophoblast (lxlO⁶ cells/2ml/ well) or JEG-3 (lxlO⁵ cells/2ml/well) were precultured for 24 hours. After removing the precultured medium, the cells were cultured in control medium or medium containing test substances. In all experiments, the media contained the same volume of CMRL-1066. In addition, in some trophoblast experiments and all of the JEG-3 cell experi¬ ments, the control medium contained the same volume of 0.01M phosphate buffered saline (PBS) (pH 7.4) as was used to dissolve the test substance in the experimental media. In the time course studies, the medium was removed and replaced with control or experimental medium each 24 hours during 72-96 hour incubation period. In other experi¬ ments, the medium in both control and experimental wells were removed at termination of the experiments after 72 hours' incubation.

The spent media were centrifuged and the super¬ natants stored at -20°C until hormone assay. In each experiment, all treatments were tested in 3-4 wells. Each variable was tested in at least two separate experiments. 3. Preparation of Decidual Conditioned Medium (DCM)

Decidual conditioned medium (DCM) was prepared by incubating decidual cells (5-7.5xl0⁶/luι_l/flask) in 75cm² cell culture flasks (Costar, Cambridge, MA) containing CMRL-1066 medium for 24-72 hours. In the time course experiments, DCM was removed each 24 hours and used for the next 24-hour treatment of trophoblast cells derived from the same placenta as were the decidual cells. In other studies, DCM was removed during the first 36-48 hours of incubation.

After the medium was decanted from decidual cells and centrifuged, the supernatant was collected for use. In dose-response experiments, the different doses of DCM were prepared by dilution with 0.01M PBS buffer.

4. Evaluation of the Active Fraction of DCM

The effect of proteolytic enzyme treatment on DCM was evaluated by incubating DCM for 16 hours at 37 " C with trypsin or chymotrypsinogen-A (Sigma, 40 ug enzyme/mg DCM protein) , followed by the addition of 5 ug α-l-antitryp- sin(Sigma)/ug trypsin to the treated DCM with incubation at 37°C for an additional 4 hours. The same weight of bovine serum albumin (BSA, Sigma) as DCM was added to CMRL-1066 medium, which served as a control, and the medium was treated as described above (60 ug enzyme/mg BSA). DCM was also tested after heating to 100°C for 30 minutes in a water bath.

The approximate molecular size of the active fraction of DCM was evaluated through ultrafiltration and gel chromatography. DCM was ultrafiltered through an Amicon Diaflo Apparatus (Amicon Corp., Lexington, MA) with a YM-10 membrane which has a molecular exclusion limit of 10,000. The greater than iϋ,U00 and the less than 10,000 Dalton fractions were tested. In some experiments, 20ml DCM was concentrated by ultrafiltration using an Amicon YM-2 membrane with a cut off 1,000 Daltons, followed by lyophilization of the concentrated DCM. The lyophilized DCM was resuspended in 0.5 ml 0.01 M PBS buffer, pH 7.4 and applied to a 1 x 25 cm Sephadex G-25 or G-50 column and eluted with the same buffer. Column characteristics were: V₀1 = 10.5 ml, V_t = 45 ml and 35 ml for G-25 and G-50, respectively. Twenty five drops per fraction were collected. Several contiguous fractions were pooled and tested at a concentration of 25% of the pool for ability to inhibit hCG in the JEG cells.

5. Negative Controls CMRL-1066 media collected following 36-48 hours of incubation with AV-3 cells (5xl0⁶ cells/lOml/flask) , or RPMI-1788 cells (5xl0⁶ cells/lOml/flask) , purchased from American Type Tissue Collection (Rockville, MD) , were used as negative controls. For positive control purposes, 0.5mM 8-bromo-cAMP (Sigma) was used to stimulate the JEG or trophoblast cells. Another positive control for the system was composed of incubation of decidual cells for 96 hours in medium derived from trophoblast culture, with measurement of prolactin (PRL) and placental protein 12 (PP12; insulin-like growth factor-I-binding protein) as indices of decidual protein production.

6. Measurements of Hormones and Proteins hCG, human placental lactogen (hPL) , PRL, and PP12 in the media were measured by homologous radioimmunoassay methods as previously described by G.D. Braunstein et al.. Am. J. Obstet. Gynecol. 131:25-32 (1978)); G.D. Braunstein et al., Am J. Obstet. Gvnecol. 138:1205-1213 (1980); P.R.P. Salacinski et al.. Anal. Biochem. 117:136-146 (1981) and E-M. Rutanen, H. Bohn, and M. Seppala, Am. J. Obstet. Gvnecol. 144:460-463 (1982). Sensitivity of the assays were 1 ng/ml for hCG and hPL, 1.7 ng/ml for PRL,and 7.8 ng/ml for PP12. The protein content of the cells and media were measured by the method of M.M. Bradford in Anal. Biochem. 72:248-254 (1976), using bovine serum albumin as a standard and Bio-Rad protein reagents (Bio-Rad Laboratories, Richmond, CA) . Concentrations of the hormones in the media were expressed as ng/mg protein or ng/well.

7. Statistical Analysis All results were presented as the mean + SEM. Student's t-test with the Bonferroni correction was used for determining the statistical significance of the dif¬ ference between control and experimental groups. A p<0.05 was considered to be significant. 8. Amino Acid Analysis

Seven separate decidual samples were grown in culture and the media combined. The decidual choriogonadotropin inhibitory protein was purified by ultrafiltration, Sepha- dex G50 column chromatography, dialysis of the fractions containing the inhibitory protein, followed by concentra¬ tion by lyophilization, and application onto a low molecu¬ lar weight SDS polyacrylamide gel. The 8,000-10,000 Dalton band was electrically transblotted to a PVDF mem¬ brane (Trans-Blot Transfer Medium; BioRad Labs, Richmond, CA 94804) and subjected to amino acid analysis by Pico Tag chemistry using pre-column reaction with PITC (Phenyl iso- thiocyanate) using the method of Matsudaira (Matsudaira, PT: A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press. 1989: pp. 5-7) . This method does not measure free asparagine, glutamine, cysteine, tryptophan, and may under represent tyrosine. (The amino acid analysis was run on a fee-for-service basis by Dr. Audree Fowler, at UCLA School of Medicine, Department of Biological Chemistry, Los Angeles, CA 90024-9842; 213-825-9842).

1. Co-Culture of Trophoblast and Decidual Cells In the co-culture of trophoblast and decidual cells over 120 hour incubation, lxlO⁶ trophoblast cells released 112.1+20.2 ng hCG/well/120 hours, while trophoblast plus an average (from 3 experiments) of 5.75xl0⁵ decidual cells released 28.2+4.1 ng hCG/well/120 hour. Thus, the tropho¬ blast cells co-cultured with decidual cells released 75% (p<0.001) less hCG than did trophoblast cultured alone. The degree of inhibition of trophoblast hCG secretion by decidual cells was dependent upon the number of decidual cells, with a progressively greater inhibition as the number of decidual cells seeded was increased (Figure 1) .

2. Trophoblast Cells Cultured in DCM-Containing Medium

Primary trophoblast cells cultured in CRML-1066 medium containing 50% DCM released 45% (p<0.005), 64% (p<0.001), 66% (p<0.005), and 70% (p<0.005) less hCG than did control cultures at 24, 48, 72 and 96 hours, respec- tively (Figure 2) . hPL release from primary trophoblast cultures exposed for 96 hours to 50% DCM was significantly increased (Table I) . Decidual cells exposed for 96 hours to 50% trophoblast cell culture medium showed significant stimulation of PRL secretion in the medium compared to control decidual cultures, but PP12 release remained unchanged (Table I) .

TABLE I

THE INTERACTION OF TROPHOBLAST AND

DECIDUAL CELL ON HORMONES AND PROTEIN RELEASE

Trophoblast cell (or decidual cell) exposed for 96 hours to 50% DCM (or trophoblast culture medium) . Each hormone level in the medium is mean±SEM of 20 wells from three different placental experiments. *p<0.005, **p<0.001 compared to control. DCM = Decidual conditioned medium.

3. JEG-Cells Cultured in DCM-Containing Medium JEG-3 cells exposed for 72 hours to 25% DCM also had a significant reduction in hCG secretion into the medium compared to control cultures, but there was neither stimu- lation nor inhibition of hPL release. Control media obtained from incubation with AV-3 cells or RPMI-1788 cells incubated under the same conditions as the decidual cells for 36-48 hours had no inhibitory effects on hCG release from the JEG-3 cells (Table II). 0.5 mM 8-Bromo- cAMP markedly stimulated h c secretion; however, DCM, but not conditioned medium from RPMI-1788 cells, significantly reduced the stimulant effect of 8-bromo-cAMP on hCG secre¬ tion in a dose-dependent manner (Figure 3) . TABLE II THE EFFECTS OF DCM OR CONTROL MEDIA ON hCG RELEASE BY JEG- 3 CELLS

Treatment hCG (ng/mg protein)

Control 103±6

25% DCM 66±12*

25% Boiled DCM 90±12

Control 162±34

25% DCM 92±10*

Medium treated by trypsin 150±41

25% DCM treated by trypsin 134124

Medium treated by chymotrypsin 139±18 25% DCM treated by chymotrypsin 156127

Control 7616

25% AV-3 cell medium 6417

25% RPMI cell medium 7316

JEG-3 cell exposed for 72 hours to DCM with different treatments, or 0.5 mM cAMP or control cell media. The hCG value in media is meaniSEM of six wells from at least two experiments. DCM = Decidual conditioned medium. *p<0.05 compared to control. 4. Dose-Dependent Inhibition of hCG Release

The inhibitory effect of DCM on hCG release by pri¬ mary trophoblast cells and JEG-3 cells was dose-dependent as depicted in Figure 4. The inhibitory activity of DCM was lost following heating at 100"C for 30 minutes or treatment with trypsin or chymotrypsin (Table II) . Fol¬ lowing ultrafiltration, the hCG inhibitory activity was found in the less than 10,000 Dalton fraction, and eluted in the 7,200-9,800 Dalton fractions following Sephadex G-50 or G-25 chromatography of the DCM (Figure 5) . This fraction contained 18.6% of the protein eluted from the column.

5. Amino Acid Analysis

The preliminary amino acid composition from 3 sepa- rate bands in the 8,000-10,000 Dalton molecular weight range obtained and analyzed as described above are as follows:

The results of our study indicate that the human decidual cell secretes a factor that inhibits hCG release by placental trophoblasts and JEG-3 cells. The inhibition was DCM dose-dependent. The inhibition was also specific for hCG, as DCM (1) did not inhibit total protein synthe¬ sis by JEG-3 cells or placental trophoblasts, and (2) stimulated hPL release from primary trophoblast cultures and did not alter hPL secretion by JEG-3 cells (failure of DCM to stimulate hPL release from JEG-3 cells may be due to the paucity of hPL production by these cells) . The specificity of this effect is supported further by the absence of hCG inhibition in JEG-3 cells incubated with negative control media.

The similar qualitative hCG secretory pattern of trophoblast cells incubated with DCM and trophoblast cells incubated with control media (Figure 2) also indicates that the effect is not due to non-specific inhibition, since the peak of hCG release occurred at 72 hours of incubation with or without DCM exposure. We and others have previously demonstrated a similar time course of hCG secretion in primary trophoblast cultures. Y. Kato and G.D. Braunstein, J. Clin. Endocrinol. Metab. 68:814-819 (1989) and J.O. Lobo and F.L. Bellino, J. Clin. Endo¬ crinol. Metab. 69:868-874 (1989).

Our preliminary characterization of the inhibitory factor indicates that it is a protein with a preliminary amino acid analysis and a molecular weight between about 7,000 and about 10,000 Daltons determined by ultrafiltra¬ tion and gel chromatography. (A molecular weight up to about 15,000 daltons was measured by SDS polyacrylamide gel electrophoresis. Garfin, D.E. "1-Dimensiona1 Gel Electro." MT-Deutch. ed. , Guide to Protein Purification- Acad. Press 1989 p. 425-441.) Yuen and associates have previously suggested that PRL inhibits hCG production in vitro and in vivo. While the decidual cell is a primary source of PRL during pregnancy, it is unlikely that PRL or a fraction of PRL accounted for our results. PRL has a molecular weight of 22,500 Daltons whereas our inhibitory factor has a molecular weight of less than about 10,000 Daltons (or 15,000 Daltons determined as described above) . Additionally, the average concentration of immunoreactive PRL in our DCM was approximately 200 ng/ml, which did not inhibit hCG release from trophoblast cells in our studies (Ren and Braunstein, unpublished observations) . Progesterone has also been found by some investi¬ gators to inhibit hCG production by placental tissue in vitro. (T. Maruo et al.. Endocrinology 119:855-864 (1986) ; E.A. Wilson, M.J. Jawad, and L.R. Dickson, Am. J. Obstet. Gynecol. 138:708-713 (1980) and E.R. Barnea and M. Kaplan, J. Clin. Endocrinol. Metab. 69:215-217 (1989).) However, progesterone is synthesized by the trophoblast rather than the decidual cells. Since the active hCG inhibitory factor in DCM is destroyed by heat and proteo- lytic enzymes it is unlikely that progesterone (a steroid hormone) is responsible for the hCG inhibition.

Our data indicate that direct decidual-trophoblast cell contact is not required for regulation of hCG production, although the exact mechanisms by which the decidual cell factor inhibits hCG production is unclear. Since DCM inhibited the stimulant effect of exogenous cAMP on hCG secretion by trophoblasts in our study, DCM may interfere with activation of the cAMP-dependent protein kinases of the placenta, or with transcription of genes encoding the a- and 0-subunits of hCG regulated by cAMP.

Trophoblast hPL secretion was stimulated by DCM, a finding that is consistent with those of T. Fay et al.,

11th Rochester Trophoblast Conference with the European

Placenta Group. Abst. #108 (1988) . The different secre- tory behavior of hCG and hPL in trophoblast cell cultures exposed to the same DCM supports previous observations that the physiological regulation of secretion of differ¬ ent placental protein hormones may differ. It is unknown whether the different action of DCM on hPL and hCG release resides in the same substance or in different substances.

Our results also confirm prior findings by

S. Handwerger et al. that placental tissue stimulates PRL production by decidual ceils. (s. Handwerger et al..

Endocrinology 112:1370-1374 (1983) and S. Handwerger et al., Biochem. Biophvs. Res. Coπuiiun. 147:452-459 (1987)). Trophoblast cell culture medium did not, however, change PP12 release from decidual cells indicating that decidual PRL and PP12 regulation are under different control.

In summary, production of hCG by trophoblast and JEG-3 cells is inhibited in a dose-dependent fashion by a protein of about 7,000 to about 10,000 daltons produced by decidual cells. This protein will be useful for investi¬ gating abnormalities of human pregnancy, and may provide the basis for diagnostic agents to detect such abnormali¬ ties, and for therapeutic agents to induce abortion by decreasing hCG production.

Other and further embodiments of the invention are readily apparent from the above description of the inven¬ tion, and these embodiments are believed to be within the scope of the invention.

Claims

1. A substance having an inhibitory effect on the production of human chorionic gonadotropin (hCG) compris¬ ing a regulatory polypeptide being characterized in that it:

(a) inhibits, in a dose dependent manner, the production of hCG by human trophoblasts in vitro;

(b) substantially inhibits the stimulation of hCG secretion by human trophoblasts caused by exogenous cyclic AMP in vitro;

(c) does not substantially inhibit the production of human placental lactogen (hPL) by human tropho¬ blasts .in vitro; and (d) exhibits a molecular weight of about 7,000 to about 10,000 daltons as determined by ultra¬ filtration and gel exclusion chromatography.

2. A substance for inhibiting the production of hCG having as an active element an effective amount of the substance of Claim 1.

3. A substance for Claim 2 used to inhibit hCG pro¬ duction by human trophoblasts.

4. The substance of Claim 2 used in vitro to inhibit hCG production by human trophoblasts.

5. The substance of Claim 2 used .in vitro to inhibit hCG production by JEG-3 choriocarcinoma cells.

6. A substance having an inhibitory effect on the production of human chorionic gonadotropin (hCG) compris¬ ing a regulatory polypeptide isolated from human decidual cells said regulatory polypeptide being characterized in that it: (a) inhibits, in a dose dependent manner, the pro¬ duction of hCG by human trophoblasts in vitro;

(b) substantially inhibits the stimulation of hCG secretion by human trophoblasts caused by exogenous cyclic AMP .in vitro;

(c) does not substantially inhibit the production of human placental lactogen (hPL) by human tropho¬ blasts in vitro; and

(d) exhibits a molecular weight of about 7,000 to about 10,000 daltons as determined by ultra¬ filtration and gel exclusion chromatography.

7. A substance for inhibiting the production of hCG having as a active element an effective amount of the sub¬ stance of Claim 6.

8. The substance of Claim 7 used to inhibit hCG production by human trophoblasts.

9. The substance of Claim 7 used in vitro to inhibit hCG production by human trophoblasts.

10. The substance of Claim 7 used in vitro to inhibit hCG production by JEG-3 choriocarcinoma cells.

11. A method for evaluating inhibition of hCG production comprising measuring the substance of Claim 1.

12. The method of Claim 11 wherein the evaluation is of in vitro inhibition of hCG production.

13. A method for evaluating inhibition of hCG pro- ;tion comprising measuring the substance of Claim 6.

14. The method of Claim 13 wherein the evaluation is of in vitro inhibition of hCG production.