Proc. Nati. Acad. Sci. USA Vol. 75, No. 1, pp. 99-102:, January 1978

Biochemistry

Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells (choriogonadotropin/hormone action/testicular tumor)

MARIO ASCOLI AND DAVID PUETT Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232

Communicated by Sidney P. Colowick, October 11, 1977

ABSTRACT Testicular tumors are generally characterized by a loss of responsiveness to gonadotropins. The M5480 Leydig cell tumor is unusual, if not unique, in that it responds to human choriogonadotropin and to lutropin via increased steroidogenesis. This report describes the identification of two variants of the original M5480 tumor that have altered steroid output both in the basal state and in response to human choriogonadotpin. One of the tumors produces mainly progesterone, which is stimulated by the choriogonadotropin; the other tumor produces about equal amounts of progesterone and testosterone, and the secretion of both is stimulated by the choriogonadotropin. The dissociation constant describing the interaction between Leydig tumor cells and 125I-labeled human choriogonadotropin is between 3 and 5 X 10-11 M. This agrees with values reported for normal Leydig cells, although the tumor cells appear to have fewer receptors. The differences noted in the two tumors and normal Leydig cells may have arisen from alterations in gene regulation, or in mutations, involving one or more enzymes in the pathway in which progesterone is converted to testosterone. Under the experimental conditions used, all the tumors studied (seven generations) responded to the choriogonadotropin both in binding and in the resultant stimulation of steroidogenesis. This property, together with the characteristic that a homogeneous cell population can be obtained without enzymatic treatment, should qualify the M5480 Leydig cell tumor(s) as a model system for further studies on the mechanism of action of gonadotropin, on hormone receptors, and on hormonally responsive tumors.

The mechanism of action of lutropin and human choriogonadotropin has been studied in some detail using collagenasedispersed rat Leydig cells (1-3). Although this system has been useful in obtaining information about the interaction of gonadotropins with their target cells (see ref. 4), there are two major disadvantages, namely, the cells are prepared by enzymatic dispersion of the tissue (1-3) and the cell population obtained contains only 10-40% Leydig cells (2, 3). Recently, however, Conn et al. (5) have reported that a homogeneous population of Leydig cells can be obtained by centrifugation of collagenase-dispersed rat Leydig cells in metrizamide gradients. Because of these inherent problems, the possibility of using Leydig cell tumors as an alternate system to study the interaction of gonadotropins with their target cells has been investigated. Of the several Leydig cell tumors available that are either transplantable or adapted to growth in tissue culture (6-8), only one, designated M5480, appears to be hormone responsive (1, 9, 10). This tumor originated in a mouse of the C57B1/6 strain and was adapted for serial transplantation by F. Dunning of the Papanicolau Cancer Research Institute. Some aspects of the in vitro interaction of lutropin with the tumor cells were studied several years ago by Moyle and coworkers (1, 9, 11-15). They

reported, however, that only some of the tumors were hormone responsive.

Recently, Neaves studied the growth, composition, and hormone responsiveness of the M5480 tumor in vivo, and showed that between 11 and 14 days after transplantation the tumor is composed mainly of Leydig cells (about 95%), most of which appeared viable and healthy by morphological criteria (10, 16, 17). Therefore, this tumor may represent a source of a nearly homogeneous population of Leydig cells. Moreover, these cells can be obtained without enzymatic treatment of the tissue (9). These characteristics make the M5480 tumor ideal for detailed investigations of hormone binding and mechanism of action. Also, such studies are useful in attempting to elucidate various parameters and characteristics of hormone-responsive tumors. In this paper, some of the characteristics of the in vitro interaction of human choriogonadotropin (hCG) with the Leydig tumor cells are described. MATERIALS AND METHODS Hormones and Supplies. Medium 199 and Trypan Blue were from Grand Island Biological Company (Grand Island, NY). Bovine serum albumin,- testosterone, and progesterone were from Sigma Chemical Co. (St. Louis, MO), and lactoperoxidase (Grade B, 23 units/mg) from Calbiochem (La Jolla, CA). Carrier-free NaI25I, [1,2,6,7-3H(N)]progesterone, [1,2,6,7-3H(N)]testosterone, and the antisera to testosterone and progesterone were from New England Nuclear (Boston, MA). All chemicals were of reagent grade or better. hCG and ovine lutropin were purified from urine of pregnant women and sheep pituitaries, respectively, and the biological activities were similar to those reported (18). Ovine thyrotropin, follitropin, and somatotropin and bovine prolactin, were obtained from the National Institute of Arthritis, Metabolism and Digestive Diseases. Tumors. M5480P (generation 120) was obtained from the Papanicolau Cancer Research Institute (Miami, FL). M5480A (generation 176) was a generous gift of William Neaves of the University of Texas Health Science Center at Dallas. Both sets of tumors originated from the same tumor (M5480) but have a slightly different history of transplantation. M5480P was maintained in the frozen state for several years and, upon request, the tumor cells were thawed and injected into mice again. M5480A was obtained by Neaves in 1973 and has been maintained by serial transplantation every 14 days (16). In this laboratory, the tumors were transplanted every 14 (M5480A) or Abbreviations: hCG, human choriogonadotropin; 125I-hCG, l25I-labeled hCG; M5480A, designation of the altered M5480 tumor which responds to gonadotropin with both increased androgen and progesterone production; M5480P, same as M5480A but with only increased progesterone production.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 99

100

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Ascoli and Puett Table 1. Characteristics of Leydig cell tumors* Viability,t Yield of Tumor Tumor % of cells, weight, age, total g X 10-8/g days

65.2 + 2.8 1.43 0.22 1.99 0.15 12-14 M5480A 78.5 i 2.4 1.32 + 0.14 16-18 1.38 + 0.10 M5480P * The mean + SE of at least 10 determinations is shown. t Determined by Trypan Blue exclusion (20).

18 (M5480P) days into 7- to 9-week-old male C57B1/6 mice by the method of Neaves (17). Isolation of Leydig Tumor Cells. Tumor-bearing mice were killed by cervical dislocation. The tumors were removed, dissected free of fat, and weighed. Cells were obtained by gently forcing pieces of tumor through a 30 mesh stainless steel grid into a petri dish containing medium 199 with 20 1Ag of gentamicin per ml. The contents of the dish were transferred to a centrifuge tube and the tumor clumps were allowed to settle for 15-20 min at 4°. The supernatant was carefully removed and centrifuged (40) at 270 X g for 10 min. The resulting pellet, which contained variable amounts of erythrocytes, was resuspended in 3 ml of distilled water for 20 sec, followed by the addition of 1 ml of 3.6% NaCl to make the solution isotonic. The volume was adjusted to 40-50 ml with isotonic saline and the suspension was centrifuged again. The resulting erythrocytefree pellet was resuspended in medium 199 containing 1 mg of bovine serum albumin and 20 ug of gentamicin per ml; the cells were counted in a Coulter Counter and diluted to the desired density in the same medium. Microscopic examination of the cell suspension revealed that it was composed almost entirely of single cells. Measurement of Hormone Binding and Steroidogenesis. hCG was iodinated with lactoperoxidase (19) to a specific activity of 50-60 cpm/pg. The labeled hormone was stable for at least 4 weeks when stored at -70°. In agreement with previous findings (19), we found that iodination had little or no effect on the biological activity of hCG. Two milliliters of tumor cells (2.5 X 106 cells per ml) in medium 199/0.1% bovine serum albumin (containing 20 Mg of gentamicin per ml) were placed in plastic vials containing 100 Mul of 125I-labeled hCG (125I-hCG) (in 10 mM sodium phosphate buffer/0.15 M NaCl/1 mg of bovine serum albumin per ml, at pH 7.4) and incubated at 370 under 95% 02/5% CO2 in a shaking-water bath (60-80 rpm). At the times indicated, the contents of the vials were quantitatively transferred to polystyrene tubes containing 1 ml of ice-cold phosphate-buffered saline and centrifuged (4°) at 1200 X g for 10 min. The supernatant was aspirated, and the cells were resuspended in 2 ml of ice-cold phosphate-buffered saline by mixing in a Vortex mixer for 20 sec. The tubes were centrifuged again, the supernatant was aspirated, and the radioactivity of the pellet was determined in a gamma counter. Nonspecific binding was determined in parallel incubation vials containing 10 Ag of hCG. Under these conditions, nonspecific binding accounts for only 2-5% of the total binding. All binding data are reported as specific. Steroid determinations were performed in incubations identical to those described above. Steroids were measured by radioimmunoassay in suitable dilutions of the incubation medium obtained by centrifugation of the cell suspension. In some cases steroids were determined in the same vials used to measure binding. This was possible because the steroid radioimmunoassays were performed on 50-100 Mul of a 1:500 dilution of the supernatant, which contains only a small amount of radioac-

M5480A

M5480P 12

12-

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&

m

0

c 3

0

4- r

E0

E 4u

2

4

6

0

4

6

Time, hr

Time, hr

FIG. 1. Kinetics of binding of 125I-hCG to Leydig tumor cells. Two milliliters of tumor cells (2.5 X 106 cells per ml) were incubated with 1251-hCG (5 ng/ml, 40,000 cpm/ng) in Medium 199/0.1% bovine serum albumin at 37°. Each point represent the average of two determinations.

tivity derived from the free 125I-hCG. The results of the radioimmunoassays were corrected for the presence of 125I radioactivity (which accounted for 0-50% of the radioactivity present in the radioimmunoassay tubes) by including tubes without any steroid tracer. This correction was shown to be valid by comparing the ability of different levels of 125I-hCG and hCG to stimulate steroid secretion. Both hormones stimulated steroid secretion in the same fashion and to the same extent.

RESULTS

Leydig tumor cells The M5480 (A and P) tumors have been transplanted in this laboratory for seven generations (about 150 mide) with 100% success. Some of the characteristics of the Leydig cell tumors and the cells derived from them are given in Table 1. Neaves has shown that, although the cell composition of this Leydig cell tumor is independent of time after transplantation, the relative proportion of healthy cells is optimal in 11- to 14-day-old tumors (17). For this reason, the M548;OA tumor was studied 12-14 days after transplantation. The M5480P tumor exhibited a slightly slower rate of growth and therefore was used 16-18 days after transplantation, when its size was similar to that of the M5480A tumors. The same number of cells was obtained from both tumors, although the percentage of viable cells was higher from the M5480P tumors. The relatively low percentage of viable cells is somewhat puzzling, although it appears to be a characteristic of the tumor rather than the isolation procedure. Ficoll/metrizamide gradients (21) were also used to separate the erythrocytes from the tumor cells without any improvement in cell viability. Likewise, enzymatic dispersion of the tissue (collagenase, 0.5 mg/ml) failed to increase cell viability and resulted in a suspension composed mainly of cell clumps. It should be noted that similar percentages of viable cells have been obtained from other solid tumors (22) and that

Trypan Blue exclusion gives erratic results with some tumor cells (23, 24). Hormone binding Fig. 1 shows the kinetics of the interaction of 125I-hCG with the tumor cells. Hormone binding reaches a maximum at 1 hr, followed by a slow decline during the next 5 hr, at which time the amount of 125I-hCG bound was 60-70% of that bound at 1 hr. Fig. 2 shows the equilibrium binding data for the interaction of 1251-hCG with the tumor cells. The equilibrium dissociation constants are given in Table 2. These data are consistent with the presence of a single class of binding sites. The Kd for the interaction of 125I-hCG with the tumor cells is

Proc. Natl. Acad. Sci.. USA 75 (1978)

Ascoli and Puett

Biochemistry:

101

60 0.0

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D

0

A(

20-

0

0-~~~

0

io io0o iooo

10

Hormone, ng

FIG. 3. Specificity of the hCG receptor in Leydig tumor cells. Two milliliters of M5480P tumor cells (2.5 X 106 cells per ml) were incubated with 10 ng of 125j-hCG (527,474 cpm) and the indicated amounts of nonlabeled hormones at 370 for 1 hr. 0, hCG; 0, ovine lutropin. The solid bar shows the results obtained with thyrotropin, follitropin, somatotropin, and prolactin.

duction over a 6-hr period are quite different for both tumors. 6

8

10

Free 1251-hCG, x1O0° M

FIG. 2. Equilibrium binding of 1251-hCG to Leydig tumor cells. Two milliliters of tumor cells (2.5 X 106 cells per ml) were incubated with increasing amounts of 1251-hCG (35,000 cpm/ng) in medium 199/0.1% bovine serum albumin for 1 hr at 37°. The results of two independent experiments are shown (0, 0). (Inset) The binding at low concentrations of 1251-hCG. Each point represents the average of two determinations.

Fig. 5 shows the relationship between binding and steroid production. Both tumors exhibit a good correlation between these parameters. The concentration of hCG required to give maximal stimulation of progesterone production is 5-10 ng/ml in both tumors; this saturates about 80% of the receptors. At lower concentrations of hCG, the correlation between binding

identical in both kinds of cells and similar to that reported for normal Leydig cells (2). The specificity of the receptor appears to be the same as that observed in normal tissues (4), i.e., the receptor recognizes both lutropin and hCG, but not other glycoprotein hormones (Fig. 3). Note that the apparent affinity of ovine lutropin for the receptor is about one-tenth that of hCG. Activation of steroidogenesis Fig. 4 shows the kinetics of steroid production by the tumor cells. Under basal conditions, M5480P produces mainly progesterone, while M5480A produces about the same amounts of progesterone and testosterone. When the cells are stimulated with hCG, M5480P responds only with increased progesterone production, while M5480A shows an increase in the production of both steroids. However, the stimulation of progesterone production in M5480A is greater than the stimulation of testosterone production. Note also that the levels of steroid pro-

0)

a,c

0

Kd, Tumor

X

1011 M

M5480P

5.16

M5480P M5480A M5480A

3.56 + 0.24 5.22 ± 0.44 3.20 0.31

0.73

c

a)

0)

Table 2. Equilibrium dissociation constants for 1251-hCG interaction with the Leydig tumor cells*

ai 0

20 XI

0 (A

HD

'Receptors/ cell 2214

61

1952 + 25 1294 + 24 1099 20

* Determined by direct analysis (25) of the binding curves shown in Fig. 2.

Time, hr

FIG. 4. Kinetics of steroid production by Leydig tumor cells. Two milliliters of tumor cells (2.5 X 106 cells per ml) were incubated with (-) or without (0) hCG (5 ng/ml). Each point represents the average of duplicate incubations. The bars extend to the individual value of each incubation. (Note the difference in ordinate scales.)

102

Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Ascoli and Puett

0.05 0.5

5.0

50

1251-hCG, ng/ml FIG. 5. Relationship between binding and steroidogenesis in Leydig tumor cells. Two milliliters of tumor cells (2.5 X 106 cells per ml) were incubated with increasing amounts of 1251-hCG for 1 hr at 37°. *, Specific binding; 0, progesterone; 0, testosterone. Each point represents the average value of two independent experiments. The bars extend to the individual values obtained in each experiment.

and activation of steroidogenesis M5480A tumor.

appears

to

be better in the

DISCUSSION This work has led to the identification of two variants of the original M5480 testicular tumor which exhibit different steroid profiles both in the basal state and in response to gonadotropin. The nature of this difference remains to be delineated. In collaboration with T. J. McKenna and A. LaCroix, steroid profiles are being measured in an effort to identify the site(s) of enzyme deficiency. (The steroid profiles obtained to date have confirmed our findings, based on radioimmunoassay of the unextracted medium, that progesterone is the major steroid secreted.) There are several differences between normal Leydig cells and the Leydig tumor cells, the most obvious being the type of steroids secreted. The Leydig tumor cells produce mainly progesterone, while normal Leydig cells produce only testosterone. This difference appears to have arisen during the course of transplantation since originally the tumors were reported to produce mainly testosterone (9). Also, the capacity of hormone binding is different. Normal Leydig cells prepared by enzymatic dispersion are reported to have about 7000 receptors per cell (2), while the tumor cells apparently have fewer receptors. Since the majority of the normal Leydig cell receptors appear to be "spare" or "nonfunctional" with respect to steroid production (2), one can speculate that the tumor cells have lost mainly these receptors, rather than those involved in evoking the steroidogenic response. However, a cautionary note is in order since the number of specific receptors per cell may be an operational parameter that is strongly influenced by the methods of cell preparation and other variables. The gonadotropin receptor present in the tumor cells has retained the specificity and affinity of that present in normal tissue. During the past several months the M5480 A and P tumors have been transplanted into about 150 mice. When cells are used at the prescribed times after transplantation, all tumors have responded to hCG both in terms of binding and increased progesterone secretion upon hormonal stimulation. This is in contrast to the earlier studies of Moyle et al. (1, 9, 11-15) on the in vitro interaction of lutropin with the M5480 tumor. Using 2- to 6-month-old tumors, they found that only some of the tumors were gonadotropin responsive. This may have resulted from the age of the tumors used, since Neaves has shown that

there is a discrete time interval during which the morphology and composition of the transplanted tumors are optimal (17). The ability of the tumor cells to bind gonadotropin and respond via increased steroidogenesis seems to be consistently retained from one generation to another when the tumors are used at a defined time period after transplantation. This characteristic, together with the property that a homogeneous cell population can be obtained without enzymatic treatment, establishes the M5480 tumor as an ideal system for studies on the mechanism of action of gonadotropins. Moreover, the nature of the molecular changes occurring with time is most intriguing, and, last, the M5480 tumor represents one of the few endocrine-responsive tumors available for study. We thank Dr. W. B. Neaves for his interest and aid in this study. This work was supported in large part by National Institutes of Health Research Grant AM-15838 and Biomedical Research Support Grant RR-05424; services and facilities provided by the Vanderbilt Population Center (HD-05797) and Training Program (HD-07043) are gratefully acknowledged. D.P. is a Research Career Development Awardee (AM-00055) and a Dreyfus Teacher-Scholar Awardee. Last, we thank the Vanderbilt University Research Council for partial support. 1. Moyle, W. R. & Ramachandran, J. (1973) Endocrinology 93, 127-134. 2. Mendelson, C., Dufau, M. L. & Catt, K. J. (1975) J. Biol. Chem. 250,8818-8823. 3. Janszen, F. H. A., Cooke, B. A., Van Driel, M. J. A. & Van der Molen, H. J. (1976) J. Endocrinol. 70, 345-309. 4. Catt, K. J., Tsuruhara, T., Mendelson, C., Ketelslegers, J.-M. & Dufau, M. L. (1970) in Hormone Binding and Target Cell Activation in the Testis, eds. Means, A. R. & Dufau, M. L. (Plenum Press, New York), pp. 145-165. 5. Conn, M. P., Tsuruhara, T., Dufau, M. & Catt, K. J. (1977) Endocrinology 101, 639-642. 6. Jacobs, B. B. & Huseby, R. A. (1968) J. Natl. Cancer Inst 41, 1141-1153. 7. Shin, S. I. (1967) Endocrinology 81, 440-448. 8. Shin, S. I., Yasumura, Y. & Sato, G. H. (1968) Endocrinology 82, 614-616. 9. Moyle, W. R. & Armstrong, D. T. (1970) Steroids 15, 681693. 10. Neaves, W. B. (1975) Cancer Res. 35, 2663-2669. 11. Moyle, W. R., Moudgal, N. R. & Greep, R. 0. (1971) J. Biol. Chem. 246, 4978-4982. 12. Moudgal, N. R., Moyle, W. R. & Greep, R. 0. (1971) J. Biol. Chem. 246, 4983-4986. 13. Pokel, J. D., Moyle, W. R. & Greep, R. 0. (1972) Endocrinology 91,323-325. 14. Moyle, W. R., Jungas, R. L. & Greep, R. 0. (1973) Biochem. J. 134,407-413. 15. Moyle, W. R., Jungas, R. L. & Greep, R. 0. (1973) Biochem. J. 134,415-424. 16. Neaves, W. B. (1973) J. Natl. Cancer Inst. 50, 1069-1073. 17. Neaves, W. B. (1975) J. Natl. Cancer Inst. 55,623-631. 18. Holladay, L. A. & Puett, D. (1975) Arch. Biochem. Biophys. 171, 708-720. 19. Papaionannou, S. & Gospodarowicz, D. (1975) Endocrinology 97, 114-124. 20. Phillips, H. J. (1973) in Tissue Culture Methods and Applications, eds. Kruse, P. F. & Patterson, M. K. (Academic Press, New York), pp. 406-408. 21. Harris, R. & Ukaejiofo, E. 0. (1970) Br. J. Haematol. 18,229235. 22. Kulczycki, A., Jr., Isersky, C. & Metzger, H. (1974) J. Exp. Med. 139,600-616. 23. Cassel, W. A. (1976) Methods Cell Biol. 14, 181-186. 24. Moore, G. E., Ito, E., Ulrich, K. & Sandberg, A. A. (1966) Cancer

19,713-732.

25. Bliss, C. I. & James, P. (1966) Biometrics 22,573-602.

Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells.

Proc. Nati. Acad. Sci. USA Vol. 75, No. 1, pp. 99-102:, January 1978 Biochemistry Gonadotropin binding and stimulation of steroidogenesis in Leydig...
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