Dehydroepiandrosterone Sulfate (DHEA-S) and 3’, 5’-Cyclic Adenosine Monophosphate (cAMP) Production in a Cultured Human Adrenocortical Carcinoma Cell Line (SW-13)
SACHIKO ITOH1), KOSHI TANAKA1), TOSHIYUKI HORIUCHI2), MUNEHITO KUMAGAE1), TOSHIO WATABE1), AKIRA KANBEGAWA3) AND NAOKATA SHIMIZU1)
Third Department of Medicine1), First Department of Medicine2) and Department of Gynecology and Obstetrics3), Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo 173, Japan
Abstract
Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3’, 5’-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were iucubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO2 and 95% air for 2 hours at 37℃. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10 ng/ml) and forskolin (10 nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10-7 M), noradrenalin (3×10-5 M), adrenalin (3×10-5 M) or a-melanocyte- stimulating hormone (a-MSH, 10-7 M), none of which was associated with cAMP production. Neither adrenocorticotropin (10-10 M) nor human chorionic gonadotropin (500 mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10-7 M), and 12-O-tetradecanoyl- phorbol-13-acetate (10-8 M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.
Address all correspondence and reprints re- quests to: SACHIKO ITOH, M. D., Third De- partment of Medicine, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo
173, Japan.
This work was supported in part by a research grant from the Japanese Ministry of Health and Welfare and by a research grant from the Japanese Ministry of Education, Science and Culture.
A number of cultured cell lines have been widely used to investigate the cellular mechanisms of action of the various hormones. Several cell lines originating in the adrenal cortex have been established and some of them are now commercially available. These include mouse adrenal cancer cell line Y1 (Morera and Saez 1980), rat adrenocortical carcinoma 494 (Ganguly et al., 1984) and SW-13. SW-13 is the only widely available cultured human adrenocor- tical cell line established from a small-cell carcinoma of the adrenal cortex (Leibovits et al., 1973). Although several morphological studies on this cell line have been reported (Johnson and Sheridan 1971; Murray et al., 1981; Silva and Gilula 1972), there are few reports on the biochemical function or steroidogenesis in this particular cell line, except that by Murray et al. (1981). They have reported that production of both cAMP and fluorogenic steroids in this cell line is not stimulated by adrenocorticotropic hormone (ACTH) or cholera toxin (CT), a receptor-independent direct stimulator of adenylate cyclase (Cassel and Pfeuffer 1978). Dehydroepiandrosterone sulfate (DHEA-S) is characteristically produced in a much larger quantity in the human adrenocortical carcinoma (Plotz et al., 1952) and in fetal adrenal cortex (Simpson et al., 1979). In order to define the function and the mechanisms of steroidogenesis in SW-13, the effects of several peptides which stimu- late steroidogenesis in normal adrenal cortex, as well as the direct stimulators of adenylate cyclase and of protein kinase C, on the production of several adrenal steroids and cAMP, were studied in this cell line.
Materials and Methods
SW-13 (ATCC, CCL-105) was provided by Dainihon Pharmaceutical Co., Ltd., Tokyo, Japan. Fetal calf serum (FCS), penicillin- streptomycin (PC-SM), fungisone and Leibovitz
L-15 medium (L-15) were purchased from Gibco Lab., NY, USA. Chorela toxin (CT), 12-O- tetradecanoyl phorbor 13-acetate (TPA), human chorionic gonadotropin (HCG), 1-noradrenalin, 1-adrenalin, bovine gamma globulin (Cohn fraction II), bovine serum albumin (BSA) and DHEA were obtained from Sigma Chemical Co., Ltd., St Louis, Mo, USA. Synthetic a-melano- cyte-stimulating hormone (a-MSH) was obtained from Peptides Inst., Osaka, Japan. Synthetic (1-24)adrenocorticotropin (ACTH) and angio- tensin II (A-II) were kindly donated by Daiichi Seiyaku Co., Ltd. Tokyo, Japan and Ciba Geigy Co., Ltd., Tokyo, Japan, respectively, and forskolin (FS) by Nippon Kayaku Co., Ltd., Tokyo, Japan. A23187 was purchased from Behring Diagnostics, Ca, USA.
SW-13 was cultured to confluent monolayers in a multiwell tissue culture dish (# 76-033-05, Flow Lab., Inc., Va., USA) in L-15 medium containing 10% FCS, 100 U/ml PC, 100 µg/ml SM and 2.5 µg/ml fungisone in an atmosphere of 5% CO2 and 95% air at 37°C. The cells (31.0×104 cells/ml) were washed twice with L-15 medium containing 0.1% BSA immediately prior to the experiment and then incubated in the L-15 medium containing 0.1% BSA with or without the reagents described above in an atmosphere of 5% CO2 and 95% air for 2 hours. After the incubation, the medium was transferred to a tube and centrifuged at 2500 g for 15 min at 4℃ to obtain the cell-free medium. To the cells, 1.0 ml H2O was added, once frozen at -20°℃ and then sonicated with a sonicator (Handy Sonic Model UR-20P, Tomy Seiko Co., Ltd., Tokyo, Japan). The sonicated cells were centrifuged at 2500 g for 15 min at 4℃ and the supernatant was transferred to another tube. Aliquots of the medium and the cell extract were stored frozen at -20℃ for later determination of the steroids and cAMP.
Aldosterone (Ald) was measured by a specific radioimmunoassay (RIA) as described previously (Horiuchi et al., 1985). The sensitivity of Ald-RIA was 0.53 ng/ml sample. Corti- costerone (B) was also determined by a direct RIA and the sensitivity was 4.1 ng/ml sample. Cortisol (F) was determined with a RIA kit obtained from Eiken Immunochemical Co., Ltd., Tokyo, Japan. The sensitivity of cortisol RIA was 12.5 ng/ml sample.
DHEA-S was measured by radioimmunoassay after converting DHEA-S to DHEA by acid
hydrosis. To a 0.7 ml sample, 1.0 ml of di- ethylether was added and vortexed for several seconds to remove steroids except DHEA-S and then the ether phase was discarded. To the water phase which contain DHEA-S, 0.7 ml of 5% H2SO4 was added and the contents were boiled for 20 min to convert DHEA-S to DHEA. 3.0 ml ether was added and vortexed to extract DHEA into the ether phase. The ether phase was transferred to another tube and 1.0 ml of distilled water was added to wash the extract. The ether phase was then transferred to another tube, dried under a nitrogen gas stream at 40℃ and reconstituted with 0.3 ml RIA buffer (0.05 M Borate buffer containing 0.5% BSA, 0.1% bovine gamma globulin and 0.01% NaN3). The re- constituted samples or standard DHEA were incubated with 3H-DHEA (New England Nuclear Products. Ma, USA) and anti-DHEA antiserum (final dilution 1: 200,000) in a final volume of 0.5 ml. At the end of incubation at 4℃ for 24 hours, 0.5 ml of saturated NH4SO4 was added to each tube, vortexed and centrifuged at 3000 g for 20 min at 4℃. 0.5 ml of the supernatant was transferred to a vial containing 3 ml of Aquasol (New England Nuclear Products, Ma, USA) and counted. Crossreactivity of the anti- serum was 100% with DHEA, 40.2% with DHEA-S, 2.0% with testosterone (T), 1.4% with dehydro-T, 19.8% with 4-androsteronedione, 11.4% with androsterone, 1.4% with progeste- rone, 1.6% with pregnenolone, 1.1% with cortisol, 6.6% with 16-OH-DHEA, 0.02% with 16-OH- DHEA-S and 3.4% with estradiol-17. Intra- and inter-assay coefficients of variation were 13.0 and 7.2%, respectively, and the sensitivity was 0.05 ng/ml sample.
cAMP was determined with a RIA kit obtained from Yamasa Shoyu Co., Ltd., Chiba, Japan. The samples were succinated (Frandsen and Krishna 1976) without extraction, according to the instruction included in the RIA kit. Inter- and intra- assay CV’s for cAMP assay were less than 15%.
Statistical analysis was performed using Student’s unpaired t-test and the results are presented as the mean and SEM, of duplicate determinations for three experiments, unless otherwise indicated.
Results
The effects of A-II (10-7 M), ACTH (10-10 M), HCG (500 mIU/ml), noradrenalin (3×10-5 M) and adrenalin (3×10-5 M) and a-MSH (10-7 M) on DHEA-S production are shown in Fig. 1. The DHEA-S con- centration in the medium was much greater than in the control (0.24±0.04 ng/ml) when the cells were incubated with A-II (0.93± 0.13 ng/ml, p<0.01), noradrenalin (2.55± 0.69 ng/ml, p<0.05), adrenalin (2.10±0.35 ng/ml, p<0.01), or a-MSH (3.11±0.61 ng/ml, p<0.01) (Fig. 1, lower panel). There was no significant increase in the intracellular DHEA-S concentration by any of these hormones (p<0.05 for all of these six hormones, Fig. 1, upper panel). Although the concentration of DHEA-S in the cells incubated with catecholamines and a-MSH appeared to be greater than in the control, the increase was not statistically significant. There was no statistically significant increase in the DHEA-S concentration in the medium by ACTH (0.62±0.28 ng/ml, p>0.05) or HCG (0.44±0.17 ng/ml, p>0.05).
cAMP production by A-II, ACTH, HCG, catecholamines and «-MSH is shown in Fig. 2. There was essentially no increase (p> 0.05 for all) in cAMP content by A-II (1.93±0.08 pmol/ml), ACTH (2.03±0.03 pmol/ml), HCG (1.86±0.03 pmol/ml), norad- renalin (1.58±0.03 pmol/ml) adrenalin (2.15 ±0.15 pmol/ml), or a-MSH (2.42±0.27 pmol /ml) when compared to the control (2.13± 0.08 pmol/ml).
The effect of CT or FS on the cAMP level in SW-13 is shown in Fig. 3. A highly significant increase in the cAMP con- centration was observed by both FS (14.81 ±0.89 pmol/ml, p<0.001) and CT (12.68± 1.21 pmol/ml, p<0.001) when compared to the control value (1.59+0.13 pmol/ml. The changes in the DHEA-S concentration in response to CT or FS are shown in Fig. 4. There was no statistically significant change
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DHEA-S AND CAMP PRODUCTION IN SW-13
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caused by CT or FS in the DHEA-S con- centration both in the medium (0.23±0.04 ng/ml, p>0.05 and 0.43±0.22 ng/ml, p> 0.05, respectively : control ; 0.24±0.04 ng/ml) and in the cells (0.33±0.04 ng/ml, p>0.05 and 0.18±0.03 ng/ml, p>0.05, respectively. control; 0.16±0.11 ng/ml).
When the cells were incubated with TPA, a significant increase in the DHEA-S concentration (0.99±0.10 ng/ml, p<0.05: control ; 0.51±0.03 ng/ml) in the medium was observed as shown in Fig. 5 (lower panel). However, the DHEA-S concentration
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in the cells was reduced significantly (1.15 ±0.04 ng/ml, p<0.01. control; 1.87±0.13 ng/ml) in the presence of TPA (Fig. 5, upper panel). Consequently, there was no significant change in the total DHEA-S concentration (cell plus medium ; 2.15±0.15 ng/ml, control; 2.39±0.10 ng/ml, p>0.05).
A23187, a Ca ionophore, induced a significant increase in the DHEA-S con- centration in the medium (1.36±0.15 ng/ ml, p<0.01. control; 0.51±0.03 ng/ml) as shown in the lower panel of Fig. 5. On the other hand, the DHEA-S concentration in the cells was reduced significantly (1.05 ±0.08 ng/ml, p<0.01, control; 1.87±0.13 ng/ml) by A23187 (Fig. 5, upper panel). The total DHEA-S concentration (cell plus medium) was essentially unchanged (2.41± 0.06 ng/ml, p>0.05) by A23187 when compared to the control value (2.39+0.10 ng/ml).
The concentrations of Ald, B and F were less than the sensitivity in all of the samples assayed simultaneously with DHEA- S.
Discussion
Significant stimulation of cAMP pro- duction was demonstrated in SW-13 by both CT and FS in the present study. cAMP is one of the important second messengers for adrenal steroidogenesis which in turn acti- vate protein kinase A in the normal adrenal cortex (Shima et al., 1980). However, no significant increase in the production of steroids was demonstrated in spite of a significant increase in the cAMP concen- tration due to these receptor-independent stimulators of adenylate cyclase in SW-13. CT is recognized as activating the catalytic unit of the adenylate cyclase by inactivat- ing GTPase in the regulatory unit of the adenylate cyclase (Cassel and Pfeuffer 1978). FS stimulates cAMP production by directly acting on the catalytic unit of the adenylate cyclase (Seamon and Daly 1981). Thus,
our present results suggest that both the regulatory and catalytic units of the adenylate cyclase are functioning and also suggest that there is some coupling defect(s) between cAMP and protein kinase A or distal to this step in SW-13. Another possible defect of this cell line may be the impaired Ca2+-influx by cAMP, although all of these possibilities still remain to be investigated. The exact reasons for the discrepancy between our present results and the report by Murray et al. (1981) who have failed to detect cAMP production by CT in the same cell line, are not clear. The difference in the culture medium used in the experiments might have resulted in a different response of the cells to CT. No details of the method for cAMP assay are given the report by Murray and co- workers (1981). Thus, another possibility is that the sensitivity of the cAMP assay they used was lower than that employed in this study so that a small increase in cAMP might have escaped detection.
The production of Ald, B, F or DHEA- S, as well as cAMP, was not increased by ACTH or HCG. The concentrations of ACTH (Pedersen and Brownie 1980; Farese et al., 1983) and HCG (Meidan et al., 1985 ; Dyer and Erickson 1985) used in this study were much higher than those which stimu- late steroidogenesis in vitro in the adrenal cortex and in the gonadal gland, respectively. Therefore, the present data indicate that SW-13 may either lack the receptors for these hormones or have some coupling defect(s) between the receptors and regula- tory unit of the adenylate cyclase, if these hormones had bound to the receptors. Since both ACTH (Shima et al., 1980; Farese et al., 1983) and HCG (McIlroy and Ryan 1983) have been reported to stimulate adeny- late cyclase, the unresponsiveness of cAMP in SW-13, in spite of functioning regulatory and catalytic units of the adenylate cyclase, favors the lack of the receptors for these hormones. The study of the binding of
these hormones to the cell surface membrane receptor in SW-13, may shed light on this matter.
a-MSH is an unique peptide in that this hormone is produced only in the fetal pituitary (Silman et al., 1976) and does not normally exist in post neonatal life except in some ectopic ACTH-producing carcinoma cells (Abe et al., 1967) in man. In the present study, significant production of DHEA-S was demonstrated in this human carcinoma cell line by a-MSH, without an increase in cAMP production. Thus, these results suggest that the receptor for a- MSH in SW-13 is not coupled to adenylate cyclase to stimulate steroidogenesis.
The biological actions of A-II (Garrison et al., 1984) and a-adrenergic receptor mechanism (Exton 1985) are mediated through the PI-DG-protein kinase C system and also Ca2+-calmodulin. On the other- hand, B-receptor is coupled with adenylate cyclase (Exton 1985). Significant production of DHEA-S was demonstrated in SW-13 by noradrenalin, adrenalin, A-II and «-MSH without demonstrable stimulation of cAMP production in this study. TPA has been known to directly stimulate protein kinase C, thus mimicking the action of DG (Castagna et al., 1982), and secretion of hormones in normal endocrine glands (Pruss et al., 1985 ; Abou-Samura et al., 1986). A23187 is a well established Ca2+ ionophore (Blackmore et al., 1978) and causes the release of steroids by normal adrenocortical cells (Capponi et al., 1984). Ca2+-calmodulin system has been reported to be a key factor in adrenal steroidogenesis (Hall et al., 1981 ; Sekimoto et al., 1984). Demonstration of significant release of DHEA-S into the medium by TPA and A23187 in SW-13 suggests that an intra- cellular signaling system which involves protein kinase C and Ca2+ is indeed functioning in this cell line. Thus, the present results indicate that SW-13 may have a-adrenergic, A-II and a-MSH receptors
which are functionally linked with the PI- DG-protein kinase C system or with acti- vation of the Ca2+-calmodulin system. How- ever, the existence of these receptors remains to be proven by the study using the appro- priate agonistic and antagonistic ligands. Further study is also required to establish the exact mechanism of DHEA-S release by TPA and A23187 in SW-13.
SW-13 was shown to produce at least DHEA-S in response to several ligands in the present study. These results imply that SW-13 probably contains a complex of enzymes, such as 17a-hydroxylase, 17, 20- lyase and which are required to produce DHEA-S. No significant production of other steroids such as Ald, B and F was demonstrated in response to any of the factors examined, and this may be accounted for by the relatively lower sensitivity of the RIA’s employed to determine these steroids in this study. However, aldosterone and corticosterone in primary culture of the rat adrenocortical cells under similar conditions using smaller number of cells than in the present study, have easily been detected (Horiuchi et al., 1985, 1987 ; Farese et al., 1983). Thus, significant amounts, if any, of these steroids except DHEA-S, are not likely to be produced in this cell line.
Iu summary, significant production of CAMP by CT and FS without steroid production, and release of DHEA-S by catecholamine, a-MSH, A-II, TPA and A23187 were demonstrated in SW-13 in the present study for the first time, although in a preliminary form. Thus, SW-13 pro- bably retains a functionally intact regulatory and catalytic unit of adenylate cyclase which has some defect(s) in mediating the intra- cellular message between cAMP formation and the production of steroids. The present results also suggest that other intracellular signaling systems such as the PI-DG-protein kinase C system, and that involve Ca2+, are functioning in SW-13. Since SW-13 is only widely available cell line established
from a human adrenocortical tissue, this cell line may provide a useful model for investigating the cellular mechanism of production and secretion of adrenal steroids in man.
Acknowledgements
We are grateful to Ms Yukiko Shimada for her excellent technical assistance. We also thank Ciba Geigy Co., Ltd., Daiichi Seiyaku Co., Ltd. and Nippon Kayaku Co., Ltd. for gener- ously supplying of A-II, (1-24)ACTH and FS, respectively.
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