METABOLIC REGULATION OF STEROIDOGENESIS IN ADRENOCORTICAL CARCINOMA CELLS OF RAT. EFFECT OF ADRENOCORTICOTROPIN AND ADENOSINE CYCLIC 3’ , 5’ -MONOPHOSPHATE ON THE INCORPORATION OF (20S)-20-HYDROXY [ 7a-”H] CHOLESTEROL INTO DEOXYCORTICOSTE- 3
RONE AND CORTICOSTERONE.
Rameshwar K. Sharma and James S. Brush With the Technical Assistance of Lynda Sutliff Department of Biochemistry University of Tennessee Medical Units Memphis, Tennessee 38163
Received November 27,1973
Summary: ACTH in the isolated adrenocortical carcinoma cell inhibits the incorporation of (20S) -20-hydroxycholesterol in- to deoxycorticosterone and corticosterone. These results are in direct contrast to those obtained with the normal isolated adrenal cell where it has been shown that ACTH stimulates the synthesis of deoxycorticosterone and corticosterone from the above precursor. Nº-02’-dibutyryl adenosine 3’ , 5’ -monophos- phate, the nucleotide, which does not stimulate steroidogene- sis from (20S)-20-hydroxycholesterol in a’ normal adrenal cell, inhibits its incorporation into deoxycorticosterone and corti- costerone. The results presented further demonstrate the uniqueness of the ACTH-sensitive control system in the tumor.
Introduction
Studies with isolated adrenocortica. carcinoma 494 cells have investigated various aspects of the markedly altered control of steroidogenesis by ACTH- and cAMP 1 (1-5). These biochemical abnormalities have been localized in the reactions both before and after the cleavage of the cholesterol side chain.
The chemical methods have demonstrated in the adrenal
Trivial Names and Abbreviations
ACTH, adrenocorticotropic hormone; cAMP, adenosine cyclic 3’ , 5’ -monophosphate; Bt2CAMP, N6-2’0-dibutyryl adenosine 3’ , 5’ -monophosphate; pregnenolone, 5-pregnen-38-ol-20-one; corticosterone, 118, 21-dihydroxy-4-pregnen-3, 20-dione; deoxycorticosterone, 21-hydroxy-4-pregnen-3, 20-dione.
tumor an ACTH- and cAMP-insensitive conversion of endo- genous precursor (presumably cholesterol) to corticoste- rone (1). More precise radioactive labeling methods have indicated that the hormone and cAMP insensitivity are probably due to the loss of the cycloheximide-inhibited part of the system which converts cholesterol to (20S) - 20-hydroxycholesterol in the normal adrenal cell (6) . It has been further demonstrated, that although the tumor cells have the capacity to synthesize corticosterone from exogenous pregnenolone, progesterone and deoxycorticoste- rone, the incorporation of pregnenolone and progesterone is inhibited by ACTH (3), whereas the incorporation of exo- genous deoxycorticosterone into corticosterone is stimulated. This latter observation has been related to an effect of the hormone upon transport mechanisms carrying deoxycorticosterone into the cell (5). The pattern of inhibition by cAMP of the incorporation exogenous pregnenolone and progesterone into corticosterone was different from that of ACTH (3). Further- more, in direct contrast to the ACTH effect, Bt,CAMP was de- void of any stimulatory effect on the incorporation of deoxy- corticosterone into corticosterone in adrenal tumor cells (5). In continuation of the above studies upon the alteration in control processes in the tumor, the effect of ACTH and Bt,CAMP on the incorporation of (20S)-20-hydroxycholesterol into deoxycorticosterome and corticosterone has been studied and is reported herein.
Materials and Methods
The isolated adrenocortical carcinoma 494 cells were prepared by trypsin digestion (1). The method of incuba-
tion of ACTH or Bt, CAMP with (20S) -20-hyroxy [7a- H] choleste- 2 3 rol was as previously described (6) : Incubation was carried out in Teflon flasks. Each flask contained 20 ml of suspended isolated adrenal tumor cells prepared from 1.5 g adrenal tumor tissue. In addition to the appropriate amount of suspended tumor cells, each flask contained 1.60 uCi of (20S) -20- hydroxy [7a-H] cholesterol and ACTH (200 microunits per 3 ml) or Bt,CAMP (1 mM). The incubation was conducted for 2 150 min and the reaction was stopped by the addition of 15 ml of distilled water and 75 ml of methylene chloride to each flask. To the reaction mixture in each flask 15 mg of deoxycorticosterone and 15 mg of corticosterone were added and the products processed identically.
Deoxycorticosterone and corticosterone were purified by thin-layer chromatograghy (3,6). The isolated deoxy- corticosterone was acetylated (3,7), further purified by thin-layer chromatography (3,6) and crystallized from acetone-n-hexane to constant specific activity. The puri- fied corticosterone was crystallized from acetone-ligroin until the specific activity was constant.
ACTH, a United States Pharmacopeia standard, was purchased from United States Pharmacopeia. (20S) -20- Hydroxy [7a- H]cholesterol (specific activity, 25 Ci per 3 mmole) was purchased from New England Nuclear, Boston, Mass.
Results and Discussion
It has been well documented, from the studies of various laboratories, that ACTH in the adrenal cell, stimulates the rate limiting step which consists of the
conversion of cholesterol to pregnenolone (8-10). That this stimulation is mediated by cAMP has been shown (8,11, 12) and supported by this laboratory (3,13,14). Recent studies (6,15) have led to the modification of the hypo- thesis of Garren, et al. (16), regarding the mode of ACTH action. According to this scheme, ACTH stimulation in the normal isolated adrenal cell is both dependent and indepen- dent of cAMP. It has been proposed that ACTH stimulates two biosynthetic steps in the conversion of cholesterol to pregnenolone. The first step consists in the conver- sion of cholesterol to (20S)-20-hydroxycholesterol and is cycloheximide sensitive; the second step stimulates the synthesis of pregnenolone from (20S)-20-hydroxycholesterol which is cycloheximide insensitive. The latter effect of ACTH is not mediated by cAMP.
The present studies conducted with the isolated adreno- cortical carcinoma cells show that in contrast to the stimu- latory effect of ACTH in the transformation of (20S) -20- hydroxycholesterol into deoxycorticosterone and corticoste- rone in the normal adrenal cell (6), the hormone inhibits the incorporation of (20S)-20-hydroxycholesterol into deoxycorticosterone and corticosterone (Table I) in the tumor cell. Bt,CAMP, which has been shown to be devoid of any stimulatory activity in the transformation of (20S) - 20-hydroxycholesterol into steroid hormones in normal adrenal cell, also inhibits the incorporation of the above precursor into deoxycorticosterone and corticosterone in adrenocorti- cal carcinoma cells (Table II). These results appear to suggest that this is an additional biochemical alteration in the control by ACTH and cAMP in the adrenal tumor.
Table I Effect of ACTH on transformation of (20S)-20-hydroxy [7a-”H] - 3 cholesterol into deoxycorticosterone and corticosterone in isolated adrenocortical carcinoma cell preparation.
3
The total ‘H-disintegrations per min of the products (and their derivatives) obtained after the incubation of (20S)-20-hydroxy [7a- H] cholesterol with isolated adrenal 3 cells. Incubation was carried out in Teflon flasks con- taining 20 ml of isolated adrenocortical carcinoma cell preparation as mentioned under “Experimental Procedure”. Each flask contained (20S) -20-hydroxy [7a- H] cholesterol 3 (1.60 uCi) and ACTH (200 microunits per ml) or Bt CAMP (1 mM) . 2 The incubation was for 2.5 hours and the reaction was stopped by the addition of 75 ml of methylene chloride to each flask. Deoxycorticosterone and corticosterone were isolated as described under “Experimental Procedure”.
| Compound | Crystallization | 3 H dpm of compound from | |
|---|---|---|---|
| Control | +ACTH | ||
| Deoxycorticosterone | Crude Product | 794,000 | 373,000 |
| acetate | 1st | 786,000 | 362,000 |
| Corticosterone | 1st | 200,000 | 98,000 |
| 2nd | 183,000 | 87,000 | |
| 3rd | 176,000 | 77,000 | |
In view of these observations, coupled with earlier ones (2,3,4), it is evident that control by ACTH and CAMP of cholesterol side chain hydroxylation and subse- quent conversion to pregnenolone is highly modified in
Table II
Effect of Bt CAMP on transformation of (20S)-20-hydroxy [7a-”H] - 3 cholesterol into deoxycorticosterone and corticosterone in isolated adrenocortical carcinoma cell preparation.
3
The total ‘H-disintegrations per min of the products (and their derivatives) obtained after the incubation of (20S) -20-hydroxy [7a- H] cholesterol with isolated adrenal cells. 3 Conditions of the experiment were similar to the experiment in Table I.
| Compound | Crystallization | 3 H dpm of Control | compound from +Bt2CAMP |
|---|---|---|---|
| Deoxycorticosterone | Crude Product | 674,000 | 133,000 |
| acetate | 1st | 668,000 | 132,000 |
| Corticosterone | 1st | 170,000 | 31,000 |
| 2nd | 155,000 | 29,000 | |
| 3rd | 141,000 | 27,000 |
the tumor. By elimination this suggests that the rate limiting enzyme (s) which converts cholesterol to (20S) - 20-hydroxycholesterol (20-hydroxylase) may be, (1) missing, (2) present but not stimulated because of a defective activating protein for the process, (3) present but not stimulated because of the lack of synthesis of the acti- vating protein. Of these possibilities it is thought the first one is unlikely for the reason that the (20S) -20- hydroxycholesterol side chain cleavage enzyme (s) and the 118-hydroxylase are present and it is probable, therefore, that the 20-hydroxylase is also present. Based on the
premises that a cAMP-dependent protein kinase is involved in hormonal action, these studies suggest that the modifi- cation in the tumor may be in one of the following factors: a) cAMP binds to the regulatory subunit of the protein kinase but does not dissociate the catalytic subunit or b) CAMP dissociates the catalytic subunit of the protein kinase but the latter does not stimulate the ATP-dependent phosphoryla- tion of ribosomes which in turn results in the lack of the translation of mRNA.
The results shown here and elsewhere (1-5) demonstrate that adrenocortical carcinoma is not unresponsive to CAMP and ACTH as originally proposed (17). Rather the tumor possesses an ACTH-sensitive control system which is uniquely different from that of normal tissue. The techniques de- scribed demonstrate a means of elucidating these systems.
Acknowledgements
This research was supported by NSF Grant GB-38162 and Damon Runyon Memorial Fund for Cancer Research DRG- 1237.
References
1. Sharma, R. K. and Hashimoto, K .: Cancer Res. 32: 666-674 (1972).
2. Sharma, R. K .: Cancer Res. 32:1734-1736 (1972) .
3. Sharma, R. K .: Europ. J. Biochem. 32:506-512 (1973) .
4. Sharma, R. K. and Brush, J. S .: Arch, Biochem. Biophys. 156:560-562 (1973) .
5. Sharma, R. K .: FEBS Letters (In Press) .
6. Sharma, R. K .: J. Biol. Chem. 248 :5473-5476 (1973) .
7. Sharma, R. K., Doorenbos, N. J. and Bhacca, N. S. : J. Pharm. Sci. 60:1677-1682 (1971) .
8. Karaboyas, G. C. and Koritz, S. B. : 4:462-468 (1965) .
9. Stone, D. and Hechter, O .: 51:457-469 (1954) .
10. Billair, R. B. and Eik-Nes, K. B .: Biochim. Biophys. Acta 104:503-514 (1965).
11. Gill, G. N .: Metabolism 21:571-588 (1972) .
12. Burstein, S. and Gut, M .: Recent Progr. Hormone Res. 27: 303-439 (1971) .
13. Kitabchi, A. E. and Sharma, R. K .: Endocrinology 88: 1109-1116 (1971) .
14. Sharma, R. K., Hashimoto, K. E. and Kitabchi, A. E .: Endocrinology 91:994-1003 (1972) .
15. Sharma, R. K .: Ninth International Congress of Biochemistry, Stockholm, Sweden, July, 1973.
16. Garren, L. D., Gill, G. N., Masui, H. and Walton, G. M. : Recent Progr. Hormone Res. 27:433-478 (1973) .
17. Ney, R. L., Hochella, N. J., Grahme-Smith, D. G., Dexter, R. N. and Butcher, R. W .: J. Clin. Invest. 48: 1733-1739 (1969) .