PREGNENOLONE BIOSYNTHESIS IN ISOLATED CELLS OF SNELL RAT ADRENOCORTICAL CARCINOMA 494
J.I. MASON and W.F. ROBIDOUX
Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545, U.S.A.
Received 12 June 1978; accepted 8 August 1978
Adrenocorticotrophin (ACTH) produced an insignificant stimulation of pregnenolone bio- synthesis from endogenous precursors in isolated cells prepared from the rat Snell adrenal carcinoma 494. On the addition of 25-hydroxycholesterol, the rate of pregnenolone synthesis increased 10-fold. These results, noting also the very low cholesterol content of the tumor cells, suggested that lack of cholesterol was responsible for the poor steroidogenic response of the cells to ACTH. Endogenous pregnenolone production was sensitive to cytochalasin B as well as cycloheximide. However, pregnenolone synthesis after the addition of 25-hydroxycholesterol was not affected by these inhibitors. Removal of cycloheximide from the cells resulted in the immediate restoration of the initial rate of pregnenolone synthesis from endogenous precursors. This suggested that cycloheximide was interfering with the action of a stable activated intra- cellular messenger.
Keywords: Isolated adrenal tumor cells; pregnenolone biosynthesis; ACTH mechanism of action; 25-hydroxycholesterol; cycloheximide; cytochalasin B.
Current evidence has indicated that adrenocorticotropin (ACTH) exerts its regulatory effect on adrenal steroidogenesis at the level of the cholesterol side-chain cleavage enzyme system. Much consideration has been given to whether this regulatory effect involves control of substrate (cholesterol) availability to the enzyme (see Mason et al., 1978) or a direct action on the enzyme (Caron et al., 1975; Koritz et al., 1977). The sensitivity of ACTH-stimulated steroidogenesis to inhibitors of protein synthesis such as puromycin (Ferguson, 1962) and cyclo- heximide (Garren et al., 1965) led to the concept that a rapidly turning-over pro- tein is involved in the regulation of steroidogenesis. Early studies suggested that the protein factor was involved in the transportation of free cholesterol released from lipid droplets to the mitochondrial site of cholesterol sidechain cleavage (Garren et al., 1971). More recent studies favor the involvement of the cyclo- heximide-sensitive factor at the level of intramitochondrial relocalization of choles- terol (Mahaffee et al., 1974; Paul et al., 1976; Mason et al., 1978). However, while ACTH-dependent stimulation of a specific peptide in cultured adrenocortical cells has been reported (Dazord et al., 1977) and addition of lutropin to rat testis
Leydig cells stimulated the synthesis of a specific protein (though this could only be detected 2 h after addition of lutropin, Janszen et al., 1976), hitherto no protein has been isolated that would fill the role of a rapidly turning-over protein factor.
Alternative mechanisms have been proposed to explain these observations. Schulster et al. (1974) proposed that although continued synthesis of some labile protein factor was necessary for the expression of the ACTH response, ACTH had the effect of activating this protein factor rather than of directly inducing it. Rubin et al. (1973) demonstrated the reversible inhibition of ACTH-induced corticosteroid release by cycloheximide and proposed the existence of a stable mediator on such evidence. Steroidogenesis from endogenous precursors in mouse adrenal tumor cells grown in tissue culture was stimulated by ACTH and was inhibited both by cycloheximide (Kowal, 1970) and by cytochalasin B, an inhib- itor of microfilament motility (Mrotek and Hall, 1977). However, using an isolated cell preparation of the Snell adrenal carcinoma 494, Sharma (1973) reported that corticosteroidogenesis in these tumor cells was insensitive to both ACTH and cycloheximide. This insensitivity has been associated with an abnormal protein kinase in these tumor cells (Sharma et al., 1977).
Since earlier work on the Snell adrenal carcinoma had indicated low rates of corticosterone synthesis (Ney et al., 1969; Sharma, 1973) which appeared associated with low rates of mitochondrial steroid hydroxylase activity (Peron et al., 1974), we have studied the involvement of a cycloheximide-sensitive factor in the regula- tion of steroid synthesis in these cells by monitoring pregnenolone formation using a sensitive radioimmunoassay procedure. The inability of cycloheximide or ACTH to influence the metabolism of 25-hydroxycholesterol in both normal isolated adrenal cells (Falke et al., 1975) and in adrenal mitochondria (Jefcoate et al., 1974), suggested that an investigation of the metabolism of this sterol in isolated adrenal tumor cells should indicate the steroidogenic potential of these cells.
MATERIALS AND METHODS
The rat adrenocortical carcinoma 494 was transplanted and maintained in 50- 70 g male Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilming- ton, Mass., U.S.A.) as described by Ney et al. (1969). Tumor tissue was implanted subcutaneously through a small incision near the base of the tail. The tumor tissue for all the reported studies was taken 3 weeks following the initial transplant. A dispersed tumor cell preparation was obtained as described previously (Kimmel et al., 1974). Viable tumor tissue was placed in a 125-ml Erlenmeyer flask con- taining 50 ml Krebs-Ringer phosphate buffer, pH 7.4, containing 0.2% glucose and 0.5% bovine serum albumin. The tissue was stirred for 15-20 min at 37℃ using a magnetic stirring bar. After the undissociated tissue settled out, the super- natant which contained dissociated cells was filtered through gauze into 50-ml centrifuge tubes. The dissociation procedure was repeated twice and the combined
supernatants were centrifuged at 600 rpm in an International Model PR-2 refrigerated centrifuge (International Equipment Co., Needham Heights, Mass., U.S.A.) for 20 min. The resulting pellets of packed cells, cellular debris and contaminating red blood cells were resuspended in 50 ml buffer and allowed to settle by gravity for 60 min. The resulting supernatant which contained most of the contaminating red blood cells and cellular debris was discarded. The loosely packed cells were resus- pended again in 50 ml buffer and gravity settling repeated at least twice. This procedure resulted in a preparation of nonaggregated tumor cells virtually free of red blood cells and other small particles that did not settle with the relatively heavy tumor cells. The nucleated cells were counted in a haemocytometer following staining with Neisser’s B stain.
Cells were prepared from normal adrenals of male Sprague-Dawley rats using a trypsin-collagenase digestion procedure exactly as described previously in this laboratory (Haksar et al., 1973).
Incubations of the isolated cells were carried out in air at 37° in the Krebs- Ringer phosphate buffer, pH 7.4, containing 0.2% glucose and 0.5% bovine serum albumin (Fraction V) in a total volume of 1 ml containing 2 X 106 cells. The incubations were terminated by addition of 4 ml methanol or rapid chilling. Each incubation contained 5 uM cyanoketone (2a-cyano-4,4,17a-trimethyl-176-hydroxy- 5-androsten-3-one), an inhibitor of 30-hydroxysteroid dehydrogenases, to prevent further metabolism of pregnenolone. Pregnenolone was determined in the samples following organic extraction into chloroform and finally ethanol using [3H] preg- nenolone as a recovery marker exactly as described for rat adrenal mitochondrial preparations (Mason et al., 1978). The method was a radioimmunoassay procedure using antisera raised in New Zealand white rabbits to pregnenolone-20-albumin. The method is based on that described by Abraham et al. (1973) and recorded in detail previously (Mason et al., 1978). In these studies it was found possible also to determine pregnenolone directly in incubations by diluting the chilled incubations with 4 ml water and assaying 10-ul aliquots in the radioimmunoassay procedure. The results obtained using this more direct assay procedure were similar to those obtained on samples processed through organic extraction. Thus the level of preg- nenolone-binding proteins (Kream and Sauer, 1977) in the tumor cells, if present, did not interfere at the dilution used in the present assay.
25-Hydroxycholesterol (5-cholestene-30,25-diol; Steraloids, Inc., Wilton, N.H., U.S.A.) was added in ethanol to the incubations as was cyanoketone and choles- terol; the ethanol content did not exceed 1%. All other additions were made in the Krebs-Ringer phosphate albumin buffer.
Cytochrome P-450 was determined in an Aminco-DW2 spectrophotometer in the split-beam mode as described by Omura and Sato (1964) using an extinction coefficient 91 mM-1 cm-1 for the absorbance change, 450-490 nm. The reducing agent was sodium dithionite.
Cholesterol levels in the isolated cells were determined using the method of Sperry and Webb (1950) using the digitonide to separate free from ester choles- terol.
ACTH used in these studies was USP corticotropin reference standard distrib- uted by United States Pharmacopeial Convention, Inc. Cycloheximide, cytochalasin B, cholesterol, bovine serum albumin (fraction V) and pregnenolone were ob- tained from Sigma Chemical Co., St. Louis, Mo., U.S.A. NEN Chemicals, Boston, Mass., U.S.A., supplied the [7a-3H]pregnenolone specific activity 17 Ci/mmol, NET 039) used in the radioimmunoassay. All other chemicals were of analytical reagent grade.
RESULTS
(i) Pregnenolone formation in isolated adrenal tumor cells
When isolated adrenal tumor cells were incubated at 37℃ in the presence of the 33-hydroxysteroid dehydrogenase inhibitor (cyanoketone) pregnenolone formation from endogenous precursors and was linear over the 2-h assay period. No preg- nenolone was detected when cyanoketone was omitted from the assay medium.
15
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Pregnenolone formation nmoles per 2 x 106 cells
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Addition of 20 nM ACTH, a concentration which produced maximal cyclic 3’,5’- AMP accumulation in this tumor-cell preparation (Peron et al., 1975), resulted in an insignificant increase in pregnenolone formation. Addition of 100 uM 25-hydroxy- cholesterol to the assay medium resulted in a marked stimulation of pregnenolone production which was not influenced by the presence of ACTH (this result not shown). The data is presented in Fig. 1.
(ii) Effect of cycloheximide on pregnenolone synthesis
Fig. 1 also illustrates the effect of the protein synthesis inhibitor, cycloheximide, on pregnenolone formation by the tumor cells from endogenous precursors and in the presence of 100 uM 25-hydroxycholesterol. Cycloheximide (25 uM) was found to inhibit almost completely pregnenolone formation from endogenous precursors in the tumor cells, whereas it had almost no effect on the stimulated rate of preg- nenolone formation in the presence of 25-hydroxycholesterol.
(iii) Reversibility of cycloheximide inhibition of steroidogenesis
Since the inhibition of ACTH-stimulated steroidogenesis by cycloheximide in normal adrenocortical cells has been thought to involve the inhibition of synthesis of rapidly turning over protein(s) required for hormone-stimulated steroidogenesis, it was of interest to study steroidogenesis in these adrenal tumor cells following exposure to the protein synthesis inhibitor. Isolated adrenal tumor cells were incubated at 37° for 60 min in the presence of 25 uM cycloheximide. These cells were then centrifuged at 100 g for 10 min and the packed cell pellet resuspended in fresh buffer (2 X 106 cells/ml), recentrifuged and finally resuspended in buffer at 2 X 106 cells/ml. Cyanoketone (5 (M) was added at this point and the cells brought up to 37º. The rate of pregnenolone formation was determined to be 0.95 ± 0.07 nmoles per 2 × 106 cells per 2 h compared to 0.88 ± 0.06 nmoles per 2 × 106 cells per 2 h in control cells treated similarly but omitting cycloheximide from the medium. The results are expressed as the means of 3 experiments + the standard deviation.
(iv) Effect of cytochalasin B on adrenal tumor-cell pregnenolone synthesis
The results presented in Fig. 2 show that cytochalasin B inhibited the basal rate of steroidogenesis from endogenous precursors in the isolated rat adrenal tumor cells, but had no effect on pregnenolone formation in the presence of 100 uM 25-hydroxycholesterol. The latter result excluded the possibility that cytochalasin B was inhibiting glucose uptake into the cells and interfering with steroidogenesis via this route.
(v) Free and esterified cholesterol levels in normal rat adrenocortical cells and rat adrenal tumor cells
Earlier reports have noted the low levels of cholesterol ester in the Snell adrenal carcinoma tissue compared to the high levels associated with normal adrenal tissue
100
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Control Activity
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6
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4
- log [Cytochalasin B, molar ]
(Lossow et al., 1965; Ney et al., 1969). Free and esterified cholesterol levels were determined in isolated cells prepared from both normal rat adrenal glands and rat adrenal tumor tissue. The results shown in Table 1 illustrate the extremely low levels of cholesterol ester in the tumor cell compared with the normal adrenal cell which correlates with the lack of the characteristic cholesterol ester-rich lipid droplets of the normal adrenal cell (Sharma and Hashimoto, 1972; Kimmel et al., 1974). The free cholesterol of the tumor cell was also low as compared with the normal.
| Tumor | Normal | ||
|---|---|---|---|
| nmoles/106 cells | |||
| Cholesterol ester | 0.7 ± 0.1 (3) | 1250 | + 97 (3) |
| Non-esterified cholesterol | 10.5 ± 0.7 (3) | 250 | + 43 (3) |
| Cytochrome P-450 | 0.015 ± 0.002 (2) | 0.230 | ± 0.018 (2) |
Cytochrome P-450, non-esterified and esterified cholesterol were determined as described in the Methods. The sterol determinations were performed on aliquots of 150 x 105 tumor cells and 3 × 105 normal adrenal cells. The results are expressed as the mean + standard deviation with the number of experiments in parenthesis.
(iv) Cytochrome P-450 content of rat adrenal carcinoma cells and normal rat adrenocortical cells
Cytochrome P-450 is the terminal oxidase component of the several steroid hydroxylase systems involved in the pathway of corticosteroidogenesis (reviewed by Simpson and Mason, 1976). Thus its level in steroidogenic cells may be taken as an approximate indication of the steroidogenic potential of the cell.
The level of cytochrome P-450 in normal adrenal cells as well as in the adrenal tumor cells is shown in Table 1. The amount in tumor cells is approximately 5% that in the normal adrenal cell. Thus one can expect the maximal steroid output of the tumor cell to be in the region of 5% of that in the normal adrenal cell.
DISCUSSION
The rate of pregnenolone synthesis from endogenous precursors in the Snell adrenal carcinoma cells was in the order of 2.2 nmoles pregnenolone per 2 X 106 cells per 2-h incubation (Fig. 1). This rate is approximately 10-fold greater than the rate of corticosterone synthesis when determined in these tumor cells previously reported by Sharma and Hashimoto (1972) and Kimmel et al. (1974). Since omis- sion of the hydroxy-steroid dehydrogenase inhibitor, cyanoketone, from the cell incubation resulted in failure to detect pregnenolone in the medium it was con- cluded that pregnenolone was converted rapidly to further steroidal products of which corticosterone represented only about 25% of the total product. A similar finding has recently been reported by Janzsen et al. (1978) in studies on steroido- genesis in a rat Leydig cell tumor. Those studies revealed that testosterone was not the major steroidal product of the tumor cell. This suggests that a characteristic of a steroidogenic tumor is the lack of a concerted intracellular control directed at synthesis of the major characteristic steroid of the specific steroid-secreting cell.
Addition of ACTH to the assay medium resulted in an insignificant stimulation in the rate of pregnenolone synthesis. This has to be compared to the 100-fold stimulation seen in normal adrenocortical cells (Sayers et al., 1971). The concen- tration of ACTH used in the assays reported in Fig. 1 was 20 nM. This dosage was chosen since it produced maximal activation of adenulate cyclase in earlier studies (Peron et al., 1975).
The addition of 25-hydroxycholesterol, an alternative substrate for the choles- terol desmolase enzyme (Jefcoate et al., 1974; Arthur et al., 1976a), resulted in a 6-fold increase in pregnenolone synthesis. It is of interest that Falke et al. (1975) showed a similar stimulation of the basal rate of corticosterone synthesis in normal rat adrenal cells upon addition of 25-hydroxycholesterol, but stimulation of the cells with ACTH produced no further increase in corticosteroidogenesis. Tentatively it is suggested, therefore, that the rate of 25-hydroxycholesterol metabolism in adrenal cells reflects the maximal rate of steroidogenesis through the cholesterol desmolase. Since the rate of pregnenolone synthesis from endogenous precursors in
either non-stimulated or ACTH-stimulated tumor cells did not attain the rate achieved in the presence of 25-hydroxycholesterol, it seems probable that avail- ability of endogenous precursor is rate-limiting for pregnenolone biosynthesis in the tumor cells. If the endogenous precursor is cholesterol, as would seem likely, the question arises whether it is cholesterol availability to the mitochondrial desmolase that is rate-limiting (Mahaffee et al., 1974; Arthur et al., 1976b; Paul et al., 1976) as it would appear to be in the normal adrenal or whether it is the extremely low levels of both free and esterified cholesterol in the tumor cell com- pared to the normal adrenal cell (Table 1). In the normal adrenal cell, mobilization of cholesterol ester from the lipid droplets under the influence of a cyclic 3’,5’. AMP-dependent protein-kinase-activated cholesterol esterase (Trzeciak and Boyd, 1973) has been proposed (Garren et al., 1971) to permit availability of free choles- terol for hormonally stimulated steroidogenesis. It would seem that this cholesterol reservoir is lacking in the tumor cell and would account for the poor response to the tropic hormone. When the rates of steroid synthesis in the presence of 25- hydroxycholesterol in the tumor cell (13.2 nmol/2 × 106 cells/2 h) are compared to that reported by Falke et al. (1975) in the normal adrenal cell (50 nmol corticosterone/2 X 106 cells/2 h), the steroidogenic activity of the tumor cell is greater than that of the normal adrenal cell if the relative levels of cytochrome P-450, the terminal oxidase of steroid hydroxylases, are considered (Table 1).
The protein synthesis inhibitor, cycloheximide, inhibited pregnenolone biosyn- thesis from endogenous precursors in the adrenal tumor cells. Sharma (1973) reported that Snell adrenal carcinoma corticosteroidogenesis was insensitive to cycloheximide. The discrepancy can be explained possibly by noting that the rate of corticosterone synthesis was very low compared to that of pregnenolone so that corticosterone biosynthesis does not reflect adequately cholesterol catabolismthe sensitivity of ACTH-stimulated corticosteroidogenesis in the adrenal gland to cycloheximide has been much studied and reviewed (Schulster et al., 1974; Simp- son and Mason, 1976). Presently, most workers support the concept that an acti- vated protein factor is involved in permitting intramitochondrial relocalization of cholesterol, facilitating its metabolism (Mahaffee et al., 1974; Arthur et al., 1976b; Paul et al., 1976). Pregnenolone synthesis in the autonomous steroid- secreting adrenal carcinoma cells would seem to involve a similar scheme of control. Kowal (1970) has also reported that basal steroidogenesis in a mouse adrenal tumor cell line was inhibited by cycloheximide.
The cycloheximide-mediated inhibition of pregnenolone formation from endoge- nous precursors was found to be readily reversible (Table 1). This rapid return to normal function suggested that the cycloheximide-sensitive process does not involve synthesis of a rapidly turning-over protein but activation of a more stable mediator as proposed by other workers (Rubin et al., 1973; Lowry and McMartin, 1974). Addition of ACTH was not required to trigger steroid production in the tumor emphasizing the autonomous nature of these cells. The tumor itself would not appear to be producing ACTH since its basal adenyl cyclase activity can be acti- vated on addition of ACTH (Peron et al., 1975).
The insensitivity of pregnenolone synthesis to cycloheximide in the presence of 25-hydroxycholesterol was similar to that observed in normal adrenocortical cells (Falke et al., 1975). Uptake and metabolism of this hydroxysterol obviously does not involve the systems involved in the mobilization of cholesterol for ste- roidogenesis in adrenal cells.
Similar results were obtained when the effect of cytochalasin B on pregnenolone synthesis from endogenous precursor and 25-hydroxycholesterol was examined (Fig. 2). Mrotek and Hall (1977) have shown previously that endogenous steroid production in the ACTH-stimulated mouse adrenal Y-1 tumor cell culture was inhibited by cytochalasin B. The similarity in inhibition patterns of cycloheximide and cytochalasin B (sensitivity of endogenous but not of 25-hydroxycholesterol- stimulated pregnenolone synthesis) raised the possibility that both compounds had a similar inhibitor locus. Microfilaments could therefore influence cholesterol avail- ability to the mitochondrial desmolase. This notion requires further experimenta- tion. The lack of effect of cytochalasin B on 25-hydroxycholesterol metabolism implied that possible cytochalasin-B-mediated effects on other cellular functions, e.g. glucose uptake, were not involved in the inhibition of endogenous preg- nenolone synthesis.
These studies have shown that autonomous pregnenolone biosynthesis in Snell adrenal carcinoma cells is subject to control by similar mediators to those in normal adrenal cells. It is suggested that although there may well be abnormalities in regula- tion of protein kinase activity in these carcinoma cells (Sharma et al., 1977), the low rate of pregnenolone synthesis in these cells is due to limiting amounts of pre- cursor, presumably cholesterol. This would agree with current models of ACTH actions involving the hormone-promoting cholesterol availability to the mito- chondrial cholesterol desmolase. Lack of an available cholesterol reservoir results presumably in a redundant ACTH-activated process. It should be noted, however, that initial experiments presenting cholesterol to the tumor cells, in a similar fashion to 25-hydroxycholesterol, failed to produce any stimulation in the rate of pregnenolone synthesis (J.I. Mason and W.F. Robidoux, unpublished experiments). It would seem that appropriate presentation of cholesterol to lipoprotein receptors (Faust et al., 1977) may be necessary to observe a stimulation of steroidogenesis.
ACKNOWLEDGEMENTS
This work was supported by award CA18635 from the National Cancer Institute, National Institutes of Health.
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