Steroidogenesis in Isolated Cells and Mitochondria of Rat Snell Adrenocortical Carcinoma 494*
J. I. MASONt AND W. F. ROBIDOUX
The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545
ABSTRACT. ACTH produced a 75% increase in pregnenolone biosynthesis from endogenous precursors in isolated cells pre- pared from the rat Snell adrenocortical carcinoma 494. On the addition of 24- and 25-hydroxycholesterol to the tumor cells, the rate of pregnenolone synthesis increased 10-fold but was insen- sitive to the presence of ACTH. Addition of lipoprotein choles- terol resulted in increased pregnenolone biosynthesis when ACTH was present. High density lipoprotein cholesterol ap- peared to be internalized and used for steroidogenesis preferen- tially to low density lipoprotein cholesterol. The cholesterol ester hydrolase activity of the cytosolic fraction of the tumor was found to be extremely low compared to that of the normal
adrenal cell. These results, noting also the low cholesterol con- tent of the tumor cells, suggested that the lack of availability of cholesterol was the factor responsible for the poor steroidogenic response of the cells to ACTH.
The major steroid product of the tumor cells was determined to be deoxycorticosterone. This correlated with the low levels of steroid 118-hydroxylase activity detected in the adrenal tumor mitochondria compared to the mitochondrial cholesterol des- molase activity. Little of the mitochondrial cytochrome P-450 appeared to function in a steroid 118-hydroxylase complex. (Endocrinology 105: 1230, 1979)
T HE SNELL rat adrenocortical carcinoma was rec- ognized in vivo originally as a corticosteroid-secret- ing tumor (1). However, in in vitro studies using isolated cell preparations of the tumor, only very low rates of corticosterone production have been found (2). Further- more, corticosterone biosynthesis in these adrenocortical carcinoma cells appeared insensitive to ACTH. Since cAMP formation in the tumor cells was stimulated by ACTH (3), previous investigators have attempted to define the cause of the apparent insensitivity of corticos- teroidogenesis to ACTH (reviewed in Ref. 4). Current evidence has indicated that ACTH exerts its regulatory effect on adrenal steroidogenesis at the level of the mi- tochondrial cholesterol side chain cleavage enzyme sys- tem (5-7). Those reports suggested that the regulatory effect involved the control of substrate (cholesterol) availability to the enzyme, in particular at the level of intramitochondrial relocalization of cholesterol (5-7). In the present study we have investigated whether substrate availability might explain the apparent ACTH insensitiv- ity of steroidogenesis in the tumor cell. Since earlier reports suggested low rates of steroid 118-hydroxylation in the tumor cells (8, 9), the rate of steroidogenesis was monitored by the determination of the rates of both pregnenolone and corticosteroid formation.
Received September 12, 1978.
* This investigation was supported by Grant CA-18635, awarded by the National Cancer Institute, DHEW.
t To whom requests for reprints should be addressed.
Materials and Methods
The rat adrenocortical carcinoma 494 was transplanted and maintained in male Sprague-Dawley rats (Charles River Breed- ing Laboratories, Inc., Wilmington, MA), as described by Ney (10). Tumor tissue was implanted sc into 35-day-old rats through a small incision near the base of the tail. The tumor tissue for all of the reported studies was taken 3 weeks after the initial transplant. A dispersed tumor cell preparation was ob- tained by a procedure similar to that described previously (9). Viable tumor tissue from two rats was placed in a 125-ml Erlenmeyer flask containing 50 ml Krebs-Ringer-phosphate buffer (omitting calcium ions), pH 7.4, containing 0.2 g/100 ml glucose and 0.5 g/100 ml bovine serum albumin (fraction V). The tissue was stirred for 15-20 min at 37 C using a magnetic stirring bar. After the undissociated tissue settled out, the supernatant, which contained dissociated cells, was filtered through gauze into 50-ml centrifuge tubes. For maximal cell yield, the dissociation procedure was repeated twice. The com- bined supernatants were centrifuged at 600 rpm for 20 min in a refrigerated centrifuge. The resulting pellets of packed cells were resuspended in 50 ml buffer and allowed to settle by gravity for 60 min on ice. The resulting supernatant, which contained most of the contaminating red blood cells and cellular debris, was discarded. The loosely packed cells were resus- pended in 50 ml buffer, and the gravity-settling process was repeated at least twice. This procedure resulted in a preparation of nonaggregated tumor cells virtually free of red blood cells and other small particles which did not settle with the relatively heavy tumor cells. The nucleated cells were counted using a hemacytometer after staining with Neiser’s B stain.
Incubations of the isolated tumor cells were carried out in air at 37 C in the isolation buffer plus 1.6 mm calcium chloride in
a total volume of 1 ml containing 2 × 106 cells. The incubations were terminated by rapid chilling. When pregnenolone was to be assayed, the incubations contained 5 AM cyanoketone,1 an inhibitor of 33-hydroxysteroid dehydrogenases, to prevent fur- ther metabolism of pregnenolone. All steroid additions were made in ethanol; the final ethanol content did not exceed 1%. All other additions were made in the Krebs-Ringer-phosphate- glucose-albumin buffer.
Pregnenolone was determined using a RIA method recorded in detail previously (9, 11) based on the procedure described by Abraham et al. (12). Antiserum to pregnenolone-20-albumin conjugate was raised in New Zealand White rabbits and used at a dilution of 1:3500. Briefly, the 1-ml chilled incubations were diluted with 4 ml water, and 10-ul aliquots were used directly in the RIA. Protein from the broken cells did not interfere with the assay at the cell dilution employed. Desmosterol, and 24- and 25-hydroxycholesterol cross-reacted at less than 1% with equimolar amounts of pregnenolone. The zero time incubation blank was subtracted from the values determined in these assays.
Corticosterone was determined fluorimetrically, using the method of Silber et al. (13), after dichloromethane extraction of the 1-ml incubation samples.
Total deoxycorticosterone plus corticosterone was deter- mined using the blue tetrazolium method described by Elliott et al. (14) which is specific for steroids with a free a-ketol side chain. The deoxycorticosterone value was obtained by subtrac- tion of the fluorimetrically determined corticosterone value from the value obtained for blue tetrazolium-reacting material.
Mitochondria were prepared from viable tumor tissue, in the manner described previously for rat adrenal mitochondria (11), using a volume ratio of tissue to 0.25 M sucrose of 1:5. The mitochondrial fraction was washed once with 0.25 M sucrose before final resuspension in the same medium. Mitochondrial protein contents were determined by the procedure of Lowry et al. (15) using bovine serum albumin as the standard protein.
The cholesterol side chain cleavage activity of the tumor mitochondria was determined using the procedure described for the rat adrenal mitochondrial enzyme, which requires de- termination of pregnenolone by RIA (11). As with the isolated cells, 5 µM cyanoketone were added to prevent further metab- olism of pregnenolone.
Tumor mitochondrial steroid 118-hydroxylase activity was assayed by monitoring corticosterone formation, which was determined using a modification (16) of the fluorimetric method (13). Deoxycorticosterone (100 µM) was added as the substrate. In both the cholesterol side chain cleavage and steroid 118- hydroxylase assays, the reactions were initiated by the addition of isocitrate (final concentration, 10 mm) and carried out at 37 C with shaking.
Cytochrome P-450 was determined in an Aminco-DW 2 spectrophotometer in the split beam mode, as described by Omura and Sato (17), using an extinction coefficient of 91
mM-1cm-1 for the absorbance change (450-490 nm). The reduc- ing agent was sodium dithionite. The interaction of steroids with tumor mitochondrial cytochrome P-450 was followed in the Aminco spectrophotometer using a cell holder maintained at 37 C. The procedure was similar to that described previously (6).
The cholesterol ester hydrolase activity of tumor cytosol was determined at 37 C using the procedure described by Gorban and Boyd (18), which is based on an earlier method (19).
The ACTH in this study was the USP corticotropin reference standard. Human high density and low density lipoproteins were generously supplied by Dr. E. R. Simpson, Cecil H. and Ida Green Center for Reproductive Biology, University of Texas Southwestern Medical School (Dallas, TX). The lipoproteins had been prepared as described previously (20). The 25-hy- droxycholesterol and desmosterol were obtained from Stera- loids (Wilton, NH). The 24(R)- and 24(S)-hydroxycholesterols were generously provided by Dr. Marcel Gut of this Institute. Cyanoketone was kindly supplied by the Sterling-Winthrop Research Institute (Rensselaer, NY). All other steroids and bovine serum albumin (fraction V) were supplied by Sigma Chemical Co. (St. Louis, MO).
The [7a-3H]pregnenolone (NET 039; 17 Ci/mmol) and cho- lesterol [1-14C]oleate were supplied by NEN Chemicals (Boston, MA). All other chemicals were of analytical reagent grade.
Results
Steroid biosynthesis from endogenous precursors in iso- lated adrenal carcinon .a cells
When isolated tumor cells were incubated at 37 C in the presence of a 30-hydroxysteroid dehydrogenase in- hibitor (cyanoketone), pregnenolone formation from en- dogenous precursors was linear over a 2-h assay period (Fig. 1). Pregnenolone was not detected when cyanoke- tone was omitted from the assay medium, indicating rapid further metabolism of this steroid. Corticosterone was found to account for only about 20% of pregnenolone catabolites. However, the blue tetrazolium-reacting ma- terial accounted for over 75% of the endogenous steroid production. In the rat, blue tetrazolium-reacting material will be corticosterone or deoxycorticosterone. The results presented in Fig. 2 provided evidence that deoxycorticos- terone was the principal corticosteroid product from endogenous precursors of the Snell rat adrenocortical carcinoma cell.
The addition of 20 nm ACTH to the isolated adrenal carcinoma cells, a concentration of tropic hormone known to produce maximal increases in cAMP produc- tion, resulted in a 75% increase in pregnenolone synthesis (in the presence of cyanoketone) and a 125% increase in deoxycorticosterone formation, while corticosterone pro- duction was not significantly altered. These results are presented in Figs. 1 and 2.
The distribution of pregnenolone between the extra- cellular medium and the tumor cells was examined. At
’ The following trivial names are used: cyanoketone; 2-cyano- 4,4,17a-trimethyl-178-hydroxy-5-androsten-3-one; desmosterol, 5,24- cholestadien-38-ol; 25-hydroxycholesterol, 5-cholesten-30,25-diol; 24(R)-hydroxycholesterol, 5-cholesten-33,24R-diol; 24(S)-hydroxycho- lesterol, 5-cholesten-33,24S-diol; 20a,22R-dihydroxycholesterol, 5-cho- lesten-38,20a,22R-triol.
2
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NMOL PREGNENOLONE PER 2x 106 CELLS
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the termination of the incubation at 37 C (in the presence of cyanoketone), the assay tubes and their contents were chilled and centrifuged at 100 x g for 10 min. The resulting supernatant and cell pellet were separated, and the pregnenolone content was analyzed by RIA. After 20- or 60-min incubations at 37 C, the newly synthesized pregnenolone was entirely in the supernatant (extracel- lular medium). Pregnenolone could not be detected in the pellet of cells. This implied that pregnenolone was not retained within the cells.
Pregnenolone biosynthesis in isolated adrenal carci- noma cells in the presence of various sterols
Figure 3 illustrates the effect on the rate of pregneno- lone formation in isolated adrenal carcinoma cells of the addition of various sterols to the cell incubation medium.
Upon the addition of 25-hydroxycholesterol or 24(S)- or 24(R)-hydroxycholesterol, a 10-fold increase in the rate of pregnenolone biosynthesis was observed com- pared to endogenous production. The addition of des- mosterol also resulted in a marked stimulation of preg- nenolone formation. However, when cholesterol was added, the rate of pregnenolone biosynthesis was de- pressed. Significant stimulation of the rate of pregneno- lone biosynthesis was seen at 10-uM concentrations of the
hydroxysterols (results not shown), but maximal biosyn- thesis was seen with concentrations of 50 UM or more. The addition of 20 nm ACTH to the incubation medium did not produce any further stimulation in pregnenolone biosynthesis in the presence of either 24(S)-hydroxycho- lesterol (Fig. 3) or the other sterols (results not shown).
Utilization of lipoprotein cholesterol for pregnenolone biosynthesis
After the addition of either human high density lipo- protein (520 µg protein and 190 µg cholesterol) or low density lipoprotein (150 µg protein, and 240 µg choles- terol) to isolated adrenal tumor cells (2 × 106 cells), no significant increase in pregnenolone synthesis was de- tected during the first hour of incubation at 37 C. During subsequent incubation periods, a stimulation in pregnen- olone production was observed in the presence of high density lipoprotein and, to a lesser extent, in the presence of low density lipoprotein. However, upon the addition of 20 nm ACTH, a marked increase in pregnenolone biosynthesis was observed in the presence of high density lipoprotein, with low density lipoprotein being less effec-
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NMOL STEROID PRODUCED PER 2 × 106 CELLS
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NMOL PREGNENOLONE PER 2 x 106 CELLS
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tive (250% and 100% increases, respectively, vs. ACTH alone). The results are presented in Fig. 4.
Adrenal tumor mitochondrial cholesterol side chain cleavage and steroid 11ß-hydroxylase activities
The relative activities of the adrenal tumor mitochon- drial cholesterol side chain cleavage enzyme system and the steroid 113-hydroxylase are presented in Fig. 5. It was observed that the specific activity of the mitochon- drial cholesterol side chain cleavage enzyme was at least 10-fold greater than that of the steroid 118-hydroxylase. Furthermore, the mitochondrial cholesterol desmolase activity was enhanced markedly upon the addition of 100 u.M cholesterol.
Spectral interaction of steroids with adrenal tumor mi- tochondrial cytochrome P-450
Using optical difference spectroscopy, the interaction of substrates of the cholesterol side chain cleavage en-
zyme and the steroid 118-hydroxylase with the cyto- chrome P-450 present in adrenal tumor mitochondria was investigated. The data are presented in Table 1. Deoxycorticosterone, the steroid 118-hydroxylase sub- strate, and progesterone produced insignificant spectral perturbations in the Soret region of the optical spectrum, characteristic of a substrate interacting with the terminal oxidase component of a steroid hydroxylase, cytochrome P-450. However, 20a,22R-dihydroxycholesterol, 25-hy- droxycholesterol, and 24(S)-hydroxycholesterol per- turbed the mitochondrial cytochrome P-450 spectrum so as to produce an increase in the optical difference spec- trum at around 387 nm and a corresponding decrease at 420 nm. Thus, a so-called type I difference spectrum, characteristic of the cytochrome P-450-substrate inter- action, was observed with the hydroxy sterols. Addition of 100 µM cholesterol produced a type I spectral pertur- bation of lesser magnitude than the other sterols. Using the spectral extinction coefficient of 130 mm-1cm~1 for the absorbance change from 387 to 420 nm, as described by Jefcoate (21), it was calculated that 20a,22R-dihy-
16
NMOL PREGNENOLONE PER 2x 10° CELLS
8
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&
0
2
4
HOURS
A
8
B
0 50
-
NMOL PREGNENOLONE
MG-1 PROTEIN
NMOL CORTICOSTERONE
MG-1 PROTEIN
t
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0.25
:
0
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5
10
15
5
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TIME (MIN)
TIME (MIN)
| AA (387-420 nm) | |
|---|---|
| 24(S)-Hydroxycholesterol | 0.021 |
| 25-Hydroxycholesterol | 0.008 |
| 20(a),22(R)-Dihydroxycholesterol | 0.032 |
| Cholesterol | 0.004 |
| Deoxycorticosterone | 0.001 |
| Progesterone | 0.000 |
The cytochrome P-450 content was 0.38 uM. Adrenal tumor mito- chondria (2.1 mg protein ml-1) were suspended in 50 mM potassium phosphate buffer, pH 7.4. Experiments were performed in duplicate at a 100-AM final concentration of the steroid at 37 C. The absorbance change (AA) from 387 nm to 420 nm was determined.
droxycholesterol produced a spin state change in 61% of the tumor mitochondrial cytochrome P-450.
Cholesterol ester hydrolase activity of tumor cytosol
The cholesterol ester hydrolase activity of the cytosol fraction prepared from adrenal tumor cell homogenate was 9 ± 3 pmol min-1 mg-1 protein (mean ± SE of triplicate determinations of three separate cytosol sam- ples). The addition of 100 UM cAMP and 100 UM ATP did not produce a stimulation of the hydrolase activity.
For comparative purposes, the cholesterol ester hydro- lase activity of cytosol from adrenal homogenates of control rats not bearing tumors was determined. This was found to be 480 ± 30 pmol min-1 mg-1 protein (mean ± SE of triplicate determinations in two separate cytosol samples).
Discussion
The results presented in Figs. 1 and 2 demonstrate that the rate of pregnenolone formation from endogenous precursors in isolated Snell rat adrenocortical carcinoma cells was significantly greater than that of corticosterone. It would appear that subsequent metabolism of pregnen- olone resulted in the formation of blue tetrazolium-posi- tive steroid products. This was likely to be mainly deox- ycorticosterone, since low levels of corticosterone had been determined using the more specific fluorimetric method.
The addition of ACTH to the adrenal cells resulted in an approximately 75% increase in pregnenolone synthe- sis. Earlier studies on the ACTH responsiveness of iso- lated rat adrenal tumor cells (2, 9) failed to show stimu- lation of steroid production by the tropic hormone. Our ability to demonstrate ACTH sensitivity of steroidogen- esis in the rat adrenal tumor cell might be related to alterations in cell function occurring between current and past studies. In the light of the observations presented in Fig. 4 demonstrating lipoprotein-supported and ACTH- sensitive pregnenolone biosynthesis, it seems probable that the stimulation of endogenous pregnenolone synthe- sis by ACTH is dependent upon the availability of a cellular reserve of steroidogenic cholesterol. One such alteration might be an increase in the steroidogenic cho- lesterol reserve. However, the free and esterified choles- terol content of cells were not significantly elevated in either the present or our earlier studies (9).
The results presented in Fig. 4 indicate that the rat adrenocortical carcinoma cells preferentially incorporate high density lipoprotein cholesterol for subsequent ste- roid biosynthesis. The data are in agreement with the recent report that isolated rat adrenocortical cells take up high density lipoprotein preferentially to low density lipoprotein (22). The lag period of less than 1 h before marked increases in pregnenolone biosynthesis being ob- served (Fig. 4) correlates well with previous studies on the time course of lipoprotein uptake and the internali- zation of the lipoprotein receptor complex (22, 23). These studies on rat adrenocortical carcinoma cells support the previous reports that high density lipoprotein could be important in the regulation of adrenocortical cholesterol metabolism in the rat (24, 25). This contrasts to the mouse adrenal tumor Y-1 cell line in which low density lipoprotein cholesterol utilization is required for steroid biosynthesis (26). The evidence presented supports the currently accepted concept that the mobilization of cho- lesterol for steroidogenesis is the ACTH-regulated step of corticosteroidogenesis (27). The addition of 24-hydrox- ycholesterol, 25-hydroxycholesterol, or desmosterol to the tumor cells provided a substrate for the mitochon-
drial cholesterol desmolase, resulting in increased preg- nenolone biosynthesis. The ACTH-regulated cholesterol mobilization step was circumvented (6, 28, 29), resulting in pregnenolone formation insensitive to ACTH (Fig. 3).
It is of interest to mention the apparent inability of the adrenal tumor cells to use extracellular cholesterol presented in an ethanolic solution, contrasting with the fate of the cholesterol analogs (Fig. 3). It would appear that specific pathways of cholesterol translocation (via specific receptor proteins) exist to direct cholesterol to the sites of cholesterol catabolism
The studies suggest that the rate of supply of plasma lipoprotein cholesterol governs the rate of steroid syn- thesis in the rat adrenocortical carcinoma cells. The lack of a large cholesterol reserve (in the form of cholesterol ester-rich lipid droplets) in the rat adrenal carcinoma cell (8-10, 30-32) is likely to account for the relatively small 2- to 4-fold increases in ACTH-mediated steroidogenesis seen in the rat adrenal tumor cell and in the mouse adrenal tumor Y-1 cell line (26, 33-35). These responses contrast sharply with the 124-fold stimulation in corti- costerone production seen with the addition of ACTH to cholesterol ester-rich rat adrenal zona reticularis cells (36). The lack of a mobilizable cholesterol reserve in the rat adrenal tumor cell is presumably due to the very low rate of cholesterol ester synthesis, which has been re- ported previously (30), as well as to the low activity of cytosolic cholesterol ester hydrolase, which is demon- strated in the present study.
In previous studies from this laboratory (8) it was suggested that the low rates of corticosterone biosyn- thesis in isolated rat adrenal tumor cells were due to the almost nondetectable levels of the mitochondrial steroid 118-hydroxylase. This conclusion was supported by the findings that the tumor homogenate transformed radio- active pregnenolone primarily to deoxycorticosterone (37). In the present study, the addition of 100 UM choles- terol to the tumor mitochondria resulted in a marked increase in mitochondrial pregnenolone biosynthesis. This further supported the notion that the availability of cholesterol was a rate-determining step for steroidogen- esis in the tumor cell. The tumor mitochondrial choles- terol desmolase activity was found to be 10-fold higher than the steroid 113-hydroxylase activity (Fig. 5). Nor- mal rat adrenal mitochondria have higher steroid 113- hydroxylase than cholesterol desmolase activities. No significant spectral interaction of deoxycorticosterone with tumor mitochondrial cytochrome P-450 was ob- served (Table 1). Adrenal mitochondrial steroid 118-hy- droxylase cytochrome P-450 is present normally as a predominantly low spin, substrate-free hemoprotein and, hence, may bind exogenous deoxycorticosterone. Thus, the results presented in Table 1 would indicate that less
than 5% of the mitochondrial cytochrome P-450 was involved in steroid 113-hydroxylation. In normal rat ad- renal mitochondria, approximately 35% of the mitochon- drial cytochrome P-450 is involved in steroid 118-hy- droxylation (21). Marked type I spectral perturbations in the Soret region were observed on the addition of 100 UM 20a,22R-dihydroxycholesterol to the adrenal mito- chondria. This compound, a substrate intermediate for the cholesterol desmolase, interacted with about 60% of the tumor mitocohondrial cytochrome P-450 using the spectral extinction criteria proposed earlier (21). Since the hydroxycholesterols do not interact with the entire cholesterol desmolase cytochrome P-450 pool at pH 7.4 (21), we might conclude that a large proportion of the tumor mitochondrial cytochrome P-450 is involved in the cholesterol desmolase rather than other steroid hydrox- ylation systems.
The low activities of steroid 113-hydroxylase and cho- lesterol ester hydrolase do not appear to be related to a deficiency of ACTH during tumor growth. A single daily sc injection of ACTH (8 U Acthar gel) during tumor growth prevents the atrophy of the animal’s adrenal glands. No significant increase in the mitochondrial ste- roid 118-hydrolase activity of tumors from ACTH- treated animals was observed (Mason, J. I., and W. F. Robidoux, unpublished observations).
Although the steroid 118-hydroxylase activity of ad- renal tumor mitochondria was low, significant conversion of exogenous deoxycorticosterone (100 (M) to cortico- sterone in isolated tumor cells was observed (730 pmol corticosterone formed/106 cells· 2 h; Fig. 2).
These studies have shown that the availability of cho- lesterol is a limiting step of steroidogenesis in the Snell rat adrenal carcinoma cell. Lipoprotein cholesterol ap- pears to be used directly for steroidogenesis. This process is facilitated by ACTH. The major steroid product of the tumor in vitro would appear to be deoxycorticosterone.
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