Metabolic Regulation of Steroidogenesis in Isolated Adrenal and Adrenocortical Carcinoma Cells of Rat. The Incorporation of (20S)-20-[7-3H]Hydroxycholesterol into Deoxycorti- costerone and Corticosterone
RAMESHWAR K. SHARMA AND JAMES S. BRUSH
Laboratories of Endocrinology and Metabolism, Veterans Administration Hospital, and Department of Biochemistry, University of Tennessee Medical Units, Memphis, Tennessee 38104
Received November 6, 1972
Previous studies from these laboratories have demonstrated the following: (1) that cyclic 3’5’-AMP (c-AMP) phosphodiesterase activity of adrenocortical car- cinoma 494 is only 20% of that found in the normal adrenal; (2) that corticosteroido- genesis in the isolated tumor cells is inhibited by ACTH, and (3) that the normal adrenal biosynthetic pathway from pregnenolone to corticosterone is intact but less active in the tumor. The present studies show that both the isolated adrenocortical carcinoma cell and the normal isolated adrenal cell of the rat have the capacity to transform (20S)-20-hydroxycholesterol into deoxycorticosterone and corticosterone. It is, therefore, proposed that the lack of stimulation by ACTH of corticosterone synthesis of the tumor cells cannot be explained by the absence of enzymes cleaving the cholesterol side chain. It is, therefore, postulated that a modified protein kinase may be present in the tumor which is not stimulated by c-AMP.
Earlier studies with isolated adrenal and adrenocortical carcinoma 494 cell prep- arations have revealed that, although nor- mal isolated adrenal cells (1, 2) are markedly stimulated by microunit quantities of ACTH to form corticosterone, the isolated tumor cells are completely devoid of such stimula- tion (3).
Previously it was shown that isolated adrenal tumor cells (3, 4) and tumor slices (5, 6) have the capacity to convert preg- nenolone to corticosterone. It was further demonstrated that the inability of the ad- renal tumor to synthesize increased amounts of corticosterone from endogenous pre- cursors in response to ACTH was not due to accelerated c-AMP breakdown (7). This unresponsiveness was not corrected by exogenous c-AMP or any of the six other cyclic 3’,5’-nucleotide monophosphates tested (3). This is in contrast to the nor-
mal isolated adrenal cell which shows a marked response to c-AMP and the 3’,5’ cyclic nucleotide monophosphates of gua- nosine and inosine (1).
The present studies have demonstrated that both the normal adrenal and adrenal tumor cells are capable of taking up exog- enous (20S)-20-hydroxycholesterol, the pos- tulated intermediate in the formation of pregnenolone from cholesterol (9-13) and converting it to deoxycorticosterone and corticosterone. This work, therefore, depicts not only the usefulness of the preparations in investigating the control of steroid syn- thesis, but helps to limit the possible causes of this control transformation in the adrenal tumor.
MATERIALS AND METHODS
Isolated adrenal and adrenocortical carcinoma cell preparations were made by the previously
described methods (1, 3). About 1.5 g of adreno- cortical carcinoma 494 tissue of rat (8) was used to prepare the trypsinized cells. An equal number of (3-5 million/ml) isolated adrenal tumor and nor- mal adrenal cells were suspended in 20 ml of Krebs- Ringer bicarbonate buffer, pH 7.4, containing 4% albumin and 0.2% glucose (KRB-AG). 1.50 uCi of (20S)-20-[7-3H]hydroxycholesterol (sp act 25 Ci/ mmole and 0.20 uCi of [4-14C]pregnenolone (sp act 40-50 mCi/mmole) were added in each incubation flask, and the reaction continued for 150 min at 37°C. The reaction was stopped by the addition of 50 ml of methylene chloride, and the deoxycorti- costerone and corticosterone purified by chroma- tography as previously described (4). The radio- active extracts of deoxycorticosterone and corti- costerone were diluted with nonradioactive com- pounds. Deoxycorticosterone was acetylated (4) and purified by tle on silica gel [benzene-ethyl acetate (1:1)]. Deoxycorticosterone acetate and corticosterone were then crystallized several times until the specific activity and 3H/14C ratio were constant (Tables I and II).
(20S)-20-[7-3H]Hydroxycholesterol, sp act 25 Ci/mmole, and [4-14C]pregnenolone, sp act 40-50 mCi/mmole, were purchased from New England Nuclear Corporation, Boston, MA, and their purities checked by tlc.
RESULTS AND DISCUSSION
The most frequently advanced scheme for the enzymatic transformation of cholesterol to pregnenolone is thought to be via the formation of (20S)-20-hydroxycholesterol (9-13). Although the precise structure(s) of the hydroxylated cholesterol intermediate(s) is a subject of controversy (12, 14), it is universally recognized that the adrenal desmolase complex of enzyme(s) in the mito- chondria has the capacity to cleave the side chain of (20S)-20-hydroxycholesterol and thus transform it to pregnenolone.
In the present investigation (20S)-20- [7-3H]hydroxycholesterol and [4-14C]pregnen- olone were incorporated into deoxycortico- sterone and corticosterone in both tumor and normal adrenal cells. The ratio of the incor- poration from the two labeled precursors is indicated in Table I for normal adrenal cells and in Table II for the tumor cells. The similarity of 3H/14C ratios in these two products is compatible with the view that they are both on the same pathway. The finding that this ratio in the tumor is again the same for these two steroids (albeit lower
| Product | Crystalli- zation | 3H/14C ratio |
|---|---|---|
| Corticosterone | 1st | 5.30 |
| 2nd | 5.31 | |
| 3rd | 5.23 | |
| Deoxycorticosterone | 1st | 4.77 |
| acetate | 2nd | 4.77 |
| Product | Crystalli- zation | 3H/14C ratio |
|---|---|---|
| Corticosterone | 1st | 3.60 |
| 2nd | 3.28 | |
| 3rd | 3.26 | |
| Deoxycorticosterone acetate | 1st | 2.88 |
| 2nd | 2.87 |
than for normal cells) argues for the existence of the same biosynthetic pathways in the tumor. The lowering of the ratios in the tumor can be explained either by a decrease in rates of plasma or mitochondrial mem- brane transport processes or by diminution of cholesterol side-chain cleavage rates in the two types of cells. The present data cannot distinguish unequivocally between the two possibilities.
The percentage incorporation of (20S)-20- [7-3H]hydroxycholesterol into deoxycorti- costerone and corticosterone by the tumor cells was 13.1 and 3.3%, respectively, and by normal adrenal cells was 11.6 and 11.4%, respectively. For the tumor these data indicate that the ratio of synthesis of deoxy- corticosterone to corticosterone is greater in the tumor than in the normal adrenal cell. This may reflect differences in relative pool sizes as an earlier report suggested (5). The finding that the normal isolated adrenal cell has the same total incorporation of radio- activity into deoxycorticosterone as corti- costerone is surprising. From the incorpora-
tion data it may be concluded that there has been a sizable reduction in the activity of the 118-hydroxylase enzyme in the tumor as compared to normal tissue.
Previously it was shown that the lack of stimulation of steroidogenesis by ACTH or exogenous cyclic nucleotide in the adrenal tumor was not due to accelerated break- down of c-AMP (7). Rather, c-AMP phos- phodiesterase activity was markedly lower in the tumor. In the present study, it is shown that the defect cannot be ascribed to the absence of the cholesterol side chain cleavage enzyme system. In the currently proposed hypothesis of the mechanism of ACTH action (15), this suggests the pos- sibility of a defect in the tumor protein kinase system. This possibility is also sup- ported by earlier studies (7). An alteration in such a system when compared to normal tissue has already been reported in a hepatoma cell line (16).
The ease with which (20S)-20-hydroxy- cholesterol can be converted to deoxycorti- costerone and corticosterone in the isolated adrenal and adrenal tumor cells is also of interest. This suggests that mechanisms exist for the transport of this precursor into both types of cell and for its subsequent metabolism. Previous studies have not evaluated these aspects of the normal ad- renal and adrenal tumor cell.
ACKNOWLEDGMENTS
This work was supported by a Veterans Admin- istration Institutional Research Grant.
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