Loss of Two Steroid 11 ;- Hydroxylating Com- ponents in a Rat Adrenocortical Carcinoma 1,2
MAX L. SWEAT and MELVIN J. BRYSON,3 Department of Obstetrics and Gynecology, University of Utah College of Medicine, Salt Lake City, Utah
SUMMARY-Examination of the Snell homologously transferred rat Adrenocarcinoma 494 indicated a loss of two components (F-40 and F-80) associated with steroid 116-hydroxylation in normal adrenal tissue. The tumor retained the mechanisms associated with 21-hydroxylation of proges- terone and C-5 isomerization and C-3 oxidation of pregnenolone .- J Nat Cancer Inst 33: 849-854, 1964.
STEROID METABOLIC STUDIES with the Snell tumor 494 (1) led Johnson et al. (2) to conclude that this homologously (rat) transferred adreno- cortical carcinoma exhibits a markedly lower capacity of 118-hydroxylation than normal adrenal tissue. The recent demonstration that the 118- hydroxylating system is resolvable into two discrete components (3) has suggested an examination of the tumor in respect to each of these factors. The present study presents evidence that both 118- hydroxylating components are absent in the tumor. These observations suggest that the tumor cells arose from an active hormone-synthesizing cell in which the mitochondrial machinery was either lost or modified, from normal ardenal cells not possessing the 116-hydroxylating species of mito- chondria, or from incompletely differentiated cells retained in the tissue as embryonic rests. The introduction of an inhibitor during the change seems unlikely, as neither of the 118-hydroxylating components is demonstrable in the tissue.
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EXPERIMENTAL
A. Preparation of Bovine Adrenal Fractions
Bovine adrenal glands collected and stored on dry ice were thawed, trimmed free from fat, and cut longitudinally (parallel to flat surface) in thin slices. Cortical tissue, 400 g, was trimmed from the medullary portion of the slices, minced on a wooden block with a battery of spaced razor blades, and suspended in cold (0-4º C) 0.25 M sucrose as a 20 percent suspension. The suspension was homogenized for 30 to 60 seconds in a Potter- Elvehjem-type homogenizer fabricated with a
1 Received April 27, 1964
2 Supported by grant AM-01803, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, and by grant P-248, the American Cancer Society, Inc.
3 We are indebted to Dr. David F. Johnson for calling our attention to this tumor, to Dr. Katharine C. Snell for making the tumor tissue available, and to Richard B. Young for valuable technical assistance.
loose-fitting Teflon pestle and centrifuged at 600 X g for 10 minutes to sediment the cell frag- ments and nuclei. A second centrifugation at 10,000 × g for 20 minutes sedimented mitochon- dria, which were washed with 0.25 M sucrose and recentrifuged. The washed mitochondria in 25 ml of cold distilled water were slowly added to 10 volumes of cold acetone (-5º C). The solid phase was allowed to settle for several minutes, after which the acetone was decanted. The process was repeated 3 times with 10 volumes of acetone each time. The mitochondrial residue was finally poured on a Büchner funnel, dried with suction, followed by vacuum desiccation. To prepare a soluble mitochondrial extract, 10 g of dry mito- chondrial powder was suspended in 100 ml of 0.01 M sodium-potassium phosphate buffer, pH 7.4, with the aid of an all-glass Potter-Elvehjem homogenizer, and centrifuged at 105,000 X g for 1 hour. The residue was resuspended in 75 ml of 0.01 M sodium-potassium phosphate buffer and again centrifuged at 105,000 X g for 1 hour. To the combined supernatant fraction, saturated ammonium sulfate solution (pH 7.4) was slowly added in progressive increments (each followed by centrifugation) to effect protein precipitations, respectively, at 0 to 30, 30 to 42, 42 to 50, 50 to 70, and 70 to 80 percent saturation. (Solid ammo- nium sulfate was added with rapid stirring to effect 80% saturation.) Before incubation, each fraction was dissolved in 0.05 M phosphate buffer (pH 7.4), dialyzed 24 hours against 0.012 M phosphate buffer at the same pH, and centrifuged 30 minutes at 10,000 X g. The protein precipitated between 30 to 42 percent was designated F-40; that pre- cipitated between 70 to 80 percent saturation was designated F-80. F-80 has been further purified (4), but only the crude fraction was used.
B. Preparation of Ammonium Sulfate Fractions of Snell Tumor 494
Tumors transplanted and grown for approxi- mately 3 months in Osborne-Mendel rats were removed from the hosts, immediately frozen, and stored on dry ice. The thawed tissue, 100 g, was homogenized and treated as in A. The respective fractions were designated T-40 and T-80.
C. Incubation and Analytical Methodology
Progesterone-4-C14 [Specific activity (S.A.) 8.8 pc/umole], 3.2 to 8.3 mumoles, 11.3 mumoles deoxycorticosterone (S.A. 1 uc/umole), 2.8 mumoles 17a-hydroxyprogesterone-4-C14 (S.A. 14.0 uc/umole), or 0.3 mumole pregnenolone-7-H3 (S.A. 689 uc/umole) was incubated with the tumor homogenate, the partially purified protein fractions, or bovine adrenal cortical mitochondrial fractions, in various combinations at 37º C for 1 hour. The incubation medium contained 0.05 M phosphate buffer (pH 7.4), 0.004 M MgCl2, and 0.025 M KCI. In several experiments 1 × 10-3 M diphos- phopyridine nucleotide or 1 × 10-3 M triphospho- pyridine nucleotide, 1 X 10-3 M glucose-6- phosphate, and 1 Kornberg unit of glucose-6- phosphate dehydrogenase were employed for coenzyme fortification or reduced triphosphopyri- dine nucleotide generation.4
The incubations were terminated by addition of 3 volumes of acetone. After the mixtures stood overnight at 0 to 4º C for flocculation of protein, they were filtered and extracted with additional portions of acetone and chloroform. The volatile solvents were removed by vacuum distillation and the water residue extracted with chloroform. Residues from the chloroform extracts were partitioned between 70 percent aqueous ethanol and hexane to remove additional lipide material. Evaporation of the 70 percent ethanol left the final residue for chromatography. Chromatography was performed with the formamide methods of Zaffaroni (5). Low polar compounds were chro- matographed in the hexane system, the inter- mediate compounds in the hexane-benzene system, and the high polar compounds in the benzene and chloroform systems. The resolved products were located by scanning with a micromil window or windowless gas-flow detector with an automatic recorder. The quantities of steroids were calcu- lated by triangulation of the radioactive areas on the chromatograph. The following data estab- lished the identity of the steroid products:
11-Deoxycorticosterone (4-pregnen-21-ol-3, 20-dione) .---- This steroid remained on the origin in the
4 Radioactive steroids were purchased from New England Nuclear Corp., Boston, Mass., and checked for purity by chromatography before use. Cofactors were purchased from Sigma Chemical Co., St. Louis, Mo.
original hexane-formamide chromatography. Upon rechromatography in the hexane-benzene-form- amide system, it migrated with an RF of 0.35. Chromatography of the acetylated product and 100 µg of carrier demonstrated the compound to migrate near the solvent front (RF 0.75, hexane- benzene). Oxidation of the free compound (and carrier) formed a high polar compound correspond-
ing to 3-keto-4-etiocholenic acid, which remained on the origin in the chloroform-formamide system. Final proof of identity was established by crystal- lization of the product with the pure carrier index to constant specific activity:
Product of progesterone-4-C14 [disintegration per minute (dpm)]:
| Solvent | Crystals | Mother liquor | |
|---|---|---|---|
| dpm per u mole | dpm per umole | ||
| Hexane: First crystallization | 4.7 | 4.7 | |
| Hexane: Second crystallization | 4.1 | 3.7 | |
| Hexane: | Third crystallization | 4.0 | 4.1 |
Product of Pregnenolone-7-H3:
| Solvent | Crystals | |
|---|---|---|
| dpm per umole | ||
| 70 Percent methanol: | First crystallization | 17.7 |
| 70 Percent methanol: | Second crystallization | 16.6 |
| 70 Percent methanol: | Third crystallization | 18.1 |
Corticosterone (4-pregnen-110,21 - diol-3,20-dione) .- This compound, a product of F-40 and F-80, was chromatographed initially in the hexane-forma -. mide system where it remained on the origin. After chromatography with carrier in the benzene- formamide system it had an RF of 0.10. Strong oxidation (0.2 mg CrO3 in 0.5 ml glacial acetic acid overnight at room temperature) of it and the carrier resulted in a high polar compound that remained on the origin in the chloroform-forma- mide system (indicative of the formation of the etiocholenic acid). Mild oxidation (0.05 mg CrO3 in 0.6 ml glacial acetic acid overnight at room temperature) formed Reichstein’s substance A. Acetylation of the unoxidized compound and carrier, followed by chromatography in the benzene-formamide system, demonstrated a single peak (RF 0.64).
11-Dehydrocorticosterone(4-pregnen-21-ol-3,11,20-trione). Chromatography in the benzene-formamide system for 7 hours resolved corticosterone and the 11- dehydro isomer (and their respective carriers) into two well-defined peaks. The 21 acetate derivatives of each were separated by 7-hour chromatography
in the hexane-benzene-formamide system. Oxida- tion of the two compounds produced high polar compounds corresponding to the etiocholenic acids that remained on the origin in chloroform- formamide after chromatography for 7 hours.
11-Deoxycortisol (4-pregnen-17a, 21-diol-3, 20- dione) .- This steroid was isolated from the incuba- tions when 17a-hydroxyprogesterone was used as a substrate. Its RF in benzene-formamide (with the carrier) was 0.1. The acetylated product and carrier 11-deoxycortisone migrated (benzene-form- amide) at identical rates (RF 0.45). Oxidation with chromic acid produced a product that migrated, in the hexane-formamide system (6 hours) and the hexane-benzene-formamide system, identically with a standard index of androstene- dione.
RESULTS
The homogenate and partially purified protein fractions of the tumor were examined separately and in combination with bovine adrenocortical mitochondrial preparations for 118-hydroxylation
VOL. 33, NO. 5, NOVEMBER 1964
| Experi- ment No. | Rat tumor tissue and purified bovine fractions* | Steroids Isolated | ||||
|---|---|---|---|---|---|---|
| Unmetabo- lized pro- gesterone (mumoles) | Deoxycorti- costerone (mpmoles) | AKt (mumoles) | BKİ (mumoles) | Unidentified high polar compounds § (mumoles) | ||
| 1 | Homogenate | 0. 5 | 6. 8 | - | - | 0. 9 |
| 2 | Homogenate + cofactors | 0.2 | 6.6 | - | - | 1.5 |
| 3 | Homogenate + cofactors + F-40 | 0. 2 | 7.0 | 0. 39 | 0. 6 | 0. 3 |
| 4 | Homogenate + cofactors + F-40 + F-80 | 0. 02 | 6.3 | 0. 2 | 1. 0 | 0. 8 |
| 5 | Homogenate + cofactors + F-80 | 0. 4 | 6.2 | - | - | 1. 7 |
| 6 | F-40 + cofactors | 1. 1 | 1.4 | - | 0. 6 | 0. 2 |
| 7 | F-40 + F-80 + cofactors | 1.0 | 0. 3 | - | 1. 6 | 0.3 |
| 8 | F-80 + cofactors | 3.2 | - | - | - | - |
*Contents of incubation flasks: (8.3 mumoles progesterone-4-C14 in experiments 1-5; 3.2 mumoles progesterone-4-C44 in experiments 6-8); 0.1 ml of respective fractions was added to either 5 X 10-2 M sodium- potassium phosphate buffer (pH 7.4) containing 2.5 X 10-1 M KCI, 5 X 104 M MgCl, in a final volume of 1.0 or 0.5 ml of a 20 percent tumor tissue homogenate containing the same buffer-medium components made up to 3.0 ml. The incubations were carried out (with shaking) 1 hour at 37º C in oxygen.
TAK: Kendall’s Compound A (4-pregnen-21-ol-3,11,20-trione).
activity (tables 1 and 2). From these data, it is apparent that the tumor did not contain significant quantities of either F-40 or F-80. The synthesis of 118-hydroxylated compounds was not as great in homogenates fortified with F-40 and F-80 as when these fractions were incubated alone- probably due to enzymic systems competing for the coenzyme. The major products obtained from the tumor tissue (either unfortified or with added cofactors-addition of cofactors increased the product yield slightly), when progesterone was employed as a substrate, were deoxycorticosterone and an unidentified high polar compound(s), possibly 6-hydroxy-11-deoxycorticosterone. A mi- nor product migrated at the same rate as aldos- terone in the benzene system. However, on subsequent chromatography and acetylation, it did not conform to a standard unlabeled aldosterone carrier added with the product. The in vitro synthesis of aldosterone by fresh tumor tissue was reported by Johnson et al.(2). From the present data it would appear that the freezing of the tumor tissue (necessary for the accumulation of sufficient material for the preparation of fractions) destroyed the capacity for synthesis of this compound.
The addition of bovine adrenal F-40 and F-80 to the tumor homogenate gave rise to corticosterone and its oxidation product, Kendall’s compound A
BE: Kendall’s Compound B (4-pregnene-118,21-diol-3,20-dione). §Calculated as mumoles progesterone. (The high polar compounds exhibited no characteristics of BK, AK, cortisol, cortisone, or their respective derivatives possessing 6 or 20 hydroxy groups.)
{The beef adrenal F-40 in these experiments contained a larger con- tamination of F-80 than most preparations of F-40; however, & sufficiently large increase of 118-hydroxylation was demonstrable upon addition of F-80 (free from F-40) to differentiate distinctly between the two factors.
(4-pregnen-21-ol-3, 11,20-trione), both of which are indicative of 118-hydroxylation. The observation that moderate quantities of Kendall’s compound A are formed by the adrenal tumor in combination with the bovine fractions is noteworthy, since only trace quantities of this oxidized compound are ob- served as a product in normal bovine adrenal tissue incubations. This suggests that the oxidation proc- ess for this reaction is increased in the rat tumor. The rat adrenal synthesizes primarily corticos- terone; the bovine adrenal synthesizes both corti- costerone and cortisol. The analogous oxidized compound, cortisone, and compound A are formed only in trace quantities in the bovine adrenal.
| Enzyme preparation* | Corticosterone (mumoles/1 hr) |
|---|---|
| Tumor T-40 | 0 |
| Tumor T-40, tumor T-80 | 0 |
| Tumor T-40, adrenal F-80 | 0 |
| Adrenal F-40 | 8. 8 |
| Adrenal F-40 + tumor T-80 | 8. 8 |
| Adrenal F-40 + adrenal F-80 | 45.2 |
*Contents of flasks: 0.1 ml of each fraction indicated was incubated with 1 X 10-$ M triphosphopyridine nucleotide, 1 X 10-3 M glucose-6- phosphate, 1 Kornberg unit glucose-6-phosphate dehydrogenase, 5 X 10-4 M MgCl2, 2 X 10-1 M sodium-potassium phosphate buffer pH 7.4, and distilled water to a final volume of 1.0 ml. Incubations were carried out (with shaking) 1 hour at 37º C in oxygen.
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Experiments with partially purified tumor frac- tions T-40 and T-80 were also carried out with deoxycorticosterone-4-C14 as the substrate (table 2). Although small radioactive areas suggesting the possible formation of trace products were observed, the tumor appeared to be essentially inactive toward deoxycorticosterone. Progesterone-4-C14 incubated with tumor fractions (T-40) gave rise to deoxycorticosterone, which indicated that the 21- hydroxylating system was intact. It is probably the formation of deoxycorticosterone that is in part responsible for the physiological manifestations in the animals with tumors, as observed by Snell (1). The block of 11-hydroxylation and accumulation of this 21-hydroxy-11-deoxy compound result in a metabolic condition suggestive of the hyperten- sive form of congenital adrenal hyperplasia (6). Incubation of T-40 with F-80 and T-80 with F-40, as with the whole homogenate, failed in the 118-hydroxylation of progesterone. From these latter and previous data, it is concluded that both 116-hydroxylating factors are absent in the Snell tumor 494. The isolation of deoxycorticosterone after incubation of 36-hydroxy-5-pregnen-20-one- 7-H3 (pregnenolone-7-H3) with the tumor tissue indicated that both the 33-ol-dehydrogenase and the 45-isomerase enzymes are functional in the tumor.
DISCUSSION
The absence of the two 118-hydroxylating en- zymes in the tumor tissue suggests that the tumor cells were derived from an adrenal cortical cell which lost these enzymic entities during tumori- genesis, although the possibility exists that their origin was from normal cells originally lacking these factors or from partially differentiated em- bryonic cells retained in the cortical tissue as an embryonic rest. A distinction cannot be made between these processes at present, since it has not been established whether partially differentiated cells exist either normally or as rests in adrenal tissue. However, due to the nature of membrane barriers (both cell and organelle) it is certain that most, if not all, normal adrenal cortical cells, to carry out the over-all concerted system of hormone steroidogenesis, likely incorporate the complete integrated mechanisms of steroid syn-
thesis in an enclosed unit. It is unlikely that an inhibitor is involved, as neither of the two 118- factors is demonstrable.
Of particular significance in these studies is the observation that the 116-hydroxylating factors, both of which are of mitochondrial origin in normal tissue, have been lost. This suggests that a partic- ular modification (or loss) of the mitochondria has occurred during tumorigenesis. It is interesting that 118-hydroxylation involves incorporation of molecular oxygen (7, 8), a process long associated with mitochondria in respect to the cytochrome oxidase system. Although the reaction of 118- hydroxylation does not seem to be directly asso- ciated with the cytochrome system, the oxygen moiety associated with the hydroxylation is undoubtedly activated at an electron-potential level common with the cytochrome system. Because oxidative processes occurring in mito- chondria of cancer tissue appear to be altered, it is possible that a general alteration of the oxidative systems concomitantly influences steroid 118- oxygen incorporation as well as the basic oxidative respiratory chain.
Studies of other endocrine tumors indicate that tumorigenesis may influence other organelles and compartments of the cell. A homologously trans- ferred testicular teratocarcinoma, which retains 17-desmolase (cleavage of 17-hydroxy chain) activity, was shown (9) to have lost both its 17-hydroxylating capacity (present in particle-free cytoplasm) and the androstenedione-aromatizing enzymes (microsomal). Further, a strain of cul- tured rabbit testis fibroblasts has been reported (10) to possess the aromatizing enzymes, but not the 17-hydroxylating enzyme(s). A human mas- culinovoblastoma has been shown to possess the 17-hydroxylating enzyme, but not the aromatizing mechanisms (11). Although it appears that carci- nogenesis may influence a number of steroid synthesis steps at various cellular sites, the process seems to have been restricted to the mitochondrial elements in the Snell tumor.
REFERENCES
(1) SNELL, K. C., and STEWART, H. L .: Variations in histologic pattern and functional effects of a trans- plantable adrenal cortical carcinoma in intact, hypophysectomized, and newborn rats. J Nat Cancer Inst 22: 1119-1155, 1959.
VOL. 33, NO. 5, NOVEMBER 1964
(2) JOHNSON, D. F., SNELL, K. C., FRANCOIS, D., and HEFT- MANN, E .: In vitro metabolism of progesterone-4-C- 14 in an adrenocortical carcinoma of the rat. Acta Endocr (Kobenhavn) 37: 329-335, 1961.
(3) SWEAT, M. L., and BRYSON, M. J .: Separation of two components of the steroid 118-hydroxylating system. Arch Biochem 96: 186-187, 1962.
(4) SWEAT, M. L .: Factor 80 and steroid 118-hydroxyla- tion. Fed Proc 21: 189, 1962.
(5) ZAFFARONI, A .: Micromethods for the analysis of adrenocortical steroids. Recent Prog Hormone Res 8: 51, 1953.
(6) EBERLEIN, W. R., and BONGIOVANNI, A. M .: Patho- physiology of congenital adrenal hyperplasia. Metabolism 9: 326-340, 1960.
(7) SWEAT, M. L., ALDRICH, R. A., DEBRUIN, C. H., FOWLKS, W. L., HEISELT, L. R., and MASON, H. S .: Incorpora-
tion of molecular oxygen into 118 position of corti- costeroids. Fed Proc 15: 367, 1956.
(8) HAYANO, M., LINDBERG, M. C., DORFMAN, R. I., HANCOCK, J. E. H., and DOERING, W. VON E .: The mechanism of C-118-hydroxylation of steroids; a study with H2O18 and O218. Arch Biochem 59: 529-531, 1955.
(9) BRYSON, M. J., and SWEAT, M. L .: Metabolism of steroids by mouse testicular embryonal carcinoma. Fed Proc 20: 238, 1961.
(10) BRYSON, M. J., and SwIM, H. E .: Metabolism of ste- roids by a strain of rabbit fibroblasts. Fed Proc 19: 385, 1960.
(11) BRYSON, M. J., DOMINGUEZ, O. V., KAISER, I. H., SAMUELS, L. T., and SWEAT, M. L .: Enzymic steroid conversions in a masculinovoblastoma. J Clin Endocr 22: 773-783, 1962.