Establishment of an adrenocortical carcinoma xenograft with normotensive hyperaldosteronism in vivo
HITOSHI YAMAZAKI,1 YOSHIYUKI ABE,’ YUKO KATOH,’ NOBUKO SAWA,2
YASUYUKI OHNISHI,2 YUJI TANAKA,3 HIRONOBU SASANO,4 YOSHIRO OSHIKA,1 TESTUJI TOKUNAGA,’ HIROSHI KIJIMA,1 NORIKAZU TAMAOKI,1 MASATO NAKAMURA1, 3 and YOSHITO UEYAMA1, 2
‘Department of Pathology, Tokai University School of Medicine, Kanagawa, 2Central Institute for Experimental Animals, Kanagawa, 3Fourth Department of Internal Medicine, University of Tokyo School of Medicine and 4Department of Pathology, Tohoku University, Japan
Yamazaki, H., Abe, Y., Katoh, Y., Sawa, N., Ohnishi, Y., Tanaka, Y., Sasano, H., Oshika, Y., Tokuna- ga, T., Kijima, H., Tamaoki, N., Nakamura, M. & Ueyama, Y. Establishment of an adrenocortical carcinoma xenograft with normotensive hyperaldosteronism in vivo. APMIS 106: 1056-1060, 1998.
We established a xenograft line of human adrenocortical carcinoma (ADR-1), and analyzed the hyper- aldosteronism induced by the xenograft in vivo. Adrenocortical carcinoma specimens from a 25-year- old woman were subcutaneously inoculated into nude mice (BALB/c-nu/nu) followed by serial pas- sages in vivo. ADR-1 retained the histopathological features (trabecular and sinusoid nests) seen in the primary carcinoma. The patient showed hyperaldosteronism (serum aldosterone >4000 pg/ml) and hypokalemia (serum K 2.1 mEq/l), but did not show hypertension. The nude rat (F344-rnu/rnu) bearing ADR-1 showed hyperaldosteronism (serum aldosterone 3320±1420 pg/ml; control 191±130 pg/ml) and hypokalemia (serum K 3.4±0.4 mEq/1; control 5.2±1.0 mEq/l) in vivo, and hypertension was not obvious. ADR-1 was shown immunohistochemically to retain production of human-specific corticosteroid synthetase. The xenograft ADR-1 will be useful to elucidate the regulatory mechanism of normotensive hyperaldosteronism.
Key words: Aldosterone; adrenocortical carcinoma; xenograft.
Yoshito Ueyama, Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan.
Primary hyperaldosteronism is a clinical con- dition attributed in 90% of cases to overpro- duction of aldosterone by a single adreno- cortical adenoma (3). Only 4% of adreno- cortical carcinomas are clinically associated with hyperaldosteronism (2), while one of the first recorded examples of primary hyperaldos- teronism was a malignant tumor (4). Many in- vestigations have suggested that the mainten- ance of blood pressure is associated with several
factors, including the renin-angiotensin system, aldosterone production and sodium retention (12). However, the precise mechanism of induc- tion of hypertension by overproduction of aldo- sterone still remains unclear.
We encountered a case of adrenocortical car- cinoma accompanied by normotensive hyperal- dosteronism. We established a xenograft line of this adrenocortical carcinoma by serial xeno- transplantation in vivo. We here report several characteristics (blood pressure, serum aldoster- one level) in the host animals bearing this xeno- graft in vivo.
CANCER XENOGRAFT WITH HYPERALDOSTERONISM
MATERIALS AND METHODS
Case report
A 25-year-old Japanese woman presented with a pitting edema on the dorsal surface of the foot, gen- eral fatigue and nocturnal polyuria (2-3 times/night). Diuretics had no marked effect on the edema. After 1 month, she complained of bilateral paresthesia of the hands and was admitted to hospital owing to se- vere hypokalemia (serum K 2.0 mEq/ml). A tumor mass was found in the right adrenal cortex. Blood pressure was 120/80 mmHg, and pulse rate 76/min. The laboratory data are summarized in Table 1. The patient showed hypokalemia (2.1 mEq/ml) with in- creased urinary excretion of potassium (41 mEq/day). Plasma and urinary excretion levels of sodium ions were within normal limits (144 mEq/ml, 142 mEq/ day). Serum aldosterone level was markedly elevated (4,000 pg/ml; control 7-12 pg/ml). Serum levels of other hormones (adrenocorticotropic hormone, corticosterone, dehydroepiandrosterone and test- sterone) were within normal limits. Plasma renin ac- tivity was not markedly decreased (0.3 ng/ml/h). Levels of urinary 17-hydroxy-corticosteroids (17- OHCS) and 17-ketosteroids (17-KS) were normal. After resection of the adrenocortical tumor lesion, she gradually recovered.
Establishment of adrenocortical carcinoma xenograft Tumor specimens were obtained from the primary lesion under sterile conditions. Tumor tissues cut and
| Plasma | Urine | |
|---|---|---|
| Na | 144 mEq/1 | 142 mEq/day |
| K | 2.1 mEq/1 | 41 mEq/day |
| CI | 101 mEq/1 | |
| Aldosterone | >4,000 pg/ml (7-12) | |
| ACTH | <20 pg/ml (<20) | |
| Corticosterone | 2.15 ng/ml | |
| (1.0-10.0) | ||
| DHEA | 3.1 ng/ml | |
| (1.2-7.5) | ||
| Testosterone | 0.8 ng/ml | |
| (0.7-2.0) | ||
| RA | 0.3 ng/ml/h (0.3-2.9) | |
| 17-OHCS | 7.2 mg/day (3.5-8.0) | |
| 17-KS | 10.1 mg/day | |
| (4.0-14.0) |
ACTH, adrenocorticotropic hormone; DHEA, de- hydroepiandrosterone; RA, renine activity; 17- OHCS, 17-hydroxycorticosteroids; 17-KS, 17-keto- steroids; ( ), normal values of hormones.
immersed in Dulbecco’s modified Eagle’s medium (DMEM) were subcutaneously inoculated into nude mice (BALB/c-nu/nu, 6 to 10-week-old female mice). The xenograft line named ADR-1 was established through serial passage in vivo.
Histological and immunohistochemical examination
Specimens of the primary cancer lesion and the xenograft ADR-1 were fixed in isotonic buffered formalin (10%, v/v) and embedded in paraffin. The deparaffinized sections were incubated with the fol- lowing five antibodies against enzymes related to the synthesis of adrenocortical hormones: anti-human cytochrome P450 side chain cleavage, P450SCC; anti- human 3ß-hydroxysteroid dehydrogenase, 3ß-HSD; anti-human cytochrome P450 CII, P450Cll; anti-hu- man cytochrome P450C17, P450C17; anti-human cytochrome P450C21, P450C21 (10). Products im- munoreactive with the streptavidin-biotin complex were visualized with 20% 3.3’-diaminobenzidine-4 HCL, 0.005% H2O2, 1M Tris-Cl buffer (pH 7.6). Sec- tions were counterstained with methyl green.
Laboratory examination of animals bearing ADR-1 in vivo
We used nude rats (F344-rnu/rnu, 6- to 8-week- old females for laboratory analysis because we could get a sufficient volume of serum. Sample plasma and sera (5 ml) were obtained from nude rats bearing the ADR-1 xenograft. Each group in the in vivo studies consisted of four animals. Plasma electrolytes (sodium, potassium, chloride) were estimated, and serum levels of various hor- mones (aldosterone, cortisol, corticosterone, di- hydroepiandrosterone, dihydroepiandrosterone-sul- fate, 17-hydroxypregnenolone) were quantified by radioimmunoassay (6, 14). The significance of dif- ferences between ADR-1-bearing and control ani- mals was assessed using Student’s t-test.
Blood pressure of the nude rats (F344-rnu/rnu) was regularly measured (Softron BP-98A) once or twice a week after subcutaneous inoculation of the tumor tissue (8).
RESULTS
Histopathology
The xenograft line named ADR-1 was estab- lished through serial passage in vivo. The tumor sizes were 241 ±80.1 mm3 (day 8) and 320±125.3 mm3 (day 22), while neither metastasis nor in- vasion was apparent in the mice.
The primary adrenocortical carcinoma showed solid trabeculae of atypical polygonal epithelial cells with focal sinusoid-like structures
B
and slight vascular stroma (Fig. 1A). The carci- noma showed focal necrosis and hemorrhage. The carcinoma cells possessed clear cytoplasm and atypical nuclei with a few mitotic figures.
The histology of the xenograft ADR-1 showed features similar to those of the primary adrenocortical carcinoma (Fig. 1B). The serially passaged xenograft ADR-1 retained the solid trabecular features of the neoplastic cells. Mi- totic figures were increased.
Immunohistochemical studies
Five antibodies against enzymes related to the synthesis of adrenocortical hormones (P450SCC, 3B-HSD, P450C11, P450C17, and P450C21) were used for immunohistochemical analysis (Table 2). The cells of primary adreno-
| Antibody* | Dilution | Primary | ADR-1 Xenograft |
|---|---|---|---|
| P450SCC | ×100 | + | + |
| 3B-HSD | ×100 | + | + |
| P450C11 | ×100 | + | + |
| P450C17 | ×100 | + | + |
| P450C21 | ×100 | + | + |
* Antibodies used were established in our laboratory (10): anti-human cytochrome P450 side chain cleav- age, P450SCC; anti-human 3ß-hydroxysteroid de- hydrogenase, 3ß-HSD; anti-human cytochrome P450 C11, P450C11; anti-human cytochrome P450C17. P450C17; anti-human cytochrome P450 C21, P450C21.
cortical carcinoma showed positive immuno- staining for all the above-mentioned antibodies.
The ADR-1 xenograft cells retained immuno- reactivity for all the above-mentioned anti- bodies, including the human-specific antibody P450C17.
Levels of plasma electrolytes/serum hormones and blood pressure in the animals bearing the xenograft ADR-1 in vivo
Laboratory analysis showed statistically sig- nificant hypokalemia in nude rats bearing ADR- 1 (3.4±0.4mEg/l; control 5.2±1.0mEq/1) (Table 3). Serum aldosterone levels were also signifi- cantly elevated in the group of ADR-1-bearing nude rats (3,320±1,420 ng/dl; control 191±130 ng/dl). Overproduction of both dehydroepiand-
| Nude rat (F344-rnu/rnu) | ||
|---|---|---|
| ADR-1 | (control) | |
| Na (mEq/I) | 148±2 | (143±4) |
| K (mEq/1) | 3.4±0.4* | (5.2±1.0) |
| Cl (mEq/1) | 99±5 | (104±2) |
| Aldosterone (pg/ml) | 3320±1420* | (191±130) |
| DHEA (ng/ml) | 2.3+1.0* | (<0.24) |
| DHEA-S (ng/ml) | 293±91* | (<200) |
DHEA, dehydroepiandrosterone; DHEA-S, dehydro- epiandrosterone-sulfate; * statistical significance be- tween ADR-1-bearing rat and control by Student’s t- test. Each group in the in vivo studies consisted of four rats.
CANCER XENOGRAFT WITH HYPERALDOSTERONISM
rosterone (DHEA 2.3±1.0 ng/ml; control <0.24 ng/ml) and dehydroepiandrosterone sulfate (DHEA-S 293±125 ng/ml: control <200 ng/ml) were also preserved in nude rats bearing ADR-1. The nude rats bearing ADR-1 showed no marked hypertension (88.67 mmHg; control 96.33 mmHg).
We analyzed corticosterone, cortisol and 17- hydroxypregnenolone in nude mice. The data for nude mice bearing ADR-1 were as follows: corticosterone 55.3 ng/ml (control 94.3); cor- tisol 4.0 ng/dl (control 7.2); 17-hydroxypregnen- olone 1.92 ng/ml (control 0.93).
DISCUSSION
We established a human adrenocortical carci- noma xenograft line (ADR-1) in immunodefic- ient animals in vivo. ADR-1 showed well-pre- served autonomous production of human aldo- sterone seen in the primary adrenocortical carci- noma. Immunohistochemical studies demon- strated reservation of the enzymes involved in the synthesis of adrenocortical hormones in the xenograft ADR-1.
Some reports have described adrenocortical carcinoma cases with hyperaldosteronism (13). Revach et al. reported an adrenocortical carci- noma in a patient with clinical and biochemical features of primary hyperaldosteronism but no evidence of excess of other adrenocortical hor- mones (9). The levels of hormones other than aldosterone were within normal limits in this case.
This patient and the immunodeficient ani- mals bearing ADR-1 had normal blood press- ure. Normotensive primary aldosteronism has been reported previously (7). The primary adrenocortical carcinoma patient and the host animals bearing the ADR-1 were considered to exhibit normotensive hyperaldosteronism. Hy- pertension is the result of many factors includ- ing the renin-angiotensin system, aldosterone production and sodium ion retention in vivo, which may affect normotensive hyperaldo- steronism in vivo.
Cytochrome P-450 specific for steroid 17a- hydroxylation (P-45017%) was demonstrated im- munohistochemically in adrenocortical dis- orders (11). We confirmed the preservation of the human-specific enzymes required for syn-
thesis of human aldosterone in the xenograft ADR-1 in vivo immunohistochemically.
Some human adrenocortical carcinoma cell lines have been reported (1, 5), while there have been few studies on tumor xenografts producing human aldosterone in vivo. The histopathology of the adrenal gland in ADR-1-bearing animals showed marked vacuolation in the cells of the reticular zone (data not shown). Some negative feed-back mechanisms of aldosterone produc- tion might exist in ADR-1-bearing animals. This xenograft ADR-1 provides a useful model for analysis of the regulatory mechanism of normotensive hyperaldosteronism in vivo.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Edu- cation, Science and Culture (Y.U. 07680921; N.T. 07457588; M.N. 06670206) and by Tokai University School of Medicine Research Aid (M.N., Y.U., H.Y.). We thank Yuichi Tada, Johbu Itoh, Kenji Kawai and Kyoko Murata for their technical assistance.
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