Original Article
Characterization of a newly established cell line derived from human adrenocortical carcinoma
MUNEHISA UENO,1 JUN NAKASHIMA,4 MASUMI AKITA,3 SHIN-ICHI BAN,2 TAKASHI NAKANOMA,1 MASAHIRO IIDA1 AND NOBUHIRO DEGUCHI1
1Department of Urology, Kidney Disease Center, 2Department of Pathology, and 3Division of Morphological Science, Biomedical Research Center, Saitama Medical School, Saitama, and 4Department of Urology, Keio University School of Medicine, Tokyo, Japan
Abstract
Background: ACT-1, a new cell line of human adrenocortical carcinoma, has been established and successfully maintained in culture. This study examined the biological characteristics of the cells. Methods: The tumor cells were isolated from a surgical specimen of the tumor thrombus and cul- tured in monolayer.
Results: Histologically, the primary tumor was composed of a solid proliferation of large polygo- nal cells. A part of the atrophic adrenal cortex remained at the periphery of the tumor. The cul- tured ACT-1 cells were spindle-shaped in morphology and grew exponentially with an approximate population doubling time of 24 h. A chromosomal analysis revealed a modal number of 61 with consistent structural abnormalities of add(3)(q11), add(9)(p11), and add(16)(q11). The expression of 3ß-hydroxysteroid dehydrogenase was observed in the ACT-1 cells as well as in normal human adrenal glands.
Conclusions: The ACT-1 cell line provides a reproducible model system which gives good insight into the oncogenesis of adrenocortical carcinoma.
Key words adrenocortical carcinoma, cell culture, 3ß-hydroxysteroid dehydrogenase.
Introduction
Adrenocortical carcinoma is a highly malignant tumor. The overall mortality rate after 5 years is 75-90%, and the mean survival time is 14.5 months.1 The only potentially curative treatment is complete surgical excision, but most patients present at an advanced tumor stage with local invasion or distant metastases and can rarely be cured.2 The present case was also shown to form thrombosis extending into the vena cava, and the patient died soon after the surgery because of multiple metastases. Adrenocortical carci-
Correspondence: Munehisa Ueno MD, Department of Urology, Kidney Disease Center, Saitama Medical School, 38 Morohongo, Moroyamamachi, Iruma, Saitama 3500495, Japan. Email: camtmune@saitama-med.ac.jp Received 28 February 2000; accepted 6 July 2000.
noma is a neoplasm so rare1 that it is difficult to per- form controlled studies to assess the impact of any therapy on survival. Furthermore, only a few human adrenal cancer cell lines that have been used for stud- ies on chemosensitivity can presently be provided.3-6
In this report we described the establishment and characterization of a human adrenocortical carcinoma cell line in culture which may prove to be a good model to investigate the oncogenesis for the tumor and develop an effective modality of treatment.
Methods
Case report
A 62-year-old man presented with low-grade fever, left-sided flank pain, and weight loss. X-ray studies disclosed a left adrenocortical tumor with thrombosis
A
B
extending into the vena cava. He underwent a radi- cal surgery including adrenalectomy, nephrectomy, splenectomy, and vena cavectomy. Grossly, a bulky tumor 13×9×8 cm in size was definitely distinguish- able from the left kidney and showed extensive areas of necrosis and hemorrhage. The left adrenal vein was completely obstructed by the thrombus. Histologically, the tumor was composed of a solid proliferation of large polygonal cells with a round to oval nucleus and an abundance of compact, eosinophilic cytoplasm that included a scattering of bizarre pleomorphic cells and multinucleated cells (Fig. 1a). A part of the atrophic adrenal cortex remained at the periphery of the tumor (Fig. 1b). These findings led to a pathologic diagnosis of adrenocortical carcinoma. The patient died approxi- mately 3 months after the surgery because of multiple distant metastases.
Cell culture
Immediately after removal of the surgical specimen, approximately 1 mL of tissue was aseptically excised from the tumor thrombus, minced, and rinsed with RPMI1640. The resulting cell suspension was passed through a Cell Strainer (FALCON, Becton Dickinson, Franklin Lakes, NJ, USA) to dissociate the cell aggre- gates. The cells were maintained in 25 cm2-culture flasks containing RPMI1640 medium supplemented with 10% fetal bovine serum, Insulin-Transferrin- Selenium-A (ITSA; GIBCO BRL, Grand Island, NY, USA), 100 units/mL of penicillin G, and 100 µg/mL of streptomycin in a humidified atmosphere of 5%
CO2-95% air at 37°C, as described previously.7 Aliquots of 105 viable cells excluding trypan blue were dispersed into 9.0 cm2 multiwells. The culture medium was renewed and the viable cells were counted in trip- licate with a hemocytometer every 3 days. The dou- bling time was determined when the cells were in the exponential growth phase. This cell line was desig- nated as ACT-1, and the cell culture was initiated on 16 October 1998.
Electron microscopy
The cells were fixed in 2.0% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) at 4℃ and postfixed with 1% OsO4 at 4℃ in the same buffer. The specimens were dehydrated and embedded in Epon 812. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with an electron microscope (JEM-1010, JEOL, Tokyo, Japan).
Cytogenetic analysis
For the chromosomal preparation, the ACT-1 cells in the logarithmic growth phase detached with trypsin were treated with 0.05 mg/mL colcemid trypsin for 2 h, subjected to hypotonic conditions for 15 min, and then fixed with methanol-acetic acid solution (3:1). Trypsin-Giemsa staining was performed and G- banded metaphases were photographed and analyzed.
Reverse transcription-polymerase chain reaction
A reverse transcription-polymerase chain reaction (RT-PCR) of 3ß-hydroxysteroid dehydrogenase (3ß- HSD) type II and cytochrome P450c17 (CYP17) was performed.
Total RNA was extracted using a guanidium isothio- cyanate kit (RNeasy Mini Kit, QIAGEN, Tokyo, Japan). The RNA contents were estimated by spectroscopy on a GeneQuant II spectrophotometer (Pharmacia, Peapack, NJ, USA) and by gel analysis. This RNA was reverse transcribed using the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (Roche Molecular Systems, Branchburg, NJ, USA). For first-strand cDNA synthesis, 1 µg of total RNA from each sample, 5’- TCTGATCCTCATTTAACCAACTTGT-3’ (for 3ß-HSD type II reverse primer) or 5’-CAGCAGATCATTTCG TATTT-3’ (for CYP17 reverse primer) were incubated with rTth polymerase at 70℃ for 10 min. For PCR, the product was incubated with 5’-TATCAGAAAACTTC CCAGCCAGATC-3’ or 5’-CAGCAGATCATTTCG TATTT-3’ as 3B-HSD type II and CYP17 forward primer,
respectively, at 35 cycles of 95℃ for 1 min, 58℃ or 55℃ for 1 min, and 72℃ for 1.5 min, followed by a final cycle at 72℃ for 10 min. The 3B-HSD type II mRNA amplification product had an expected length of 279 bp,8 and the CYP17 amplification product had an expected length of 698 bp.9 After PCR, aliquots of the reaction were analyzed on a 1.2% agarose gel with ethidium bro- mide (0.5 mg/mL).
Results
The ACT-1 cells could not be transplanted into nude mice. The cultured cells were spindle-shaped in morphology when examined under a phase contrast microscope (Fig. 2). In electron microscopy, the nuclear envelopes of the cells commonly showed shal- low or deep infoldings (cytoplasmic invaginations). The cells possessed a small number of microvilli and a moderate number of mitochondria that were tubular rather than vesicular. Fair amounts of rough and smooth-surfaced endoplasmic reticulum and lipid droplets were contained within the cells (Fig. 3).
A
*
B
C
1000
Cell number ( x104 cells / 9.0 cm2-well )
100
10
0
3
6
9
12
15
Time in culture (days)
Figure 4 illustrates the exponential growth of the ACT-1 cell population, which approximately doubled every 24h at the 26th passage. The cells did not release any detectable amounts of cortisol, aldosterone, progesterone or prognenolone in culture flasks.
The chromosomes were analyzed in the ACT-1 cells
from the 10th subculture and 10 metaphases were
counted. The number of chromosomes varied from 58
to 65 and the modal number was 61. The representa-
tive G-banded karyotype was 5965<3n>, X, -X[10],
-Y[10], +1[10], add(1)(p11)[3],add(1)(p11)[9],add(1)
(p21)[9], -2[10], -2[10], add(3)(p11)[9], add(3)(p11)
[10], -4[10], add(4)(p21)[8], ?add(5)(p15)[2], add(6)
(p21)[9], +7[9], add(8)(p11)×2[10], add(8)(q1?)[9],
-9[10], add(9)(p11)[10], add(9)(p1321)[9], -10[10],
-10[10], -11[10], add(11)(p11)[9], -12[9], -12[3],
add(12)(q13)[7], -13[4], -14[10], -14[9], add(14)
(p11)[9], -15[10], add(15)(q22)[6], +16[4], add
(16)(q11)[10], add(16)[2], -17[10], -17[6], -18[8],
add(18)(q23)[9], -19[10], -19[10], -19[8], -20[6],
1
18
2
3ft
41
5 1
6
1
7
18tt
19t
10
11}
121
É
1
13
141
15
1611
17
18
1
,
19
20
21
22
x
Y
Marker chromosomes
1
2
3
4
36-HSD
-279 bp
P450c17
-698 bp
-21[10], -21[4], -22[10], -22[10], -22[10], +13~18mar (Fig. 5).
To examine whether ACT-1 had retained the capa- city to express adrenal steroidogenic enzymes, RT-PCR was used to investigate the expressions of 3B-HSD type II and CYP17, both of which have been shown to be expressed in normal adrenal glands. As a result, the ACT-1 were found to express 3B-HSD type II but not CYP17 (Fig. 6). Two different renal carcinoma cell lines, ACHN10 and KU-2,11 were used as controls. No bands were observed in the KU-2 cells, whereas
the ACHN cells were detected to have mRNA of 3B- HSD type II.
Discussion
Most patients with adrenocortical carcinoma present with metastases, a complication associated with poor prognosis. To make things worse, chemotherapy and radiotherapy for the metastatic lesions are generally ineffective, possibly because of multidrug resistance (MDR). The agent mitotane [1-(o-chlorophenyl)-1- (p-chlorophenyl)-2,2-dichloroethane], which is report- ed to modulate a P-glycoprotein (Pgp) and to probably reverse the Pgp-related drug resistance,1 may be suc- cessful in reducing hypercortisolism but has very limited antitumor efficacy.12 Feller et al. reported that treatment with a combination of multiple-resistance drugs plus mitotane yielded no significant effects in vivo.1 Therefore, we must analyze the oncogenesis of adrenocortical carcinoma more deeply and develop a good model to help us establish an effective new treatment.
We established a new cell line of adrenocortical carcinoma designated ACT-1. Ultrastructurally, lipid droplets were abundant in the cultured ACT-1 cells. Lipid droplets are known to be a prominent feature of adrenocortical cells, especially in species in which most of the cholesterol is derived from lipoproteins.13 The cytoplasm contained prominent smooth-surfaced endoplasmic reticulum, a feature typical of steroid- secreting activity. Together these findings suggest that ACT-1 cells may be able to produce steroids via the synthesis of cholesterol. ACT-1 has retained the capac- ity to express the mRNA of 3B-HSD type II, an adrenal steroidogenic enzyme. The initial steps of the steroido- genic pathway are the side chain cleavage of cho- lesterol, catalyzed by cytochrome P450 side chain cleavage (P450scc) to form pregnenolone and conver- sion of the pregnenolone into progesterone by 3ß-HSD. In the adrenal cortex, the subsequent 17a-hydroxyla- tion of pregnenolone or progesterone is a branch point for the formation of cortisol and adrenal androgens. The activities of 17a-hydroxylase and 17,20-lyase are catalyzed by microsomal cytochrome P450c17.8,14 In humans, there are two 3ß-HSD isoenzymes chronologi- cally designated as types I and II. The type I gene is predominantly expressed in the placenta and peripheral tissues, including the mammary gland and the skin, while 3B-HSD type II gene was originally isolated from a human adrenal cDNA library15 and is almost exclu- sively expressed in the adrenals, ovary, and testis.16 The results from RT-PCR analysis indicate that ACT-1
originated from the adrenals. In addition, the cells have not expressed mRNA of P450c17, a cytochrome essen- tial for the biosynthesis of cortisol,14 that takes place predominantly in the zona fasciculata of the cortex. The absence of this genetic component reflects the inability of ACT-1 cells to produce cortisol.
The microscopic findings showed that this cell line was derived from the adrenal cortex because the tumors were adjacent to the atrophic adrenals and the cell line was obtained from the thrombus extending through the adrenal vein. When the tumor burden of adrenal carcinoma is extremely great, histologic diag- nosis with hematoxylin and eosin stain is often impos- sible because the pathologic features of the cells are often similar to those of renal cell carcinoma (RCC). Immunohistochemical staining using specific antibod- ies for P450scc or 3ß-HSD is effective in differential diagnosis. As these antibodies are not commercially available, RT-PCR techniques using specific primers seemed to be convenient and reliable. However, in the present study, ACHN, an RCC cell line, expressed mRNA of the 3B-HSD type II to the same extent as normal human adrenals. Other RCC cell lines that were formerly established in our laboratory were proven not to express this mRNA (data not shown). The 3B-HSD activity is also found in the kidney in other species such as rodents.14 Although ACHN is thought to be derived from an exceptional RCC, RT- PCR with specific primers of 3B-HSD type II is not sufficient to diagnose adrenal tumors. Further investi- gation should be carried out to develop a new means for the diagnosis of adrenocortical carcinoma probably using our newly established cell line.
Acknowledgments
The authors are grateful to Ms R Hirata at the Kidney Disease Center and Ms K Tanaka at the Division of Morphological Science at the Biomedical Research Center of the Saitama Medical School for their excel- lent technical assistance.
References
1 Feller N, Hoekman K, Kuiper CM et al. A patient with adrenocortical carcinoma: Characterization of its biological activity and drug resistance profile. Clin. Cancer Res. 1997; 3: 389-94.
2 Søreide JA, Braband K, Thoresen SØ. Adrenal cortical carcinoma in Norway, 1970-1984. World J. Surg. 1992; 16: 663-8.
3 Leibovitz A, McCombs WM, Johnston D, McCoy CE, Stinson JC. New human cancer cell culture cell lines. I. SW-13, small-cell carcinoma of the adrenal cortex. J. Natl Cancer Inst. 1973; 51: 691-7.
4 Fang VS. Establishment and characterization of a strain of human adrenal tumor cells that secrete estrogen. Proc. Natl Acad. Sci. USA 1977; 74: 1067-71.
5 Moffett RB. Expression of an endogeneous renin- angiotensin system by an adrenal cortical tumor cell line in culture. Fed. Proc. 1987; 46: 648.
6 Gazdar AF, Oie HK, Shackleton CH et al. Establish- ment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 1990; 50: 5488- 96.
7 Ueno M, Seferynska I, Beckman B, Brookins J, Nakashima J, Fisher JW. Enhanced erythropoietin secretion in hepatoblastoma cells in response to hypo- xia. Am. J. Physiol. 1989; 257: C743-9.
8 Rheaume E, Simard J, Morel Y et al. Congenital adren- al hyperplasia due to point mutations in the type II 3ß- hydroxysteroid dehydrogenase gene. Nat. Genet. 1992; 1: 239-45.
9 Shibata H, Ando T, Suzuki T et al. COUP-TFI expres- sion in human adrenocortical adenomas: Possible role in steroidogenesis. J. Clin. Endocrinol. Metab. 1998; 83: 4520-23.
10 Kochevar J. Blockage of autonomous growth of ACHN cells by anti-renal cell carcinoma monoclonal antibody 5F4. Cancer Res. 1990; 50: 2968-72.
11 Hayakawa M, Osawa A, Nagakura K, Kikuchi H. Heterogeneity of a KU-2 cell line derived from human renal cell carcinoma. Acta Urol. Jap. 1985; 31: 763-8.
12 Luton JP, Cerdas S, Billaud L et al. Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mitotane therapy. N. Engl. J. Med. 1990; 322: 1195-201.
13 Nussdorfer GG. Cytophysiology of the adrenal cortex. In: Bourne GH, Danielli JF, Jeon KW (eds). Interna- tional Review of Cytology Series, Vol. 98. Academic Press, Orlando, 1986; 319-30.
14 Simard J, Durocher F, Mebarki F et al. Molecular biology and genetics of the 3ß-hydroxysteroid dehydro- genase/45-44-isomerase gene family. J. Endocrinol. 1996; 150: S189-207.
15 Rheaume E, Lachance Y, Zhao HF et al. Structure and expression of a new complementary DNA encoding the almost exclusive 30-hydroxysteroid dehydrogenase/ 45-44-isomerase in human adrenals and gonads. Mol. Endocrinol. 1991; 5: 1147-57.
16 Simard J, Rheaume E, Mebarki F et al. Molecular basis of 3ß-hydroxysteroid dehydrogenase deficiency. J. Steroid Biochem. Mol. Biol. 1995; 53: 127-38.