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Overexpression of Glutathione-S-Transferase A1 in Benign Adrenocortical Adenomas from Patients with Cushing’s Syndrome

Departments of Endocrinology and Metabolism and Teratology and Genetics (S.H.), Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; and Department of Surgery II, Nagoya University School of Medicine (T.I., H.F.), 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Dr. Hisao Seo, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: hseo{at}riem.nagoya-u.ac.jp

	Abstract

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Benign adrenocortical adenoma is a major primary cause of Cushing’s syndrome. Although numerous studies have been performed, the molecular mechanism of adrenocortical adenoma is yet to be elucidated. In this study we endeavored to identify genes differentially regulated in adrenocortical adenoma by suppression PCR-based complementary DNA (cDNA) subtractive hybridization. The cDNA population in atrophied nontumorous adrenal gland adjacent to the adenoma was subtracted from that in the adenoma. Then adenoma-specific cDNAs were amplified by PCR. We cloned several cDNAs that are selectively up-regulated in the adenoma, one of which was identified to encode glutathione-S-transferase A1 (GSTA1). Northern blot analysis revealed that GSTA1 messenger ribonucleic acid was abundantly expressed in the adenoma compared with that in the adjacent atrophied nontumorous gland. Western blot analysis and immunohistochemistry showed high expression of GSTA1 also at the protein level. In concordance with this finding, GST activity was significantly higher in the adenoma than in the adjacent atrophied nontumorous gland. To clarify the role of GSTA1 in adrenocortical cells, GST activity in the H295R human adrenocortical cell line was inhibited by ethacrynic acid. Inhibition of GSTs interfered with proliferation of the cells. We, therefore, hypothesize that overexpression of GSTA1 in adrenocortical adenomas might be involved in the growth of tumor cells. We also speculate that this overexpression might be an adaptive response to excess cortisol production.

	Introduction

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ALTHOUGH NUMEROUS studies have been carried out, the exact molecular events leading to the pathogenesis of benign functioning adrenocortical adenoma are yet to be elucidated. ACTH is the major regulator of cortisol production and adrenocortical growth (1, 2). ACTH binds to its receptor and activates the G_s -subunit of heterotrimeric G_s protein (3). This stimulates adenyl cyclase-mediated cAMP production, which acts as the second messenger to augment adrenocortical growth and steroidogenesis. However, in patients with benign adrenocortical adenoma, abnormalities in the post-ACTH signal transduction pathway such as activating mutations in ACTH receptor or mutations in G_s, that might explain the pathogenesis have not been identified (4, 5, 6, 7). Mutations in tumor suppressor gene p53, overexpression of insulin-like growth factor II, and high inhibin-like immunoreactivity have been reported in the adenoma (8, 9, 10, 11, 12). However, either these findings could not be demonstrated by separate studies or the significance of these findings is not yet known. In view of this background, we endeavored to identify genes that are selectively up-regulated in benign cortisol-producing adenoma and that might have some role in the process of tumorigenesis.

We employed suppression PCR-based complementary DNA (cDNA) subtractive hybridization to isolate genes differentially up-regulated in the adenoma compared with the atrophied nontumorous adrenal gland. This procedure allows the isolation of differentially expressed cDNAs among two different cDNA populations, called Tester and Driver (13, 14). Usually, the Driver cDNA population is subtracted from the Tester cDNA population by hybridization. Thus, cDNAs present only in the Tester are enriched and amplified by PCR. In our study we employed cDNA derived from the adenoma tissue as the Tester and that from the adjacent atrophied nontumorous gland as the Driver. We cloned several cDNAs that are selectively up-regulated in the adenoma. Sequence analysis revealed that one of them encodes glutathione-S-transferase A1 (GSTA1).

GSTs (EC 2.5.1.18) constitute a family of related proteins that play critical roles as intracellular detoxification enzymes (15). GSTs can be broadly divided into two groups: cytosolic GSTs and membrane-bound GSTs. In humans, the cytosolic GSTs are divided into four classes, namely , µ, , and . Within each GST class there are multiple protein subunits, each encoded by a distinct gene. Each functional GST enzyme exists as a homodimer or heterodimer consisting of two protein subunits within the same class. Human GST class subunits are encoded by six genes, for GSTA1, -A2, -A3, -A4, -9.9, and - (15). The major biological function of GSTs is to provide protection against cellular oxidative stress through neutralization of a wide range of hydrophobic and electrophilic endogenous compounds or xenobiotics by catalyzing their conjugation to reduced glutathione (16). GSTs also reduce organic hydroperoxides in a nonselenium-dependent pathway. Other functions of GSTs include binding of steroids (initially described as ligandin), bilirubin, carcinogens, and organic anions and ⁵-3-ketosteroid isomerase activity (17).

In the present study we observed high expression of GSTA1 in benign cortisol-producing adrenocortical adenomas at both messenger ribonucleic acid (mRNA) and protein levels. We also demonstrated that GSTs are necessary for the proliferation of human adrenocortical cells. We thus hypothesize that GSTs are involved in the molecular events of adrenocortical tumorigenesis or are overexpressed as a result of excess cortisol production.

	Case Subjects

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Eleven patients with Cushing’s syndrome were studied. Written informed consent was obtained from all patients for the use of tissue samples obtained at operation. All patients presented with typical signs and symptoms of Cushing’s syndrome, such as moon face, buffalo hump, central obesity, hypertension, osteoporosis, hirsutism, urolithiasis, psychological disorders, and amenorrhea. Ultrasonography, computed tomography, or magnetic resonance imaging revealed unilateral adrenal tumor in all the patients. The patients were treated by unilateral adrenalectomy. The clinical data of the patients are summarized in Table 1.

Blood was drawn at 0600, 1200, 1800, and 2400 h to determine the diurnal variations in plasma ACTH and serum cortisol. In all patients, plasma ACTH levels were completely suppressed (<5 pg/mL) throughout the day. Serum cortisol did not show diurnal variations.

A dexamethasone suppression test was carried out by the administration of 0.5 mg dexamethasone (Decadron, Banyu, Tokyo, Japan), orally, every 6 h for 2 days, and blood was drawn on the third morning. Dexamethasone (2 mg every 6 h) was continued for another 2 days, blood was drawn on the fifth morning, and plasma ACTH and serum cortisol were determined (18). When the ACTH level was determined before and after dexamethasone administration, the levels were less than 5 pg/mL. Serum cortisol was not suppressed after 2 or 8 mg dexamethasone administration, suggesting an adrenal origin of the Cushing’s syndrome. Indeed, benign adrenocortical adenoma was demonstrated in all patients (Table 1).

	Materials and Methods

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Sample collection

After adrenalectomy, a portion of resected adenoma and adjacent atrophic adrenal tissue were immediately frozen in liquid nitrogen and kept at -80 C. For immunohistochemistry, formalin-fixed paraffinembedded sections were used.

Extraction of RNA and cDNA synthesis

Total RNA was extracted using the QIAGEN RNA/DNA kit according to the manufacturer’s protocol (QIAGEN, Hilden, Germany). Total RNA from six patients (170 ng each) were pooled for the adenoma and the adjacent atrophic tissue. Thus, 1 µg total RNA each from the adenoma and the adjacent atrophic tissue was used to prepare double stranded cDNA using the SMART PCR cDNA synthesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s protocol.

Suppression PCR-based cDNA subtractive hybridization

Double stranded cDNA was used to perform suppression PCR-based cDNA subtractive hybridization using the PCR-Select cDNA subtraction kit (CLONTECH Laboratories, Inc.). The cDNAs from the adenoma and the adjacent atrophic tissue were regarded as Tester and Driver, respectively. The cDNAs were digested with RsaI (15 U) for 3 h. Two Tester populations were created by ligating two sets of adaptor oligonucleotides (Table 2) separately. Tester cDNA with adaptor 1 was mixed with an excess amount of denatured Driver cDNA, and hybridization was carried out at 68 C for 8 h. Tester cDNA with adaptor 2 and freshly denatured Driver cDNA were added to the first hybridization mixture, and second hybridization was carried out at 68 C overnight. Then Tester-specific cDNAs were amplified with two rounds of PCR using the primers that correspond to the sequences of the adaptor oligonucleotides. The amplified products were cloned into pGEM-Teasy vector (Promega Corp., Madison, WI), which was used to transform JM109 Escherichia coli.

After transformation, the colonies were picked up, and plasmids were purified using Wizard Plus Minipreps DNA Purification Systems (Promega Corp.). The purified plasmids (1 µg each) were digested with EcoRI (Roche Molecular Biochemicals, Mannheim, Germany), the site of which is present in the multiple cloning site of the vector. The vector along with the digested products were transferred to a GeneScreen Plus nylon membrane (NEN Life Science Products. Boston, MA) by Southern blotting according to standard procedures (19). Two sets of membranes were thus prepared. The cDNA populations (500 ng each) from Driver and Tester were labeled with [-³²P]deoxy-CTP (NEN Life Science Products) and were used to hybridize the membranes separately. The radioactivity was analyzed by the BAS 2000 Bioimage analyzing system (Fuji Photo Film Co., Ltd., Tokyo, Japan), and the bands showing significant differences in intensity were identified. The cloned fragments corresponding to the bands were sequenced by the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer Corp., Foster City, CA). A sequence similarity search of the cloned cDNAs was performed against the sequence information database in GenBank using the Basic Local Alignment Search Tool (BLAST; http://www.blast.genome.ad.jp.).

Northern blot analysis

Fifteen micrograms of total RNA from adenoma and the adjacent atrophic gland were subjected to Northern blot analysis as described previously (20). The membrane was hybridized with human GSTA1 cDNA, one of the cDNA fragments cloned by subtractive hybridization labeled with [-³²P]deoxy-CTP (NEN Life Science Products). The same membrane was rehybridized with rat glyceryldehyde-3-phosphate dehydrogenase (GAPDH), GSTM1, and GSTP1 cDNA probes. The cloning of rat GAPDH was reported previously (21). A 486-bp fragment of human GSTM1 cDNA was cloned by RT-PCR from human adrenal mRNA using the following primers: sense, 5'-TGG ACT TTC CCA ATC TGC CCT AC-3'; and antisense, 5'-TGC CCC AGA CAG CCA TCT TTG AG-3'. A 327-bp fragment of human GSTP1 cDNA was cloned by RT-PCR using the following primers: sense, 5'-GTG AAT GAC GGC GTG GAG GAC C-3'; and antisense, 5'-GTA CTC AGG GGA GGC CAG GAA G-3'. Analysis of radioactivity was carried out using the BAS 2000 Bioimage analyzing system (Fuji Photo Film Co., Ltd.). GSTA1 mRNA levels were normalized by GAPDH mRNA levels.

Western blot analysis

Tissue samples (100 mg) were homogenized in 300 µL RIPA buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in phosphate-buffered saline (PBS) with protease inhibitor cocktail (Roche Molecular Biochemicals)] and centrifuged at 15,000 rpm for 20 min at 4 C. The supernatant was used as the total cell lysate. One hundred micrograms of total cell lysate from each sample were run in a 15% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane (Hybond-C pure, Amersham International, Aylesbury, UK) using an electroblotting apparatus (Milliblot, Millipore Corp., Marlborough, MA). The membrane was blocked with Blotto A [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 5% skimmed milk, and 0.05% Tween-20] for 20 min at room temperature and incubated with anti-GSTA1 antibody (1:1000; Calbiochem, La Jolla, CA) in Blotto A for 1 h at room temperature. The membrane was washed three times for 5 min each time with 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween-20 and incubated with alkaline phosphatase-conjugated goat antirabbit IgG (1:2000; Zymed Laboratories, Inc., San Francisco, CA) in Blotto A for 45 min. The membrane was washed three times for 5 min each time with 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween-20 and once for 5 min with TBS [10 mmol/L Tris-HCl (pH 8.0) and 150 mmol/L NaCl]. Color development was carried out using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt ready to use tablets (Roche Molecular Biochemicals).

Immunohistochemistry

Immunohistochemistry was performed using the Vectastatin Universal Quick Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s protocol with several modifications. Formalin-fixed paraffin-embedded sections were deparaffinized by serial incubations in xylene and graded ethanol. The sections were incubated in 0.1% Triton-X in PBS at room temperature for 30 min. After washing in PBS the sections were incubated in 3% H₂O₂ in water for 5 min at room temperature to quench endogenous peroxidase. Then the sections were incubated in a blocking solution containing 2.5% normal horse serum (Vector Laboratories, Inc.) for 1 h at room temperature and in anti-GSTA1 antibody (Calbiochem) diluted 1:1000 in the blocking solution containing 1.5% normal horse serum at 4 C overnight. The sections were washed in PBS and incubated in biotinylated universal secondary antibody (Vector Laboratories, Inc.) at room temperature for 1 h, washed again in PBS, and incubated in streptavidin/peroxidase complex solution (Vector Laboratories, Inc.) at room temperature for 1 h. After washing in PBS the sections were incubated in peroxidase substrate solution containing diaminobenzidine tetrahydrochloride (200 µg/mL; Sigma, St. Louis, MO) and H₂O₂ (0.006%) at room temperature.

Measurement of GST activity

Tissues were homogenized in 100 mmol/L potassium phosphate buffer (pH 6.5), sonicated for 20 s, and centrifuged at 15,000 rpm for 30 min at 4 C. The supernatant was used as the cell lysate. The assay for GST activity was based on the conjugation reaction between 1-chloro-2,4-dinitrobenzene (Sigma) and glutathione (Sigma) in the cell lysate, according to the method of Habig et al. (22). GST activity was monitored at 340 nm at 25 C and expressed as nmol 1-chloro-2,4-dinitrobenzene conjugated per min/mg protein.

Cell culture

The human adrenocortical cell line H295R (American Type Culture Collection, Manassas, VA; CRL-2128) was cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 0.00625 mg/mL insulin, 0.00625 mg/mL transferrin, 6.25 ng/mL selenium, 1.25 mg/mL BSA, and 0.00535 mg/mL linoleic acid [in the form of ITS+1 liquid medium supplement (Sigma, St. Louis, MO)] and 2.5% Nu-Serum I (Collaborative Biomedical Products, Becton Dickinson and Co., Bedford, MA).

Cell viability assay

Cells (5 x 10³/well) were plated in a Falcon 96-well plate (Becton Dickinson and Co., Franklin Lakes, NJ). After 24 h, the cells were treated, or not, with various concentrations of a specific GST inhibitor, ethacrynic acid (EA; Sigma), and cultured from 1–8 days (23, 24). Cell viability was determined by a cell counting kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. The data were expressed as the mean ± SD (n = 8).

Statistical analysis

Statistical analysis was carried out using one-way ANOVA, followed by Fisher’s protected least significant difference analysis.

	Results

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After subtractive hybridization, several cDNAs, selectively up-regulated in the adenoma, were identified by the screening with Southern blotting. Cloning of the cDNAs and subsequent sequence analysis revealed that most of the clones were independent, suggesting the efficacy of subtractive hybridization. BLAST search revealed that the nucleotide sequence of one clone of 456 bp was completely identical to the region from 321–776 bp of GSTA1 cDNA (GenBank accession no. S49975). The flanking RsaI site at the 3'-end lies in GST A1 cDNA, whereas that at the 5'-end resides in the adaptor oligonucleotide used for cDNA synthesis. In the screening experiment, as described in Materials and Methods, GSTA1 showed the maximum difference in the expression level between the adenoma and the adjacent atrophic tissue compared with the other clones identified. In addition, GSTA1 was identified to be overexpressed in the adenoma in two independent screenings, and 4% of the clones screened overall were identified as GSTA1. As such we tried to characterize the expression of GSTA1 in adrenocortical adenomas.

To check for the authenticity of the preferential expression of GSTA1 in the adenoma we performed Northern blot analysis. As shown in Fig. 1A, GSTA1 mRNA was detected as a single band of 0.8 kb, which corresponds to the previous report (25). In 11 patients studied, GSTA1 mRNA was abundantly expressed in the adenoma, whereas its expression was either very low or undetectable in the adjacent atrophied nontumorous gland. In contrast, the expression of the housekeeping gene GAPDH was similar in these two tissues. We also analyzed the expression levels of other GST mRNAs, namely GSTM1 and GSTP1. However, we found no difference in their expressions between the adenoma and the adjacent gland (data not shown).

View larger version (47K): [in this window] [in a new window]   Figure 1. GSTA1 is expressed more in the adenoma than in the adjacent atrophied nontumorous adrenal gland at mRNA and protein levels. A, Expression of GSTA1 and GAPDH mRNAs in the adenoma and adjacent normal adrenal gland was determined by Northern blot analysis as described in Materials and Methods. The numbers at the top correspond to the patient numbers in Table 1. B, Expression of GSTA1 protein in the adenoma and adjacent normal adrenal gland was determined by Western blot analysis using anti-GSTA1-antibody as described in Materials and Methods. The numbers at the right denote the molecular mass markers. The numbers at the top correspond to the patient numbers in Table 1. C, Formalin-fixed paraffin-embedded section containing both the adenoma (T) and the adjacent adrenal gland (N) was stained with anti-GSTA1-antibody as described in Materials and Methods. The middle panel shows x100 and the lowest panel shows x1000 magnification views. T, Adenoma; N, adjacent atrophied nontumorous gland.

To strengthen our observation from Western blotting further, we performed immunohistochemistry on formalin-fixed paraffin-embedded sections using anti-GSTA1 antibody. The uppermost panel in Fig. 1C shows a macroscopic view of a section containing both the adenoma (T) and the adjacent atrophied nontumorous adrenal gland (N). Uniform and low level expression of GSTA1 could be detected in the adjacent atrophied nontumorous gland in all layers (Fig. 1C, middle right panel). No immunoreactivity was detected in the capsule. In the adenoma, however, GSTA1 was abundantly expressed in the tumor cells (Fig. 1C, middle left panel). The expression level was not uniform in all cells, and some variations were observed among the cell populations. Views at higher magnification (x1000) clearly showed the cytoplasmic distribution of GSTA1 (lowest panel; arrows) and confirmed the abundant expression in the adenoma.

We measured GST activity in the cell lysate from adenomas and adjacent atrophied nontumorous adrenal glands from five patients. As shown in Table 3, GST activity was 1.7- to 3.45-fold higher in the adenoma than in the adjacent atrophied gland (P < 0.0001).

View larger version (28K): [in this window] [in a new window]   Figure 2. GSTs are necessary for the growth of human adrenocortical cells. H295R human adrenocortical cells were cultured in the absence or presence of the indicated concentration of EA for 1–8 days. Cell growth was determined as described in Materials and Methods. The data represent the mean ± SD (n = 8). *, Significant differences from day 0 (P < 0.0001).

	Discussion

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GSTs are implicated in cell proliferation. GSTs are identified in proliferating fetal tissues and in the proliferative zones of many mature epithelia, including the basal layer of the cervix and crypts of colon (26, 27). Inhibition of GSTs by EA resulted in prevention of growth in human myelomonocytic and rat C6 astroglioma cell lines (28, 29). Proliferation of human Jurkat T cells was inhibited by treatment with EA, and the cells underwent apoptotic change (35). In accordance with this finding, overexpression of GST A4–4 prevented apoptosis of K562 human erythroleukemia cells (36). In this study we also observed that inhibition of GSTs by EA impaired the growth of human adrenocortical cells. At low concentrations, EA had a cytostatic effect. At high concentrations, it predominantly showed a cytotoxic effect. This could be due to nonspecific effect of EA, because it has been shown that EA at higher concentrations inhibits diverse enzymes involved in cell metabolism, including sodium/potassium-dependent adenosine triphosphatase, glyceraldehyde phosphate, lactate, succinate, malate, and -ketoglutarate dehydrogenases (37, 38). The abundant expression of GSTA1 in the adenoma and the results of the study using adrenocortical cells indicate that GSTs might be required for the proliferation, survival, and sustenance of adrenocortical adenoma cells.

Several studies have shown that GSTs, especially the class, are abundantly expressed in human adrenal cortex (39, 40, 41). GSTs are also highly expressed in other steroidogenic tissues, e.g. ovaries, testes, and placenta (42). In the testes and ovaries, the pattern of GST (ligandin) expression during the postpartum development of rat was remarkably similar to the pattern of serum levels of the corresponding steroid hormones, namely testosterone and progesterone (43). In addition, the ligandin concentration in the developing rat adrenal showed similar concordance with the serum corticosterone levels (44). These findings indicate that GSTs might be related to the process of steroidogenesis. GSTs are reported to have ⁵-3-ketosteroid isomerase activity. In the porcine ovary, GSTA1 activity copurified with the isomerase activity in affinity column chromatography (45). Thus, GSTs might be directly involved in steroid synthesis. GSTs also act as ligandins, binding to steroid hormone metabolites. Recently, the C-terminal helix 9 of GST A1–1 has been shown to serve the ligandin function (46). In addition, during the process of steroidogenesis, lipid peroxides and other free oxygen radicals are generated (47). GSTs are thus required to detoxify these molecules in tissues actively engaged in steroidogenesis. In this respect it is likely that GSTA1 expressed in the adenoma is involved in the increased synthesis of glucocorticoids. Its reduced expression in the adjacent normal gland may support this hypothesis.

The major class of GSTs that is overexpressed in a variety of malignancies is GSTP1 (15). More importantly, GSTP1 is not expressed in normal hepatocytes, but is highly expressed during the progression of hepatocarcinogenesis (15). In addition, it has been shown that the expression of GSTs, especially those belonging to class µ, is modulated by ACTH in rats and in the mouse adrenocortical cell line Y1 (48, 49). We, therefore, examined whether GSTs belonging to these classes, namely GSTP1 and GSTM1, show differential expression in the adenoma. Interestingly, we could not detect any difference in their expression levels between the adenoma and the adjacent normal tissue (data not shown). Thus, among GSTs, it is GSTA1 that has preferential association with adrenocortical adenoma.

In this study we show for the first time that GSTA1 is overexpressed in benign functioning adrenocortical adenoma. We also show that inhibition of GSTs interferes with the growth of human adrenocortical cells in vitro. Thus, GSTs might be directly involved in the pathogenesis of adenoma formation by facilitating cell proliferation and steroidogenesis, or they might be overexpressed as an adaptive mechanism for the cells in response to excess production of cortisol. Further studies are needed to clarify these issues.

Received July 17, 2000.

Revised November 15, 2000.

Accepted December 22, 2000.

	References

Top Abstract Introduction Case Subjects Materials and Methods Results Discussion References

 

1. Simpson ER, Waterman MR. 1983 Regulation by ACTH of steroid hormone biosynthesis in the adrenal cortex. Can J Biochem Cell Biol. 61:692–707.[Medline]
2. Gill GN. 1972 Mechanism of ACTH action. Metabolism. 21:571–588.[CrossRef][Medline]
3. Mountjoy KG, Robbins LS, Mortrud MT, Cone RD. 1992 The cloning of a family of genes that encode the melanocortin receptors. Science. 257:1248–1251.[Abstract/Free Full Text]
4. Latronico AC, Reincke M, Mendonca BB, et al. 1995 No evidence for oncogenic mutations in the adrenocorticotropin receptor gene in human adrenocortical neoplasms. J Clin Endocrinol Metab. 80:875–877.[Abstract]
5. Light K, Jenkins PJ, Weber A, et al. 1995 Are activating mutations of the adrenocorticotropin receptor involved in adrenal cortical neoplasia? Life Sci. 56:1523–1527.[CrossRef][Medline]
6. Sarkar D, Imai T, Shibata A, Funahashai H, Seo H. Expression of adrenocorticotropin receptor (ACTH-R) gene in adrenocortical adenomas from patients with Cushing’s syndrome: possible contribution for the autonomous production of cortisol. Proc of the 82nd Annual Meet of The Endocrine Soc., Toronto, Canada, 2000; p. 458.
7. Reincke M, Karl M, Travis W, Chrousos GP. 1993 No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms. J Clin Endocrinol Metab. 77:1419–1422.[Abstract]
8. Lin SR, Lee YJ, Tsai JH. 1994 Mutations of the p53 gene in human functional adrenal neoplasms. J Clin Endocrinol Metab. 78:483–491.[Abstract]
9. Reincke M, Wachenfeld C, Mora P, et al. 1996 p53 mutations in adrenal tumors: Caucasian patients do not show the exon 4 "hot spot" found in Taiwan. J Clin Endocrinol Metab. 81:3636–3638.[Abstract]
10. Reincke M. 1998 Mutations in adrenocortical tumors. Horm Metab Res. 30:447–455.[Medline]
11. Munro LM, Kennedy A, McNicol AM. 1999 The expression of inhibin/activin subunits in the human adrenal cortex and its tumours. J Endocrinol. 161:341–347.[Abstract]
12. Nishi Y, Haji M, Takayanagi R, Yanase T, Ikuyama S, Nawata H. 1995 In vivo and in vitro evidence for the production of inhibin-like immunoreactivity in human adrenocortical adenomas and normal adrenal glands: relatively high secretion from adenomas manifesting Cushing’s syndrome. Eur J Endocrinol. 132:292–299.[Abstract/Free Full Text]
13. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA. 1995 An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23:1087–1088.[Free Full Text]
14. Diatchenko L, Lau YF, Campbell AP, et al. 1996 Suppression subtractive hybridization: a method for generating differentially regulated or tissuespecific cDNA probes and libraries. Proc Natl Acad Sci USA. 93:6025–6030.[Abstract/Free Full Text]
15. Hayes JD, Pulford DJ. 1995 The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 30:445–600.[Medline]
16. Hayes JD, Strange RC. 1995 Potential contribution of the glutathione S-transferase supergene family to resistance to oxidative stress. Free Rad Res. 22:193–207.[Medline]
17. Mannervik B, Danielson UH. 1988 Glutathione transferases: structure and catalytic activity. Crit Rev Biochem. 23:283–337.[Medline]
18. Imai T, Seo H, Murata Y, et al. 1991 Dexamethasone-nonsuppressible cortisol in two cases with aldosterone-producing adenoma. J Clin Endocrinol Metab. 72:575–581.[Abstract/Free Full Text]
19. Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor: Cold Spring Harbor Laboratory.
20. Sarkar D, Kambe F, Hirata A, Iseki A, Ohmori S, Seo H. 1999 Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin. FEBS Lett. 462:430–434.[CrossRef][Medline]
21. Miyazaki T, Sato M, Murata Y, Maeda K, Seo H. 1995 Factor(s) present in sera from patients on long-term hemodialysis increase(s) mRNAs for collagenase and stromelysin in synovial cells. Am J Nephrol. 15:48–56.[Medline]
22. Habig WH, Pabst MJ, Jakoby WB. 1974 Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 249:7130–7139.[Abstract/Free Full Text]
23. Ahokas JT, Davies C, Ravenscroft PJ, Emmerson BT. 1984 Inhibition of soluble glutathione S-transferase by diuretic drugs. Biochem Pharmacol. 33:1929–1932.[CrossRef][Medline]
24. Ploemen JH, van OB, van BP. 1990 Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate. Biochem Pharmacol. 40:1631–1635.[CrossRef][Medline]
25. Eickelmann P, Morel F, Schulz WA, Sies H. 1995 Turnover of glutathione S-transferase alpha mRNAs is accelerated by 12-O-tetradecanoyl phorbol-13-acetate in human hepatoma and colon carcinoma cell lines. Eur J Biochem. 229:21–26.[Medline]
26. Carder PJ, Al-Nafussi A, Rahilly M, Lauder J, Harrison DJ. 1990 Glutathione S-transferase detoxication enzymes in cervical neoplasia. J Pathol. 162:303–308.[CrossRef][Medline]
27. Strange RC, Fryer AA, Hiley C, Bell J, Cossar D, Hume R. 1990 Developmental expression of GST in human tissues. In: Hayes JD, Pickett CB, Mantle TJ, eds. Glutathione S-transferases and drug resistance. Oxford: Taylor and Francis; 262–271.
28. Sato Y, Fujii S, Fujii Y, Kaneko T. 1990 Antiproliferative effects of glutathione S-transferase inhibitors on the K562 cell line. Biochem Pharmacol. 39:1263–1266.[CrossRef][Medline]
29. Senjo M, Ishibashi T. 1988 Possible involvement of glutathione S-transferases in the cell growth of C6 astroglioma cells. J Neurochem. 50:163–166.[CrossRef][Medline]
30. Harrison DJ, Kharbanda R, Bishop D, McLelland LI, Hayes JD. 1989 Glutathione S-transferase isoenzymes in human renal carcinoma demonstrated by immunohistochemistry. Carcinogenesis. 10:1257–1260.[Abstract/Free Full Text]
31. Di Ilio C, Del Boccio G, Aceto A, Casaccia R, Mucilli F, Federici G. 1988 Elevation of glutathione transferase activity in human lung tumor. Carcinogenesis. 9:335–340.[Abstract/Free Full Text]
32. Moscow JA, Townsend AJ, Goldsmith ME, et al. 1988 Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Natl Acad Sci USA. 85:6518–6522.[Abstract/Free Full Text]
33. Moorghen M, Cairns J, Forrester LM, et al. 1991 Enhanced expression of glutathione S-transferases in colorectal carcinoma compared to non-neoplastic mucosa. Carcinogenesis. 12:13–17.[Abstract/Free Full Text]
34. Kitahara A, Satoh K, Nishimura K, et al. 1984 Changes in molecular forms of rat hepatic glutathione S-transferase during chemical hepatocarcinogenesis. Cancer Res. 44:2698–2703.[Abstract/Free Full Text]
35. McCaughan FM, Brown AL, Harrison DJ. 1994 The effect of inhibition of glutathione S-transferase P on the growth of the Jurkat human T cell line. J Pathol. 172:357–362.[CrossRef][Medline]
36. Cheng JZ, Singhal SS, Saini M, et al. 1999 Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells. Arch Biochem Biophys. 372:29–36.[CrossRef][Medline]
37. Duggan DE, Noll RM. 1965 Effects of ethacrynic acid and cardiac glycosides upon a renal membrane adenosine triphosphatase of renal cortex. Arch Biochem Biophys. 109:388–396.[CrossRef]
38. Beyer KH, Baer JE, Michaelson JK, Russo HF. 1965 Renotropic characteristics of ethacrynic acid: a phenoxyacetic saluretic-diuretic agent. J Pharmacol Exp Ther. 147:1–22.[Abstract/Free Full Text]
39. Campbell JA, Corrigall AV, Guy A, Kirsch RE. 1991 Immunohistologic localization of , µ, and class glutathione S-transferases in human tissues. Cancer. 67:1608–1613.[CrossRef][Medline]
40. Meikle I, Hayes JD, Walker SW. 1992 Expression of an abundant -class glutathione S-transferase in bovine and human adrenal cortex tissues. J Endocrinol. 132:83–92.[Abstract/Free Full Text]
41. Sundberg AG, Nilsson R, Appelkvist EL, Dallner G. 1993 Immunohistochemical localization of alpha and pi class glutathione transferases in normal human tissues. Pharmacol Toxicol. 72:321–331.[Medline]
42. Rabahi F, Brule S, Sirois J, Beckers JF, Silversides DW, Lussier JG. 1999 High expression of bovine alpha glutathione S-transferase (GSTA1, GSTA2) subunits is mainly associated with steroidogenically active cells and regulated by gonadotropins in bovine ovarian follicles. Endocrinology. 140:3507–3517.[Abstract/Free Full Text]
43. Eidne KA, Bass NM, Sherman M, Millar RP, Kirsch RE. 1984 Ligandin concentrations in the steroidogenic tissues of the rat during development. Biochim Biophys Acta. 801:424–428.[Medline]
44. Vallette G, Delorme J, Benassayag C, et al. 1982 Developmental patterns of levels of corticosterone and of corticosterone binding in the serum of female rats: effects of ovariectomy and adrenalectomy. Acta Endocrinol (Copenh). 101:442–451.[Abstract/Free Full Text]
45. Keira M, Nishihira J, Ishibashi T, Tanaka T, Fujimoto S. 1994 Identification of a molecular species in porcine ovarian luteal glutathione S-transferase and its hormonal regulation by pituitary gonadotropins. Arch Biochem Biophys. 308:126–132.[CrossRef][Medline]
46. Dirr HW, Wallace LA. 1999 Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1–1. Biochemistry. 38:15631–15640.[CrossRef][Medline]
47. Hornsby PJ, Crivello JF. 1983 The role of lipid peroxidation and biological antioxidants in the function of the adrenal cortex. Part 2. Mol Cell Endocrinol. 30:123–147.[CrossRef][Medline]
48. Mankowitz L, Castro VM, Mannervik B, Rydstrom J, DePierre JW. 1990 Increase in the amount of glutathione transferase 4–4 in the rat adrenal gland after hypophysectomy and down-regulation by subsequent treatment with adrenocorticotrophic hormone. Biochem J. 265:147–154.[Medline]
49. Mankowitz L, Staffas L, Bakke M, Lund J. 1995 Adrenocorticotrophic-hormone-dependent regulation of a µ-class glutathione transferase in mouse adrenocortical cells. Biochem J. 305:111–118.

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