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Katsuhiko Yoshimoto, Chisato Tanaka, Shozo Yamada, Takehiko Kimura, Hiroyuki Iwahana, Toshiaki Sano and Mitsuo Itakura

Abstract

The p16INK4A and p15INK4B genes on chromosome 9p21 encode the p16 and p15 inhibitors of cyclin D/cyclin-dependent kinase 4 complexes respectively. Mutations and deletions of the p16INK4A gene have been found in melanomas and many other types of tumors. To assess the role of the p16INK4A and p15INK4B genes in tumorigenesis of the pituitary gland, 31 sporadic pituitary adenomas and 2 pituitary adenomas in familial acrogigantism were examined for loss of heterozygosity on 9p21–22 and screened for mutations in the p16INK4A and p15INK4B genes. To identify pituitary adenomas which had lost which had lost 9p21–22, pituitary adenomas were genotyped with markers flanking the p16INK4A and p15INK4B loci. The frequency of mutations in coding regions of the p16INK4A and the p15INK4B genes in pituitary adenomas was determined with polymerase chain reation-single strand conformation polymorphism analysis and sequencing of variants. Of the 33 pituitary adenomas, two revealed loss of 9p21–22 sequences, but none of them had tumor-specific mutations. We conclude that mutations of the p16INK4A and p15INK4B genes are not required for tumorigenesis of the pituitary gland.

European Journal of Endocrinology 136 74–80

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Emiko Hosoi, Yutaka Yokogoshi, Eiji Hosoi, Hidetaka Horie, Toshiaki Sano, Shozo Yamada and Shiro Saito

We investigated the prevalence of Gs α gene mutations in growth hormone (GH) secreting pituitary adenomas from Japanese patients with acromegaly. Forty-five GH-secreting adenomas were examined for the presence of point mutations in codons 201 or 227 of the Gs α gene using the polymerase chain reaction-direct sequencing method and deoxyribonucleic acid extracted from paraffin-embedded tumor specimens. Mutation of codon 227 of the Gs α gene was not observed in any of the tumors, but a mis-sense mutation of codon 201 was identified in two tumors (4.4%). One lesion was a densely granulated GH cell adenoma in a patient with adenomatous goiter and breast cancer. The other was a mixed GH cell-prolactin cell adenoma in a patient with multiple endocrine neoplasia type 1 associated with parathyroid hyperplasia and a pancreatic islet cell tumor. The Gs α gene detected in parathyroid tissue and pancreatic tumor tissue was of the wild type in this second patient, and the mutation was specific to the pituitary tumor. These results suggest that point mutations of codons 201 or 227 of the Gs α gene may not be important mediators of oncogenesis for GH-secreting pituitary adenomas in Japan.

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Junko Igarashi-Migitaka, Akira Takeshita, Noriyuki Koibuchi, Shozo Yamada, Ritsuko Ohtani-Kaneko and Kazuaki Hirata

Objective: Androgens are critical for the development and maintenance of male sexual characteristics. Their action is mediated through the androgen receptor (AR). Ligand-bound AR interacts with coactivator proteins that mediate transcriptional activation. Such coactivators include three members of the 160 kDa proteins (p160s): SRC-1, TIF2/GRIP1, and p/CIP/RAC3/ACTR/AIB1/TRAM-1. The aim of this study was to investigate the expression of the three p160 coactivators and their association with AR in testis and epididymis.

Methods: We determined the localization of these three p160 coactivators in immature and mature rat testis, and epididymis by immunohistochemistry using the specific monoclonal antibodies. We also performed double immunofluorescence staining to examine whether p160s are colocalized with AR in these tissues.

Results: In seminiferous tubules of mature rat testis, SRC-1 and TRAM-1 immunoreactivity was found predominantly in spermatogonia and spermatocytes. In contrast, TIF2 was expressed predominantly in Sertoli cells. AR was coexpressed with TIF2 in this cell type. In immature rat testis, however, all three coactivators were expressed in both germ cells and Sertoli cells. In the epididymis, SRC-1 and TIF2 immunoreactivities were localized in nuclei of epithelial cells. However, TRAM-1 immunostaining was observed in the luminal portion of the cytoplasm with greater intensity than in the nucleus, especially in the caput epididymidis.

Conclusions: The cell-type-specific expression of p160 coactivators suggests specific roles in male reproductive organs. Further, the strong cytoplasmic localization of TRAM-1 protein in epithelial cells of epididymis suggests that TRAM-1 may have additional role(s) in transcriptional regulation.

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Yuka Kinoshita, Hiroko Fujii, Akira Takeshita, Manabu Taguchi, Megumi Miyakawa, Kenich Oyama, Shozo Yamada and Yasuhiro Takeuchi

Objective

Impaired glucose metabolism is common in acromegaly, but it is not clear how glucose metabolism is impaired or what predicts its restoration after cure of the disease. To identify factors involved in the impairment of glucose metabolism in acromegaly, we evaluated clinical parameters before and immediately after surgical cure of the disease.

Design and methods

We retrospectively analyzed clinical data of 92 consecutive Japanese patients with acromegaly who underwent successful pituitary surgery. Patients who had received medical therapy for acromegaly or insulin treatment for diabetes were excluded. We evaluated insulin resistance (IR) and pancreatic β-cell function in addition to GH and IGF1 levels before and after surgery.

Results

In this study of Japanese patients with acromegaly, average body mass index (BMI) was 23.4, and no patient had a BMI>30. IR was involved in the impairment of glucose metabolism, which was restored upon surgical cure of acromegaly if β-cell function was preserved. Insufficient β-cell function did not improve after normalization of GH/IGF1 and was associated with impaired glucose metabolism before and after surgery. Results of receiver operating characteristic analysis of preoperative clinical parameters suggest that insulinogenic index (IGI) >0.50 best predicts restoration of normal glucose metabolism upon cure of acromegaly in Japanese patients.

Conclusions

IR impairs glucose metabolism in acromegaly. Once β-cell function is impaired, abnormal glucose metabolism persists even after cure of acromegaly. IGI>0.50 indicates that β-cell function is preserved in non-obese Japanese patients with acromegaly.

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Yuji Tani, Naoko Inoshita, Toru Sugiyama, Masako Kato, Shozo Yamada, Masayoshi Shichiri and Yukio Hirata

Objective

Cushing's disease (CD) is usually caused by ACTH-secreting pituitary microadenomas, while silent corticotroph adenomas (SCA) are macroadenomas without Cushingoid features. However, the molecular mechanism(s) underlying their different tumor growth remains unknown. The aim of the current study was to evaluate and compare the gene expression profile of cell cycle regulators and cell growth-related transcription factors in CD, SCA, and non-functioning adenomas (NFA).

Design and methods

Tumor tissue specimens resected from 43 pituitary tumors were studied: CD (n=10), SCA (n=11), and NFA (n=22). The absolute transcript numbers of the following genes were quantified with real-time quantitative PCR assays: CDKN2A (or p16 INK4a), cyclin family (A1, B1, D1, and E1), E2F1, RB1, BUB1, BUBR1, ETS1, and ETS2. Protein expressions of p16 and cyclin D1 were semi-quantitatively evaluated by immunohistochemical study.

Results and conclusion

CDKN2A gene expression was about fourfold greater in CD than in SCA and NFA. The gene expressions of cyclins D1, E1, and B1, but not of A1, in CD were significantly suppressed than those in NFA. Cyclin D1 gene expression positively correlated with cyclins B1 and E1. The gene expressions of E2F1, RB1, BUB1, BUBR1, ETS1, and ETS2 did not differ between each group. Positive immunostaining for p16 and negative immunostaining for cyclin D1 were more frequent in CD than in NFA; there were positive correlations between mRNA and protein expressions of p16 and cyclin D1. Thus, it is suggested that upregulated CDKN2A with the concomitant downregulated cyclin gene family is partly involved in the small size of ACTH-secreting adenoma.

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Shozo Yamada, Noriaki Fukuhara, Hiroshi Nishioka, Akira Takeshita, Hisanori Suzuki, Megumi Miyakawa and Yasuhiro Takeuchi

Objective

The aim of this study was to determine the frequency and characteristics of severe GH deficiency (sGHD) in patients after treatment of acromegaly by surgery alone.

Design and methods

One hundred and eighty-six patients fulfilling the criteria for cure of acromegaly were examined by GH-releasing peptide-2 stimulation test or arginine stimulation test as well as oral glucose tolerance test (GTT). In addition, the Japanese adult hypopituitarism questionnaire was completed to determine the quality of life (QoL).

Results

sGHD was found in 17 patients (9.1%; the GH-deficient group), and not found in 169 patients (90.9%; the GH-sufficient group). There were no significant differences in preoperative serum GH levels, IGF1 levels, incidence of hyperprolactinemia, tumor volumes, or incidence of microadenoma between the two groups. Upon follow-up examination, IGF1 levels and Z-scores of IGF1 levels were significantly lower in the GH-deficient group than in the GH-sufficient group, whereas neither basal GH levels nor nadir GH levels during 75 g GTT were significantly different between the two groups. Moreover, sGHD patients had a substantially higher incidence of multiple pituitary failures (17.6 vs 2.4%) and dyslipidemia (60 vs 16.2%). sGHD patients had a substantially poorer condition-related QoL.

Conclusions

This is the first large-scale, single-center, clinical study to evaluate sGHD in patients after cure of acromegaly by surgery alone. This study found that sGHD occurred in ∼9% of patients and assessment of GHD by stimulation tests is critical after successful treatment of acromegaly by surgery.

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Satoko Shimazu, Akira Shimatsu, Shozo Yamada, Naoko Inoshita, Yuko Nagamura, Takeshi Usui and Toshihiko Tsukada

Objective

Dopamine agonists normalize prolactin (PRL) levels and reduce tumour size in responsive prolactinoma. However, several cases have shown resistance to dopamine agonists upon initial treatment. Infrequently, prolactinoma initially responds, but then becomes refractory to prolonged treatment (secondary resistance). We investigated the possible mechanisms of resistance to dopamine agonists.

Subjects and methods

Twelve cases of prolactinoma were surgically resected and classified according to the responsiveness of PRL levels and tumour size to dopamine agonists: good responders (n=5), poor responders (n=5), or secondary resistance (n=2). We examined the expression of dopamine D2 receptor (D2R) isoform (short: D2S and long: D2L) mRNA and protein. We investigated DNA methylation patterns in the promoter region of the DRD2 gene.

Results

The predominant D2R isoform expressed in prolactinoma was D2L. Levels of D2L mRNA were significantly lower in secondary resistance and poor responders than in good responders. Expression of D2R protein was variable among cases. Almost no CpG sites of the DRD2 gene promoter region were methylated.

Conclusion

Resistance of prolactinoma to dopamine agonists is correlated with a reduction in D2L isoform mRNA levels. Silencing of the DRD2 gene by methylation in the promoter region is unlikely to play a role in dopamine agonist resistance in prolactinoma.

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Toru Tateno, Hajime Izumiyama, Masaru Doi, Takanobu Yoshimoto, Masayoshi Shichiri, Naoko Inoshita, Kenichi Oyama, Shozo Yamada and Yukio Hirata

Objective

Differential expression of several genes between ACTH-secreting pituitary tumors causing Cushing' disease (CD), silent corticotroph adenoma (SCA), and non-functioning pituitary tumors (NFT) was investigated.

Design and methods

We used tissue specimens from 35 pituitary tumors (12 CD, 8 SCA, and 15 NFT). Steady-state mRNA levels of the genes related to proopiomelanocortin (POMC) transcription, synthesis, processing, and secretion, such as neurogenic differentiation 1 (NeuroD1), T-box 19 (Tpit), corticotropin releasing hormone receptor (CRHR), vasopressin receptor 1b (V1bR), prohormone convertase (PC) 1/3 and PC2, 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2, glucocorticoid receptor α (GRα), annexin A1, histone deacetylase 2 (HDAC2), and BRM/SWI2-related gene 1, were determined by real-time RT-PCR.

Results and conclusion

POMC and Tpit mRNA levels were greater in CD and SCA than those in NFT. NeuroD1 mRNA levels were less in CD than those in NFT, but almost comparable between SCA and NFT. PC1/3 mRNA levels were greater in CD, but less in SCA than those in NFT. PC2 mRNA levels in CD and SCA were less than those in NFT. CRHR, V1bR, and 11β-HSD2 mRNA levels in CD were greater than those in SCA and NFT. HDAC2 mRNA levels in CD and SCA were lower than those in NFT. In conclusion, our study demonstrated that the genes related to transcription, synthesis, processing, and secretion of POMC are differentially regulated in ACTH-secreting pituitary tumors causing CD and SCA compared with those in NFT. This may partly explain the development of clinically active and inactive CD.

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Akira Takeshita, Naoko Inoshita, Manabu Taguchi, Chikao Okuda, Noriaki Fukuhara, Kenichi Oyama, Kenichi Ohashi, Toshiaki Sano, Yasuhiro Takeuchi and Shozo Yamada

Context

Crooke's cell adenoma (CCA), characterized by massive Crooke's hyaline change in corticotroph adenoma, causes a rare subtype of Cushing's disease. In contrast to ordinary corticotroph adenomas, CCAs are generally aggressive and present as invasive macroadenomas, which are refractory to both surgery and radiotherapy and have a high-recurrence rate. Moreover, some patients with CCA present with distant or craniospinal metastases. Currently, there are no effective standard therapies for CCA.

Objective

We report a patient with Crooke's cell carcinoma who presented with local invasion and liver metastases, which was refractory to conventional therapeutic modalities including transsphenoidal surgery, radiosurgery, medications, and hepatic transcatheter arterial embolization. After all these treatments failed, the patient had monthly temozolomide administrations, resulting in gradual clinical improvement and biochemical data that were consistent with tumor shrinkage. In glioblastoma, low O6-methylguanine DNA methyltransferase (MGMT) expression is associated with epigenetic gene silencing and predicts a better response to temozolomide.

Methods

We thus investigated MGMT expression, immunohistochemically, in seven CCAs (five invasive macroadenomas and two invasive microadenomas) and 17 ordinary-type adenomas (OTAs; three noninvasive macroadenomas, 12 noninvasive microadenomas, and two invasive microadenomas) from patients with Cushing's disease.

Results

In seven CCAs, all five invasive macroadenomas exhibited low MGMT expression, defined as <5% nuclear MGMT staining. In 17 OTAs, only one adenoma showed low MGMT expression.

Conclusion

In Cushing's disease, invasive macroadenomas including CCA usually have low-MGMT expression. Temozolomide thus may be a new therapeutic option for invasive macroadenomas such as CCA particularly when conventional treatments are ineffective.

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Akira Sugenoya, Shinya Kobayashi, Yoshio Kasuga, Hiroyuki Masuda, Minoru Fujimori, Makoto Komatsu, Shozo Takahashi, Shiro Yokoyama, Tadahiro Shimizu, Takashi Yamada and Futoshi Iida

To clarify the intrathyroidal accumulation of TSH receptor antibody (TR-ab) produced in the thyroid, adrenalin was injected directly into the thyroid artery of patients with Graves' disease during surgery, allowing serial determination of the TR-ab levels in the thyroidal venous blood. Nine surgical patients (3M and 6F) with Graves' disease and receiving anti-thyroid drugs preoperatively for a period of one to four years were enrolled in this study. Comparison between pre- and post-treatment TR-ab levels revealed continuous increments from the pretreatment levels within 15 min of the injection in five (55.6%) of the nine patients. In three of the five patients, TR-ab levels that had been negative before adrenalin injection became positive 1 min after injection. It is assumed that the increase in the TR-ab level is due to adrenalin-induced constriction of the capillaries in the thyroid tissues, resulting in a voluminous flow of the TR-ab retained in the thyroid into the vein. These findings indicate that the TR-ab level in the peripheral blood does not necessarily reflect precisely the abnormal immunological condition in the Graves' thyroid.