Analysis of gene alterations of mitochondrial DNA D-loop regions to determine breast cancer clonality.

BACKGROUND: It has been a challenge to determine breast cancer clonality accurately. The aim of the present study was to assess methods using formalin-fixed paraffin-embedded (FFPE) tissue to differentiate new primary tumours from true recurrences that are associated with poorer prognoses and often require more aggressive treatment. METHODS: We investigated the novel method of analysing gene alterations of mitochondrial DNA D-loop region (GAMDDL) and compared it with the conventional method of analysing the X-chromosome-linked human androgen receptor (HUMARA). The FFPE sections of primary and secondary breast cancers, the non-neoplastic mammary gland, and lymph nodes were examined. RESULTS: Informative rates for HUMARA, GAMDDL, and combined analyses were 42.1%, 76.9%, and 89.5%, respectively. All of the 10 contralateral breast cancers were determined to be non-clonal. In contrast, 3 out of 8 (37.5%) of the ipsilateral secondary tumours shared a clonal origin with the primary tumour and were classified as true recurrences, whereas 4 out of 8 (50%) were classified as new primary tumours. CONCLUSION: GAMDDL analysis represents a novel and useful molecular method for examining the precise cell lineages of primary and secondary tumours, and was more accurate than HUMARA in determining clonality.

Pituitary blastoma: a unique embryonal tumor.

Pituitary blastoma, a recently described tumor of the neonatal pituitary, exhibits differentiation to Rathke epithelium and adenohypophysial cells of folliculostellate and secretory type, a reflection of arrested pituitary development and unchecked proliferation (Scheithauer et al. in Acta Neuropathol 116(6):657-666, 2008). Herein, we report the pathologic features of three additional cases, all ACTH-producing. One involved a 9-month-old male presenting with progressive right ophthalmoplegia, MRI findings of a large suprasellar mass with cavernous sinus invasion, and elevated plasma ACTH levels. The second was nonfunctioning and occurred in a 13-month-old female with right third nerve palsy. The third had been previously published as a "pituitary adenoma" in a 2-year-old female (Min et al. in Pathol Int 57(9):600-605, 2007). The subtotally resected tumors were subject to histochemical, immunohistochemical and, in two cases, ultrastructural study. Histologically, the complex tumors consisted of glands of varying from rosettes to glandular structures resembling Rathke epithelium, small undifferentiated-appearing cells (blastema), and large secretory cells. Mucin-producing goblet cells were noted in case 3. Cell proliferation was high in two cases and low in case 3. Immunoreactivity of the secretory cells included synaptophysin, chromogranin, various keratins and, to a lesser extent, ACTH and beta endorphin. MGMT immunolabeling was 40-60%. Mitotic activity was moderate to high in cases 1 and 2 and was low in case 3. The same was true for MIB-1 labeling. Germ cell markers were lacking in all cases. One tumor ultrastructurally consisted of three cell populations including (a) small, polyhedral, primitive-appearing cells (blastema) with scant cytoplasm, abundant glycogen and few organelles, (b) folliculostellate cells and (c) large corticotroph cells containing rough endoplasmic reticulum, golgi membranes, spherical, 150-400 nm secretory granules and occasional perinuclear, intermediate filament bundles. A second example (case 3) lacked a blastema and glandular component. The clinical and morphologic features of our three cases were those of pituitary blastoma. The finding of cellular elements of adenohypophysial development is consistent with a diagnosis of pituitary blastoma and aligns it with blastomas of other organs. It also suggests an underlying specific genetic abnormality. Marked variations in cellular proliferative activity suggest blastomas occur in low- and higher-grade form. Variable MGMT reactivity suggests an incomplete response to temozolomide therapy. Literature regarding similar morphologically complex, infantile, Cushing disease-associated lesions is briefly reviewed.

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio > 0.31 indicates an enlarged pituitary, whereas a P/B ratio ≤ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ± SD Ki-67 labeling index (LI) was 0.55% ± 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ± 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ± 2.2% and 10.9% ± 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P < 0.01 and P < 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P < 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r = 0.820, P < 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r = 0.560, P = 0.03) and the MCM7 LI (r = 0.854, P < 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors.

A case of oncocytic carcinoma of the endometrium.

We report an unusual case of endometrial adenocarcinoma in a 80-year-old woman who underwent mastectomy for breast cancer at 68 years of age. The tumor diffusely involved the entire thickness of the myometrium. Atypical cells throughout the tumor contained abundant oxiphilic cytoplasm and were arranged in a solid or solid-tubular nests in a focal papillary manner. Components of the carcinoma were focally observed in situ. The tumor was classified according to the International Federation of Gynecology and Obstetrics (FIGO) as grade 2 and stage IIIa. Immunohistochemistry revealed that the tumor cells were positive for p53 but negative for vimentin, estrogen, and progesterone receptors, GCDFP-15, and mammaglobin. They were positive for antimitochondrial antigen and thyroid transcription factor-1. The Ki-67 labeling index was approximately 50%. Immunostaining revealed endometrial oncocytic carcinoma. Distinguishing between primary uterine neoplasm and carcinoma caused by metastasis of breast cancer appears important.

Immunohistochemical expression of somatostatin type 2A receptor in neuroendocrine carcinoma of uterine cervix.

INTRODUCTION: Somatostatin and its analogues inhibit the growth of various kinds of endocrine and exocrine cells via the somatostatin receptor (SSTR). Expression of SSTR2A has been reported in many well-differentiated neuroendocrine tumors and small numbers of poorly differentiated neuroendocrine carcinomas. SSTR2A-positive neuroendocrine tumors may be treatable by SS analogues. Uterine cervical neuroendocrine carcinoma is generally associated with poor prognosis. MATERIALS AND METHODS: We examined the expression of SSTR2A and other neuroendocrine markers [neural cell adhesion molecule (NCAM), chromogranin A, and synaptophysin] by immunohistochemical methods in seven neuroendocrine tumors of the uterine cervix: five small cell carcinomas and two large cell neuroendocrine carcinomas. RESULTS: SSTR2A was expressed in one small cell carcinoma and two neuroendocrine large cell carcinomas. The cell membrane of cells in these carcinomas showed strong immunohistochemical reactivity for SSTR2A. SSTR2A-positive cases are also frequently positive for the expression of chromogranin A. Treatment with SSTR2A may be effective for endocrine tumors. The results indicate the usefulness of SSTR2A analogs for the treatment of some uterine neuroendocrine carcinomas.

Intracisternal neurinoma of the C1 posterior root.

We report a rare intracisternal C1 posterior root neurinoma in a 35-year-old man without neurofibromatosis who presented with headache, nuchal pain, bilateral motor weakness of the upper extremities, and numbness in the right distal upper extremity. CT and MRI study showed a 20-mm intracisternal lesion at the foramen magnum. At surgery, there was an anastomosis between the C1 posterior root and a spinal accessory nerve at the site of the tumor; the root from the collateral sulcus of this C1 root was absent. Postoperatively, the patient remains free of symptoms. Foramen magnum neurinomas have been described as accessory nerve tumors. We present new anatomical consideration regarding this lesion.

Loss of heterozygosity alterations associated with progesterone therapy in endometrial hyperplasia and adenocarcinoma.

Loss of heterozygosity (LOH) was analyzed in four patients with endometrial hyperplasia (EH) with atypia (two patients) and without atypia (two patients) and in five patients with endometrial adenocarcinoma (EAC) to clarify the clinicopathologic relationship between genetic alterations and hormone therapy. Each patient was initially administered high-dose medroxyprogesterone acetate (MPA) as a uterine-sparing treatment. The five microsatellite markers used to analyze LOH were at chromosomal loci 8p22.1, 8p21, 8p21.3, 8p22, and 8p22. DNA was extracted from paraffin-embedded sections before, during, and after MPA therapy using laser capture microdissection. As a result, LOH was more frequently detected after MPA therapy (overall ratios were 16, 17, and 29% before, during, and after MPA therapy, respectively). LOH is more easily detected in EH loci than in EAC loci before MPA. For EAC, initial LOH detection on chromosome 8 may be related to an incomplete response to MPA, but negative LOH does not guarantee a favorable treatment outcome. For EH or atypical endometrial hyperplasia, it is unknown whether LOH alteration associated with MPA therapy is related to atypia of the disease.

Exogenous expression of Pit-1 in AtT-20 corticotropic cells induces endogenous growth hormone gene transcription.

The pituitary-specific POU-homeodomain transcription factor, Pit-1, is known to regulate the expression of the GH gene in somatotropes, prolactin (PRL) in lactotropes, and TSH in thyrotropes. It is not normally expressed in corticotropes or gonadotropes. We addressed the question of whether exogenous Pit-1 was sufficient to induce ectopic transcription of the GH gene in the corticotropic cell line, AtT-20, or the gonadotropic cell line, alpha T3-1. A fusion gene composed of enhanced green fluorescent protein gene and human Pit-1 cDNA was transfected into AtT-20 and alpha T3-1 cells. The endogenous mouse GH mRNA was induced in three of nine AtT-20 cell lines and one of three alpha T3-1 cell lines containing the fusion gene. A small amount of GH protein was also detected in these cell lines. These data indicate that transfected Pit-1 is capable of inducing transcription of the GH gene in AtT-20 cells and alpha T3-1 cells. These data also suggest that synergistic co-factors might be required to transcribe the GH gene effectively for translation into GH protein. Furthermore, our findings support the hypothesis that the function of anterior pituitary cells is determined by the combinatorial action of specific transcription factors.

Immunohistochemical analysis of RCAS1 in human pituitary adenomas.

It has been reported that RCAS1 (receptor-binding cancer antigen expressed on SiSO cells) acts as a ligand for a receptor present on normal peripheral lymphocytes and induces apoptotic cell death. It is expressed in uterine and ovarian carcinomas, especially in invasive cancers. This immunohistochemical study is aimed to elucidate the expression of RCAS1 in human pituitary adenomas in order to clarify its role in their proliferative regulation and invasiveness. Five normal pituitary glands, 50 human pituitary adenomas, and one malignant glioma were subjected to immunohistochemical studies. In normal pituitary glands, immunostaining of RCAS1 and MIB-1 was not found. In malignant glioma, large numbers of cell nuclei were positive for MIB-1 (MIB-1 index: 28%), and RCAS1 was detected both in the cytoplasm and on the membrane of the tumor cells. Expression of RCAS1 was noted in 48% of pituitary adenomas immunohistochemically (60.0% of growth hormone-secreting adenomas, 60.0% of prolactin-secreting adenomas, 42.9% of adrenocorticotrophin-secreting adenomas, 40.0% of thyroid-stimulating hormone-secreting adenomas, 33.3% of nonfunctioning adenomas, and 44.4% of gonadotropin-subunit-positive adenomas). It showed no correlation with tumor type, size, and invasiveness. The statistically significant relationship between RCAS1 and MIB-1 positivity was identified in our study. These results suggest that expression of RCAS1 as well as MIB-1 positivity predict the growth potential of individual pituitary adenomas.

Neuroendocrine marker expression in thyroid epithelial tumors.

Tissue sections from 50 cases with thyroid tumors, composed of 11 follicular adenomas, 10 follicular carcinomas, 14 papillary carcinomas, 10 anaplastic carcinomas, and 5 medullary carcinomas, were immunohistochemically analyzed for representative neuroendocrine markers. Immunoexpression ratios of these neuroendocrine markers were as follows: Follicular adenomas, neuron-specific enolase (NSE)63.6%, synaptophysin (SynP) 45.5%, Leu7 27.3%, NCAM 45.5%, chromogranin A (CgA) 0%, SNAP25 0%; follicular carcinomas, NSE 90.0%, SynP 80.0%, Leu7 80.0%, NCAM 0%, CgA 0%, SNAP25 0%; papillary carcinomas, NSE 85.7%, SynP 78.6%, Leu7 100%, NCAM 7.0%, CgA 0%, SNAP25.0%; anaplastic carcinomas, NSE 10.0%, SynP 0%, Leu7 0%, NCAM 0%, CgA 0%, SNAP25 0%; medullary carcinomas, NSE 100%, SynP100%, Leu7 80.0%, NCAM 40.0%, CgA 100%, SNAP25 100%. The two follicular carcinomas, which were morphologically characterized by "insular" (or "alveolar") arrangements, showed distinct immunoexpression of NSE and SynP at the same time. By in situ hybridization (ISH), expression of mRNA for NSE was confirmed in cases with marked immunoexpression of NSE. Although no endocrine granules were found, our results suggested that a specific type of follicular carcinoma, i.e., insular variant, may be immaturely neuroendocrine-differentiated.

Expression of chromogranin/secretogranin mRNA in spontaneous mammary tumors in aging Fischer-344 rats.

There is a type of human breast cancer showing a neuroendocrine differentiation. Little is known, however, about the cell origin of this cancer or the process by which it expresses neuroendocrine features. Rat mammary tumors, either spontaneous or induced, have not been subjects for the investigation of aspects regarding the neuroendocrine differentiation of mammary epithelial cells. The aim of the present study was to show the potential of rat mammary tumors for expressing chromogranin (Cg)/secretogranin (Sg) mRNA. We examined CgA, SgI and SgII mRNA expression by reverse transcription-polymerase chain reaction in rat mammary adenocarcinoma and fibroadenoma which had arisen spontaneously in aging Fischer-344 rats. CgA and SgII mRNA were expressed in both mammary tumors, but SgI mRNA was not detected in either. The results of the present study show that rat mammary tumors can express chromogranin genes.

Immunohistochemical analysis of p27 (Kip1) in human pituitary glands and in various types of pituitary adenomas.

p27 (Kip1) plays regulatory roles in the cell cycle by inhibiting the activity of cyclin dependent kinases (CDKs). This immunohistochemical study is aimed at elucidating the expression of p27 in human pituitary and in various types of pituitary adenomas in order to clarify its role in the regulation of proliferation. Sixteen normal pituitary glands and 179 human pituitary adenomas were used for immunohistochemical studies. The tissues were fixed in 10% formalin and embedded in paraffin. Indirect peroxidase method was performed after heat-induced antigen retrieval using a monoclonal antibody against p27 protein. p27 protein was expressed in the nuclei of all 16 normal human pituitary glands. p27 protein was also expressed in 128 of 179 cases of pituitary adenomas (71.5%). A marked decrease of p27 expression was noted in ACTH-secreting adenomas, 8/20 (40.0%), compared with other types of pituitary adenomas--GH-secreting adenomas, 35/46 (76.1%); PRL-secreting adenomas, 22/33 (66.7%); TSH-secreting adenomas, 8/11 (72.7%); and nonfunctioning adenomas, 55/69 (79.7%). These results suggest that p27 may play some role in the regulation of proliferation in all types of pituitary adenomas. The lower levels of p27 in ACTH-secreting adenoma is of particular interest with respect to the intermediate lobe-derived pituitary tumor developed in p27 knockout mice.

Expression and localization of leptin receptor in the normal rat pituitary gland.

Leptin receptor (leptin-R) is a polypeptide consisting of a single transmembrane-spanning component. Recent studies performed by reverse transcriptase polymerase chain reaction (RT-PCR) have shown the production of leptin-R in various tissues including the pituitary, hypothalamus and reproductive organs. The localization of leptin-R protein in the pituitary gland, however, has not been extensively studied. This study deals with the expression of leptin-R in the normal rat pituitary gland, which was disclosed primarily in the plasma membrane fraction by immunoblotting and immunohistochemical staining methods. Double immunohistochemical staining revealed that the colocalization of leptin-R and anterior pituitary hormone expression was seen mainly in growth hormone (GH)-secreting cells (97.4 +/- 1.3%; GH-positive cells/leptin-R-positive cells), but in less than 1% of prolactin (PRL)-, adrenocorticotropic hormone (ACTH)-, thyroid-stimulating hormone-beta (TSH beta)- and follicle-stimulating hormone-beta (FSH beta)/luteinizing hormone-beta (LH beta)-positive cells. In contrast, leptin was localized most frequently in FSH beta/LH beta- and less frequently in TSH beta-positive cells. The above findings suggest that, in the rat anterior pituitary gland, there are paracrine relationships between leptin-producing cells and cells with leptin-R, which may regulate the function of GH cells.

Accumulation of p53 in esophageal squamous cell carcinoma.

p53 is one of the most important tumor suppressor genes. Mutation of the p53 gene can be detected immunohistochemically as over-expression of its protein in the nucleus. The p53 gene product is known to regulate cell growth and proliferation. Genetic alterations related to the carcinogenesis or progression of esophageal cancer are poorly understood. We examined accumulation of p53 protein in esophageal squamous cell carcinomas including early-stage cancers, and its clinicopathological significance. p53 immunoreactivity in the cancer tissues was found in 61 (79.2%) of 77 esophageal squamous cell carcinomas. Over-expression of p53 protein (diffusely and focally positive staining) was seen in 70.1% (54/77). p53 over-expression was detected not only in the cases of in situ or intramucosal carcinomas, but also in invasive carcinomas. No significant correlations were found between p53 over-expression and clinicopathological features such as depth of tumor invasion, lymph node metastasis or venous/lymphatic invasion. These results suggested that p53 mutations may be closely associated with the early-stage of pre-invasive esophageal carcinoma, and p53 gene mutations may play an important role in the carcinogenesis of human esophageal cancer.

Expression of neuro D1 in human normal pituitaries and pituitary adenomas.

Neuro D1 is a basic helix-loop-helix transcription factor expressed in the endocrine cells of pancreas and in a subset of neurons as they undergo terminal differentiation. In the adult pituitary gland, Neuro D1 is expressed in corticotroph cells and contributes to the corticotroph-specific pro-opiomelanocortin (POMC) transcription by interacting with Pituitary homeobox 1 (Ptx 1) transcription factor. In the present study, we investigated the expression of Neuro D1 in human normal pituitaries and different types of human pituitary adenomas using the RT-PCR and immunohistochemical techniques. Using RT-PCR, Neuro D1 mRNA was found to be expressed in ACTH-secreting adenomas (n = 3) and 6 of 8 non-functioning adenomas. On the other hand, GH-secreting adenomas (n = 5) and PRL-secreting adenomas (n = 3) were completely negative for Neuro D1 mRNA. Immunohistochemically, Neuro D1 was expressed in all ACTH-secreting adenomas (n = 10), and in 14 of 20 nonfunctioning adenomas. In contrast, 3 of 10 PRL-secreting adenomas and 2 of 10 GH-secreting adenomas showed positive Neuro D1 staining in the nuclei. The above results suggest that Neuro D1 contribute to the functional expression and the differentiation of ACTH-secreting adenomas. It also appears from our study that Neuro D1 might play a role in the differentiation of non-functioning adenomas, the mechanism of which remains to be further investigated. This is the first study on Neuro D1 in case of human pituitary adenomas.

In situ expression of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) genes in human skin.

Systemic stresses induce corticotropin-releasing hormone (CRH) expression in hypothalamus. CRH is released to the pituitary gland, where it stimulates proopiomelanocortin (POMC) production acting via the CRH receptor (CRH-R). CRH and POMC peptides are also detected in sites outside of the central nervous system (CNS), such as the skin. However, it has not been elucidated whether these peptides detected in the skin are derived from CNS or are produced locally. Using immunohistochemical and in situ reverse-transcription (RT)-PCR techniques, we demonstrated coexpression of CRH and POMC mRNAs in the epidermis and pilosebaceous units of the human skin. This coexpression was confirmed by the combination of laser-capture microdissection (LCM) with RT-PCR, analyzing mRNA expressions in captured sebaceous cells. Immunoreactivities and expressions of CRH and POMC mRNAs were strong in inflammatory lesions, melanocytic nevus, seborrheic keratosis, and also in the periphery of the benign tumor. These findings suggest that CRH and POMC peptides are produced locally in the skin and are regulated by inflammatory cells as well as by autocrine mechanisms. The skin may have "a local stress response system," whose activity is mediated by CRH and POMC peptides, in an equivalent to hypothalamus-pituitary adrenal axis.

Bcl-2 expression in breast cancer is down-regulated by trans-arterial administration of chemotherapeutic agents.

Bcl-2 is one of the cytoplasmic oncoproteins, and has been shown to suppress apoptotic cell death. In this study, we investigated the relationship between Bcl-2 expression and effects of chemotherapeutic agents on human breast cancer cells. We examined 26 surgically resected breast tumors with preoperative trans-arterial administration of chemotherapeutic agents and 30 control cases using immunohistochemical methods. In all 26 cases in the chemotherapy group, the breast cancer cells were focally degenerated to various degrees, associated with inflammation and stromal desmoplastic changes. Bcl-2 expression was found in 46% (12/26) of the chemotherapy group and in 67% (20/30) of controls. Of the 12 Bcl-2-positive cases in the chemotherapy group, 5 were diffusely positive [Bcl-2(2+)] and 7 were focally positive [Bcl-2(+)]. Of the 20 Bcl-2-positive cases in the control group, 18 were diffusely positive and 2 were focally positive. We speculate that Bcl-2 expression was down-regulated by trans-arterial administration of chemotherapeutic agents and was associated with apoptosis and degeneration of breast cancer cells.

Expression of interleukin-6, interleukin-6 receptor (gp80), and the receptor's signal-transducing subunit (gp130) in human normal pituitary glands and pituitary adenomas.

Interleukin-6 (IL-6) is an important cytokine in cell proliferation and differentiation in several organs. It has also been reported that IL-6 plays a role in secretion or release of pituitary hormones in pituitary hormone-secreting cells and pituitary adenomas, but convincing data in situ have not yet been reported. In this study, we examined the participation of IL-6 in the production of pituitary hormones and the differences between human normal pituitary glands and pituitary adenomas by determination of the localization or expression of IL-6, IL-6 receptor (IL-6R, gp80), and the signal-transducing subunit (gp130) of the receptor using immunohistochemical staining and RT-PCR. IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells in normal pituitary glands, as shown by double staining. gp 80 and gp130 were coexpressed in almost all GH- and PRL-secreting cells and in approximately 30% of FSH/LH-secreting cells. RT-PCR showed that IL-6 mRNA was expressed in only one of all the pituitary adenomas examined, whereas gp 80 and gp 130 mRNAs were detected in all these pituitary adenomas. In conclusion, IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells, and the receptors were expressed in GH-, PRL- and FSH/LH-secreting cells in human normal pituitary glands. Furthermore, our data emphasized that the mechanism of IL-6 function in human pituitary adenoma cells is distinct from that in normal pituitary cells.

Ghrelin and growth hormone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adenomas.

OBJECTIVE: The level of growth hormone (GH), growth hormone secretogogue (GHS) and GHS receptor (GHSR) messenger ribonucleic acid (mRNA) expression has been reported as being higher in GH-producing pituitary adenomas than in other types of pituitary adenomas. Recently, ghrelin, an endogenous ligand specific for GHSR, was isolated. Therefore, we attempted to clarify whether ghrelin mRNA is expressed in various types of human pituitary adenoma by competitive reverse transcription-polymerase chain reaction (RT-PCR). We also examined the relationship between the levels of ghrelin or GHSR mRNA and hormonal and tumour characteristics in patients with pituitary adenomas. PATIENTS: Pituitary adenoma tissue was obtained at surgery from 13 patients with acromegaly, 4 with prolactinomas, 5 with gonadotrophin (Gn)-producing adenomas, 4 with non-functioning adenomas, 2 with ACTH-producing adenomas and 2 with TSH-producing adenomas. METHODS: The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. RESULTS: Ghrelin mRNA was detected in all pituitary adenoma tissues examined, with the highest mean level detected in non-functioning adenomas, a moderate level in GH-producing adenomas and Gn-producing adenomas, and the lowest level in prolactinomas. The level of ghrelin mRNA expression in GH-producing adenomas correlated negatively with the size of the adenoma (n = 13) (r = - 0.756, P = 0.0028). Furthermore, the mean level of ghrelin mRNA expression in high-grade (III and IV of Hardy classification) GH-producing adenomas was significantly lower than that in low-grade (I and II) GH-producing adenomas (P = 0.0016). GHSR mRNA was also detected in all pituitary adenomas with the highest mean level in GH-producing adenomas, a moderate level in nonfunctioning adenoma, and the lowest level in prolactinoma and Gn-producing adenomas. CONCLUSIONS: Ghrelin mRNA, in addition to GHSR mRNA, is expressed in various types of pituitary adenoma with different levels of expression in each type. Our findings suggest that ghrelin produced in pituitary adenoma may play some role in the mechanism underlying the development of adenoma cells through autocrine and/or paracrine pathways.

Characterization of 5'-flanking region of rat somatostatin receptor sst2 gene: transcriptional regulatory elements and activation by Pitx1 and estrogen.

The sst2 somatostatin receptor mediates the inhibitory effects of somatostatin on secretive and proliferative processes. We previously showed that sst2 is one of the major subtypes expressed in the rat pituitary, and its messenger RNA level is up-regulated by chronic treatment with estrogen. To investigate the molecular mechanisms regulating sst2 gene expression, we cloned the upstream region (9.5 kb) from the translation initiation codon of the rat sst2 gene. It contained a single intron (5.0 kb) at the 5'-untranslated region, lacked TATA and CCAAT boxes, and had multiple transcriptional start sites. Transient transfection analysis with deleted mutants of a luciferase reporter construct showed that the promoter activity was regulated negatively and positively in the distal and proximal promoter regions, respectively. The promoter activity of each construct was more efficient in GH(3) pituitary cells than in nonpituitary cells. The construct (-77/+172/luc) containing a cAMP response element (CRE; -54/-47) provided maximum promoter activity, but a further 5'-deleted construct dramatically reduced the activity. Competitive gel shift and supershift assays indicated that Sp2 and Sp3 were bound to an Sp1 site (-40/-31), and activating transcription factor-2 and c-Jun were bound to a CRE site. Both Sp1 and CRE sites were essential for the full promoter activity. Overexpression of the pituitary homeoprotein Pitx1 activated the promoter activity of the -4066/+172/luc construct, and mapping analysis indicated the existence of two Pitx1 response sites, including the CRE site. Estrogen also increased the promoter activity of -77/+172/luc in GH(3) cells or in HeLa cells overexpressing both the estrogen receptor and c-Jun. These studies demonstrated the nature of the rat sst2 gene and the functional importance of both Sp1 and CRE sites in regulating sst2 gene expression and suggest that the CRE site mediates, at least partly, the promoter activity activated by Pitx1 or estrogen.