Evaluating the usefulness of plasma chromogranin A measurement in cyclic ACTH-dependent Cushing's syndrome.

Cushing's syndrome, a clinical condition characterized by hypercortisolemia, exhibits distinct clinical signs and is associated with cyclic cortisol secretion in some patients. The clinical presentation of cyclic Cushing's syndrome can be ambiguous and its diagnosis is often challenging. We experienced a 72-year-old woman with cyclic ACTH-dependent Cushing's syndrome caused by a pulmonary carcinoid tumor. Diagnosis was challenging because of the extended trough periods, and the responsible lesion was initially unidentified. A subsequent follow-up computed tomography revealed a pulmonary lesion, and ectopic ACTH secretion from this lesion was confirmed by pulmonary artery sampling. Despite the short peak secretion period of ACTH (approximately one week), immunostaining of the surgically removed tumor confirmed ACTH positivity. Interestingly, stored plasma chromogranin A levels were elevated during both peak and trough periods. The experience in evaluating this patient prompted us to investigate the potential use of plasma chromogranin A as a diagnostic marker of ACTH-dependent Cushing's syndrome. A retrospective study was conducted to determine the efficacy of plasma chromogranin A in three patients with ectopic ACTH syndrome (EAS), including the present case, and six patients with Cushing's disease (CD) who visited our hospital between 2018 and 2021. Notably, plasma chromogranin A levels were higher in patients with EAS than in those with CD. Additionally, a chromogranin A level in the present case during the trough phase was lower than that in the peak phase, and was similar to those in CD patients. The measurement of plasma chromogranin A levels could aid in differentiating EAS from CD.

Preoperative and long-term efficacy and safety of lanreotide autogel in patients with thyrotropin-secreting pituitary adenoma: a multicenter, single-arm, phase 3 study in Japan.

Somatostatin analogs are recommended for pharmacotherapy of TSH-secreting pituitary adenoma (TSHoma). A multicenter clinical trial was conducted to evaluate the efficacy and safety of lanreotide autogel treatment for TSHoma. A total of 13 Japanese patients with TSHoma were enrolled from February to December 2018 and treated with lanreotide autogel 90 mg every 4 weeks, with dose adjustments to 60 mg or 120 mg. Analysis was performed on data from patients receiving preoperative treatment (n = 6) up to 24 weeks and from those receiving primary or postoperative treatment (n = 7) up to 52 weeks. The primary efficacy endpoints were serum concentrations of TSH, free triiodothyronine (FT3), and free thyroxine (FT4). The secondary efficacy endpoints were pituitary tumor size and clinical symptoms. The serum concentrations of TSH, FT3, and FT4 decreased with treatment, and euthyroid status was maintained until final assessment. FT4 at final assessment was within reference ranges in 10/13 patients. The median (interquartile range) percent change in pituitary tumor size from baseline at final assessment was -23.8% (-38.1, -19.8). The clinical symptoms were also improved. The patients receiving preoperative treatment did not develop perioperative thyroid storm. Regarding safety, adverse events were observed in 12/13 patients, but none discontinued treatment. The common adverse events were gastrointestinal disorders (12/13 patients) and administration site reactions (5/13 patients). Lanreotide autogel may be effective for controlling thyroid function and reducing the pituitary tumor size, and is tolerable in patients with TSHoma (Japic Clinical Trials Information; JapicCTI-173772).

Impairment of the Hypothalamus-Pituitary-Thyroid Axis Caused by Naturally Occurring GATA2 Mutations In Vitro.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus-pituitary-thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone ß (TSHß). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHß promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHß promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood.

Food intake and body weight regulation depend on proper expression of the proopiomelanocortin gene (Pomc) in a group of neurons located in the mediobasal hypothalamus of all vertebrates. These neurons release POMC-encoded melanocortins, which are potent anorexigenic neuropeptides, and their absence from mice or humans leads to hyperphagia and severe obesity. Although the pathophysiology of hypothalamic POMC neurons is well understood, the genetic program that establishes the neuronal melanocortinergic phenotype and maintains a fully functional neuronal POMC phenotype throughout adulthood remains unknown. Here, we report that the early expression of the LIM-homeodomain transcription factor Islet 1 (ISL1) in the developing hypothalamus promotes the terminal differentiation of melanocortinergic neurons and is essential for hypothalamic Pomc expression since its initial onset and throughout the entire lifetime. We detected ISL1 in the prospective hypothalamus just before the onset of Pomc expression and, from then on, Pomc and Isl1 coexpress. ISL1 binds in vitro and in vivo to critical homeodomain binding DNA motifs present in the neuronal Pomc enhancers nPE1 and nPE2, and mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to hypothalamic POMC neurons in transgenic mice and zebrafish. ISL1 is necessary for hypothalamic Pomc expression during mouse and zebrafish embryogenesis. Furthermore, conditional Isl1 inactivation from POMC neurons impairs Pomc expression, leading to hyperphagia and obesity. Our results demonstrate that ISL1 specifies the identity of hypothalamic melanocortin neurons and is required for melanocortin-induced satiety and normal adiposity throughout the entire lifespan.

Partially redundant enhancers cooperatively maintain Mammalian pomc expression above a critical functional threshold.

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

HDR syndrome in a Japanese girl with biliary atresia: a case report.

BACKGROUND: Hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder. We report the first detailed case of hypoparathyroidism complicated by biliary atresia. CASE PRESENTATION: A 1-year-old Japanese girl was admitted to our hospital for living donor liver transplantation. She suffered from obstructive jaundice owing to biliary atresia. She also had persistent hypocalcemia. Despite oral calcium and abundant vitamin D supplementation, a laboratory test showed hypocalcemia (1.4 mmol/l) and hyperphosphatemia (2.6 mmol/l). The intact parathyroid hormone level was normal (66 ng/l) with severe vitamin D deficiency (25-hydroxy vitamin D: undetectable levels). There were no rachitic changes in metaphysis on X-rays. Her family history showed that her mother had sensorineural deafness, a low serum calcium level (2.1 mmol/l), hypoplastic left kidney, and a past history of an operation for right vesicoureteral reflux. We suspected that this patient and her mother have hypoparathyroidism, sensorineural deafness, and renal dysplasia syndrome. A heterozygous GATA3 gene mutation (c.736delGinsAT) was found in this patient and her mother, but not in her father. CONCLUSION: This familial case confirms the importance of family history in the diagnosis of HDR syndrome. Regardless of marked vitamin D deficiency, the complication of hypoparathyroidism prevented the onset of vitamin D deficiency rickets in our patient.

Urinary Cross-linked N-terminal Telopeptide of Type I Collagen Levels of Infants with Osteogenesis Imperfecta and Healthy Infants.

The urinary cross-linked N-terminal telopeptide of type I collagen (uNTx) levels in infantile osteogenesis imperfecta (OI) have not been well studied. Here we investigated the levels of uNTx in infants with OI and healthy infants. We collected spot urine samples from 30 infants with OI (male/female, 14/16; Sillence classification, I/II/III/IV: 15/3/6/6; age, 5.2±4.4 months) and 120 healthy infants (male/female, 75/45; age, 5.1±4.1 months) for the measurement of uNTx levels. The uNTx levels of the OI infants were significantly lower than those of the healthy infants (mean±SD, 1,363.7±530.1 vs. 2,622.2±1,202.6 nmol BCE/mmol Cr; p＜0.001). The uNTx levels of the infants with type I OI were significantly lower than those of the age-matched healthy infants, although an overlap was observed between the 2 groups. Among the 1-month-old infants, the uNTx levels of the infants with types I, III or IV OI were significantly lower than those of the healthy infants, without overlap (1,622.5±235.8 vs. 3,781.0±1,027.1 nmol BCE/mmol Cr; p＜0.001). These results indicate that uNTx levels are significantly lower in infants with OI than in healthy infants, and they suggest that uNTx might be useful as a reference for diagnosing OI.

A Case of 17α-hydroxylase/17,20-lyase Deficiency Diagnosed at 45 Years of Age with Hyperaldosteronism.

17α-hydroxylase deficiency is a type of congenital adrenocortical hyperplasia that is typically diagnosed in childhood or adolescence. It manifests as hypertension with gonadal dysfunction as the primary symptom. We herein report 17α-hydroxylase/17,20-lyase deficiency (17OHD) diagnosed at the age of 45 years. The patient presented with hypertension, irregular menstruation, and hyperaldosteronism. The clinical manifestations of 17OHD vary based on the specific variant pattern of CYP17A1. In this case, the variant was c.157_159 TCC del p. Phe53del, which has been frequently reported in Japan. The enzymatic deficiency due to this variant is partial, leading to a delay in making a correct diagnosis.

Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity.

Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH-1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r = 0.421, p < 0.001), and CD68-positive TAMs (r = 0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = -0.259, p = 0.002) and CD4-positive TILs (r = -0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunoreactivity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heterogeneity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.

[Relationships of Exercise and Dietary Intake with Bone Density in Young Adult Women].

The aim of this study was to investigate the relationships of dietary intake, exercise, and menstrual regularity with bone density. In 81 female university students, the osteo-sono-assessment index (OSI) was determined by quantitative ultrasonography. In addition, a questionnaire on the intake of calcium, vitamin D, and phosphorus, exercise experiences in junior high school and high school days, and menstrual regularity was administered. The OSI was higher in the group that had the habit of exercising in junior high school and high school. Furthermore, the higher OSI was associated with higher vitamin D intake and lower phosphorus intake. These findings suggest the importance of exercise and dietary intake for improving bone density.

Early childhood exposure to maternal smoking and obesity: A nationwide longitudinal survey in Japan.

Involuntary exposure to tobacco smoke is suspected to be one of the risks factors that are associated with obesity in children. The purpose of this study was to examine the relationship between early childhood exposure to tobacco smoke and the risk of obesity and overweight in Japan. This study utilized a nationwide, population-based longitudinal survey. The participants were restricted to 32 081 children who had available information on maternal smoking history as well as childhood height and weight. We conducted a binomial log-linear regression analysis with children of non-smoking mothers as the reference group. The children with mothers who were smokers had a higher risk of developing obesity or being overweight compared to the children with mothers who were nonsmokers. The risk ratios were 1.20 (95% confidence interval [CI]: 1.09-1.32) for overweight and 1.17 (95% CI: 0.95-1.44) for obesity. Early exposure to maternal smoking increases the risk of being overweight and having obesity during childhood. The increased risk is more pronounced among children with mothers, smoked heavily, or parents, who were smokers.

Nutrients Associated with Sleep Bruxism.

BACKGROUND: The purpose of the present research was to identify nutrients related to sleep bruxism and to establish a hypothesis regarding the relationship between sleep bruxism and nutrients. METHODS: We recruited 143 Japanese university students in 2021 and assigned them to sleep bruxism (n = 58) and non-sleep bruxism groups (n = 85), using an identical single-channel wearable electromyography device. To investigate nutrient intakes, participants answered a food frequency questionnaire based on food groups. We assessed differences in nutrient intakes between the sleep bruxism and non-sleep bruxism groups. RESULTS: Logistic regression modeling showed that sleep bruxism tended to be associated with dietary fiber (odds ratio, 0.91; 95% confidence interval, 0.83-1.00; p = 0.059). In addition, a subgroup analysis selecting students in the top and bottom quartiles of dietary fiber intake showed that students with sleep bruxism had a significantly lower dietary fiber intake (10.4 ± 4.6 g) than those without sleep bruxism (13.4 ± 6.1 g; p = 0.022). CONCLUSION: The present research showed that dietary fiber intake may be related to sleep bruxism. Therefore, we hypothesized that dietary fiber would improve sleep bruxism in young adults.

Neuropeptide W stimulates adrenocorticotrophic hormone release via corticotrophin-releasing factor but not via arginine vasopressin.

Neuropeptide W (NPW) was isolated as an endogenous ligand for NPBWR1, an orphan G protein-coupled receptor localized in the rat brain, including the paraventricular nucleus. It has been reported that central administration of NPW stimulates corticosterone secretion in rats. We hypothesized that NPW activates the hypothalamic-pituitary-adrenal (HPA) axis via corticotrophin-releasing factor (CRF) and/or arginine vasopressin (AVP). NPW at 1 pM to 10 nM did not affect basal or ACTH-induced corticosterone release from dispersed rat adrenocortical cells, or basal and CRF-induced ACTH release from dispersed rat anterior pituitary cells. In conscious and unrestrained male rats, intravenous administration of 2.5 and 25 nmol NPW did not affect plasma ACTH levels. However, intracerebroventricular (icv) administration of 2.5 and 5.0 nmol NPW increased plasma ACTH levels in a dose-dependent manner at 15 min after stimulation (5.0 vs. 2.5 nmol NPW vs. vehicle: 1802 ± 349 vs. 1170 ± 204 vs. 151 ± 28 pg/mL, respectively, mean ± SEM). Pretreatment with astressin, a CRF receptor antagonist, inhibited the increase in plasma ACTH levels induced by icv administration of 2.5 nmol NPW at 15 min (453 ± 176 vs. 1532 ± 343 pg/mL, p<0.05) and at 30 min (564 ± 147 vs. 1214 ± 139 pg/mL, p<0.05) versus pretreatment with vehicle alone. However, pretreatment with [1-(ß-mercapto-ß, ß-cyclopentamethylenepropionic acid), 2-(Ο-methyl)tyrosine]-arg-vasopressin, a V1a/V1b receptor antagonist, did not affect icv NPW-induced ACTH release at any time (p>0.05). In conclusion, we suggest that central NPW activates the HPA axis by activating hypothalamic CRF but not AVP.

Genetic analysis in Japanese patients with osteogenesis imperfecta: Genotype and phenotype spectra in 96 probands.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare connective-tissue disorder characterized by bone fragility. Approximately 90% of all OI cases are caused by variants in COL1A1 or COL1A2. Additionally, IFITM5 variants are responsible for the unique OI type 5. We previously analyzed COL1A1/2 variants in 22 Japanese families with OI through denaturing high-performance liquid chromatography screening, but our detection rate was low (41%). METHODS: To expand the genotype-phenotype correlations, we performed a genetic analysis of COL1A1/2 and IFITM5 in 96 non-consanguineous Japanese OI probands by Sanger sequencing. RESULTS: Of these individuals, 54, 41, and 1 had type 1 (mild), type 2-4 (moderate-to-severe), and type 5 phenotypes, respectively. In the mild group, COL1A1 nonsense and splice-site variants were prevalent (n = 30 and 20, respectively), but there were also COL1A1 and COL1A2 triple-helical glycine substitutions (n = 2 and 1, respectively). In the moderate-to-severe group, although COL1A1 and COL1A2 glycine substitutions were common (n = 14 and 18, respectively), other variants were also detected. The single case of type 5 had the characteristic c.-14C>T variant in IFITM5. CONCLUSION: These results increase our previous detection rate for COL1A1/2 variants to 99% and provide insight into the genotype-phenotype correlations in OI.

Retroperitoneal Paraganglioma With Asymptomatic Follicular Lymphoma: A Case Report.

Paraganglioma (PGL) is a rare tumor originating from extra-adrenal paraganglionic chromaffin tissues, and most sympathetic PGLs have excessive catecholamine secretion. However, nonfunctional PGLs are sometimes found. Although malignant PGL is defined by metastasis to nonchromaffin tissues, it is difficult to predict malignancies due to the lack of reliable markers of potential malignancies. We report the case of a 69-year-old Japanese woman with an incidental retroperitoneal tumor and multiple enlarged mesenteric lymph nodes simultaneously. The patient had no subjective symptoms and there were no laboratory findings suggesting catecholamine hypersecretion. Both the retroperitoneal tumor and the enlarged mesenteric lymph nodes showed high accumulation of fluorodeoxyglucose (FDG), whereas metaiodobenzylguanidine (MIBG) was accumulated only at the retroperitoneal tumor. Although a retroperitoneal tumor was diagnosed as nonfunctional PGL by examination including MIBG scintigraphy, the cause of enlarged mesenteric lymph nodes could not be diagnosed by imaging and biochemical tests. As a result of retroperitoneal tumor resection and mesenteric lymph nodes sampling, histopathological examination revealed that a retroperitoneal tumor was PGL and enlarged mesenteric lymph nodes were follicular lymphoma. To reveal an underlying genetic factor, we performed whole exome sequencing of genomic DNA, and we identified 2 possible candidate variants in SDHD and DLST, but the pathogenicity of these variants remains uncertain in the present case. This rare case reinforces the importance of histopathological diagnosis of nonchromaffin tissue lesions in patients with PGL for the appropriate treatment strategy.

Possible contribution of 2-aminoethoxydiphenyl-borate-sensitive Ca2+ mobilization to adrenocorticotropin-induced glucocorticoid synthesis in rat adrenocortical cells.

Cytoplasmic calcium ([Ca(2+)](i)) provided through voltage-dependent Ca(2+) channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca(2+)](i) supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca(2+)](i) supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10(-11) M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 microM 2APB by 51.8% (6.71 +/- 0.97 vs. 3.23 +/- 0.05 ng/mL/4 hours; p<0.05). Furthermore, 2APB significantly decreased the 10(-11) M ACTH-stimulated [Ca(2+)](i). ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10(-13) M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca(2+) from the ER or the subsequent store-operated Ca(2+) entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca(2+) supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca(2+)](i) used for steroidogenesis may be derived from a 2APB-sensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.

CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant.

BACKGROUND: Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. METHOD: DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. RESULTS: Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. CONCLUSION: We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors.

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells.

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.

A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance.

Broad-spectrum drug resistance is a major obstacle in cancer treatment, which is often caused by overexpression of ABC transporters the levels of which vary between individuals due to single-nucleotide polymorphisms (SNPs) in their genes. In the present study, we focused on the human ABC transporter ABCC4 and one major non-synonymous SNP variant of the ABCC4 gene in the Japanese population (rs11568658, 559 G > T, G187W) whose allele frequency is 12.5%. Cells expressing ABCC4 (G187W) were established using the Flp-In™ system based on Flp recombinase-mediated transfection to quantitatively evaluate the impacts of this non-synonymous SNP on drug resistance profiles of the cells. Cells expressing ABCC4 (WT) or (G187W) showed comparable ABCC4 mRNA levels. 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that the EC50 value of the anticancer drug, SN-38, against cells expressing ABCC4 (G187W) was 1.84-fold lower than that against cells expressing ABCC4 (WT). Both azathioprine and 6-mercaptopurine showed comparable EC50 values against cells expressing ABCC4 (G187W) and those expressing ABCC4 (WT). These results indicate that the substitution of Gly at position 187 of ABCC4 to Trp resulted in reduced SN-38 resistance.