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Serum dehydroepiandrosterone sulfate (DHEA-S) levels reflect the state of adrenocorticotropic hormone (ACTH) secretion. However, it is difficult to use serum DHEA-S to diagnose hypothalamic-pituitary-adrenal (HPA) axis insufficiency due to its non-normal and highly skewed distribution. In this study, we focused on HPA insufficiency caused by hypothalamic and/or pituitary dysfunction and evaluated the usefulness of the standard deviation score of log-transformed DHEA-S (ln DHEA-S SD score), which was calculated from the established age- and sex-specific reference values. We retrospectively reviewed the medical records of 94 patients suspected of having HPA insufficiency, in whom serum DHEA-S measurement and the rapid ACTH stimulation test were performed, and included 65 patients who met our criteria in this study. The ln DHEA-S SD scores were distributed more normally than measured DHEA-S levels and were significantly higher in patients with a peak cortisol level ≥18 μg/dL than in those below this value, suggesting that this score is a legitimate and strong indicator of adrenocortical function. The optimal cut-off value for impaired HPA function was -0.853, with a sensitivity of 70.3% and a specificity of 100%. Among the 37 patients whose peak cortisol levels were below 18 μg/dL, 11 patients with ln DHEA-S scores ≥-0.853 exhibited significantly higher basal ACTH and basal and peak cortisol levels than the 26 patients with scores

The orphan nuclear receptor DAX1 (dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1; NR0B1) has been known for its various roles in human development, specifically sex determination and steroidogenesis. Its expression has been reported in endocrine and sex steroid-dependent neoplasms such as human adrenocortical, pituitary, endometrial, and ovarian tumors. Prostate cancer is also sex steroid-dependent tumor in which androgens play important roles in the pathogenesis and development via androgen receptor (AR). DAX1 is also reported to repress AR activity in human prostate cancer cell line (LNCaP) but its biological roles have remained unclear in the human prostate cancer. The aim of this study is to examine the expression of DAX1 in human prostate cancer using immunohistochemistry in order to evaluate its possible biological and/or clinical significance. In this study, we examined the DAX1 immunoreactivity in human prostate cancer obtained from surgery (n = 40), and correlated the findings with clinicopathological features of the patients. Twenty-one cases were defined as positive cases for DAX1 immunoreactivity (53%). Immunoreactivity for DAX1 was inversely and significantly correlated with Gleason score (P

It is well-known that estrogens are closely involved in the growth of human breast carcinomas, and that the great majority of breast carcinoma express estrogen receptors. Recent studies have demonstrated that estrogens are locally produced and act on the breast carcinoma tissue. Among these pathways, aromatase is a key enzyme for intratumoral production of estrogens in breast carcinomas, and aromatase inhibitors are currently used in the breast carcinoma in postmenopausal women as an estrogen deprivation therapy. This review summarizes the results of recent studies on the expression and regulation of aromatase in breast carcinoma tissues, and discusses the potential biological and/or clinical significance of aromatase. Aromatase is abundantly expressed in various cell types, such as carcinoma cells, intratumoral stromal cells, and adipocytes adjacent to the carcinoma, in breast carcinoma tissues. Further, a key regulator for aromatase expression differed according to cell type. In addition, aromatase suppressed in situ production of bioactive androgen, 5alpha-dihydrotestosterone (DHT), in breast carcinoma. Aromatase inhibitors may thus have additional antiproliferative effects through increasing local DHT concentration with estrogen deprivation.

Adrenocortical carcinoma (ACC) is a rare, highly malignant tumor. The aim of the present study is to evaluate the prognostic relevance of a proliferation marker Ki67/MIB1 by immunohistochemistry in 17 cases who underwent resections of the primary tumors and diagnosed to have ACC at Tohoku University Hospital based on the criteria of Weiss during the period from 1976 to 2005. The follow-up periods ranged from 221 days to 10659 days (about 29 years) with the median of 1895 days. The median age at diagnosis was 46 years old, and the mean size of the primary tumors was 7.1 cm with the minimal of 3.5 cm. Ki67/MIB1 labeling index (Ki67/MIB1LI) ranged from 1% to 26%. Kaplan-Meier analysis revealed that patients with Ki67/MIB1LI of 7% or more were associated with significantly shortened disease-free survival (P = 0.0037). The evaluation with Weiss criteria revealed that the median score of Weiss criteria was five, and 13 patients (76.5%) presented positive findings in the criteria of mitotic rate. The survival analysis with Weiss score showed that patients with the scores of 6 or more had both significantly shortened disease-free survival (P = 0.0001) and overall survival (P = 0.0063). The present study has suggested that Ki67/MIB1LI, as well as Weiss score, is a useful predictor for tumor recurrence after resection of the primary tumors in patients with ACC.

Context: The steroidogenic acute regulatory protein (StAR) is essential for the production of steroid hormones. The mutations in the StAR gene typically cause congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by severe adrenal insufficiency in both sexes and complete female external genitalia in genetic males. Affected 46, XX females feminize at puberty and menstruate but have progressive hypergonadotropic hypogonadism. It has been hypothesized that the cholesterol accumulation in the steroidogenic cells destroys the residual steroidogenic capacity and progressive ovarian failure occurs (two-hit model). Additionally, ovulation and luteinization in the patients is supposed to be impaired. However, those hypotheses have not been confirmed histologically. Objective: We examined whether pathological findings of the ovary in a patient of lipoid CAH corresponded with two-hit model, and whether ovulation and luteinization occurred or not in the patient. Subject: The ovary in an adult 46, XX female with a homozygous nonsense mutation (Q258X) in the StAR gene was examined. When the patient was age 22 yr, the ovary was resected because of enlargement with polycysts and subsequent torsion. Result: The affected ovary demonstrated remarkable lipoid deposition and changes of the mitochondrial ultrastructure. Immunohistochemical examination showed decrease of steroidogenic enzymes such as P450 cholesterol side-chain cleavage (P450scc). Additionally, we detected corpus albicans in the affected ovary. Conclusion: This is the first detailed report on ovarian histology in an adult 46, XX female with a null type mutation of the StAR gene (Q258X), which indicates the evidence of the impairment of ovarian StAR-independent steroidogenesis by lipoid deposition.

Nucleobindin 2 (NUCB2) is a multifunctional protein containing several functional domains, and associated with wide variety of biological process such as food intake and energy homeostasis. Recently, NUCB2 has been implicated in not only normal human tissues but also some kinds of human malignancies. However, its clinical and/or biological significance has largely remained unknown in endometrial carcinomas. We therefore immunolocalized NUCB2 protein in 87 endometrial carcinoma tissues and examined its clinical significance. NUCB2 immunoreactivity was detected in 19 out of 87 (22%) of endometrial carcinoma cases examined, and positively correlated with Ki67 labeling index, while there was no significant correlation between NUCB2 and stage, histological grade, and progesterone receptor status. Furthermore, NUCB2 immunoreactivity was significantly correlated with increased risk of recurrence and worse clinical outcome regardless of stage or histological grade. Subsequent multivariate analyses did reveal that NUCB2 immunoreactivity was an independent prognostic factor for both disease-free survival and endometrial cancer specific survival. In vitro experiments demonstrated that knockdown of NUCB2 using specific siRNA for NUCB2 significantly impaired cell proliferation and migration of the endometrial carcinoma cell lines, Ishikawa and Sawano cells, and that nesfatin-1 treatment significantly promoted cell proliferation and migration in Ishikawa cells. These findings possibly suggested that NUCB2 and/or nesfatin-1 had pivotal roles in the progression of endometrial carcinomas. Immunohistochemical NUCB2 status may therefore serve as a potent biomarker for endometrial carcinomas.

A right adrenal tumor was incidentally discovered on abdominal computed tomography performed on a 53-year-old Japanese man, who had been hospitalized with diabetic ketoacidosis. Normal values were obtained for adrenal hormones in the morning after an overnight fast and urinary cortisol excretion after treatment of diabetic ketoacidosis with insulin. However, overnight dexamethasone administration with 1 mg or 8 mg did not completely suppress serum cortisol levels. There were no remarkable physical findings related to Cushing's syndrome. The patient was diagnosed as having preclinical Cushing's syndrome (PCS). Histological examination of the adrenalectomy specimen demonstrated adrenal black adenoma. Blood glucose levels subsequently improved after adrenalectomy, and the patient never developed adrenal insufficiency after hydrocortisone withdrawal. The patient was treated with diet therapy alone, and maintained good glycemic control. However, the patient still showed a diabetic pattern in an oral glucose tolerance test. It seems that the existence of PCS in addition to the underlying type 2 diabetes mellitus contributed to aggravation of blood glucose levels. Although there are many aspects of the natural course of PCS that have not been thoroughly elucidated, it is necessary to remain aware that a PCS patient with abnormal glucose metabolism may develop diabetic ketoacidosis by environmental agents.

We reported a rare case of simultaneous primary aldosteronism and preclinical Cushing's syndrome due to unilateral double adrenocortical adenomas in a 57 year-old woman who had had hypertension for the last 10 years. Abdominal computed tomography showed double tumors in her right adrenal gland. Physical findings revealed simple obesity and hypertension, but no other abnormal findings were detected. Laboratory findings demonstrated that serum potassium was 3.8 mmol/l; plasma renin activity, 0.3 ng/ml/h; plasma aldosterone, 100 pg/ml, and aldosterone renin ratio (ARR), 33. Serum cortisol was 15.7 microg/dl. There was no circadian rhythm of serum cortisol, and no suppression of serum cortisol in response to exogenous dexamethasone administration. Right adrenalectomy was performed under laparoscopy. Two well-circumscribed tumors, whose sizes were 21 and 19 mm in greatest diameter, were detected. They were macroscopically composed of a golden-yellow portion admixed with a brown portion, which corresponded to clear cells and compact cells, respectively. Immunohistochemical staining for steroidogenic enzymes demonstrated the presence of all the enzymes involved in corticosteroidogenesis in these two adenomas, indicating that the two adenomas produced both cortisol and mineralocorticoid. Specifically, one adenoma mainly caused excessive production of cortisol as compared to the other one. These findings indicate that overproduction of both cortisol and mineralocorticoid was evident in the two adenomas of the right adrenal gland in immunohistochemical study for steroidogenic enzymes, whereas there was less clinical manifestation of primary aldosteronism and Cushing's syndrome in the present patient.

Progesterone is involved in various functions of the cardiovascular system, including those of vascular smooth muscle cells (VSMCs) via progesterone receptor (PR). Progesterone has also been postulated to be involved in inhibition of VSMC proliferation via PR. However, the details of PR expression have remained largely unknown in human cardiovascular VSMCs. Therefore, we first examined the relative levels of PR isoform (PR-A and PR-B) expression in VSMCs, using both immunohistochemistry and quantitative RT-PCR analysis. PR-B was equally expressed between male and female aorta, but PR-A was more abundant in female than in male aorta. This finding demonstrated that the status of PR subtype expression was associated with the difference of genders.

Somatostatin is well known to inhibit the hormone secretion of various peptides. This action has been considered to be generally mediated via six different specific somatostatin receptors (sstr), sstr1, sstr2A, sstr2B, sstr3, sstr4, and sstr5. It then becomes very important to demonstrate the localization of these sstr subtypes in order to elucidate the possible biological and/or clinical significance of somatostatin actions. These sstr subtypes have been demonstrated to be expressed throughout the human body, including the central nervous system, gastrointestinal tract, pancreas, kidney, and other organs, but its details, especially its systemic distribution and localization in tissue compartments, have yet to be examined thoroughly in human. Therefore, in this study, we examined the systemic localization of all six somatostatin receptors in normal human organs using immunohistochemistry with recently developed specific antibodies against these receptor subtypes. In all of the human tissues examined, various sstr subtypes were detected not only in parenchymal cells but also in various stromal cells such as lymphocytes, fibroblasts, and endothelial cells. Among human tissues in which the presence of sstr has not been previously reported, the parotid gland demonstrated immunoreactivity for sstr2B and sstr5, bronchial gland for sstr1, 2B, 3, 4, 5, parathyroid gland for sstr1, 3, 4, and duodenum for all subtypes immunoreactivity. The great majority of other organs examined demonstrated results consistent with those of previously reported biochemical studies. In pancreatic islet cells, only sstr2A was positive in all the cases but other sstr subtypes were associated with marked intraislet heterogeneity in their distribution. In stomach, all subtypes of receptor were detected in various cell types of the mucosa, but none in ECL cells of fundic gland. These findings demonstrated the broad systemic actions of somatostatin in non-endocrine cells.

3beta-Hydroxysteroid dehydrogenase (3beta-HSD) is known to be involved in steroid production and/or metabolism and to be expressed in many tissues including adrenal cortex. Expression of this enzyme has also been elucidated in human cardiovascular system but its details remain largely unknown. Therefore, in this study, we examined the status of 3beta-HSD in postmortem human aorta utilizing RT-PCR and immunohistochemical analysis. Both mRNAs and immunoreactivity for the enzyme were detected predominantly in female aorta, and in those with mild atherosclerotic changes. In addition, immunohistochemical study demonstrated that immunoreactivity for 3beta-HSD was detected in vascular smooth muscle cells (VSMCs) of aorta. These findings all indicated that steroidogenesis via 3beta-HSD may occur in VSMCs of human aorta, possibly related to gender differences and/or the degree of atherosclerosis.

We have previously reported that the urine of patients with Cushing's syndrome, including pituitary adenoma cases and adrenal adenoma cases, consistently show a conspicuous peak in the chromatographical analysis of 17-ketosteroid fraction but not in the urine of control subjects. The substance emerges just before 11beta-hydroxy-androsterone (11beta-OH-A) in capillary gas chromatography. In the present study, we have identified an "unknown peak substance" observed in the urine of Cushing's syndrome patients using gas chromatography-mass spectrometry (GC/MS). Trimethylsilylether (TMS)-derivative of the substance was found to have a molecular weight (MW) of 448, which is similar to that of 11-OH-A (MW: 450). From these findings, we hypothesized that the substance had the structure of a C-19 steroid with two hydroxyl groups at positions C-3 and C-11, one keto-group at C-17 and a double bond between C-4 and C-5 of the A ring. We hypothesized that the unknown peak substance was 3alpha,11beta-dihydroxy-4-androsten-17-one (3alpha,11beta-DH-A). To confirm this speculation we synthesized 3alpha,11beta-DH-A and compared the elution pattern of it with that of the "unknown peak substance" using GC and GC/MS. We found that both substances were indistinguishable by GC and GC/MS analysis. These results suggest that the unknown substance observed in the urine of patients with Cushing's syndrome is 3alpha,11beta-DH-A.

Adrenal incidentalomas have recently increased in incidence, and thus it has become important to establish clinical management of these patients. It is also important to evaluate whether these tumors are different from preclinical or overt Cushing's syndrome in their steroidogenesis. In this study, we therefore examined steroidogenesis of hormonally inactive adrenal incidentalomas via short-term culture of tumor specimens, in addition to an immunohistochemical study of steroidogenic enzymes. Five patients (two men and three women) diagnosed with adrenocortical incidentaloma without any clinical signs of adrenocortical hormonal excess except for hypertension and disturbed glucose tolerance, were recruited for this study. Hormonal findings, including circadian rhythms for cortisol and ACTH secretion, the response of ACTH to CRH infusion and results of dexamethasone suppression test were all within normal limits in these patients. Immunoreactivity for all steroidogenic enzymes involved in cortisol production was detected in tumor cells in all cases examined. Results of in vitro steroidogenesis analysis using short-term culture revealed that levels of cortisol secretion varied among the cases. There were no differences in the immunolocalization of steroidogenic enzymes and/or the levels of cortisol secretion between these hormonally inactive tumors and preclinical and/or overt Cushing's syndrome. Dehydroepiandrosterone-sulfotransferase (DHEA-ST) immunoreactivity in nonneoplastic regions was suppressed in one case in which the tumor secreted cortisol similar to preclinical and/or overt Cushing's syndrome. These results demonstrate that the levels of in vitro steroid production and/or the immunolocalization of steroidogenic enzymes in hormonally inactive adrenocortical tumors vary markedly and are not overtly different from those of preclinical and/or overt Cushing's syndrome.

We developed a new method for measuring an unidentified ketosteroid glucuronide (US-G) detected by the method of Iwata et al. for measuring 17-ketosteroid glucuronides by reversed phase HPLC on a Capcell-Pak C8 column with three kinds of mobile phase solutions (Iwata method; Clin Chem 35: 795-799, 1989). The Iwata method inadequately separated US-G and two hydroxy 17-ketosteroides, 11β-hydroxyetiocholanolone and 11β-hydroxyandrosterone, and it exhibits insufficient sensitivity for measuring traces of US-G in the urine of healthy subjects. We solved these problems by developing a new method which measures US-G in urine, as a free type by hydrolyzing the glucuronide type enzymatically, by normal phase HPLC on a Capcell-Pak Silica column with one kind of mobile phase solution. By this method, the levels of US excreted as a glucuronide in the urine of healthy subjects and of patients with Cushing's syndrome were determined as proportions of the levels of 11β-hydroxyandrosterone. The average daily urinary excretion of US was 971μg (125-4, 995μg) in patients with Cushing's syndrome (n=22: two males and 20 females aged 26 to 65 years), and 34μg (0-141μg) in healthy subjects (n=63: 49 males, and 14 females aged 21 to 54 years), and the differences were clearly significant. However, there were no differences between the urinary US levels of patients with pituitary adenoma and patients with adrenal adenoma. Furthermore, no US was detected in the urine of patients with aldosteronism (two males and eight females aged 34 to 61 years). The daily level of urinary US excretion in two of the patients with polycystic ovary syndrome was 159 and 142μg, but no US was detected in the other two patients.

Steroids in fetal circulation are known to exist in a sulfoconjugated form. To investigate the contribution of human fetal liver to the sulfoconjugation of steroids, and the properties of steroid sulfotransferase, an attempt was made to separate the specific sulfotransferases toward estrone (E-ST) and 3β-hydroxysteroid (3β-ST). For the purification procedure, successive chromatography was used:(1) DEAE-Sepharose CL-6B chromatography (2) Chromatofocusing and (3) Adenosine 3', 5'-diphosphateagarose affinity chromatography. Enzymatic activity was obtained with 3H-estrone (E1), 3H-pregnenolone (P) and 3H-dehydroepiandrosterone (DHA) as substrates. Two active fractions of steroid sulfotransferase were collected from the eluate of a CL-6B column. Fractions 88-120 found to be specific for E1, were purified approximately 1, 800 fold by further chromatographic procedures. The affinity column condition used was set at pH 7.2. The apparent Km and Vmax values were 1.66 μM and 35.6 nmol/mg protein/min, respectively. Fractions 130-162 specific for P were further purified approximately 144 fold. The affinity column condition used was set at pH 6.0. The apparent Km and Vmax values were 0.25 μM and 4.36 nmol/mg protein/min, respectively. Both enzymes were found to be a single band and had almost the same molecular weight of approximately 36 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both enzymes equally catalyzed DHA to form DHA-sulfate. In the present study, E-ST and 3β-ST were separated and purified in human fetal liver for the first time, with kinetic analysis for both enzymes.

The patient was a 26-year-old man with Cushing's disease who underwent transsphenoidal microscopic surgery for a pituitary microadenoma. His postoperative course was uneventful, but he died suddenly five years after the operation. At autopsy, a ruptured dissecting aneurysm with marked atherosclerosis was observed in the aorta. In the pituitary, a small focus of adrenocorticotropic hormone (ACTH) producing adenoma, possibly residual adenoma, was detected and Crooke's degeneration was observed in the non-tumorous pituitary gland. But immunohistochemical patterns of pituitary hormones in the non-tumorous pituitary gland were normal and the adrenal cortex was unremarkable. In the hypothalamus, corticotropin-releasing hormone immunoreactivity was not detected and arginine vasopressin was sporadically positive. Considering these findings, this patient may have developed subclinical hypercortisolism due to the residual adenoma at the time of autopsy, despite clinical remission. Cushing's syndrome is considered to be a risk factor dissecting aneurysm, and in this case the metabolic changes in Cushing's disease may have influenced the development of the dissecting aneurysm. Periodic cardiovascular re-evaluations should therefore be performed when there is clinical remission of Cushing's syndrome.

Transcription factor GATA-6 has been demonstrated to be expressed in the human fetal and adult adrenal cortex and has been postulated to play an important role in adrenal steroid biosynthesis. However, the status for GATA-6 expression has not been examined in detail especially in relation to adrenal development and aging. Therefore, in this study, we analyzed GATA-6 expression in 11 human fetal adrenals and 19 adrenal glands after birth using immunohistochemistry. In the fetal adrenals, the status of GATA-6 immunoreactivity in the definitive zone was significantly and directly correlated with ages of development (P

Sex steroids have been postulated to influence pathophysiology of human skin through various skin appendages. The presence of sex steroid receptors has been also reported in adnexal tumors but its details still remained unknown. Therefore, in this study, we immunolocalized sex steroid receptor protein (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR)A, PRB and androgen receptor (AR)) in 23 cases of non-pathological skin (male: 10, female: 13) and in 50 cases of skin adnexal tumors (male 24, female 26; 38 benign and 12 malignant). ERalpha immunoreactivity was detected exclusively in basal cells of sebaceous glands of non-pathological skin. AR and PRB immunoreactivity was detected in both differentiated and basal cells of sebaceous gland. AR and ERbeta immunoreactivity was also detected in sebaceous and eccrine sweat glands but not in outer root sheath of hair follicles. In sebaceous gland neoplasms, the number of ERalpha positive cases was significantly lower in skin appendage neoplasms than non-pathological skin. ERbeta immunoreactivity was not detected in any of sebaceous gland neoplasms examined. There were no significant differences in PRA, PRB and AR immunoreactivity between non-pathological sebaceous gland and its neoplasm. In sweat gland neoplasms, the number of AR positive cases was significantly lower in benign neoplasms than their non-pathological counterpart. Therefore sex steroids are considered to play important roles in regulation of non-pathological skin appendage function and pathogenesis and/or development of its neoplasm. In addition, the status of the great majority of sex steroid hormone receptors was maintained throughout the process of neoplastic transformation of skin appendages, except for AR and ERalpha in sweat and sebaceous gland neoplasms.

Several cardiovascular risk factors correlate with adiponectin levels. It is not known whether total homocysteine, folate and gamma-glutamyl transferase levels correlate with adiponectin. We cross-sectionally analyzed the association between adiponectin and these cardiovascular risk factors in diabetic patients. One hundred and two male inpatients with type 2 diabetes without overt nephropathy or insulin use were studied. In a regression analysis of the quartiles of adiponectin, plasma levels of adiponectin were associated positively with HDL-cholesterol and age, and inversely with body mass index and HbA1c, but not with total homocysteine, folate or gamma-glutamyl transferase. Non-traditional cardiovascular risk factors such as homocysteine and folate levels were not associated with adiponectin levels in male type 2 diabetic patients who are not subject to insulin therapy.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) behaves predominantly as an oxoreductase converting the receptor-inactive glucocorticoids to their active forms in vivo, while the type 2 isoform (11beta-HSD2) possesses only dehydrogenase activity and inactivates cortisol in human or corticosterone in rat. We determined enzyme activity of 11beta-HSD in rat lungs from fetus to adult, and examined whether 11beta-HSD1 exists in alveolar type II cells, the most important site for the synthesis of pulmonary surfactant in mature lungs, by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activity of 11beta-HSD1 and 2 in lung tissue homogenate were determined as NADP(+)- and NAD(+)-dependent conversion of corticosterone to 11-dehydrocorticosterone, respectively. We found that 11beta-HSD1 activity was increased progressively from 21 days gestation to 7 weeks after birth. 11beta-HSD2 activity was significantly lower than that of 11beta-HSD1 throughout gestation and after birth. Immunoreactivity for 11beta-HSD1 was detected in the cytoplasm of the cells in the alveolar region of adult rats. Some of these expressing 11beta-HSD1 were considered to be alveolar type II cells, because of their cuboid shape and localization at the corner of the alveoli. RT-PCR demonstrated 11beta-HSD1 mRNA in isolated alveolar type II cells. Our results suggest that alveolar type II cells enhance intracellular glucocorticoid availability via 11beta-HSD1. 11beta-HSD1 in alveolar type II cells is thought of as an autocrine amplifier of glucocorticoid action in the lung.

Progesterone markedly inhibits the functions of human macrophages and T lymphocytes, and acts as an immunosuppressant during pregnancy. It is important to examine progesterone metabolites to understand the overall bioactive properties of this sex steroid. However, progesterone metabolism has not been examined in human immune cells. The human leukemic monoblast U937 cell line exhibits monocytic lineage and provides a valuable model to analyze monocyte-macrophage differentiation. Therefore, in this study, we analyzed progesterone metabolism in U937 cells by thin-layer chromatography. Progesterone was metabolized to 5alpha-pregnan-3beta,6alpha-diol-20-one via 5alpha-dihydroprogesterone and 5alpha-pregnan-3beta-ol-20-one, and 5alpha-pregnan-3beta,20alpha-diol was also detected as a final metabolic product via 20alpha-dihydroprogesterone and 5alpha-pregnan-20alpha-ol-3-one. 5alpha-reduction (5alpha-reductase type 1) and 20alpha-reduction were involved in the first step of metabolism. To identify the enzyme responsible for the 20alpha-reduction, we screened an U937 cDNA library, and obtained a clone (1.2 kb), which was identical to the human hepatic bile acid-binding protein or 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). 293 cells transfected with this cDNA demonstrated marked 20alpha-reduction of progesterone to 20alphaDHP, but 20alpha-oxidative, 3alpha-HSD or 17beta-HSD activity was found to be negligible. In experimental animals, the importance of 20alpha-HSD has been reported to be involved in the protection of immune cells from the toxic effects of progesterone. Therefore, our present data suggest that 20alpha-HSD plays an important role in the regulation of progesterone actions in human immune cells.

We report an 11-year-old girl with growth failure caused by long-term administration of 13-cis-retinoic acid after bone marrow transplantation for neuroblastoma. Her growth velocity was 1-2 cm/year after 13-cis-retinoic acid administration. Her endocrinological findings were normal except for peak growth hormone levels of 6.4 ng/ml (clonidine) and 9.7 ng/ml (arginine). IGF-1 and IGFBP-3 were normal. It is not possible to conclude that her severe growth failure was caused by partial growth hormone deficiency, but premature epiphyseal closure was seen on radiographic examination. We concluded that the growth failure was caused by pediatric cancer therapy for the musculoskeletal system but not by endocrinological disturbance.