The complete mitochondrial DNA sequence of the pig (Sus scrofa)

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disorder caused by a germline inactivating mutation of the adenomatous polyposis coli (APC) gene. Patients with FAP sometimes develop various extracolonic manifestations including adrenocortical neoplasms. We present a 14-year-old boy with FAP who had an adrenocortical tumor with atypical histopathologic features, ie, sex-cord-like differentiation. Immunohistochemical studies of adrenal 4 binding protein (Ad4BP) and steroidogenic enzymes showed the capacity of these tumor cells to produce steroids. Genetic analysis of the tumor disclosed a two-hit mutation in APC: a germline 5-base pair deletion accompanied by a loss of the normal allele. Because there were no reports of genetic alterations in adrenocortical tumors developed in FAP patients, we examined 10 sporadic adrenal tumors (four carcinomas and six adenomas) for mutations in APC. However, no mutations were found in these 10 sporadic adrenal tumors. These results suggest that mutation of APC is also responsible for some fraction of the adrenocortical tumors: the tumor in this case is included.     

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.     

Nutritional support for the patient with pancreatobiliary disease

The mechanisms which regulate adrenocortical steroidogenesis in differentiated parenchymal cells have been studied in great detail. However, the stem cells that are responsible for regeneration of the adult cortex have never been identified or isolated, and their characteristics are unknown. We have developed a tissue culture system that supports the simultaneous proliferation and differentiation of steroidogenic adrenocortical cells. Utilizing density gradient separation, a cell population composed of a mixture of stromal, endothelial and parenchymal cells (MIX) was isolated from the adult rat adrenal cortex. In primary culture, MIX populations formed high saturation density multilayers from which rounded cells emerged. These cells proliferated, contained lipid, and expressed the steroidogenic enzymes delta 5,3 beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochrome P-450scc. After selective passaging, rounded MIX-derived cells retained their steroidogenic potential, even in the absence of trophic hormone treatment. In contrast, parenchymal cells obtained from the zonae fasciculata (FASC) and glomerulosa (GLOM) respectively, formed homogeneous monolayers in primary culture, gradually de-differentiated, and no longer responded to trophic hormone treatment after passaging. Thus, primary MIX cultures provided a microenvironment that resulted in the production of adrenocortical cells with stem cell-like qualities. These cultures provide for the first time, a system for the identification of specific inducers that are responsible for both adrenocortical cytogenesis and its associated proliferation and steroidogenic differentiation.     

Forskolin treatment directs steroid production towards the androgen pathway in the NCI-H295R adrenocortical tumour cell line

The human adrenocortical tumour cell line, NCI-H295, secretes steroids on the mineralocorticoid, glucocorticoid and adrenal androgen pathways. We have investigated the effects of 96 h treatment of cells in monolayer culture with either forskolin (10 microM) (a direct activator of adenylate cyclase), angiotensin II (10 nM) or no agonist ('control') on the steroidogenic phenotype of this cell line. Androstenedione, cortisol and corticosterone secreted into the medium in response to a subsequent 4 hour treatment with angiotensin II (10nM) indicated that the steroidogenic phenotype of NCI-H295 cells changes away from 17-deoxysteroid biosynthesis towards adrenal androgen production in response to forskolin. The NCI-H295R cell line therefore serves as a useful model for investigation of the differential regulation of the steroidogenic pathways in the human adrenal cortex.     

Interleukin 2 in the treatment of acute leukemia

Recent immunohistochemical analysis of cell cycle-related proteins such as p27, a cell cycle inhibitory protein, and Ki-67, a proliferation marker, indicated their possible values in predicting the biologic behavior of various human neoplasms. In this study, we performed an immunohistochemical analysis of p27 and Ki-67 in 42 adrenocortical neoplasms (12 adrenocortical carcinomas, 24 adrenocortical adenomas) and 6 normal adrenal glands to evaluate their possible values in diagnosing adrenocortical malignancy and in predicting the biologic behavior of carcinomas. We detected Ki-67 and p27 immunoreactivity in the nuclei of all of our cases, and we observed a significant negative correlation (r = -0.572, P < .001) between the p27 and Ki-67 labeling indexes (LIs). The LIs of p27 and Ki-67 were 61.7+/-2.6 and 0.28+/-0.08 in the normal adrenal cortex and 59.4+/-6.5 and 0.33+/-0.11 in the adenomas, respectively, with no significant differences between the LIs of the adenomas and normal adrenals. The LIs of p27 and Ki-67 in the carcinomas were 48.9+/-7.5 and 630+/-6.21, respectively. The LI of p27 in the carcinomas was significantly lower than that in the adenomas. The LI of Ki-67 in the carcinomas was significantly higher than that in the adenomas (P < .01). Among carcinoma cases, the Ki-67 LI in living cases tended to be lower than that in deceased cases, and the p27 LI in living cases tended to be higher than that in deceased cases, but these differences did not reach statistical significance. These results indicated that decreased p27 protein expression might cause increased cell proliferation in adrenocortical carcinoma cells in combination with other positive and/or negative regulators of the cell cycle. These results also suggested that immunohistochemical analysis of p27 and Ki-67 might be useful in distinguishing between adrenocortical adenoma and carcinoma     

Smad3 is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on StAR expression

Transforming growth factor betas (TGFbetas) constitute a family of dimeric proteins that regulate growth and differentiation of many cell types. TGFbeta1 is also a potent autocrine regulator of adrenocortical steroidogenesis. We have recently shown that in primary cultures of bovine fasciculo-reticularis cells, the main target of TGFbeta is the steroidogenic acute relay protein (StAR), a key protein necessary for intramitochondrial cholesterol transport. Here, we show that StAR expression is also inhibited by TGFbeta1 in the human adrenocortical carcinoma cell line NCI-H295R. This inhibitory effect is mediated by Smad proteins. Indeed, we found that overexpression of wild-type Smad3 inhibited endogenous StAR mRNA expression while overexpression of a dominant negative Smad3 protein reversed the inhibitory effect of TGFbeta1 on StAR mRNA expression. Taken together, these results demonstrate that the Smad3 protein is involved in TGFbeta-dependent regulation of steroidogenesis.     

Serum bilirubin distribution and its relation to cardiovascular risk in children and young adults

The discovery of an asymptomatic adrenal mass (incidentaloma) during the investigation of an unrelated condition is relatively common. In this study, we report the clinical, radiologic, and endocrine evaluation of 38 patients (22 women and 16 men aged 24 to 84 years) with adrenal incidentaloma (size, 1 to 12 cm). The patients underwent basal and dynamic evaluation of the hypothalamic-pituitary-adrenal (HPA) axis, renin-angiotensin-aldosterone system, and adrenomedullary function. Moreover, computed tomograpy (CT) scan and 131I-6beta-iodomethyl-19-norcholest-5(10)-en-3beta-ol(NP-59) and/or 131I-metaiodobenzylguanidine (MIBG) scintigraphy were performed. The endocrine evaluation indicated two cases of pheochromocytoma and four cases of preclinical Cushing's syndrome, three of which underwent surgery with histologic diagnosis of two adrenocortical adenomas and one carcinoma. Low levels of serum dehydroepiandrosterone sulfate (DHEA-S), associated with a markedly increased 17-hydroxyprogesterone (17-OHP) response to a corticotropin (ACTH) test, were found in patients with incidentaloma. On the basis of endocrine and morphologic data, 13 patients underwent surgical treatment: five adrenocortical adenomas (two functioning), two pheochromocytomas, two ganglioneuromas, one cortisol-secreting adrenal carcinoma, one lymphangiomatous cyst, one myelolipoma, and one hemorrhage were found. Careful diagnostic assessment of incidentally discovered adrenal masses must be performed to exclude the presence of malignant and/or functioning lesions and to verify the possibility that patients with incidentaloma have a genetic or acquired deficit of adrenal steroidogenic activity.     

Frog chromaffin and adrenocortical cell co-cultures: a model for the study of medullary control of corticosteroidogenesis

Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin-adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin-adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co-cultures. In these co-cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10(-10) M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10-40-fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co-cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.