No evidence for relevant QT interval prolongation in mitotane-treated patients with adrenocortical carcinoma.

OBJECTIVE: We designed a single-center retrospective study to assess the QT interval duration and to describe cardio vascular events among patients treated with mitotane for a adrenocortical carcinoma (ACC). DESIGN: We selected 14 patients (6 males and 8 females) that met the following criteria: ACC treated with mitotane, for whom an electrocardiogram (ECG) at baseline (before mitotane initiation) was available and for whom at least one ECG was available during the course of mitotane therapy together with a concomitant mitotane plasma level determination. RESULTS: Mean mitotane plasma level at baseline and after treatment showed a significant increase (mean level increased from 0 to 14.9±2 mg/l). At baseline and before mitotane was initiated all QTc intervals were <450 msec for men and <460 msec for women. During the treatment phase with mitotane, no QTc>470 msec was found in any patients respectively for men and women. In addition, no patient showed any significant QTc prolongation (>5% or >10 msec) at any time during mitotane treatment. During a mean follow-up of 15.9±3.5 months (range 2-45 months). No cardiovascular deaths or hospitalization for cardiovascular events was documented. No torsades de pointes were documented on ECG. No syncope, dizziness, heart failure were observed during follow up. Six out of 14 patients died during the follow-up, in five cases due to the progression of the disease, one patient died suddenly at home during followup. CONCLUSION: This short and retrospective series shows no evidence that mitotane induce any QT prolongation, even when plasma levels are well above the therapeutic window.

Risk factors for conversion and complications after unilateral laparoscopic adrenalectomy.

BACKGROUND: Laparoscopic adrenalectomy (LA) is the procedure of choice for surgical management of most benign adrenal tumours, with a reported overall complication rate around 10 per cent. The aim of this study was to determine predictive factors for postoperative complications and conversion to open surgery after unilateral LA. METHODS: From 1994 to 2009, consecutive patients undergoing unilateral LA by the lateral transabdominal approach were analysed from a prospectively maintained database. A mass larger than 12 cm in diameter and suspected primary adrenal carcinoma were considered contraindications to LA. Predictive factors for postoperative complications and conversion to open surgery were analysed. RESULTS: Some 462 patients were analysed. There were no postoperative deaths. Postoperative complications occurred in 53 patients (11·5 per cent), medical complications in 28, and surgical complications in 33 patients. Six patients underwent reoperation for complications. Multivariable logistic regression analysis showed that conversion to open surgery (odds ratio (OR) 6·20, 95 per cent confidence interval 2·08 to 18·53; P = 0·001) and left-sided tumour (OR 1·89, 1·02 to 3·52; P = 0·044) were independent predictive factors for overall complications. Conversion to open surgery was the only independent predictive factor for medical complications (OR 12·88, 4·21 to 39·41; P = 0·001), and left-sided LA was the only predictive factor for surgical complications (OR 2·22, 1·01 to 4·89; P = 0·047). No factor was predictive of conversion to open surgery. CONCLUSION: In this single-institution study, conversion to open surgery and left-sided tumours were independent predictive factors for overall complications, but none of the variables analysed was predictive of conversion.

[Biology of primary hyperparathyroidism: selective venous sampling]. / Biologie de l'hyperparathyroïdie primaire: prélèvements veineux étagés.

The diagnosis of primary hyperparathyroidism (PHP) is chemical: high level of Parathormone (PTH) in conjunction with hypercalcaemia. In borderline cases with sub-normal plasma PTH and calcium, an oral calcium load test could allow a differential diagnosis from other causes of high PTH. Imaging is required only for PHP. Selective venous sampling can help in localizing a parathyroid adenoma in difficult cases by PTH cartography in the following situations: imaging in favour of an ectopic mediastinal gland or a deep cervical adenoma, persistent or recurrent PHP after first failed surgery with negative neck exploration or unsatisfactory in case of another hypersecreting gland, PHP well diagnosed with indeterminate imaging, symptomatic PHP with normal PTH and negative imaging. Venous blood sampling performed in a vascular radiological department with a quick PTH assay can reveal an area of maximum secretion potentially linked to a nodule localized by previous ultrasound coupled to scintigraphy, giving thus a "biological imaging" study. The association of imaging and biology is an efficient procedure enabling localization of an area of abnormal PTH secretion and characterization of the level of PTH secretion. The area with the highest gradient of PTH concentration can help to protocol CT and MR examination.

Treatment of adrenocorticotropin-dependent Cushing's syndrome: a consensus statement.

OBJECTIVE: Our objective was to evaluate the published literature and reach a consensus on the treatment of patients with ACTH-dependent Cushing's syndrome, because there is no recent consensus on the management of this rare disorder. PARTICIPANTS: Thirty-two leading endocrinologists, clinicians, and neurosurgeons with specific expertise in the management of ACTH-dependent Cushing's syndrome representing nine countries were chosen to address 1) criteria for cure and remission of this disorder, 2) surgical treatment of Cushing's disease, 3) therapeutic options in the event of persistent disease after transsphenoidal surgery, 4) medical therapy of Cushing's disease, and 5) management of ectopic ACTH syndrome, Nelson's syndrome, and special patient populations. EVIDENCE: Participants presented published scientific data, which formed the basis of the recommendations. Opinion shared by a majority of experts was used where strong evidence was lacking. CONSENSUS PROCESS: Participants met for 2 d, during which there were four chaired sessions of presentations, followed by general discussion where a consensus was reached. The consensus statement was prepared by a steering committee and was then reviewed by all authors, with suggestions incorporated if agreed upon by the majority. CONCLUSIONS: ACTH-dependent Cushing's syndrome is a heterogeneous disorder requiring a multidisciplinary and individualized approach to patient management. Generally, the treatment of choice for ACTH-dependent Cushing's syndrome is curative surgery with selective pituitary or ectopic corticotroph tumor resection. Second-line treatments include more radical surgery, radiation therapy (for Cushing's disease), medical therapy, and bilateral adrenalectomy. Because of the significant morbidity of Cushing's syndrome, early diagnosis and prompt therapy are warranted.

[Management of hypercortisolism]. / Prise en charge d'un hypercorticisme.

PURPOSE: Cushing's syndrome is a rare but frequently considered disease. Its diagnosis can lead to some difficulties, including confirming the effective endogenous hypercortisolism and determining its cause. The severity of this disease, the diversity of its complications and the multiple therapeutic options make its management challenging. The aim of this review is to present the most recent data about management of Cushing's syndrome, especially diagnostic approaches and therapeutic options. Our references were obtained by screening MEDLINE database from 1996 to 2006. We also included some anterior reviews and consensus statements. MAIN POINTS: We retained the following points: midnight salivary cortisol is a useful tool in the diagnosis of Cushing's syndrome; the desmopressin test can help to distinguish between Cushing's syndrome and "pseudoCushing's" due to alcohol consumption or psychiatric disorders; cavernous sinus and inferior petrosal sinus sampling is indicated in the evaluation of ACTH-dependent Cushing's syndromes when pituitary imaging is normal or equivocal or when dynamic tests are contradictory; multislice computed-tomography of the chest and the abdomen and somatostatin analogue scintigraphy, eventually combined, are the best imaging procedures in occult ectopic ACTH syndromes; patients with Cushing's disease should be referred to a neurosurgeon experienced in corticotroph adenomas surgery; metabolic consequences of Cushing's syndrome, such as cardiovascular risk factors and osteoporosis need an aggressive treatment. PERSPECTIVES: The incidence of Cushing's syndrome is only 1/100000 per year. However, hypercortisolism is diagnosed by systematic evaluation in 2 to 5% of patients with poorly controlled type 2 diabetes and adrenal incidentalomas. Endocrinological management of the disease improves metabolic disorders in these patients. If these results are confirmed, screening for Cushing's syndrome should be systematically performed in these populations.

MANAGEMENT OF ENDOCRINE DISEASE: Can we cure Cushing's disease? A personal view.

One of today's challenges in endocrinology is the treatment of Cushing's disease: Although pituitary surgery has the potential to 'cure' the patient and restore a completely normal pituitary adrenal axis, there are immediate failures and late recurrences that will ultimately require alternate therapeutic approaches. Their high number is in direct correlation with their serious limitations and they all appear to be 'default options'. This 'personal view' tries to shed some light on the inescapable difficulties of the current treatments of Cushing's disease and to provide some optimistic view for the future where the pituitary adenoma should be the 'reasonable obsession' of a successful therapeutist.

Simultaneous assay of immunoreactive beta-lipotropin, gamma-lipotropin, and beta-endorphin in plasma of normal human subjects, patients with ACTH/lipotropin hypersecretory syndromes, and patients undergoing chronic hemodialysis.

We have studied the relative concentrations of the human immunoreactive (IR) peptides gamma-lipotropin (hgammaLPH, [1-58]hbetaLPH), beta-lipotropin (hbetaLPH), and beta-endorphin (hbetaEND, [61-91]hbetaLPH) using gel exclusion chromatography together with a specific radio-immunoassay (RIA) for hgammaLPH and a RIA that (because hbetaEND is the COOH-terminus of the hbetaLPH molecule) measures both hbetaEND and hbetaLPH on an equimolar basis. In normal subjects, basal plasma IR-hgammaLPH was often undetectable (<12.5 fmol/ml), but ranged up to 21 fmol/ml, and IR-hbetaEND/hbetaLPH was 10.8+/-0.7 fmol/ml; previous studies by others suggest that most of the IR-hbetaEND/hbetaLPH was probably hbetaLPH. Both IR-hgammaLPH and IR-hbetaEND/hbetaLPH were significantly elevated (P < 0.001) in patients undergoing chronic hemodialysis (101.5+/-12.7 and 23.8+/-2.0 fmol/ml, respectively). Their IR-hgammaLPH coeluted with standard hgammaLPH as a single peak, and IR-hbetaEND/hbetaLPH coeluted with hbetaLPH; no distinct peak of IR-hbetaEND was observed. In patients with ACTH/LPH hypersecretion due to Addison's disease, Nelson's syndrome, or ectopic ACTH syndrome, IR-hgammaLPH and IR-hbetaEND/hbetaLPH were both elevated, and IR-hbetaEND/hbetaLPH eluted as two peaks, one coeluting with hbetaLPH and the other with hbetaEND. The molar concentrations of all three peptides were significantly correlated with one another. The lower concentrations of endogenous IR-hbetaEND observed may be due in part to its apparent shorter plasma half-life, as estimated in an Addison's patient given a cortisol infusion. The biologic significance of these three peptides in circulating blood is still unknown. The increased levels of hbetaLPH and hgammaLPH in plasma of patients with chronic renal failure suggest that the kidney may be an important organ for their metabolism.

Pituitary-like proopiomelanocortin transcripts in human Leydig cell tumors.

Proopiomelanocortin is a polypeptide precursor molecule, the processing of which generates ACTH, beta-endorphin, the beta- and gamma-lipotropins, the joining peptide, and the NH2-terminal fragment. Anterior pituitary corticotrophs are the major site of proopiomelanocortin gene expression in man and the predominant, if not sole source of circulating ACTH. Recent data have established that proopiomelanocortin gene expression also occurs in various normal nonpituitary tissues, one of the best studied being the testis. In this latter organ the dominant gene products are short transcripts of approximately 800 nucleotides, which lack the first two exons of the gene and cannot encode a complete proopiomelanocortin molecule. In this report we show that the mode of proopiomelanocortin gene expression is occasionally modified in human Leydig cell tumors: a 1,200-nucleotide mRNA species identical to that in the pituitary is produced. It results from the usual (pituitary) start site of transcription and thus can encode the complete proopiomelanocortin molecule. In two out of six tumors, large amounts of the 1,200-nucleotide transcript led to a dramatic increase of approximately 1,000-fold in proopiomelanocortin peptide concentrations as compared with the normal and peritumoral testis. Proopiomelanocortin processing in these tumors generates various peptide fragments including ACTH. These results may help to understand the mechanism of proopiomelanocortin expression in nonpituitary tumors and have implications for the more general phenomenon of ectopic hormone secretion.

Altered proopiomelanocortin gene expression in adrenocorticotropin-producing nonpituitary tumors. Comparative studies with corticotropic adenomas and normal pituitaries.

In order to assess the mechanisms of proopiomelanocortin (POMC) gene expression in human ACTH-producing tumors, we performed the simultaneous evaluation of POMC products and messenger RNA (mRNA) in tissue fragments obtained from two corticotropic adenomas, five nonpituitary tumors, and two normal human pituitaries. The POMC products were examined using a combination of gel exclusion chromatography and four different radioimmunoassays directed against gamma 3 melanocyte stimulating hormone (gamma 3MSH), ACTH, gamma-lipotropin (gamma LPH), and beta-endorphin. The POMCmRNA was detected and analyzed by dot and northern blot hybridization using a single-stranded genomic DNA probe corresponding to the coding region of the human POMC gene. Tissue concentrations of POMC products and mRNA showed parallel distributions. Immunoreactive gamma 3MSH and gamma LPH patterns revealed only 16-kD fragment- and gamma LPH-like peptides in normal and tumoral pituitaries; additional gamma 3MSH- and/or beta MSH-like peptides were found in all five nonpituitary tumors. A single POMCmRNA of approximately 1,200 bases (b) was detected in normal and tumoral pituitaries; a single identical POMCmRNA was also found in four nonpituitary tumors. A thymic carcinoid tumor, in addition to the 1,200-b POMCmRNA, contained equal amounts of a second larger POMCmRNA of approximately 1,450 b. It is concluded that POMC gene expression appears qualitatively unaltered in corticotropic adenomas. In nonpituitary tumors, in contrast, abnormal POMC processing is frequent; in addition, an extra POMCmRNA was detected in a thymic tumor with a greater length than the normal mRNA; the mechanisms and pathophysiological implications of these modifications remain to be elucidated.

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome.

A novel mutation in DAX1 causes delayed-onset adrenal insufficiency and incomplete hypogonadotropic hypogonadism.

Mutations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). In affected boys, primary adrenal insufficiency occurs soon after birth or during early childhood; HHG is recognized at the expected time of puberty. In this report, we describe the novel phenotype of a man who presented with apparently isolated adrenal insufficiency at 28 years of age. Examination revealed partial pubertal development and undiagnosed incomplete HHG. Gonadotropin therapy did not improve his marked oligospermia, suggesting a concomitant primary testicular abnormality. Genomic analysis revealed a novel missense mutation, I439S, in DAX1. The mutant DAX-1 protein was studied for its ability to function as a transcriptional repressor of target genes. Consistent with the patient's mild clinical phenotype, the I439S mutation conferred intermediate levels of repressor activity of DAX-1 when compared with mutations associated with classic AHC. This unique case extends the clinical spectrum of AHC to include delayed-onset primary adrenal insufficiency in adulthood and milder forms of HHG. Furthermore, in accordance with findings in Ahch (Dax1) knockout mice, the clinical features in this patient suggest that DAX-1 function is required for spermatogenesis in humans, independent of its known effects on gonadotropin production.

Luteinizing hormone pulsatility in patients with major ovarian hyperandrogenism.

Hyperandrogenism and ovulatory dysfunction are common in women with either polycystic ovary (PCOS) or ovarian virilizing tumor. However, contrasting with the numerous studies that have extensively described gonadotropin secretory abnormalities, principally increased LH pulse amplitude and frequency, few studies have concerned gonadotropin secretion in patients with ovarian virilizing tumors; low gonadotropin levels have occasionally been reported, but never extensively studied. The goal of the present study was to further evaluate the pulsatility of LH secretion in women with ovarian virilizing tumor compared with that of PCOS patients. Eighteen women with major hyperandrogenism (plasma testosterone level >1.2 ng/ml) were studied (5 women with ovarian virilizing tumor, 13 women with PCOS, and 10 control women). Mean plasma LH level, LH pulse number and amplitude were dramatically low in patients with ovarian tumors when compared to both PCOS (p<0.001) and controls (p<0.001). In case of major hyperandrogenism, LH pulse pattern differs markedly between women with ovarian virilizing tumor or PCOS, suggesting different mechanisms of hypothalamic or pituitary feedback.

From benign adrenal incidentaloma to adrenocortical carcinoma: an exceptional random event.

New European guidelines for the management of adrenal incidentalomas were recently released. One of the most novel recommendations is to stop following patients when they present a typical, small and non-secreting adenoma. We report here the case of a 71-year-old man with such an adenoma, who developed an adrenocortical carcinoma (ACC) fourteen years later, with subsequent metastases and death. Clinically, he had a normal blood pressure and no sign of hormonal hypersecretion. The hormonal work-up showed no hormone excess: urinary free cortisol level was normal, the diurnal cortisol rhythm was respected and urinary catecholamine metabolites levels were normal. Computed tomography (CT) scan showed a homogeneous lesion, with a low density. The lesion remained unchanged during the five years of follow-up. Eight years after the last CT, a large right heterogeneous adrenal mass was incidentally discovered during an ultrasound examination. On CT scan, it was a 6 cm heterogeneous tumor. On hormonal work-up, there was no secretion. The patient was operated of an adrenalectomy, and the histology described an ACC with a Weiss score at 8, with no benign contingent. To our knowledge, this is the first case of an ACC occurring in a patient with prior adrenal imaging showing a typical benign adenoma.

RT-PCR analysis of corticotroph-associated genes expression in carcinoid tumours in the ectopic-ACTH syndrome.

OBJECTIVE: ACTH is frequently produced in non-pituitary tumours, leading to the ectopic-ACTH syndrome, but the molecular mechanisms of its expression remain obscure. This study was aimed at understanding the transcription mechanisms of the ACTH-precursor gene in carcinoid tumours of the lung or thymus. DESIGN: Transcripts coding for a series of corticotroph-associated transcription factor genes were detected, together with markers of the corticotroph phenotype. We studied a series of 41 carcinoid tumours including 15 with proven ectopic-ACTH syndrome. METHODS: Specific RT-PCR reactions were designed for each gene including alternatively spliced isoforms. RESULTS: The markers of the corticotroph phenotype were detected in all ACTH-positive tumours. Expression of the Tpit and Pitx1 genes were not restricted to ACTH-positive tumours but were also detected in many ACTH-negative carcinoids. Only a subset of ACTH-negative tumours expressed NAK-1/Nur77, and NeuroD1 expression was detected in approximately 50% of the tumours regardless of their secretory status. The glucocorticoid receptor alpha was detected in every tumour in contrast to its beta isoform detectable in a few tumours only. Chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and peroxisome proliferator-activated receptor (PPAR) gamma2 were expressed in 50% of the tumours of each group whereas PPARgamma1 was expressed in almost every tumour. CONCLUSIONS: ACTH-positive carcinoids do not share a characteristic expression pattern of the corticotroph-associated transcription factor genes, suggesting that the transcriptional mechanisms of the ACTH-precursor gene differ from those in normal pituitary corticotrophs. Expression of Tpit and Pitx1 genes in most carcinoids suggests that some aspects of the pituitary corticotroph phenotype may belong to general carcinoid differentiation.

High levels of tyrosine phosphorylated proto-ret in sporadic phenochromocytomas.

Pheochromocytomas are tumors originating from chromaffin cells, the large majority of which are sporadic neoplasms. The genetic and molecular events determining their tumorigenesis continue to remain unknown. On the other hand, RET germ-line mutations cause the inheritance of familial tumors in multiple endocrine neoplasia (MEN)-2 diseases, which account for a minority of pheochromocytomas. We investigated the expression of the RET gene in 14 sporadic tumors harboring no activating mutations. A subset of highly RET-expressing tumors (50%) could be distinguished. They showed RET transcript, protein amounts as well as Ret-associated phosphotyrosine levels similar to those measured in MEN-2A-associated pheochromocytomas. We also determined the GDNF and GDNF family receptor alpha (GFRalpha)-1 transcript levels in tumors and in normal tissues. Whereas the GFRalpha-1 transcripts were detected at similar levels in normal tissues and in tumors, GDNF was frequently found expressed in sporadic tumors at levels several times higher than in controls. These results led us to propose the existence of an autocrine or paracrine loop leading to chronic stimulation of the Ret signaling pathway, which could participate in the pathogenesis of a number of sporadic pheochromocytomas.

Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors.

Genetic alterations, such as loss of heterozygosity (LOH) at the 17p13 and 11p15 loci and overexpression of the insulin-like growth factor (IGF)-II gene, are associated with the malignant phenotype in sporadic adrenocortical tumors. A high risk of recurrence after surgery for adrenocortical tumors is predicted in cases with regional invasion or distant metastases. However, patients with localized tumors also have a high risk of recurrence. Reliable prognostic markers are required to identify subjects at high risk of recurrence. The aim of this study was to assess the prognostic value of three molecular markers (17p13 LOH, 11p15 LOH, and overexpression of the IGF-II gene) by assessing disease-free survival in a large series of adult patients with sporadic adrenocortical tumors. Adult patients (114) were prospectively followed up from diagnosis of the disease to June 1999 or to death. Malignancy was initially diagnosed in 18 patients (McFarlane stage III: n = 1 and stage IV: n = 17). The remaining 96 patients with localized adrenal disease at diagnosis (stage I: n = 60 and stage II: n = 36) were at risk of recurrence. Histological grade was assessed according to Weiss criteria, and tumors were classified into two groups (Weiss score or=4). Tumor samples were analyzed for LOH at the 17p13 and 11p15 loci and for IGF-II gene mRNA content. 17p13 LOH was a strong predictor of shorter disease-free survival in univariate analysis (P = 0.001; relative risk, 27), as were histological grade (Weiss score >or=4; P = 0.00001; relative risk, 15), 11p15 LOH (P = 0.004; relative risk, 9), tumor size (size >5 cm; P = 0.006; relative risk, 18), and overexpression of the IGF-II gene (P = 0.01; relative risk, 5). In a Cox proportional hazards regression model, histological grade (P = 0.04; relative risk, 4.2) and 17p13 LOH (P = 0.009; relative risk, 21.5) were independently associated with recurrence. Molecular markers, particularly 17p13 LOH, are predictive of long-term outcome in patients with sporadic adrenocortical tumors. In patients who have undergone curative surgery, routine assessment of these tumor markers is a useful complement to histological scoring for predicting recurrence and guiding decisions for subsequent follow-up and management.

Analysis of the human proopiomelanocortin gene promoter in a small cell lung carcinoma cell line reveals an unusual role for E2F transcription factors.

The small cell lung carcinoma (SCLC) cell line DMS-79 has been used as a model for studying the molecular mechanism underlying the ectopic ACTH syndrome. We previously showed that two domains of the human Proopiomelanocortin (POMC) gene promoter were specifically active in DMS-79 cells. The present study focuses on the more distal one, Domain IV (-376/-417). DNaseI footprinting experiments identified a single binding site for DMS-79 cell proteins in this domain. Gel-shift and sequence analysis indicated that E2F proteins might bind this site. Indeed, proteins from DMS-79 cells which bind this site (i) have in vitro DNA binding properties indistinguishable from those of E2F proteins (ii) form, like E2F proteins, multiprotein complexes which can be dissociated by sodium deoxycholate and (iii) are recognized by antibodies directed against E2F proteins. Further, we show that the rat POMC distal promoter domain contains a homologous sequence which constitutes a natural mutant of the human POMC E2F binding site, since it does not bind E2F. We show by transient transfection that this natural mutant, in the context of the rat POMC promoter, is not active in DMS-79 cells by contrast to the human POMC E2F binding site. We conclude that E2F binding is required for the activity of Domain IV in DMS-79 cells and contributes to the expression of the POMC gene in SCLC. Further studies are required to elucidate the role of E2F factors in POMC gene transcription in SCLC cells, but our results have identified mechanisms different from those in pituitary corticotroph cells that are used by these SCLC tumor cells.

Human joining peptide: a proopiomelanocortin product secreted as a homodimer.

The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules.

Variable modes of proopiomelanocortin gene transcription in human tumors.

We studied the mechanism of POMC gene expression in human nonpituitary tumors that is responsible for the ectopic ACTH syndrome. All tumors contained a 1200-nucleotide (nt) POMC mRNA species identical to that in normal and tumoral pituitaries. In two of six nonpituitary tumors, equivalent amounts of a larger, ca. 1450-nt POMC mRNA species were also present. S1 mapping studies with probes encompassing the three exons of the gene revealed that this larger POMC mRNA species was 5' extended; the other regions were identical to that in the 1200-nt POMC mRNA. In order to analyze the 5'-end of the larger POMC mRNA species, a genomic clone starting at 3.0 kilobases upstream from the usual (pituitary) cap site was obtained, and single-stranded DNA probes were used for S1 mapping studies. They showed several upstream start sites of transcription located at -369, -217, and -108. Analysis of the human genomic sequence showed TATA and GC box-like motifs preceding the -369 and -217 sites and a GC-rich region preceding the -108 site. S1 mapping with a DNA probe, encompassing exon 1 and 93 nt of its 5'-flanking region, allowed quantitative determinations, which showed that the 5'-extended POMC mRNA species accounted for variable proportions of the overall POMC transcripts in different tissues: 0.3% or less in two normal pituitaries, 0.5-3% in five tumoral pituitaries, and up to 35% and 40% in two of six nonpituitary tumors. These results show that variable modes of human POMC gene expression are induced by upstream promotors whose relative activities appear increased in some nonpituitary tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cushing's syndrome and adrenal adenoma. Two surprising associations]. / Maladie de Cushing et adénome cortico-surrénalien. Deux associations étonnantes.

INTRODUCTION: Cushing's syndrome has a very low incidence (1-10 cases/million/year), and familial cases are even more rare. We report on two situations involving different causes of Cushing's syndrome. CASES: In the first case, we describe the case of a patient with an adrenal adenoma 20 years before the occurrence of Cushing's disease related to the pineal gland. In the second case, two members of the same family were diagnosed almost simultaneously with adrenal cortical adenoma (mother) and Cushing's disease (daughter). DISCUSSION: These cases lead us to consider the known causes of familial Cushing's syndrome, which were not found here.