Efficacy of etanercept in combination with methotrexate in moderate-to-severe rheumatoid arthritis is not dependent on methotrexate dosage.

OBJECTIVE: To evaluate the impact of methotrexate (MTX) dosage on clinical, functional and quality of life outcomes in patients with rheumatoid arthritis (RA) from two previous etanercept (ETN) trials after 24 months of treatment. METHODS: Patients with active RA in the ETN+MTX combination treatment arms of the Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes (TEMPO) and COmbination of Methotrexate and ETanercept in Active Early Rheumatoid Arthritis (COMET) studies were pooled in this post hoc analysis and stratified by MTX dosage at 24 months, having MTX monotherapy groups as control: low dose, <10.0 mg/week; medium dose, 10.0-17.5 mg/week; and high dose, >17.5 mg/week. Data from these patient subgroups were included in descriptive summaries of demographic and disease characteristics at baseline. The following outcomes at 24 months were also evaluated for each subgroup: Disease Activity Score in 28 joints (DAS28) low disease activity (LDA) and remission; American College of Rheumatology 20%, 50% and 70% improvement criteria (ACR20, 50 and 70) responses; and changes from baseline in DAS28, Health Assessment Questionnaire Disease Index (HAQ-DI) and EuroQol 5-dimensions visual analogue scale (EQ-5D VAS). RESULTS: Baseline demographics were similar between the low, medium and high MTX dose groups in the ETN+MTX combination and MTX monotherapy arms, with the exception of disease duration (ETN+MTX low 5.5; medium 5.1; high 0.8 years vs MTX low 8.3; medium 4.7; high 0.8 years). Responses to ETN+MTX combination therapy at 24 months were consistently high across MTX dosage groups, with very similar rates of DAS28 LDA/remission and ACR20/50/70. Improvements in DAS28, HAQ-DI and EQ-5D VAS were also not dependent on MTX dosage in the combination treatment arm. CONCLUSIONS: Patients with RA in the TEMPO and COMET trials who received ETN+MTX showed similar efficacy outcomes at 24 months, regardless of MTX dosage. TRIAL REGISTRATION NUMBERS: NCT00195494 (COMET) and NCT00393471 (TEMPO).

Fine-needle aspiration cytology of adrenal masses: a re-assessment with histological confirmation.

BACKGROUND: Fine-needle aspiration (FNA) of adrenal masses is a method currently indicated in lesions suspected of being extra-adrenal in origin; even though its diagnostic reliability has already been determined in many studies, few have used histological examination obtained after adrenalectomy for diagnostic confirmation. AIM: To analyze the diagnostic performance of adrenal FNA in subjects with an available histological confirmation. SUBJECTS AND METHODS: Fifty subjects (26 benign adrenal lesions, 9 primary malignant lesions, and 15 metastatic lesions) who had undergone ultrasound (US)-guided adrenal FNA and then adrenalectomy were re-analyzed retrospectively. RESULTS: FNA guaranteed a sensitivity of 85.7% and a specificity of 100% in all subjects; after having divided the subjects into oncologic and non-oncologic groups, the sensitivity of the test in oncologic patients (100%) increased significantly compared to non-oncologic (57.1%) with no difference in specificity (100% in both groups). Considering also non-diagnostic samples in our analysis (no.=11; 22% of all samples studied), FNA correctly diagnosed malignancy only in 75% of the cases and benignancy only in 66.6%; however, even after including non-diagnostic samples, the percentage of correct malignancy diagnosis remained significantly higher in oncologic (93.3%) than in non-oncologic patients (44.4%) without significant statistical difference between the 2 groups regarding the percentage of correct benignancy diagnosis (respectively 100% and 63.6%). CONCLUSIONS: Our study, based on histological confirmation, underlines the low discriminant value of US-guided adrenal FNA, though the method may have value in oncologic patients.

AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation. AMPK is a alphabetagamma heterotrimer, where alpha is the catalytic subunit. RT-PCR analysis of AMPK subunits in ROS17/2.8 osteoblastic cells and in mouse tibia showed that the AMPKalpha1 subunit is the dominant isoform expressed in bone. We analysed the bone phenotype of 4month-old male wild type (WT) and AMPKalpha1-/- KO mice using micro-CT. Both cortical and trabecular bone compartments were smaller in the AMPK alpha1-deficient mice compared to the WT mice. Altogether, our data support a role for AMPK signalling in skeletal physiology.

Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors.

Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

Role of AMP-activated protein kinase in the control of appetite.

AMP-activated protein kinase is a key enzyme in the regulation of energy metabolism. Its activation has pleiotropic effects in multiple tissues, including increased fatty acid oxidation, glucose uptake and glycolysis, as well as the inhibition of fatty acid and glycogen synthesis and gluconeogenesis, and stimulation of mitochondrial biogenesis. Recently, the AMP-activated protein kinase (AMPK) has also emerged as a regulator of appetite, contributing to the control of energy metabolism at both cell and the whole body levels. Pharmacological and genetic activation or inhibition of hypothalamic AMPK lead to increased or reduced food intake, respectively. AMPK appears to play a role in hypothalamic glucose and nutrient sensing and numerous studies have suggested a role for AMPK in mediating the orexigenic or anorexigenic effects of various endogenous and exogenous substances.

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

Diagnosis and complications of Cushing's syndrome: a consensus statement.

In October 2002, a workshop was held in Ancona, Italy, to reach a Consensus on the management of Cushing's syndrome. The workshop was organized by the University of Ancona and sponsored by the Pituitary Society, the European Neuroendocrine Association, and the Italian Society of Endocrinology. Invited international participants included almost 50 leading endocrinologists with specific expertise in the management of Cushing's syndrome. The consensus statement on diagnostic criteria and the diagnosis and treatment of complications of this syndrome reached at the workshop is hereby summarized.

Cyclical Cushing's syndrome in a patient with a bronchial neuroendocrine tumor (typical carcinoid) expressing ghrelin and growth hormone secretagogue receptors.

A 56-yr-old woman was referred with a diagnosis of Cushing's disease. Hypertension and severe hypokalemia were present and high urinary free cortisol/cortisone ratio was detected, raising a suspicion of an ectopic ACTH syndrome. Inferior petrosal sinus sampling, thoracic computed tomography, and octreotide scans were negative. Remission and relapse periods lasting 3-4 months were observed during the 3.5 yr of follow-up. Finally a thoracic computed tomography scan showed a basal paracardic nodule in the left lung. After surgery, a well-differentiated neuroendocrine tumor (typical bronchial carcinoid) was diagnosed, staining positively for ACTH. RT-PCR revealed expression of proopiomelanocortin, CRH receptor, and V3 vasopressin receptor. Somatostatin receptor type 1, 2, 3, and 5 mRNA was detected only in tumoral tissue. Interestingly, we observed the simultaneous presence of ghrelin and both GH secretagogue (GHS) receptors (1a and 1b) mRNA in tumoral tissue but not in the normal lung. This finding correlates with the in vivo ACTH hyperresponsiveness to hexarelin (a GHS). This is the first report of a cyclical ectopic ACTH-secreting tumor with an in vivo ACTH response to hexarelin coupled with the tumoral expression of ghrelin and GHS receptors. This finding might imply an autocrine/paracrine modulatory effect of ghrelin in bronchial ACTH-secreting tumors.

Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.

Is ovarian and adrenal venous catheterization and sampling helpful in the investigation of hyperandrogenic women?

OBJECTIVE: To audit our practice of performing ovarian and adrenal venous catheterization and sampling in hyperandrogenic women who fail to suppress their elevated androgen levels following a 48-h low-dose dexamethasone suppression test (LDDST). We considered the technical success rate of catheterization, the extra information obtained in addition to the standard biochemical tests and imaging findings, and the impact of sampling on management decisions. DESIGN: A retrospective analysis of the results of all ovarian and adrenal venous catheterizations performed at St Bartholomew's Hospital, London, in the years 1980-1996. PATIENTS AND METHODS: Baseline ovarian and adrenal androgens were measured in all women presenting with symptoms and signs of hyperandrogenism. Those patients who failed to suppress their elevated testosterone (T), androstenedione (A4) and/or dehydroepiandrosterone-sulphate (DHEAS) levels following a LDDST to within the normal range or to less than 50% of the baseline value were investigated further with adrenal computed tomography (CT), ovarian ultrasound, and ovarian and adrenal venous catheterization and sampling. RESULTS: Results were available in 38 patients. The overall catheterization success rate was: all four veins in 27%, three veins in 65%, two veins in 87%. The success rate for each individual vein was: right adrenal vein (RAV) 50%, right ovarian vein (ROV) 42%, left adrenal vein (LAV) 87% and left ovarian vein (LOV) 73%. Eight patients were found to have tumours by means of imaging (adrenal CT and ovarian ultrasound), three adrenal and five ovarian, seven of which underwent operation. In six of these patients the clinical presentation was suggestive of the presence of a tumour; in addition, the combination of imaging findings allowed the detection of suspicious adrenal and ovarian masses in all eight cases. The five patients with ovarian tumours had serum testosterone levels > 4.5 nmol/l. In a further eight patients, laparotomy was performed based on a combination of diagnostic and therapeutic indications; in two of these patients the catheterization results were suggestive of an ovarian tumour. All these eight patients were shown histologically to have polycystic ovarian syndrome (PCOS), and no occult ovarian tumour was identified. None of the patients with nontumourous hyperandrogenism had a baseline testosterone level in excess of 7 nmol/l (median 4.4 nmol/l, range 2.5-7 nmol/l). CONCLUSIONS: Our results suggest that ovarian and adrenal venous catheterization and sampling should not be performed routinely in women presenting with symptoms and signs of hyperandrogenism, even if they fail to suppress their elevated androgen levels to a formal 48-h LDDST. All patients presenting with symptoms and signs of hyperandrogenism and elevated androgen levels, and where the suspicion of an androgen-secreting tumour is high, should have adrenal CT and ovarian ultrasound imaging to detect such a tumour. Venous catheterization and sampling should be reserved for patients in whom uncertainty remains, as the presence of a small ovarian tumour cannot be excluded on biochemical and imaging studies used in this series alone. Its use should be restricted to units with expertise in this area.

Macrophage migration inhibitory factor expression is increased in pituitary adenoma cell nuclei.

Macrophage migration inhibitory factor (MIF) is an essential regulator of the macrophage responses to endotoxin. MIF also has the ability to override the anti-inflammatory actions of glucocorticoids during an immune response, and is thus an important pro-inflammatory factor. The presence of MIF in cells of the anterior pituitary has been described, and high levels of MIF in other rapidly proliferating tIssues have also been demonstrated. It has been hypothesised that MIF release from these cells is influenced by the hypothalamo-pituitary-adrenal axis, and that ACTH and MIF are released simultaneously to exert counter-regulatory effects on cortisol. However, another intracellular role for MIF has also been suggested as it has been shown that MIF exerts an effect on the inhibitory cell cycle control protein p27 through an interaction with Jab1, a protein implicated in p27 degradation. We studied MIF expression in different normal and adenomatous human pituitary samples using immunohistochemistry and RT-PCR. There was evidence of co-immunoprecipitation of MIF with Jab1, suggesting an interaction of the two proteins. Our results showed that there is increased expression of MIF protein in the nuclei of all pituitary adenomas compared with normal tIssue (P=0.0067), but there was no statistically significant difference in nuclear MIF expression between the different adenoma types. Nuclear MIF expression correlated positively with p27 and its phosphorylated form in normal tIssue (P=0.0028 and P<0.0001); however, this relationship was not seen in the adenoma samples. Cytoplasmic expression of MIF was found to be variable both in normal and adenomatous samples, with no consistent pattern. MIF mRNA was demonstrated to be present in all tumour and normal samples studied. Somatotroph tumours showed higher MIF mRNA expression compared with normal pituitary or other types of adenomas. In conclusion, MIF is expressed in cell nuclei in pituitary adenomas to a greater extent than in normal pituitary tIssue. We speculate that it may play a role in the control of the cell cycle, but whether its higher level in adenomas is a cause or a consequence of the tumorigenic process remains to be clarified.

Treatment of advanced neuroendocrine tumours using combination chemotherapy with lomustine and 5-fluorouracil.

OBJECTIVE: Combination chemotherapy with the two agents streptozotocin (SZT), which is a nitrosurea, and 5-fluorouracil (5-FU), an alkylating agent, has a long-established role in the treatment of neuroendocrine tumours; however, it is often accompanied by considerable toxicity, and it has not been assessed in a comparative manner with other current chemotherapy regimens. In order to assess the therapeutic response and adverse effects using an alternative nitrosurea, lomustine (CCNU), which has a different side-effect profile, in combination with 5-FU, we have reviewed all patients with neuroendocrine tumours who received this form of treatment in our department. DESIGN: Retrospective analysis of the case notes of patients with metastatic neuroendocrine tumours who received treatment with the combination of CCNU and 5-FU, and who were followed up according to a defined protocol in a given time frame. PATIENTS: Thirty-one patients with metastatic neuroendocrine tumours (18 with carcinoid tumours, five islet-cell tumours, five chromaffin-cell tumours and three medullary carcinoma of the thyroid) treated with the combination of CCNU and 5-FU, and when necessary additional therapy, over a 22-year period, were included in this analysis. MEASUREMENTS: The symptomatic, hormonal and tumoural responses before and after chemotherapy with the combination of CCNU and 5-FU over a median follow-up duration of 25 months (range 9-348 months) were recorded. Of the 31 patients (16 males; median age 52 years, range 20-86 years), eight (four males; median age 61 years, range 30-74 years) were treated with the combination of CCNU and 5-FU alone (Group 1), whereas the other 23 patients (12 males; median age 47 years, range 20-86 years) received additional therapy with other chemotherapeutic regimens, somatostatin analogues, alpha-interferon or radiolabelled meta-iodobenzylguanidine (131I-MIBG) therapy (Group 2). RESULTS: A total of 121 therapeutic cycles was administered (mean 3.9, range 1-14 cycles). None of the patients obtained a complete tumour response. A partial tumour response (not a complete but a 50% or greater reduction of all measurable tumour) was seen in six out of the 29 patients (21%) (four out of eight in Group 1 and two out of 21 in Group 2, respectively). There was no tumour progression in eight out of the 29 patients (27.5%) (one out of eight in Group 1 and seven out of 21 in Group 2, respectively). The median survival over the period of the study was 48 months (95% confidence interval, CI, 22-74 months). The overall 5-year survival rate was 42% (95% CI, 17-67%) for all patients and 50% (95% CI, 18-83%) for the carcinoid group alone, according to Kaplan-Meier analysis. A complete or partial symptomatic response was obtained in 12 out of 27 (44%) patients who presented with symptoms (four out of eight in Group 1 and eight out 19 in Group 2, respectively) and a complete or partial hormonal response in eight out of 19 patients (42.1%) who presented with hormonally active disease (two out of four in Group 1 and six out of 15 in Group 2, respectively). Nine out of the 15 (60%) patients with carcinoid tumours who presented with symptoms obtained a symptomatic response, five out of 10 patients (50%) a hormonal response, and four out of 16 (25%) patients a partial tumoural response, respectively. The combination of CCNU and 5-FU was safe and well tolerated. Serious side-effects necessitating the termination of CCNU and 5-FU were seen only in two patients, and mainly consisted of reversible bone marrow suppression. No chemotherapy-related death was recorded. CONCLUSIONS: Chemotherapy with CCNU and 5-FU, either alone or in combination with other therapeutic modalities, produces considerable symptomatic and hormonal improvement and moderate tumour regression/stabilization according to currently accepted WHO criteria, particularly in patients with metastatic gastroenteropancreatic neuroendocrine tumours with minimal adverse effects. However, long-term survival was still relatively poor. It may therefore be a valuable additional therapl was still relatively poor. It may therefore be a valuable additional therapeutic option, particularly for well-differentiated carcinoid and islet-cell tumours, but mainly reserved for when there is no response or progression of the disease after currently available first-line treatment with somatostatin analogues or radiopharmaceuticals.

Ocular metastases secondary to carcinoid tumors: the utility of imaging with [(123)I]meta-iodobenzylguanidine and [(111)In]DTPA pentetreotide.

Ocular metastases from carcinoid tumors are considered rare. They can be the primary presentation of a carcinoid tumor or develop during the course of the disease. The extent of distant metastases from carcinoid tumors correlates with poor prognosis and survival; early detection of metastasis may change the overall management. Radiopharmaceutical-labeled imaging techniques have been widely applied for the detection and localization of such lesions based on isotope uptake by neuroendocrine tumors. Routine application of these imaging modalities may reveal previously unsuspected lesions and may also be used to help stage the disease and to identify patients who may be treated with radiopharmaceuticals. Of 40 patients with carcinoid tumors reviewed in our department since we started routine scanning, we identified 6 (15%) who demonstrated ocular metastases: 5 with obvious lesions and 1 with presumed metastasis according to the results of nucleotide scanning. All 6 were negative on screening with [(123)I]meta-iodobenzylguanidine, whereas 3 of 4 who were screened with [(111)In]octreotide showed positive uptake. All patients responded well to radiotherapy and chemotherapy and did not require surgical treatment. The orbit and its contents appear to be a common site for carcinoid metastasis, and radiopharmaceutical imaging with labeled octreotide is useful in identifying many of these lesions.

Transforming growth factor beta1: implications in adrenocortical tumorigenesis.

TGFbeta1, a multifunctional growth modulator, inhibits the proliferation of epithelial cells. TGFbeta1 signaling is dependent on the heterodimerization of the TGFbeta1 receptor II (TGFbeta1RII) with the TGFbeta1 receptor I (TGFbeta1RI). The cytoplasmic proteins Smads are the mediators of the TGFbeta1 signal. TGFbeta1 regulates adult and fetal adrenal growth and function. Previously we have shown by Northern analysis that TGFbeta1mRNA was well expressed in normal adrenal and in adrenocortical adenomas but reduced in carcinomas. To investigate whether TGFbeta1 receptors may act as tumor suppressors of adrenal tumorigenesis, 16 adenomas and 12 carcinomas were studied. We have used SSCP analysis to scan for inactivating mutations in carcinomas. All tumor samples were negative for somatic alterations of both genes. A competitive RT-PCR system was developed to compare the levels of expression of TGFbeta1, TGFbeta1R-I and TGFbeta1R-II, Smad-2 and Smad-4 genes in all tumors. In our study, we confirmed the presence of reduced levels of TGFbeta1 in carcinomas. On the contrary, Smad-4 gene levels were elevated in carcinomas when compared to that of adenomas. No significant differences were observed in gene expression of TGFbeta1RI and Smad-2. Our results suggest that mutations of TGFbeta1 receptors appear not to be involved in adrenal tumorigenesis. Adrenal carcinomas showed a significant reduction of the TGFbeta1 mRNA levels but on the contrary Smad 4 mRNA levels were significantly increased.