[Ultrasound versus MRI in preventive examinations - a retrospective analysis of 833 patients]. / Stellenwert der Sonografie im Rahmen präventiver Untersuchungen im Vergleich zur Ganzkörper-MRT - eine retrospektive Untersuchung bei 833 Patienten.

PURPOSE: The benefit of ultrasound in comparison with full-body MRI during a medical checkup in preventive health care was examined with regard to the detection of cardiovascular risk factors, metabolic syndrome, malignant tumors and further relevant findings. MATERIALS AND METHODS: 833 consecutive patients (266 f/567 m, age: 19 - 93 y, mean age: 56.6 y) underwent both ultrasound (extracranial carotid arteries, thyroid, abdominal ultrasound and echocardiography) and whole-body MRI (whole-body MR angiography, head, thorax, abdomen and virtual colonoscopy). For ultrasound examinations, DEGUM level III devices were used (Siemens Acuson Antares, Siemens G60, Siemens, Erlangen). MRI examinations were performed using a 1.5 Tesla MRI device (Siemens Avanto, Siemens, Erlangen). All patients were reviewed retrospectively based on the written reports. RESULTS: Ultrasound was much more sensitive in detecting early atherosclerotic changes than MRI angiography. In 33 % of the patients, manifestations of atherosclerosis were found. Thoracic (3) and abdominal aortic and mesenteric artery aneurysms (3) were diagnosed by both methods. Hepatic steatosis as an important risk factor of metabolic syndrome was only found by ultrasound in 20.4 % of our patients. Malignant tumors were rare in this population (1.4 %): all abdominal tumors except one renal oncocytoma were found using both methods. MRI and ultrasound were equally sensitive with respect to the detection of small liver foci. As expected, MRI was less sensitive than ultrasound in the diagnosis of thyroid nodes. For intracranial diagnoses, malignant intrathoracic findings and colonic polyps, ultrasound is not the method of choice. CONCLUSION: For the detection of lifestyle-dependent diseases such as atherosclerosis and metabolic syndrome, ultrasound examination was more sensitive than MRI, and the same was true for the early detection of thyroid diseases. For the detection of malignant abdominal tumors, both methods were equally sensitive. Whole-body MRI can additionally detect pathological changes in the head, lungs and colon.

Very high prevalence of thyroid nodules detected by high frequency (13 MHz) ultrasound examination.

BACKGROUND: The prevalence of thyroid nodules in a healthy population is high: in the German Papillon study, nationwide ultrasound screening of more than 90 000 people using 7.5 MHz scanners revealed the presence of thyroid nodules in 33% of the normal population. A study employing more sensitive 13 MHz scanners has not been conducted so far. MATERIALS AND METHODS: Six hundred and thirty-five consecutive patients (33% female, 67% male, mean 56.7 years) presenting for a preventive health check up underwent ultrasound screening of the thyroid gland (Siemens Acuson Antares, 13 MHz-linear scanner, B-mode and Power mode) and measurement of the basal TSH (thyroid stimulating hormone) value. Size and degree of vascularization of the thyroid gland and of nodules were determined and analysed retrospectively. RESULTS: In 432 of 635 patients, thyroid nodules could be detected with an increasing incidence with age, in 338 without goiter. Mean thyroid size was 12.3 mL for women and 20.5 mL for men correlating strongly with body weight. Fifty-three percentage of the nodules were smaller than 5 mm. Incidence of thyroid dysfunction was only 4%. No cancerous lesions could be found. CONCLUSIONS: Using the 13 MHz technology, we found a substantially higher prevalence of thyroid nodules (68%) than the Papillon study (33%). Even if our population is older than in Papillon, the difference remains in comparable age groups. This is due to the higher sensitivity of 13 MHz scanning. Our study underlines the clinical significance of iodine deficiency and should renew the discussion on routine iodine supplementation.

Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids.

Glucocorticoids play an essential role in maintaining basal and stress-related homeostasis, and lack of glucocorticoid action is incompatible with life in primates. Most known effects of glucocorticoids are mediated by the intracellular GR. The magnitude of a cell's response to glucocorticoids depends both on the hormone level it is exposed to and on its glucocorticoid sensitivity, i.e. the efficiency of GR-mediated signal transduction. In this review, we have summarized the multiple endogenous and exogenous factors that have been shown to be involved in this signaling cascade and, thus, to alter glucocorticoid sensitivity.

Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans.

Alternative splicing of the human glucocorticoid receptor (hGR) pre-mRNA generates two highly homologous isoforms, termed hGR alpha and hGR beta. hGR alpha is a ligand-activated transcription factor which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element (GRE) DNA sequences. In contrast, hGR beta does not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGR beta is able to inhibit the effects of hormone-activated hGR alpha on a glucocorticoid-responsive reporter gene in a concentration-dependent manner. [3H]-Dexamethasone binding studies indicate that hGR beta does not alter the affinity of hGR alpha for its hormonal ligand. The presence of hGR beta in nuclear extracts and its ability to bind to a radiolabeled GRE oligonucleotide suggest that its inhibitory effect may be due to competition for GRE target sites. Reverse transcription-PCR analysis shows expression of hGR beta mRNA in multiple human tissues. These results indicate that hGR beta may be a physiologically and pathophysiologically relevant endogenous inhibitor of glucocorticoid action, which may participate in defining the sensitivity of target tissues to glucocorticoids. They also underline the importance of distinguishing between the two receptor isoforms in all future studies of hGR function and the need to revisit old data.

Expression of urocortin in the extravillous human trophoblast at the implantation site.

Urocortin (UCN) is a 40 amino acid peptide which is closely related to corticotropin-releasing hormone and binds with high affinity to both CRH type 1 and type 2 receptors. UCN is expressed in human reproductive tissues including endometrium, ovary, and placenta. This study was designed to investigate the cellular localization of UCN at the implantation site of the human blastocyst, as well as the regulation of the UCN promoter by two major intracellular signaling pathways, the cAMP/PKA and diacylglycerol/PKC pathways, in cells of placental origin. For this reason, immunohistochemistry was performed on tissue sections from paraffin-embedded human first trimester placentas and freshly isolated human invasive extravillous trophoblast cells (EVT) were analyzed for UCN expression using RT-PCR and immunofluorescence. Finally, UCN promoter activity was analyzed in the JEG3 human choriocarcinoma cell line. Immunohistochemistry revealed expression of UCN in the cytotrophoblast, the EVT and decidual cells. Both UCN mRNA and peptide were detectable in freshly isolated EVT. Finally, a human UCN promoter luciferase reporter construct transfected into JEG3 cells was significantly inducible by phorbol ester plus ionomycin, but not by phorbol ester alone or by forskolin. Collectively, the present study reports the expression of UCN in EVT and the activation of the UCN gene promoter by the diacylglycerol/PKC pathway. The functional significance of urocortin for the physiology of EVT requires further investigation.

[Prevention and anti-aging in endocrinology]. / Prävention und "Anti-aging" mit Hormonen Ersetzen, was im Alter fehlt?

The aging process is associated with a characteristic decline in the levels of certain hormones. In both sexes, growth hormones, melatonin, dehydroepiandrosterone (DHEA) and its sulfate compound DHEAS reach their maximum levels in the third decade of life, and then decline progressively. In addition, a constant decrease in the production of biologically active free testosterone of approximately 1% per year is observed in men. The abrupt cessation of sex hormone production seen in women is not observed in men. Irrespective of the hormone being supplemented, it should always be remembered that not merely the hormone-producing organ, but also the target tissue has aged, and may thus manifest a different reaction to the substituted hormone than youthful tissue.

Expression of the glucocorticoid receptor in the human adrenal cortex.

Glucocorticoids produced in the adrenal cortex act by binding to a specific intracellular protein, the glucocorticoid receptor (GR), which then modulates gene transcription in target tissues. Whether the adrenal cortex itself is a glucocorticoid target tissue has not been analyzed as yet. Since the presence of GR would be a prerequisite for such "intracortical" glucocorticoid action, this study was designed to analyze GR expression in the normal human adrenal gland using RT-PCR, Western blot, and immunohistochemistry. RT-PCR revealed the presence of GR mRNA in adrenal cortex as well as in NCIh295 cells. These results were confirmed at the protein level by Western blot employing a specific anti-human GR antibody. Immunohistochemically, weak GR staining was observed in the adrenal medulla. In contrast, GR was strongly expressed in the adrenal cortex with the zona reticularis showing the most intense staining. Transfection of a GR-responsive luciferase reporter gene into NCIh295 cells resulted in dexamethasone-dependent induction of luciferase activity, indicating that GR is functional in this tissue. In this study, we show for the first time that GR is expressed in the human adrenal cortex. Its preferential expression in the zona reticularis may indicate a functional role in the regulation of adrenal androgen biosynthesis.

The glucocorticoid receptor is specifically expressed in the stromal compartment of the human endometrium.

The human endometrium is a classical target tissue for steroid hormones. While the expression pattern and functional roles of both the estrogen receptor (ER) and the progesterone receptor (PR) are well defined, expression of the glucocorticoid receptor (GR) in this tissue has not been described so far. In the present study, we used immunohistochemistry to analyze the expression of GR in the normal human endometrium throughout the menstrual cycle. The expression of GR was compared to that of ER and PR, which were analyzed in parallel. We show that GR is expressed in the human endometrium with a pattern that markedly differs from the expression patterns of ER and PR. ER and PR are expressed in the nuclei of endometrial glands, whereas GR is completely absent from these structures. However, GR is strongly expressed in the stromal compartment of the endometrium throughout the cycle. Both stromal fibroblasts and lymphocytes are GR-positive. In addition GR expression is also observed in the endothelium of small endometrial blood vessels, which are ER- and PR-negative. Western blot analysis performed on endometrial tumor cell lines of glandular (HEC-1B) and mesodermal (SKUT-1B) origin, respectively, showed GR expression only in the latter. In summary, we demonstrate that GR is expressed in fibroblasts, lymphocytes and endothelial cells of the human endometrial stroma, while it is absent from the glandular compartment. The specific expression pattern of GR within the human endometrium points to a possible functional role of glucocorticoids in the process of decidualization which occurs primarily in the stromal compartment.

Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells.

The Fas (Apo-1/CD95) ligand (FasL) belongs to the tumor necrosis factor family and acts through its receptor (FasR/ Apo-1/CD95) to induce apoptosis in target cells. FasL is expressed in several immunologically privileged sites. Induction of apoptosis by FasL in invading lymphocytes acts as a mechanism of immune privilege and is important in preventing graft rejection. Furthermore, FasL is expressed in certain malignancies and it has been implicated as a possible key mechanism in immune privilege of these tumors. Since the invading placental trophoblast is another very important site with a privileged immune status, we investigated whether FasL is expressed in the normal and tumoral human placenta. For this purpose, mRNA was extracted from first and third trimester human placental samples as well as from JEG3 choriocarcinoma cells and reverse transcribed to obtain cDNAs. These were used as templates for PCR analysis of FasL expression, in which specific primers were employed to amplify an 853 bp fragment spanning the whole FasL coding region. A product of the appropriate length was amplified from normal placenta as well as from the choriocarcinoma cells. Expression of FasL protein was confirmed by Western Blot and was localized to trophoblast by immunohistochemistry using a FasL-specific antibody. Expression of FasL in the human placenta indicates that induction of apoptosis in lymphocytes by the invading trophoblast could be an important mechanism implicated in the immune tolerance of the fetal semi-allograft.

Human lymphocytes produce urocortin, but not corticotropin-releasing hormone.

Hypothalamic corticotropin-releasing hormone (CRH) is the principal regulator of the hypothalamus-pituitary-adrenal axis in mammals. In addition, immunoreactive CRH is also present at peripheral sites, where it is thought to act as a proinflammatory peptide. However, the source of peripheral CRH has remained obscure. Human lymphocytes were shown to produce immunoreactive CRH, yet the data on CRH mRNA expression in these cells are equivocal. More recently, Vaughan et al. discovered a new member of the CRH family, termed urocortin. Urocortin was shown to act through the same receptors as CRH. The current study was designed to investigate both mRNA and protein expression of CRH and urocortin in human lymphocytes. Using a commercial CRH(1-41) radioimmunoassay, we demonstrate that normal human lymphocytes and Jurkat T lymphoma cells produce significant amounts of immunoreactive peptide. However, no CRH mRNA was detectable by RT-PCR in these cells. In contrast, a band of the correct size and sequence was amplified with urocortin-specific primers. Immunocytochemical analysis of human lymphocytes using antibodies that could distinguish between CRH and urocortin revealed significant expression of urocortin but not of CRH, consistent with our RT-PCR data. We conclude that human lymphocytes produce urocortin, but not CRH.

Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes.

The immunosuppressive effects of glucocorticoids (GC) have led to their wide application in the treatment of inflammatory and autoimmune states. However, long term GC treatment is associated with severe side-effects. The development of agents displaying a more favorable ratio of wanted and unwanted GC effects, is, therefore, a major goal of pharmacological and clinical research. In this study, the progesterone receptor agonist medroxyprogesterone acetate (MPA), which also binds to the glucocorticoid receptor (GR), was tested with regard to its immunosuppressive properties. Using a recently established electroporation protocol, we show that MPA (but not progesterone) can suppress a human interleukin-2 (IL-2) promoter-luciferase construct to the same extent as the synthetic GC dexamethasone in normal human lymphocytes. MPA also markedly suppressed IL-2 (as well as IL-1 and IL-6) release, as assessed by specific enzyme-linked immunosorbent assays. In contrast, a highly dexamethasone-inducible glucocorticoid response element-driven promoter construct was only marginally stimulated by MPA in both normal human lymphocytes and HeLa cells. RT-PCR and Western blot analysis of normal human lymphocytes revealed that they do not express progesterone receptor messenger ribonucleic acid and protein, respectively. In contrast, the GR protein was clearly detectable in all samples and was shown to mediate the effects of MPA in transfected Jurkat T lymphoma cells. Our data indicate that 1) MPA can transrepress the human IL-2 gene in normal human lymphocytes in the absence of significant trans-activation; and 2) this effect is mediated by GR. Because of its dissociative GC activity, MPA is a highly promising substance for the treatment of inflammatory/autoimmune states.

Expression of pit-1 messenger ribonucleic acid and protein in the human placenta.

It is well established that the human placenta produces a wide range of hormones similar to those secreted by the pituitary and hypothalamus. However, the physiological role and regulation of placental hormone synthesis and release are still largely unknown. GH (GH-N) is expressed in the pituitary, where it requires the tissue-specific transcription factor Pit-1. Chorionic somatomammotropin A (CS-A) and CS-B as well as the placental GH variant (GH-V), which also belong to the GH gene family and are located in the same chromosomal cluster, are expressed in the placental syncytiotrophoblast. The presence of Pit-1-binding sites in the CS-A and GH-V promoter regions predicts that Pit-1 may be expressed in the placenta. However, this has not yet been demonstrated. To examine possible similarities in the regulation of these genes in the pituitary and placenta, we studied the expression of pit-1 messenger ribonucleic acid (mRNA) in the human placenta, transformed human placental cells, and the JEG-3 choriocarcinoma cell line. Polymerase chain reaction (PCR) products of the expected size were amplified from first and third trimester placentas, transformed placental cells, and JEG-3 complementary DNA by reverse transcription-PCR. The pit-1-specific sequence was confirmed by restriction endonuclease digestion, Southern hybridization, and DNA sequencing. Human pituitary tissue was used as a positive control; no PCR product was obtained from hippocampus (negative control). In situ hybridization of placental tissue sections revealed the presence of pit-1 mRNA in first and third trimester syncytiotrophoblast. Pit-1 protein was localized by immunohistochemistry with the same tissue distribution and a nuclear localization pattern. These data demonstrate expression of pit-1 mRNA and Pit-1 protein in the human placenta, thus questioning its role as a pituitary-specific regulator of GH-N gene transcription. The expression of Pit-1 in the placenta, together with its previously demonstrated capability to bind to and activate the CS-A and the GH-V promoters, suggests that it may play a role in the regulation of hormones belonging to the GH gene family in both pituitary and placenta.

Expression patterns of the cell-cycle inhibitor p27 and the cell-cycle promoter cyclin E in the human placenta throughout gestation: implications for the control of proliferation.

The rapid development of the placenta necessitates a high proliferative potential and cell-division rate. This, coupled with a high capacity for invasion, could confer on the placental tissue a tumour-like character. To exclude this, tight mechanisms of control are necessary for both proliferation and invasiveness. Despite their importance, very little is known about the molecular basis of these mechanisms. The present study was thus designed to investigate the molecular mechanisms implicated in the control of proliferation in the human placenta. We used immunohistochemistry to study the expression of two cell-cycle controlling molecules with opposing effects: the cell-cycle inhibitor, p27, which belongs to the Kip/Cip family of CDK inhibitors and can mediate G1 arrest, and cyclin E, a G1-cyclin esential for G1/S progression. Expression was studied throughout pregnancy in a total of 41 normal human placental samples. In addition, immunohistochemistry for Ki-67 was performed as a control for proliferation. The cell-cycle inhibitor p27 was expressed in the differentiated, non-dividing syncytiotrophoblast, while expression of cell-cycle promoter cyclin E was localized to the nuclei of the cytotrophoblast and correlated well with expression of Ki-67. No cyclin E expression was observed in the syncytiotrophoblast. In conclusion, strong expression of the cell-cycle inhibitor p27 and absence of expression of cyclin E in the syncytiotrophoblast might represent an important control mechanism in placental proliferation. This differentiates it from the proliferation of malignant tumours, where p27 has been shown to be frequently downregulated while cell cycle promoters are overexpressed.

Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin.

The effects of mineralocorticoids and glucocorticoids are mediated by the intracellular mineralocorticoid glucocorticoid receptor (MR) and glucocorticoid receptor (GR), respectively. Several studies suggest that hormone binding and, thus, receptor activation depend on the association of both MR and GR with the 90-kDa heat shock protein (hsp 90). However, there are few reports analyzing the functional relevance of this association in vivo. The present study was designed to determine how the new hsp 90-binding agent geldanamycin, which was previously shown to disrupt the formation of steroid receptor/hsp complexes, interferes with MR- and GR-mediated transactivation in intact cells. We show that geldanamycin inhibits aldosterone-dependent transactivation of a mineralocorticoid-responsive reporter genes in a concentration-dependent manner. Similar effects were observed for the dexamethasone-activated GR. However, geldanamycin did not affect transcription from a retinoic acid-dependent reporter gene. Inhibition of GR-mediated transactivation was observed both in HeLa cells expressing endogenous GR and in COS-7 cells transfected with a GRa expression vector. Binding studies indicate that geldanamycin disrupts receptor function by reducing hormone binding affinity without lowering intracellular receptor protein levels. Our data support the current model of hsp 90-dependent steroid receptor activation. Furthermore, we show for the first time that MR function also depends on the interaction with hsp 90 in intact cells. Finally, we demonstrate that the function of endogenous is thought to keep the receptor protein in an inactive, yet ligand-activable state (9-17). Ligand binding induces a conformational change in the receptor molecule, which causes it to dissociate from the hsp complex, to translocate to the cell nucleus, and, finally, to interact with specific hormone response elements in the promoter regions of hormone-responsive genes (6-8). Both MR and GR bind as homodimers to identical palindromic sequences on the target DNA, termed glucocorticoid response elements (GREs) (18). The formation of GR/MR heterodimers has also been described (19,20) and may have profound functional consequences (21). The current model of MR and GR function holds that these receptors are unable to bind their respective hormones as long as they are not associated with the hsp complex (9-17). However, experimental support for this model is mainly based on in vitro work. There are few reports analyzing the functional relevance of GR/hsp interactions in mammalian cells. In the most recent study, Whitesell et al. showed that the hspE90-binding agent geldanamycin can specifically disrupt GR/hsp association, thus inhibiting glucocorticoid-mediated transcriptional activation (22). MR is even less well studied in this respect. To our knowledge, there have not been any data supporting a functional role for proper MR/hsp interaction in intact cells. In this study, we show for the first time that MR function depends on the interaction with hsp 90 in intact human cells. Furthermore, we demonstrate that geldanamycin inhibits GR-mediated transcriptional activation in two human cells lines, confirming the results by Whitesell et al. and extending them to transfected as opposed to endogenous GR.

Transcriptional regulation of the human 'leukemia inhibitory factor' gene: modulation by glucocorticoids and estradiol.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite its possible importance as a therapeutic target, very little is known about the regulation of human LIF. In particular, its regulation at the promoter level has not been studied so far, and was, therefore, the focus of the present work. After showing that Jurkat T lymphoma cells can be induced to express endogenous LIF mRNA, we used this cell line as a model to study the regulation of the human LIF promoter in transient transfection assays. For this purpose, a 666 bp fragment of the human LIF 5'-flanking region, amplified from genomic DNA by nested polymerase chain reaction (PCR), was used for the construction of a luciferase reporter plasmid (hLIF666-Luc). In unstimulated Jurkat cells, the human LIF promoter showed low constitutive activity. The promoter was induced upon stimulation with phorbol ester (TPA). Combined stimulation with TPA and the calcium ionophore ionomycin resulted in strong synergistic induction of luciferase activity from the LIF promoter. Transfection experiments with deletion constructs (hLIF274-Luc and hLIF82-Luc) located the region required for this induction to a 192 bp portion of the promoter, which carries two putative c-ets binding sites. We then investigated the effect of glucocorticoids and estradiol by cotransfecting the respective receptors. Both hormones strongly inhibited the stimulation of the LIF promoter by TPA and ionomycin. Since LIF is implicated in the pathogenesis of inflammatory and degenerative conditions, such as rheumatoid arthritis and osteoporosis, the finding that therapeutic agents employed in the treatment of such conditions, i.e. glucocorticoids and estrogens, can modulate the induction of LIF at the transcriptional level, is of particular interest.

Regulation of the human interleukin-2 gene by the alpha and beta isoforms of the glucocorticoid receptor.

The immunosuppressive effects of glucocorticoids are largely due to transcriptional inhibition of immunologically relevant genes, such as the interleukin-2 (IL-2) gene. These effects are mediated by the intracellular glucocorticoid receptor (GR). In humans, alternative splicing of the GR precursor mRNA gives rise to two receptor isoforms, termed GRalpha and GRbeta. We previously demonstrated that GRbeta could antagonize GRalpha-mediated transactivation of a glucocorticoid-responsive element (GRE)-driven reporter gene in COS-7 cells. The present study was designed to analyze the roles of the two GR isoforms on glucocorticoid-mediated transrepression of the IL-2 gene. Using a recently developed transfection technique, we demonstrate that in primary human lymphocytes, stimulation of a 548 bp IL-2 promoter-luciferase reporter construct by phorbol ester and calcium ionophore is reversed by dexamethasone to a similar extent as in Jurkat T lymphoma cells transfected with a GRalpha expression vector. Transfection of a GRbeta expression vector alone did not result in IL-2 promoter repression in response to glucocorticoids. Furthermore, GRbeta did not antagonize the repressive effects of GRalpha on IL-2 promoter activity. Surprisingly, overexpression of GRbeta in Jurkat cells did not cause significant inhibition of GRalpha-induced transactivation of a GRE-dependent luciferase reporter gene either. We conclude that the transrepressive effect of glucocorticoids on IL-2 gene transcription is exclusively mediated by GRalpha. GRbeta can neither antagonize GRalpha-mediated transactivation nor transrepression in Jurkat cells, indicating a cell type-specific pattern of GRbeta-mediated antiglucocorticoid activity.

Expression of leukemia inhibitory factor (LIF) and LIF receptor (LIF-R) in the human adrenal cortex: implications for steroidogenesis.

It is well established that steroidogenesis in the adrenal cortex is regulated by extraadrenal factors, such as ACTH and angiotensin II. However, over the last years, it has become increasingly clear that paracrine and autocrine mechanisms are also important for steroid synthesis in the adrenal gland. The current study was designed to analyze whether the pleiotropic cytokine leukemia inhibitory factor (LIF) and/or its receptor (LIF-R) are expressed in the normal human adrenal cortex, and whether they may play a role in regulating steroidogenesis. Using LIF- and LIF-R-specific primers, we show by RT-PCR that both mRNAs are expressed in this tissue, as well as in the NCI-H295 adrenal carcinoma cell line. The correct sequences of the PCR products were verified by restriction enzyme analysis and DNA sequencing. Immunohistochemistry, employing specific antibodies against LIF and LIF-R, reveals expression of both proteins in the normal human adrenal cortex. Finally, we show that LIF can significantly enhance basal and ACTH-induced production of cortisol and aldosterone in NCI-H295 cells. In summary, we show for the first time that LIF and its receptor are expressed in the normal human adrenal cortex. Our stimulation experiments indicate that the intraadrenal LIF/LIF-R system may participate in regulating adrenal steroidogenesis.

Protein kinase C (PKC) isoenzyme expression pattern as an indicator of proliferative activity in uterine tumor cells.

The protein kinase C (PKC) signal transduction pathway is the prototype of a growth factor-responsive intracellular signaling system, which is activated by various cytokines, growth factors and tumor promoters, such as the phorbol ester 12-O-tetradecanoyl-phorbol acetate (TPA). To date, a large number of different PKC isoforms has been identified, the physiological relevance of which is unknown. Moreover, the expression pattern of PKC isoforms in uterine cells has not been studied as yet. To study the functional role of differential PKC isoform expression in uterine tumor progression, we have compared the proliferative response to TPA, changes in cell morphology induced by TPA, and the PKC isoform expression pattern in two uterine tumor cell lines of different origin. The moderately differentiated endometrial HEC-1-B adenocarcinoma cell line showed a marked increase in proliferative activity and a profound morphological change in response to TPA. In contrast, TPA did not induce cell proliferation and/or morphological changes in the well-differentiated SKUT-1-B mixed mesodermal cell line. Analysis of the PKC isoform expression profile by Western blot revealed that PKC alpha, betaI, delta, epsilon, and zeta were expressed at a much higher level in HEC-1-B as compared to SKUT-1-B cells. PKC beta11 was the only isoenzyme to exhibit a higher expression level in SKUT-1-B cells. This is the first study analyzing the PKC isoform expression profile in uterine tumor cells. Our data demonstrate that the proliferative response to TPA correlates with the expression levels of the majority of PKC isoforms in these cells. Overexpression of PKC isoforms indicates a higher proliferative capacity, and may, thus, represent an important step in the pathogenesis of certain uterine malignancies.

Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas.

The molecular mechanisms leading to increased cellular proliferation rates and, thus, tumor formation in the anterior pituitary gland are poorly understood. The cyclin-dependent kinase inhibitor p27Kip1 is a key molecule regulating the G1 phase of the cell cycle in many cell types. Furthermore, it was shown that p27 knock-out mice develop pro-opiomelanocortin-positive pituitary tumors. In an effort to clarify the role of p27 in the normal and tumorous human pituitary, we studied the expression of p27 by immunohistochemistry, using a highly specific mouse monoclonal anti-human p27 antibody. Normal pituitaries and 54 pituitary adenomas (twelve somatotrope adenomas, nine prolactinomas, twelve corticotrope adenomas, three TSH-producing tumors, six gonadotrope adenomas, six null cell adenomas, and six oncocytomas) were analyzed. p27 expression was determined semiquantitatively with regard to both the percentage of positive cells and the intensity of the staining. Normal human pituitaries showed strong expression of p27 in most nuclei. In contrast, the levels of p27 were reduced in the majority of the tumors analyzed. Twenty-two tumors (six somatotrope adenomas, five prolactinomas, four corticotrope adenomas, two TSH-producing tumors, two gonadotrope adenomas, and three null cell adenomas) were completely p27-negative. In 18 tumors, p27 expression was found in < or = 10% of the cells. In the other ten tumors, 11-80% of the cells were p27-positive. In summary, we were able to demonstrate reduced expression levels of the cell-cycle inhibitor p27 in tumors derived from all pituitary cell types. Our data indicate that p27 may be an important regulator of cellular proliferation in the anterior pituitary, the underexpression of which could play a role in pituitary tumorigenesis.

Inhibition of mineralocorticoid activity by the beta-isoform of the human glucocorticoid receptor.

Mineralocorticoids and glucocorticoids are important regulators of electrolyte homeostasis and arterial blood pressure. Their effects are mediated by the mineralocorticoid (MR) and the glucocorticoid receptor (GR), respectively. The present study was designed to determine how the two isoforms of the human GR, the "classic" GR alpha and the non-hormone-binding GR beta, interfere with the transcriptional effects of the hormone-activated human MR. COS-7 monkey kidney cells were transfected with different mineralocorticoid-responsive reporter plasmids and a vector expressing the human MR protein. Different amounts of either control, GR alpha, or GR beta plasmid were co-transfected, and luciferase activity was measured after stimulation with aldosterone and/or dexamethasone. MR-mediated stimulation of transcription was enhanced by co-transfection of the GR alpha expression vector. In contrast, MR-mediated stimulation of transcription was strongly inhibited by co-transfection of equal amounts of the GR beta expression vector. Reverse transcription-polymerase chain reaction (RT-PCR) showed expression of both GR isoforms as well as of MR in the human kidney. These data indicate that the two isoforms of the human GR exert opposite effects on mineralocorticoid activity. We conclude that the ratio between GR alpha and GR beta can define the sensitivity of mineralocorticoid target tissues to aldosterone. Imbalances of this ratio may participate in clinical syndromes of impaired or augmented mineralocorticoid sensitivity, such as certain cases of pseudohypoaldosteronism or, possibly, primary arterial hypertension.