Christoph M. Bamberger

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.

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.

Strongly reduced expression of the cell cycle inhibitor p27 in endometrial neoplasia

Abstract In the present study we investigated the expression of the cell cycle inhibitor p27 in endometrial neoplasia using immunohistochemistry with a p27-specific antibody. Expression of p27 in endometrial carcinomas was compared with expression in the normal endometrium throughout the cycle. Normal endometrial cells showed strong nuclear expression of p27. Expression was present throughout the cycle and was stronger during the secretory phase. We found strongly reduced or abolished expression of p27 in endometrial carcinoma (85.3% of cases). The 41 tumours analysed were classified according to p27 staining intensity and percentage of positive cells into the following categories of p27 expression: negative/very low (56.0%); low (29.3%); moderate (14.7%) and high (0.0%). All the p27-positive tumours were well-differentiated endometrioid carcinomas of malignancy grade G1. Comparison with the p53 status showed that all tumours with strong p53 expression had low/negative p27 staining, while those that were positive for p27 had negative/low p53 staining. Reduced or absent p27 levels were also observed by Western blot analysis both in tumour samples and in HEC-1B endometrial adenocarcinoma cells. It thus seems that p27 expression is essential for the control of normal endometrial proliferation, and reduced or absent p27 expression may be an important step in endometrial carcinogenesis.

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.

Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma.

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin αvβ3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin αvβ3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI‐h295 cells (>six‐fold increase, p < 0.001). Transfection with integrin αvβ3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti‐integrin β3 antibody, indicating a functional relationship of OPN and integrin αvβ3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan‐Meier analysis including 111 patients with primary tumours graded for OPN staining and follow‐up data available. In conclusion, our in vitro data indicate that OPN and integrin αvβ3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue. Copyright

PKC Isoenzyme expression and cellular responses to phorbol ester in JEG-3 choriocarcinoma cells

Protein kinase C (PKC) is a key regulatory enzyme involved in the transduction of extracellular growth signals to the cell nucleus. It occurs in several isoforms, the exact functional roles of which have not been established as yet. The tumor-promoting agent 12-O-tetradecanoyl-phorbol acetate (TPA) is the classic activator of PKC and modulates the activity of the activating protein-1 (AP-1) transcription factor complex via this pathway. AP-1, in turn, induces cell proliferation in many tissues. In the present study, the PKC isoenzyme expression pattern in JEG-3 choriocarcinoma cells was analyzed. The results were compared with those obtained in HEC-1B endometrium adenocarcinoma cells, which had previously been characterized in this respect. To gain insight into the possible functional consequences of different PKC expression patterns, cell proliferation rates and AP-1 activity in response to TPA in both cell lines was studied. Western blot analysis of the PKC isoenzyme expression pattern revealed that JEG-3 cells are deficient in the PKC α, δ, and ε isoforms. These isoenzymes are strongly expressed in HEC-1B cells, with the α and δ being constitutively active. As opposed to HEC-1B cells, JEG-3 cells did not show an enhanced proliferation rate in response to TPA. Furthermore, TPA-treated JEG-3 cells did not exhibit any change in cell shape and refractility as observed in HEC-1B cells. AP-1 activity, as determined by a transfected AP-1 luciferase reporter plasmid, was induced 10-fold by TPA in JEG-3 cells, yet only threefold in HEC-1B cells. It is concluded from these data that differential expression of a subset of PKCs, e.g., the α, δ, and ε isoforms, may serve as an indicator of the proliferative potential in response to growth factors and mitogens. Furthermore, our data indicate that the inducibility of AP-1 activity does not necessarily reflect the proliferative capacity of a given cell type in response to classical tumor promoters such as phorbol ester.

[Mesenterial lymphangiolipoma - a rare finding in an asymptomatic patient].

BACKGROUND Lymphangioma is an uncommon tumor, an intraperitoneal lymphangiolipoma is exceedingly rare. These tumors are principally benign, but lead to complications due to their size and localization. CASE REPORT A 46 year old male patient presented for a regular medical check up. Apart from a hearing loss 2006 and 2008 he reported no previous or chronic diseases. An extensive health examination had been performed two years ago and had been without pathological results. Abdominal ultrasound revealed a large polycystic lesion in the right middle and lower abdomen, approximately 12x10x7 cm in size. There was no vascularisation in the septae. In MRI, the tumor appeared cystic as well without communication to the intestinal wall. Laboratory values including echinococcus serology was without pathological results. An explorative laparotomy was done with right hemicolectomy and subsequent ileotransversostomy. Histologically, a lymphangiolipoma was diagnosed, as well as a chronic appendicitis and chronic lymphangitis of the ileocolic lymph nodes. Postoperatively, the patient recovered without any complications. CONCLUSION Lymphangiomas, especially lymphangiolipomas, are an extremely rare differential diagnosis of intraabdominal cystic tumors. Potential complications included ileus, intussusception or an immuring growth. Abdominal ultrasound can reveal important pathological findings even in symptom- free patients.ZusammenfassungHintergrund:Lymphangiome, speziell Lymphangiolipome sind extrem seltene Tumoren im Mesenterium. Es sind benigne Raumforderungen mit vorwiegender Manifestation in der Kindheit, die aber aufgrund ihrer Größe, eines ummauernden Wachstums und ihrer Rezidivneigung häufiger zu Problemen führen können.Fallbeschreibung:Ein 46-jähriger Patient stellte sich beschwerdefrei zu einer internistischen Vorsorgeuntersuchung vor. Bis auf zwei Hörstürze 2006 und 2008 war die Vorgeschichte leer. Eine umfassende Voruntersuchung 2 Jahre zuvor war ohne auffälligen Befund geblieben. In der abdominellen Sonographie fiel eine große (ca. 12x10x7 cm) cystische mehrfach septierte Raumforderung mit echofreiem Inhalt ohne Vaskularisation im rechten Mittel- und Unterbauch auf. Ein umfassendes internistisches Routinelabor incl. Echinococcusserologie (der Patient war Jäger) war unauffällig. Auch in der abdominellen MRT zeigte sich eine zystisch-liquide Raumforderung ohne Vaskularisation der Septen, eine Verbindung zum Darm oder zu anderen Organen war nicht nachweisbar. Es erfolgte die explorative Laparatomie mit Hemicolektomie rechts und Anlage einer Ileotransversostomie. Die Histologie ergab die Diagnose eines Lymphangiolipoms, einer chronischen Appendicitis sowie einer chronischen Lymphadenitis der ileocolischen Lymphknoten ohne Hinweis für Malignität oder Spezifität. Der postoperative Verlauf war unkompliziert.Schlussfolgerung:Das Lymphangiom bzw. die Subspezifität Lymphangiolipom ist eine seltene Differentialdiagnose cystischer intraabdomineller Tumoren. Mögliche Komplikationen sind Ileus, Invaginationen und auch ein ummauerndes Wachstum. Abdomineller Ultraschall erhebt in der Vorsorge wichtige Befunde auch bei beschwerdefreien Patienten.AbstractBackground:Lymphangioma is an uncommon tumor, an intraperitoneal lymphangiolipoma is exceedingly rare. These tumors are principally benign, but lead to complications due to their size and localization.Case Report:A 46 year old male patient presented for a regular medical check up. Apart from a hearing loss 2006 and 2008 he reported no previous or chronic diseases. An extensive health examination had been performed two years ago and had been without pathological results. Abdominal ultrasound revealed a large polycystic lesion in the right middle and lower abdomen, approximately 12x10x7 cm in size. There was no vascularisation in the septae. In MRI, the tumor appeared cystic as well without communication to the intestinal wall. Laboratory values including echinococcus serology was without pathological results. An explorative laparotomy was done with right hemicolectomy and subsequent ileotransversostomy. Histologically, a lymphangiolipoma was diagnosed, as well as a chronic appendicitis and chronic lymphangitis of the ileocolic lymph nodes. Postoperatively, the patient recovered without any complications.Conclusion:Lymphangiomas, especially lymphangiolipomas, are an extremely rare differential diagnosis of intraabdominal cystic tumors. Potential complications included ileus, intussusception or an immuring growth. Abdominal ultrasound can reveal important pathological findings even in symptom- free patients.

MOLEKULARE UND KLINISCHE ENDOKRINOLOGIE DES ENDOMETRIUMS

ZusammenfassungDie Sexualhormone Östradiol und Progesteron wirken auf molekularer Ebene über intrazelluläre Rezeptoren, die nach Bindung ihrer Liganden in den Zellkern translozieren und dort an die Promotorregion verschiedener Zielgene binden. Die Folge ist eine veränderte Transkriptionsrate und damit eine veränderte Produktion der entsprechenden Proteine. Zu den Zielgenen der Sexualhormone gehören u.a. Zytokine und Wachstumsfaktoren wie z.B. TGF-β, IL-1, CSF-1 und LIF. Die zeitliche Gangart und Aktivität der Steroidogenese, Rezeptormodulation und Transkription spiegeln sich morphologisch in den bekannten Proliferations- und Differenzierungsvorgängen im Endometrium wider. Umgekehrt gehen diese und deren Störungen mit biochemisch faßbaren, quantitativen und/oder qualitativen Veränderungen einher. Noch ist aber der endokrinologische Laborbefund weit weniger als die Vaginalsonographie Ausgangspunkt der Entscheidung zur Endometriumbiopsie oder Abrasio. Große Bedeutung kommt diesem Zusammenhang in der Perimenopause zu. In ≥90% der Frauen, die wegen des sonographischen Verdachts auf ein Endometriumkarzinom abradiert werden, läßt sich morphologisch nur ein „klimakterisches Übergangsendometrium” feststellen. Eine Ausnahmesituation besteht allerdings bei Frauen unter Behandlung mit Tamoxifen. Wegen des erhöhten Karzinomrisikos sollten diese ab einer sonographischen Endometriumdicke von ≥5 mm grundsätzlich histologisch untersucht werden.SummaryThe ovarian steroid hormones estradiol and progesterone exert their effect by interacting with their intracellular receptors, which, after ligand binding translocate to the nucleus and bind to the promoter regions of target genes. The consequence is a change in the transcription rate of the target genes, followed by a change in production of the corresponding proteins. Target genes of the sexual steroid hormoness include cytokines and growth factors, among them CSF-1, TGF-β and LIF. The rhythm and activity of steroidogenesis, receptor modulation and transcription are reflected by cycle-specific proliferation and differentiation processes in the endometrium. Quantitative and/or qualitative molecular endocrinology is of increasing interest for better definition of morphological changes, although, as yet, the pathological laboratory test is of much less practical consequence than a suspicious vaginal sonography. In spite of the high standard of ultrasound techniques, however, most cases with slightly increased endometrial thickness show histologically benign changes of the endometrium rather than endometrial precancer or cancer. This is especially true for perimenopausal women with no other clinical findings. Yet, the cancer risk is increased in women under tamoxifen therapy. Hence, as a rule, these cases, when endometrial thickness exceeds 5 mm, need a diagnostic biopsy or abrasio.

Mesenteriales Lymphangiolipom – eine Rarität als Zufallsbefund

BACKGROUND Lymphangioma is an uncommon tumor, an intraperitoneal lymphangiolipoma is exceedingly rare. These tumors are principally benign, but lead to complications due to their size and localization. CASE REPORT A 46 year old male patient presented for a regular medical check up. Apart from a hearing loss 2006 and 2008 he reported no previous or chronic diseases. An extensive health examination had been performed two years ago and had been without pathological results. Abdominal ultrasound revealed a large polycystic lesion in the right middle and lower abdomen, approximately 12x10x7 cm in size. There was no vascularisation in the septae. In MRI, the tumor appeared cystic as well without communication to the intestinal wall. Laboratory values including echinococcus serology was without pathological results. An explorative laparotomy was done with right hemicolectomy and subsequent ileotransversostomy. Histologically, a lymphangiolipoma was diagnosed, as well as a chronic appendicitis and chronic lymphangitis of the ileocolic lymph nodes. Postoperatively, the patient recovered without any complications. CONCLUSION Lymphangiomas, especially lymphangiolipomas, are an extremely rare differential diagnosis of intraabdominal cystic tumors. Potential complications included ileus, intussusception or an immuring growth. Abdominal ultrasound can reveal important pathological findings even in symptom- free patients.ZusammenfassungHintergrund:Lymphangiome, speziell Lymphangiolipome sind extrem seltene Tumoren im Mesenterium. Es sind benigne Raumforderungen mit vorwiegender Manifestation in der Kindheit, die aber aufgrund ihrer Größe, eines ummauernden Wachstums und ihrer Rezidivneigung häufiger zu Problemen führen können.Fallbeschreibung:Ein 46-jähriger Patient stellte sich beschwerdefrei zu einer internistischen Vorsorgeuntersuchung vor. Bis auf zwei Hörstürze 2006 und 2008 war die Vorgeschichte leer. Eine umfassende Voruntersuchung 2 Jahre zuvor war ohne auffälligen Befund geblieben. In der abdominellen Sonographie fiel eine große (ca. 12x10x7 cm) cystische mehrfach septierte Raumforderung mit echofreiem Inhalt ohne Vaskularisation im rechten Mittel- und Unterbauch auf. Ein umfassendes internistisches Routinelabor incl. Echinococcusserologie (der Patient war Jäger) war unauffällig. Auch in der abdominellen MRT zeigte sich eine zystisch-liquide Raumforderung ohne Vaskularisation der Septen, eine Verbindung zum Darm oder zu anderen Organen war nicht nachweisbar. Es erfolgte die explorative Laparatomie mit Hemicolektomie rechts und Anlage einer Ileotransversostomie. Die Histologie ergab die Diagnose eines Lymphangiolipoms, einer chronischen Appendicitis sowie einer chronischen Lymphadenitis der ileocolischen Lymphknoten ohne Hinweis für Malignität oder Spezifität. Der postoperative Verlauf war unkompliziert.Schlussfolgerung:Das Lymphangiom bzw. die Subspezifität Lymphangiolipom ist eine seltene Differentialdiagnose cystischer intraabdomineller Tumoren. Mögliche Komplikationen sind Ileus, Invaginationen und auch ein ummauerndes Wachstum. Abdomineller Ultraschall erhebt in der Vorsorge wichtige Befunde auch bei beschwerdefreien Patienten.AbstractBackground:Lymphangioma is an uncommon tumor, an intraperitoneal lymphangiolipoma is exceedingly rare. These tumors are principally benign, but lead to complications due to their size and localization.Case Report:A 46 year old male patient presented for a regular medical check up. Apart from a hearing loss 2006 and 2008 he reported no previous or chronic diseases. An extensive health examination had been performed two years ago and had been without pathological results. Abdominal ultrasound revealed a large polycystic lesion in the right middle and lower abdomen, approximately 12x10x7 cm in size. There was no vascularisation in the septae. In MRI, the tumor appeared cystic as well without communication to the intestinal wall. Laboratory values including echinococcus serology was without pathological results. An explorative laparotomy was done with right hemicolectomy and subsequent ileotransversostomy. Histologically, a lymphangiolipoma was diagnosed, as well as a chronic appendicitis and chronic lymphangitis of the ileocolic lymph nodes. Postoperatively, the patient recovered without any complications.Conclusion:Lymphangiomas, especially lymphangiolipomas, are an extremely rare differential diagnosis of intraabdominal cystic tumors. Potential complications included ileus, intussusception or an immuring growth. Abdominal ultrasound can reveal important pathological findings even in symptom- free patients.

The efficacy of forceful ankle and toe exercises to increase venous return: A comprehensive Doppler ultrasound study

Objective Venous stasis is a risk factor for venous thromboembolism. We aimed to determine the efficacy of forceful foot exercises for actuation of the calf muscle pump to counteract stasis. Methods We examined 20 seated healthy subjects. The peak systolic velocity at the level of the popliteal vein was assessed by Doppler ultrasound. Results The mean peak systolic velocity measurements (in cm/s) were as follows: baseline = 5.6; ankle plantar flexion with toe flexion = 91.0; toe touch heel lift = 107.4; ankle dorsiflexion with toe extension = 193.6; isolated flexion of all toes = 118.8; ankle plantarflexion with 100 and 250 Newton forefoot force = 89.9 and 154.5, respectively. Conclusion All exercises achieved significant increases in peak systolic velocity compared to baseline. Ranking showed that forceful ankle dorsiflexion, plantarflexion with 250 Newtons and forceful flexion of all toes yielded the highest mean peak systolic velocity values (193.6, 154.5, and 118.8 cm/s, respectively).