Recently, evidence is accumulating pointing to a function of the COP9 signalosome (CSN) in regulation of ubiquitination by specific ubiquitin ligases. Here, we demonstrate by mammalian two-hybrid analysis that the transcriptional regulators and substrates of the ubiquitin system Id1 and Id3, but not Id2 and Id4, bind to the CSN subunit CSN5. Pull-down experiments revealed that Id3 physically interacts with the CSN complex. Additional far Western and pull-down studies with Id3 support our two-hybrid data and show that the transcription regulator can bind to CSN5 and CSN7. Recombinant Id3 is not phosphorylated by the CSN-associated kinases CK2 and PKD. However, it inhibits c-Jun and CSN2 phosphorylation by the isolated CSN complex and by the recombinant CK2. The inhibitors of CSN associated kinases, curcumin and emodin, significantly induce ubiquitination and proteasome-dependent degradation of transiently expressed Id3 in HeLa cells. Proteasome-dependent degradation of endogenous Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin. Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-ubiquitin cDNA. Curcumin increased Id3-ubiquitin conjugate formation, as shown by Western blotting and His-pull-downs. In addition, overexpression of CSN2 leads to stabilization of Id3 protein. On the basis of these data, it is speculated that CSN-mediated phosphorylation inhibits ubiquitination of Id1 and Id3.

Kleinkalibrige ePTFE Prothesen mit einem Durchmesser von kleiner als 6 mm zeigen hohe Verschlussraten in vivo, wenn sie als arteriovenöser (AV) Shunt zur Hämodialyse eingesetzt werden. Der Schwerpunkt verschiedenster Untersuchungen zur Senkung der Verschlussraten lag einerseits auf der Verbesserung von Implantationstechniken, andererseits auf dem Tissue Engineering. Neueste Untersuchungen haben gezeigt, dass man das Lumen von ePTFE Prothesen unter Fluss mit autologen Endothelzellen beschichten kann, die den Scherkräften des Blutflusses in vivo wiederstehen. Das Ziel dieser Studie war die Regenertaionsfähigkeit der Endothelzellen in vitro nach wiederholter Punktion zu untersuchen.

Hintergrund. Die Inhibition der Tumorangiogenese könnte einen wichtigen Beitrag in der additiven Therapie von soliden Tumoren darstellen. Leflunomid, ein in Deutschland zur Therapie der rheumatoiden Arthritis zugelassenes Medikament, hemmt in vitro die Wirkung verschiedener Wachstumsfaktoren. Ziel der Untersuchung war die Wirkung des Medikaments auf die Tumorangiogenese im Nacktmausmodell.

Das Keloid des Ohrläppchens ist eine benigne, fibröse, proliferative Erkrankung, die nach chirurgischer Exzision eine hohe Rezidivquote von bis zu 80% zeigt. Ursächlich sind Verletzungen des Ohrläppchens durch Unfälle mit Kopfverletzungen, Stichinzisionen, Verbrennungen oder chirurgische Interventionen. Neben der alleinigen chirurgischen Exzision und Rekonstruktion des Ohrläppchens werden verschiedene adjuvante Methoden in der Literatur zur Rezidivprophylaxe beschrieben. Wir berichten über den Fall einer Patientin mit einer ausgedehnten Keloidbildung nach Ohrlochstechen beider Ohren. Dieser Bericht gibt eine Übersicht über die Prinzipien und Komplikationen der verschiedenen relevanten Behandlungsmodalitäten in der Therapie des Keloid des Ohrläppchens und diskutiert die Frage der Aufklärungspflicht über dieses Risiko vor dem Ohrlochstechen.

Earlobe keloids are benign, fibrous proliferations that show a high rate of recurrence of up to 80% following surgical excision. Traumas to the earlobe such as ear piercing, burns or surgical interventions are important in the pathogenesis of the disease. In addition to surgical keloid excision and reconstruction of the earlobe, several adjuvant therapeutic concepts have been described to prevent recurrence. Here we present the case of a female patient who suffered from severe bilateral keloid development after piercing of both ears. The report gives an overview of the relevant therapeutic concepts in the treatment of earlobe keloids and their possible complications. In addition, the question of written informed consent before ear piercing is discussed.

The inhibition of tumorangiogenesis may be of importance in the additive treatment of various cancers. Leflunomide, a drug which has been approved in Germany for the therapy of rheumatoid arthritis, inhibits the activity of several growth factors in vitro. The aim of this study was to investigate the effects of the drug on tumor angiogenesis in a nude mouse model.A total of 40 nude mice were injected with human colon carcinoma cells. Following randomization in 4 groups, therapy started on day five. Group 1 was treated daily with orally administered Leflunomide (35 mg/kg) dissolved in 1.5% Carboxymethylcellulose (CMC). Group 2 served as a control group and received 1 ml CMC orally per day. The animals of group 3 were treated daily with 35 mg Leflunomide/kg KG and 500 mg Uridine/kg dissolved in 1 ml Nacl 0.9% intraperitoneally. The 4th group again served as a control group and received only 500 mg Uridine/kg intraperitoneally each day. The main outcome criterion was the angiogenesis score (AS). In addition, the tumor volume and tumor weight were also assessed. The AS was determined by immunohistochemistry using an antibody against factor VIII related antigen.All animals tolerated the procedure well. In the Leflunomide and the Leflunomide/Uridine group the angiogenesis score (p < 0.01), the tumor volume (p < 0.01) and the tumor weight (p < 0.01) were lower compared to the respective control groups.The administration of Leflunomide leads to a significant reduction of tumor weight and tumor volume following subcutaneous injection of human colon carcinoma cells in a nude mouse model. This could be due to the reduction of tumor angiogenesis. Following further experimental and clinical studies, Leflunomide may come to play a role in the additive treatment of colonic carcinoma.

Photodynamic therapy is an effective palliative treatment of esophageal cancer. Minor complications associated with this therapy include pleural effusions, fever or esophageal strictures. In addition to this major complications such as respiratory-esophageal fistula and bronchus perforation have been described. We report here our experience with a patient who developed a complete esophageal necrosis and perforation of the left main bronchus following photodynamic therapy. The surgical and intensive care management of the patient is described and the literature discussed.

Clinical report Long-term survival of a patient with small cell-lung cancer (SCLC) following treatment with thalidomide and combination chemotherapy JW Mall, AW Philipp, W. Mall & C. Pollmann  … TECHNICAL REPORT A computer image analysis system for microvessel density measurement in solid tumours Jonathan Charles Goddard, Christopher Derek Sutton, Peter N. Furness, Roger Clive Kockelbergh & Kenneth John O'Byrne … ORIGINAL ARTICLES Human vascular endothelial cells with extended life spans: In vitro cell response, protein expression, and angiogenesis Edith Gagnon, Paola Cattaruzzi, May Griffith, Lea Muzakare, Katell LeFlao, Robert Faure Richard BØliveau, Sabah N. Hussain, Michael Koutsilieris & Charles J. Doillon … A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model Wei Zhang, Sophia Ran, M …

Objective: Vascular endothelial growth factor (VEGF) is an important cytokine in the process of angiogenesis. Elevated serum levels of the cytokine may determine cancer patients who will benefit from adjuvant anti‐angiogenic therapy in the future. To correlate serum levels of VEGF with tumour stage and established prognostic markers in patients with small cell lung cancer (SCLC), a prospective study was performed on 70 patients. Methodology: From August 1999 to May 2000, 70 consecutive patients (51 male, 19 female) with histologically proven SCLC were enrolled into the study. Staging of the disease included clinical investigation, bronchoscopy, chest X‐ray, thoracic computed tomography and ultrasound. The patients were grouped into five stages according to the Marburg classification (very limited disease (VLD), limited disease (LD), extensive disease I (EDI), extensive disease II (EDII) and extensive disease III (EDIII)). Prior to treatment, a 10 mL serum sample from each patient was examined by ELISA to quantify levels of VEGF and neuron‐specific enolase (NSE). Lactate dehydrogenase (LDH) and albumin levels were determined by photomorphometric analysis. Statistical analysis was performed using the Waller–Duncan k ratio t ‐test and Pearson’s correlation test. Results: Serum VEGF levels correlated well with tumour stage ( P < 0.0001). Albumin levels were not correlated with tumour stage, but levels of NSE and LDH increased with stage progression. When patients were divided into two groups (VLD and LD vs EDI–III), VEGF levels were significantly lower in the initial stages of the disease compared with extensive disease ( P < 0.0001). Serum levels of VEGF correlated better with tumour stage than did concentrations of NSE, LDH or albumin. Conclusion: Serum VEGF levels may serve as an additional prognostic marker in the course of patients with SCLC. Further studies are needed to determine whether these patients may benefit from additional anti‐angiogenic therapy in the future.

Thalidomide, an angiogenesis inhibitor is currently used in clinical trials in the US and Europe in combination with chemotherapy for the treatment of various solid tumors. The prognosis of patients suffering from small-cell lung cancer (SCLC) is poor. A 73-year-old female with extensive disease of SCLC was given six courses of chemotherapy with adriamycine, cyclophasphamide and oncovine, which led to complete remission of the disease. Following written informed consent, the patient has been treated with thalidomide 200 mg orally on a daily basis for 2 years and 5 months. There has been no sign of tumor recurrence during the follow-up. This case underlines the possible role of additional treatment with angiogenesis inhibitors in combination with traditional chemotherapy in the therapy of SCLC. Although there is no proof that thalidomide contributed to this good outcome and no conclusions can be drawn from this treatment in a single patient, further studies may determine the role of thalidomide as an adjuvant antiangiogenic agent in the therapy of SCLC.

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.

Angiogenesis is a prerequisite for solid tumor growth and metastasis. Elucidation of the signaling pathways that control tumor angiogenesis constitutes the basis for a rational antiangiogenic tumor therapy. Here we show that the production of vascular endothelial growth factor (VEGF) in HeLa and HL-60 cells is directed by the constitutive photomorphogenesis 9 signalosome (CSN). The CSN is a kinase complex that cooperates with the ubiquitin/26S proteasome system in regulating the stability of proteins involved in signal transduction. VEGF expression is controlled by the transcription factors activator protein (AP)-1, AP-2, SP-1, and hypoxia-inducible factor 1. Inhibition of CSN kinase activity by 50 microM curcumin for 2 h decreases the cellular c-Jun concentration, resulting in a reduction of the VEGF production by approximately 75%. The removal of the inhibitor from the cells led to a time-dependent recovery of endogenous c-Jun that is paralleled by increasing VEGF production. Elevated cellular CSN activity induced by CSN subunit 2 overexpression causes increased VEGF production in HeLa cells. A competitor of CSN-dependent c-Jun phosphorylation, the NH(2)-terminal c-Jun fragment Deltac-Jun(1-226), inhibits VEGF production in HeLa cells. The transcription factors AP-2 and SP-1 act independently of the CSN. They contribute less than a quarter to basal VEGF production. Under our experimental conditions, hypoxia-inducible factor 1alpha protein was not detected. Overexpression of the tumor suppressor p53 reduces VEGF production in HeLa cells. p53 competes with c-Jun for CSN-specific phosphorylation with the consequence of c-Jun destabilization. We conclude that CSN-directed c-Jun signaling mediates high VEGF production in HeLa and HL-60 cells. The data provide an explanation for the known antiangiogenic and antitumorigenic activities of curcumin. Because the CSN regulates the major part of VEGF production in the tested tumor cells, it constitutes a potentially important target for tumor therapy.

UNLABELLEDHemorrhagic proctitis is a rare, but severe complication of radiation therapy in the treatment of several pelvic malignancies. Administration of topical steroids, anti-inflammatory agents or laser therapy and rectal instillation of 4% formalin have been described as a method of treating this complication. A dog model was established to study the safest volume and duration of administration of formalin, the histological changes in the rectal mucosa, and rectal compliance following this treatment.METHODTwenty-one mongrel dogs were assigned randomly to seven groups. Three dogs received a rectal formalin bolus of 400 ml for 1 h; in the other six groups formalin was instilled in 30 ml aliquots to a total volume of 400 ml. Serum levels of formalin were obtained at designated time intervals, rectal compliance was evaluated pre- and post-formalin instillation, and rectal mucosa was analyzed for blood-vessel density and mucosal injury at different time points.RESULTSSerum formalin in the bolus group reached toxic levels, while sequential instillation caused no serum toxicity in any dog. Rectal compliance and mucosal thickness were not affected by formalin, but there was a decrease in the angiogenesis score, and mild proctitis was seen in the acute and 1-week group.