Johann Schönberger

Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML‐1. Cultured on a three‐dimensional clinostat, ML‐1 cells formed three‐dimensional MCTSs (MCTS diameter: 0.3±0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up‐regulated the thyroid‐stimulating hormone‐receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML‐1 cells grown on the clinostat showed elevated amounts of the apoptosis‐associated Fas protein, of p53, and of bax but showed reduced quantities of bcl‐2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase‐mediated dUTP digoxigenin nick end labeling, 4′, 6‐diamidino‐2‐phenylindole staining, DNA laddering, and 85‐kDa apoptosis‐related cleavage fragments. These fragments resulted from enhanced 116‐kDa poly(ADP‐ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

Glucose Transporter 1 Gene Expression is Related to Thyroid Neoplasms with an Unfavorable Prognosis: An Immunohistochemical Study

PURPOSE An accelerated rate of glucose metabolism mediated by overexpression of key regulatory glycolytic enzymes and glucose transporters is among the most characteristic biochemical marker of malignant transformed cells. In thyroid neoplasms, however, an increased uptake of glucose [measured by 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET)] seems to be restricted to more aggressive and high-grade tumors, whereas tumors with favorable prognosis demonstrate no significant tracer uptake. We therefore studied the expression of glucose transporters in thyroid carcinomas with different grades of malignancy. METHODS Sections of formalin-fixed and paraffin-embedded tissue obtained from 45 patients with thyroid cancer (5 anaplastic, 20 papillary and 20 follicular tumors) were investigated. Polyclonal rabbit antiglucose transporter antibodies, reactive with glucose transporters 1-5 (GLUT1-5), were used after heat pretreatment of the sections. Staining was performed by the avidin-biotin conjugate immunoperoxidase reaction and evaluated semiquantitatively. RESULTS Expression of GLUT1 transporter on the cell membrane was closely related to the grade of malignancy in thyroid neoplasms (Fisher exact test p < 0.05). All anaplastic tumors showed a high level of GLUT1 expression in the cytoplasm and on the cell membrane. Positive membranous staining in differentiated tumors was detected predominantly in neoplasms with unfavorable prognosis, e.g., in widely invasive follicular or metastatic tumors, whereas low or no immunoreactivity could be seen in well-differentiated tumors or in normal thyroid epithelium. CONCLUSIONS These data indicate that overexpression of GLUT1 on the cell membrane of thyroid neoplasms is closely related to tumors demonstrating a more aggressive biological behavior. Therefore, determination of GLUT1 expression in thyroid cancer tissue may be a prognostic marker, and FDG-PET may be a helpful technique in identifying patients at a higher risk.

Establishment and characterization of the follicular thyroid carcinoma cell line ML-1

Abstract.The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.

Application of free-flow IEF to identify protein candidates changing under microgravity conditions.

Using antibody‐related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s‐μg). In this study, we performed proteome analysis in order to identify more gravity‐sensitive thyroid proteins. Cells cultured under 1g or s‐μg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free‐flow IEF. The fractions obtained were further separated by SDS‐gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free‐flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s‐μg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.

CME: Papilläres Mikrokarzinom und papilläres Karzinom der Schilddrüse ≤1cm: Modifizierte Definition der WHO und therapeutisches ‧Dilemma

Aims: Major controversies exist regarding the treatment of papillary microcarcinoma of the thyroid (PMC). Prior to 2003 PMC was defined by the WHO as a papillary carcinoma of 1cm or less in diameter. In 2004 that definition changed, with the new classification requiring that the tumour also must be found incidentally. Patients, methods: In this study we reviewed the clinical records of 67 patients with papillary tumours of the thyroid ≤1 cm, taking into account the new WHO definition (54 pts. with incidentally found PMC, median age: 53 years, 13 pts. with suspicion of thyroid neoplasm before resection, median age: 38 years). Clinical presentation, surgical treatment, further therapy and follow-up are presented. Results: Median tumour size was 7 mm in both groups (1.10 mm). Multicentric tumours were found in 15 pts. (22%), 8 had more than one PMC on the same side, and 7 displayed PMC bilaterally. Eleven (16%) of the primary tumors had metastatic involvement of regional lymph nodes at the time of initial surgery or during follow-up. Two patients showed distant metastases. No correlation between tumour size and multifocality or the presence of lymph node metastases could be seen. The gender of patients was the only significant independent variable for all patients; age and lymph node involvement was significantly different between incidentally and non-incidentally found PMC. Conclusions: Despite the majority of patients with PMC having an excellent outcome, there are also cases showing an unfavorable course. Currently no predictive parameter exists to anticipate the course and long-term outcome for an individual patient. Until this problem is solved, each patient should have the option to decide for him or herself whether to be treated similarly or differently than for conventional thyroid cancer.

Retinoid- and sodium-butyrate– induced decrease in heat shock protein 70 membrane-positive tumor cells is associated with reduced sensitivity to natural killer cell lysis, growth delay, and altered growth morphology

Abstract Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX−) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 μM, 1 μM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX− tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.

Nachweis einer Aortitis im 18F–FDG–PET

E 54-jährige Patientin wurde uns zur Abklärung einer rheumatischen Grunderkrankung stationär zuverlegt. Sie berichtete über multiple uncharakteristische Beschwerden wie generalisierte Schmerzen, eine reduzierte Leistungsfähigkeit sowie rezidivierende Übelkeit und Erbrechen. In einem auswärtigen Haus war bei typischer Schmerzsymptomatik, BSG-Beschleunigung, latenter Depression und Gewichtsverlust der Verdacht auf eine Polymyalgia rheumatica geäußert worden. Die trotz fehlender Augensymptome vorgenommene Biopsie der Arteria temporalis ergab das histologische Bild einer Riesenzellarteriitis. Die entsprechende Therapie mit 60 mg Prednisolon pro Tag über 14 Tage erbrachte subjektiv keine sofortige Besserung der Symptomatik, jedoch kam es zu einer anfänglichen Appetitsteigerung, Herzrasen, Panikattacken und Taubheitsgefühl im ganzen Körper. Deshalb lehnte die Patientin eine Fortsetzung der Steroidtherapie ab. Laborchemisch ergaben sich bei Aufnahme bis auf ein erhöhtes CRP (45,6 mg/l, Normwert < 5 mg/l) und eine deutlich erhöhte BSG von 41/67 mm (n.W.) sowie ein leicht erhöhtes Kreatinin im Serum (1,11 mg/dl, Normwert 0,50–0,90 mg/dl) keine pathologischen Laborparameter. ANATiter und Rheumafaktoren waren negativ. In der weiteren durchgeführten Diagnostik zeigte sich im 18F-FDG-PET (Abbildungen 1a und 2a) eine intensive Aktivitätsanreicherung im gesamten Aortenbogen, die bis zur Aorta abdominalis reichte, so dass sich hieraus der Verdacht auf eine Arteriitis der großen Gefäße ableiten ließ. Nachdem die Patientin die Therapie der ersten Wahl mit Steroiden bzw. Azathioprin explizit ablehnte, wurde nach entsprechender Aufklärung der Patientin hinsichtlich der potentiellen Nebenwirkungen als „Escape-Medikation“ eine Endoxanstoßtherapie begonnen. Darunter entwickelte die Patientin zunehmend pustulöse Effluoreszenzen, so dass die Behandlung ebenfalls abgebrochen werden musste. Die anschließend durchgeführte Therapie mit Ciclosporin A wurde von der Patientin gut toleriert; es kam zu einer Besserung der Symptomatik, einer Normalisierung der Laborparameter (mit CRP 8,42 mg/l) und einer verminderten Anreicherung der 18F-markierten Deoxyglucose im angefertigten Kontroll-18F-FDG-PET nach 4 Monaten (Abbildungen 1b und 2b). Dieser Fall unterstreicht die potentielle Bedeutung von nuklearmedizinischer Bildgebung zur Diagnosestellung und Verlaufskontrolle bei klinisch okkulten Vaskulitiden [1–7].