HERG1 gene expression as a specific tumor marker in colorectal tissues.

INTRODUCTION: Colorectal carcinomas exhibit a frequent recurrence after curative surgery, which may partially be due to histopathologically inconspicuous minimal residual disease. Reliable markers for tumor cells in colorectal tissue are still missing. Therefore, in this study we compared the predictive value of the putative tumor markers carcinoembryonic antigen (CEA), cytokeratin-19 (CK19) and cytokeratin-20 (CK20) to that of a novel marker, the human ether-a-go-go-related gene (HERG1) K(+) channel, a suggested regulator of tumor cell proliferation. MATERIALS AND METHODS: Using RT-PCR we studied HERG, CEA, CK19 and CK20 expression in colorectal carcinomas and non-carcinoma controls. HERG1 immunhistochemistry was performed in a total of 66 specimens, in colorectal carcinoma (n = 23), in matched histopathologically negative samples (n = 23) taken near the excision site from the same tumor patients and in healthy control biopsies (n = 20). In order to verify the relevance of HERG1 for tumor proliferation we studied the effect of HERG1 inhibition in the Colo-205 colon cancer carcinoma cell line using the MTT-assay. RESULTS: HERG1 was expressed in all tumor samples regardless of their stage and in adenomas larger than 0.4 cm, but absent in small adenomas, sigmadiverticulitis specimen and healthy histopathologically negative samples, except for one which developed a tumor recurrence. In contrast, CEA, CK19 and CK20 were absent in some tumors. The selective HERG1 inhibitor E-4031 dose-dependently impaired tumor growth in the proliferation assays. DISCUSSION: Our data indicate that HERG1, but not CEA, CK19 or CK20, is a highly sensitive and reliable tumor biomarker that may constitute a novel molecular target for tumor treatment.

Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts.

Studies with multidrug resistance modifiers indicate that perturbations of the cell membrane structure may influence P-glycoprotein (P-gp)-mediated drug transport. We describe studies of plasma membrane order using electron-paramagnetic resonance (EPR) in resistant (CH(R)C5) and sensitive (AUXB1) chinese hamster ovary cells treated with R-verapamil and bile salts. Cell growth rates were determined in presence of doxorubicin mitomycin and cisplatin. The plasma membrane order in untreated resistant cells was higher than in the sensitive cells. Both the bile salt taurochenodeoxycholate (TCDC; 0.2-1.6 mM) and R-verapamil (1-3 microM) lowered the membrane order in the CH(R)C5 cells to that in the sensitive cells and reversed the resistance to doxorubicin and mitomycin. The bile salt tauroursodeoxycholate (TUDC; 0.2-3 mM) did not lower membrane order and did not sensitise CH(R)C5 cells. Neither R-verapamil, TCDC nor TUDC reduced the membrane order of the sensitive cells AUXB1 cells. These results support the view that changes in multidrug resistance in Chinese hamster ovary cells and P-gp function are associated with alterations in the fluidity of the plasma membrane.

Panhypopituitarism associated with severe retroperitoneal fibrosis.

A 43-year-old man, with a history of central diabetes insipidus diagnosed 3 years previously, complained about reduced libido. An MRI scan showed a suprasellar lesion just below the supraoptic recess of the third ventricle. A stereotactically guided biopsy revealed fibrous glia, but no other specific tissue and no inflammatory cells. Two months later the patient presented with fatigue and muscular weakness. Tertiary adrenal failure and hypothyroidism were diagnosed by endocrine function tests and therapy with levothyroxine and hydrocortisone was started. Another 2 months later the patient was admitted with giddiness, nausea, peripheral oedema and oliguria. Radiological imaging and an open transperitoneal kidney exploration showed severe fibrosis around both ureters. Histological examination confirmed the diagnosis of idiopathic retroperitoneal fibrosis. Presumably the suprasellar tumour was the first manifestation of retroperitoneal fibrosis. Once the diagnosis 'idiopathic retroperitoneal fibrosis' is confirmed, fibrotic manifestations and complications involving extra-retroperitoneal tissues including the endocrine system, should be sought.

Resistance modulation in CHO cells by R-verapamil and bile salts is associated with physical and chemical changes in the cell membrane.

OBJECTIVES: Changes in multidrug resistance by resistance modifiers such as R-verapamil cause changes in fluidity of the cell membrane. The extent to which these changes involve structural alterations in membrane lipids has been investigated in CHO cells. METHODS: Sensitive (AUXB1) and resistant (CH(R)C5) chinese hamster ovary cells (CHO) were grown in culture. Incubations were carried out with R-verapamil (0-10 microM) or the membrane perturbing agents tauro-cheno-deoxycholate (0-1.6 mM, TCDC) and tauro-urso-deoxycholate (0-3.5mM, TUDC). Cell membrane fluidity was determined by electron-paramagnetic resonance spectroscopy and membrane lipids by HPLC and TLC. RESULTS: The resistant CH(R)C5 subline had a higher cell membrane order (lower fluidity, S = 0.7234) in the interface region of the cell membrane than sensitive AUXB1 cells (S = 0.6984) determined using EPR. The MDR-modulator R-verapamil and TCDC, but not TUDC, lowered cell membrane order in a concentration-dependent manner and increased membrane fluidity of the resistant CH(R)C5 subline. TCDC and R-verapamil were without effect on the cell membrane fluidity of AUXB1 cells. These changes were accompanied by alterations in the fatty acid composition of the plasma membrane. Untreated sensitive AUXB1 cells had higher levels of unsaturated fatty acids than resistant CH(R)C5 cells. In CH(R)C5 cells, R-verapamil increased the content of poly-unsaturated fatty acids and TCDC, but not TUDC, increased the content of mono-unsaturated fatty acids. CONCLUSIONS: The results demonstrate that resistance modifiers such as verapamil may influence cytostatic drug action by producing structural changes to lipid domains in the plasma membrane.

Cytostatic sensitivity and MDR in bladder carcinoma cells: implications for tumor therapy.

The clinical success generally seen in chemotherapy of advanced bladder carcinoma is far from optimal. The mechanism of resistance development is unclear and the expression of P-170 glycoprotein is generally low. The aim of this study, carried out in vitro in sensitive and cisplatin-resistant cell lines, was to examine sensitivity modulation using R-verapamil and cell membrane perturbing agents. Cell growth rates and changes in the order of the cell membrane, determined using electron-paramagnetic resonance spectrometry, were recorded. R-verapamil increased the toxic effect of doxorubicin in the cisplatin-resistant cell line which showed the highest membrane order. Linolenic acid had a similar effect and also increased sensitivity to cisplatin and methotrexate. Bile salts (tauro-cheno-deoxycholate,TCDC, and tauro-urso-deoxycholate TUDC), had little effect on cytotoxicity. These results indicate that R-verapamil and linolenic acid can act as sensitivity modulators in bladder carcinoma cells and that the action of these agents may involve membrane fluidity changes, a phenomenon noted previously in regard to sensitivity modulation in chinese hamster ovary cell lines.

Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity.

Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen. PicoGreen has been shown to detect as little as 0.5 ng pure DNA or 10(2) cells (interassay SD < 10%, intraassay SD < 5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen, cells were digested with papain for 20 h at 60 degrees C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.

Dedifferentiation of human hepatocytes by extracellular matrix proteins in vitro: quantitative and qualitative investigation of cytokeratin 7, 8, 18, 19 and vimentin filaments.

BACKGROUND/AIMS: Liver cirrhosis and carcinogenesis are accompanied by an alteration in extracellular matrix material. Histological studies reveal upregulation of the intermediate filaments cytokeratins 8 and 18 and de novo synthesis of vimentin, and cytokeratin 7 or 19 in hepatocytes. The aim of this study was to investigate how these two processes are linked. METHODS: Human hepatocytes were seeded: (i) on the matrix components collagen I, IV, laminin, or fibronectin; (ii) on stoichiometrically different complete matrices, derived from human placenta (matrix I) or the Englebreth-Holm-Swarm tumor (matrix II), and (iii) inside a three-dimensional collagen I sandwich. Filament expression and assembly were measured by cytofluor analysis or confocal laserscan microscopy. RESULTS: The matrix components or complete matrices triggered enhancement of cytokeratins 8 and 18 and de novo synthesis of cytokeratins 7, 19 and vimentin in a characteristic way. Confocal images demonstrated a dense and uniform network of cytokeratin 18 in freshly isolated cells, which was "replaced" by a few, thick protein bundles within 20 days. Interestingly, newly synthesized cytokeratin 19 structurally resembled the cytokeratin 19 organization in biliary epithelial cells. Marked cytokeratin alterations could be partially prevented when hepatocytes were grown in a three-dimensional collagen sandwich. CONCLUSIONS: Pathological alterations to the chemical composition, molecular structure, or spatial arrangement of the liver matrix lead to specific changes in the intermediate filament pattern in human hepatocytes. We assume that degradation of the matrix results in pathological alterations to the hepatocyte-receptor matrix-ligand ratio, followed by a switch from physiological to pathological cell-activation.

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K.

Differentiated human teratocarcinoma cell lines produce the human teratocarcinoma-derived virus (HTDV) particles encoded by the human endogenous retrovirus sequence HERV-K. We screened almost 2,000 human sera for antibodies against this endogenous human retrovirus, HTDV/HERV-K. Specificity of the immunofluorescence reactions using particle producing teratocarcinoma cells was confirmed by immunoelectron microscopy of ultrathin frozen sections. Immunoblot analyses using lysates of HTDV-producing cells revealed a 80-kDa HERV-K Gag precursor and a 90-kDa putative viral Env protein after incubation with positive sera. No processed Gag protein could be observed. Virus-specific bands were not detected in lysates of nonproducing cells. High antibody titers were found in about 60% of male patients with germ cell tumors. Antibody reactivity declined after tumor removal. In healthy blood donors, anti-HTDV reactivity was found only at low titers in a small percentage (3.9%) of individuals. A slightly elevated but statistically significant percentage of HTDV positivity was also observed for sera of pregnant women, whereas human immunodeficiency virus-positive individuals exhibited no peculiarity compared to normal blood donors. Our results provide evidence that HTDV particles are expressed in vivo and that the immune reaction against HTDV/HERV-K is specific for defined viral proteins.

Loss of in vitro cytotoxicity of cisplatin after storage as stock solution in cell culture medium at various temperatures.

BACKGROUND: Cisplatin (cis-dichlorodiammineplatinum [II]) is one of the most potent and widely used cytotoxic drugs in cancer therapy. However, in vitro cytotoxicity assays often yield diverging results. Therefore, it is believed by many investigators that cisplatin is not effective in vitro. The authors investigated whether the experimental conditions used could be responsible for the diverging results. METHODS: In vitro cytotoxicity assays were performed using the original cisplatin solution as distributed by the manufacturer or a 5-fold dilution of this in cell culture medium after storage for 2 weeks or 1 month at various temperatures (room temperature, 4 degrees C, and -20 degrees C). RESULTS: The original solution of cisplatin retained the full cytotoxic potency for at least 1 month under all storage conditions tested. The highest loss of cytotoxicity resulted from storage of cisplatin in cell culture medium in which a >7-fold increase in the concentration that inhibits 50% occurred after 2 weeks' storage at room temperature. Solutions stored in culture medium at -20 degrees C retained full cytotoxic potency. CONCLUSIONS: Cisplatin is a potent cytotoxic drug in vitro when stored under appropriate conditions. These findings are of relevance for in vitro studies with cisplatin.

Tumour imaging of bladder carcinomas and their metastases with 111indium-labelled monoclonal anti-CEA antibody BW 431/26.

Fifty-one patients with muscle-infiltrating bladder carcinoma (T2-T4, N0-3, M0-1) were studied with a new imaging technique using murine monoclonal antibody directed against the carcinoembryonic antigen (CEA). A total number of 67 investigations were performed. The intact 111indium-labelled antibody (BW 431/26, Behringwerke Marburg) detected 86% of primary tumours, 93% of local and 75% of distant metastases whether there was an elevated CEA level in serum or not. Immunohistologically (avidin-biotin-peroxidase method) positive frozen tissue sections from tumour biopsies stained with the same monoclonal anti-CEA antibody, thus confirming the presence of the CEA antigen in vitro. The method was of much higher sensitivity in detecting even very small metastases than X-ray computed tomography (86% versus less than 30%). The specificity was in the region of 90%. The response to chemotherapy (MVEC regimen) was shown by repeated studies demonstrating reduced uptake (partial remission) or no accumulation (complete remission) in the second immunoscan. We suggest immunoscintigraphy of bladder tumours and their metastases as an additional method in preoperative staging and postoperative care.

Endoscopic intubated ureterotomy.

On the basis of our experience with 9 cases, endoscopic ureterotomy for ureteral stenoses represents an alternative to open reconstructive surgery. In cases of acquired ureteral stenoses, the transurethroureteral access appears to be the method of choice. In contrast to open surgery, particularly after previous operations, surgical trauma is minimal. A primary attempt at ureteroscopic ureterotomy is warranted, since it does not appear to pose any problems for open surgery should this prove necessary later.

[Application of the ultrasonic aerosol apparatus "TuR" USI50 for the test of the bronchial reactivity--comparative studies with "TuR" USI3 (author's transl)]. / Einsatz des Ultraschall-Aerosolgerätes "TuR" USI50 bei der Prüfung der bronchialen Reaktivität--vergleichende Untersuchungen zum "TuR" USI3.

By comparative studies with the ultrasonic aerosol apparatus "TuR" USI3 and "TuR" USI50 was shown that their applicability for tests of the bronchial reactivity leads to corresponding test results. Based on long experiences with the ultrasonic aerosol method the USI50 apparatus may recommended also for use of the acetylcholine test (0.01% solution) in clinics and ambulances. Detailed references are given for use of the application technique to guarantee that uniform conditions of investigation are present.