[P32-labelling of nucleic acids and the role of phosphate in the nucleic acid metabolism of Euglena greacilis].
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Chromatography↗
Biomedical subjects
E Spiess
Publications and source records attributed to E Spiess.
[Isolation and characterization of rapidly labelled RNA from Euglena gracilis by means of analytical and preparative electrophoresis in polyacrylamide gels].
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Acrylates↗
[The nucleic acids of green and bleached cells of Euglena gracilis].
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Chromatography↗
Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors.
The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.
Carcinoma, Non-Small-Cell Lung↗
Cell surface glycosylation and characterization of a differentially expressed glycoprotein in metastatic and non metastatic cell lines of the rat BSp73 tumor.
The highly (ASML) and non metastatic (AS) variants of the rat tumor BSp73 were compared with respect to cell surface carbohydrate proteins. Fluorescence labelling with lectins (ConA, MPA, PNA, SBA, UEA-I, WGA) revealed a differentiated carbohydrate pattern at the cell surface of these cell lines. The highly metastatic variant was significantly more glycosylated with respect to galactosyl, mannosyl and N-acetylgalactosylamine residues; fucosyl residues were exclusively expressed in the metastatic variant. Examination of isolated plasma membrane fractions showed quantitative differences with respect to glycosylated proteins separated on polyacrylamide gels. A 30 kDa glycoprotein (GP30-ASML) dominant in the metastatic variant was further characterized. Various detergents (CHAPS, Nonidet, SDS, Triton X-100) and urea extracted it exclusively from the highly metastatic variant. GP30-ASML is a predominantly O-glycosylated single polypeptide chain with terminal N-acetylgalactosamine and galactosyl residues; its molecular weight determined by SDS-PAGE is 30 kDa and its isoelectric point is 7.8. Immunofluorescence localization experiments with monoclonal antibodies specific for GP30-ASML and polyclonal antibodies raised against GP30-ASML showed this protein at the cell surface and in the lysosomal compartment of both cell lines; exclusively in the non metastatic variant it was also found in the nuclear membrane. The function of this protein is still unknown.
Adenocarcinoma↗
Cathepsin B, plasminogenactivator-inhibitor (PAI-1) and plasminogenactivator-receptor (uPAR) are prognostic factors for patients with non-small cell lung cancer.
To evaluate the possible role of cysteine proteases and serine proteases, as well as their respective inhibitors and receptors, as new prognostic factors in NSCLC, we examined, for the first time, 10 biological parameters related to three proteolytic systems within a homogeneous collective of 147 cases of NSCLC. Activities (cath B(AT), cath B(A7.5)) and protein levels of cath B(C), cath L(C), uPA, PAI-1, uPAR [measured by three different assays uPAR (ADI), uPAR (HD13), uPAR (IIIF10)] and TF were measured in homogenates of lung tumour tissue and corresponding non-malignant lung parenchyma. Total cath B activity (cath B(AT)) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B(A7.5)), were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The concentrations of cath B(C), cath L(C), uPA, PAI-1, uPAR and TF were determined by ELISAs. uPAR was determined using three different ELISA formats. The median levels of cath B(AT) (5.1-fold), cath B(A7.5) (2.5-fold), cath B(C), (8.5-fold), cath L(C) (6.6-fold), uPA (6.5-fold), PAI-1 (4.2-fold), uPAR (ADI) (2.2-fold), uPAR (HD13) (4.0-fold) and uPAR (IIIF10) (2.6-fold) were higher in tumour tissue compared to the lung parenchyma. Cath B(AT), cath B(A7.5) and cath B(C) in primary tumours correlated with lymph node metastases. Regarding histologies, the concentration of PAI-1 seems to be associated with the histological cell types of NSCLC. We found the highest values of PAI-1 in large cell carcinoma > SCC, AC > carcinoid and lowest values in metastases of primary tumours of other organs. Only PAI-1 was significantly increased in poorly-differentiated cells (G3) compared to well- and moderately- differentiated cells (G1/G2). PAI-1 significantly correlated with cath B(AT) and cath B(A7.5) with uPAR (ADI), uPAR (HD13), uPAR (IIIF10) with uPA, and only weakly with TF, but not with cath B(C) and cath L(C). Significant correlations with overall survival in the total population of NSCLC patients were observed in univariate analysis for cath B(AT), cath B(C), PAI-1, uPAR (ADI), uPAR (HD13), and uPAR (IIIF10). Cath L(C) was not significantly associated with poor prognosis. Regarding the histological tumour type, only in patients with squamous cell carcinomas did cath B(A7.5) and PAI-1 remain significant prognostic factors. In multivariate survival analysis only two proteolytic factors, PAI-1 and uPAR (III101F), stayed significant. In conclusion, among 10 biological parameters evaluated within the same cohort of patients, only PAI-1, uPAR (ADI), uPAR (HD13), uPAR (IIIF10), cath B(AT) and cath B(C) are prognostic factors for overall survival of NSCLC patients. Moreover, PAI-1 and uPAR (IIIF10) add independent prognostic information with regard to established clinical and histomorphological factors in NSCLC.
Adolescent↗
Ultrastructural studies on the lung colonization by nonmetastatic rat tumor cells.
The events during the settlement of BSp73AS (AS) tumor cells in the syngeneic rat lung are described. Although AS cells show highly invasive behavior in vitro, subcutaneous primary tumors grow solidly without detectable metastatic spreading. However, AS cells when applied to the syngeneic rats via the tail vein give rise to lung colonies which grow rapidly at the site of the cells' primary arrest in the capillaries. The colonization comprises formation of microemboli, penetration of the endothelium including the basal lamina, and invasion of the lung tissue. Within two weeks, large colonies develop, thereby compressing, invading, and destroying their surroundings without detectable preference in direction. This establishment of AS tumors in the lung reflects the high invasive potential of AS cells and displays many of the morphological features observed during the formation of colonies of metastatic cell lines. Thus, we conclude that a nonmetastatic tumor cell line, such as AS, may possess almost the whole set of properties necessary for successful metastasis.
Animals↗
Amplification, expression and localization of the c-myc gene in BSp73 rat tumor cell lines.
Metastatic (ASML 14-1, ASmv) and non metastatic (AS17-4) cell lines of the rat BSp73 pancreatic adenocarcinoma were investigated for amplification and expression of oncogene DNA. The c-myc gene was amplified, but only in one metastatic variant, ASML. The degree of amplification (3.5-fold) increased after prolongued in vitro cultivation (17.5-fold). All three tumor cell lines expressed c-myc and ras mRNA. Ras expression was at the same level as in rat liver. C-myc expression was considerably above the level in rat liver, but also differed considerably between the metastatic variants. In the metastatic ASML cells the c-myc gene was localized by in situ hybridization on a marker chromosome derived from chromosome 7. The karyotypes of the metastatic variants are different and have no common marker chromosomes. Our results obtained with the BSp73 tumor model do not support a role of the c-myc gene in the metastatic process.
Adenocarcinoma↗
An in vitro model study of BSp73 rat tumour cell invasion into endothelial monolayer.
In order to study the process of invasion in more detail we developed an in vitro model of the vessel wall. Rat tumour cells derived from an adenocarcinoma of the pancreas, BSp73 AS--of high invasive but low metastatic capacity--and BSp73 ASML--not invasive but highly metastatic--were compared for their mode of invasion into confluent monolayers of endothelial cells. Corneal as well as vascular endothelial cells were plated alternatively onto the basal lamina-like bovine lens capsule that was mounted in a combi-ring dish or reconstituted extracellular matrix (Basement Membrane Matrigel) as substrata. The endothelial monolayers were confronted with AS- and ASML-tumour cells. The interaction of the various cell types was followed by scanning and transmission electron microscopy. The invasive cell type AS was able to force the endothelial cells to retract and subsequently undermined the endothelial cell layer. In the noninvasive cell population ASML most cells remained in the typical roundish morphology and did not interact with the endothelial cell layers. Only a very minor fraction of ASML populations was able to attach to and also invade into the endothelial cell monolayer. It could be shown that AS-cells individually and as small groups penetrated the endothelial cell layer. The results of transmission and scanning electron microscopy suggest that endothelial cell retraction and underlapping of adjacent endothelial cells by tumour cells play an important role in invasion and extravasation through blood vessels. Against all expectations, the nonmetastasizing tumour cell variant (AS-cells) exhibited a dramatic invasive behaviour whereas the highly metastatic ASML-variant mostly retained its spherical shape and showed invasive activity only in exceptional cases.
Adenocarcinoma↗
Ultrastructural analysis of experimentally induced invasion in the rat lung by tumor cells metastasizing lymphatically.
The colonization of the lung by the rat tumor cells BSp73 ASML which have the ability to metastasize via the lymphatic system was studied at the ultrastructural level. Tumor cells arriving in the lung after i.v. injection become transiently embolized; within hours, however, they begin to extravasate from the blood capillaries. Swelling cellular protrusions open a limited area between endothelium and basal lamina through which tumor cells erupt. Tumor cells then form metastases in the interstitial tissue and, in an apparently lymphotropic action, intravasate the lymphatic vessels in a similar manner to a reverse diapedesis-like process. Within the lympatic system they settle, spread, and build up extensive tumor foci particularly in the subpleural region.
Animals↗
Experimental approaches to problems of invasion and metastasis.
The highly metastasizing ASML cells and the non-metastasizing AS cells, arisen as spontaneous tumors of the rat, were confronted with rat lung tissue in vitro. Small cubes of the lung were allowed to heal their cut edges, then tumor cells were added. Both tumor cell types adapted their shape to the environment, penetrated the superficial layer of lung cells, either of epithelial or of fibroblastoid character and settled on the basal lamina, which, however, was not pierced. In a second set of experiments the tumor cells were inoculated intravenously into the living animal. The lung loaded with tumor cells was excised and cut into cubes which were then incubated in vitro. Here also both tumor cell types exhibited an invasive behavior but the basal lamina of the vessels in which the tumor cells have been arrested was not penetrated. These data indicate that tumor cell behavior is strongly dependent on the environment and the complete invasion or extravasation must be considered as an inducible process.
Animals↗
Invasive activities of metastasizing and nonmetastasizing tumor cell variants in vitro. II. Studies on confrontations with aorta, vein, ductus thoracicus, diaphragm, and lung.
In continuation of a previous paper (1), we have prolonged the time of confrontation of two rat tumor cell variants (BSp73 AS and ASML) with normal epithelial cells and broadened the spectrum of confrontation partners. In addition to the aorta we have also used small veins, the ductus thoracicus as a lymphatic vessel, the diaphragm, and lung fragments. The organ sections were preserved for a longer period of time by incubating them in a gyratory shaker. Under these conditions the vessel material and diaphragm remained morphologically intact for up to 12 hrs; lung cubes concealed the cut surfaces by immediate wound healing, and were preserved intact for up to 40 days. All endothelia and the mesothelium of the diaphragm were destroyed by the nonmetastasizing AS cells, but the basal lamina remained intact by morphological criteria even after 18 hrs of exposure to the AS cells. The metastasizing ASML cells attached by filopodia mostly to the basal lamina of the vessels, but were unable to destroy neither the endothelia nor the basal lamina. The superficial cell layers of the lung cubes, however, were penetrated by the cells of both tumor lines, but to a different degree.
Animals↗
Invasive activities of metastasizing and nonmetastasizing tumor cell variants in vitro.
Pieces of the endothelium of the aorta of BDX rats were confronted with two syngeneic-tumor-cell variants. While the AS variant is non-metastasizing, the ASML cells metastasize spontaneously via the lymphatic vessels. By means of scanning-electron microscopy the adhesion phenomena and the various stages of invasive activity of the non-metastasizing variant (AS) as well as the retraction of the endothelial cells depending on the time of confrontation were studied. Though the metastasizing variant (ASML) adhered firmly to the endothelium, we found neither signs of invasive activity of these tumor cells nor retraction phenomena of the endothelial cells. Aggregates of AS- and clusters of ASML-cells, respectively, behaved in exactly the same way as single cells: while the ASML-clusters remained inactive, the AS-aggregates exhibited invasive activities. Those cells which were in intimate contact with the endothelium started to leave the aggregate, thereby forming a foot-like layer beneath the rest of the aggregate; these cells began to invade.
Animals↗
Cathepsin B in tumors, normal tissue and isolated cells from the human lung.
Human lung tumors of different histologic cell types and adjacent normal lung parenchyma, purified lung parenchyma, purified lung macrophages and three human lung-derived cell lines were investigated in an attempt to identify tumor specific premature and mature cathepsin B species. By polyacrylamide gel electrophoresis and immunoblotting techniques with specific antibodies we detected mature cathepsin B forms with molecular masses of 31 kDa and 32 kDa in tissues. Reductive conditions revealed in these populations single and double chain 31/32 kDa forms. The latter were recognized by their heavy part, the 26/27 kDa molecule forms. Qualitative differences in the pattern of cathepsin B species between tumor and corresponding normal material or between different histologies of lung tumors were not found. However, tumor material is considerably richer in cathepsin B activity than normal material. Isolated alveolar macrophage populations and established cell lines showed the same cathepsin B pattern as tissues. All these cells released cathepsin B proforms of 44 to 46 kDa into the culture medium, indicating that the release of pro-cathepsin B cannot be considered a tumor-specific mechanism. The secreted proforms were sensitive to in vitro activation by pepsin.
Cathepsin B↗
Prognostic value of increased lung tumor tissue cathepsin B.
In the study of 65 matched pairs of lung tumor tissue and normal lung parenchyma, cathepsin B (CB) activity was found to be increased about 4.6-fold, when regarding median levels. CB activity was found to be insignificantly higher in adenocarcinoma compared to the other histologic cell types. CB levels did not correlate with tumor stages (TNM) nor with cell differentiation, while increased CB activity was related to shorter survival rates of the patients. It is concluded that lung tumor CB is of some prognostic value for the outcome of the malignant disease.
Adult↗
Charge polymorphism in human lung cell pro-cathepsin B.
Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 human lung-fibroblast cells was pre-purified by ion exchange chromatography and investigated by 2D gel electrophoresis. Four (Wi-38), six (HS-24) and ten (alveolar macrophages) polypeptides differing in charge, but with the same molecular mass of 45 kDa, were found. The isoelectrical points of these polypeptides ranged from 5.43 to 6.57. Deglycosylation reduced the mass (7 kDa) but did not change the charge pattern. This investigation established cell type-specific patterns of secreted pro-cathepsin B-forms, but only parts of these may be cell type-specific forms depending on differentiated expression of mRNA and post-translational modification.
Carcinoma, Squamous Cell↗