Browsing by Author "Klünder, Irene"

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Open Access
β-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures
(1993) Klünder, Irene; Hülser, Dieter F.
We have investigated whether three-dimensional cultivation of cells to multicell spheroids influences the expression of a transfected gene. Ltk- cells (mouse fibroblasts. thymidine kinase negative) have been transfected with a bacterial lacZ gene which was coupled to a β-actin promoter. The transfected cells synthesize β-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl β-D-galactoside and for cytometry with fluorescein di(β-D-galactopyranoside). As we have shown with monolayer cells, β-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from ~98% to ~2% within a week. This reduction is also independent of cell density, medium condition, and cell cycle. Nonproducing multicell spheroid cells, however, regained their ability to synthesize β-galactosidase within a few days when the cells were recultivated as monolayers. Since the lacZ gene was not lost, its expression might have been regulated by its β-actin promoter. We, therefore, investigated whether the endogenous synthesis of β-actin was similarly regulated. A correlation between the distinct reduction in β-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.
Open Access
β-galactosidase production in two- and threedimensionally cultivated cell cultures
(1991) Klünder, Irene; Hülser, Dieter F.
In these studies, we demonstrate that the threedimensional arrangement of the cells in spheroids influences the β-galactosidase activity of transfected ltk--cells. In contrast to cells grown in monolayer culture cells growing as spheroids lost their β-galactosidase activity within few days. The reduction of β-galactosidase activity in cells grown as spheroids was not caused by a lost of the lacZ-gene as can be seen from the recovery of the β-galactosidase production when cells were retransferred in monolayer culture. As we showed in cells cultivated as monolayer, β-galactosidase activity seems to be not impaired by gradients of nutrient supply or cell cycle. These measurements are confirmed by histological sections, where the highest β-galactosidase activity was found in the viable center of the spheroids.
Open Access
Bioproduktion in dreidimensional wachsenden Zellkulturen
(1989) Hülser, Dieter F.; Klünder, Irene; Brenner, Joachim
Die biotechnologische Produktion von Substancen wie t-PA, monoklonalen Antikörpern, lnterleukinen, Wachstumsfaktoren, Interferonen und Hormonen sollte vorzugsweise mit Säugetierzellen erfolgen, weil Bakterien diese Substanzen nicht in ihrer nativen Form herstellen können. Die dazu benötigten großen Mengen von Säugerzellen werden - je nach Zelltyp - in Monolayer- oder Suspensionskultur gezüchtet. Für Monolayerkulturen müssen große Oberflächen (Roller Bottles, Microcarrier) zur Verfügung gestellt werden, was viel Raum und Materlal beansprucht und damit kostenintensiv ist. In Suspension wachsen die Zellen voneinander isoliert, wodurch Zellkooperation unterbunden wird und interzellulärer Informationsaustausch nicht mehr möglch ist. Es gibt jedoch eine weitere Möglichkeit, Eurokaryonten zu kultivieren, welche die Nachteile der beiden anderen Methoden aufhebt. Diese bisher vorwiegend in der Krebsforschung oder bei der Untersuchung von Regulationsmechanismen von Zellen verwendete Technik läßt die Zellen zu dreidimensional angeordneten kugelförmigen Zellaggregaten - den Multizell-Sphäroiden - heranwachsen.
Open Access
Hydrogen ion-mediated enhancement of cytotoxicity of bis-chloroethylating drugs in rat mammary carcinoma cells in vitro
(1989) Jähde, Eckhard; Glüsenkamp, Karl-Heinz; Klünder, Irene; Hülser, Dieter F.; Tietze, Lutz-F.; Rajewsky, Manfred F.
Aerobic glycolysis, a metabolic characteristic of malignant cells, can be exploited to increase the concentration of lactic acid selectively in tumor tissues in vivo by systemic administration of glucose (E. Jähde and M. F. Rajewsky, Cancer Res., 42: 1505-1512, 1982). To investigate whether a more acidic microenvironment can enhance the effectiveness of cytocidal drugs, we have analyzed the colony-forming capacity of M1R rat mammary carcinoma cells exposed to bis-chloroethylating agents in culture as a function of extracellular pH (pHe). At pHe 6.2 the cytotoxicity of 4-hydroperoxycyclophosphamide, as measured by inhibition of colony formation, was potentiated by a factor of ∼200 as compared to pHe 7.4. Similar results were obtained with mafosfamide, nitrogen mustard, nornitrogen mustard, melphalan, and chlorambucil; not, however, with ifosfamide. As indicated by experiments using the ionophor nigericin for rapid equilibration of pHe and intracellular pH (pHi; measured with pH-sensitive microelectrodes), modulation of drug action by varying pHe primarily resulted from the concomitant decrease in pHi. The acidic microenvironment enhanced cytotoxicity most effectively during the phase of cellular drug uptake and monofunctional alkylation of DNA. DNA cross-link formation appeared to be less affected by pH, and lowering of pHe during the phase of cross-link removal was only marginally effective.