Beta-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures.
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 beta-actin promoter. The transfected cells synthesize beta-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside and for cytometry with fluorescein di-(beta-D-galactopyranoside). As we have shown with monolayer cells, beta-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from approximately 98% to approximately 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 beta-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 beta-actin promoter. We, therefore, investigated whether the endogenous synthesis of beta-actin was similarly regulated. A correlation between the distinct reduction in beta-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.
Klünder
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I
Abteilung Biophysik, Universität Stuttgart, Germany.
Hülser
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DF
D000199:Actins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002450:Cell Communication; D002452:Cell Count; D002461:Cell Line, Transformed; D015972:Gene Expression Regulation, Neoplastic; D007763:Lac Operon; D051379:Mice; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D000199:Actins; D001616:beta-Galactosidase