β-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures

Abstract

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.