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

Exp Cell Res. 1993 Jul;207(1):155-62. doi: 10.1006/excr.1993.1175.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / analysis
  • Actins / biosynthesis
  • Actins / genetics
  • Animals
  • Cell Communication
  • Cell Count
  • Cell Line, Transformed
  • Gene Expression Regulation, Neoplastic*
  • Lac Operon
  • Mice
  • Transfection
  • beta-Galactosidase / biosynthesis*
  • beta-Galactosidase / genetics

Substances

  • Actins
  • beta-Galactosidase