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.

Hydrogen ion-mediated enhancement of cytotoxicity of bis-chloroethylating drugs in rat mammary carcinoma cells in vitro.

Aerobic glycolysis, a metabolic characteristic of malignant cells, can be exploited to increase the concentration of lactic acid selectivity 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 approximately 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.