SELECTING AGENT HYGROMYCIN-B ALTERS EXPRESSION OF GLUCOSE-REGULATED GENES IN TRANSFECTED CACO-2 CELLS

Incorporation into plasmids of genes conferring resistance to aminogly coside antibiotics such as hygromycin B is currently utilized for sele ction in experiments involving gene transfer in eukaryotic cells. Usin g a subclone of Caco-2 cells stably transfected with an episomal plasm id containing the hygromycin resistance gene, we observed that transfo rmed cells subcultured in the presence of hygromycin B exhibit, compar ed with the same cells subcultured in antibiotic-free medium, a sixfol d increase in the rates of glucose consumption and lactic acid product ion and dramatic changes, at mRNA and protein level, of the expression s of sucrase-isomaltase and hexose transporter GLUT-2, which are downr egulated, contrasting with an upregulation of hexose transporter GLUT- 1. This occurs without significant modifications of the differentiatio n status of the cells, as demonstrated by the normal expression of vil lin, ZO-1, dipeptidyl peptidase IV, or Na+-K+-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured i n the presence of hygromycin B. These results draw attention to the ne ed to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.