Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells.
Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na(+)-K(+)-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.
Rodolosse
A
A
Unité de Recherches sur la Différenciation Cellulaire Intestinale, Institut National de la Santé et de la Recherche Médicale, Villejuif, France.
Barbat
A
A
Chantret
I
I
Lacasa
M
M
Brot-Laroche
E
E
Zweibaum
A
A
Rousset
M
M
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018938:Caco-2 Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D018628:Gene Dosage; D005786:Gene Expression Regulation / Q000187:drug effects*; D005947:Glucose / Q000378:metabolism / Q000502:physiology*; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D006801:Humans; D006921:Hygromycin B / Q000494:pharmacology*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009834:Oligo-1,6-Glucosidase / Q000378:metabolism; D010957:Plasmids / Q000187:drug effects; D013393:Sucrase / Q000378:metabolism; D014162:Transfection / Q000502:physiology
D000900:Anti-Bacterial Agents; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D006921:Hygromycin B; D009834:Oligo-1,6-Glucosidase; D013393:Sucrase; D005947:Glucose