Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells

DONADIO AC, LOBO C, TOSINA M, DE LA ROSA V, MARTÍN-RUFIÁN M, CAMPOS-SANDOVAL JA, MATÉS JM, MARQUEZ J, ALONSO FJ, SEGURA JA

JOURNAL OF CELLULAR BIOCHEMISTRY

Liss

Lugar: United States; Año: 2008 vol. 103 p. 800 - 811

0730-2312

Glutamine behaves as a key nutrient for tumours and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates well with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells in order to study its effect on the hexosamine pathway and the pattern of protein Oglycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells and showed a more differentiated phenotype. ORF19 cells had an 80 % reduction in glutamine:fructose-6- P amidotransferase activity, the rate-limiting step of hexosamine pathway. Hexosamine pathway provides UDP-GlcNAc, necessary for the O-linked Nacetylglucosamine modification of proteins. Although the overall cellular protein Oglycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase, the enzyme that links Nacetylglucosamine to proteins, was 5-fold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins allowed us to detect two new O-GlcNAc transferase target proteins: the chaperonin TCP-1 è and the oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. è and the oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell. è and the oncogene Ets-related protein isoform 7. Taken together, our results support hexosamine pathway and the O-glycosylation of proteins as a sensor mechanism of the nutritional and energetic states of the cell.