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

• Published in: Journal of cellular biochemistry Vol. 103; no. 3; pp. 800 - 811
• Main Authors: Donadio, Ana C., Lobo, Carolina, Tosina, Marta, de la Rosa, Vanessa, Martín-Rufián, Mercedes, Campos-Sandoval, José A., Matés, José M., Márquez, Javier, Alonso, Francisco J., Segura, Juan A.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 15.02.2008
• Subjects: Animals; antisense mRNA; Breast Neoplasms - enzymology; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Gene Expression - genetics; glutaminase; Glutaminase - antagonists & inhibitors; Glutaminase - genetics; Glutaminase - metabolism; Glutamine - metabolism; Hexosamines - biosynthesis; Hexosamines - metabolism; Humans; Mice; N-Acetylglucosaminyltransferases - metabolism; O-GlcNAc transferase (OGT); O-glycosylation; Protein Processing, Post-Translational - genetics; Proteomics; RNA Interference; Sp1; Sp1 Transcription Factor - biosynthesis; Sp1 Transcription Factor - metabolism; Tumor Cells, Cultured; Uridine Diphosphate N-Acetylglucosamine - metabolism
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.21449

Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine‐utilizing enzyme in these cells, and its activity correlates 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 O‐glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose‐6‐P amidotransferase activity, which is the rate‐limiting step of the hexosamine pathway. Although the overall cellular protein O‐glycosylation did not change, the O‐glycosylation status of several key proteins was altered. O‐glycosylation of O‐GlcNAc transferase (OGT), the enzyme that links N‐acetylglucosamine to proteins, was fivefold 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 permitted the detection of two new OGT target proteins: the chaperonin TCP‐1 θ and the oncogene Ets‐related protein isoform 7. Taken together, our results support the hexosamine pathway and the O‐glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell. J. Cell. Biochem. 103: 800–811, 2008. © 2007 Wiley‐Liss, Inc.