Decrease of glutaminase expression by interferon-gamma in human intestinal epithelial cells.
BACKGROUND
Glutaminase, the principal enzyme of glutamine hydrolysis, breaks down glutamine to supply energy and intermediates for cell growth and is present in high concentrations in replicating tissues such as intestinal epithelium and malignant tumors. In the host with cancer, glutaminase activity in the gut mucosa diminishes as the tumor grows, but the regulation of this response is unknown. Because cytokines may regulate the altered glutamine metabolism that is characteristic of the host with cancer, we studied the effects of cytokines on gut mucosal glutaminase expression in vitro using the human enterocytic Caco-2 cell line.


METHODS
Differentiated confluent cells were incubated with interleukin (IL)-1, IL-6, tumor necrosis factor, or interferon-gamma (IFN-gamma). After a 12-h incubation, glutaminase-specific activity and kinetic parameters (maximal enzyme activity [Vmax] and enzyme affinity [Km]) were determined. Glutaminase protein concentration was determined by Western blot analysis using a rabbit antirat polyclonal antibody. Total cellular RNA was extracted for Northern hybridization and radiolabeled with a glutaminase cDNA probe.


RESULTS
Of the cytokines studied, only IFN-gamma altered glutaminase activity. Kinetic studies indicated a decrease in activity secondary to a 25% decrease in Vmax with no change in Km, consistent with a reduction in the number of glutaminase molecules rather than a change in enzyme affinity. Glutaminase protein was decreased 50% in IFN-gamma-treated cells when compared with controls. This decrease was dose-independent and was associated with a concomitant 75% decrease in glutaminase messenger RNA levels. These reductions in message and protein translated into a 60-80% decrease in functional glutaminase-specific activity.


CONCLUSIONS
This IFN-gamma-mediated decrease in glutaminase activity may be one mechanism by which gut glutamine metabolism is diminished as the tumor grows and becomes the principal organ of glutamine use.
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
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P
Sarantos
Z
Z
Abouhamze
E M
EM
Copeland
W W
WW
Souba
D000230:Adenocarcinoma / Q000201:enzymology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000201:enzymology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005972:Glutaminase / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007422:Intestines / Q000187:drug effects / Q000201:enzymology*; D012333:RNA, Messenger / Q000187:drug effects; D013053:Spectrophotometry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D015850:Interleukin-6; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D005972:Glutaminase