Steve F. Abcouwer

Expression of Angiogenic Factors Vascular Endothelial Growth Factor and Interleukin-8/CXCL8 Is Highly Responsive to Ambient Glutamine Availability Role of Nuclear Factor-κB and Activating Protein-1

Vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8) are prominent pro-angiogenic and pro-metastatic proteins that represent negative prognostic factors in many types of cancer. Hypoxia is thought to be the primary environmental cause of VEGF and IL-8 expression in solid tumors. We hypothesized that a lack of nutrients other than oxygen could stimulate the expression of these factors and previously demonstrated that expression of VEGF and IL-8 is responsive to amino acid deprivation. In the present study, we examined the effect of glutamine availability on the expression of these factors as well as the role of transcription factors NFκB and activating protein-1 (AP-1) in the response of TSE human breast carcinoma cells to glutamine deprivation. VEGF and IL-8 secretion and mRNA levels were dramatically induced by glutamine deprivation. mRNA stabilization contributed to this response. Glutamine deprivation increased NFκB (p65/p50) and AP-1 (Fra-1/c-Jun+JunD) DNA-binding activities. Blocking NFκB and AP-1 activation with curcumin as well as expression of dominant inhibitors, inhibitor of nuclear factor-κB (IκB) super repressor (IκBM), and a mutant form of c-Fos (A-Fos) demonstrated that the activation of NFκB and AP-1 transcription factors was necessary for the induction of IL-8 expression but dispensable for the induction of VEGF expression. A macro-array containing 111 NFκB target genes identified a total of 17 that were up-regulated 2-fold or more in response to glutamine deprivation. These included growth regulated oncogene α (GROα/GRO1/CXCL1), another neutrophil chemoattractant implicated in tumor angiogenesis and metastasis.

Mechanisms Governing the Expression of the Enzymes of Glutamine Metabolism—Glutaminase and Glutamine Synthetase

Whether on the scale of a single cell, organ or organism, glutamine homeostasis is to a large extent determined by the activities of glutaminase (GA, EC 3.5.1.2) and glutamine synthetase (GS, EC 6.3.1.2), the two enzymes that are the focus of this report. GA and GS each provide examples of regulation of gene expression at many different levels. In the case of GA, two different genes (hepatic- and kidney-type GA) encode isoforms of this enzyme. The expression of hepatic GA mRNA is increased during starvation, diabetes and high protein diet through a mechanism involving increased gene transcription. In contrast, the expression of kidney GA mRNA is increased post-transcriptionally by a mechanism that increases mRNA stability during acidosis. We found recently that several isoforms of rat and human kidney-type GA are formed by tissue-specific alternative RNA splicing. Although the implications of this post-transcriptional processing mechanism for GA activity are not yet clear, it allows for the expression of different GA isoforms in different tissues and may limit the expression of GA activity in muscle tissues by diverting primary RNA transcripts to a spliceform that produces a nonfunctional translation product. The expression of GS enzyme is also regulated by both transcriptional and post-transcriptional mechanisms. For example, the GS gene is transcriptionally activated by glucocorticoid hormones in a tissue-specific fashion. This hormonal response allows GS mRNA levels to increase in selected organs during catabolic states. However, the ultimate level of GS enzyme expression is further governed by a post-transcriptional mechanism regulating GS protein stability. In a unique form of product feedback, GS protein turnover is increased by glutamine. This mechanism appears to provide a means to index the production of glutamine to its intracellular concentration and, therefore, to its systemic demand. Herein, we also provide experimental evidence that GS protein turnover is dependent upon the activity of the 26S proteosome.

Determinants of glutamine dependence and utilization by normal and tumor‐derived breast cell lines

A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cells need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor‐derived cells. The rates of CO2 production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32–80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system X  −C These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis. J. Cell. Physiol. 176:166–178, 1998.

Glutamine deprivation induces the expression of GADD45 and GADD153 primarily by mRNA stabilization.

The expression of the growtharrest- and DNA damage-inducible genes, GADD45 and GADD153/CHOP(C/EBP-homologous protein), as well as GRP78 (glucose-regulatedprotein of 78 kDa) was examined in several human breast cell lines subjected to acute glutamine (GLN) deprivation. GLN deprivation caused rapid elevation of GADD45 andGADD153/CHOP mRNA levels in cells that were highly dependent upon GLN for growth and viability. BothGADD mRNAs were rapidly elevated up to several hundred-fold. In contrast, GRP78 expression was elevated by no more than 4-fold by GLN deprivation. The magnitude ofGADD up-regulation roughly correlated with the extent of GLN dependence of each cell line. The levels of all three mRNAs were responsive to alterations of ambient GLN content in a physiologically relevant concentration range that corresponded to the affinities of cellular GLN transporters. Provision of GLN-derived metabolites partially inhibited the induction of GADDexpression in GLN-deprived cells. Nuclear run-on assays and mRNA decay studies suggested that the primary mechanism leading to increasedGADD mRNA levels was not transcriptional, but rather that GADD45 and GADD153/CHOPexpression were up-regulated in response to GLN deprivation via marked stabilization of these mRNAs. These results suggest that the expression of GADD genes contributes to growth arrest and/or protection from metabolic damage during GLN-poor conditions.

Expression of the pro-angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 by human breast carcinomas is responsive to nutrient deprivation and endoplasmic reticulum stress

BackgroundThe expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-κB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-κB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines.ResultsWe found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153).ConclusionThese results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

Identification of glucocorticoid-responsive elements that control transcription of rat glutamine synthetase.

Basal expression of glutamine synthetase (GS) is very low in rat lung and muscle and remarkably enhanced by glucocorticoid hormones during trauma and catabolic states. Although this response is believed to be transcriptionally regulated, the genetic elements responsible for tissue-specific glucocorticoid induction of GS expression have not been identified. A rat lung epithelial cell line (L2) and a glucocorticoid receptor-deficient human prostate cancer cell line (PC3), together with GS reporter gene constructs, were utilized in gene transfer experiments to identify two regions within the rat genomic clone gGS3 that imparted dexamethasone (Dex) responsiveness to both the homologous GS promoter and the heterologous herpes simplex virus thymidine kinase promoter in glucocorticoid receptor-dependent fashions. One region lies nearly 6 kb upstream of the GS transcription initiation site, and the other lies within the first intron of the GS gene. Dex responsiveness was localized to a 325-bp fragment of the intron region containing a canonical glucocorticoid response element and to a 225-bp fragment of the far-upstream region containing three separate glucocorticoid response element half-sites. The GS promoter exhibited relatively high basal activity that was repressed by inclusion of the far-upstream or the intron glucocorticoid-responsive region. Dex treatment negated this repression. A model is suggested in which the glucocorticoid-receptor unit causes derepression of lung and muscle GS transcription during trauma and catabolic states.

Glutamine synthetase expression in muscle is regulated by transcriptional and posttranscriptional mechanisms

Skeletal muscle exports glutamine (Gln) and increases the expression of the enzyme glutamine synthetase (GS) in response to physiological stress. Acute stress or direct glucocorticoid administration raises muscle GS mRNA levels dramatically without a parallel increase in GS protein levels. In the lung, this discrepancy is caused by feedback destabilization of the GS protein by its product Gln. It was hypothesized that muscle GS protein levels increase during stress only when the intracellular Gln pool has been depleted. Adult male rats were injected with the glucocorticoid hormone dexamethasone (DEX) to mimic the acute stress response and with the GS inhibitor methionine sulfoximine (MSO) to deplete muscle Gln stores. DEX increased GS mRNA levels by 2.8-fold but increased GS protein levels by an average of only 40%. MSO diminished muscle GLN levels by 68% and caused GS protein levels to rise in accordance with GS mRNA. Chronic stress was mimicked using 6 days of MSO treatment, which produced anorexia, 23% loss of body weight, and 64% decrease in muscle Gln levels, as well as pronounced increases in both muscle GS mRNA (26-fold) and protein levels (35-fold) without elevation of plasma glucocorticoid levels. Calorie-restricted pair-fed animals exhibited lesser increases in muscle GS mRNA (8-fold) and protein levels (5-fold) without a decline in muscle Gln content. Thus regulation of GS expression in both acute and chronic stress involved both transcriptional and posttranscriptional mechanisms, perhaps affected by muscle Gln content.

Glutamine synthetase expression in rat lung is regulated by protein stability

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500-700%, GS activity levels rise only 20-45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% ( P < 0.01) and decreased lung tissue Gln levels by 70% ( P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold ( P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% ( P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold ( P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction ( P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.

Regulation of glutamine synthetase in human breast carcinoma cells and experimental tumors

BACKGROUND Acute deprivation of extracellular glutamine causes up-regulation of glutamine synthetase (GS) expression by a mechanism involving an increase in GS protein stability. This study examines GS expression in a highly glutamine-dependent and tumorigenic human breast cancer cell line, TSE cells, in response to acute and chronic glutamine deprivation in culture and during tumor formation. METHODS TSE cells were subjected to acute glutamine deprivation, adapted to growth in low glutamine concentrations, and subcutaneously injected into nude mice. GS protein and mRNA levels were assayed by Western and Northern blotting, and intracellular glutamine levels were evaluated by using a colorimetric assay. RESULTS GS protein levels increased, but GS mRNA levels were unchanged in response to acute glutamine deprivation. Chronic glutamine deprivation in vitro and tumor growth in vivo caused an increase in both GS protein and mRNA levels. Large tumors exhibited lower intracellular glutamine, higher GS protein, and relatively unchanged GS mRNA levels relative to small tumors. CONCLUSIONS TSE tumors exhibit up-regulation of GS protein and mRNA levels and declines in intracellular glutamine content, suggesting that growth in vivo causes a chronic and progressive glutamine deprivation. Up-regulation of GS expression may contribute to adaptation to a nutrient-poor intratumor environment.

Induction of cytokine-induced neutrophil chemoattractant (CINC) mRNA in the lungs of septic rats

OBJECTIVE To study cytokine-induced neutrophil chemoattractant (CINC) mRNA induction in lungs of normal, neutropenic, and adrenalectomized rats after intraperitoneal Escherichia coli lipopolysaccharide (LPS) administration and in cultured rat pulmonary cell lines after exposure to mediators of the septic response. MATERIALS AND METHODS Northern blotting was used to assay relative CINC mRNA levels and a colorimetric myeloperoxidase assay was used as a measure of neutrophil infiltration. RESULTS After a single dose of LPS, rapid induction of CINC mRNA coincided with neutrophil infiltration into lungs, a response that lasted approximately 12 to 24 hours. Multiple LPS treatments resulted in a similar CINC response, but a more prolonged myeloperoxidase response. CINC mRNA induction in lungs was heightened 30% in adrenalectomized animals and 400% in neutropenic ones. LPS and cytokines induced CINC mRNA in cultured endothelial and epithelial cells. CONCLUSIONS Induction of CINC mRNA expression in pulmonary endothelial and/or epithelial cells by systemic LPS or cytokines may play a role in mediating neutrophil infiltration into lungs during sepsis. Markedly increased CINC induction in the lungs of neutropenic animals suggests that neutrophils may act to inhibit expression of this chemoattractant via a negative feedback mechanism.