Different molecular mechanisms are responsible for primary and secondary imatinib resistance in GISTs. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.

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The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Hypoxic/ischemic conditions provoke activation of the hypoxia-inducible factor-1 (HIF-1), which functions as a transcription factor. HIF-1 is composed of the HIF-1␣ and -␤ subunits, and stability regulation occurs via accumulation/degradation of HIF-1␣ with the notion that a prolyl hydroxylase accounts for changes in protein level. In addition, there is evidence that HIF-1 is up-regulated by diverse agonists during normoxia. We investigated the impact of inflammatory mediators nitric oxide (NO) and tumor necrosis factor-␣ (TNF-␣) on HIF-1␣ regulation. For comparison, LLC-PK 1 cells were exposed to hypoxia, stimulated with desferroxamine (DFX, known to mimic hypoxia), and the thiol-cross- The transcription factor hypoxia-inducible factor-1 (HIF-1) 1 is a heterodimer composed of the helix-loop-helix/Per-Arnt-Sim protein HIF-1␣ and the aryl hydrocarbon nuclear translocator, also known as HIF-1␤ (1-3). An active HIF-1 complex accumulates in the nucleus; binds to a specific DNA sequence, the HIF-1 binding site within the hypoxia response element (HRE); and enhances transcription of hypoxia-inducible genes, such as erythropoietin or vascular endothelial growth factor. The availability of HIF-1 is mainly determined by the presence/absence of HIF-1␣ (4, 5). In many cell types, both HIF-1␣ and HIF-1␤ appear to be constitutively expressed at the mRNA level, whereas, on protein level, HIF-1␣ is degraded under normoxic conditions, which contrasts with permanently expressed HIF-1␤. Studies in von Hippel-Lindau-defective renal cell carcinomas indicated that the von Hippel-Lindau protein fulfills a critical function in HIF-1␣ degradation, thus accounting for the extremely short protein half-life (6). However, accumulation of HIF-1␣ that promotes active HIF-1 complex formation by hypoxia is not fully understood. Oxygen species such as superoxide (O 2 Ϫ ) or hydrogen peroxide (H 2 O 2 ) have been proposed to limit HIF-1␣ stability (7). A postulated source for these species is a NAD(P)H-metabolizing membrane-bound type b cytochrome quite similar to the respiratory burst oxidase in phagocytes. In addition to these intracellular redox changes, phosphorylation cascades such as mitogen-activated protein kinases have been ascribed to stabilize HIF-1␣, but precise signaling mechanisms and their cross-talk have not been not fully defined (8 -10).Activation of HIF-1 as an adaptive response was first described for conditions of decreased oxygen pressure. Therefore, most mechanistic and functional studies on HIF-1 regulation refer to hypoxic conditions. More recent evidence suggests that HIF-1 can be activated by growth factors, cytokines, hormones, or nitric oxide (NO) as well with very little information on signal transduction pathways being involved (11-15). Zhong et al. (11) verified a role of phosphatidylinositol 3-kinase (PI3K), Akt phosphorylation, and FRAP activation for HIF-1␣ induction in response to hypoxia and epithelial growth factor treatment. This pathway is negatively regulated by PTEN (phosphatase and tensin homologue de...

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RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 g/ml) and interferon-␥ (100 units/ml), respond with endogenous NO ⅐ formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO ⅐ -derived apoptosis was blocked by the NO ⅐ -synthase inhibitor N Gmonomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-␥, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO ⅐ , released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO ⅐ -mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.Nitric oxide (NO) 1 is recognized for its participation in diverse biological processes in nearly all aspects of life (1-3). The formation of NO ⅐ occurs under both physiological and pathophysiological settings. The molecule is synthesized by a family of enzymes termed NO ⅐ synthases (NOS), which utilize arginine as their substrate in the generation of NO ⅐ and stoichiometric amounts of citrulline (4). For convenience, two types of NOS are recognized; constitutive isoforms, which are active for a relatively short time in response to intracellular Ca 2ϩ fluctuations, and a cytokine-inducible isoform. For the latter to be active, mRNA translation and protein synthesis are required. The inducible NOS generates large amounts of NO ⅐ for an extended period. However, once NO ⅐ is produced by the action of NOS, it is extremely susceptible to both oxidation and reduction. This results in the concomitant formation of species with NO ϩ -like activity (nitrosonium ion) or NO Ϫ (nitroxyl anion), respectively (5). In addition to reacting with oxygen, superoxide, and transition metals, NO ⅐ causes biological signaling via interactions with sulfhydryl groups. Because multiple NO surrogates are formed, transduction pathways are classified as either cyclic GMP-dependent or -independent. Cyclic GMP formation is initiated by NO ⅐ binding to the heme group of soluble guanylyl cyclase, thus causing enzyme activati...

During proliferative glomerulonephritis, the early phase of mesangiolysis is linked to increased nitric oxide (NO) production. NO. as well as superoxide (O2-) are inflammatory mediators that are generated by mesangial cells (MC) after cytokine stimulation. Added individually, both radicals induce MC apoptosis. However, the co-existence of a defined NO./O2- ratio is cross-protective. Apoptosis is characterized by specific features such as chromatin condensation, DNA strand breaks, and the occurrence of apoptotic regulating proteins. The tumor suppressor p53 and Bax (Bcl-2 associated protein x) are considered to be classical death promotors, which accumulate after toxic insults. To study p53 and Bax protein accumulation in NO. and/or O2(-)-induced apoptosis, we used the NO-donor S-nitrosoglutathione (GSNO) and the redox cycler 2,3-dimethoxy-1,4-naphtoquione (DMNQ). Both agonists initiated DNA fragmentation in a concentration dependent manner associated with transient p53 and Bax up-regulation. Co-generation of NO./O2- resulted not only in reduced DNA fragmentation, but also in decreased Bax accumulation. Comparable to the NO./O2- co-generation, cytokines failed to induce apoptosis. In contrast, cytokines in combination with pyrrolidine dithiocarbamate, which blocks endogenous superoxide dismutase, allowed p53 and Bax accumulation as well as DNA fragmentation. Our results demonstrate p53 and Bax as early components in NO. and O2(-)-induced rat MC apoptosis and point to the NO./O2- interaction as a naturally occurring cell defense mechanism.

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