The inhibition of NF-kappaB activation pathways and the induction of apoptosis by dithiocarbamates in T cells are blocked by the glutathione precursor N-acetyl-L-cysteine

Nuclear factor-kappaB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-kappaB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-kappaB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-kappaB inhibitor, can actually enhance rather than inhibit IkappaB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-kappaB inhibition. This was observed at the level of IkappaB degradation, NF-kappaB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-kappaB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.     

Human immunodeficiency virus type-1 Tat protein induces nuclear factor (NF)-κB activation and oxidative stress in microglial cultures by independent mechanisms

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro-inflammatory cytokine interleukin-1beta and tumour necrosis factor-alpha in a nuclear factor (NF)-kappaB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-kappaB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF(2alpha) accumulation without affecting NO and cytokine production. Consistently, Tat-induced IkappaBalpha degradation - an index of NF-kappaB activation - was not affected by free radical scavengers, but was prevented by an NF-kappaB-specific inhibitor. Our observations indicate that NF-kappaB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-kappaB activation induced by Tat occur by independent mechanisms.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.     

NF-kappaB activation and IkappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells

This review will discuss the recent literature on the molecular mechanism of NF-kappaB activation, with special focus on IkappaB alpha dynamism involved in iNOS- and chemokine-induction in glial cells. NF-kappaB, a heterotrimer composed of p50, p65 (Rel A) and IkappaB alpha, has been shown to be activated by elimination of the regulatory subunit IkappaB alpha from the heterotrimer. The elimination of IkappaB alpha (formation of active NF-kappaB, p50-p65) is due to phosplorylation of serines 32 and 36 of IkappaB alpha, followed by polyubiquitination and 26S proteasomal degradation of IkappaB alpha. Experiments using stable clones of rat C6 glioma cells transfected with dominant negative IkappaB alpha (serines 32 and 36 replaced by alanine) suggest that NF-kappaB activation (phosphorylation of IkappaB alpha) is involved in LPS/IFNgamma- or IL-1beta/IFNgamma-induced iNOS expression. Furthermore, the time courses of phosphorylation, ubiquitination of IkappaB alpha and proteasome activity after IL-1beta treatment also suggest that 26S proteasomal degradation of IkappaB alpha is more crucial for chemokine expression in glial cells.     

Pyrrolidine dithiocarbamate inhibits TNF-alpha-dependent activation of NF-kappaB by increasing intracellular copper level in human aortic smooth muscle cells

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC and another antioxidant, N-acetyl-l-cysteine (NAC), on TNF-alpha-dependent activation of NF-kappaB in human aortic smooth muscle cells (HASMC). Treatment of the cells with TNF-alpha induced the activation of p65/p50 heterodimer NF-kappaB and increased the mRNA levels of monocyte chemoattractant protein (MCP)-1. Pretreatment with PDTC markedly suppressed the NF-kappaB activation and expression of MCP-1 by inhibiting IkappaB-alpha degradation. In contrast, NAC had no effect. PDTC concomitantly increased the intracellular levels of copper, and bathocuproinedisulfonic acid, a non-cell-permeable chelator of Cu(1+), inhibited the PDTC-induced increase in intracellular copper level and reversed the PDTC effects on IkappaB-alpha, NF-kappaB, and MCP-1. These results indicate that TNF-alpha-dependent expression of MCP-1 in HASMC is tightly regulated by NF-kappaB and that intracellular copper level is crucial for the TNF-alpha-dependent activation of NF-kappaB in HASMC.     

Inhibitory effect of DCDC on lipopolysaccharide-induced nitric oxide synthesis in RAW 264.7 cells

In the present study we have examined the effect of DCDC (2',5'-dihydroxy-4-chloro-dihydrochalcone) on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to DCDC inhibited the nitrite accumulation in culture medium. DCDC also concentration-dependently inhibited LPS-stimulated increase of iNOS expression; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory action to DCDC is mainly due to the inhibition on iNOS expression rather than iNOS enzyme activity. DCDC significantly inhibited LPS-evoked degradation of IkappaB-alpha and the nuclear translocation of NF-kappaB; it also exhibited the activity of scavenging the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DCDC also inhibited cyclooxygenase-2 activity in RAW 264.7 cells with an IC50 of 3.0 microM; furthermore, it also significantly decreased LPS-induced mortality rate in mice. Taken together, we demonstrate that DCDC exhibits inhibitory effects on nitric oxide production through the inhibition of IkappaB-alpha degradation and NF-kappaB activation, and therefore the suppression of iNOS expression. DCDC also shows the antioxidant activity and COX-2 inhibitory action. Moreover, it improves survival in a murine model of endotoxaemia suggesting that DCDC may be potential in the therapy of septic shock.