The Rgr oncogene (homologous to RalGDS) induces transformation and gene expression by activating Ras, Ral and Rho mediated pathways.

The effects of the 5'-truncated Rgr oncogene, a previously shown specific guanine exchange factor for Ral in vitro, in stimulating proliferation, cell transformation and gene expression were investigated. We have established TetRgr cell lines in which expression of Rgr can be inhibited by the presence of tetracycline in the medium. Using this system, we show that Rgr overexpressing cells are morphologically transformed and grow in a disorganized manner. At the transcriptional level, Rgr enhances the activity of the serum response element and c-Jun. Rgr induces phosphorylation of ERKs, p38 and JNK kinases, and increases the levels of the GTP-bound forms of Ral and Ras. Ras activation could account for the broad spectra of effects displayed by Rgr. The important role of these pathways is confirmed by experiments in which the transcriptional activation events can be blocked by dominant negative versions of Ras, Ral and Rho. Among all the Rgr-induced pathways, the Ras-Raf-MEK-ERK cascade is essential for the transforming properties of Rgr. Additional analysis has shown that the activation of this pathway by Rgr is not due to a feed back mechanism mediated by the Grb2 adaptor protein. Oncogene (2000).

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells.

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.

Tumor necrosis factor-alpha increases ATP content in metabolically inhibited L929 cells preceding cell death.

The effects of tumor necrosis factor-alpha (TNF) on ATP levels were studied in metabolically inhibited L929 cells. Treatment of these cells with TNF in the presence of actinomycin D or cycloheximide induces cyclic changes in the intracellular ATP content preceding cell death. After 3 h of incubation, the intracellular ATP content increased by 48 +/- 6% (p < 0.001), but at 4 h, it decreased to the control level. Two hours later, it increased again by 23 +/- 5% over the control level (p < 0.001). Coinciding with cell death, ATP content decreased progressively until almost complete depletion. These changes in ATP content were associated with parallel alterations in the respiratory coupling and with increased generation of reactive oxygen species. The mechanism by which TNF/actinomycin D or TNF/cycloheximide increased cellular ATP seemed to be dependent on the mitochondrial ATP synthesis and related to the cytotoxic effect of TNF, since blockade of mitochondrial electron transport prevented the increase in cellular ATP, the formation of reactive oxygen species, and the apoptotic cell death caused by TNF. We suggest that the TNF/actinomycin D- or TNF/cycloheximide-induced changes in intracellular ATP levels may be involved in the cytotoxic effect of TNF in metabolically inhibited L929 cells.

Tumor necrosis factor alpha inhibits collagen alpha 1(I) gene expression in rat hepatic stellate cells through a G protein.

BACKGROUND & AIMS: Tumor necrosis factor alpha (TNF-alpha) inhibits collagen gene expression in cultured fibroblasts. By binding to cell surface receptors, TNF-alpha promotes signals within the cells. The purpose of this study was to investigate the role played by G proteins in TNF-alpha-induced inhibition of collagen gene expression. METHODS: Effect of TNF-alpha on collagen alpha 1(I) messenger RNA (mRNA) level was measured in cultured hepatic stellate cells in basal condition and after inhibiting or activating G proteins or the major intracellular signal transduction pathways. RESULTS: TNF-alpha significantly decreased the level of alpha 1(I) collagen mRNA. Treatment of cells with pertussis toxin inhibited this effect, whereas blocking adenylate cyclase or protein kinase A had no effect. Likewise, blocking phospholipase A2, phospholipase C1 calcium channels, calmodulin, or protein kinase C did not eliminate the inhibitory effect of TNF-alpha on collagen mRNA. On the other hand, C2-ceramide and sphingomyelinase reproduced the effect of TNF-alpha on collagen gene expression, and TNF-alpha did not increase the effect of sphingomyelinase. CONCLUSIONS: TNF-alpha-induced inhibition of alpha 1(I) collagen gene expression in a hepatic stellate cell line may be mediated by a pertussis toxin-sensitive G protein. TNF-alpha may inhibit this gene by using sphingomyelin/ceramide as an intracellular signal transduction pathway.

Toxic oil stimulates collagen synthesis acting at a pretranslational level in cultured fat-storing cells.

BACKGROUND/AIMS: The toxic oil syndrome appeared in Spain in 1981 as a result of ingestion of rapeseed oil denatured with aniline. Some patients developed scleroderma-like skin lesions and liver cirrhosis. Mechanisms of these fibrotic lesions are not known. The present study was designed to investigate the effect of toxic oils on collagen metabolism. METHODS: We measured the relative rate of collagen production, absolute rate of collagen synthesis, production, secretion, and degradation, proline transport, steady-state levels of procollagen alpha 1(l)-messenger RNA (mRNA) in cultured fat-storing cells, and chloramphenicol acetyltransferase activity in transfected cells. RESULTS: Toxic oils increased collagen synthesis, procollagen alpha 1(l)-mRNA levels, and chloramphenicol acetyltransferase activity in cultured fat-storing cells. Effect on collagen production correlated with lipid peroxide content in oils. Cycloheximide, alpha-tocopherol, and methylene blue prevented the increase in procollagen alpha 1(l)-mRNA. Oleylanilide and linoleylanilide, markers for toxic oils, reproduced the stimulatory effects of toxic oils on collagen production and procollagen alpha 1(l)-mRNA. CONCLUSIONS: Toxic oils increased collagen synthesis by acting on the promoter of procollagen alpha 1(l) gene, probably through lipid peroxides derived from acylanilides. We suggest that toxic oil may have stimulated procollagen gene expression through the formation of adducts of aldehydes with some transcription factor.

Reversal of carbon tetrachloride induced changes in microviscosity and lipid composition of liver plasma membrane by colchicine in rats.

Colchicine is beneficial in the treatment of cirrhotic patients, it prevents changes in plasma membrane bound enzymes induced by CCl4 intoxication. In this study, lipid composition and microviscosity were measured in liver plasma membranes isolated from rats given CCl4. Microviscosity values increased in rats given CCl4 for six weeks but fell considerably in those given CCl4 for 10 weeks. Both these changes were absent when colchicine was given with CCl4. The cholesterol/phospholipid molar ratios and lipid peroxide values increased but plasma membrane phospholipids, the length of fatty acyl chains, and the unsaturation index fell significantly after CCl4 intoxication. Colchicine treatment also prevented these changes. Changes in the lipid composition of liver plasma membranes were significantly correlated with lipid peroxidation. Colchicine prevents changes in the physicochemical properties of liver plasma membranes induced by longterm CCl4 treatment, probably by blocking peroxidation of unsaturated fatty acids.

Diminished cytochrome b content and toxic oxygen metabolite production in circulating neutrophils from patients with Crohn's disease.

Phagocytic, chemotactic, and oxidative metabolic capacity of circulating neutrophils was studied in 20 patients with Crohn's disease. In vitro tests of chemotaxis and phagocytosis of isolated neutrophils from patients did not differ from that of healthy controls. However, superoxide anion production by phorbol-myristate-acetate and formylmethionyl-leucyl-phenylalanine-stimulated neutrophils from patients with Crohn's disease was significantly diminished compared with controls. Measurement of cytochrome b559 in total membranes of neutrophils from patients showed that it was significantly lower than in controls. Disease activity did not correlate either with the production of superoxide anion or with the cytochrome b559 content. It is concluded that oxidative metabolism is impaired in neutrophils from patients with Crohn's disease and that this defect could be caused by a reduced content in membrane b-type cytochrome. Although this defective neutrophil function may contribute to granuloma formation, other factors have to be implicated in disease inflammatory activity.