Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P₄) were transfected by lipofection with c-Ha-ras^EJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-ras^EJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor-age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. α-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-ras^EJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential. © 1994 Wiley-Liss, Inc.     

Investigation of the mechanism underlying the inhibitory effect of heterologous ras genes in plant cells

The ras genes from yeast and mammalian cells were fused to plant expression promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development. All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues. This is consistent with earlier findings that heterologous ras genes were highly lethal to protoplasts following direct DNA uptake. These effects could not be reversed by increasing exogenous or endogenous cytokinin levels. These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras proteins such as effector-binding and membrane-binding sites. Similarly, co-transformation with the genes encoding the Ras-negative regulators, GTPase-activating protein and neurofibromin did not affect the ras inhibitory effect, indicating that the mechanism of ras inhibition of plant cells is not related to normal ras cellular functions. This conclusion was supported by further studies in which ras gene expression was modified using various promoters and antisense constructs. The introduced ras sequences remained fully inhibitory regardless of which promoters (inducible or tissue-specific) or which orientations (sense or antisense) were tested. This strongly suggests that the ras DNA sequence itself, rather than the Ras protein or ras mRNA, is directly involved in the inhibitory effect. The mechanism underlying this novel phenomenon remains unknown. Introduced ras genes may inhibit plant cell growth by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth.     

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.     

Effects of oxygen free radicals on articular chondrocytes in culture: C-myc and c-Ha-ras messenger RNAs and proliferation kinetics

Since oxygen free radicals are believed to play an important role in cartilage degradation, we studied the effects of these radicals generated by the hypoxanthine-xanthine oxidase system on rabbit articular chondrocytes in culture. Among the damages induced by these radicals, cell proliferation inhibition and G2 arrest were observed. To elucidate the mechanisms involved in this phenomenon, the expression of c-myc and c-Ha-ras genes whose products are associated with cell growth control was studied. Results showed that in chondrocytes, c-myc and c-Ha-ras expression was particularly important during the G1 phase of the cell cycle and that oxygen reactive species, especially H₂O₂, induced an important decrease of c-myc and c-Ha-ras mRNA levels. Chondrocytes cell cycle analysis revealed an accumulation of cells in G2 phase. It led us to suggest that the chondrocyte cell cycle perturbations observed after oxygen free radicals treatment could be associated with the decrease of c-myc and c-Ha-ras expression.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Yeast RAS2 affects cell viability,mitotic division and transient gene expression in Nicotiana species

Overexpression of the budding yeast RAS2 gene in Nicotiana plumbaginifolia cells revealed that RAS2 acted as a suicide gene in freshly isolated protoplasts from leaves and blocked cell proliferation in cell suspension-derived protoplasts. Among a series of genes tested (such as npt II, CDC35, PDE2), RAS2 was the only one to block the expression of the cat gene, as measured in a transient gene expression assay. Another ras gene, v-Ha-ras, had similar effects. Furthermore, the RAS2 effect was species-specific and depended on the modulation of hormonal metabolism in the transfected cells, while no differences were noticed between the normal and the activated val19 gene. Transfected plant cells are shown to synthesize a RAS2 protein of the same electrophoretic mobility as the yeast RAS2 product. The results are discussed in the broader context of the evolutionarily conserved ras genes involved in vital cellular functions.     

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12–0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair. © 1994 Wiley-Liss, Inc.     

Vanadium chemistry and biochemistry of relevance for use of vanadium compounds as antidiabetic agents

The stability of 11 vanadium compounds is tested under physiological conditions and in administration fluids. Several compounds including those currently used as insulin-mimetic agents in animal and human studies are stable upon dissolution in distilled water but lack such stability in distilled water at pH7. Complex lability may result in decomposition at neutral pH and thus may compromise the effectiveness of these compounds as therapeutic agents; Even well characterized vanadium compounds are surprisingly labile. Sufficiently stable complexes such as the VEDTA complex will only slowly reduce, however, none of the vanadium compounds currently used as insulin-mimetic agents show the high stability of the VEDTA complex. Both the bis(maltolato)oxovanadium(IV) and peroxovanadium complexes extend the insulin-mimetic action of vanadate in reducing cellular environments probably by increased lifetimes under physiological conditions and/or by decomposing to other insulin mimetic compounds. For example, treatment with two equivalents of glutathione or other thiols the (dipicolinato)peroxovanadate(V) forms 9dipicolinato)oxovanadate(V) and vanadate, which are both insulin-mimetic vanadium(V) compounds and can continue to act. The reactivity of vanadate under physiological conditions effects a multitude of biological responses. Other vanadium complexes may mimic insulin but not induce similar responses if the vanadate formation is blocked or reduced. We conclude that three properties, stability, lability and redox chemistry are critical to prolong the half-life of the insulin-mimetic form of vanadium compounds under physiological conditions and should all be considered in development of vanadium-based oral insulin-mimetic agents.Abbreviations ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - ADP-V adenosine 5-diphosphate-vanadate - bpV bis(peroxo)oxovanadium(V) - (bpV)2 bis(peroxo)oxovanadium(V) dimer - bpVpic bis(peroxo)picolinatooxovanadate(V) - ¹³C carbon-13 - EDTA ethylenediaminetetraacetic acid - EPR electron paramagnetic resonance - EXSY exchange spectroscopy - ¹H proton - HSG glutathione - NAD -nicotinamide adenine dinucleotide - NADP -nicotinamide adenine dinucleotide phosphate - NADV -nicotinamide adenine dinucleotide vanadate - NMR nuclear magnetic resonance (also referred to as magnetic resonance imaging) - pVdipic (dipicolinato)peroxovanadate(V) - Vcit (citrato)dioxovanadate(V) - VEDTA (ethylenediaminetetraacetato)dioxovanadate(V) - Vmalto bis(maltolato)-oxovanadium(IV) - Voxal bis(oxalato)dioxovanadate(V) - ⁵¹V vanadium-51 - V₁ vanadate monomer - V₂ vanadate dimer - V₄ vanadate tetramer - V₅ vanadate pentamer - UV-vis spectroscopy ultraviolet-visible spectroscopy     

Reduction of vanadium(V) by <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> EV-SA01 isolated from a South African deep gold mine

Dissimilatory reduction of vanadium(V) by Enterobacter cloacae EV-SA01, isolated from a gold mine at 1.6 km below surface, is shown to occur anaerobically as well as aerobically. Growth rates were unaffected by up to 2 mM V₂O₅. Reduction of vanadium(V) was growth phase-dependent and resulted in cell deformities and precipitation of the vanadium in its lower oxidation states. The vanadate reductase activity was membrane-associated and coupled the oxidation of NADH to the reduction of vanadate.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Comparative Effects of Direct Cadmium Contamination on Gene Expression in Gills, Liver, Skeletal Muscles and Brain of the Zebrafish (Danio rerio)

The effects of cadmium (Cd) on gene expression were examined in four organs (gills, liver, skeletal muscles and brain) of the zebrafish. Adult male fish were subjected to three different water contamination pressures over periods of 7 and 21 days: control medium (C₀: no Cd added) and two contaminated media (C₁: 1.9 ± 0.6 μg Cd l⁻¹, and C₂: 9.6 ± 2.9 μg Cd l⁻¹). Fourteen genes involved in antioxidant defences, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair and apoptosis were selected and their expression levels investigated by quantitative real-time PCR. Cadmium concentrations were determined in the four organs and metallothionein (MT) protein levels investigated in brain, liver and gills. Although skeletal muscle was a poor Cd-accumulating tissue, many genes were up-regulated at day 7: mt1, cyt, bax, gadd and rad51 genes. Three additional genes, c-jun, pyc and tap, were up-regulated in muscles at day 21 whereas bax, gadd and rad51 had returned to basal levels. Surprisingly, mt1 and c-jun were the only genes displaying a differential induction after 21 days in liver, although this organ accumulated the highest cadmium concentration. In brain, only mt1, mt2 and c-jun genes were up-regulated after 21 days. In gills, the highest response was observed after 7 days, featuring the differential expression of oxidative stress-response hsp70 and mitochondrial sod genes, along with genes involved in mitochondrial metabolism and metal detoxification. Then, after 21 days, the expression of almost every genes returned to basal levels while both mt1 and mt2 genes were up-regulated.     

Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

Vanadyl-and Vanadate-Induced Lipid Peroxidation in Mitochondria and in Phosphatidylcholine Suspensions

Vanadyl (V_(IV)) was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V_(V)) to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect.

Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene.

All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.