Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.     

Control of nucleolar growth during interphase in higher plant meristem cells

Summary The evolution of nuclear and nucleolar sizes throughout interphase have been studied in synchronous caffeine-labeled binucleate cells of onion root meristems by using silver impregnation and stereological methods over semithin sections. Nucleus and nucleolus grow independently, since nucleolus enlarges at its fastest rate in G 1, while nucleus grows mostly in two periods: onset of replication and G 2. Nucleolar size in the cycle seems to be a genecontrolled function, hardly affected by protein synthesis inhibition. Hence, there is a biphasic response to cycloheximide (CHM) in the fast growing nucleoli of both early and late G 1 with an initial stimulation later counterbalanced by a depressed rate, so that nucleolar size in S was similar to control shortly afterwards the start of the CHM treatment. The initial enlargement under CHM was due to an increase of all nucleolar structural components, i.e., fibrillar, granular, vacuolar, and lacunar regions. No cycloheximide effect whatsoever was detected in S and G 2 nucleoli.Abbreviations CHM cycloheximide - F fibrillar component - G granular component - L lacunae - V vacuoles - VN nuclear volume - VNu nucleolar volume - VvNu volume density of the nucleoli     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid–soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid-soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

14C]serine from phosphatidylserine labels ceramide and sphingomyelin in L929 cells: evidence for a new metabolic relationship between glycerophospholipids and sphingolipids

After incubation of L929 cells with [14C]serine and various effectors an inverse correlation between label in ceramide and phosphatidylserine (PS) was displayed. This surprising behavior of the two metabolites prompted us to check whether serine of PS could be a source for ceramide synthesis. We therefore incubated L929 cells for 30 min in serum-free medium with L-phosphatidyl-L-[3-14C]serine in the presence or in the absence of cycloserine, an established inhibitor of serine palmitoyltransferase. During this short period L-phosphatidyl-L-[3-14C]serine labeled ceramide and this label was suppressed by cycloserine. Then L929 cells were grown for 16-18 h in the presence of L-phosphatidyl-L-[3-14C]serine. After this period the label was seen in sphingomyelin. Labeling of ceramide and sphingomyelin by serine from PS provides evidence for a new metabolic relationship between glycerophospholipids and sphingolipids.     

Fragmentation of condensed chromatin in nuclei of a plant sperm,pteridium aquilinum (L.) Kuhn

Nuclei of pteridium sperm have been dispersed by turbulence in natural or slightly alkaline buffer after stripping off the cytoplasm with nonionic detergent. The nuclei tended to break up into fragments arranged in a linear order. These fragments fluoresced brightly with acridine orange as did intact nuclei. Grounds are given for identifying the smaller fragments with chromosomes. It is proposed that the sperm nucleus of British Pteridium, possibly an autotetrapolid, consists of a sequence of paired homologues.     

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.     

Apoptosis-suppressing and autophagy-promoting effects of calpain on oridonin-induced L929 cell death

Calpain, calcium-dependent cysteine protease, is reported here to impose the crucial influence on oridonin-induced L929 cell apoptosis and autophagy. We found that inhibition of calpain increased oridonin-induced Bax activation, cytochrome c release and PARP cleavage, indicating that calpain plays an anti-apoptotic role in oridonin-induced L929 cell apoptosis. To explore this potential anti-apoptotic mechanism, we inhibited calpain and proteasome activity in oridonin-induced L929 cell apoptosis, and discovered that the inducible IκBα proteolysis was partially blocked by the inhibition of either calpain or proteasome, but completely blocked by the inhibition of both. It demonstrated that calpain and proteasome were two distinct pathways participating in IκBα degradation. To further study the role of calpain in oridonin-induced L929 cell autophagy, we discovered that calpain inhibitor decreased oridonin-induced autophagy, as well as Beclin 1 activation and the conversion from LC3-I to LC3-II. Moreover, Inhibition of autophagy by 3-MA increased oridonin-induced apoptosis. In conclusion, besides suppressing apoptosis, calpain promotes autophagy in oridonin-induced L929 cell death, and inhibition of autophagy might contribute to up-regulation of apoptosis.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

A short note on the nucleolar size and density in apoptotic leukemic granulocytic precursors (HL-60 cells)

The present study was undertaken to provide more information on the nucleolar size and density in mononuclear blastic granulocytic precursors represented by HL-60 cells the proliferation of which was blocked by photodynamic treatment (PDT) which induced apoptotic process without preceding terminal maturation. Both the nucleolar size and density did not change in apoptotic cells in comparison with controls. Thus, large and dense nucleoli in apoptotic cells are not necessarily related to the nucleolar biosynthetic or cell proliferation activity.     

The significance of telomeric aggregates in the interphase nuclei of tumor cells

Telomeres are TTAGGG repetitive motifs found at the ends of vertebrate chromosomes. In humans, telomeres are protected by shelterin, a complex of six proteins (de Lange [2005] Genes Dev. 19: 2100-2110). Since (Müller [1938] Collecting Net. 13: 181-198; McClintock [1941] Genetics 26: 234-282), their function in maintaining chromosome stability has been intensively studied. This interest, especially in cancer biology, stems from the fact that telomere dysfunction is linked to genomic instability and tumorigenesis (Gisselsson et al. [2001] Proc. Natl. Acad. Sci. USA 98: 12683-12688; Deng et al. [2003] Genes Chromosomes Cancer 37: 92-97; DePinho and Polyak [2004] Nat. Genetics 36: 932-934; Meeker et al. [2004] Clin. Cancer Res. 10: 3317-3326). In the present overview, we will discuss the role of telomeres in genome stability, recent findings on three-dimensional (3D) changes of telomeres in tumor interphase nuclei, and outline future avenues of research.     

3D chromatin architecture and epigenetic regulation in cancer stem cells

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation.During this process,a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis.Such characteris-tics allow these cancer stem cells (CSCs) to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics.Cancer devel-opment and progression are demarcated by transcrip-tional dysregulation.In this article,we explore the epigenetic mechanisms shaping gene expression dur-ing tumorigenesis and cancer stem cell formation,with an emphasis on 3D chromatin architecture.Comparing the pluripotant stem cell state and epigenetic repro-gramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics.We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.     

In vitro biocompatibility of dextrin: the addition of a low concentration of dextrin in the medium promotes the cell activity of L929 mouse fibroblasts

To develop a bone substitute with shape-generating properties, we focused our attention on dextrin, which has a low viscosity. After considering methods of evaluation for research and development, we started by using cells that are widely used for safe biological evaluations in the field of dentistry and conducted in vitro evaluations. In this experiment, we variously added concentrations of 0.1, 1.0 and 10 mmol/l of dextrin to a culture medium in order to examine the effects on L929 mouse fibroblasts in vitro. As a result, the proliferative activity of the L929 cells was promoted during the culture period as the concentration of added dextrin became lower, and in particular, the 0.1 and 1 mmol/l addition group showed higher values than those of the control group. From the above results, it was revealed that the addition of a low concentration of dextrin in a medium promotes the cell proliferative activity.     

Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei

A gene density-related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119-1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal–limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenyarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3–4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.     

The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.