Effects of reduced cell volume and water content on glycolysis in L-929 cells

Mouse L-929 cells were subjected to increasing concentrations of sorbitol, which remove cell water and reduce volume osmotically. The rate of lactate production from glucose was significantly higher in osmotically perturbed cells than in controls, both in monolayers and in suspensions. L cells can apparently use sorbitol as a glycolytic substrate; however, studies using other solutes (trehalose and sucrose) and permeabilized cells showed that the major effect of sorbitol on glycolysis in intact cells is mediated through a reduction in cell water content and volume. It is possible to explain some of these results by an increase in the chemical potentials of dissolved components of the glycolytic pathway caused by water loss; however, the relationship between water loss and glycolytic rate increase in not a simple linear one, suggesting that the situation is more complex than would result merely from increased concentrations of pathway components. Whatever the complete explanation might be, these studies show that glycolysis continues in an orderly fashion in cells that have lost about 85% of their original water content, suggesting that the operation of this pathway is not unduly sensitive to events taking place in the bulk aqueous phase.     

Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.     

Quality control testing of surfaces for mammalian cell culture using cells propagated in a protein-free medium

Mouse NCTC clone 929 L (L-929) cells were propagated continuously for 3 years as monolayers in a protein-free chemically-defined medium. These cells, designated L-929-WS, were used for quality control testing of the surfaces of commercially available cell culture plastic flasks. Differences in attachment and saturation density of L-929-WS cells in a protein-free culture medium were taken to define various levels of quality of the culture vessels tested. The rate of attachment and growth of L-929-WS cells on a surface of a given quality correlated directly with that of human embryonal fibroblasts and embryonal epithelial cells grown in a serum-free medium supplemented with growth factors and hormones. L-929-WS cells propagated continuously in a protein-free medium provide a simple and sensitive assay system for more general quality control testing of surfaces used for the culture of monolayer cells.     

A substance in L-929 cell extracts which replaces the ascorbate requirement for prolyl hydroxylase in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and the level of prolyl hydroxylase activity in these cells

Reductant used as cofactor for the prolyl hydroxylase reaction, was measured by a tritium release assay modified from an enzyme assay by making all components of the assay system saturating except for the reductant, but including prolyl hydroxylase. Reduced glutathione (6 mm), which had little activity as a cofactor, and thymol (0.1 mm), an antioxidant which exhibited no cofactor activity at all, were required for optimal proline hydroxylation dependent on reducing cofactor, with thymol fulfilling the previously described requirement for catalase. Ascorbate, cysteine and 6,7-dimethyltetrahydropterin were active as cofactors, in descending order of activity at equimolar concentrations, and activity was concentration dependent for all of these compounds. Sonicates of stationary phase L-929 cells which exhibit ascorbate-independent proline hydroxylation in culture contained reducing cofactor which could replace ascorbate in the cofactor assay, while sonicates of log phase cells which exhibit an ascorbate requirement in culture contained about one-third or less of that amount. NADH and NADPH, which themselves have little or no activity as cofactor, increased the cofactor activity of log phase cell sonicates but had relatively little effect on the activity of stationary cell sonicates suggesting that the cofactor is in a more reduced state in stationary phase. Within 24 h after replating dense, stationary phase cell cultures at low density, conditions where cells return to ascorbate dependence, prolyl hydroxylase activity had decreased to one-fifth the original activity while the concentration of functional reducing cofactor had decreased to less than 1% of its original concentration, largely as a result of oxidation. Ascorbate was not present in L-929 cells sonicates and the levels of tetrahydropterin and cysteine in sonicates could not account for the amount of cofactor activity exhibited by the sonicates in the assay system. Treatment of L-929 cultures with aminopterin did not decrease ascorbate independence, suggesting that tetrahydrofolate did not contribute significantly to cellular proline hydroxylation. These results suggest that an unidentified reductant present in L-929 cells can account for ascorbate-independent proline hydroxylation and also regulate prolyl hydroxylase activity in these cells and that cellular levels of reduced pyridine nucleotides may regulate the reduction state of this substance.     

Spontaneous expulsion of micronuclei by enucleation in the micronucleus assay

The effect of enucleation on the frequency of micronuclei induced by mitomycin C (MMC) and vincristine (VCR) was examined in mouse L-929 cells enucleated with cytochalasin B (Cyt-B). Approximately 30% of the L-929 cells became enucleated cells during the 8-h incubation in medium containing 8 micrograms/ml of Cyt-B. Using this enucleation technique, we estimated the reduction rate of 2 mutagen-induced micronuclei by enucleation. Treatment with MMC caused a dose-dependent induction of micronuclei in L-929 cells, with the reduction rate being 38.6% at the lowest dosage (0.0125 microgram/ml), which induced mostly mono-micronuclei in L-929 cells, and 6.8% at the highest dosage (0.1 microgram/ml), which induced many multi-micronuclei. Furthermore, VCR also induced micronuclei in a dose-dependent way in L-929 cells, and the same tendency for micronucleus reduction as with MMC was observed. The reduction rate of micronucleated cells by enucleation was estimated to be about 31-39% when the micronucleated cells contain mono-micronuclei. Therefore, the rate of reduction is affected by the number of micronuclei per cell, and the reduction depends on the increase in the number of micronuclei per cell.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells

L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization.     

31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium

The origin of the nuclear magnetic resonance (NMR)-measurable ATP in equilibrium Pi exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP in equilibrium Pi exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO2) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the Pi----ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the Pi----ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. Upon elimination of the glycolytic component, the measured Pi----ATP rates displayed a linear dependence on MVO (micromoles of O consumption rate) with a slope of 2.36 +/- 0.15 (N = 8, standard error of the mean). This linear relationship is expected if the rate determined by CST is the net rate of ATP synthesis by the oxidative phosphorylation process, in which case the slope must equal the P:O ratio. The ATP----Pi rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO2 levels were equal to the corresponding Pi----ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP in equilibrium Pi exchange is inhibited or is specifically shown not to exist, the myocardial Pi in equilibrium ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO2 levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H+-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the "operative" P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value of 3.     

Dynamics of spreading of cells of L-929 line after the mitosis

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".     

Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells

L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phosphoglycerate (PGA) added to DSP cells reduces the specific activity of lactate coming from [14C]glucose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type.     

The establishment and characterization of mouse L-929 cells in protein-free Eagle's Minimal Essential Medium

The mouse cell line L-929 was established in protein-free Eagle's Minimal Essential Medium. The cells have been 'adapted' to continuous growth in the medium using stepwise reductions in the concentration of fetal bovine serum. The cells designated L-929-WS have now been propagated in protein-free Eagle's Minimal Essential Medium for two years. The population-doubling time was about 37 h. The addition of serum stimulated cell growth only slightly, but the saturation density was significantly increased. Morphological examination, a study of the secretion of colony stimulating activity and cytochemical investigations for acid phosphatase and alkaline phosphatase showed that L-929-WS cells, grown in protein-free Eagle's Minimal Essential Medium, did not differ markedly from cells propagated in medium containing serum. The cells provided a simple model for the study of cell growth in the absence of serum or the other macromolecular substances usually added to cell cultures. The general application of the cells for purposes in which the addition of serum or growth factors might interfere, is suggested.     

Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA

Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1-, S-, and G2-Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7-48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L-929 cells in medium without serum reduced their RNA content down to one-third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.     

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.     

Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.     

Dynamics of L-929 cell spreading after mitosis

Changes in the cell shape of L-929 (NCTC, clone 929) during the cell cycle were analyzed with time-lapse microscopy. It was found that the cells pass through three spreading stages. The maximal cell spreading was observed during the first 1.5 h after mitosis. In this period, the cell area increases in correspondence with the sigmoid dependence and enlarges by approximately 3.0–3.5 times. After a short plateau, the cell area begins to increase, also correspondence with the sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division), with an additional 1.5-fold increase in the cell size. Later, cell area enlargement continues linearly up to the beginning of the next mitosis.     

Immunocytochemical localization of glucocorticoid receptors in cells, cytoplasts, and nucleoplasts

A monoclonal antibody has been used to assess the intracellular localization of the glucocorticoid receptor in rodent L-929 fibroblasts and GH3 pituitary tumor cells. Whole cells from both cell lines showed immunoreactivity in the cytoplasm and nucleus. However, when cytoplasts and nucleoplasts of these cells were examined, only L-cells showed strong antibody binding in both fractions; in contrast, GH3 cells exhibited nuclear staining and slight cytoplasmic staining. These results are discussed in terms of the current findings regarding the intracellular location of steroid hormone receptors.     

The position of cleavage furrow in cultured <Emphasis Type="Italic">L-929</Emphasis> and <Emphasis Type="Italic">CHO</Emphasis> cells

The position of the cleavage furrow (random or otherwise) was studied on cultured L-929 (NCTC, clone 929) and CHO cells. CHO cells were seeded uniformly on the surface of Petri dishes; L-929 cells were grown as colonies so that migrating cells could be watched. Cell behavior was registered by time-lapse imaging. Two parameters were analyzed on captured images: the angle between the cell polarization axis and cleavage furrow and the angle between the cell polarization axis or cleavage furrow and the horizontal axis of the image field. It was shown that the position of CHO cells in the dish plane and the value of the angle between the cell polarization axis and the cleavage furrow were random. The L-929 cells migrating from the colony were orientated such that their polarization axis was directed to the colony center and the cleavage furrow was perpendicular to this axis. The nonrandom position of cultivated cells during mitosis and their cleavage furrow during the telophase are discussed.     

Actin and tubulin synthesis in monolayer and suspension cultures of murine L cells

The rates of total protein, actin and tubulin synthesis were studied for monolayer (L-929) and suspension (LS) cultures of mouse L cell. Data on pulse 34S-label incorporation into the cellular protein pool show that LS characterized by a short cell cycle have, comparatively to L-929, higher rates of protein synthesis and phosphorylation. According to PAGE data, the level of actin and tubulin synthesis in suspension line exceeds that in monolayer one. The correlation between growth conditions, biosynthetic parameters and dynamics of cytoskeleton is discussed.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.