Namalva Cells Research Articles

STUDIES OF MAGNESIUM AND PHOSPHORUS COMBINED MEDICATION BASED ON CASEIN

Aim. The Department of Biochemistry and Physiology of Animals, named after Academician Guly NUBIP of Ukraine, developed magnesium and phosphorus combined medication based on casein. Our aim was to test its bioavailability based on the ability to be hydrolyzed by a mixture of pancreatic digestive enzymes trypsin and chymotrypsin, also check the absence of cytotoxic effects on cell cultures. Methods. To assess bioavailability, we used hydrolysis of the medication with a mixture of trypsin and chymotrypsin, followed by detection of hydrolysis products by polyacrylamide gel electrophoresis. A standard MTT-test performed on both MT-4 and Namalva cell lines was used to assess cytotoxic effects. Results. Based on electrophoresis data, it was found that despite chemical modifications of the natural casein, the medication based on it is characterized by a high ability to hydrolyze by digestive enzymes under the same conditions as casein. Also, an MTT-test demonstrates that the medication has no cytotoxic properties against cell lines MT-4 and Namalva. Conclusions. Since the negative effects of the drug associated with its digestibility and toxicity have not been observed, it is recommended to continue the study of its effects on living organisms.

Interferon-regulating activity of the CelAgrip drug and its influence on the formation of reactive oxygen species and expression of innate immunity genes in Burkitt’s lymphome cell cultures

Interferons (IFN) and IFN inducers are effective in suppressing viral reproduction and correcting of the innate immunity mechanisms. The aim of the study was to test the hypothesis of the possible involvement of the IFN inducer CelAgrip (CA) as an activator or suppressor of antiviral effects in Burkitt's lymphoma (LB) cell cultures with different ability to produce Epstein-Barr virus antigens (EBV). The kinetic analysis of the dynamics of reactive oxygen species (ROS) production and determination of gene group expression by real-time PCR in response to CA treatment were done in human cell lines LB P3HR-1 and Namalva, spontaneously producing and not producing EBV antigens. When treating CA in Namalva cells, a decrease in the ROS activation index was found; in P3HR-1 cells, an increase was observed. After treatment with CA, there was no reliable activation of the IFN-α, IFN-β and IFN-λ genes in Namalva cells, but the expression of the ISG15 and P53(TP53) genes was increased more than 1200 times and 4.5 times, respectively. When processing the CA of P3HR-1 cells, the expression of IFN-α genes increased by more than 200 times, IFN-λ - 100 times, and the ISG15 gene - 2.2 times. The relationship between IFN-inducing action of CA and the activity of ISG15 and ROS in LB cell cultures producing and not producing EBV antigens is supposed. In Namalva cells that do not produce EBV antigens the treatment of CA results in suppression of ROS generation and activation of the expression of genes ISG15 and P53 (TP53); in P3HR-1 cells producing EBV antigens, the opposite picture is observed - the formation of ROS and the expression of the IFN-α and IFN-λ genes are activated and the activity of the ISG15 and P53 (TP53) genes is suppressed.

Intracellular immunoglobulins in Namalva and U266 cells cocultivated with mesenchymal stromal cells

The data concerning the influence of mesenchymal stromal cells (MSCs) on immunoglobulin (Ig) production are contradictory. Most results were obtained using MSC derived from bone marrow. The properties of MSCs obtained from other tissues are not well studied. In the present work, MSC cultures have been established from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSCs from all these sources exhibited similar surface markers. We assayed the influence of MSC cocultivation at exponential or stationary growth phases on IgM content in Namalva and IgE content in U266 cells. Bone marrow MSCs from healthy donors did not affect IgM and IgE production. Proliferating MSCs from patients with Crohn’s disease and multiple sclerosis stimulated Ig production. Exponentially growing MSCs derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSCs at stationary cultures enhanced IgM production in Namalva (cells) and suppressed IgE synthesis in U266 cells. Thus, MSCs from various tissues with common phenotypes differed in their capacity to modulate Ig production by B-lymphoid cells. The effect of MSCs depends on their growth stage and may be different for lymphoblastoid and myeloma cells.

The influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis

A number of publications contain contradictory data about influence of mesenchymal stromal cells (MSC) on B-lymphocyte growth, differentiation and production of immunoglobulins (Ig). The aim of the study was to investigate the influence of MSC derived from adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of lymphoblastoid cell line Namalva and myeloma cell line U266. Co-cultivation of Namalva cells with MSC stimulated their proliferation, decreased the doubling time and the minimal effective seeding dose and therefore made cloning of these lymphoblastoid cells possible. The presence of MSC supported the survival and proliferation of Namalva cells cultivated in growth factor deficient medium. MSC also stimulated proliferation of U266 myeloma cells. Both MSC derived from adipose tissue from the healthy donors and from patients with breast cancer effectively stimulated B-cell lines proliferation. Presence of MSC in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells respectively. Co-cultivation of Namalva or U266 with MSC resulted in the formation of close intercellular contacts between cells of both types.

The role of P2X7 receptor in ATP-mediated human leukemia cell death: calcium influx-independent

Activation of the P2X7 receptor leads to a rapid, bidirectional flux of cations, causing broad range of biological responses including cytotoxicity. However, the mechanism of P2X7-mediated cytotoxicity remains largely unexplored. In our previous study, the lack of P2X7-mediated calcium response under normal conditions was found in P2X7(+) hematopoietic cell lines. In this study, the P2X7-mediated cytotoxicity in different type of cells (P2X7(-), P2X7(+) with calcium response, and P2X7(+) without calcium response) was investigated. Our results showed that P2X7 agonists, adenosine 5'-triphosphate (ATP) or 2',3'-O-(4 benzoylbenzoyl)-ATP, dose-dependently reduced the cell viability in all P2X7(+) cells tested, including J6-1, LCL, and Namalva cells which are negative for P2X7-mediated calcium response, although these effects were lower than those observed in KG1a cells which has normal P2X7 functions. The cytotoxic effect could be blocked by P2X7 antagonists, oxidized ATP and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine. In addition, externalization of phosphatidylserine could be detected in a time-dependent manner and apoptotic morphological changes could be observed after the activation of P2X7 receptor in J6-1 cells. Furthermore, P2X7-mediated pore formation could be detected in KG1a and J6-1 cells under low-ionic conditions, but not under low-divalent conditions. These effects could not be observed in P2X7(-) Ramos cells. These results suggested that P2X7 receptor-mediated cytotoxic effects may occur independent of calcium response.

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells.

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.

The influence of glutathione and detoxifying enzymes on DNA damage induced by 2-alkenals in primary rat hepatocytes and human lymphoblastoid cells.

The reaction of 2-alkenals with GSH to form GSH conjugates by Michael addition is a major detoxification pathway. The reaction proceeds at a much higher rate under catalysis by glutathione S-transferase (GST) than the non-enzymatic reaction. Oxidation of 2-alkenals to the corresponding acids by cytosolic and microsomal fraction of rat liver also contributes to detoxification. Primary rat hepatocytes rich in GSH and proficient for GST and other metabolizing enzymes consume much more alkenal than human lymphoblastoid cells (Namalva cells), that are poor in GSH and in metabolic activities. In Namalva cells DNA single strand breaks were induced by much lower concentrations of acrolein, crotonaldehyde and (E)-2-hexenal than in primary rat hepatocytes. In both cell systems intracellular GSH depletion by 2-alkenals proceeds in a dose dependent manner, approaching about 20% of pretreatment level before DNA damage becomes detectable. GSH conjugates of (E)-2-hexenal and (2E,6Z)-2,6-nonadienal induce DNA damage in Namalva cells at high concentrations (1.5 mM). In the absence of GSH these conjugates decompose slowly into aldehyde and GSH. Although the rate of decomposition is only about 10(-4) times that of Michael adduct formation, such GSH conjugates could potentially function as transport molecules for 2-alkenals, if they reach tissues low in GSH and GST.

Adenovirus-Mediated Transfer of the Amphotropic Retrovirus Receptor cDNA Increases Retroviral Transduction in Cultured Cells

The presence of functional amphotropic receptors on the cell surface is necessary for amphotropic retrovirus-mediated gene transfer. A recombinant adenoviral vector that expresses the receptor for amphotropic retrovirus (RAM) was constructed and used to express the receptor cDNA in different cell types in culture. Transfer of the RAM cDNA increased amphotropic retroviral-mediated transfer from 0 to 60% in Chinese hamster ovary cells. RAM expression increased retroviral transduction four- to eight-fold from 2-4% to 18%-35% in HeLa, Namalva, and X63 cells, but had no effect on 208F and HepG2 cells which have high baseline retroviral transduction rates of about 50%. For the purpose of application to ex vivo gene therapy, primary mouse hepatocytes were studied in a similar manner. Hepatocytes had a baseline transduction efficiency of about 40% and did not have increased rates of retroviral-mediated gene transfer with expression of recombinant RAM. This recombinant adenoviral vector conferred infection of amphotropic retrovirus into cells that were relatively resistant to infection, thus offering a rapid and easy method to stably introduce genes into these cell lines.

Double-Tailed Perfluoroalkylated Glycolipids as Components for Drug Delivery and Targeting Systems. Preliminary Biocompatibility Results

Vesicles are being investigated as drug carriers, especially for enhancing the therapeutic effectiveness of the drug while minimizing its side effects. Drug targeting can be achieved if there is a specific recognition of the vesicle's outer wall by specific cells. With these objectives in mind new glycolipids fitted with fluorinated, hydrogenated or mixed, single and double-tails, containing either a gluco- or a galactopyranose residue in their hydrophilic head, were synthesized and their ability to achieve self-organized supramolecular systems was assessed. Replacement of hydrogen by fluorine in these glycolipids was found to enhance biological tolerance. Thus, a fluorinated single-tailed glycolipid displayed no action on red blood cells at concentrations as high as 50 g/l while its hydrogenated analog was hemolytic at 5 g/l. 100% of survival was obtained one month after intravenous or intraperitoneal injection into mice of isotonic dispersions of single and double-tailed glycolipids at a dose of 500 mg/kg. These glycolipids were innocuous on Namalva cell cultures at a concentration of 0.1 g/l.

Amphiphilic Sugar Phosphates with Single or Double Perfluoroalkylated Hydrophobic Chains for Use in Oxygen and Drug Delivery Systems

New anionic amphiphiles with a phosphate ester junction between the fluorophilic-lipophilic tail and the sugar-based hydrophilic head were synthesized and evaluated. The single hydrophobic chain surfactants 1 a, b and 2a allowed the preparation of stable and fine highly concentrated emulsions of perfluorodecalin or perfluoroocytl bromide, either when used alone or in conjunction with egg yolk phospholipids (EYP). Surfactants 3d, 5d, 6d and 6e, with two hydrophobic chains, one fluorinated the other not, gave liposomal structures, and displayed encapsulation properties for carboxyfluorescein. The phosphodiesters tested cause no significant inhibition of the growth and viability of Namalva cell cultures (0.1-1 g/L range). Single chain phosphodiesters manifest no detectable hemolytic activity (at 100 g/L for 1a) whereas double chain compounds do moderately (ca 20% hemolysis at 20 g/L). The maximum tolerated dose compatible with the survival of all of a series of 10 intravenously injected mice is in 130 mg/kg body weight range.

Morphine effect on proliferation of normal and tumor cells of immune origin

The influence of morphine on proliferation of human tumor K562 and lymphoid cells was studied and compared with that on the mitogen-induced proliferation of human peripheral blood mononuclear cells (PBMC). Morphine was shown to act as a suppressor of both cellular DNA synthesis (50% and more as compared to control) and the cellular population growth of mitogen-induced PBMC, B-lymphoma Namalva cells and EBV-transformed lymphocytes. Morphine activated proliferation of myeloid K562 and T-lymphoma Yurkat cells 1.5-fold. It is supposed that the opposite effects of morphine on proliferation of cell lines of immune origin reveal the difference in modulation of diverse immune cell types by morphine.

Perfluoroalkylated fatty acid monoesters of trehalose and sucrose for biomedical applications: Remarkable emulsifying properties of 6‐O‐[3′‐(perfluorooctyl) propanoyl]‐trehalose

A new series of perfluoroalkylated fatty acid monoesters of α,α‐trehalose and sucrose has been evaluated with respect to their physicochemical and biological properties for possible biomedical use. These water‐soluble compounds strongly reduce the water surface tension and fluorocarbon/water interfacial tension. As co‐surfactants in perfluorodecalin/Pluronic F‐68 type emulsions they significantly increase the stability of these emulsions. Remarkably stable concentrated perfluorodecalin‐in‐water (50% w/v) emulsions were obtained when the C8F17CH2CH2C(O)‐α,α‐trehalose monoester was used as the sole surfactant, while no emulsion could be obtained with its maltoside analogue. No significant effect on the growth and viability of Namalva cell cultures and no hemolytic activity on human erythrocytes at concentrations up to 50 g/L were detected for these amphiphiles in spite of their high surface activity. The LD50 was found to be in the range of 250–375 mg/kg of body weighti.v. in mice.

Synthesis and preliminary evaluation of perfluoroalkylacyl carnitines as surfactants for biomedical use

A series of acyl carnitines, 4 of which bearing a terminal perfluoroalkyl fragment, was obtained from carnitine in one step in a high grade of purity in yields ranging from 44–81%. The F-alkyl derivatives display high solubilities in water, strong surface activity and a significant emulsifying power towards fluorocarbons in water. In spite of their strong surface activity, all the biocompatibility tests performed ( in vitro toxicity on Namalva cells cultures, hemolysis on human red blood cells, and iv injections in mice) indicate a significantly better biological tolerance than their hydrocarbon analogs, provided the F-alkyl moiety is larger than the alkyl moiety in the hydrophobic tail.

Characterization of Marker Chromosomes in Namalva Cells by Chromosomal In Situ Suppression (CISS) Hybridization and R‐Banding

Chromosomal in situ suppression (CISS) hybridization was used to investigate the distribution of material of chromosomes 1 and 5 present in marker chromosomes of Namalva cells. The Namalva cell line, established from a Burkitt's lymphoma, exhibits a highly variable female karyotype with a large number of marker chromosomes. Libraries from sorted human chromosomes 1 and 5 were used to delineate material of these chromosomes present in the Namalva karyotype. We used the DAB/peroxidase reaction and reflection contrast microscopy for detection of biotinylated hybrid molecules. Identification of chromosomes was achieved by fluorescent R-banding after CISS hybridization, which allowed the assignment of hybridized regions to the particular marker chromosomes. After CISS hybridization with a chromosome I library, the normal chromosome I was labelled as well as a large marker MI and the long arm of marker M3. Using a chromosome 5 library it could be shown that the distal part of the long arm of one chromosome 5 was translocated to marker M2. The normal chromosome 5 was completely labelled. The present investigation demonstrates the advantage of combining CISS hybridization and banding for the identification of complex, rearranged tumor karyotypes.

Investigations on organ-specific metabolism and genotoxic effects of the urinary bladder carcinogen N-nitrosobutyl-3-carboxypropylamine (BCPN) and its analogs N-nitrosodibutylamine (NDBA) and N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA)

N-Nitrosodibutylamine (NDBA) and its ω-oxidized metabolites N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA) and N-nitrosobutyl-3-carboxypropylamine (BCPN) are potent urinary bladder carcinogens. To study putative organ specific activation of BCPN, its α-oxidation by liver and urinary bladder microsomal fractions was investigated in comparison to NDBA and 4-OH-NDBA. Additionally, induction of DNA single strand breaks (SSB) was monitored in hepatocytes and in a human lymphoblastoid cell line (Namalva) in the presence and absence of external metabolic activation, including N-nitroso- t-butyl- n-butylamine as a negative control. BCPN was α-hydroxylated and dealkylated at both alkyl chains in small rates (about 1 nmol × mg protein −1 × 60 min −1) by microsomes from rat liver and pig urinary bladder epithelium. NDBA and 4-OH-NDBA were dealkylated at similarly low rates by pig urinary bladder microsomes, in strong contrast to the high debutylation rates observed for rat liver microsomes. Correspondingly, SSB induction by NDBA and 4-OH-NDBA was observed in Namalva cells with NDBA and 4-OH-NDBA in the presence of PB-induced rat liver microsomes but not with urinary bladder microsomes or without external activation. BCPN did not induce DNA-damage in Namalva cells (with or without external activation) or in rat hepatocytes. Significant induction of sister chromatid exchanges (SCEs) and micronuclei, however, was observed in Namalva cells after incubation w with NDBA and BCPN.

Induction of interferon messenger RNA in human lymphoblastoid cells (Namalva)

Human B lymphoblastoid cells Namalva were induced by Sendai virus and the levels of α-Interferon (IFN-α)mRNA, and IFN-α protein were measured. The level of IFN-αmRNA reached a maximum level 6 h after induction and then slowly declined. The interferon titer reached its maximum level 18 to 24 h after induction. Northern blot analysis of induced Namalva cell poly(A)+ mRNA with an interferon probe revealed a transcript of 1.2 kb. No IFN-α mRNA was detected in uninduced Namalva cells under the conditions used. This study adds to our understanding of the events which lead to interferon production in vitro.

Interferon gamma is active on human lymphoblastoid Namalva cells without inducing an antiviral state

We demonstrate the presence of high affinity receptors specific for interferon-γ (IFN-γ) in human lymphoblastoid Namalva cells. The presence of these receptors, whose binding affinity and cross-linking characteristics were not distinguishable from those of the corresponding receptors in sensitive cells, was not consistent with the lack of responsiveness of Namalva cells to IFN-γ as regards growth inhibition, induction of 2′–5′ oligoadenylate synthetase activity and inhibition of virus multiplication. Nevertheless, IFN-γ enhanced the expression of two genes, HLA class II and c-myc. Although the mechanism of these IFN-γ-mediated modifications is not understood, these results provide evidence that the IFN-γ receptors present in Namalva cells are functional.

Establishment of Namalva cell lines which grow continuously in glutamine-free medium

Glutamine has been shown to be a preferred energy source for some established cell lines and cancer cells in culture (Kovacevic, 1971; Kovacevic, 1972; Lavietes, 1974). Empirically, glutamine is the most abundant amino acid in most of the culture media developed. The major end product of glutamine metabolism is ammonia. Ammonia build up is one of the limiting factors in the proliferation of mammalian cells in higher density culture and is directly related to the initial glutamine concentration. The susceptibility of glutamine to thermodecomposition prevents the heat sterilization of glutamine-enriched media and this significantly increases the cost of medium preparation at the industrial scale. In an attempt to overcome these drawbacks, a population of Namalva cells capable of growing in glutamine-free media was established. The adapted cells were found to contain a higher level of glutamine synthetase activity which enable them to synthesize sufficient amounts of glutamine for their growth.

Corticosteroids modulate the binding of recombinant interferons alpha and gamma in Namalva cells.

To investigate possible mechanisms of interaction between corticosteroids and interferons (IFNs), the specific binding of recombinant human IFNs alpha 2 and alpha in Namalva cells after 72 h culture with dexamethasone (10(-8) M to 10(-6) M) was evaluated. Exponentially growing cells were incubated with different concentrations of the radiolabelled IFNs, with or without an excess of unlabelled IFN. The parameters of the interaction between each IFN and its specific receptor were analyzed by the Scatchard method. In the dose range tested, dexamethasone induced a dose-dependent inhibition of Namalva cells growth, which reached about 35% at 10(-6) M. The specific binding of IFN-alpha 2 was decreased to a maximum of 40%, for dexamethasone concentrations greater than or equal to 10(-7) M. The decrease in binding induced by the corticoid was additive with the down-regulation induced by IFN-alpha 2 itself. On the contrary, the specific binding of IFN-alpha was increased by dexamethasone in a dose-dependent fashion within the tested range. The maximal increase in the number of sites per cell was about 60%, with a slight decrease in affinity. These results suggest that complex interactions might arise between corticosteroids and IFNs in the course of their clinical use.

Vasoregulin, a glucocorticoid-inducible vascular permeability inhibitory protein.

A vascular permeability inhibitory protein 'Vasoregulin' was induced by dexamethasone and other glucocorticoids in Namalva cells. Precipitates produced by trichloroacetic acid from cultured supernatant (crude released vasoregulin) suppressed serotonin-induced paw edema of mice (ID30 = 750 micrograms/mouse) when dosed 30 min. before serotonin. Carrageenan paw edema of rats was also suppressed (ID30 = 9 mg/rat, s.c. and i.p., not by oral dosing) when the compound was injected at the same time as carrageenan and the edema determined after 3 hrs. The possible effect of glucocorticoid itself being contained in the vasoregulin preparation was excluded. The presence of Cu, Zn-superoxide dismutase (SOD), spermidine and spermine also gave good yields of vasoregulin in culture; SOD protected the inactivation of vasoregulin from superoxide radical in vitro. Vasoregulin partially purified by Sephadex G-75 columns had no malonaldehyde-formation inhibitory capacity. Thus, vasoregulin is a glucocorticoid-inducible anti-inflammatory protein which differs from lipocortin.