Autocrine factors in defense of cytotoxic CTLL-2 cells from oxidative stress

The role of pyruvate and autocrine polypeptide factors (APF) secreted by cytotoxic IL-2-dependent CTLL-2 cells in cell defense from oxidative stress was investigated. The addition of a conditioned medium (CM) containing pyruvate and APF into CTLL-2 cell cultures significantly increased the cell survival under oxidative stress conditions induced by hydrogen peroxide (H₂O₂). The kinetics of (H₂O₂) removal from cell cultures with added CM has been registered. It has been shown that, at the beginning of oxidative stress (less than 15 min), H₂O₂ was mostly removed by means of its reaction with pyruvate contained in CM. Pyruvate content in CM was estimated as 138 ± 7 μM. Gel filtration on a column with Bio-Gel P-10 was used to eliminate pyruvate from CM. Gel filtration resulted in three CM fractions (A, B, and C) corresponding to three chromatogram peaks. Pyruvate was not detected in any fraction. The fraction A was the first to be eluted from the column and contained the largest molecules. In the cell survival test, fraction B had the highest protective ability for CTLL-2 cells under oxidative stress. Fraction A supported cell survival to a lesser degree and fraction C did not show any protective abilities. Fraction B added to cells under oxidative stress kept intracellular ATP content at a significantly higher level then in control cells. Moreover, it was found that APF from fraction B was able to react with H₂O₂ directly and inactivate it in the absence of cells. APF from fraction A did not have such properties.     

Effects of an autocrine factor deficit on the survival and energy metabolism of CTLL-2 cells under oxidative stress conditions in culture

An increasing body of data shows that survival of mammalian cells is under the control of growth factors and autocrine survival factors (AF). We studied the effects of an AF deficit on the survival, intracellular ATP content and the transmembrane potential of mitochondria in IL-2-dependent CTLL-2 cells following oxidative stress. We show that cells cultivated under conditions of AF deficit became more susceptible to oxidative injury in comparison with CTLL-2 cells cultivated without an AF deficit (the control cells); their death occurred at lower H₂O₂ concentrations than in the case of control cells. The ATP content in CTLL-2 cells decreased under an AF deficit even without stress; treatment of these cells with hydrogen peroxide led to an additional significant decrease of ATP content, which was accompanied by injury of the cell membrane (blebbing) and by a sharp fall in mitochondrial potential. Cell death after oxidative stress under conditions of AF deficit was shown to proceed along both the apoptosis and necrosis pathways.     

Influence of autocrine factor deficit in culture on survival and energy metabolism of CTLL-2 cells under oxidative stress

A lot of data has shown recently that survival of mammalian cells is under a control of growth factors and autocrine survival factors (AF). We studied the influence of AF deficit on survival, intracellular ATP content, and transmembrane potential of mitochondria of IL-2-dependent CTLL-2 cells under oxidative stress. CTLL-2 cells cultivated under deficit of AF have been shown to be more susceptible to oxidative injury in comparison with the cells cultivated without deficit of AF (control); they died at smaller concentrations of H2O2 than control cells did. The ATP content in CTLL-2 cells was decreased under AF deficit conditions even without any stress and treatment of the cells by hydrogen peroxide resulted in additional large decrease of it. ATP depression was accompanied by disruption of cell membrane (blebbing) and drop of mitochondrial potential. Cell death under oxidative stress in the presence of AF deficit has been shown to proceed by both apoptosis and necrosis.     

Survival of T-lymphocytes under alkalosis and autocrine factor deficiency

Alkalosis associated with elevated pH is characteristic of many clinical pathologies. Respiratory alkalosis is a result of hyperventilation, i.e., reduced partial CO₂ pressure in alveolar air and blood. Yet another type of alkalosis, i.e., metabolic alkalosis, is associated with an absolute or relative increase in the levels of alkaline compounds in the organism. Despite high toxicity of the latter, mechanisms whereby these compounds exert their toxic effects remain obscure. In multicellular organisms, cell survival is controlled by a vast variety of factors, such as autocrine survival factors (AF) specifically targeted at cells that secrete them. Our previous studies (Lutsenko and Diachkova, 2003) demonstrated that AF control cell survival and energy metabolism in T-lymphocytes. In this study, combined effects of AF deficiency and alkalosis (pH 8.3) on cell survival, intracellular content of ATP and mitochondrial transmembrane potential of T-lymphocytes were studied using an IL-2-dependent cell line CTLL-2. It was found that in the absence of AF deficiency, alkalosis had no effect on survival of cultured CTLL-2 cells. The main mechanism of protection of CTLL-2 cells against cytotoxic effects of alkalosis was an enhanced anaerobic glycolysis and consequential increase in the lactate production. In contrast, alkalosis combined with AF deficiency caused a substantial decrease of cell survival, which lowered down to 53% after 6 h and to about 10 % after 20 h of culturing under these conditions. The ATP content dropped down sharply under the AF deficiency even at pH 7.3 but gradually restored to the initial level within the next 2-3 h; cell survival was at a high level under these conditions. Alkalosis combined with the AF deficiency notably worsened the functional state of the cells; ATP content in them remained at a low level over the whole period of the alkaline stress. After a 2-h incubation under alkalosis and AF deficiency, 23% of cells contained depolarized mitochondria; lactate production was notably suppressed. The data obtained suggest that the reduction of the intracellular ATP level in CTLL-2 cells under alkalosis and AF deficiency are due to inhibition of anaerobic glycolysis and mitochondrial dysfunction. Cell death developed predominantly via the necrotic rather than the apoptotic pathway.     

Choice of DMEM, formulated with or without pyruvate, plays an important role in assessing the in vitro cytotoxicity of oxidants and prooxidant nutraceuticals

There is much interest in the positive health effects of nutraceuticals, in particular, polyphenols, which have both antioxidant and prooxidant characteristics. Pyruvate, a scavenger of hydrogen peroxide, is a component in some, but not in all, commercial formulations of cell culture media, Dulbecco’s modified Eagle’s medium in particular. This study showed that the cytotoxicities to human fibroblasts of hydrogen peroxide, tert-butyl hydroperoxide, and various prooxidant nutraceuticals were lessened in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to the same medium but formulated without pyruvate. Intracellular glutathione was unaffected in cells treated with hydrogen peroxide in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to medium formulated without pyruvate. In these studies, intracellular glutathione was analyzed in acid-soluble cell extracts by determining the oxidation of reduced glutathione by 5,5′-dithiobis(2-nitrobenzoic acid) to glutathione disulfide, with the formation of the yellow chromagen, 5-thio-2-nitrobenzoic acid, measured spectrophotometrically at 412 nm and by the visualization of reduced glutathione in cells stained with the fluorescent dye, Cell Tracker? Green 5-chloromethylfluorescein diacetate. A survey of various cell culture media, formulated with and without pyruvate, confirmed that the level of added hydrogen peroxide was greatly lessened in those media formulated with pyruvate. This study suggested that the pyruvate status of Dulbecco’s modified Eagle’s medium be specified in the experimental design, especially in studies involving oxidative stress.     

The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli

Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.     

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.     

Pyruvate reduces DNA damage during hypoxia and after reoxygenation in hepatocellular carcinoma cells

Pyruvate is located at a crucial crossroad of cellular metabolism between the aerobic and anaerobic pathways. Modulation of the fate of pyruvate, in one direction or another, can be important for adaptative response to hypoxia followed by reoxygenation. This could alter functioning of the antioxidant system and have protective effects against DNA damage induced by such stress. Transient hypoxia and alterations of pyruvate metabolism are observed in tumors. This could be advantageous for cancer cells in such stressful conditions. However, the effect of pyruvate in tumor cells is poorly documented during hypoxia/reoxygenation. In this study, we showed that cells had a greater need for pyruvate during hypoxia. Pyruvate decreased the number of DNA breaks, and might favor DNA repair. We demonstrated that pyruvate was a precursor for the biosynthesis of glutathione through oxidative metabolism in HepG2 cells. Therefore, glutathione decreased during hypoxia, but was restored after reoxygenation. Pyruvate had beneficial effects on glutathione depletion and DNA breaks induced after reoxygenation. Our results provide more evidence that the alpha-keto acid promotes the adaptive response to hypoxia followed by reoxygenation. Pyruvate might thus help to protect cancer cells under such stressful conditions, which might be harmful for patients with tumors.     

Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T. ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25kDa, as well as precursor forms above 48kDa. Metalloproteinase bands below the main band at 48kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor dl-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T. ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.     

Characterization of a low molecular weight suppressor of lymphocyte proliferation from guinea pig L2C leukemia cells

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.     

Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts

Cell survival was significantly decreased in primary cultured rat calvarial osteoblasts in vitro at Day 0, 1, and 3 by replacement of the standard culture medium (alpha-modified minimum essential medium; alpha-MEM) with Dulbecco's modified eagle's medium (DMEM). Decreased cell survival was also observed following medium replacement in cultures of murine calvaria-derived osteoblastic cell line MC3T3-E1. Staining with Hoechst33342 revealed apoptotic cells with fragmented or condensed nuclei, while a fraction of the cell culture was stained with propidum iodide, indicating necrosis. Marked increases in DNA binding of both activator protein-1 and nuclear factor-kappaB were found in nuclear extracts of cells following medium replacement. The addition of either pyruvate or cysteine at each concentration found in alpha-MEM almost entirely prevented cell death associated with medium replacement at Day 3. These results suggest that pyruvate and cysteine may be essential factors for cell growth and survival in osteoblast cultures at the proliferative phase.     

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants — the “Skulachev-ion” derivatives

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.     

Differential expression of αB-crystallin causes maturationdependent susceptibility of oligodendrocytes to oxidative stress

Oligodendrocyte precursor cells (OPCs) are most susceptible to oxidative stress in the brain. However, the cause of differences in susceptibility to oxidative stress between OPCs and mature oligodendrocytes (mOLs) remains unclear. Recently, we identified in vivo that αB-crystallin (aBC) is expressed in mOLs but not in OPCs. Therefore, we examined in the present study whether aBC expression could affect cell survival under oxidative stress induced by hydrogen peroxide using primary cultures of OPCs and mOLs from neonatal rat brains. Expression of aBC was greater in mOLs than in OPCs, and the survival rate of mOLs was significantly higher than that of OPCs under oxidative stress. Suppression of aBC by siRNA transfection resulted in a decrease in the survival rate of mOLs under oxidative stress. These data suggest that higher susceptibility of OPCs than mOLs to oxidative stress is due, at least in part, to low levels of aBC expression. [BMB Reports 2013; 46(10): 501-506]     

Protective effects of tetramethylpyrazine on rat retinal cell cultures

The retinal degeneration characterized with death of retinal ganglion cells is a pathological hallmark and the final common pathway of various optic neuropathies. Thus, there is an urgent need for identifying potential therapeutic compounds for retinal protection. Tetramethylpyrazine has been suggested to be neuroprotective for central neurons by acting as an antioxidant and a calcium antagonist. In this study, we tested the effects of tetramethylpyrazine on the viability of both neuronal and non-neuronal cells in mixed rat retinal cell cultures during a long-term cultivation or following hydrogen peroxide treatments. Cellular and biochemical analyses demonstrated that 50 microM tetramethylpyrazine significantly preserved neuronal morphology and survival in retinal cell cultures following 4-week in vitro cultivation as well as lethal exposures to hydrogen peroxide (10 microM or 50 microM for 24h). Hydrogen peroxide treatments induced remarkable increases in lipid peroxidation and mitochondrial reactive oxygen species (ROS) generation paralleled by the loss of mitochondrial membrane potential, microtubule-associated protein-2 (MAP-2) in neuronal soma and rattin peptide in cultured cells. Addition of tetramethylpyrazine in the cultures efficiently attenuated the signs of oxidative stress and retained abundance of MAP-2 and rattin in association with cell survival. In addition, siRNA-mediated downregulation of MAP-2 or rattin significantly increased the vulnerability of retinal neurons or the number of degenerating cells in the cultures, respectively, whereas exogenous humanin peptide, an analog of rattin, promoted cell survival in cultures under hydrogen peroxide attacks. These results suggest that tetramethylpyrazine protect retinal cells through multiple pathways and might be a potential therapeutic candidate for retinal protection in certain optic neuropathies.     

Oxidative stress decreases extracellular homocysteine concentration in human hepatoma (HepG2) cell cultures

BACKGROUND: Mild hyperhomocysteinemia is associated with premature vascular disease. The mechanism behind the vascular injuries is, however, still unknown. Homocysteine may be catabolized in the trans-sulfuration pathway to cysteine. Cystathionine beta-synthase, which catalyses the first step in the trans-sulfuration pathway is redox-sensitive. We have therefore investigated total extracellular homocysteine turnover in the presence of oxidative stress in human cell lines. METHODS: The turnover of total extracellular homocysteine in HeLa and hepatoma cell cultures has been investigated in the presence of hydrogen peroxide. Furthermore, the effect of hydrogen peroxide on the removal of high amounts of exogenously added homocysteine was also studied. RESULTS: Total extracellular homocysteine concentration in hepatoma cell cultures decreased in the presence of hydrogen peroxide, whereas the extracellular homocysteine concentration in HeLa cell cultures was not influenced. There was no significant change of intracellular homocysteine in any type of cell cultures. Furthermore, the presence of hydrogen peroxide did not increase the removal of exogenously added homocysteine. CONCLUSION: The presence of hydrogen peroxide probably increases the activity of the trans-sulfuration pathway in hepatoma cell cultures, which increases the intracellular use of homocysteine and lowers its extracellular release. Consequently this mechanism might tend to lower total plasma homocysteine concentration in oxidative stress.     

INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS-PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.     

Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.     

Deregulation of catalase, not MnSOD, is associated with necrotic death of p53-defective DF-1 cells under antimycin A-induced oxidative stress

One of distinct genetic alterations in spontaneously immortalized DF-1 cells was found to be dysfunction of p53 and E2F-1 as well as altered antioxidant gene expression (upregulation of MnSOD and downregulation of catalase). We have characterized the cellular responses of primary and immortal DF-1 cells to oxidative stress and found that DF-1 cells were more sensitive to oxidative stress than their primary counterparts when treated with antimycin A. The increased DF-1 cell death by oxidative stress was accompanied by an increase in the levels of intracellular superoxide anions and hydrogen peroxide. The cell death in DF-1 cells by antimycin A showed none of the hallmarks of apoptosis, but displayed a significantly increased necrotic cell population. Anti-apoptotic Bcl-2 failed to inhibit oxidative-induced necrotic cell death in the DF-1 cells. However, this necrotic cell death was significantly decreased by treatment with hydrogen peroxide scavengers such as sodium pyruvate and N-acetyl-cysteine. Interestingly, overexpression of human catalase in DF-1 cells endowed cells resistant to the oxidative stress by antimycin A treatment, although the downregulation of MnSOD by an antisense strategy showed no evident change in the cytotoxic effect caused by antimycin A. Taken together, the present study might provide new therapeutic approach for tumor cells having the loss of p53 function and the altered antioxidant functions.     

Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes

The effect of phorbol esters on the proliferation and survival of interleukin-2(IL-2)-dependent cells was studied using an IL-2-dependent T cell line (CTLL-2) and blasts of BALB/c mouse spleen cells stimulated with Concanavalin A. Addition of phorbol 12,13-dibutyrate (PDBu) to CTLL-2 or ConA blasts induces a mitogenic response which is 25-40% of that elicited by IL-2. Interleukin 2 deprivation leads to a marked decline in the number of viable cells (50% of CTLL-2 cells have died after 8-10 hours incubation in IL-2-free medium). The mechanism of cell death seems to correspond to the suicide process known as apoptosis since an early degradation of DNA into oligonucleosome-size fragments could be observed after removal of the growth factor. When present, PDBu inhibits both the activation of the endonuclease and the development of the cell death process in CTLL-2 cells and ConA-blasts deprived of IL-2. Taken together, our results suggest that the tumor promoters phorbol esters inactivate in T cells the mechanism of cell elimination triggered by IL-2 deprivation and may help to explain why transformation of T cells decreases or even abolishes their requirements of IL-2 for survival and growth.     

Nicotinamide influence on pancreatic cells viability

The study was undertaken to investigate the modulating effect of nicotinamide (NAm) in different concentrations and under different glucose concentrations on the viability and oxidative stress induced by streptozotocin (STZ, 5 mmol/l) and hydrogen peroxide (H2O2, 100 micromol/l) on isolated rat pancreatic cells of the Langerhans islets in vitro. Cell viability did not depend on the concentration of glucose in the range of 5-20 mmol/l, and in subsequent studies we used glucose in concentration of 10 mmol/l to protect cells against its hypo- and hyperglycemic action. Cytoprotective effect of NAm in concentrations from 5 to 20 mmol/l on cells survival was the same. It was found that the destructive action of STZ and H2O2 during 24 hours on isolated cells of the pancreas resulted in the significant cell death. It was revealed that NAm in concentration of 5 mmol/l not only had cytoprotective effects against STZ and H2O2 but also partially reduced the level of oxidative stress in the investigated cells induced by these compounds. High concentration of NAm, 35 mmol/l, causes cytotoxic effect on the viability of pancreatic islet cells and increase of oxidative stress induced by STZ and H2O2. Most likely these effects could be associated with direct modulatory action of NAm on important effector mechanisms involved in cell death, including PARP-dependent processes, or/and indirectly, through metabolic and antioxidant effects of the compound.