Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione

Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media. The cells entered the resting state during culture when they were seeded at high cell densities. They remained viable and were mostly present in the G1 or G0 phase. In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol. In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly. Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis. DNA synthesis slowly increased, reaching a maximum after a lag period.     

The synergistic action of the copper chelator bathocuproine sulphonate and cysteine in enhancing growth of L1210 cells in vitro

The growth stimulating effect of a copper-specific chelator, 2,9-dimethyl-4,7-diphenyl-1,10-phenonthroline-sulfonic acid on mouse lymphoma L1210 cells in vitro has been studied. Since they are defective in cystine transport, these cells require cysteine for their growth in vitro. However, addition of cysteine does not greatly enhance cell growth because it is rapidly oxidized to cystine. We have observed that the copper chelator potently inhibited oxidation of cysteine in culture medium and that simultaneous addition of cysteine and the chelator greatly enhanced cell growth. The chelator alone stimulated cell growth slightly by stabilizing a small amount of cysteine effluxed from the cells to the medium. The chelator also enhanced the growth promoting activity of 2-mercaptoethanol by stabilizing cysteine produced in the medium during culture. These results suggest that the chelator stimulates cell growth by inhibiting copper mediated oxidation of cysteine in culture medium.     

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.     

Use of 2-mercaptoethanol in cell culture]

Survival and growth in in vitro cultivation of lymphocytes, lymphoma cells and some other cells including human carcinomas are profoundly improved by 2-mercaptoethanol. These cells hardly take up cystine, an essential nutrient in the culture medium, but in the presence of 2-mercaptoethanol they can utilize cystine. Recently it has been found that 2-mercaptoethanol is effective in the in vitro cultivation of pathogenic trypanosomes and in the in vitro development of bovine embryos. The mechanisms by which 2-mercaptoethanol improves the survival and growth of these cells are described.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.

The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.     

Influence of sodium selenite and selenomethionine on DNA/RNA synthesis and BaP binding to spleen lymphocytes in culture

In the present study, an attempt was made to provide some information regarding the effects of organic and inorganic selenium compounds on DNA/RNA synthesis and benzo(a)pyrene uptake in cultured lymphocytes from mice spleen. It was clear from the results that there was a significant inhibition of DNA/RNA synthesis with increasing concentration of either form of selenium from 0.1ΜM to 1 mM in culture medium. However, when used at the same level as selenite with respect to selenium content, selenomethionine exerted more DNA/RNA synthesis inhibitory effect than selenite. Benzo(a)pyrene uptake by proliferating lymphocytes was also significantly reduced with increasing selenium concentration (0.1 ΜM to 1 mM). However, both forms of selenium at the same selenium concentration showed almost the same inhibitory effect on the cellular uptake of benzo(a)pyrene, which indicated that some factor(s) other than the DNA synthesis are also involved in the interaction between benzo(a)pyrene and cells. Involvement of the changes in the carcinogen metabolism and glutathione level has been discussed. Present studies show that organic selenium as a source of selenium is a more potent chemopreventive compared to the inorganic one. This information may have a useful therapeutic potential.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

Redox imbalance in cystine/glutamate transporter-deficient mice

Cystine/glutamate transporter, designated as system x(-)(c), mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT(-/-) mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT(-/-) mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT(-/-) mice was lower than that in the xCT(-/-) mice. The embryonic fibroblasts derived from xCT(-/-) mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT(-/-)-derived cells and permitted growth. These results demonstrate that system x(-)(c) contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.     

Biosynthesis of ergothioneine from endogenous hercynine in Mycobacterium smegmatis

Ergothioneine was synthesized and accumulated in growing cultures of Mycobacterium smegmatis when the medium was adequately supplied with sulfur. In a low sulfur medium, the accumulation was sharply limited although growth of the organism was apparently normal. Synthesis of hercynine, the precursor of ergothioneine, was unaffected by low sulfur levels and was markedly increased by addition of l-histidine, the precursor of hercynine. Resting-cell pellicle experiments, performed with cells grown on the low sulfur high histidine medium, showed that ergothioniene was synthesized from endogenous hercynine, when cysteine or compounds readily converted to cysteine (such as cystine, lanthionine, cystathionine, and thiazolidine carboxylic acid) were added. Homocysteine and djenkolic acid allowed for minimal synthesis of betaine, whereas methionine, S-methylcysteine, sodium sulfate, and sodium thiosulfate were unable to donate sulfur for ergothioniene synthesis under the experimental conditions employed. Addition of cysteine to a resting pellicle preparation caused the formation of 100 to 200 mug of ergothioneine per g of dry cells in 2.5 to 3 hr. A modified procedure for isolating ergothioneine and hercynine, employing a 75% ethyl alcohol extraction of wet organisms, followed by a single alumina column separation of the compounds, is described.     

Effects of arsenic on DNA synthesis in human lymphocytes stimulated by phytohemagglutinin

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. It was also shown that both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes, and immediately stimulated or inhibited DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of β-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, β-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing. © 1996 Wiley-Liss, Inc.     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.     

Mercury, silver, and gold inhibition of selenium-accelerated cysteine oxidation

In vivo, cysteine in proteins or glutathione is the major amino acid involved in sulfhydryl oxidation-reduction reactions. An in vitro model of cysteine oxidation accelerated by selenium compounds was used to study the interaction of selenocystine and sodium selenite with metal ions. The interaction of metal ions with selenium compounds inhibited cysteine oxidation. The ionic forms of three toxic soft-acid metals, mercury, silver, and gold, were the most effective inhibitors. The antiarthritic gold drugs, aurothiomalate and aurothioglucose, were of particular interest as they inhibit the activity of selenium-glutathione peroxidase. The effect of gold ligands on gold(I) inhibition of selenocystine-accelerated cysteine oxidation was tested. Sodium cyanide partially reversed inhibition and potassium iodide had no effect. Inhibition of selenium-accelerated oxidation-reduction reactions by soft-acid metal ions may be of biological relevance during toxicities or during antiarthritic gold therapy.     

Involvement and relative importance of at least two distinct mechanisms in the effects of 2-mercaptoethanol on murine lymphocytes in culture

2-Mercaptoethanol (2-ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2-ME. Furthermore, 2-ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2-ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2-ME. As expected, 2-ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2-ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2-ME not accounted for by antioxidant action could be accounted for by enhanced protein synthesis.     

Effect of selenium compounds on selenium content, growth and 35S-cystine metabolism of skin fibroblasts from normal and cystinotic individuals.

Kidney samples from children with the inborn metabolic disease cystinosis contain 4 times more selenium (Se) than do kidney samples from normal individuals (p = 0.1). However, when cultured skin fibroblasts from cystinotic patients and normal control individuals are incubated in Se-D,L-methionine, Se-D,L-cystine, Se-cystamine X HCl, Se-urea, selenite or in medium without added selenium, only the cystinotic fibroblasts grown in Se-urea or selenite (SeO3=) contain more selenium than do the corresponding normal cells (p less than 0.05). In both types of cultured fibroblasts, the order of descending toxicity per ppm selenium is: Se-urea greater than Se-cystamine greater than Se-cystine greater than or equal to SeO3= much greater than Se-methionine. High (apparently toxic) concentrations of Se-urea and Se-cystamine lower the elevated intracellular free (nonprotein) cystine content of cystinotic fibroblasts to less than 60% of control values; at lower concentrations, these compounds raise the cystine content of these cells to over 140% of control values. Appropriate concentrations of SeO3=, Se-cystine and Se-methionine also elevate the free cystine content of the cystinotic cells. During a 75 minute incubation in 35S-cystine, the incorporation of 35S into the acid precipitable (protein) fraction of both cell types is significantly inhibited by Se-cystamine (approximately 55% control; p less than 0.05). The incorporation of 35S-cystine into glutathione is inhibited by Se-cystine (approximately 40% control) in both fibroblast types (p less than 0.05). In cystinotic cells, Se-cystamine significantly reduces incorporation of 35S-cystine into the cystine pool (40% control) as does SeO3= (67% control; p less than 0.05). Protein and glutathione synthesis in cystinotic fibroblasts are more strongly inhibited by Se-cystine and SeO3=, respectively, than in normal fibroblasts (p less than 0.05). These studies demonstrate that selenium compounds exhibit a different sequence of toxicity in fibroblasts than in the intact animal and that some previously unreported metabolic effects (i.e. inhibition of glutathione synthesis) may contribute to their toxicity.     

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamidetreated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-ME_ox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [³⁵S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [³⁵S]cystine with 2-ME_ox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-ME_ox and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.     

Low membrane transport activity for cystine in resting and mitogenically stimulated human lymphocyte preparations and human T cell clones.

In order to determine whether the cysteine requirement of human T lineage cells is met primarily by extracellular cysteine or by cystine, amino-acid-transport activities were measured in resting and mitogenically stimulated human peripheral blood lymphocytes (PBL) and several human T cell clones and T cell tumors. The transport activity of the small neutral amino acids cysteine and alanine (ASC system) and the transport of the cationic amino acid arginine (y+ system) were found to be markedly increased after stimulation of PBL by the T cell mitogen phytohemagglutinin from Phaseolus vulgaris. The anionic transport activity for cystine and glutamate (Xc- system), in contrast, was extremely weak in both resting and activated human PBL and also in all human T cell lines under test. The weak system Xc- activity of human T lineage cells was further confirmed by an independent line of experiments showing that an increase of the extracellular concentration of glutamate, i.e. a competitive inhibitor of cystine transport, causes a decrease in the intracellular cystine levels in cells of the promonocytic line U937, but not in T lineage cells (Molt-4). A third set of experiments showed that the rate of DNA synthesis in mitogenically stimulated human PBL is strongly influenced by variations of the extracellular cysteine level, even in cultures with relatively high and approximately physiological concentrations of cystine. Cysteine cannot be replaced in this case by the addition of corresponding amounts of cystine or methionine. This demonstrates an important functional consequence of the weak cystine transport activity of human lymphocytes. The results may be relevant for the pathogenetic mechanism of the acquired immunodeficiency syndrome, since the mean plasma cysteine concentration of human-immunodeficiency-virus-1-seropositive persons was found to be strongly decreased in comparison with that of healthy blood donors, and since the cysteine level even of healthy persons is extremely low in comparison with all other protein-forming amino acids.     

Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine

Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+-dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bathocuproinedisulfonate, a nontoxic copper-specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25-400 microM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide-activated cells, however, cysteine enhanced the cellular glutathione contents in a dose-dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.