Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Regulation by glutathione of the effect of lymphokines on differentiation of primary activated lymphocytes. Influence of glutathione on cytotoxic activity of CD3-AK-.

By activating murine lymphocytes with anti-CD3 antibodies for 1 to 2 days, we generated a subset of activated killer cells, namely CD3-AK-. CD3-AK- mediated the slow lysis (20-h 125I-UdR release assay) of allogeneic P815 but had little effect on syngeneic HFL/b cells. Addition of IL-2 (murine or human) or an IL-2 inducer such as PMA in the assay medium induced the cytolytic activity of CD3-AK- on HFL/b. The activating effect of murine IL-2 and PMA on CD3-AK- was decreased by anti-murine IL-2 mAb. Although anti-murine IL-4 mAb alone did not show any effect, it enhanced the inhibitory effect of anti-IL-2 mAb, suggesting that IL-2 and IL-4 may have a synergistic effect on the cytolytic activity of CD3-AK-. Incubation of CD3-AK- with L-buthionine-(SR)-sulfoximine (BSO), an inhibitor of de novo glutathione (GSH) synthesis, decreased cellular GSH levels and inhibited the cytolytic activity of CD3-AK-, in a concentration-dependent manner. This inhibitory effect of BSO was not primarily due to a general cytotoxic effect and was positively correlated with the requirement for IL-2 for the CD3-AK(-)-mediated killing of the target cells. Incubation of CD3-AK- with GSH or 2-ME, which increased the level of cellular GSH, reversed the inhibitory effect of BSO. These results suggest that cellular GSH may regulate the effect of lymphokine(s) such as IL-2 and thus affect the differentiation of activated primary cytotoxic lymphocytes.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

The mode of cisplatin-induced cell death in CYP2E1-overexpressing HepG2 cells: modulation by ERK, ROS, glutathione, and thioredoxin

In a previous study, E47 HepG2 cells that overexpress human CYP2E1 were shown to be more sensitive to cisplatin than C34 cells that do not express CYP2E1. In this study, we found that this sensitivity was due to an earlier activation of ERK in the E47 cells compared to the C34 cells. Glutathione depletion by L-buthionine sulfoximine (BSO) enhanced cisplatin cytotoxicity via increasing production of reactive oxygen species (ROS) and activation of ERK. In contrast, elevation of glutathione by glutathione ethyl ester (GSHE) decreased cisplatin/BSO cytotoxicity by decreasing ROS production and ERK activation. Inhibition of ERK activation by U0126 protected against cisplatin/BSO cytotoxicity via inhibiting ROS production but not restoring intracellular glutathione content. Examination of the mode of cell death showed that U0126 inhibited cisplatin-induced necrosis but not apoptosis. Cisplatin-induced apoptosis was caspases-dependent; BSO switched cisplatin-induced apoptosis to necrosis via decreasing activity of caspases, and GSHE switched cisplatin/BSO-induced necrosis back to apoptosis through maintaining activity of caspases. Similar to GSHE, U0126 partially switched cisplatin/BSO induced necrosis to apoptosis via restoring activity of caspases. Cisplatin lowered levels of thioredoxin, especially in the presence of BSO. Although U0126 failed in restoring intracellular glutathione levels, it restored thioredoxin levels, which maintain the activity of the caspases. These results suggest that thioredoxin can replace glutathione to promote the active thiol redox state necessary for caspase activity, and thus glutathione and thioredoxin regulate the mode of cisplatin toxicity in E47 cells via redox regulation of caspase activity.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Cyclosporin A blocks calcium-dependent pathways of gene activation.

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.     

The role of low molecular weight thiols in T lymphocyte proliferation and IL-2 secretion

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.     

Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression

Ultraviolet A (UVA) irradiation causes human skin aging and skin cancer at least partially through the activation of matrix metalloproteinases (MMPs). MMP-1, the interstitial collagenase, is responsible for the degradation of collagen and is involved in tumor progression in human skin. The present study uses human skin fibroblast cells (FEK4) to investigate the involvement of lipid peroxidation and the role of peroxides as possible mediators in MMP-1 activation by UVA. Preincubation with the antioxidants butylated hydroxytoluene and Trolox reduced UVA-dependent MMP-1 upregulation, suggesting that peroxidation of membrane lipids is involved. Blocking the iron-driven generation of lipid peroxides and hydroxyl radicals by different iron chelators led to a decrease in UVA-induced MMP-1 mRNA accumulation. Moreover, modulation of glutathione peroxidase activity by use of the specific inhibitor mercaptosuccinate (MS) or by the depletion of glutathione (using buthionine-S, R-sulfoximine, BSO), enhanced the UVA-dependent MMP-1 response. Finally, UVA irradiation generated a significant increase in intracellular peroxide levels which is augmented by pretreatment of the cells with BSO or MS. Our results demonstrate that lipid peroxidation and the production of peroxides are important events in the signalling pathway of MMP-1 activation by UVA.     

Oxidative burst and metallothionein as a scavenger in macrophages

The role of metallothionein (MT) in the scavenging of superoxide radicals (*O2-) generated by macrophages has been examined. The present work has focused on the effects of added cadmium, a known inducer of MT biosynthesis, on determined amounts of superoxide radicals produced by in vitro cultured rat peritoneal macrophages on their stimulation with phorbol-12-myristate-13-acetate (PMA). The levels of superoxide radicals (*O2-) have been found to decrease when cadmium was added to cells exposed to PMA. However, substantially lower levels of MT have been determined in this case compared to cells untreated with PMA. This effect could be reversed by incubation of the PMA and cadmium-treated cells with a reducing agent, 2-mercaptoethanol (2-ME). Results suggest that *O2- caused thiolate oxidation and subsequent metal loss, thus reducing the cellular MT content as quantified by the silver saturation METHOD: This conclusion is supported by cell-free experiments in which the oxidation of rabbit MT-I by a xanthine/xanthine-oxidase system could be reversed by its subsequent reduction with 2-ME. The data presented provide direct evidence of the involvement of MT in scavenging superoxide radicals in living cells.     

Increased metal tolerance in Salix by nicotinamide and nicotinic acid

We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of N-acetyl-cystein (NAC) and l-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se.     

Modulatory function of protein kinase C in the activation of ornithine decarboxylase and in cAMP production in rat osteoblasts

The effect of activation of protein kinase C on stimulation of ornithine decarboxylase (ODC) activity and cAMP production was studied in fetal rat osteoblasts. Both phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, and 4 alpha-phorbol, ineffective in activating protein kinase C, failed to stimulate ODC activity and cAMP production. We tested the effect of protein kinase C on stimulation of ODC activity by parathyroid hormone (PTH) and forskolin. In contrast to PTH-stimulated ODC activity, which was not affected by PMA, forskolin-stimulated (1 and 10 microM) ODC activity was dose dependently reduced. PMA (400 nM) reduced both 1 and 10 microM forskolin-stimulated ODC activity to the same level, approximately 3 nmol CO2/mg protein, which suggests a controlling role of protein kinase C in forskolin-stimulated ODC activity. The study of the effect of protein kinase C on PTH- and forskolin-stimulated cAMP production also revealed differences between PTH and forskolin. When PMA was added simultaneously with PTH (4 and 20 nM) or forskolin (1 and 10 microM) the PTH-stimulated cAMP production was dose-dependently potentiated by PMA, whereas forskolin-stimulated cAMP production was not affected. However, both PTH- and forskolin-stimulated cAMP production was dose-dependently augmented when PMA was added 3 min prior to PTH or forskolin. With increasing preincubation periods (up to 24 h) with PMA instead of a potentiation an inhibition was observed. This inhibition is not due to PTH receptor desensitization, although, on basis of the present results desensitization can not completely be excluded. In all cases 4 alpha-phorbol was without effect. The present results show that protein kinase C modulates stimulation of ODC activity and cAMP production in fetal rat osteoblasts. The modulation of both ODC activity and cAMP production appears to be dependent on the nature of the stimulator. The present data suggest a role for protein kinase C in limiting the cAMP-mediated stimulation of ODC activity in these cells. Furthermore, it is suggested that protein kinase C can interfere at more than one site in the cAMP-generating system.     

Differential effects of some novel synthetic oestrogen analogs on oxidative PC12 cell death caused by serum deprivation

Oestrogens with no or reduced oestrogen receptor (ER) binding properties are reported to have neuroprotective functions. However, we have previously shown that the hormonally inactive isomer of 17β-estradiol (17β-E), 17α-estradiol (17α-E), down-regulates glutathione (GSH) synthesis, and fails to rescue serum deprivation-induced cell death in the rat pheochromocytoma cell line PC12 in micromolar concentration. The present study examined cellular protective effects of new 17β-E analogs and 2-methoxyestradiol (2-ME) analogs with no or little oestrogen activity. 17β-E, 17α-E, 2-ME, and an antagonist of the G protein-coupled oestrogen receptor (GPER), G36, were also included. Both 17α-E and 2-ME protected against deprivation-induced cell death in PC12 cells at 1?nM, but they enhanced the deprivation-induced cell death accompanied by caspase 3 activity and decreased intracellular GSH levels during deprivation at 10?µM. In addition, 10?μM 17α-E activated the p38 mitogen activated protein kinase pathway, which was linked to the enhanced death and reduced GSH levels. Analogs of 2-ME modified with a 6-isoquinoline moiety (6iq) protected against deprivation-induced cell death at 1?nM and did not interfere with the GSH levels nor increase p38 protein levels at 10?µM. The promoter activity of the catalytic subunit of the rate-limiting enzyme, glutamate cysteine ligase (GCLC) in GSH synthesis as well as protein levels of GCLC and Nrf2, increased with the 2-ME analogs at 10?µM. In conclusion, the steroids have differential protective effects, and modifying 2-ME may give the steroid more favourable properties than 17α-E, 2-ME, and G36 in regard to GSH regulation.     

Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate

Since the enhancement of the activity of the natural glutathione (GSH)-dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we have compared the effectiveness of diverse intracellular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L-ornithine carboxylase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report at a 2-mM concentration, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine, and L-2-oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 microM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA-treated cells and is the most potent inhibitor of TPA-decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA-induced ODC activity, suggesting that the potential antitumor-promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation-promotion protocol.     

Interleukin-2 regulates the activity of ornithine decarboxylase in a cloned murine T lymphocytic cell line: evidence for a protein kinase C-dependent pathway

The role of protein kinase C (PKC) in the regulation of ornithine decarboxylase (ODC) activity during interleukin-2 (IL-2)-dependent cell growth was investigated. A large biphasic increase in the activity of ODC was observed after treatment of IL-2-deprived CTLL-2 cells with recombinant human IL-2 (rec IL-2). The PKC activators phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD), but not the inactive analog 4 alpha-PDD, induced ODC activity in exponentially growing cultures. Unlike IL-2, however, phorbol esters were poor inducers of IL-2-depleted cultures. H-7, a potent inhibitor of PKC and cyclic nucleotide-dependent protein kinases (CN-PK), suppressed the IL-2-induced ODC activity, while HA1004, a more potent inhibitor of CN-PK than of PKC, had opposite effects depending on its concentration. The results suggest that activation of PKC is involved in but is not the sole mechanism for the induction of ODC by rec IL-2. At concentrations which suppressed the induction of ODC activity by IL-2, H-7 inhibited DNA synthesis and HA1004 did not.     

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.     

Modulation of intracellular glutathione concentrations alters lymphocyte activation and proliferation

Glutathione (GSH) has been implicated in lymphocyte activation and differentiation, as well as in protection from radiation damage. Since [3H]thymidine ([3H]TdR) at high concentrations in the nucleus causes radiation damage to the cells, it is important to rule out the possibility that changes in [3H]TdR uptake by mitogen-activated lymphocytes are not caused by 3H-induced cell injury following alterations in intracellular GSH concentration. In this study, flow-cytometric analysis of cell cycle was used to measure lymphocyte activation. Intracellular GSH levels were enhanced using 2-L-oxothiazolidine-4-carboxylate (OTC) and 2-mercaptoethanol (2ME), which deliver cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO) which inhibits gamma-glutamylcysteine synthetase. Enhancement of intracellular GSH concentrations in lymphocytes with 2-oxothiazolidine-4-carboxylate or 2-mercaptoethanol augments mitogen-induced lymphocyte activation, and proliferation, while suppression of intracellular GSH levels by buthionine sulfoximine inhibits the progression of cellular proliferation--but not activation, as measured by flow cytometry. There was a linear relationship between intracellular GSH concentration and conA-activated cells by flow cytometry and between GSH concentration and [3H]TdR incorporation as measured at 24 h. We conclude that alterations of intracellular GSH concentrations may be one way to modulate lymphocyte activation and differentiation.     

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.