The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis.

TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated L-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.

[CELLS FORM AND THEIR SENSITIVITY TO LYTIC ACTIVITY OF NATURAL KILLER CELLS UNDER THE ANTIOXIDANT ACTION].

The present paper is an attempt to estimate the influence of cell surface morphology changes to functional activity under the effect of antioxidant, N-acetylcysteine (NAC), and alpha-lipoic asid (ALA). Two experimental parameters were used to characterize transformed fibroblasts 3T3-SV40 status. The functional one was the cell sensitivity to lysis by natural killer (NK) mouse splenocytes, and morphology index (cell form index) was a cell area. We showed that addition of NAC or ALA to the cell medium caused fast decrease of cell area and changes of cell form. On the other hand, their sensitivity to lysis NK cells gradually and significantly decreased. Then we compared NAC or ALA effect with the effects of other substances, which were non-antioxidants but caused cell responses which concurred with of antioxidants, at least partly. They were: latrunculin B, desorganizing actin filaments (as both antioxidants), OTZ reducing ROS level in the cell (as NAC), BSO (inhibitor of glutathione synthesis), increasing ROS level in the cell (as ALA), antibodies to gelatinases, MMP-2 and MMP-9 inactivating their activities (as both antioxidants). The results obtained showed a correlation between changes of morphology index and functional activity, sensitivity to lysis by NK cells. We suppose that geometry of cell surface might be a functional indicator of cell reaction to the antioxidant.

Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light.

This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage.

NG as a novel nitric oxide donor induces apoptosis by increasing reactive oxygen species and inhibiting mitochondrial function in MGC803 cells.

NG, O(2)-(2,4-dinitro-5-{[2-(12-en-28-ß-D-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl] amino} phenyl) 1-(N-hydroxyethylmethylamino) diazen-1-ium-1,2-diolate, was identified in our laboratory as a novel nitric oxide-releasing prodrug with antitumor effects. A previous study showed that NG inhibited cell growth, and induced apoptosis in HepG2 cells. In this study, the inhibitory effects of NG on the viability of MGC803 cells were examined using methylthiazolyl tetrazolium biomide (MTT) assay, neutral red assay and trypan blue exclusion test. The results showed that NG had strong cytotoxicity to induce apoptosis, which was characterized by a significant externalization of phosphatidylserine, nuclear morphological changes and enhanced Bax-to-Bcl-2 ratio. Moreover, the release of cytochrome c (Cyt c) from mitochondria and the activation of caspase-9/3 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. NG induced mitochondrial dysfunction in MGC803 cells by altering membrane potential (△Ψm), the inhibition of complexes I, II and IV consequently decreasing ATP level. Furthermore, the treatment of MGC803 cells with NG caused a marked rise in oxidative stress as characterized by accumulation of reactive oxygen species (ROS), excessive malondialdehyde (MDA) production and a reduction in glutathione hormone (GSH) level and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. In addition, pretreatment with N-acetylcysteine (NAC), a GSH synthesis precursor, was partially protective against the NG-induced ROS generation and cell apoptosis. In contrast, pretreatment of MGC803 cells with L-buthionine-S, R-sulfoximine (BSO), a GSH synthesis inhibitor, increased the ROS levels, and aggravated cell apoptosis by NG. These results suggest that NG-induced apoptosis in MGC803 cells is mediated, at least in part, by the increase in ROS production, oxidative stress and mitochondrial dysfunction.

A role for glutathione, independent of oxidative stress, in the developmental toxicity of methanol.

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6h later in embryonic ROS formation, measured by 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2'-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.

Modulation of glutathione intracellular levels alters the spontaneous proliferation of lymphocyte from HTLV-1 infected patients.

The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with neoplasias and inflammatory diseases, such as adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected individuals present a spontaneous T lymphocyte proliferation. This phenomenon is related to the HTLV-1-proviral load and the persistence of the infection. Viral proteins induce many cellular mediators, which can be associated with the abnormal cellular proliferation. The intracellular levels of glutathione (GSH) are important to modulate the cellular proliferation. The aim of this study was to investigate the correlation between the modulation of intracellular GSH levels and the spontaneous lymphocyte proliferation during the HTLV-1 infection. Intracellular GSH level can be modulated by using dl-buthionine-[S,R]-sulfoximine (BSO, GSH synthesis inhibitor) and N-acetylcysteine (NAC, peptide precursor). Our results demonstrated that BSO was capable of inducing a decrease in the spontaneous proliferation of PBMC derived from HTLV-1 carriers. On the other hand, the GSH precursor induces an increase in mitogen-stimulated cellular proliferation in infected and uninfected individuals. Similar results were observed by the inhibition of ABCC1/MRP1 protein, augmenting the mitogen-induced proliferation. This effect can be related with an increase in the GSH levels since ABCC1/MRP1 transports GSH to the extracellular medium. There was a significant difference on the expression of CD69 and CD25 molecules during the lymphocyte activation. We did not observe any alterations on CD25 expression induced by BSO or NAC. However, our results demonstrated that NAC treatment induced an increase in CD69 expression on unstimulated CD8(+) T lymphocytes obtained from HTLV-1 infected individuals, healthy donors and HTLV carriers. Therefore, our results suggest that the cellular proliferation promoted by the infection with HTLV-1 and the activation phenotype of CD8(+) T lymphocytes can be regulated by changing the intracellular GSH levels; suggesting the modulation of these intracellular levels as a new approach for the treatment of pathologies associated with the HTLV-1 infection.

Bioactive sulfoximines: syntheses and properties of Vioxx analogs.

The syntheses and biological profiles of sulfoximine-based Vioxx analogs 2 are described. Interesting data have been obtained for 2a, which shows a selective COX-2 inhibition (albeit not as strong as Vioxx itself) exhibiting reduced hERG activity compare to the parent sulfone Vioxx (1).

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.

Glutathione depletion in immortalized midbrain-derived dopaminergic neurons results in increases in the labile iron pool: implications for Parkinson's disease.

Glutathione depletion is one of the earliest detectable events in the Parkinsonian substantia nigra (SN), but whether it is causative for ensuing molecular events associated with the disease is unknown. Here we report that reduction in levels of glutathione in immortalized midbrain-derived dopaminergic neurons results in increases in the cellular labile iron pool (LIP). This increase is independent of either iron regulatory protein/iron regulatory element (IRP/IRE) or hypoxia inducible factor (HIF) induction but is both H(2)0(2) and protein synthesis-dependent. Our findings suggest a novel mechanistic link between dopaminergic glutathione depletion and increased iron levels based on translational activation of TfR1. This may have important implications for neurodegeneration associated with Parkinson's disease in which both glutathione reduction and iron elevation have been implicated.

Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression.

Induction of cytochrome P4502E1 (CYP2E1) is considered to be an important mechanism by which ethanol can cause toxicity related to oxidative stress both in vivo and in vitro. In the current study, we used HeLa cells with doxycycline-regulated CYP2E1 expression to test the hypothesis that induction of CYP2E1 could lead to secondary DNA oxidation that could potentially contribute to the carcinogenicity of ethanol in vivo. Overexpression of CYP2E1 protein was not associated with oxidative stress per se as assessed by markers of lipid peroxidation (cis-parinaric acid oxidation), glutathione depletion and elevation of intracellular reactive oxygen species (dichlorofluoroscin oxidation) in the presence or absence of ethanol substrate (10 mM, 24 h). Furthermore, there was no evidence of elevation of frequency of DNA strand breaks as assessed by the comet assay. In contrast, however, after pre-incubation of cells with L-buthionine-(S,R)-sulphoximine (BSO, 10 microM) which caused a 75% reduction in intracellular reduced glutathione (GSH) levels, CYP2E1 expression resulted in oxidative stress as assessed by all of these markers and DNA strand breaks but only in the presence of ethanol (10 mM). No effect was observed under these conditions in control cells not expressing CYP2E1. Furthermore, these effects could be attenuated by co-incubation with 1-aminobenzotriazole (0.5 mM), a suicide inhibitor of P450 activity. In conclusion, in this in vitro model CYP2E1-mediated interaction with ethanol results in the intracellular oxidative stress and the formation of DNA strand breaks which are detectable in cells pre-sensitized by depletion of intracellular levels of GSH.

Intracellular glutathione status regulates mouse bone marrow monocyte-derived macrophage differentiation and phagocytic activity.

Although a redox shift can regulate the development of cells, including proliferation, differentiation, and survival, the role of the glutathione (GSH) redox status in macrophage differentiation remains unclear. In order to elucidate the role of a redox shift, macrophage-like cells were differentiated from the bone marrow-derived monocytes that were treated with a macrophage colony stimulating factor (M-CSF or CSF-1) for 3 days. The macrophagic cells were characterized by a time-dependent increase in three major symptoms: the number of phagocytic cells, the number of adherent cells, and the mRNA expression of c-fms, a M-CSF receptor that is one of the macrophage-specific markers and mediates development signals. Upon M-CSF-driven macrophage differentiation, the GSH/GSSG ratio was significantly lower on day 1 than that observed on day 0 but was constant on days 1-3. To assess the effect of the GSH-depleted and -repleted status on the differentiation and phagocytosis of the macrophages, GSH depletion by BSO, a specific inhibitor of the de novo GSH synthesis, inhibited the formation of the adherent macrophagic cells by the down-regulation of c-fms, but did not affect the phagocytic activity of the macrophages. To the contrary, GSH repletion by the addition of NAC, which is a GSH precursor, or reduced GSH in media had no effect on macrophage differentiation, and led to a decrease in the phagocytic activity. Furthermore, we observed that there is checkpoint that is capable of releasing from the inhibition of the formation of the adherent macrophagic cells according to GSH depletion by BSO. Summarizing, these results indicate that the intracellular GSH status plays an important role in the differentiation and phagocytosis of macrophages.

Polymer-coated polyethylenimine/DNA complexes designed for triggered activation by intracellular reduction.

BACKGROUND: Site-specific gene delivery requires vectors that combine stability in the delivery phase with substantial biological activity within target cells. The use of biological trigger mechanisms provides one promising means to achieve this, and here we report a transfection trigger mechanism based on intracellular reduction. METHODS: Plasmid DNA was condensed with thiolated polyethylenimine (PEI-SH) and the resulting nanoparticles surface-coated using thiol-reactive poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) with 2-pyridyldisulfanyl or maleimide groups, forming reducible disulphide-linked or stable thioether-linked coatings, respectively. RESULTS: Both sets of polymer-coated complexes had similar size and were stable to a 250-fold excess of the polyanion poly(aspartic acid) (PAA). Reduction with dithiothreitol (DTT) allowed complete release of DNA from disulphide-linked coated complexes, whereas complexes with thioether-linked coating remained stable. Disulphide-linked complexes showed 40-100-fold higher transfection activity than thioether-linked ones, and activity was selectively further enhanced by boosting intracellular glutathione using glutathione monoethyl ester or decreased using buthionine sulfoximine. The chloroquine- and serum-independent transfection activity of disulphide-linked coated complexes suggests this system may provide a viable trigger mechanism to enable site-specific transfection in complex biological settings. CONCLUSIONS: Linkage of hydrophilic polymer coating to PEI/DNA complexes via reducible disulphide bonds offers a means of fulfilling the contradictory requirements for extracellular stability and intracellular activity.

Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.

Modulation of the stress response during apoptosis and necrosis induction in cadmium-treated U-937 human promonocytic cells.

Treatment for 2 h with 200 microM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent L-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5 degrees C) or with 50 microM cadmium, which caused apoptosis. The administration of N-acetyl-L-cysteine (NAC) at the beginning of recovery after BSO/200 microM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 microM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 microM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation.

Association of glutathione with flowering in Arabidopsis thaliana.

In order to study the relationship between GSH and flowering, wild-type and late-flowering mutant, fca-1, of Arabidopsis thaliana were treated with L-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, under long-day conditions. BSO treatment of the fca-1 mutant starting at 17 d after imbibition promoted flowering. However, when the treatment was started at 12 d after imbibition, BSO treatment at 10(-4) M resulted in an inhibition of flowering. This inhibitory effect of BSO on flowering was abolished by GSH treatment at 10(-4) M, although GSH treatment at an increased concentration of 10(-3) M clearly delayed flowering. In contrast, BSO treatment of wild-type plants starting at 12 d after imbibition promoted flowering, whose effect was abolished by GSH application. In the fca-1 mutant, whose endogenous GSH levels were high, chilling treatment lowered the GSH levels and promoted flowering, as was the case in the BSO treatment. An A. thaliana mutant, cad2-1, which has a defect in GSH biosynthesis also exhibited late flowering. The late-flowering phenotype of this mutant tended to be strengthened by BSO and abolished by GSH treatment. These results suggest that flowering is associated with the rate of GSH biosynthesis and/or the levels of GSH in A. thaliana.

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.

Glutathione deficiency potentiates manganese toxicity in rat striatum and brainstem and in PC12 cells.

Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), glutathione (GSH), ascorbic acid (AA), dehydroascorbic acid (DHAA) and uric acid (UA) were determined in the striatum and/or in the brainstem of 3-month-old male Wistar rats after subchronic oral exposure to MnCl2 (20 mg kg-1 daily) alone or associated to buthionine (S,R)sulphoximine-ethyl ester (BSO-E), an inhibitor of GSH synthesis. The NA, DA, DOPAC, GSH and glutathione disulphide (GSSG) concentrations were also determined in PC12 cells incubated with Mn alone or associated with either BSO-E or AA. When PC12 cells were incubated with AA, cellular AA and DHAA concentrations were also determined. It was found that BSO-E: (a) decreased GSH levels in the striatum and in the brainstem; (b) potentiated the Mn-induced increase in AA oxidation and uric acid formation in both brain regions; and (c) potentiated the Mn-induced DA and NA depletion in the brainstem. Moreover, the changes in striatal DA metabolism induced by the BSO-E association with Mn (decrease in DA, DOPAC and HVA levels and in the DOPAC + HVA/DA ratio) are consistent with the hypothesis of a loss of dopaminergic neurons. In PC12 cells, BSO-E decreased GSH and GSSG levels and potentiated the Mn-induced decrease-in DA and NA concentrations. On the contrary, AA antagonised the Mn-induced DA and NA depletion. AA antagonised also the Mn- and MN+ BSO-induced decrease in PC12 cells viability. In conclusion, the impairment of neuronal antioxidant system activity plays a permissive role in the oxidative stress-mediated Mn neurotoxicity.

Defective antigen processing correlates with a low level of intracellular glutathione.

Previously, we reported that Chinese hamster ovary (CHO) cells transfected with murine mouse major histocompatibility complex class II genes, exhibit a unique antigen (Ag) processing defect whereby these cells are impaired in processing only Ag with disulfide bonds. Here, we examined various aspects of the intracellular reducing environment in the CHO cells to understand the underlying mechanism causing the defect. A cell hybrid generated by the fusion of CHO cells and L cell fibroblasts was used for comparison due to their competency in processing Ag. The transport pathway of cysteine within the CHO cells appeared normal. However, these cells had a significantly lower level of glutathione, a major physiological reducing thiol, compared to the cell hybrid. Treatment of the CHO cells with N-acetyl-L-cysteine did not augment their glutathione content nor their ability to process Ag. When the cell hybrid was treated with L-buthionine-(S,R)-sulfoximine (BSO), which significantly decreased their glutathione level, the hybrid poorly processed hen egg lysozyme (HEL) and ovalbumin, which have disulfide bonds. In contrast, BSO treatment did not affect the capacity of the hybrid to process pigeon cytochrome c and carboxymethylated HEL, which lack disulfide bonds. Therefore, low intracellular glutathione levels in antigen-presenting cells correlated with defective processing of Ag with disulfide bonds, indicating that this thiol may be a critical factor in regulating productive Ag processing.

Lack of a role of glutathione in cellular nonenzymatic activation of BMS-181174, a novel analogue of mitomycin C.

Recent studies, using a cell-free system, have suggested that thiol-dependent nonenzymatic bioactivation may be responsible for the superior antitumor activity of the mitomycin C analogue BMS-181174 [N-7-[2-(4-nitrophenyldithio)ethyl]mitomycin C] when compared to the parent compound. If operational in tumor cells, this pathway could have enormous clinical implications since tumor cell resistance to a variety of anticancer agents is often associated with increased glutathione (GSH) levels and BMS-181174 may be used to reverse this mechanism of resistance. The present study was undertaken to determine the role of GSH in cellular activation of BMS-181174 using a pair of well-characterized human bladder cancer cells (J82 and SCaBER) as a model. A 20-h pretreatment of J82 and SCaBER cells with a nontoxic concentration of D,L-buthionine-S,R-sulfoximine (BSO) caused about 80-88% reduction in cellular GSH levels. Surprisingly, the sensitivity of both cells to BMS-181174 was increased, not reduced, by BSO-induced GSH depletion. On the other hand, the cytotoxicity of BMS-181174 was significantly reduced in both cells by a 4-h pretreatment with 1 mM GSH. Like BSO, a 4-h pretreatment with another thiol compound (cysteine) resulted in a statistically significant sensitization of both cells to BMS-181174. Cellular GSH levels were not affected in either of the cell lines by pretreatment with GSH or cysteine. In conclusion, the results or the present study argue against a role of GSH in cellular nonenzymatic activation of BMS-181174 in J82 and SCaBER cells.

Thiol-mediated redox regulation of neutrophil apoptosis.

BACKGROUND: Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state. METHODS: Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting. RESULTS: Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation. CONCLUSIONS: Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represents a novel therapeutic target for the regulation of cellular function.