Homocysteine and cysteine - albumin binding in homocystinuria: assessment of cysteine status and implications for glutathione synthesis?

Summary. Measurement of plasma total cysteine rather than free dimeric cystine gives a better indication of cysteine status in homocystinuric patients. This is the result of displacement of cysteine from albumin by homocysteine and is related to the plasma homocysteine concentration. In control subjects the free/bound cyst(e)ine ratio was independent of albumin and total cysteine concentrations. In homocystinuric (HCU) patients both free and total cyst(e)ine values differed significantly from control values (P < 0.001) but whilst free cystine considerably overlapped control values the total cysteine concentrations were almost invariably lower. The possible consequences of this on glutathione synthesis was explored by assay of plasma total glutathione but no evidence for glutathione deficiency was found. Measurement of total cysteine, rather than free cystine, provides a better indication of cysteine status in HCU. Received February 1, 2001 Accepted November 13, 2001     

Unique characteristics of rat spleen lymphocyte, L1210 lymphoma and HeLa cells in glutathione biosynthesis from sulfur-containing amino acids

Suspensions of rat spleen lymphocyte, murine L1210 lymphoma and HeLa cells were partially depleted of glutathione (GSH) with diethyl maleate and allowed to utilize either [35S]methionine, [35S]cystine or [35S]-cysteine for GSH synthesis. Lymphocytes preferentially utilized cysteine, compared to cystine, at a ratio of about 30 to 1, which was not related to differences in the extent of amino acid uptake. Only HeLa cells displayed a slight utilization of methionine via the cystathionine pathway for cysteine and GSH biosynthesis. HeLa and L1210 cells readily utilized either cystine or cysteine for GSH synthesis. The three cell types accumulated detectable levels of intracellular cysteine glutathione mixed disulfide when incubated in a medium containing a high concentration of cystine. Various enzyme activities were measured including gamma-glutamyl transpeptidase, GSH S-transferase and gamma-cystathionase. These results support the concept of a dynamic interorgan relationship of GSH to plasma cyst(e)ine that may have importance for growth of various cell types in vivo.     

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.     

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

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.     

Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells

Objectives:  The aim of this study was to investigate mechanisms involved in the growth inhibitory effect of silymarin, in humanhepatocellular carcinoma.
Materials and Methods:  The human hepatocellular carcinoma cell line HepG2 was utilized and the MTT assay was performed to study the antiproliferative effect of silymarin. Dual staining was undertaken for ethidium bromide/acridine orange, propidium iodide staining and DNA fragmentation studies were executed to confirm the presence of apoptosis. Cell-cycle analysis was revealed by flow cytometry and mitochondrial transmembrane potential was measured by uptake of the mitochondrial-specific lipophilic cationic dye rhodamine 123. Western blotting analysis for cytochrome c, p53, Bax, Bcl-2, APAF-1, caspase-3, survivin, β-catenin, cyclin D1, c-Myc and PCNA was carried out.
Results:  Silymarin inhibited population growth of the hepatocellular carcinoma cells in a dose-dependent manner, and the percentage of apoptotic cells was increased after treatment with 50 and 75 µg/ml silymarin for 24 h. Silymarin treatment increased the proportion of cells with reduced DNA content (sub-G₀/G₁ or A₀ peak), indicative of apoptosis with loss of cells in the G₁ phase. Silymarin also decreased mitochondrial transmembrane potential of the cells, thereby increasing levels of cytosolic cytochrome c while up-regulating expression of pro-apoptotic proteins (such as p53, Bax, APAF-1 and caspase-3) with concomitant decrease in anti-apoptotic proteins (Bcl-2 and survivin) and proliferation-associated proteins (β-catenin, cyclin D1, c-Myc and PCNA).
Conclusions:  Our results demonstrate that silymarin treatment inhibited proliferation and induced apoptosis in the human hepatocellular carcinoma cell line HepG2.     

Anti-proliferative effects of γ-tocotrienol on mammary tumour cells are associated with suppression of cell cycle progression

Objectives:  Previous studies have shown that γ-tocotrienol induces potent anti-proliferative effects on +SA mammary tumour cells in culture; here, investigations have been conducted to determine its effects on intracellular signalling proteins involved in regulating cell cycle progression.
Materials and methods:  +SA cells were maintained in mitogen-free defined media containing 0 or 4 μ m γ-tocotrienol, for 48 h to synchronize cell cycle in G₀ phase, and then they were exposed to 100 ng/ml EGF to initiate cell cycle progression. Whole cell lysates were collected at various time points from each treatment group and were prepared for Western blot analysis.
Results and conclusions:  Treatment with 4 μ m γ-tocotrienol significantly inhibited +SA cell proliferation over a 4-day culture period. Moreover, this treatment resulted in a relatively large reduction in cyclin D1, cyclin dependent kinase (CDK)4, CDK2 and CDK6 levels, between 4 and 24 h after EGF exposure. Tocotrienol treatment also resulted in a relatively large increase in CDK inhibitor (CKI) p27, prior to and after EGF exposure, but had little effect on levels of CKIs, p21 and p15. Tocotrienol treatment also induced a large relative reduction in retinoblastoma (Rb) protein phosphorylation at ser780 and ser807/811. These findings strongly suggest that anti-proliferative effects of γ-tocotrienol are associated with reduction in cell cycle progression from G₁ to S, as evidenced by increased p27 levels, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6 and phosphorylated Rb levels.     

Sample processing alters glutathione and cysteine values in blood

The accurate assessment of glutathione status of blood is essential for its use as an index of health and aging. A major variable in glutathione analysis is sample processing, and identification of optimal standard conditions is needed. Thus our objective was to evaluate several methods to determine which one yields maximal levels of free and bound glutathione and cyst(e)ine in blood. Reduced glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), and cystine were analyzed specifically by an HPLC-dual electrochemical method. The highest GSH levels were found in ultrafiltrates of hemolysates, which were 58% greater than those in acid extracts of whole blood, and accounted for 96% of the free and bound GSH in borohydride-reduced samples; GSSG was undetected. The next highest values were in acid extracts of hemolysates which were 13% greater than in extracts of whole blood; both extracts contained GSH and GSSG. Their GSSG contents expressed in GSH equivalents comprised 7-9% of GSH + GSSG. Cys levels were highest in ultrafiltrates which were 11-fold greater than in acid extracts of whole blood, accounting for 62% of the total cyst(e)ine pool. In summary, the results indicate that ultrafiltration of hemolysates is the blood processing method of choice to obtain maximal values of free and bound GSH and cyst(e)ine.     

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10⁻⁵M, 10⁻⁶M or 10⁻⁷M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10⁻⁵ M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G₀/G₁ phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G₁/S transition by prolonging G₂/M and through decreasing the DNA synthesis rate     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Cell kinetic status of haematopoietic stem cells

The haematopoietic stem cell (HSC) population supports a tremendous cellular production over the course of an animal's lifetime, e.g. adult humans produce their body weight in red cells, white cells and platelets every 7 years, while the mouse produces about 60% of its body weight in the course of a 2 year lifespan. Understanding how the HSC population carries this out is of interest and importance, and a first step in that understanding involves the characterization of HSC kinetics. Using previously published continuous labelling data (of Bradford et al. 1997 and Cheshier et al. 1999 ) from mouse HSC and a standard G₀ model for the cell cycle, the steady state parameters characterizing these HSC populations are derived. It is calculated that in the mouse the differentiation rate ranges between about 0.01 and 0.02, the rate of cell re-entry from G₀ back into the proliferative phase is between 0.02 and 0.05, the rate of apoptosis from the proliferative phase is between 0.07 and 0.23 (all units are days⁻¹), and the duration of the proliferative phase is between 1.4 and 4.3 days. These values are compared with previously obtained values derived from the modelling by Abkowitz and colleagues of long-term haematopoietic reconstitution in the cat ( Abkowitz et al. 1996 ) and the mouse ( Abkowitz et al. 2000 ). It is further calculated using the estimates derived in this paper and other data on mice that between the HSC and the circulating blood cells there are between 17 and 19.5 effective cell divisions giving a net amplification of between ~170 000 and ~720 000.     

Stimulation of colonic mucosal growth associated with oxidized redox status in rats

Limited data in animal models suggest that colonic mucosa undergoes adaptive growth following massive small bowel resection (SBR). In vitro data suggest that intestinal cell growth is regulated by reactive oxygen species and redox couples [e.g., glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) redox]. We investigated the effects of SBR and alterations in redox on colonic growth indexes in rats after either small bowel transection (TX) or 80% midjejunoileal resection (RX). Rats were pair fed +/- blockade of endogenous GSH synthesis with buthionine sulfoximine (BSO). Indexes of colonic growth, proliferation, and apoptosis and GSH/GSSG and Cys/CySS redox potentials (E(h)) were determined. RX significantly increased colonic crypt depth, number of cells per crypt, and epithelial cell proliferation [crypt cell bromodeoxyuridine (BrdU) incorporation]. Administration of BSO markedly decreased colonic mucosal GSH, GSSG, and Cys concentrations in both TX and RX groups, with a resultant oxidation of GSH/GSSG and Cys/CySS E(h). BSO did not alter colonic crypt cell apoptosis but significantly increased all colonic mucosal growth indexes (crypt depth, cells/crypt, and BrdU incorporation) in both TX and RX groups in a time- and dose-dependent manner. BSO significantly decreased plasma GSH and GSSG, oxidized GSH/GSSG E(h), and increased plasma Cys and CySS concentrations. Collectively, these data provide in vivo evidence indicating that oxidized colonic mucosal redox status stimulates colonic mucosal growth in rats. The data also suggest that GSH is required to maintain normal colonic and plasma Cys/CySS homeostasis in these animal models.     

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na⁺-independent, glutamate-sensitive route previously found in various types of cells and designated as system x⁻_c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.     

3-O-methylfunicone, a metabolite of Penicillium pinophilum, inhibits proliferation of human melanoma cells by causing G2 + M arrest and inducing apoptosis

Objectives:  Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3- O -methylfunicone (OMF), a metabolite of Penicillium pinophilum , has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines.
Materials and methods:  Cell proliferation and apoptosis were analysed by flow cytometry, DNA fragmentation, caspase-3 and caspase-9 activation, and PARP-1 cleavage.
Results:  We demonstrated that OMF affected cell proliferation in a time- and dose-dependent manner, reaching the best effect at concentration of 80 µg/ml for 24 h. Flow cytometry revealed that OMF caused significant G₂ phase arrest, which was associated with marked decrease in cyclin B1/p34^cdc2 complex and p21 induction. OMF also induced marked decrease of survivin expression. Reduced levels of apoptosis were evident after silencing p21 expression in both cell lines. Finally, the effect exercised by OMF on hTERT and TEP-1 gene expression confirmed the ability of this molecule to interfere with replicative ability of cells.
Conclusions:  The results reported here seem to suggest that OMF as a promising molecule to include in strategies for treatment of melanoma.     

Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death

The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.