Aldose reductase-catalyzed reduction of acrolein: implications in cyclophosphamide toxicity

Acrolein, a highly cytotoxic aldehyde, is a metabolic by-product of the antineoplastic agent cyclophosphamide and is responsible for the development of hemorrhagic cystitis, a serious side effect of cyclophosphamide therapy. Aldose reductase (EC 1.1.1.21), a member of the aldo-keto reductase superfamily, catalyzes the NADPH-dependent reduction of acrolein to allyl alcohol (Km = 80 microM, kcat = 87 min-1). Aldose reductase is expressed at different levels in individuals. This suggests that individual differences in the reductive metabolism of acrolein may be a determinant of acrolein toxicity. In addition to being a substrate, acrolein also produces a time-dependent 7-20-fold increase in the activity of aldose reductase toward a variety of substrates. This involves initial binding of acrolein to a second site (Ks = 58 microM). Acrolein activation of aldose reductase results not only in higher kcat values for all substrates but also in higher Km values and decreased catalytic efficiencies. Acrolein activation of aldose reductase reduces its affinity for aldose reductase inhibitors.     

Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells

Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.     

Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.     

Effects of the antiproliferative cyclopentenone prostaglandin A1 on glutathione metabolism in human cancer cells in culture

Homeostatic mechanisms for the maintenance of glutathione (GSH) are fundamental in the provision of a cellular defense against electrophilic/oxidant challenges. Cyclopentenone prostaglandins (CP-PGs) are powerful antiproliferative endogenous substances that may act as electrophilic regulating compounds, by virtue of the presence of an alpha,beta-unsaturated carbonyl group in the cyclopentane ring. Nevertheless, differential resistance to CP-PG cytotoxic/cytostatic effect has been reported in different cell types. It is reported that the activity/expression of gamma-glutamylcysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis) can be inducibly activated by electrophiles, including CP-PGs. The response of the human cancer strains HEp-2 (larynx carcinoma) and HL-60 (promyelocytic leukemia) cells to treatment with the CP-PG PGA1 in culture was investigated by evaluating the time-course of GSH synthesis and activity of enzymes of GSH metabolism, other than gamma-GCS, after PGA1 addition. HEp-2 cells, being more resistant to PGA1 cytotoxic and cytostatic effects, have basal GSH levels that were 2.4-fold higher than that of HL-60 cells. The activities of GSH S-transferase (GST), glutathione reductase (GSRd) and glutathione peroxidase (GSPx) are constitutively higher in HL-60 cells than in HEp-2 cells (respectively, 17.0-, 28.5- and 12.3-fold). When challenged with PGA1, both cell types exhibited a dose-dependent rise in GSH content that was maximal 18 h after PGA1 addition and was preceded by a rise in GST and GSRd activities in both cell types (at 12 h). GSPx activity increased only in HEp-2 (PGA1 evoked a 93.4%-inhibition in HL-60 cells). Moreover only HEp-2 cells exhibited early capacity to enhance GSH content (1-2 h just after PGA1 addition). These results and earlier data showing that leukemia cells are sensitive to CP-PG treatment suggest that deficiencies in GSH metabolism may be strategically in therapeutic approaches to the treatment of human leukemias.     

Thiol and disulfide metabolites of the radiation protector and potential chemopreventive agent WR-2721 are linked to both its anti-cytotoxic and anti-mutagenic mechanisms of action

The ability of the potential chemopreventive agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) to protect against radiation-induced mutagenesis at the hprt locus and cell killing was studied using CHO-AA8 cells incubated for 30 min at 37 degrees C in growth medium containing its active thiol 2-[(aminopropyl)amino]ethane-thiol (WR-1065). In parallel experiments, the thiol and disulfide forms of the drug present in cells and incubation medium were determined in order to identify which, if either, of the components were associated with the observed protective effects. Treatment with 4 mM WR-1065 produced significant intracellular levels of the thiol (WRSH) and disulfide (WRSS) forms of the drug, but also caused dramatic elevation of cellular glutathione (GSH) and cysteine levels, accompanied by marked protection against 60Co gamma-photon- and neutron-induced cell killing and mutagenesis. When drug-treated cells were transferred to drug-free medium and incubated for 4 h at 37 degrees C, levels of WRSH and WRSS and protection against cell killing decreased markedly, whereas levels of GSH and cysteine and protection against mutagenesis showed little change. GSH and cysteine levels were not associated with protection against radiation-induced mutagenesis, as established by experiments performed with buthionine sulfoximine to block GSH synthesis. These data do not support the hypothesis that modulation of GSH or cysteine levels by WR-1065 is a major mechanism accounting for protection. Protection against mutagenesis was seen for cells incubated in medium with concentrations of added WR-1065 as low as 10 microM, where cellular levels of WRSH and WRSS became difficult to measure (< or = 5 microM) and no protection against cell killing was found. An unexpected observation was that cells incubated in 40 microM WR-1065 incorporated the drug much more rapidly than expected for uptake by passive diffusion and concentrated the drug to a marked degree; this indicates that a cell-mediated transport system is involved in the uptake of WR-1065 at low drug concentrations.     

Protective effects of glutathione isopropyl ester on the sensitivity of cultured cells to UVB irradiation

The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.     

Modulation of intracellular glutathione and cysteine metabolism in bovine oviduct epithelial cells cultured In vitro

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.     

三种缝线材料对人肺腺癌细胞A549增殖和细胞周期的影响

Objective: The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods: Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results: Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05). The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung adenocarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05).Conclusion: Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.     

Vaccination for experimental gliomas using GM-CSF-transduced glioma cells

Within the pituitary, folliculostellate (FS) cells are considered to regulate the intercellular communication between endocrine cells by paracrine mechanisms. One of the possible paracrine factors involved, could be interleukin-6 (IL-6) which is produced by these cells. Since IL-6 has been shown to be a growth factor of pituitary cells, we have determined whether IL-6 can also influence FS cell proliferation. To test this, a FS cell-like mouse pituitary cell line (TtT/GF cells) was used that exhibits most characteristics of normal FS cells. Under serum-free conditions the proliferation of TtT/GF cells is critically dependent on the initial seeding density: cells seeded at low density do not grow at all whereas cells seeded at high density proliferate with maximal doubling times of 34 hrs. Mouse IL-6 (mIL-6) stimulated only low density cell cultures in a dose and time dependent manner. For cells seeded at high density, exogenously added IL-6 may have failed to stimulate growth because of endogenously produced mIL-6. Conditioned medium from TtT/GF cells, in which mIL-6 concentrations up to 1017 pg/ml were measured, stimulated the proliferation of TtT/GF cells, indicating an autocrine growth stimulatory mechanism. A neutralizing mIL-6 antibody partially suppressed the growth of TtT/GF cells seeded at high density and partially reduced the growth stimulatory activity of TtT/GF conditioned medium. Thus, IL-6 is one but not the only factor that is involved in the autocrine growth stimulatory loop. The relevance of this mechanism for normal FS cells and its physiological consequence needs to be elucidated.     

Effect of ifosfamide treatment on glutathione and glutathione conjugation activity in patients with advanced cancers

Several studies have suggested that the glutathione/glutathione S-transferase (GSH/GST) system is involved in resistance of tumors toward ifosfamide and other cytostatic agents. Besides, ifosfamide metabolites (in vitro) as well as ifosfamide treatment (in vivo) have been shown to decrease cellular GSH availability. In the present study, the in vivo effects of three different ifosfamide treatment schedules on the GSH/GST system were studied in patients with advanced cancers (n = 24): continuous i.v. infusions of 1300 mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The GSH/GST system was characterized by administering bromisoval, a probe drug to assess GSH conjugation activity in vivo, as well as by daily monitoring of GSH concentrations in blood cells and plasma. Bromisoval pharmacokinetics was assessed before and at the end of the ifosfamide treatment. Blood cell GSH levels decreased significantly (P < 0.05) during the 3- and 10-day ifosfamide treatment schedules; the 24-h treatment had no effect. The ifosfamide treatment schedules had only minimal effects on bromisoval pharmacokinetics. Assuming that the kinetics of the probe drug provide an accurate reflection of enzyme activity, this suggests that GST activity remains unchanged. Because GSH conjugation of bromisoval enantiomers requires both GST activity and GSH availability, these results also indicate that, despite the 35% decrease in GSH in blood cells of two patient groups, the GSH availability of the cancer patients was not rate-limiting for GSH conjugation of bromisoval enantiomers. If GSH levels in blood cells reflect those in tumors/other tissues, the present results indicate that ifosfamide may be used clinically to decrease GSH levels. However, whether a 35% decrease is sufficient to increase tumor sensitivity toward (other) cytostatics remains uncertain.     

Methodological aspects of observations of rapid tympanic membrane movements due to changes in static pressure (Valsalva maneuver) using digital high speech video recordings

Conditions of oxidative stress lead to down-regulation of glutathione (GSH) and glutathione peroxidase (GPO), which could be responsible for tyrosinase induction in pigment cells. To address this question, the effects of selective modulation of GSH metabolism on melanogenic parameters of slightly and highly melanized melanoma cells were examined. Under standard culture conditions (100 microM cystine, 100 microM tyrosine), the levels of GSH and the activities of glutathione reductase (GR) and GPO were found to be directly related to the pigmentation of melanoma cells. Exposure to 50 microM buthionine sulfoximine for 72 h decreased tyrosinase activity by 30-50% and GSH levels by more than 95%. In contrast, inhibition of GR activity with bis(chloroethyl)nitrosourea or stimulation of GPO activity with sodium selenite did not affect tyrosinase activity nor pigment formation in the melanoma cells tested. Since cysteine (CysH) is a precursor of the GSH tripeptide, the modulation of tyrosinase and GPO activity by the extracellular cystine concentration was also examined. When the cystine concentration was increased from 0 to 200 microM, a dose-dependent decrease in tyrosinase activity was associated with dose-dependent increases in GPO activity and in cell levels of CysH and GSH. The results indicate that cellular thiols coregulate the activities of tyrosinase and GPO in opposite directions. These interdependent processes could provide melanoma cells with protection against oxidative stress at low as well as at high thiol concentration.     

Determination of kynurenic acid by capillary electrophoresis with laser-induced fluorescence detection

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR.     

T cell stimulation via CD47: agonistic and antagonistic effects of CD47 monoclonal antibody 1/1A4

CD47/integrin-associated protein has been extensively studied on various cell types. The function of CD47 on T cells, however, remained poorly understood. We demonstrate here that our CD47 mAb 1/1A4 has both inhibitory as well as costimulatory effects in terms of T cell activation. Soluble, not cross-linked, CD47 mAb 1/1A4 blocks allogeneic MLRs. This effect is predominantly observed with suboptimal numbers of stimulator cells. In contrast, cross-linking of CD47 in the presence of CD28 mAb or phorbol ester induces vigorous T cell proliferation that is sensitive to cyclosporin A. Cross-linking, but not immobilization, of the CD47 mAb 1/1A4 is an essential requirement for the CD28- or phorbol ester-dependent induction of T cell mitogenesis. Furthermore, CD47 mAb 1/1A4 induces T cell proliferation when coimmobilized with a CD3 mAb to the same surface. Ligation with cross-linked 1/1A4 mAb induces an increase in intracellular free calcium levels and leads to tyrosine phosphorylation of various cellular proteins and, in the presence of suboptimal concentrations of plate-bound CD3 mAb, to enhanced IL-2 promotor activity in T cells. Furthermore, we present evidence that upon cross-linking of CD47 with mAb 1/1A4, purified T cells acquire responsiveness for several T cell growth factors. IL-1beta and/or IL-6 dramatically augment this CD47-induced cytokine responsiveness. Our results suggest that the novel activation pathway via CD47 might be critically involved in Ag-dependent and Ag-independent T cell functions.     

沉默COX-2抑制A549细胞的恶性增殖

Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cyclooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control.The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.     

Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress

Expression of the MRP1 gene encoding the GS-X pump and of the gamma-GCSh gene encoding the heavy (catalytic) subunit of the gamma-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like gamma-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1, 4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably gamma-GCSh-transfected cell lines down-regulated endogenous MRP1 and gamma-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and gamma-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and gamma-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and gamma-GCSh expression.     

Effects of thiols on topoisomerase-II alpha activity and cell cycle progression

Thiol containing compounds exhibiting antioxidant properties are currently being evaluated for use in cytoprotection and chemoprevention. Many of these have also been found to be effective in inhibiting cell cycle progression and cellular proliferation. N-Acetyl-L-cysteine (L-NAC), along with its nonmetabolically active stereoisomer N-acetyl-D-cysteine (D-NAC), together with captopril and dithiothreitol (DTT) were investigated to assess their effects on cell cycle progression as determined by flow cytometry. Topoisomerase-IIa (topo-II alpha) activity, an enzyme involved in DNA synthesis, was also monitored as a function of drug dose using a kinetoplast DNA (kDNA) decatenation assay. Chinese hamster ovary (CHO) AA8 cells were exposed to each thiol at concentrations ranging from 4 microM to 4 mM for a period of 3 h. Following the removal of the thiols, cell cultures were followed for an additional 5 h to assess changes in cell cycle progression. L-NAC, which also serves as a precursor for glutathione (GSH) synthesis, effectively inhibited topo-IIa activity by at least 50% at all concentrations tested. Associated with this reduction in enzyme activity was a sixfold increase in the relative number of cells accumulating in G2phase. D-NAC, which is unable to participate in GSH synthesis, was only half as effective as L-NAC at each concentration tested in inhibiting topo-IIa activity as well as perturbing cell progression through G2. In comparison, captopril, an inhibitor of angiotensin converting enzyme (ACE), had little effect on the progression of cells into G2 phase. In contrast to the repressive effects of L-NAC and D-NAC, it enhanced topo-IIa activity over control values by approximately 20%. DTT, a well characterized thiol known to be capable of reducing disulphides in proteins, was observed to be relatively ineffective in either perturbing cell cycle progression or affecting topo-IIa activity. This suggests an involvement of a mechanism(s) in addition to thiol mediated affects on reduction/oxidation processes. The inhibitory effects of L-NAC and D-NAC on topo-IIa activity, in contrast to the other two thiols, may be due in part to the presence of amine groups which could allow for their participation in polyamine related processes. The difference in the magnitude of the effect exhibited by L-NAC, as compared to D-NAC, on the repression topo-IIa activity also suggests a role for GSH in this process. Inhibition of cellular progression and proliferation by thiols can therefore be mediated by diverse mechanisms which include both cycle-phase specific (i.e. L-NAC and D-NAC) and non cell cycle specific (i.e. captopril) processes.     

Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.     

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.