The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall.

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Purification and characterization of a 4-hydroxynonenal metabolizing glutathione S-transferase isozyme from bovine pulmonary microvessel endothelial cells.

Previous studies have suggested that a group of structurally and immunologically related mammalian glutathione S-transferases (GSTs) which utilize 4-hydroxynonenal (4-HNE) as the preferred substrate and show glutathione peroxidase activity towards phospholipid hydroperoxides may be important for the defense of cells against lipid peroxidation. In present studies we have purified and characterized GST isozymes of bovine pulmonary microvessel endothelial (BPMVE) cells. The results of these studies indicate that BPMVE cells express relatively high amounts of a GST isozyme which utilizes 4-HNE as the preferred substrate. This GST isozyme purified to homogeneity from BPMVE cells showed remarkably high specific activity towards 4-HNE (48.3 units/mg protein) and had similar immunological, kinetic, and structural characteristics as reported for mouse enzyme mGSTA4-4 and other mammalian GSTs of this group. Since the endothelial cells are exposed to constant oxidative stress, we suggest that this GST isozyme may be important for the defense of these cells against lipid peroxidation.

Transfection of a 4-hydroxynonenal metabolizing glutathione S-transferase isozyme, mouse GSTA4-4, confers doxorubicin resistance to Chinese hamster ovary cells.

It has previously been suggested that adriamycin (doxorubicin, DOX), which is used to treat various types of malignancies, exerts its cytotoxic effects through interactions with DNA and by accelerating membrane lipid peroxidation. Our previous studies have indicated that a mouse glutathione S-transferases isozyme, mGSTA4-4, which shows high activity toward 4-hydroxyalkenals and the hydroperoxides formed during lipid peroxidation, may play an important role in defense mechanism against lipid peroxidation. In order to test the hypothesis that the enhanced protection against lipid peroxidation confers DOX-resistance to cells, we have compared the cytotoxicity of DOX to Chinese hamster ovary cells transfected with mGSTA4-4 with that of controls cells transfected with the vector alone. The results of these studies show that mGSTA4-4-transfected cells have a twofold higher resistance to DOX as compared to the controls. Upon exposure to DOX, the transfected cells showed about 40% less membrane lipid peroxidation as compared to the controls, indicating a positive relationship between DOX-cytotoxicity and lipid peroxidation. These results suggest that mGSTA4-4 provides protection to cells against lipid peroxidation and that DOX cytotoxicity, at least in part, is mediated through oxidative stress resulting in peroxidation of membrane lipids which would explain the results of previous studies showing that DOX can kill cells without being internalized (T.R. Tritton and G. Yee (1982) Science 217, 248-250).

Characteristics of harringtonine-resistant human leukemia HL60 cell.

AIM: To study the mechanisms of the resistance to harringtonine (Har) in the HL60 cells. METHODS: Growth inhibition, karyotype analysis, flow cytometry, Western blotting and polymerase chain reaction. RESULTS: The Har-resistant HL60 cell line, named HR20, showed cross resistance to homoharringtonine, doxorubicin, daunorubicin, vincristine, and colchicine. The growth doubling time and the cell numbers in G1 phase were increased. The accumulation of cellular daunorubicin in the resistant cells was obviously reduced, but distinctly increased by tetrandrine and verapamil. The numbers of telocentromeric chromosome increased and the chromosomal aberration more occured in the resistant cells. The resistant cells overexpressed multidrug resistant mdr-1 gene and P-glycoprotein 150 kDa. CONCLUSION: The Har-resistant HL60 cell strain belonged to a multidrug resistance strain, overexpressing mdr-1 gene and P-glycoprotein.

Several closely related glutathione S-transferase isozymes catalyzing conjugation of 4-hydroxynonenal are differentially expressed in human tissues.

A human acidic glutathione S-transferase, hGST 5.8, was isolated from heart, pancreas, and brain by a procedure involving immunoadsorption chromatography on immobilized antibodies raised against mouse mGSTA4-4. The human hGST 5.8 enzymes isolated from these tissues had similar pI (5.8) and subunit M(r) (24.5 kDa) values, showed about 17- to 20-fold higher specific activities for 4-hydroxynon-2-enal than that for 1-chloro-2,4-dinitrobenzene, and expressed glutathione peroxidase activity toward phospholipid hydroperoxides. In this respect, the enzymes belong together with rat GST 8-8 and mouse mGSTA4-4 to a subgroup of GSTs involved in the detoxification of lipid peroxidation products. Partial sequencing of CNBr-peptide fragments of hGST 5.8 proteins isolated from various human tissues revealed significant similarity to mGSTA4-4 and the existence of several distinct isoforms differing in their primary structures. These isoforms had similar but nevertheless clearly distinguishable catalytic properties. These results indicate the existence of multiple hGST 5.8-related genes in the humans, which is consistent with our previous studies showing the presence of several closely related genes for the mouse ortholog mGSTA4-4 (Zimniak et al., J. Biol. Chem., 1994, 269, 992-1000).

[Enhancement of antitumor activity of harringtonine in human leukemia-60 cells in vitro by verapamil].

The effect of harringtonine (Har) alone and in combination with verapamil (Ver) on the proliferation of human leukemia-60 (HL-60) cells in vitro were studied. IC50 of Har alone to the cells was about 49 ng.ml-1 which was reduced to its 1/3.3 and 1/4.5 when used with Ver 1 and 2 micrograms.ml-1, respectively. In colony forming test, the survival fraction of the HL-60 cells treated with Har 15 and 30 ng.ml-1 plus Ver 2 micrograms.ml-1 was reduced to 1/3.3 and 1/8 of the cells as when treated with Har alone, respectively. The results suggested that Ver enhanced the antitumor activity of Har in vitro and may used as an enhancer of Har in vivo.

[Mechanism of enhancement of bleomycin A5 antitumor activity by verapamil].

The mechanism of enhancement of Bleomycin A5 antitumor activity by verapamil was explored by flow cytometry and tracing the radiolabelled bleomycin A5 in vivo. Verapamil was found to increase the G2 blocking effect of bleomycin A5 prominently in mouse S-180 and human HEP-2 cell lines. The distribution of 57Co-bleomycin A5 in mice bearing S-180 sarcoma was changed by verapamil and accumulation of the drug in tumor was increased. In contrast, the labelled drug in the lung was decreased. It seems that the effects of verapamil in enhancing the antitumor activity of bleomycin A5 are to increase the accumulation of the drug in tumor cells and enhance the G2 blocking effect of the drug in cell cycle.

[Effect of bleomycin A5 with calmodulin inhibitor on the proliferation of S-180 cells in vitro].

The effect of bleomycin A5 (BLM) alone and along with calmodulin inhibitor N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13) on the proliferation of S-180 cells in vitro were studied. IC50 of BLM alone to the cells was about 2.63 micrograms/ml, which was decreased to 1/3.8 and 1/9.5 of 2.63 micrograms/ml when plus W-13 1, 5 micrograms/ml respectively. The results indicated that nontoxic doses of W-13 enhanced the inhibition of cell proliferation under the condition of BLM 0.5-2.5 micrograms/ml. In colony forming test, the survival fraction of S-180 cells treated with BLM plus W-13 was decreased to 1/87-240 of the cells treated with BLM alone. The results suggest that W-13 can enhance antitumor activity of BLM in vitro and may be used as an enhancer of BLM in vivo.

[Enhancement of antitumor activity of bleomycin A5 in mouse sarcoma 180 cells in vitro and in vivo by verapamil].

Verapamil (Ver), a calcium influx blocker, enhanced the cytotoxicity of bleomycin A5 (BLM) in cultured S-180 cells. IC50 of BLM to the cells was about 2.6 micrograms/ml when the cells were treated with BLM alone, but when the cells were treated with BLM plus Ver 1, 5, 10 micrograms/ml, the IC50 were 1/1.2, 1/5.2, 1/7.4 of the cells treated with BLM alone, respectively. In colony test, the survival fractions of the cells treated with BLM 20, 60, 100 micrograms/ml plus Ver 60 micrograms/ml were 1/7, 1/8, 1/11 of the cells treated with BLM alone, respectively. Ver enhanced the growth inhibitory actions of BLM in mice bearing S-180 sarcoma by 60% or over. The mean survival the growth inhibitory actions of BLM in mice bearing S-180 sarcoma by 60% or over. The mean survival time of the mice bearing S-180 ascites sarcoma treated with BLM plus Ver was prolonged 21% from that of the mice treated with BLM alone. The results suggest that Ver may be used clinically as an antitumor enhancer of BLM.