Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells.

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic mitochondria-dependent pathway. BIM (BCL-2-interacting mediator of cell death), a BCL-2 homology 3-only pro-apoptotic protein, is upregulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia cells and has an essential role in Dex-induced apoptosis. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however, the molecular mechanisms including responsible transcription factors are unclear. In this study, we found that Dex treatment induced transcription factor Runx2 and c-Jun in parallel with BIM induction. Dex-induced BIM and apoptosis were decreased in cells harboring dominant-negative c-Jun and were increased in cells with c-Jun overexpression. Cells harboring short hairpin RNA for Runx2 also decreased BIM induction and apoptosis. On the Bim promoter, c-Jun bound to and activated the AP-1-binding site at about -2.7 kb from the transcription start site. Treatment with RU486, a GC receptor antagonist, blocked Dex-induced Runx2, c-Jun and BIM induction, as well as apoptosis. Furthermore, pretreatment with SB203580, a p38-mitogen-activated protein kinase (MAPK) inhibitor, decreased Dex-induced Runx2, c-Jun and BIM, suggesting that p38-MAPK activation is upstream of the induction of these molecules. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, and targeting these molecules may be an alternative approach to overcome GC-resistance in leukemia treatment.

[Possible mechanisms of antioxidant activity of glycyrrhizic acid].

Possible mechanisms of antioxidant activity of glycyrrhizinic acid (GA) were studied. GA did not exhibit antiradical properties at the range of concentrations 1-100 mM as was shown in the experiments with stable radical 1,1-Diphenyl-2-picrylhydrazyl. These data were conformed by the study of GA influence on chemiluminescence of luminol in cell-free system with hydrogen peroxide. However, GA decreased generation of reactive oxygen species by PMA-FMLF-activated neutrophils. Addition of GA did not ifluence free radical level in neurons, however, cell preincubation with GA resulted in the decrease of free radicals production and the increase of intracellular glutathione level.

[Effect of perfluorocarbon emulsions on function of enzyme systems responsible for generating active forms of oxygen in neutrophils]. / Vliianie émul'sii perftoruglerodov na funktsionirovanie fermentnykh sistem, otvetstvennykh za generatsiiu aktivnykh form kisloroda v neitrofilakh.

The ability of the emulsion of perfluoroorganic compounds stabilized with proxanol 268 to affect the functions of peritoneal neutrophils was evaluated. The functional activity of neutrophils was estimated from the intensity of generation of reactive oxygen species using the method of chemiluminescent analysis. The emulsion was shown to suppress the neutrophil responses to phorbol-12-myristate-13-acetate in a dose-dependent manner. No inhibition of the activity of neutrophils in the presence of the emulsion was observed in N-formylmethionylleucylphenylalanine stimulated cells. The data obtained indirectly confirm the suggestion that the perfluoride emulsion inhibits neutrophil NADPH oxidase activity. In the presence of the perfluoride emulsion, myeloperoxidase plays a more important role in the generation of luminescent responses in both N-formylmethionylleucylphenylalanine- and phorbol-12-myristate-13-acetate-stimulated neutrophils. The effect of perfluoride emulsion results in the preferential myeloperoxidase-produced generation of reactive oxygen species in the neutrophil respiratory burst.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

Variations of the effect of insulin on neutrophil respiratory burst. The role of tyrosine kinases and phosphatases.

The priming effect of insulin on the fMLP-induced respiratory burst of mouse neutrophils as well as the involvement of tyrosine protein kinases and phosphatases in this process have been studied. Peritoneal evoked neutrophils of NMRI strain mice were incubated with 0.01-100 nM insulin for 1-60 min at 22, 30, or 37 degrees C and activated by 0.1-50 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP). The production of reactive oxygen species (ROS) by neutrophils was monitored by luminol-dependent chemiluminescence. We found that 125I-labeled insulin binding by mouse neutrophils occurred with saturation and high affinity. Insulin itself did not change the basal level of the ROS production but could modulate fMLP-induced respiratory burst. The effect of insulin depended on temperature and duration of pretreatment of the neutrophils with insulin and the concentration combination of the insulin and fMLP. The tyrosine kinase inhibitor tyrphostin 51 decreased the fMLP-induced respiratory burst significantly. Insulin did not change the fMLP response of neutrophils pretreated with tyrphostin. However, the effect of tyrphostin on the response to 50 microM fMLP was considerably decreased in neutrophils treated with insulin. There was no such effect during activation by 5 microM fMLP, for which the priming effect of insulin was not observed. Insulin did not increase the fMLP-induced respiratory burst in neutrophils treated with the protein phosphatase inhibitors orthovanadate and pyrophosphate. If the inhibitors were added after insulin, the combined effect was nearly additive. It is possible that priming by insulin of the fMLP-induced respiratory burst is triggered by tyrosine phosphorylation, realized with its participation, and involves the signaling pathways initiated by tyrosine phosphorylation but subsequently is not dependent on the latter. The role of protein phosphatases in priming by insulin is of little importance. The data indirectly confirm the idea that priming of the neutrophil respiratory burst is a result of crosstalk of signaling pathways of the insulin and fMLP receptors with the participation of tyrosine phosphorylation.

Cardioselective antiischemic ATP-sensitive potassium channel (K(ATP)) openers. 6. Effect of modifications at C6 of benzopyranyl cyanoguanidines.

The effect on potency and selectivity of modifications at the C6 position of the cardioprotective K(ATP) opener BMS-180448 (2) is described. Structure-activity studies show that a variety of electron-withdrawing groups (ketone, sulfone, sulfonamide, etc.) are tolerated for cardioprotective activity as measured by EC(25) values for an increase in time to the onset of contracture in globally ischemic rat hearts. Changes made to the sulfonamido substituent indicate that compounds derived from secondary lipophilic amines are preferred for good cardioprotective potency and selectivity. The diisobutyl analogue 27 (EC(25) = 0.04 microM) is the most potent compound of this series. The cardiac selectivity of 27 results from a combination of reduced vasorelaxant potency and enhanced cardioprotective potency relative to the potent vasodilating K(ATP) openers (e.g., cromakalim). The diisobutylsulfonamide analogue 27 is over 4 orders of magnitude more cardiac selective than cromakalim (1). These results support the hypothesis that the cardioprotective and vasorelaxant properties of K(ATP) openers follow distinct structure-activity relationships. The mechanism of action of 27 appears to involve opening of the cardiac K(ATP) as its cardioprotective effects are abolished by the K(ATP) blocker glyburide.

A method for verifying treatment times for simple high-dose-rate endobronchial brachytherapy procedures.

An empirical method for verifying the total treatment time for either a one- or a two-catheter high-dose-rate procedure has been developed. The method can be performed quickly and allows for easy verification of the accuracy of the treatment time arrived at by a computerized planning system. The method is designed to confirm the treatment time to within 10%.

Quinoxaline N-oxide containing potent angiotensin II receptor antagonists: synthesis, biological properties, and structure-activity relationships.

A series of novel quinoxaline heterocycle containing angiotensin II receptor antagonist analogs were prepared. This heterocycle was coupled to the biphenyl moiety via an oxygen atom linker instead of a carbon atom. Many of these analogs exhibit very potent activity and long duration of effect. Interestingly, the N-oxide quinoxaline analog was more potent than the nonoxidized quinoxaline as in the comparison of compounds 5 vs 30. In order to improve oral activity, the carboxylic acid function of these compounds was converted to the double ester. This change did result in an improvement in oral activity as represented by compound 44.

Dihydropyrimidine angiotensin II receptor antagonists.

The discovery of the nonpeptide angiotensin II (AII) receptor antagonist losartan, previously called DuP 753, has stimulated considerable interest in the synthesis of novel analogs of this compound. Our efforts in this area have resulted in the discovery of dihydropyrimidines as potent AII receptor antagonists. The chemistry leading to this novel class of AII antagonists and their biological properties are reported in this publication. Structure-activity studies showed that a variety of substituents are tolerated on the dihydropyrimidine ring, indicating that the AII receptor is permissive in accepting this region of the nonpeptide antagonists. As reported for imidazole-based AII antagonists, the tetrazolyl dihydropyrimidine analogs were found to be more potent than the corresponding carboxylic acids. Our studies show that dihydropyrimidine analogs 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5-yl)[1, 1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester (Ki = 8.3 nM), 2-butyl-4-chloro-1,6-dihydro-6-methyl-1- [[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-5- pyrimidinecarboxylic acid (Ki = 1.0 nM), and 2-butyl-6-chloro-1,4-dihydro-4,4-dimethyl-1-[[2'-(1H-tetrazol-5-yl )[1,1'- biphenyl]-4-yl]methyl]-5-pyrimidinecarboxylic acid, ethyl ester (Ki = 1.1 nM), display affinities for the AII receptor which are comparable to or better than losartan (Ki = 9.0 nM). One of these derivatives, 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5- yl)[1,1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester, showed antihypertensive activity on oral administration to spontaneously hypertensive rats. These results demonstrate that the imidazole of losartan can be successfully replaced with a dihydropyrimidine ring.

cis-diamineplatinum (II) complexes containing phosphono carboxylate ligands as antitumor agents.

A series of platinum complexes of the form cis-M[PtA2(PC)] (I) has been prepared and tested for antitumor activity in mice. Compounds in this series contain either two monodentate amine ligands (A), such as NH3 or isopropylamine, or one bidentate diamine (A2), such as ethylenediamine, 1,2-diaminopropane, or 1,2-diaminocyclohexane. The PC ligand is a bidentate, O-bound, phosphono carboxylate chelate of the form -O2C(CR1R2)nPO3-, where n = 0 or 1 and R1 and R2 are chosen from H, methyl, ethyl, propyl, butyl, phenyl, or pentanoic acid substituents. The resulting complexes (I) were prepared as the free acids (M = H) or as sodium salts (M = Na). Members of this series have demonstrated good activity in a number of tumor screens. A total of 18 platinum-phosphono carboxylate (Pt-PC) complexes were tested against Sarcoma 180 ascites (S180a) in CFW mice, with 13 analogues showing activity above the 50% ILS level. Antitumor activity was also observed vs L1210 leukemia in CDF1 mice, where six of the 12 compounds tested gave ILS values in the 60-160% range, and vs M5076 reticulum cell sarcoma (sc tumor, iv drug), where four of the four compounds tested gave ILS and T-C values comparable to that of cisplatin. Each of the Pt-PC complexes was characterized by NMR (195Pt, 13C, and 31P), HPLC, and elemental analysis. These compounds, which are anionic at neutral pH, display excellent solubility and stability in aqueous media, such as phosphate-buffered saline and fetal calf serum. On the basis of a comparative study of BUN and serum creatinine levels in treated mice, representative complexes from this series are also less kidney toxic than cisplatin. The results of these studies demonstrate that the platinum-phosphono carboxylate complexes are a promising new class of antitumor agents.

Autoantibodies in juvenile arthritis.

Sera from 104 children with JA with different onset-types of disease were evaluated for 19S IgM RF by the LFT , hidden 19S IgM RF by the hemolytic assay, ANA by HEp-2 cell substrate, and levels of IC by the C1qSPA . Their relationship to active disease was determined. Classical 19S IgM RF were detected by the LFT in only seven patients. All were late-onset polyarticular females. Hidden 19S IgM RF were detected by the hemolytic assay in the separated IgM-containing fraction in 55 patients of all onset-types. Clinical activity correlated with the presence of hidden 19S IgM RF in 82% of cases. ANA, using the HEp-2 cell substrate, were found in 61 patients, the majority showing a speckled, immunofluorescent pattern. ANA were noted in all RF positive patients and in nine of 10 patients with iridocyclitis. IC were found in 39 patients, and correlation with clinical activity occurred in 54% of cases. A search for positive associations among the four parameters showed no statistically significant correlations except for the concordance of ANA positivity in all seven RF positive patients. The presence of hidden RF correlated more closely with disease activity (P less than 0.001) than did that of ANA or IC. The significance of these data and previous studies remains to be determined. We have demonstrated that in the average JA population 7% have 19S IgM RF and about 60% have hidden RF, ANA, or elevated levels of IC. The present findings of 98 of 104 patients with at least one of the abnormal immunoproteins , the association of ANA in patients with iridocyclitis or with RF positivity, of hidden RF with disease activity, and the presence of 19S IgM RF in isolated IC suggest a possible immunologic etiology for JA.