Rational Design,Synthesis, and Biological Evaluation of Third Generation α-Noscapine Analogues as Potent Tubulin Binding Anti-Cancer Agents

Systematic screening based on structural similarity of drugs such as colchicine and podophyllotoxin led to identification of noscapine, a microtubule-targeted agent that attenuates the dynamic instability of microtubules without affecting the total polymer mass of microtubules. We report a new generation of noscapine derivatives as potential tubulin binding anti-cancer agents. Molecular modeling experiments of these derivatives 5a, 6a-j yielded better docking score (-7.252 to -5.402 kCal/mol) than the parent compound, noscapine (-5.505 kCal/mol) and its existing derivatives (-5.563 to -6.412 kCal/mol). Free energy (ΔG _bind) calculations based on the linear interaction energy (LIE) empirical equation utilizing Surface Generalized Born (SGB) continuum solvent model predicted the tubulin-binding affinities for the derivatives 5a, 6a-j (ranging from -4.923 to -6.189 kCal/mol). Compound 6f showed highest binding affinity to tubulin (-6.189 kCal/mol). The experimental evaluation of these compounds corroborated with theoretical studies. N-(3-brormobenzyl) noscapine (6f) binds tubulin with highest binding affinity (K_D, 38 ± 4.0 µM), which is ~ 4.0 times higher than that of the parent compound, noscapine (K_D, 144 ± 1.0 µM) and is also more potent than that of the first generation clinical candidate EM011, 9-bromonoscapine (K_D, 54 ± 9.1 µM). All these compounds exhibited substantial cytotoxicity toward cancer cells, with IC₅₀ values ranging from 6.7 µM to 72.9 µM; compound 6f showed prominent anti-cancer efficacy with IC₅₀ values ranging from 6.7 µM to 26.9 µM in cancer cells of different tissues of origin. These compounds perturbed DNA synthesis, delayed the cell cycle progression at G2/M phase, and induced apoptotic cell death in cancer cells. Collectively, the study reported here identified potent, third generation noscapinoids as new anti-cancer agents.     

A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing 'real' centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the 'cytotoxicity-window' of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some 'apoptosis-evasive' cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.     

Endomorphin1 and endomorphin2, endogenous potent inhibitors of electrical field stimulation (EFS)-induced cholinergic contractions of rat isolated bronchus

In the present study, we determined whether endomorphin1 (EM1) and endomorphin2 (EM2), selective endogenous mu-opioid receptor (MOR) agonists, inhibited the response to EFS in rat isolated bronchus in a concentration- and frequency-dependent manner. EM1 (1 microM) produced significant inhibition at relatively low frequencies (< 5 Hz) (74.02 +/- 5.53%, 56.16 +/- 10.24% and 37.64 +/- 5.92% inhibition at 1, 2 and 4 Hz, respectively, p < 0.05 versus control), but no significant inhibition at 8, 16, 32 and 64 Hz (17.15 +/- 9.4%, 14.51 +/- 4.23%, 9.11 +/- 2.38% and 5.93 +/- 3.5%, respectively, p > 0.05 versus control). Similar modulations were observed in response to EM2 (1 microM). It is therefore considered that the inhibition effects of EM1 and EM2 may take place at frequencies under physiological conditions. Furthermore, EM1 and EM2 (0.01-10 microM) induced inhibition of cholinergic constriction in a dose-dependent manner at 1, 2 and 4 Hz. The inhibitory effect on EFS was blocked by the opioid receptor antagonist naloxone (10 microM), indicating that opioid receptors were involved. Neither EM1 nor EM2 (1 microM) had an effect on the contractile response to exogenous acetylcholine, indicating a prejunctional effect. All the results indicate that EM1 and EM2 are potent inhibitors of EFS-induced cholinergic bronchoconstriction. These also imply that EM1 and EM2 may modulate cholinergic bronchoconstriction under physiological conditions and that these tetrapeptides could have therapeutic potential in the treatment of airway diseases.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Flow cytometric evaluation of antiviral agents against human herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.     

Energy-dependent changes in conformation and catalytic activity of the chloroplast ATP synthase.

Energy-dependent activation of the chloroplast ATP synthase (CF0CF1) has been elucidated by investigating the conformational changes, the ADP effect, and the catalytic cooperativity of ATP hydrolysis. Conformational change was observed by measuring the reactivity of Lys-109 of the epsilon subunit of chloroplast coupling factor 1 with pyridoxal 5'-phosphate. In the postillumination dark, the Lys-109 reactivity decreased biphasically with half-times of less than 1 and 17 s. NH4Cl accelerated the slow phase decrease. Addition of ADP (0.2 microM) in the postillumination dark inactivated CF0CF1 (0.05 microM) with a half-time of 12 s. At high concentration of CF0CF1 (1.2 microM), inactivation occurred without exogenously added ADP with a half-time of 12 s. Accompanying the inactivation, the positive catalytic cooperativity of ATP hydrolysis decreased. Addition of 10 mM NH4Cl before ADP (0.2 microM) decelerated the ADP-induced inactivation to a half-time of 64 s. Throughout this inactivation, the positive catalytic cooperativity was maintained at a high level. These results suggest three distinct conformations of CF0CF1, EH, EM, and EL, and their ADP binding forms EM-ADP and EL-ADP. EH, EM, and EL have a low affinity for ADP, a high affinity for ADP, and low accessibility to ADP, respectively. EM and EL exhibit highly cooperative ATP hydrolysis. ATP hydrolysis catalyzed by EM-ADP exhibits no cooperativity. EL-ADP is inactive.     

Apoptotic activity of frog Bombina maxima skin albumin

Albumin, the most abundant protein components of blood plasma, is synthesized and secreted by liver cells in vertebrates. Recently, it was demonstrated that frog Bombina maxima albumin is also expressed in skin. Both B. maxima albumins from skin and serum (BmA-skin and BmA-serum) have similar biochemical characteristics except that the former contains haem b. Present studies showed that BmA-skin exhibited cytotoxic activity on H9 and C8166 cells. Pretreated with hemin to induce erythroid differentiation, K562 cells lost their resistance to cytotoxicity of BmA-skin. After treating cells with BmA-skin for 48 h, 50 percentage cytotoxic concentrations (CC(50)) of BmA-skin on H9, C8166 and hemin-treated K562 cells were 1.31+/-0.09, 1.59+/-0.08 and 2.28+/-0.06 microM, respectively. The cell death induced by BmA-skin was mediated by apoptosis of the tested cell lines, as demonstrated by nuclear morphological changes, DNA fragmentation and DNA hypodiploidy of apoptosis cells. At BmA-skin concentration of 2 microM, 27.3%, 19.7% and 17.8% of H9, C8166 and hemin-treated K562 cells were found to be apoptotic. In contrast, BmA-serum possessed no cytotoxic and apoptosis-inducing activity on all the cell lines tested, even with concentration used up to 15 microM. These results indicated that bound haem b in BmA-skin contributed significantly to its cytotoxic and apoptosis-inducing activity on the cell lines assayed.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase

Diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.     

Beauvericin cytotoxicity to the invertebrate cell line SF-9

The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM, for different periods of time (from 30' to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 microM concentration. This effect became gradually more evident at higher concentrations (approximately equal to 28% at 30 microM, approximately equal to 50% at 100 microM, approximately equal to 68% at 300 microM). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 microM beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations approximately equal to 23% at 3 microM, approximately equal to 47% at 10 microM, approximately equal to 65% at 30 microM, approximately equal to 90% at 100 microM, approximately equal to 99% at 300 microM). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 microM and 10 microM, respectively. In time-course experiments, no effect of beauvericin (30 microM) on cell viability could be seen after exposure for periods of time as long as 30', 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 microM. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.     

Abnormal modulation of cholinergic neurotransmission by endomorphin 1 and endomorphin 2 in isolated bronchus of type 1 diabetic rats

To assess whether diabetes alters the regulatory effects of mu-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 microM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 microM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Bloom's syndrome and EM9 cells in BrdU-containing medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption

Bloom's syndrome (BS) and EM9 cells both display elevated frequencies of sister chromatid exchange (SCE) following growth for two rounds of DNA replication in bromodeoxyuridine (BrdU)-containing medium. To learn whether hyperresponsiveness to BrdU itself might play a role in causing the SCE elevation, the effects of BrdU on two other parameters, cellular proliferation and chromosome disruption, were examined, comparing the responses of BS and normal lymphoblastoid cells and of EM9 and CHO cells. BS and normal cells responded similarly with respect to growth for 4 days in BrdU-containing medium (0, 1, 3, and 5 g/ml). Chromosome aberrrations were increased only slightly in the BS and normal cells after 2 days in BrdU. CHO cells responded to growth in BrdU-containing medium like BS and normal cells; however, little growth of EM9 was detected at any of the BrdU concentrations employed. CHO and EM9 cells also exhibited strikingly different amounts of chromosome damage following growth in BrdU. After 2 days in 1, 3, and 5 g/ml BrdU 21%, 46%, and 50%, respectively, of the CHO cells had chromosome aberrations in contrast to 92%, 96%, and 98% of the EM9 cells. Most of the aberrations in the BrdU-treated CHO cells consisted of what appeared to be polycentric and ring chromosomes or chromosomes exhibiting telomere association. Acentric fragments were absent from most cells with polycentric and ring chromosomes, indicating either that the abnormal chromosomes were formed during an earlier cell cycle or that the abnormal chromosomes represent a form of association in which the telomeres are apposed so tightly that the juncture between chromosomes cannot be identified microscopically. EM9 cells treated with BrdU exhibited many chromatid and isochromatid gaps and breaks as well as numerous quadriradial, triradial, and complex interchange configurations. In addition, the types of aberrations present in CHO cells also were increased greatly in number. The different responses of BS and EM9 cells to growth in BrdU suggest that the molecular defects in the two cell types are different.     

Kinetics of rat liver GM2 ganglioside-beta-D-N-acetylhexosaminidase

The kinetics of beta-D-N-acetylhexosaminidase against GM2 ganglioside were examined. We used a crude preparation of rat liver as the enzyme source because purification of beta-D-N-acetylhexosaminidase results in a decrease in specific activity against GM2 ganglioside. Kinetic plots were not linear but showed a break. At substrate concentrations less than 50 microM the Vmax was 6 pmol GM2 hydrolyzed per hour per micromole 4-MU-GlcNAc hydrolyzed per hour (pmol GM2/mumol 4-MU-GlcNAc) and the Km was 5 microM.At substrate concentrations greater than 50 microM, the Vmax was 7 pmol GM2/mumol 4-MU-GlcNAc and the Km was 14 microM. The critical micelle concentration of GM2 ganglioside was 20-25 microM as determined by spectral shifts of the dye pinacyanol chloride in association with GM2, and 10-15 microM from electrical conductivity measurements which also showed the end of the monomer-micelle transition to occur at 40-50 microM GM2. The increasing excess of micellar substrate at greater than 50 microM GM2 explains the discontinuity in the kinetic plots. Sodium taurocholate had a critical micelle concentration of 9-11 mM using pinacyanol chloride and 2.5-3 mM using electrical conductivity. When included in the assay mixture at a concentration of 10 mM, sodium taurocholate produced a linear kinetic plot. This is probably due to the formation of mixed micelles of detergent and GM2 ganglioside. The Vmax was 200 pmol GM2/MUmol 4-MU-GlcNAc and the Km was 93 microM. The data suggest that ganglioside hydrolysis occurs more readily when the substrate is incorporated into a membrane-like environment.     

Novel tacrine derivatives that block neuronal calcium channels

A new series of tacrine (9-amino-1,2,3,4-tetrahydroacridine) derivatives were synthesized and their effects on 45Ca(2+) entry into bovine adrenal chromaffin cells stimulated with dimethylphenylpiperazinium (DMPP) or K(+), studied. At 3 microM, compound 1 did not affect (45)Ca(2+) uptake evoked by DMPP. Compounds 14, 15 and 17 inhibited the effects of DMPP by 30%. Compounds 3, 9 and tacrine blocked the DMPP signal by about 50%. Compounds 5 and 12 were the most potent blockers of DMPP-stimulated 45Ca(2+) entry (90%); the rest of the compounds inhibited the effects of DMPP by 70-80%. Compounds 1, 3, 4, 8, 10, 11, 13, 16, 17 and tacrine inhibited 45Ca(2+) uptake induced by K(+) about 20%. Compounds 6, 14 and 15 inhibited the K(+) effects by 10% or less. Compounds 7, 9, 12 and 18 blocked the K(+) signal by 30% and, finally, compounds 2 and 5 inhibited the K(+)-induced 45Ca(2+) entry by 50%. None of the new compounds was as effective as diltiazem (IC(50)=0.03 microM) in causing relaxation of the rat aorta precontracted with 35 mM K(+); the most potent was compound 7 (IC(50)=0.3 microM). Compounds 5, 6, 8, 9, 10 and 13 had IC(50)s around 10 microM and compounds 3, 4, 11 and 12 around 20 microM. Blockade of Ca(2+) entry through neuronal voltage-dependent Ca(2+) channels, without concomitant blockade of vascular Ca(2+) channels, suggests that some of these compounds might exhibit neuroprotectant effects but not undesirable hemodynamic effects.     

Interaction of alkylating oligonucleotide derivatives with mammalian cells. A study of the uptake mechanism of derivative cells

Interaction of alkylating deoxyribooligonucleotide derivatives, bearing 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residues at their 5'-terminal phosphates, with mouse fibroblasts L929 and with ascite carcinoma cells Krebs 2 has been investigated. It was found, that the derivatives are taken up by the cells according to the endocytosis mechanism. At high concentration of the oligonucleotide derivatives in the cultivation medium (greater than 10 microM), the fluid phase endocytosis is the major pathway of uptake; binding of the derivatives by the cells is partially reversible and their intracellular mean concentration approaches 1/20 of their extracellular concentration. At low concentration of the oligonucleotide derivatives, the predominant mechanism is the more efficient adsorption endocytosis; at concentration of the derivatives less than 0.5 microM, their mean intracellular concentration exceeds that in the culture medium. Stability of the oligonucleotide derivatives in cells depends on their nucleotide composition. Their nucleolytic degradation rate is low enough to allow them to react with cellular biopolymers.     

Cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin B1 in the SF-9 insect cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.     

A novel carbazole derivative, BMVC: a potential antitumor agent and fluorescence marker of cancer cells

We have investigated a novel compound, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole diiodide (BMVC), for inhibiting telomerase activity and distinguishing human lung H1299 and oral Ca9-22 cancer cells from lung IMR90 and skin Detroit-551 normal fibroblast cells. The telomeric repeat amplification protocol (TRAP) assay shows that the concentration of BMVC that inhibits 50% of the telomerase activity (IC50) is ca. 0.05 microM. On the other hand, the cell-viability assay indicates that the cytotoxicity was less than 15% to the H1299 and Ca9-22 cancer cells, and almost negligible to the MRC-5 and Detroit-551 normal cells after incubation with 0.5 microM BMVC for 72 h. The low concentration of 0.05 microM of BMVC can inhibit telomerase activity but does not have general toxic effects to normal cells, implying that BMVC is a promising telomerase inhibitor. Moreover, wide-field fluorescence images of 0.1 microM BMVC-treated cells show bright fluorescence spots in the nuclei of the most H1299 and Ca9-22 cancer cells. Interestingly, similar fluorescence spots are hardly observed in the nuclei of the IMR90 and Detroit-551 normal cells, implying that BMVC might be a useful marker to distinguish tumor cells and normal cells.