Biomedical subjects

B G Feuerstein

Publications and source records attributed to B G Feuerstein.

At least 55 records · Page 3Linked to original sources

PubMed1994

Effect of 1,19-bis(ethylamino)-5,10,15-triazanonadecane on human tumor xenografts.

The polyamine analogue 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), 5 mg/kg i.p., was given twice daily on days 0-3 and 7-10 (cycle 1) to nude mice with human malignant gliomas (SF-767 and U-87 MG), lung adenocarcinoma (A549), and colon carcinomas (HCT116 and HT29). A second cycle of drug was given to mice with SF-767 and A549 tumors on days 42-45 and 49-52. The maximum animal weight loss varied between 4 and 12%, which was observed 10-15 days following the initiation of treatment, but no overt toxic reactions were noted. The SF-767 brain tumors were extremely responsive to BE-4-4-4-4 alone (3 of 8 complete regressions after 2 cycles); however, the growth of the U-87 MG brain tumor was only slightly inhibited by BE-4-4-4-4 treatment. There was significant inhibition of tumor growth after treatment with one cycle of BE-4-4-4-4 in animals carrying the A549, HCT116, and HT29 tumors. At day 73, the growth of the A549 tumor was inhibited by 78 and 89% following one or two cycles of BE-4-4-4-4, respectively. The mitotic index of A549 tumors was 18 times greater in control mice than in those treated with BE-4-4-4-4 for one or two cycles 99 days after initiation of treatment. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was given to mice carrying the U-87 MG or A549 tumors on day 4 (cycle 1) and day 46 (cycle 2) in the maximal tolerated dose of 50 mg/kg for BCNU alone and 40 mg/kg for BCNU plus BE-4-4-4-4. BCNU alone significantly inhibited the growth of U-87 MG tumors but not the growth of A549 tumors. Treatment with the combination of BCNU and BE-4-4-4-4 was significantly better than BCNU alone for A549 tumors and better than BE-4-4-4-4 alone for U87 tumors. However, in both animal groups treated with the combination, there was a significant weight loss, which was not observed for animals treated with either agent alone. These data suggest a role for BE-4-4-4-4 in the treatment of brain, lung, and colon tumors.

Animals↗

PubMed1994

Pretreatment with the polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) inhibits etoposide cytotoxicity in U-251 MG (NCI) human brain tumor cells.

We studied whether pretreatment of U-251 MG human brain tumor cells with the polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) affected the cytotoxicity of the topoisomerase II inhibitor etoposide. We found that BE-4-4-4-4 protected cells from the cytotoxic effects of etoposide. Possible mechanisms for this protection may be related to enhanced DNA-nuclear matrix association in analog-treated cells.

Brain Neoplasms↗

PubMed1993

Interaction of a polyamine analogue, 1,19-bis-(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), with DNA and effect on growth, survival, and polyamine levels in seven human brain tumor cell lines.

Computer graphics modeling and physicochemical studies of spermine-DNA interactions, as well as experiments in cell culture, indicate that a polyamine analogue with strong affinity for nucleic acids but poor ability to condense and aggregate DNA in vitro should act as an antiproliferative agent if it can enter cells. On the basis of our studies of polyamine-DNA interactions, we designed a pentamine, 1,19-bis(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), that had these characteristics. Measurement of melting temperature and ultraviolet light scattering studies show that the affinity of this analogue for calf-thymus DNA is about 4 times higher than that of spermine, whereas its ability to aggregate DNA is slightly poorer than that of spermine. Studies in U-87 MG, U-251 MG, SF-126, SF-188, SF-763, SF-767, and DAOY human brain tumor cells in tissue culture showed that treatment for more than 96 h with concentrations of 5 microM BE-4-4-4-4 or greater inhibited growth; decreased levels of putrescine, spermidine, and spermine; and decreased colony-forming ability in all cell lines. The cytotoxicity of the analogue varied among cell lines; DAOY and SF-767 were the most sensitive and the most resistant lines, respectively. In SF-763 cells, growth inhibition by BE-4-4-4-4 could be partially reversed by the addition of putrescine, spermidine, or spermine 1 day after BE-4-4-4-4 addition, but in U-251 MG cells, growth inhibition was reversed only by spermine and not by other polyamines. When any of the naturally occurring polyamines was added simultaneously with BE-4-4-4-4, growth inhibition was completely blocked. The data suggest that a threshold intracellular concentration of BE-4-4-4-4 is needed to manifest the growth-inhibitory and cytotoxic effects. In most cell lines, once that threshold level is reached, the growth-inhibitory and cytotoxic properties of the analogue are manifest irrespective of cellular polyamine levels. Further increases in the BE-4-4-4-4 concentration or incubation time reduce the intracellular polyamine levels but do not significantly increase growth inhibition. In U-87 MG and DAOY cells, however, prolonged incubation with higher concentrations of BE-4-4-4-4 causes additional growth inhibition along with depletion of intracellular polyamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain Neoplasms↗

PubMed1993

Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide.

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Ethidium↗

PubMed1993

Effects of ciglitazone on blood pressure and intracellular calcium metabolism.

Ciglitazone is the prototype of the thiazolidinedione class of compounds currently being developed for the treatment of insulin resistance and non-insulin-dependent diabetes. The effects of thiazolidinediones on blood pressure and cell calcium metabolism are not well defined. In the obese Zucker rat, a widely studied model of insulin resistance associated with mild hypertension, we investigated the effects of ciglitazone on plasma insulin levels and mean arterial pressure. We also evaluated the effects of ciglitazone on the changes in cytosolic calcium induced by platelet-derived growth factor in A172 human glioblastoma cells and rat A10 vascular smooth muscle cells. Oral administration of ciglitazone, approximately 45 mg/kg per day for 4 weeks, induced significant reductions in plasma insulin levels (p < 0.001) and blood pressure (p < 0.05). Ciglitazone was also found to significantly attenuate the capacity of platelet-derived growth factor BB homodimer to induce sustained increases in intracellular free calcium. These findings suggest that thiazolidinediones may offer a novel pharmacological approach to the treatment of hypertension, and raise the possibility that these compounds may affect blood pressure not only by affecting insulin metabolism but also by modifying the cell calcium response to pressor agents, growth factors, or both.

Animals↗

PubMed1992

alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.

alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.

Biogenic Polyamines↗

PubMed1992

Effect of polyamine depletion on chromatin structure in U-87 MG human brain tumour cells.

The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I. Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine. BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates. Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests. (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from changes in the lengths of nucleosomal or linker DNA, from blocks in cell-cycle progression, or from growth inhibition caused by DFMO or BE-4-4-4. Thus, because the limit digest is highest in cells with the lowest polyamine levels, it seems clear that the enhanced enzymic digestion of nuclei is caused by polyamine depletion and its possible effect on chromatin structure.

Brain Neoplasms↗

PubMed1991

Effect on N1,N14-bis-(ethyl)-homospermine (BE-4-4-4) on the growth of U-251 MG and SF-188 human brain tumor cells.

We studied the effects of the spermine analogue N1,N14-bis-(ethyl)-homospermine (BE-4-4-4) on growth, survival and polyamine levels in cultured U-251 MG and SF-188 human brain tumor cells. After 48 hr of treatment at concentrations of 1 microM or higher, BE-4-4-4 accumulated in cells with a concomitant decrease in intracellular putrescine, spermidine and spermine concentrations. Growth inhibition by 10 microM BE-4-4-4 began at 6 hr and peaked between 16 and 24 hr. The analogue was also increasingly cytotoxic with doses between 1 and 10 microM and with treatment times between 16 and 48 hr. Polyamines added 1 day after BE-4-4-4 lowered the intracellular concentrations of the analogue but did not reverse its growth-inhibitory activity. When added simultaneously with the analogue, however, polyamines caused a decrease in analogue concentration that was accompanied by a block to the growth inhibition. BE-4-4-4 has a higher affinity for DNA than spermine has, but is less able to aggregate DNA. Its growth-inhibitory and cytotoxic effects support our hypothesis that polyamine analogues that enter cells and replace natural polyamines at DNA binding sites, without fulfilling their biologic functions, should act as antiproliferative agents.

Antineoplastic Agents↗

PubMed1991

1H and 31P nuclear magnetic resonance studies of spermine binding to the Z-DNA form of d(m5CGm5CGm5CG)2. Evidence for decreased spermine mobility.

The binding of spermine to the d(m5CGm5CGm5CG) duplex has been studied by proton and phosphorus nuclear magnetic resonance techniques in order to investigate the mobility and nature of spermine bound to the resulting Z-DNA complex. A characterization of the B to Z transition as a function of increasing spermine concentration demonstrated doubling of the non-exchangeable proton and the phosphorus peaks at a ratio of about 1:1 (spermine/duplex) and a re-simplification of the spectrum at 2:1 (spermine/duplex) where about 90% or the DNA was fully converted into the Z-form. However, some of the Z-DNA proton chemical shifts differed between the 1:1 and 2:1 titration points. Since these differences involved primarily the exchangeable terminal imino and amino protons, they could result from end effects. Discrepancies were generally not observed with the non-terminal proton shifts nor with the phosphorus shifts. These proton and phosphorus chemical shift changes are fully consistent with a B to Z transition. Complexed spermine peaks appear about 0.1 parts per million upfield from the uncomplexed form. The spermine and both the B and Z-DNA hexamer signals are noticeably broadened at the 1:1 ratio but the remaining signals re-sharpen at the 2:1 ratio. Both one-dimensional and two-dimensional studies revealed negative nuclear Overhauser effect (NOE) contacts between each spermine proton. Therefore, spermine has a longer correlation time than that observed for unbounded spermine. These results are contrasted with the positive NOE contacts observed for the B-DNA-spermine complexes reported by Wemmer et al. using the dodecamer d(CGCGAATTCGCG)2 and reported here using the hexamer d(ATGCAT)2. While the mobility of spermine in the Z-DNA complex is significantly less than that of the B-DNA complex, no clear evidence of intermolecular spermine-DNA proton NOE contacts is observed.

Base Sequence↗

PubMed1991

Attachment of A172 human glioblastoma cells affects calcium signalling: a comparison of image cytometry, flow cytometry, and spectrofluorometry.

The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.

Calcium↗

PubMed1991

Implications and concepts of polyamine-nucleic acid interactions.

Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.

Crystallography↗

PubMed1991

Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Animals↗

PubMed1991

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Calcium↗

PubMed1990

Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes.

The effects of spermine and spermine analogues on the B-Z transition of poly(dG-me5dC) and on the aggregation and 'melting' temperature of calf thymus DNA were studied by spectroscopic methods. The association constants of these polyamines with double- and single-stranded calf thymus DNA were calculated from their effects on the melting temperature. The effect of these compounds on the release of ethidium bromide (EB) from an EB-DNA complex were measured by a spectrofluorimetric method. This efficiency of the polyamine-induced B-Z transition strongly depended on the length of the central carbon chains of the compounds and on the functional groups attached to the carbon chains. Both the terminal primary amino groups and the length of the central carbon chain affected the aggregation of DNA. The affinity of the analogues for DNA increased as the number of n-butyl groups increased, but decreased with either an increase or a decrease in the length of the central carbon chain. The effect of spermine and spermine analogues on the release of EB from an EB-DNA complex did not always correlate with the affinities of analogues for calf thymus DNA. In particular, tetra-amines with more than one n-butyl group bound better to DNA than did spermine, but released bound EB and induced aggregation of DNA less well than did spermine. We postulate that either a bend and/or other localized conformational changes of DNA are responsible for the spermine-induced aggregation of DNA and the release of EB from the EB-DNA complex.

Animals↗

PubMed1990

Effect of N1,N14-bis(ethyl)homospermine on the growth of U-87 MG and SF-126 human brain tumor cells.

The effect of the spermine analogue N1,N14-bis(ethyl)homospermine on the growth, polyamine levels, and survival of U-87 MG and SF-126 human brain tumor cells was examined in tissue culture. At concentrations of 10 mumols and above, N1,N14-bis(ethyl)homospermine inhibited growth significantly, caused a marked decrease in intracellular levels of the naturally occurring polyamines putrescine, spermidine, and spermine, and had a considerable cytotoxic effect on both cell lines after more than 96 h of treatment. In earlier studies we showed that the affinity of the analogue for calf thymus DNA was higher than the affinity of spermine, but that it did not aggregate DNA or release bound ethidium bromide from DNA as efficiently as spermine does. Therefore, the growth-inhibitory and cytotoxic effects of N1,N14-bis(ethyl)homospermine support our hypothesis that polyamine analogues that can enter cells, deplete intracellular levels of natural polyamines, and replace the natural polyamines from their binding sites on DNA without replacing function should act as antiproliferative agents.

Brain Neoplasms↗

PubMed1990

Molecular mechanics of the interactions of spermine with DNA: DNA bending as a result of ligand binding.

We used energy minimization of a molecular mechanical force field to evaluate spermine interactions with B-form DNA oligomers with either alternating purine/pyrimidine or homopolymeric sequences. Four different positions for spermine docking--within, along, and bridging the minor groove and bridging the major groove--were assessed for each sequence. Interaction at the major groove of alternating purine/pyrimidine sequences appears to be the most favorable of all models assessed, and are associated with significant bending of DNA. Interactions at the major groove of homopolymers were less favorable than those of heteropolymers and showed little or no bending. Interactions with the minor groove were most favorable for spermine positioned near the base of the groove, and became less favorable as spermine was moved toward the top of the groove. Association along the phosphate backbone alone was the least favorable of the interactions.

Chemical Phenomena↗

PubMed1990

Cytoplasmic microinjection of immunoglobulin Gs recognizing RNA helices inhibits human cell growth.

We report here that nucleolar and cytoplasmic RNA in mammalian cells is recognized specifically by both experimentally induced monoclonal IgG unique for left-handed Z-RNA and by autoimmune mouse monoclonal IgG specific for ribosomal RNA. Nucleolar Z-RNA synthesis, like nucleolar ribosomal RNA synthesis, is inhibited by actinomycin D treatment and dimethylsulfoxide-induced differentiation. Immune anti-Z-RNA IgGs microinjected into living nuclei bind nucleolar RNA, and these complexes appear to be removed from the nucleus within minutes. Cytoplasmically microinjected monoclonal or polyclonal anti-Z-RNA IgGs specifically bind cytoplasmic RNA and inhibit cell multiplication. Microinjection of antibodies directed against double-stranded RNAs. Elevated ionic conditions, which in energy-minimized models can cause the walls of the groove in Z-RNA (but not Z-DNA) to approach each other and close, also prevent antibody binding to specific synthetic or cellular Z-RNA determinants. Our antibodies binding unique Z-RNA structures probably recognize antigens determined by the exposed 2'-OH ribose sugar-phosphate groups.

Animals↗

PubMed1989

Correlation between the effects of polyamine analogues on DNA conformation and cell growth.

Effects of a number of synthetic analogues of the natural polyamines on the B-Z transition of poly(dG-me5dC) and on the aggregation of calf thymus DNA in solution were studied using circular dichroic and UV spectroscopy. The efficiency of induction of the B-Z transition decreased with a decrease in the length of the central alkyl chain of the analogues, and the ability of analogues to aggregate DNA was markedly reduced for compounds ethylated at the terminal amines. Both structural variations appear to have important effects on the biological functions of polyamines. Most analogues studied depleted intracellular levels of natural polyamines, but only those that did not readily induce the B-Z transition and/or aggregate DNA were good inhibitors of cell growth. All but one of the analogues studied were able to rescue cells--at least in part--from the growth-inhibitory effects of alpha-difluoromethylornithine. The single analogue that was unable to effect rescue also failed to induce both the B-Z transition and the aggregation of DNA.

Animals↗