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ABSTRACT: The structure and conformation of nucleic acids are influenced by metal ions, polyamines, and the microenvironment. In poly(purine) · poly(pyrimidine) sequences, triplex DNA formation is facilitated by metal ions, polyamines and other ligands. We studied the effects of mono- and di-valent metal ions, and ammonium salts on the stability of triple- and double-stranded structures formed from poly(dA) and poly(dT) by measuring their respective melting temperatures. In the presence of metal ions, the absorbance versus temperature profile showed two transitions: Tm1 for triplex to duplex and single stranded DNA, and Tm2 for duplex DNA melting to single stranded DNA. Monovalent cations (Li+, Na+, K+, Rb+, Cs+ and [Formula: see text] ) promoted triplex DNA at concentrations ≥150 mM. Tm1 varied from 49.8 °C in the presence of 150 mM Li+ to 30.6 °C in the presence of 150 mM K+. [Formula: see text] was very effective in stabilizing triplex DNA and its efficacy decreased with increasing substitution of the hydrogen atoms with methyl, ethyl, propyl and butyl groups. As in the case of monovalent cations, a concentration-dependent increase in Tm1 was observed with divalent ions and triplex DNA stabilization decreased in the order: Mg2+ > Ca2+ > Sr2+ > Ba2+. All positively charged cations increased the melting temperature of duplex DNA. Values of Δn (number of ions released) on triplex DNA melting were 0.46 ± 0.06 and 0.18 ± 0.02, respectively, for mono- and di-valent cations, as calculated from 1/Tm1 versus ln[M+,2+] plots. The corresponding values for duplex DNA were 0.25 ± 0.02 and 0.12 ± 0.02, respectively, for mono- and di-valent cations. Circular dichroism spectroscopic studies showed distinct conformational changes in triplex DNA stabilized by alkali metal and ammonium ions. Our results might be useful in developing triplex forming oligonucleotide based gene silencing techniques.
Biochimie 02/2013; · 3.02 Impact Factor
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ABSTRACT: Viral and nonviral condensing agents are used in gene therapy to compact oligonucleotides and plasmid DNA into nanostructures
for their efficient transport through the cell membranes. Whereas viral vectors are best by the toxic effects on the immune
system, most of the nonviral delivery vehicles are not effective for use in clinical system. Recent investigations indicate
that the supramolecular organization of DNA in the condensed state is liquid crystalline. The present level of understanding
of the liquid crystalline phase of DNA is inadequate and a thorough investigation is required to understand the nature, stability,
texture and the influence of various environmental conditions on the structure of the phase. The present study is mainly concerned
with the physicochemical investigations on the liquid crystalline transitions during compaction of DNA by cationic species
such as polyamines and metallic cations. As a preliminary to the above investigation, studies were conducted on the evolution
of mesophase transitions of DNA with various cationic counterion species using polarized light microscopy. These studies indicated
significant variations in the phase behaviour of DNA in the presence of Li and other ions. Apart from the neutralization of
the charges on the DNA molecule, these ions are found to influence selectively the hydration sphere of DNA that in turn influences
the induction and stabilization of the LC phases. The higher stability observed with the liquid crystalline phases of Li-DNA
system could be useful in the production of nanostructured DNA. In the case of the polyamine, a structural specificity effect
depending on the nature, charge and structure of the polyamine used has been found to be favoured in the crystallization of
DNA.
Pramana 04/2012; 65(4):723-729. · 0.57 Impact Factor
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Irina Nayvelt,
Shali John,
Hui-Chen Hsu,
Pingar Yang,
Wensheng Liu,
Gokul Das,
Mervi T Hyvönen,
Leena Alhonen,
Tuomo A Keinänen,
Akira Shirahata,
Rajesh Patel,
Thresia Thomas, T J Thomas
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ABSTRACT: BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 μM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.
Amino Acids 08/2011; 42(2-3):899-911. · 3.25 Impact Factor
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ABSTRACT: A novel approach was developed to efficiently package and deliver nucleic acids with low generation polypropylenimine (PPI) dendrimers by using Au nanoparticles as a "labile catalytic" packaging agent. The gold nanoparticles (Au NPs) helped low generation dendrimers to package nucleic acids into discrete nanoparticles but are not included in the final DNA/siRNA complexes. Therefore it becomes possible to eliminate the potential toxic problems associated with Au NPs by selectively removing the Au NPs from the resulting nucleic acid complexes before their delivery to targeted cells. This is a new concept in using inorganic engineered nanoparticles in nucleic acid packaging and delivery applications. Furthermore, compared to the siRNA nanostructures (mainly randomly aggregated nanofibers) fabricated by low generation dendrimer alone (Generation 3), the siRNA nanoparticles packaged using this novel approach (by Au NPs modified with G3 PPI) can be internalized by cancer cells and the delivered siRNAs can efficiently silence their target mRNA. The efficiency of mRNA silencing by this novel approach is even superior to higher generation dendrimers (Generation 5).
ACS Nano 07/2010; 4(7):3679-88. · 10.77 Impact Factor
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ABSTRACT: Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of α,α′-dimethylated spermine [(R,R)-Me2Spm, (S,S)-Me2Spm, (R,S)-Me2Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me2Spm displayed more efficient condensing ability than spermine, with significantly lower EC50 (concentration for 50% compaction) values (p ≤ 0.01). However, spermine exerted slightly more duplex stabilization than Me2Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me2Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV−C irradiation. Our findings indicate that Me2Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.
11/2009;
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ABSTRACT: Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.
Biomacromolecules 11/2009; 11(1):97-105. · 5.48 Impact Factor
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ABSTRACT: Beclin 1 is an essential mediator of autophagy and a regulator of cell growth and cell death. We examined the effect of Beclin 1 overexpression on the action of estradiol (E(2)) and two antiestrogens, raloxifene and 4-hydroxytamoxifen, in estrogen receptor alpha (ERalpha)-positive MCF-7 breast cancer cells. [(3)H]-thymidine incorporation studies showed that Beclin 1-overexpressing cells (MCF-7 x beclin) had a lower proliferative response to E(2) compared with cells transfected with vector control (MCF-7 x control). There was only a 35% increase in [(3)H]-thymidine incorporation, after 24 hours of E(2) treatment of MCF-7 x beclin cells compared with untreated cells, whereas this increase was 2-fold for MCF-7 x control cells. E(2)-induced changes in the expression of early-response genes were examined by real-time quantitiative PCR. There were significant differences in the pattern of expression of E(2)-induced genes c-myc, c-fos, Erg-1, and Nur77 between MCF-7 x beclin and MCF-7 x control cells two hours after treatment. Although E(2)-induced growth of MCF-7 x control cells was completely inhibited by 500 nmol/L raloxifene or 500 nmol/L 4-hydroxytamoxifen, these concentrations of antiestrogens had no significant effect on the growth of MCF-7 x beclin cells. Confocal microscopic and coimmunoprecipitation studies showed evidence for colocalization and association of Beclin 1 and ERalpha. In addition, E(2) caused a decrease in Akt phosphorylation in MCF-7 x beclin cells, compared with a 3-fold increase in MCF-7 cells, five minutes after treatment. These results indicate that Beclin 1 can down-regulate estrogenic signaling and growth response, and contribute to the development of antiestrogen resistance. This observation might be useful to define and overcome antiestrogen resistance of breast cancer.
Cancer Research 11/2008; 68(19):7855-63. · 7.86 Impact Factor
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ABSTRACT: The ability of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cu(2+), Cd(2+), Al(3+), V(4+), Hg(2+), Pd(2+), Au(3+), and Pt(4+) to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li(+), Na(+), K(+), Mg(2+), and Ca(2+) binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.
Biomacromolecules 08/2008; 9(7):1860-9. · 5.48 Impact Factor
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ABSTRACT: Abstract. We developed a novel and convenient method for the synthesis of a potentially safe non-viral gene delivery vehicle
based on the cationic block copolymer of spermine and aspartic acid (ASSP) and coupled it with polyethylene glycol
(PEG). The copolymer ASSP was prepared by direct polycondensation in the ionic liquid, butylmethylimidazolium hexafluorophosphate,
using triphenyl phosphite as the condensing agent under mild reaction conditions. The highly hydrophobic
ASSP was transformed into a water soluble hydrophilic micelle by coupling ASSP with polyethylene glycol (PEG)
using the same ionic liquid and 1,1-carbonyl diimidazole as the condensing agent without harsh conditions. The polycationic
ASSP-PEG was then used to condense calf thymus and plasmid deoxyribonuclceic acids (DNAs) in Tris-HCl buffer
(pH 7.4) to get a series of block ionomer complexes with various charge ratios. The physicochemical properties of the
copolymer micelle and the DNA polyplexes were studied using fourier transform-infrared (FTIR), nuclear magnetic resonance
(NMR) and circular dichroism (CD) spectroscopy, matrix assisted laser desorption/ionization-time of flight mass
spectrometry (MALDI-TOF MS), differential scanning calorimetry (DSC), transmission electron microscopy (TEM) and
particle size measurements. It was observed that the DNA was condensed to compact particles by its interaction with the
copolymer. Since DNA condensation to nano/micrometer sized particles is essential for gene delivery, our results indicate
a potential use of the copolymer for gene delivery applications.
eXPRESS Polymer Letters 01/2008; 2:330. · 1.77 Impact Factor
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ABSTRACT: 2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.
Cancer Letters 07/2007; 250(2):311-22. · 4.24 Impact Factor
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ABSTRACT: Estrogen receptors (ERalpha and ERbeta) are ligand-activated transcription factors. We examined the effects of estradiol (E2), 4-hydroxytamoxifen (HT), and the estrogen response element (ERE) on the helical content and thermal unfolding of ERbeta. A circular dichroism (CD) spectrum of ERbeta showed changes at 210 and 222 nm that were due to the presence of E2, which is indicative of partial unfolding. In contrast, HT did not alter the CD spectrum of ERbeta. The addition of E2 + ERE caused an increase in the alpha-helical content and an increase in the temperature midpoint of folding transition (TM) from 39 +/- 0.7 degrees C to 57.2 +/- 1 degrees C. The addition of E2 + mutant ERE, or E2 + control oligonucleotide, increased the TM of ERbeta to 45 +/- 2 degrees C only. In the presence of HT, ERbeta yielded similar TM values (55-58 degrees C) with ERE, mutant ERE, or control oligodeoxynucleotide. The binding affinity of ERbeta for ERE increased 125.7-fold as a result of the presence of E2, but only 4-fold as a result of HT. These results demonstrate coupled effects of E2 and ERE on ERbeta stability and binding affinity. The increased thermal stability of HT-ERbeta-ERE was associated with reduced specificity of ERbeta-ERE recognition, illustrating profound differences in conformational states of ERbeta induced by E2 and HT.
Biochemistry and Cell Biology 03/2007; 85(1):1-10. · 2.67 Impact Factor
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ABSTRACT: We studied the effectiveness of trilysine (Lys3), tetralysine (Lys4), pentalysine (Lys5), and poly-l-lysine (PLL) (MW 50000) on lambda-DNA nanoparticle formation and characterized the size, shape, and stability of nanoparticles. Light scattering experiments showed EC50 (lysine concentration at 50% DNA compaction) values of approximately 0.0036, 2, and 20 micromol/L, respectively, for PLL, Lys5, and Lys4 at 10 mM [Na+]. Plots of log EC50 versus log [Na+] showed positive slopes of 1.09 and 1.7, respectively, for Lys4 and Lys5 and a negative slope of -0.1 for PLL. Hydrodynamic radii of oligolysine condensed particles increased (48-173 nm) with increasing [Na+], whereas no significant change occurred to nanoparticles formed with PLL. There was an increase in the size of nanoparticles formed with Lys5 at >40 degrees C, whereas no such change occurred with PLL. The DNA melting temperature increased with oligolysine concentration. These results indicate distinct differences in the mechanism(s) by which oligolysines and PLL provoke DNA condensation to nanoparticles.
Biomacromolecules 03/2007; 8(2):477-84. · 5.48 Impact Factor
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ABSTRACT: Estrogen receptors (ERα and ERβ) are ligand-activated transcription factors. We examined the effects of estradiol (E2), 4-hydroxytamoxifen (HT), and the estrogen response element (ERE) on the helical content and thermal unfolding of ERβ. A circular dichroism (CD) spectrum of ERβ showed changes at 210 and 222nm that were due to the presence of E2, which is indicative of partial unfolding. In contrast, HT did not alter the CD spectrum of ERβ. The addition of E2+ ERE caused an increase in the α-helical content and an increase in the temperature midpoint of folding transition (TM) from 39± 0.7°C to 57.2± 1°C. The addition of E2+ mutant ERE, or E2+ control oligonucleotide, increased the TM of ERβ to 45± 2°C only. In the presence of HT, ERβ yielded similar TM values (55-58°C) with ERE, mutant ERE, or control oligodeoxynucleotide. The binding affinity of ERβ for ERE increased 125.7-fold as a result of the presence of E2, but only 4-fold as a result of HT. These results demonstrate coupled effects of E2 and ERE on ERβ stability and binding affinity. The increased thermal stability of HT-ERβ-ERE was associated with reduced specificity of ERβ-ERE recognition, illustrating profound differences in conformational states of ERβ induced by E2 and HT.Les récepteurs d'estrogène (REα et β) sont des facteurs de transcription activés par un ligand. Nous avons examiné les effets de l'estradiol (E2), du 4-hydroxytamoxifen (HT) et d'un élément de réponse aux estrogènes (ERE) sur le contenu en hélices et le dépliement thermique du REβ. Le spectre par dichroïsme circulaire du REβ a mis en évidence des changements à 210 et 222 nm en présence de E2, indiquant un dépliement partiel. Au contraire, le HT n'a pas modifié le spectre en DC du REβ. L'ajout de E2 en présence d'un ERE a causé une augmentation du contenu en hélices-α et une augmentation de la température de dénaturation (TM) de 39 ± 0.7 °C à 57.2 ± 1 °C. L'ajout de E2 en présence d'un ERE mutant ou de E2 en présence d'un oligonucléotide (ODN) contrôle a augmenté la TM du REβ à 45 ± 2 °C seulement. En présence de HT, les TM du REβ en présence d'un ERE, d'un ERE mutant ou d'un ODN contrôle étaient similaires (55-58°C). L'affinité de liaison du REβ pour l'ERE a augmenté de 125.7 fois en présence de E2, mais de 4 fois seulement en présence de HT. Ces résultats démontrent des effets couplés du E2 et d'un ERE sur la stabilité et l'affinité de liaison du REβ. L'augmentation de la stabilité thermique du complexe HT-REβ-RE a été associée à une diminution de la spécificité de la reconnaissance REβ-ERE, illustrant les différences profondes entre les états de conformation du REβ induits par le E2 ou le HT.
Biochemistry and Cell Biology 01/2007; 85(1):1-10. · 2.67 Impact Factor
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ABSTRACT: We studied the efficacy of five generations of polypropyleneimine (PPI) dendrimer to provoke nanostructure formation from a 21-nucleotide antisense oligodeoxynucleotide (ODN). Nanostructure formation was observed with all generations of dendrimer by light scattering and microscopic techniques. The efficacy of the dendrimers increased with generation number. Atomic force microscopy (AFM) was used to study the morphology of the structures at different condensation stages. Based on the observed nanostructures, we propose a zipping condensation mechanism, which is very different from the condensation pathways of high molecular weight DNA polymers. Electron microscopy showed the presence of toroidal nanoparticles. Confocal microscopic analysis showed that the nanostructures formed with G-4 and G-5 dendrimers could undergo facile cellular uptake in a breast cancer cell line, MDA-MB-231, whereas nanostructures formed with G-1 to G-3 dendrimers lacked this ability. Nanoparticles formed with G-1 to G-3 dendrimers showed significantly lower zeta potential (5.2–6.5 mV) than those (12–18 mV) of particles formed with G-4 and G-5 dendrimers. These results show that the structure and charge density of the dendrimers are important in ODN nanoparticle formation and cellular transport and that G-4 and G-5 dendrimers are useful in cellular delivery of antisense ODN.
Nanotechnology 10/2006; 17(21):5449. · 3.98 Impact Factor
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ABSTRACT: The purpose of this investigation is to determine the effects of physiologic levels (10-50 nmol/L) of 2-methoxyestradiol (2ME) on the growth of estrogen receptor (ER)-positive breast cancer cells and provide insights into its mechanism(s) of action.
Using the ERalpha-positive breast cancer cells, we studied the effects of 2ME on cell proliferation and cell signaling. Our hypothesis is that 17beta-estradiol (E(2)) and 2ME can affect shared cell signaling pathways, leading to different outcomes in cell proliferation, depending on the absence/presence of E(2).
E(2) stimulated the growth of MCF-7 and T-47 D cells and induced Akt phosphorylation, a nongenomic signaling pathway. In the absence of E(2), 10 to 50 nmol/L of 2ME enhanced cell growth and Akt phosphorylation. However, in the presence of E(2), 2ME inhibited E(2)-induced cell growth and prevented E(2)-induced Akt phosphorylation. Confocal microscopic studies showed that 2ME inhibited subcellular distribution of ERalpha in response to E(2) in MCF-7 and T-47D cells. 2ME also down-regulated E(2)-induced increases in cyclic AMP and ornithine decarboxylase activity. In addition, treatment of MCF-7 cells with 2ME in the presence of E(2) resulted in a decrease in ERalpha level by 72 hours. Accelerated down-regulation of ERalpha may contribute to growth inhibition in the presence of E(2)/2ME combinations. In contrast, a concentration of up to 2.5 mumol/L 2ME had no effect on the growth of ER-negative SK-BR-3 cells, either in the presence or absence of E(2).
Our results provide evidence for the nongenomic action of 2ME in ER-positive cells. In the presence of E(2), 2ME suppressed E(2)-induced cell growth, Akt signaling, and generation of cyclic AMP, whereas it acted as an estrogen in the absence of E(2). The intriguing growth-stimulatory and growth-inhibitory effects of 2ME on breast cancer cells suggests the need for its selective use in patients.
Clinical Cancer Research 05/2006; 12(7 Pt 1):2038-48. · 7.74 Impact Factor
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ABSTRACT: Estrogenic regulation of gene expression is mediated by the binding of the hormone to its receptors (ERalpha and ERbeta) followed by their binding to estrogen response element (ERE). Previous studies showed that natural polyamines -- putrescine, spermidine, and spermine -- facilitated ERalpha.ERE recognition. We determined the effects of natural and synthetic polyamines on the bending of a 27-mer oligonucleotide (ODN) harboring the ERE (ERE-ODN), using fluorescence resonance energy transfer (FRET) technique. Complementary strands of the ERE-ODN were labeled with fluorescein and tetramethylrhodamine, as donor and acceptor, respectively. The ERE-ODN was intrinsically bent with an end-to-end distance of 76 +/- 2 Angstrom, compared to a theoretical value of 98 Angstrom. The end-to-end distance of the ERE-ODN was reduced to 64 Angstrom in the presence of 250 microM spermine. A control ODN with scrambled sequence did not show intrinsic bending or spermine-induced bending. Alkyl substitution at the pendant amino groups reduced the ability of spermine to bend the ERE-ODN. Both ERalpha and ERbeta decreased the end-to-end distance of the ERE-ODN, although ERalpha was more efficient than ERbeta in inducing ERE bending. Spermine-induced bending of the ERE-ODN was significantly increased by ERalpha. Fluorescence anisotropy measurement showed that the equilibrium association constant of ERalpha-ERE binding increased by 12-fold in the presence of 250 microM spermine compared to control. The free energy change (Delta G) of ERalpha.ERE complex formation was -13.1 kcal/mol at 22 degrees C in the presence of spermine. Our results suggest that polyamine-induced bending of the ERE might be a mechanism for enhancing ERalpha-ERE binding affinity and thereby fine-tuning the transcriptional response of estrogen-responsive genes.
The International Journal of Biochemistry & Cell Biology 02/2006; 38(7):1191-1206. · 4.63 Impact Factor
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ABSTRACT: Polyamines are positively charged under physiological ionic and pH conditions. Therefore, the negatively charged macromolecules
in the cell, DNA, RNA, and certain proteins are natural targets for their interaction. Polyamine interaction with DNA was
considered to be electrostatic in nature and was theoretically interpreted in terms of the counterion condensation theory.
Early studies suggested stabilization of duplex DNA by natural and synthetic polyamines, independent of the chemical structure
of the polyamine or the sequence of the DNA. More recent studies have revealed polyamine structural specificity, as well as
DNA sequence specificity, in addition to the overriding electrostatic interaction. Studies using photoaffinity probes indicate
preferential binding of polyamines to bent adenine tracts and TATA elements, suggesting their involvement in the regulation
of gene expression. Molecular modeling and experimental studies also indicate sequencespecific binding to GC-rich major grooves.
DNA sequence-specific binding of polyamines might also be important in polyamine-induced facilitation of DNA-protein interactions,
observed with several transcription factors. The unique contact sites in the interactions between DNA and polyamines are attested
by polyamine structural specificity evident in DNA conformational transitions, DNA nanoparticle formation, triplex DNA stabilization,
and DNA·RNA hybrid stability. Both polyamine structural specificity and DNA sequence specificity may find applications in
polyamine-based drug design, bionanotechnology, and in understanding the mechanism of gene regulation.
12/2005: pages 91-122;
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ABSTRACT: Estrogen receptors (ER alpha and ER beta) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ER alpha and ER beta, we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (T(M)) of ER alpha and ER beta were 39+/-1 and 40+/-2 degrees C, respectively. Estradiol had no significant effect on the T(M) of ER alpha or ER beta. In contrast, binding of the estrogen-response element increased the T(M) of ER alpha and ER beta by 10 degrees C. Thermal unfolding of estradiol-bound ER alpha and ligand-free ER beta showed two-step transitions, with the formation of intermediates that were stable between 36-48 and 34-42 degrees C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ER beta intermediate consisted of discrete globular particles, whereas the ER alpha intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ER alpha and ER beta. Binding of estradiol to ER beta exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ER alpha. Our results illustrate the differential effects of ligands on the unfolding of ER alpha and ER beta, and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ER alpha and ER beta may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.
Journal of Molecular Endocrinology 11/2005; 35(2):211-23. · 3.48 Impact Factor
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04/2005: pages 251 - 287; , ISBN: 9783527603473
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ABSTRACT: We studied the effects of 2-methoxyestradiol (2-ME2) and 16alpha-hydroxyestrone (16alpha-OHE1), two metabolites of estradiol (E2), on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E2 and 16alpha-OHE1 stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E2 and 16alpha-OHE1 stimulated the progression of cells from G1 to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E2 and 16alpha-OHE1 increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16alpha-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E2 or 16alpha-OHE1 showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the cAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E2 and 16alpha-OHE1 respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.
Journal of Molecular Endocrinology 03/2005; 34(1):91-105. · 3.48 Impact Factor
