Biomedical subjects

T J Thomas

Publications and source records attributed to T J Thomas.

At least 19 recordsLinked to original sources

PubMed2006

Synergistic apoptosis of MCF-7 breast cancer cells by 2-methoxyestradiol and bis(ethyl)norspermine.

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.

2-Methoxyestradiol↗

PubMed2006

Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17beta-estradiol in human breast cancer cells.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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). RESULTS: 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). CONCLUSIONS: 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.

2-Methoxyestradiol↗

PubMed2006

Bending of the estrogen response element by polyamines and estrogen receptors alpha and beta: a fluorescence resonance energy transfer study.

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.

Allosteric Regulation↗

PubMed2005

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability.

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.

Base Sequence↗

PubMed2005

Differential effects of 16alpha-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells.

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.

2-Methoxyestradiol↗

PubMed2005

Conformational dynamics of estrogen receptors alpha and beta as revealed by intrinsic tryptophan fluorescence and circular dichroism.

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.

Acrylamide↗

PubMed2005

Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2.

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Activating Transcription Factor 2↗

PubMed2005

Bis(ethyl)norspermine potentiates the apoptotic activity of the pure antiestrogen ICI 182780 in breast cancer cells.

We studied the effects of ICI 182780 and bis(ethyl)norspermine (BE-3-3-3) on cell growth and apoptosis of estrogen receptor-positive MCF-7 breast cancer cells. Combination treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 for 6 days inhibited cell growth by 74.3+/-8.4% in MCF-7 cells, compared to that of 25.4+/-5.8 and 45.8+/-12.2%, respectively, when ICI 182780 and BE-3-3-3 were used as single agents. Treatment with 100 nM ICI 182780 and 5 microM BE-3-3-3 as single agents resulted in 9.1+/-1.0% and 35.1+/-4.5% apoptosis, respectively, as measured by APO-BRDU assay. When ICI 182780 and BE-3-3-3 were used in combination, the percentage of apoptosis was 60.6+/-3.8%. Improved efficacy of ICI 182780 and BE-3-3-3 combination on growth inhibition was observed for T-47D cells also. Western blot analysis showed that combinations of ICI 182780 and BE-3-3-3 caused down-regulation of the anti-apoptotic Bcl-2 and Bcl-XL proteins and increased the level of the pro-apoptotic Bax protein. Combination treatment also increased caspase-8 activation. Analysis of polyamine levels 48 h after combination treatment showed that spermidine and spermine levels were down regulated significantly. These studies indicate a potentially effective combination strategy for breast cancer treatment. Our results also link the down-regulation of polyamine pathway to apoptotic cell death and regulation of mediators of cell death.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2004

Enhanced cellular uptake of a triplex-forming oligonucleotide by nanoparticle formation in the presence of polypropylenimine dendrimers.

We used polypropylenimine dendrimers for delivering a 31 nt triplex-forming oligonucleotide (ODN) in breast, prostate and ovarian cancer cell lines, using 32P-labeled ODN. Dendrimers enhanced the uptake of ODN by approximately 14-fold in MDA-MB-231 breast cancer cells, compared with control ODN uptake. Dendrimers exerted their effect in a concentration- and molecular weight-dependent manner, with generation 4 (G-4) dendrimer having maximum efficacy. A similar increase in ODN uptake was found with MCF-7 and SK-BR-3 (breast), LNCaP (prostate) and SK-OV-3 (ovarian) cancer cells. The dendrimers had no significant effect on cell viability at concentrations at which maximum ODN uptake occurred. [3H]Thymidine incorporation showed that complexing the ODN with G-4 significantly increased the growth-inhibitory effect of the ODN. Western blot analysis showed a significant 65% reduction of c-myc protein level in ODN-G-4 treated cells compared with that of ODN-treated/control cells. Gel electrophoretic analysis showed that ODN remained intact in cells even after 48 h of treatment. The hydrodynamic radii of nanoparticles formed from ODN in the presence of the dendrimers were in the range of 130-280 nm, as determined by dynamic laser light scattering. Taken together, our results indicate that polypropylenimine dendrimers might be useful vehicles for delivering therapeutic oligonucleotides in cancer cells.

Cell Line, Tumor↗

PubMed2004

Formation of DNA nanoparticles in the presence of novel polyamine analogues: a laser light scattering and atomic force microscopic study.

We synthesized a pentamine (3-3-3-3) and two hexamine (3-3-3-3-3 and 3-4-3-4-3) analogues of the natural polyamine, spermine (3-4-3) and studied their effectiveness in condensing pGL3 plasmid DNA, using light scattering and atomic force microscopic (AFM) techniques. The midpoint concentration of the polyamines on pGL3 condensation (EC50) was 11.3, 10.6, 1.5, 0.49 and 0.52 micro M, respectively, for 3-4-3, norspermine (3-3-3), 3-3-3-3, 3-3-3-3-3 and 3-4-3-4-3 in 10 mM Na cacodylate buffer. Dynamic laser light scattering study showed a decrease in hydrodynamic radii of plasmid DNA particles as the number of positive charges on the polyamines increased. AFM data showed the presence of toroids with outer diameter of 117-191 nm for different polyamines, and a mean height of 2.61 +/- 0.77 nm. AFM results also revealed the presence of intermediate structures, including those showing circumferential winding of DNA to toroids. The dependence of the EC50 on Na+ concentration suggests different modes of binding of spermine and its higher valent analogues with DNA. Our results show a 20-fold increase in the efficacy of hexamines for DNA condensation compared to spermine, and provide new insights into the mechanism(s) of DNA nanoparticle formation. These studies might help to develop novel nonviral gene delivery vehicles.

Lasers↗

PubMed2004

Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases.

Estrogen receptors (ERs) are proteins that mediate the action of estradiol and a series of natural and synthetic chemicals that mimic the estradiol structure. Estrogenic action was initially attributed to a single type of ER, now known as ERalpha, but ERbeta was discovered in 1995. Tissue specific distribution and the intensity of expression of these proteins determine the first response of tissues to estrogenic compounds. Estrogens and ERs play a major role in the origin and progression of breast cancer, and antiestrogens that block ER function are useful for breast cancer prevention and treatment. Estrogen mimetics, however, do not fall into distinct categories of agonists and antagonists, since their action is regulated by tissue-specific expression of a number of auxiliary proteins called coactivators or corepressors. In addition, small molecules such as polyamines, fattyacids, and thioredoxin may modulate ER function. Estrogenic functions encompass multiple organ systems, including the reproductive, skeletal, cardiovascular, and nervous system. Estrogens are critical for bone remodeling and mineralization so that estrogen replacement therapy is proven to strengthen bone health in post-menopausal women. Ideally, selective blockade of ER function in breast epithelial cells should be accompanied by growth support on bone and cardiovascular systems. The details of estrogenic function in different organs are to be fully realized, in order to better utilize selective estrogen receptor modulators (SERMs) to fight not only breast cancer but also osteoporosis and cardiovascular diseases. Current research on SERMs points toward accomplishing this goal by exploiting ER as a versatile target against multiple diseases.

Animals↗

PubMed2002

DNA nanoparticles and development of DNA delivery vehicles for gene therapy.

DNA transport through the cell membrane is an essential requirement for gene therapy, which utilizes oligonucleotides and plasmid DNA. However, membrane transport of DNA is an inefficient process, and the mechanism(s) by which this process occurs is not clear. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major problem. Therefore, several laboratories are involved in the development of nonviral DNA delivery vehicles. These vehicles include polyamines, polycationic lipids, and neutral polymers, capable of condensing DNA to nanoparticles with radii of 20-100 nm. Although the structural and energetic forces involved in DNA condensation have been studied by physical biochemists for the past 25 years, this area has experienced a resurgence of interest in recent years because of the influx of biotechnologists involved in developing gene therapy protocols to combat a variety of human diseases. Despite an intense effort to study the mechanism(s) of DNA condensation using a variety of microscopic, light scattering, fluorescence, and calorimetric techniques, the precise details of the energetics of DNA nanoparticle formation and their packing assembly are not known at present. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.

Animals↗

PubMed2002

Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA.

The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines.

Animals↗

PubMed2002

Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics.

DNA undergoes condensation, conformational transitions, aggregation and resolubilization in the presence of polyamines, positively charged organic molecules present in all cells. Under carefully controlled environmental conditions, DNA can also transform to a liquid crystalline state in vitro. We undertook the present work to examine the ability of spermidine, N4-methylspermidine, spermine, N1-acetylspermine and a group of tetramine, pentamine and hexamine analogs of spermine to induce and stabilize liquid crystalline DNA. Liquid crystalline textures were identified under a polarizing microscope. In the absence of polyamines, calf thymus DNA assumed a diffused, planar cholesteric phase with entrapped bubbles when incubated on a glass slide at 37 degrees C. In the presence of spermidine and spermine, the characteristic fingerprint textures of the cholesteric phase, adopting a hexagonal order, were obtained. The helical pitch was 2.5 micro m. The final structures were dendrimeric and crystalline when DNA was treated with spermine homologs and bis(ethyl) derivatives. A cholesteric structure was observed when DNA was treated with a hexamine at 37 degrees C. This structure changed to a hexagonal dendrimer with fluidity on prolonged incubation. These data show a structural specificity effect of polyamines on liquid crystalline phase transitions of DNA and suggest a possible physiological function of natural polyamines.

Animals↗

PubMed2002

Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon.

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.

Cesium↗

PubMed2002

Development of polyamine analogs as cancer therapeutic agents.

Natural polyamines (putrescine, spermidine, and spermine) are aliphatic cations with multiple functions in cell growth and differentiation. Alterations in the polyamine structure provide a strategy to synthesize analogs that can interfere with the cellular functions of natural polyamines. Analogs of spermine are particularly effective in modifying the synthesis, catabolism, and uptake of natural polyamines. The increased requirement of natural polyamines in cancer cell growth makes it possible to utilize the polyamine pathway as a therapeutic target in cancer cells. Because polyamine functions extend from membrane phospholipid structure and signal transduction to DNA structure and conformational transitions, it is likely that the action of polyamine analogs also permeates to these sites of polyamine action. For the same reason, toxicity of polyamine analogs might be considerable. However, it is possible to design polyamine analogs that target a specific function of polyamines in cancer cells, thereby enhancing selectivity for inducing apoptosis of cancer cells. Alternatively, polyamine analogs may potentiate the action of other anticancer agents and become an effective tool in cancer chemotherapy. In either case, further research into the action of polyamine analogs will open up new opportunities in the fight against cancer.

Animals↗

PubMed2001

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.

Bacteriophage lambda↗

PubMed2001

A molecular beacon strategy for the thermodynamic characterization of triplex DNA: triplex formation at the promoter region of cyclin D1.

We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer, upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formation by these oligonucleotides. In low salt buffer (10 mM Na(+)), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22 degrees C), the equilibrium association constant (K(a)) calculated in the presence of 1 microM spermine and 10 mM Na(+) was 3.2 x 10(8) M(-1). The K(a) value was 1.0 x 10(9) M(-1) in the presence of 150 mM Na(+), and it increased by 10-fold by the addition of 1 mM spermine. Delta H, Delta S, and Delta G of triplex DNA formation, calculated from the temperature dependence of K(a) in the range of 20--45 degrees C, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, respectively, in the presence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol.K), and -13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyamines exerted different degrees of triplex DNA stabilization, as determined by binding constant measurements. Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant, whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67 degrees C. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the Delta H, Delta S, and Delta G values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.

Cyclin D1↗