T. J. Thomas

Biogenic and Synthetic Polyamines Bind Bovine Serum Albumin

Biogenic polyamines are found to modulate protein synthesis at different levels, while polyamine analogues have shown major antitumor activity in multiple experimental models, including breast cancer. The aim of this study was to examine the interaction of bovine serum albumin (BSA) with biogenic polyamines, spermine and spermidine, and polyamine analogues 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333) and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333) in aqueous solution at physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind BSA via both hydrophilic and hydrophobic interactions. Stronger polyamine-protein complexes formed with biogenic than synthetic polyamines with overall binding constants of K(spm) = 3.56 (+/-0.5) x 10(5) M(-1), K(spmd) = 1.77 (+/-0.4) x 10(5) M(-1), K(BE-333) = 1.11 (+/-0.3) x 10(4) M(-1) and K(BE-3333) = 3.90 (+/-0.7) x 10(4) M(-1) that correlate with their positively charged amino group contents. Major alterations of protein conformation were observed with reduction of alpha-helix from 63% (free protein) to 55-33% and increase of turn 12% (free protein) to 28-16% and random coil from 6% (free protein) to 24-17% in the polyamine-BSA complexes, indicating a partial protein unfolding. These data suggest that serum albumins might act as polyamine carrier proteins in delivering polyamine analogues to target tissues.

Regulation of Estrogenic Effects by Beclin 1 in Breast Cancer Cells

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.

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.

Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1.

We examined the effects of epidermal growth factor (EGF) on MDA‐MB‐468 cells to understand its mechanism of action in an EGF receptor‐rich breast cancer cell line. EGF inhibited the growth of MDA‐MB‐468 cells with an IC50 of 1.5 ± 0.5 nM, as determined by measurements of DNA content of cells in culture over a period of 4 to 6 days. This growth inhibition included apoptosis 24 h after EGF addition, as detected by an enzyme‐linked immunosorbent assay (ELISA) and Hoechst 33342 staining. In EGF‐treated cells, peak activities of two key enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S‐adenosylmethionine decarboxylase (SAMDC), were reduced by 57% and 83%, respectively. EGF treatment also caused a 30 to 50% decrease in cellular putrescine at all time points tested (12 to 48 h). EGF‐induced inhibition of DNA synthesis was also partially reversed by the addition of putrescine or spermidine, but not by spermine. Western blot analysis of cell cycle regulatory proteins showed that EGF‐mediated growth inhibition was associated with the induction of p21, an inhibitor of cyclin‐dependent kinases. However, EGF had no significant effect on the expression of cyclin D1 or cyclin E. Furthermore, putrescine reversal of EGF effects was associated with the down‐regulation of EGF‐induced p21. These results suggest that the mechanism of growth inhibition by EGF in MDA‐MB‐468 cells include a down‐regulation of polyamine biosynthesis and the induction of p21. Identification of growth regulatory pathways in breast cancer cells might be useful in the development of novel targets for therapeutic intervention. J. Cell. Physiol. 179:257–266, 1999.

Regulation of Estrogen Receptor mRNA by 2,3,7,8-Tetrachlorodibenzo-p-dioxin as Measured by Competitive RT-PCR

Environmental contaminants, such as 2,3,7,8‐tetrachlorodibenzo‐p ‐dioxin (TCDD), cause alterations in gene expression. In this study, we measured the regulation of estrogen receptor (ER) mRNA in female CD‐1 mice by competitive RT‐PCR. Previous work suggests that ER protein levels are affected by TCDD, but how this is regulated is uncertain. These studies found no significant changes in ER mRNA levels, but the methods used (Northern blot analysis and RNase protection assays) lack sensitivity for measuring the low levels of RNA transcript, such as ER mRNA. The method described here offers an excellent alternative for quantifying the changes in mRNA levels. Internal competitors were created with gene‐specific primers for ER and β‐actin by PCR reactions at low annealing temperatures. For each sample, the mRNA levels of ER and β‐actin were determined. Using competitive RT‐PCR, the relative changes in ER mRNA from TCDD‐treated and control animals were determined after normalization with the levels of β‐actin mRNA. The ER mRNA from female CD‐1 mice treated with TCDD (single dose 5 μg/kg, ip, 4 days) was found to be significantly suppressed as compared with the vehicle control in all tissues examined. TCDD decreased ER mRNA in the liver (30.1%) as expected. However, the greatest effect was in the reproductive tissues, with a 64.2% reduction in ER mRNA in the ovary. This is the first demonstration that TCDD causes tissue‐specific downregulation of ER mRNA. These effects may contribute to the tissue‐specific toxicity of TCDD.

Locating the binding sites of antitumor drug tamoxifen and its metabolites with DNA

We located the binding sites of antitumor drugs tamoxifen, 4-hydroxytamoxifen and endoxifen with calf-thymus DNA. FTIR, CD, UV-vis and fluorescence spectroscopic methods as well as molecular modeling were used to characterize the drug binding sites, binding constant and the effect of drug binding on DNA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bind DNA via hydrophobic and hydrophilic interactions with overall binding constants of K(tam-DNA)=3.5 (±0.2)×10⁴ M⁻¹, K(4-hydroxytam-DNA)=3.3 (±0.4) × 10⁴ M⁻¹ and K(endox)-DNA=2.8 (±0.8)×10⁴ M⁻¹. The number of binding sites occupied by drug is 1 (tamoxifen), 0.8 (4-hydroxitamoxifen) and 1.2 (endoxifen). Docking showed the participation of several nucleobases in drug-DNA complexes with the free binding energy of -3.85 (tamoxifen), -4.18 (4-hydroxtamoxifen) and -3.74 kcal/mol (endoxifen). The order of binding is 4-hydroxy-tamoxen>tamoxifen>endoxifen. Drug binding did not alter DNA conformation from B-family structure, while major biopolymer aggregation occurred at high drug concentrations. The drug binding mode is correlated with the mechanism of action of antitumor activity of tamoxifen and its metabolites.

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.

Liquid Crystalline Phase Behavior of High Molecular Weight DNA: A Comparative Study of the Influence of Metal Ions of Different Size, Charge and Binding Mode

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.

Induction of p21 (CIP1/WAF1/SID1) by estradiol in a breast epithelial cell line transfected with the recombinant estrogen receptor gene : A possible mechanism for a negative regulatory role of estradiol

Estrogens stimulate the growth of a majority of estrogen receptor (ER)-positive breast cancer cells. In contrast, estradiol exerted a 75% inhibition of DNA synthesis in the MCF-10AEwt5 cell line, obtained by the transfection of the ER gene into a normal breast epithelial cell line, MCF-10A. The estradiol-mediated growth inhibitory effect was reversed by ICI 164384, a pure anti-estrogen. Analysis of cell cycle by flow cytometry showed a significant increase of G1 cells by estradiol treatment compared to controls. To understand the mechanism of action of estradiol on MCF-10AEwt5 cells, we examined the level of a cyclin dependent kinase inhibitor (CKI), p21, by Western blot analysis. Our results showed a 5- to 10-fold increase in the level of p21 in estradiol-treated MCF-10AEwt5 cells compared to controls. ICI 164384 reversed estradiol-mediated induction of p21. Northern blot analysis of p21 mRNA indicated that estradiol stimulated its message in MCF-10AEwt5 cells. Analysis of a panel of 6 breast cancer cell lines showed the absence of p21 protein, whereas it was present at a very low level in MCF-10A cells. Comparison of p21 in MCF-10A and MCF-10AEwt5 cells showed an abundance of p21 in the ER-transfected cells. However, this p21 appears to be inactive in the absence of estradiol. These results suggest a p21-mediated pathway as a possible mechanism for the growth inhibitory effects of estradiol on at least a subset of ER-transfected cell lines.

Structural Specificity of Polyamines in Modulating the Binding of Estrogen Receptor to Potential Z-DNA Forming Sequences

Estrogen receptor (ER) is a gene-regulatory protein that mediates the action of estradiol. In order to examine the role of conformational dynamics of DNA in estrogenic regulation of gene expression, we studied the binding of ER to poly(dA-dC).poly(dG-dT) which undergoes transition to a left-handed Z-DNA form. This type of dinucleotide repeats are widely distributed in mammalian genome and are present in estrogen response elements. Binding affinity of ER for the polynucleotide was assessed by its ability to release ER bound to DNA-cellulose. ER binding by poly(dA-dC).poly(dG-dT) was enhanced in the presence of an endogenous polyamine, spermidine, H2N(CH2)4NH(CH2)3NH2. The concentration of spermidine required for facilitating 50% elution of ER (EC50) was 75 microM. This EC50 increased to 500 microM for a spermidine homolog, H2N(CH2)8NH(CH2)3NH2, demonstrating polyamine structural specificity. Spectroscopic measurements showed that the presence of 100-200 microM spermidine initiated changes in the conformation of the polynucleotide indicative of Z-DNA form, but a major alteration to Z-DNA spectrum occurred only at 300 microM concentration. These data suggest that ER favors DNA sequences poised for Z-DNA transition. The efficacy of spermidine homologs in facilitating ER-DNA interaction may be important in predicting their efficiency to replace cellular functions of spermidine.