Single-chain estrogen receptors (ERs) reveal that the ERalpha/beta heterodimer emulates functions of the ERalpha dimer in genomic estrogen signaling pathways.

The effects of estrogens, particularly 17beta-estradiol (E2), are mediated by estrogen receptor alpha (ERalpha) and ERbeta. Upon binding to E2, ERs homo- and heterodimerize when coexpressed. The ER dimer then regulates the transcription of target genes through estrogen responsive element (ERE)-dependent and -independent pathways that constitute genomic estrogen signaling. Although ERalpha and ERbeta have similar ERE and E2 binding properties, they display different transregulatory capacities in both ERE-dependent and -independent signaling pathways. It is therefore likely that the heterodimerization provides novel functions to ERs by combining distinct properties of the contributing partners. The elucidation of the role of the ER heterodimer is critical for the understanding of physiology and pathophysiology of E2 signaling. However, differentially determining target gene responses during cosynthesis of ER subtypes is difficult, since dimers formed are a heterogeneous population of homo- and heterodimers. To circumvent the pivotal dimerization step in ER action and hence produce a homogeneous ER heterodimer population, we utilized a genetic fusion strategy. We joined the cDNAs of ERalpha and/or ERbeta to produce single-chain ERs to simulate the ER homo- and heterodimers. The fusion ERs interacted with ERE and E2 in a manner similar to that observed with the ER dimers. The homofusion receptors mimicked the functions of the parent ER dimers in the ERE-dependent and -independent pathways in transfected mammalian cells, whereas heterofusion receptors emulated the transregulatory properties of the ERalpha dimer. These results suggest that ERalpha is the functionally dominant partner in the ERalpha/beta heterodimer.

Binding of estrogen receptor beta to estrogen response element in situ is independent of estradiol and impaired by its amino terminus.

The functions of 17beta-estradiol (E2) are mediated by estrogen receptor (ER) alpha and beta. ERs display similar DNA- and ligand-binding properties in vitro. However, ERbeta shows lower transcriptional activity than ERalpha from the estrogen response element (ERE)-dependent signaling. We predicted that distinct amino termini contribute to differences in transcription efficacies of ERs by affecting in situ ER-ERE interactions. We used chromatin immunoprecipitation and a novel in situ ERE competition assay, which is based on the ability of ER to compete for ERE binding with a designer activator that constitutively induces transcription from an ERE-driven reporter construct. Interference of activator-mediated transcription by unliganded or liganded ERs was taken as an indication of ER-ERE interaction. Results revealed that ERs interacted with ERE similarly in the absence of E2. However, E2 enhanced the ERE binding of ERalpha but not that of ERbeta. The removal of the amino terminus increased the ERbeta-ERE interaction independent of E2. The ERbeta amino terminus also prevented E2-mediated enhancement of the chimeric ERalpha-ERE interaction. Thus, the amino terminus of ERbeta impairs the binding of ERbeta to ERE. The abrogation of ligand-dependent activation function 2 of the amino-terminally truncated ERbeta resulted in the manifestation of E2 effect on ERbeta-ERE interaction. This implies that E2-mediated enhancement of ERbeta-ERE interaction is masked by the activation function 2, whereas the intact amino terminus is a dominant region that decreases the binding of ERbeta to ERE. Thus, ERbeta-ERE interaction is independent of E2 and is impaired by its amino terminus. These findings provide an additional explanation for differences between ERalpha and ERbeta functions that could differentially affect the physiology and pathophysiology of E2 signaling.

Molecular basis of therapeutic strategies for breast cancer.

The development of breast cancer is the consequence of uncontrolled growth and division of breast-ductal epithelial cells. While many factors contribute to its etiology, estrogen hormones within the context of many interrelated growth signaling pathways play critical roles for the initiation and development of breast cancer. The effects of estrogens are primarily mediated by the estrogen receptors (ERs) alpha and beta. ER mediates a complex array of genomic and non-genomic events that orchestrate cellular metabolism, mitogenesis, morphogenesis, motogenesis, and apoptosis. The current modalities for the treatment of breast cancer have centered on the development of agents with diverse pharmacology to reduce/ablate the circulating estrogens or to alter/prevent ER function. Approaches to perturb the estrogen environment are successful usually in the remission of established tumors. However, many breast tumors are not responsive or eventually develop resistance to endocrine therapies. Despite considerable effort, the mechanism for the non-responsiveness and acquisition of resistance remains unclear. The establishment of hormone responsiveness is one of the current approaches for the development of an effective therapeutic modality for de novo resistant breast tumors. Re-establishment of loss of ER synthesis/function, on the other hand, constitutes a primary therapeutic goal for acquired resistance neoplasms. We have recently engineered transregulatory proteins that specifically targeted and robustly regulated estrogen responsive genes independent of ligand, ER-subtype and cell-context. The targeted regulation of estrogen responsive gene networks by these designer transregulators could provide a basis for the development of novel approaches for experimental biology and medicine.

Differences in the abilities of estrogen receptors to integrate activation functions are critical for subtype-specific transcriptional responses.

Estrogen receptors (ER) alpha and beta are members of a superfamily of nuclear receptors and mediate estrogen [17beta-estradiol (E2)] signaling. ERbeta has considerably less transcription potency than ERalpha in heterologous expression systems that use E2 response elements (ERE) in tandem as the trans-acting unit. We show here that despite similar intracellular characteristics, ERbeta, in contrast to ERalpha, fails to induce gene transcription synergistically in response to E2 from tandem EREs. Moreover, our results indicate that ERalpha-specific partial agonistic activity of antagonists occurs additively. Although synergy contributes, it is not sufficient for differences in the transcription potencies between the ER subtypes. We demonstrate here that differences in the abilities of ERs to integrate activation functions through functional interactions between amino and carboxyl termini are critical for the transcriptional strength of ER subtypes.

The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ER alpha and ER beta.

Estrogen signaling is mediated by ER alpha and -beta. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ER alpha and ER beta bind to an ERE independently from ER ligands. We found that although the binding affinity of ER beta for an ERE is 2-fold lower than that of ER alpha, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ER alpha preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ER alpha is more transcriptionally active than ER beta. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ER alpha to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.

Water soluble, core-modified porphyrins. 3. Synthesis, photophysical properties, and in vitro studies of photosensitization, uptake, and localization with carboxylic acid-substituted derivatives.

Water soluble, core-modified porphyrins 1-5 bearing 1-4 carboxylic acid groups were prepared and evaluated in vitro as photosensitizers for photodynamic therapy. The 21,23-core-modified porphyrins 1-5 gave band I absorption maxima with lambda(max) of 695-701 nm. The number of carboxylic acid groups in the dithiaporphyrins 1-4 had little effect on either absorption maxima (lambda(max) of 696-701 nm for band I) or quantum yields of singlet oxygen generation [phi((1)O(2)) of 0.74-0.80]. Substituting two Se atoms for S gave a shorter band I absorption maximum (lambda(max) of 695 nm) and a smaller value for the quantum yield for generation of singlet oxygen [phi((1)O(2)) of 0.30]. The phototoxicity of 1-5 was evaluated against R3230AC cells. The phototoxicities of dithiaporphyrin 2, sulfonated thiaporphyrin 30, HPPH, and Photofrin were also evaluated against Colo-26 cells in culture using 4 J cm(-2) of 570-800 nm light. Compound 2 was significantly more phototoxic than sulfonated dithiaporphyrin 30, HPPH, or Photofrin. Cellular uptake was much greater for compounds 1, 2, and 5 relative to compounds 3 and 4. Confocal scanning laser microscopy and double labeling experiments with rhodamine 123 suggested that the mitochondria were an important target for dithiaporphyrins 1 and 2. Inhibition of mitochondrial cytochrome c oxidase activity in whole R3230AC cells was observed in the dark with compounds 1 and 30 and both in the dark and in the light with core-modified porphyrin 2.

In vitro photodynamic properties of chalcogenopyrylium analogues of the thiopyrylium antitumor agent AA1.

Several series of chalcogenopyrylium dyes were prepared with one or two 4-anilino substituents at the 2- and 6-positions and with phenyl, 4-N,N-dimethylanilino, or 4-(N-morphilino)phenyl substituents at 2- and/or 4-positions. The dye series are all related in structure to AA1, a thiopyrylium dye that targets mitochondria. The chalcogenopyrylium nuclei included sulfur, selenium, and tellurium at the 1-position. Key intermediates in the dye synthesis were the corresponding Delta-4H-chalcogenopyran-4-ones. All of the dyes of this study were evaluated for dark and phototoxicity toward Colo-26 cells in vitro. There was no correlation of dark toxicity with either the reduction potential of the chalcogenopyrylium dye or the n-octanol/water partition coefficient, log P. Several of the dyes of this study (thiopyrylium dyes 1-S and 13-S, selenopyrylium dyes 1-Se, 2-Se, 3-Se, 4-Se, 13-Se, 14-Se, and 27-Se, and telluropyrylium dye 13-Te) showed added phototoxicity upon irradiation. Dyes with the highest therapeutic ratio as measured by dark toxicity/phototoxicity (15 J cm(-2) of 360-800-nm light) had values of log P of 1.0-1.2. Studies of cytochrome c oxidase activity in whole R3230AC cells suggested that dyes 1-S and 3-Se, with values of log P of 2.2 and 1.7, respectively, were localized in the mitochondria. Cytocrome c oxidase activity in whole cells was inhibited by 1-S and 3-Se in the dark. Chalcogenopyrylium dyes 2-Se, 4-Se, 13-Te, and 14-Se inhibited whole-cell cytochrome c oxidase activity only following irradiation, which suggests that these dyes relocalized to mitochondria following irradiation.

Water-soluble, core-modified porphyrins as novel, longer-wavelength-absorbing sensitizers for photodynamic therapy. II. Effects of core heteroatoms and meso-substituents on biological activity.

Water-soluble, core-modified porphyrins were prepared and evaluated as sensitizers for photodynamic therapy (PDT). The addition of an aromatic aldehyde to 2,5-dilithiothiophene or -selenophene gave diol 3 as a nearly equimolar mixture of meso and d,l diastereomers, which gave a single diastereomer following careful recrystallization. The condensation of pyrrole with a diol 3 using catalytic BF(3)-etherate gave bispyrrolochalcogenophenes (4). Condensation of a diol 3 with 4 in the presence BF(3)-etherate gave 21,23-dichalcogenaporphyrins (5). 21-Thiaporphyrins (6) were prepared by condensation of a diol 3 with excess pyrrole and benzaldehyde in the presence of tetrachlorobenzoquinone and catalytic BF(3)-etherate. Sulfonation of 5 and 6 with concentrated sulfuric acid at 100 degrees C gave sulfonated derivatives 7-15. Bis-4-methoxy-21,23-dithiaporphyrins 5h and 5l were demethylated with BBr(3), and the resulting phenols were alkylated with ethyl bromoacetate. Saponification gave 21,23-dithiaporphyrin dicarboxylate salts 16 and 17. The 21,23-core-modified porphyrins gave band I absorption maxima (lambda(max) of 689-717 nm) at longer wavelengths than band I for the corresponding 21-core-modified porphyrins, but both classes had band I maxima at longer wavelengths than either TPPS(4) or Photofrin (lambda(max) of 630 nm for both). The core heteroatoms had little effect on either absorption maxima or quantum yields of singlet oxygen generation in 7-17. The meso substituents had a greater impact on absorption maxima. Compounds 7-17 were evaluated for phototoxicity against Colo-26 cells in culture using 4 J cm(-2) of 570-800 nm light. Compounds 8-12, 14, 16, and 17 gave a 50% cell kill in vitro at a lower concentration than Photofrin [5.7 mg (9 micromol)/kg]. Compounds 14, 16, and 17 gave a 50% cell kill with 4 J cm(-2) of light and submicromolar concentrations of sensitizer. Sensitizers 8 and 11 showed no toxicity or side effects in BALB/c mice observed for 90 days following a single intravenous injection of 10 mg/kg of sensitizer. Distribution studies show that sensitizer 8 accumulates in the tumors of BALB/c mice. PDT with 8 at 0.125 mg (0.13 micromol)/kg or 11 at 2.5 mg (2.5 micromol)/kg and 135 J cm(-2) of 694 nm light was comparable to PDT with Photofrin at 2.5 mg (4 micromol)/kg and 135 J cm(-2) of 630 nm light against Colo-26 tumors in BALB/c mice.

Targeting estrogen responsive elements (EREs): design of potent transactivators for ERE-containing genes.

The estrogen hormone (E2) plays an important role in the physiology and pathophysiology of target tissues. The effects of E2 are conveyed by the estrogen receptors (ER) alpha and beta. The E2-ER complex mediates an array of genomic and non-genomic events that orchestrate the expression of a number of genes involved in the regulation of cell proliferation and differentiation. The interaction of with the regulatory DNA sequence, estrogen responsive element (ERE), of each responsive gene constitutes a critical genomic signaling pathway. However, the relative importance of ERE-dependent E2-ER signaling in cell proliferation remains to be elucidated. To address this issue, we engineered ERE-binding activators (EBAs) that specifically and potently regulate ERE-containing genes. The modular nature of ER allowed us to initially design a monomeric ERE-binding module by genetically joining two DNA-binding domains with the hinge domain. Integration of strong activation domains from other transcription factors into this module generated constitutively active EBAs. These transactivators robustly induced the expression of only ERE-containing promoter constructs in transfected cells independent of ligand, dimerization, ER-subtype and -status. Moreover, EBAs altered cell cycle progression in breast cancer cell lines in a manner similar to E2-ER. These results demonstrate the importance of ERE-containing genes in the regulation of cell proliferation. These novel ERE-binding transregulators could also be a basis for the targeted regulation of ERE-containing genes, the identification of estrogen responsive gene networks, and the development of alternative/complementary therapeutic approaches for estrogen target tissue cancers.

Structural regions of ERalpha critical for synergistic transcriptional responses contain co-factor interacting surfaces.

Most highly estrogen responsive genes are synergistically activated by multiple copies of estrogen responsive elements (EREs) capable of binding to the estrogen receptor (ER). We examined here the structural features of the receptor necessary to interact with co-regulatory proteins and to produce a synergistic pattern of activation from multiple EREs. Using full length and truncated variants of ERalpha, we show in transfected mammalian cells that although the carboxyl (AF-2) and the amino (AF-1) terminal activation domains are functionally integrated to induce transcription, AF-1 is critical for mediating synergy. Partial characterization of AF-1 sub-domains revealed that both Box-1 and Box-2 regions (amino acids 41-64 and 87-108, respectively) are essential for a synergistic response to estrogen. We show that members of the p160 family of co-factors and TIF-1 interact with the AF-2 domain of ERalpha. We also found that TIF-2, a member of the p160 family, can interact with the Box-1 region of AF-1. Apparently, structural regions required for the ability of ERalpha to induce transcription synergistically from tandem ERE sequences are also critical for the interaction of the receptor with the co-regulatory proteins.

Gene expression profiling reveals that the regulation of estrogen-responsive element-independent genes by 17 beta-estradiol-estrogen receptor beta is uncoupled from the induction of phenotypic changes in cell models.

Estrogen hormone 17beta-estradiol (E(2)) is involved in the physiology and pathology of many tissues. E(2) information is conveyed by the transcription factors estrogen receptors (ER) alpha and beta that mediate a complex array of nuclear and non-nuclear events. The interaction of ER with specific DNA sequences, estrogen-responsive elements (EREs), constitutes a critical nuclear signaling pathway. In addition, E(2)-ER regulates transcription through interactions with transfactors bound to their cognate regulatory elements on DNA, hence the ERE-independent signaling pathway. However, the relative importance of the ERE-independent pathway in E(2)-ERbeta signaling is unclear. To address this issue, we engineered an ERE-binding defective ERbeta mutant (ERbeta(EBD)) by changing critical residues in the DNA-binding domain required for ERE binding. Biochemical and functional studies revealed that ERbeta(EBD) signaled exclusively through the ERE-independent pathway. Using the adenovirus infected ER-negative cancer cell models, we found that although E(2)-ERbeta(EBD) regulated the expression of a number of genes identified by microarrays, it was ineffective in altering cellular proliferation, motility, and death in contrast to E(2)-ERbeta. Our results indicate that genomic responses from the ERE-independent pathway to E(2)-ERbeta are not sufficient to alter the cellular phenotype. These findings suggest that the ERE-dependent pathway is a required signaling route for E(2)-ERbeta to induce cellular responses.

Mitochondria are targets of photodynamic therapy.

Photodynamic Therapy (PDT) is an evolving cancer treatment that depends on three known and variable components: photosensitizer, light and oxygen. Optimization of these variables yields reactive oxygen species, mainly singlet oxygen, that damage cellular components leading to cytotoxicity. Our research has demonstrated that porphyrin sensitizers, in particular, significantly inhibit the inner mitochondrial membrane enzymes cytochrome c oxidase and F(0)F(1) ATP synthase. These results were obtained from an in vivo-in vitro experimental protocol that exposes sensitizers to metabolic and pharmacokinetic events. The resulting inhibition of oxidative phosphorylation was expected to reduce ATP levels, which were quantitated in cells and were confirmed by (31)P-NMR spectroscopy of tumors in situ in animals treated with PDT. Based on these findings, and more recent investigations of apoptosis, there is little doubt that mitochondria are critical targets in the actions of PDT.

A tale of two estrogen receptors (ERs): how differential ER-estrogen responsive element interactions contribute to subtype-specific transcriptional responses.

The interaction of ERalpha and ERbeta with ERE constitutes the initial step in the canonical nuclear E2 signaling in which E2-ERbeta is a weaker transactivator than E2-ERalpha. This perspective summarizes recent findings to discuss potential mechanisms that contribute to ER subtype-specific transcriptional responses.

Core-modified porphyrins. Part 4: Steric effects on photophysical and biological properties in vitro.

21,23-Dithiaporphyrins (2-10) were designed and prepared as analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (1) to examine the impact of steric bulk at the 5- and 20-meso positions as well as the impact of symmetry. Changes at the meso positions had minimal impact on the UV-vis-near-IR absorption spectra, quantum yields for the generation of singlet oxygen, and quantum yields for fluorescence and some impact on values of the octanol/water partition coefficient. Of the compounds 1-10, 5-phenyl-20-(2-thienyl)-10,15-bis-(4-carboxylatomethoxy-phenyl)-21,23-dithiaporphyrin (3) showed the greatest phototoxicity toward cultured R3230AC cells, with 68% cell kill at 1 x 10(-7)M and irradiation with 5J cm(-2) of 350-750 nm light. Results in this study suggest that smaller substituents on the meso ring and less symmetrical compounds are more effective as photosensitizers than compounds with two bulky substituents at adjoining meso sites and a higher symmetry. The mitochondria appear to be involved in the process of phototoxicity as determined by the inhibition of whole cell cytochrome c oxidase activity in cells treated with 3 and light. No impact upon mitochondrial cytochrome c oxidase activity was observed in cells treated with 3 and no light. Fluorescence microscopy studies suggest that the mitochondria are not initial sites of accumulation of 3.

Comparison of the dark and light-induced toxicity of thio and seleno analogues of the thiopyrylium dye AA1.

2,6-Bis(4-anilino)-4-(4-N,N-dimethylanilino)thiopyrylium chloride (AA1) and -selenopyrylium chloride (AA1-Se) and 2,6-bis(4-anilino)-4-(4-N-morpholinophenyl)thiopyrylium chloride (1) and -selenopyrylium chloride (2) were prepared via the addition of 4-N,N-dimethylanilino magnesium bromide and 4-N-morpholinophenyl magnesium bromide to chalcogenopyranones 3 followed by treatment with HCl gas then water. Cellular uptake of these dyes varied from 12+/-3fmol/cell for AA1 to 150+/-40 fmol/cell for AA1-Se. upon exposure to 5 x 10(-5) M solutions of the dyes for 3 h. Exposure of cell cultures to 1.8 J/cm2) of 360-750-nm light following incubation with 1 x 10(-6) M of either AA1, 1, or 2 for 24h resulted in no significant additional phototoxicity while AA1-Se showed a significant (p < 0.05) reduction in cell viability from 81% to 46%. Thiopyrylium dyes AA1 and 1 showed significant dark toxicity relative to selenopyrylium dyes AA1-Se and 2, respectively. AA1 was the only one of the four dyes to show inhibition of whole-cell mitochondrial cytochrome c oxidase activity in the dark. Irradiation of whole cells or mitochondrial suspensions treated with AA1, AA1-Se, or 2 gave inhibition of mitochondrial cytochrome c oxidase activity. Studies of JC-1-efflux indicated that all four cationic dyes accelerated the loss of JC-1 from the mitochondria, which suggests that all four dyes target the mitochondria.

Analogues of tetramethylrosamine as transport molecules for and inhibitors of P-glycoprotein-mediated multidrug resistance.

Tetramethylrosamine and its thio- and seleno- analogues (TMR-O, TMR-S, and TMR-Se, respectively) were examined for their ability to be transported by Pgp into chemo-resistant CR1R12 cells. Verapamil (7 x 10(-6)M) enhanced the uptake of TMR-O and TMR-S into CR1R12 cells compared to those cultures not previously exposed to verapamil. The uptake of TMR-O and TMR-S in CR1R12 cells in the presence of 7 x 10(-6)M verapamil was equivalent to its uptake in the chemo-sensitive parent cell line AUXB1 in the absence or presence of verapamil. None of the TMR analogues were effective alone as photosensitizers of CR1R12 cells. However, when either TMR-S or TMR-Se was added to CR1R12 cells after 7 x 10(-6)M verapamil exposure for 2h, irradiation of cultures with 5.0J cm(-2) of 350-750 nm light caused significant phototoxicity. TMR-O showed no significant phototoxicity in the presence of verapamil. Chemo-sensitive AUXB1 cells are equally susceptible to phototoxicity using TMR-Se with or without previous exposure to verapamil. The Pgp modulators verapamil and CsA increased the uptake of CAM into CR1R12. Exposure of CR1R12 cells to TMR-S or TMR-Se for 2h in the dark resulted in no significant change in the intracellular accumulation of CAM. However, 1h of light exposure after incubation of cells with TMR-S or TMR-Se resulted in an up to 2-fold increase in CAM uptake.

Synthesis, properties, and photodynamic properties in vitro of heavy-chalcogen analogues of tetramethylrosamine.

Thio and seleno analogues of tetramethylrosamine were prepared by the directed-metalation/cyclization of the corresponding N,N-diethyl 2-(3-dimethylaminophenylchalcogeno)-4-dimethylaminobenzamide to the 2,7-bis-(N,N-dimethylamino)-9H-chalcogenoxanthen-9-one followed by the addition of phenylmagnesium bromide, dehydration, and ion exchange to the chloride salt. The thio and seleno tetramethylrosamines had longer wavelengths of absorption and higher quantum yields for the generation of singlet oxygen than tetramethylrosamine. Both the thio and selenoanalogues of tetramethylrosamine were efficient photosensitizers against R3230AC rat mammary adenocarcinoma cells in vitro.