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Supplementary Methods, Figures 1-3 from Harnessing Competing Endocytic Pathways for Overcoming the Tumor-Blood Barrier: Magnetic Resonance Imaging and Near-Infrared Imaging of Bifunctional Contrast Media

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)–based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells. [Cancer Res 2009;69(13):5610–7]

Embryo implantation requires execution of highly synchronized processes at the feto-maternal interface, initiated by blastocyst attachment to the endometrial epithelium. Hyaluronan is a major ECM component known to regulate adhesion-associated biological processes in various physiological settings. We hypothesized that hyaluronan may facilitate blastocyst attachment. In order to test our hypothesis, we characterized the blastocyst expression of hyaluronan synthesizing and degrading enzymes, as well as the expression of hyaluronan receptors during attachment. The functional impact of hyaluronan was challenged by the use of mouse transgenic blastocysts, in which genes encoding for hyaluronan synthesizing enzymes were deleted using lentiviral incorporation of Cas-9 endonuclease alongside specific short-guide RNAs into the embryonic trophectoderm. Embryos with transgenic trophectoderm were tested for their attachmentin vitro, or assessed for implantationin vivo, upon transfer to foster dams. Deletion of the trophectoderm hyaluronan biosynthesis significantly reduced the number of blastocysts attached to human uterine epithelium cellsin vitro. Reduced attachment was also observedin vivo, in pregnant mice carrying blastocysts with hyaluronan-depleted trophectoderm. In agreement, trophectoderm expression of osteopontin, was downregulated upon depletion of hyaluronan. MRI measurements revealed a decrease in uterine blood vessels permeability. Uterine expression of VEGF-A, PTGS-2 and uterine osteopontin, which constitute the immediate response to blastocyst attachment was also reduced. Furthermore, impaired implantation, associated with a decrease in hyaluronan synthesis in the mural trophectoderm, obtained upon tamoxifen treatment, has been recovered by LIF administration. These results demonstrate that estrogen-regulated hyaluronan-synthesis in the trophectoderm is indispensable for mouse blastocysts attachment to the uterine epithelium.

Background Sorafenib improves progression-free survival in patients with progressive radioactive iodine-refractory differentiated thyroid carcinoma, but causes severe side effects. Estrogens may accelerate thyroid carcinoma cell growth. Our group recently reported that isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine (cD-tboc), a novel anti-estrogenic compound, retards the growth of both thyroid carcinoma cell lines and cultured human carcinoma cells. Vitamin D receptor (VDR) is expressed in malignant cells and responds to 1,25 dihydroxyvitamin D3 (1.25D) by decreased proliferative activity in vitro. The purpose of this study was to examine the effects of vitamin D metabolites (VDM) on the expression of estrogen receptors (ERs), VDR, and 1OHase mRNA, and to evaluate the inhibitory effect of low doses of sorafenib in combination with cDtboc and VDM on cell proliferation in cultured human papillary thyroid carcinoma (PTC). Methods In 19 cultured PTC specimens and 19 normal thyroid specimens, harvested during thyroidectomies from the same patients, expression levels of ERα, ERβ, VDR, and 1 alpha-hydroxylase (1OHase) mRNA (by quantitative real-time PCR) were determined at baseline and after treatment with VMD. Cell proliferation was determined by measurement of 3[H] thymidine incorporation after treatment with sorafenib alone, sorafenib with added 1.25D or cD-tboc, and sorafenib with both 1.25D and cD-tboc added. Results 1,25D increased mRNA expression of all tested genes in the malignant and normal thyroid cells, while the ERα mRNA of the normal cells was unaffected. 1.25D dose-dependently inhibited cell proliferation in the malignant cells. The inhibitory effect of sorafenib on cell proliferation in the malignant cells was amplified after the addition of cDtboc and 1.25D, such that the maximal inhibition was not only greater, but also had been attained at a 10-fold lower concentration of sorafenib (20 μg/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200 μg/ml) alone. Conclusions The demonstration that low concentrations of cDtboc and 1.25D markedly amplify the inhibitory effect of sorafenib on the growth of human PTC supports the use of a 10-fold lower concentration of sorafenib. The findings may promote a new combination treatment for progressive radioactive iodine-refractory PTC.

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Objective: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast–enhanced-magnetic resonance imaging. Dynamic contrast–enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis. Conclusions: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE.

a b s t r a c t Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) mRNA expression in TT cells was more abundant than ER, with a ratio of 48:1. Estradiol-17 (E2) increased DNA synthesis in a dose dependent manner. (3H)-thymidine incorporation was also stim- ulated by the ER agonist DPN and the ER agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase spe- cific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on ( 3 H)-thymidine incorporation could be blocked by the ER antagonist PTHPP but not by the ER antagonist MPP, suggesting that the antiproliferative effect of cD- tBoc on these cells is mediated through ER. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.

Hormone replacement therapy (HRT) for post-menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post-menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen-mimetic peptide 1 (EMP-1) (VSWFFE) in comparison to estrogen (E(2)) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP-1-like E(2) inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP-1 did not affect E(2) stimulation of DNA, but inhibited E(2) inhibition of cell proliferation at high concentration. All effects by the combination of EMP-1 and E(2) were abolished at hg. A44-stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co-incubated with E(2) at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E(2) stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45-stimulated CK-specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP-1. EMP-1 and A44 similar to E(2)-stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP-1 provided it acts similarly in vivo can replace E(2) for treatment of post-menopausal women in hyperglycemia due to diabetes.

One of the major difficulties in the treatment of epithelial ovarian cancer (EOC) is the high rate of recurrent disease. This is thought to be due to the survival of a population of chemo-resistant cells within the tumor, the ovarian cancer stem cells (OCSCs), that are able to regenerate the tumor following chemotherapy. Therefore, the identification of a compund that can target the OCSCs is one of the main steps in improving overall survival of ovarian cancer patients. The objective of this study was to determine the effect of N-t-boc-Daidzein, a novel daidzain derivative, on OCSCs. The efficacy of this compound was evaluated in OCSC and mature ovarian cancer cell (mOCC) lines isolated from malignant ovarian cancer asicites. Cells were treated with increasing concentrations of N-t-boc-Daidzein (0.003-10 microM) and cell growth was monitored by "real time in vitro micro-imaging" using the IncuCyte system. Cell viability was measured using the CellTiter 96 Assay. Apoptosis was determined by Caspase-Glo 3/7, 8 and 9 assays. The components of the apoptotic cascade were characterized by western blot analysis. N-t-boc-Daidzein was able to significantly inhibit cell growth and decrease cell viability of OCSC as well as mOCC cells in a dose and time dependent maner. This effect was due to the induction of apoptosis, which is characterized by caspase activation, XIAP and AKT degradation, and mitochondrial depolarization. This study describes a novel compound that can target the OCSCs. These findings may provide vital aide in improving overall survival in patients with EOC.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.

The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and to raloxifene (Ral), whereas male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. In cells derived from pre-menopausal women, E(2), G, D and Ral stimulated CK to higher extent compared to post-menopausal bone cells, whereas quecertin (Qu), carboxy-biochainin A (cBA) and carboxy-genistein (cG) stimulated CK in both age groups similarly, and biochainin A (BA) stimulated post-menopausal cells to a bit higher extent than pre-menopausal cells. Since the skeletal protective effects of estrogens are not discernable in diabetic women, we tested in this study, the effects of high glucose concentration in the growth medium, on the effects of estrogenic compounds on CK in human-derived bone cells (hObs). Female-derived hObs were grown either in normal (4.5 g/l; 22 mM, NG) or high glucose (9.0 g/l; 44 mM, HG) for 7 days. HG increased constitutive CK, but attenuated E(2)- and DHT-induced CK in female or male hObs, respectively. HG also inhibited genistein (G) and daidzein (D) stimulated CK in female hObs, but not the effects of biochainin A (BA), quecertin (Qu) or Ral. Intracellular, mainly nuclear binding of (3)[H]E(2) was characteristic of the different phytoestrogens in female hObs, was abolished by HG. Membranal binding of Eu-Ov-E(2), was displaced only by E(2)-Ov, ICI, cG-Ov or cD-Ov but decreased total binding of Eu-Ov-E(2) in both age groups and completely abolished the competition with E(2)-Ov or ICI in both age groups, but the competition with cD-Ov and cG-Ov was decreased only slightly but not statistically significant. HG also abolished Eu-BSA-T, which bound similarly male-derived hObs. All hObs expressed mRNA for ERalpha and ERbeta with higher abundance of ERalpha. HG increased mRNA for both ERs in female-derived hObs, but decreased mRNA for both ERs in male-derived hObs. Hence, human bone cells, which express specific nuclear and membranal binding sites for estrogenic compounds, are modulated by HG, leading to altered hormonal responsiveness, which might block important effects of estrogenic compounds, contributing probably to their decreased skeletal preserving properties under hyperglycemia.

Carboxy derivatives of isoflavones that exhibit oestrogenic/anti-oestrogenic properties were used as carriers for affinity drug targeting to H295R adrenocortical carcinoma cells that express transcripts of oestrogen receptor (ER) alpha and beta. These derivatives were prepared by introducing a carboxymethyl group at the 6-position of genistein and of biochanin A, yielding 6CG and 6CB respectively. In transactivation assays, 6CG displayed mixed agonist/antagonist activity for ERalpha, whereas 6CB displayed only weak antagonist activity. Low concentrations of oestrogen, 6CG and 6CB were capable of inducing proliferation in H295R cells and of stimulating creatine kinase (CK) specific activity, suggesting that these cells were sensitive to oestrogenic compounds. In in vivo experiments, both 6CG and 6CB were capable of inhibiting oestrogen-induced CK specific activity in rat tIssues. For affinity drug targeting, the cytotoxic drug daunomycin was coupled to 6CB and 6CG, yielding 6CB-Dau and 6CG-Dau respectively. These conjugates were tested for their antiproliferative ability to inhibit DNA synthesis as assessed by incorporation of [(3)H]thymidine in H295R cells. A dose-dependent cytoxicity was observed with both conjugates. At 0.3-3 nM, both conjugates were 10 to 30 times more potent than daunomycin. At 30 nM these conjugates were two to three times more potent than daunomycin. At concentrations ranging between 300 and 3000 nM, no difference in cytotoxicity was observed between the conjugates and daunomycin. When the cells were treated over a wide range of concentrations with a combination of 6CG plus daunomycin, the observed cytotoxicity was less than with daunomycin alone. When non-transformed rat enterocytes, which do not express ER, were treated with 6CG-Dau or daunomycin, the antiproliferative effect of 6CG-Dau was the same as that of daunomycin over the concentration range tested. These pilot studies suggest that the ready availability of oestrogenic binding sites in H295R cells can be exploited for site-directed chemotherapy.

Background: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria. Methods: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0–8 days at room temperature and for 0–10 freeze-thaw cycles. Results: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were Conclusions: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

A monoclonal antibody (MAb) raised against sulfamethazine (21C7) was applied in an optical biosensor (Biacore Q) to develop a rapid biosensor immunoassay (BIA) for the detection of several sulfonamides in chicken serum. The performance of this MAb was compared with two polyclonal antibodies (PAbs) raised against sulfamethazine (Qflex sulfamethazine binding protein (SBP) and RIKILT 464b). Using these PAbs, the limits of detection (LODs) in 10 times diluted chicken serum were approximately 30 ng ml −1 and the two BIAs were found to be specific for sulfamethazine. Using MAb 21C7, the LOD for sulfamethazine in 10 times diluted chicken serum was lower (10 ng ml −1 ), high cross-reactivities were measured for sulfisoxazole (149%), sulfachlorpyridazine (112%), sulfachlorpyrazine (94%), sulfamerazine (87%), sulfadiazine (56%), sulfatroxazole (56%) and sulfathiazole (50%) and low cross-reactivities (11–25%) were measured with six other sulfonamides. Compared with the polyclonal antibodies, the MAb-based BIA resulted in a better sensitivity and was found suitable for the detection of 8 sulfonamides in 10 times diluted chicken serum with LODs between 7 and 20 ng ml −1 . The total run time for each cycle was 7 min.

Background The aim of this study was to assess the effect of phytoestrogens on the human vascular wall in vitro. Methods We compared the effects of E2 to those of genistein (G), daidzein (D), biochanin A (BA), equol (EQ), and quecertin (Qu) on 3[H] thymidine incorporation and creatine phosphokinase (CK) activity in human vascular smooth muscle cells (VSMC) and in a human endothelial cell line (E304). Results In VSMC, E2, the estrogen antagonist raloxifene (RAL), G, and D stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at higher concentrations. In contrast, BA and EQ had a monophasic stimulatory effect on 3[H] thymidine incorporation (87% ± 9% and 54% ± 17%, respectively) whereas Qu had only an inhibitory effect (−36 ± 16% at 30 nmol/L). In E304 cells, all phytoestrogens stimulated DNA synthesis in a dose-related manner. In both cell types E2, RAL as well as all phytoestrogens increased CK-specific activity. The administration of phytoestrogens to immature female rats resulted in increased CK in the aorta (Ao) (60% to 220%) and in the left ventricle of the heart (Lv) (45% to 160%). Similar increases in Ao and Lv CK were also induced by E2 and all five phytoestrogens in ovariectomized (OVX) female rats. RAL antagonized phytoestrogen-induced CK activity in human vascular cells and in the rat Ao and Lv tissue but did not block phytoestrogen effects on DNA synthesis in human VSMC. Conclusions Although phytoestrogens have estrogen-mimetic effects on cell growth and CK in cultured human vascular cells and on CK in rat vascular tissues in vivo, the effects on human VSMC replication are highly dependent on the concentration and the particular phytoestrogen under investigation.

We report the generation of an anti-idiotypic antibody, clone 12E12, against antisulfamethazine (SMZ) as a surrogate ligand for SMZ in immunoassay procedures. This has been achieved by using the primary monoclonal anti-SMZ antibody as an immunogen in hybridoma technology followed by screening of the resulting hybridromas for binding to the SMZ site of the primary anti-SMZ antibody. Characterization of a strong anti-idiotypic antibody of the betatype, clone 12E12, capable of mimicking SMZ, permitted the development of immunoassay formats for SMZ based on the idiotypic and anti-idiotypic approach and the direct measurement of SMZ content in diluted pig urine. The availability of the strong betatypic anti-idiotypic antibody acting as a surrogate ligand for SMZ enables antibodies to be labeled instead of ligands and offers interesting possibilities for the development of competitive type non-isotopic immunoassays for SMZ.

Background Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D5. The stained cells were analyzed by flow cytometry. Results The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80–99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. Conclusions Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis. Cytometry 41:109–114, 2000 © 2000 Wiley-Liss, Inc.

Phytoestrogenic isoflavones that are abundant in soybeans may be an important group of natural products that could play a critical role in preventing several chronic human diseases. To facilitate studying the relationship of soya exposure and chronic diseases, we report a simple method for measuring an isoflavone, genistein, in human urine and plasma. The method is a competitive enzyme-linked immunoassay that utilizes a conjugate of horseradish peroxidase (HRP) and genistein as tracer and a monoclonal antibody to genistein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, competes with HRP-genistein conjugate for the binding sites of anti-genistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubation, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with concentrations of genistein over the range of 0.1-32 ng/well. The sensitivity limit of the method is 0.5 ng of genistein per well or 0.5 ng per 10 microliters of urine and plasma. Urine and plasma levels of genistein measured by this immunoassay correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. This method can be used to assess soya exposure in humans and could facilitate epidemiological studies of the relationship of soya diets and chronic diseases, including cancer.

In most patients with growth hormone (GH) secreting pituitary adenomas and clinically nonfunctioning pituitary tumors (NFPT) the intravenous injection of thyrotropin releasing hormone (TRH) augments the secretion of GH and subunits of gonadotropin hormones respectively. Similar hormone responses to TRH have been detected in rat pituitary cell lines and in primary human pituitary tumor cultures in vitro. Nevertheless the TRH effect on tumor hormonal secretion has not been well characterized. In the present study we examined TRH-induced hormone secretion in GH secreting tumors and in NFPT in vitro. Cultured cells secreted betaLH and betaFSH (NFPT) or GH (GH secreting adenomas) up to 14 days in culture. In NFPT TRH (10(-8) mol/l) elicited peak betaLH and betaFSH secretion at 60 to 90 min, with no further increase at 24 h. TRH-stimulated GH secretion peaked at 90-120 min, and decreased after 3 h, but a secondary rise occurred after 24 h of incubation. Chronic daily exposure to TRH followed by an acute TRH challenge resulted in a further increase of GH secretion after one hour. In contrast, acute TRH administration following chronic exposure did not elicit increased P-subunits secretion in NFPT. Coadministration of cycloheximide did not change TRH induced beta-subunits secretion in NFPT. However, when it was administered 24 h prior to TRH, it blocked both basal and TRH induced beta-subunits levels in NFPT. Cycloheximide had no effect on basal or stimulated GH secretion when administered concomitantly or 24 h before TRH. Incubation of cultured GH secreting tumors with cycloheximide during 5 days blocked both basal and TRH stimulated GH secretion, thus indicating dependency on protein synthesis during the chronic, secondary phase. Since the acute secretion was not affected by coadministration of cycloheximide, these early increases in hormone levels apparently reflect the release of stored hormone. In summary, GH secreting adenomas and NFPT differ significantly in their hormonal response to continuous exposure to TRH. The mechanisms underlying the sustained effect of TRH on GH secretion in vitro remain to be investigated. If endogenous TRH exerts a similar continuous effect it may contribute to the disregulated GH secretion in acromegaly.

OBJECTIVE Clinically nonfunctioning pituitary adenomas (NFA) are mostly of gonadotroph origin. However, increased levels of circulating hormones or subunits in patients with NFA usually do not cause clinical symptoms, nor are they used as biological tumour markers. In this study we assessed the value of measuring β subunits of gonadotrophin hormones in the post-operative follow-up of patients bearing these tumours. DESIGN Patients harbouring NFA were studied before and three months after transphenoidal pituitary surgery. β-LH and β-FSH levels were measured before and following TRH administration on the two occasions. Hormone levels were analyzed in relation to imaging studies performed before and after surgery. PATIENTS Twenty four patients operated at the Tel Aviv-Sourasky Medical Centre for NFA. RESULTS Pathological β-FSH and β-LH levels were detected in 79% and 60% of patients respectively. β-LH levels decreased after surgery but there were no significant changes in β-FSH levels. There was a tendency for tumours with high basal β-LH levels to be larger and to have a poor surgical outcome. Normalization of β-LH levels post-operatively was usually associated with a decrease in tumour mass or complete removal of the tumour. Persistent pathological responses of β-LH to TRH after surgery were common in patients with residual tumours on imaging. Nevertheless there were exceptions to this pattern, rendering post-operative β-LH levels insufficiently reliable as a marker for the presence of residual tumour. CONCLUSION Although there appears to be a relationship between β-LH levels, tumour size and surgical outcome, this association is presently insufficient to allow the routine use of either basal or TRH induced β-LH responses in the post-surgical follow-up of clinically nonfunctioning pituitary adenomas.

We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3α-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 μL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 μL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 μL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 μL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5–8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.

The expression of estrogen receptor (ER) in thymocytes was studied in young, middle-aged, and old (2, 12, and 24 months, respectively) female and male C57BL/6J mice. Western immunoblots prepared from the thymocytes of females of all age groups showed the presence of a 67-kD protein band, which has been associated with the apparent MW of denatured ER. Flow cytometry analysis o,f cells stained with a monoclonal anti-ER antibody (clone 13H2) disclosed ER expression in both females and males of all age groups.In vivotreatment with estradiol (E2) led to an increase in the specific activity of thymic creatine kinase (CK) in the female mice, whereas the male thymocytes responded with an increase in CK activity only on treatment with dihydrotestosterone (DHT). The data show no differences in ER expression between male and females, but the receptor appears not to be functional in males. Interestingly, when estradiol was applied to co-cultures of lymphoid-depleted fetal thymus (FT) explants and bone-marrow cells, or thymocytes, from young and old females, it resulted in increased cellularity of cultures containing cells of the young, and not those of the old. The proportion of CD4/CD8 phenotypes of the developing cells in these cultures was not affected by E2treatment. These observations provide a new insight into ER expression and function in T-cell development in relation to gender and age.

We have demonstrated previously that rat adipose tissue showed sex and depot-specific responses to gonadal steroids. The epididymal fat pad in males responded exclusively to androgens by increased specific activity of the brain type isozyme of creatine kinase (CK). In females, the parametrial adipose tissue responded exclusively to estrogens. The present study was undertaken to follow the responsiveness to steroid hormones, and the presence of estrogen receptors (ER), in 3T3L1 cells during their differentiation from pre-adipocytes to adipocytes. In pre-adipocytes in which the basal CK specific activity is low, there was no CK response to 17β estradiol (E 2 ) or dihydrotestosterone (DHT). Differentiation of the cells into adipocytes was accompanied by increased basal CK activity which was stimulated by E 2 , but not by DHT. Responsiveness to E 2 began 5 days after switching pre-adipocytes to differentiation medium. Upon differentiation, ER became demonstrable in the cell nuclei by staining with FITC labeled anti-idiotypic antibody (clone 1D5) directed against the steroid binding domain of ER. The response to E 2 was time-dependent and blocked completely by cycloheximide or actinomycin D. 1D5 itself, which has an estrogen mimetic effect, stimulated CK activity in the cells similarly to E 2 . The antiestrogen tamoxifen which also stimulated CK activity in the adipocytes, completely blocked E 2 action. The ‘pure’ antagonist of E 2 , ICI 164,384 and the tissue-selective antiestrogens, raloxifene or tamoxifen methiodide were also complete antagonists with no agonistic effects. The response of the 3T3L1 adipocytes to E 2 was upregulated by 1,25(OH) 2 D 3 . Moreover, IGF1 was also a potent stimulator of CK in these cells, and therefore may mediate partially the stimulation by E 2 . Transient transfection of the pre-adipocytes with ER permitted E 2 induction of CK. Thus, the appearance of ER and concomitant responsiveness to E 2 is another hormone-related change occurring in 3T3L1 cells during differentiation, in addition to changes such as development of insulin responsiveness. The interactions in this system provide a useful in vitro model for investigating the development of responsiveness to E 2 .

CGβ subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHβ gene into the reading frame. Although CTP-like sequences are encrypted in the LHβ genes of several mammals, the CGβ subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHβ gene (boCTP) possesses key characteristics of the human (h) CGβ-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGβ-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) significantly increased the specific activity of creatine kinase (CK) activity in the rat uterus, and in vitro in skeletal cells capable of responding to oestradiol (E2), suggesting that the antibody has oestrogenic-like activity. Moreover, the F(ab′)2 dimer of clone 1D5 acted like an antagonist and completely inhibited the increase in CK specific activity by either E2 or clone 1D5 in these skeletal cells. In the present study, we examined the in vivo effects of clone 1D5 and its proteolytic fragment, the F(ab′)2 dimer, E2 and dihydrotestosterone (DHT) on CK specific activity in the epiphyseal cartilage, diaphyseal bone, uterus, prostate, thymus and pituitary of immature or gonadectomized female and male rat animal models. In the intact immature animals, clone 1D5 caused an increase in CK in all organs of the female except in the pituitary. In the diaphyseal bone and prostate of male rats there was no stimulation by 1D5. The CK response in the uterus, epiphysis, and diaphysis of immature female rats was dose-dependent and was blocked by either the anti-oestrogen tamoxifen or the F(ab′)2 dimer of clone 1D5. E2, DHT, as well as clone 1D5, stimulated CK specific activity in both the diaphysis and epiphysis of ovariectomized female and castrated male rats, whereas sex specificity in the CK response was observed also in the uterus and the prostate of gonadectomized animals. Collectively, these results suggest that, as in cell culture, an intact antibody is necessary for the observed stimulation of CK specific activity and the F(ab′)2 dimer can act as an antagonist. Furthermore, the observed biological effects of clone 1D5 which are absolutely parallel to E2, imply that the anti-idiotypic antibody is able to penetrate the cell and reach the nuclear oestrogen receptor and transduces a signal to the nucleus, by as yet uncharacterized mechanisms. Journal of Endocrinology (1996) 149, 305–312

Estradiol17β (E2) and the phytoestrogens genistein (G), and daidzein (D) increase creatine kinase (CK) specific activity in primary cell cultures of human female to a greater extent in cells from pre-menopausal than post-menopausal women. Pretreatment with the non-calcemic analog of Vitamin D, JK 1624 F2-2 (JKF), upregulated this estrogenic response at all ages. In contrast, biochainin A (BA) and quercertin (Qu) increased CK with no age dependence or modulation by JKF pretreatment. Both ERα and ERβ present in the cells were upregulated by pretreatment with JKF, as measured by Western blot analysis. Real time PCR showed no significant change in ERα mRNA but a marked decrease in ERβ mRNA in both age groups after JKF treatment. Cells from both age groups had surface binding sites for E2, shown by assays using cell impermeable Europium labeled ovalbumin-E2 conjugate (Eu-Ov-E2). Binding of [ 3 H ]- E 2 to intracellular E2 receptors (ERs) was similar in both age groups with differences in phytoestrogenic competition. JKF pretreatment increased nuclear but decreased membranal binding in both age groups. These results provide evidence for membranal, in addition to nuclear estrogen receptors which are differentially modulated by a Vitamin D analog.

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.

To use a direct immunofluorescence technique employing a fluorescein labeled anti-idiotypic antibody that recognizes the estrogen receptor (ER) order to assess the distribution of ER in the uteri of normal women throughout the normal menstrual cycle and of a woman exposed prenatally to diethylstilbestrol (DES).Included in the study were 25 women aged between 35 and 50 years and an amenorrheic patient diagnosed as "DES Syndrome".Localization of ER expression in frozen sections of uterine tissue was achieved by direct immunofluorescence using a fluorescein labeled anti-idiotypic antibody that interacts with ER.Analysis of the immunofluorescence staining indicated that in the normal human endometrium the intensity of ER staining varied according to the phase of the cycle as well as according to the cell type. On the other hand, endometrial ER evaluation of the patient with DES syndrome showed minimal expression of ER and after treatment with conjugated estrogens, endometrial biopsy revealed a significant increase in ER expression.These findings indicate that the fluorescein labeled anti-idiotypic antibody can be used to detect ER in normal and pathological human endometrium and to monitor changes in ER expression in the endometrium during hormonal therapy.

Estrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor β. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy.In vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and β (ERα and ERβ), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed.First, all cell types thus harvested and grown in culture expressed both ERα and ERβ, with a variably higher abundance of ERβ over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17β (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERβ agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (∼-30%).This study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.

Novel thiophilic ligands based on sulfone structures were synthesized for the fast high-performance liquid chromatographic (HPLC) purification of antibodies. In order to better characterize these new structures, affinity constants were measured as well as the influence of pH and salt on adsorption and elution. Efficient and fast purification of antibodies from ascites and serum was achieved. The purification of antibodies from cell culture supernatant proved difficult due the indicator pheno-red of the growth media. Two thiophilic structures turned out to be protein adsorbents, which adsorbed all proteins loaded and from which no protein could be eluted under normal conditions.

Estriol (E3) and progesterone (P) concentrations in saliva were determined by direct chemiluminescence immunoassays, using solid-phase monoclonal antibodies bound to the wells of microtitre plates, and isoluminol-labelled steroids conjugates. Saliva samples were obtained from women during pregnancy, from 34 to 1 weeks before delivery. The smoothed median and mean salivary E3:P ratios in normal pregnancy rose gradually from 0.45 at -34 weeks to 1.5 at -1 week. In twin pregnancy a similar rise was observed. However, in preterm delivery the smoothed mean ratio rose more slowly, from 0.6 at -30 weeks to 1.1 at -1 week. In pregnancy associated with intra-uterine growth retardation, the smoothed mean ratio did not reach a value of 1 towards the end of pregnancy. In all instances high and low E3:P ratios were observed shortly before spontaneous delivery. This raises the question of whether the E3:P ratio could be used as a predictor providing useful information in relation to the onset of labour.

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Human cultured female osteoblasts (Obs) respond age-dependently to estradiol-17β (E2) and to phytoestrogens by increased DNA synthesis (DNA) and creatine kinase specific activity (CK). ERα mRNA expression is higher than ERβ mRNA in Obs. Pre-menopausal Ob (prOb) reveals higher expression of ERα than post-menopausal Ob (poOb), but similar intracellular and membranal E2 bindings. ERα mRNA is stimulated in prOb and inhibited in poOb by different estrogens. ERβ mRNA in prOb is stimulated by 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4ʹ,4ʺ-[4-Propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) and raloxifene (Ral) and inhibited by genistein (G) and daidzein (D) while in poOb only E2 and DPN inhibited it. All phytoestrogenic carboxy- derivatives stimulated ERα mRNA in both Ob, while the protein bound- carboxy were ineffective. All compounds except carboxybiochainin A (cBA) had no effect on ERβ. There is higher expression of 1α 25 hydroxy- vitamin D (1OHase) mRNA in poOb, whereas 1,25(OH)2D3 (1,25) production is similar, but all compounds increased 1OHase mRNA and 1,25 to higher extent in prOb. Obs express 15 and 12 lipoxygenase (12LO and 15LO) mRNA and produce 12 and 15 HETE (12H and 15H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. Both LOs are increased to higher extent in poOb by all estrogens except Ral and PPT. 12H is increased by DPN, PPT and carboxy- derivatives similarly in both Obs, while 15H is increased by biochainin A (BA), Ral and carboxyderivatives. DNA synthesis and CK are stimulated by all compounds in both Obs, but to higher extent in prOb. The 1,25 less-calcemic analog JK 1624F2-2 (JKF) up-regulated DNA and CK response to all estrogens except BA and the carboxy- derivatives in both Obs. JKF stimulated intracellular binding of E2 similarly by all compounds except BA, but inhibited its membranal binding in both Obs. In conclusion Obs respond age-dependently to estrogens in a yet unknown mechanism and the beneficial outcome for human female bones is not yet clear.

In order to investigate the possible role of estrogen in the development of cartilage and bone we studied by immunofluorescence immunohistochemistry and autoradiography 26 human embryos and fetuses 7-22 weeks in gestational age associated with pregnancy interrupted for non-medical reasons. In order to demonstrate the presence of estrogen receptors (ERs) in human fetal cartilage, cryostat sections of long bones and lumbar and thoracic vertebrae were prepared for (1) fluorescent immunocytochemistry using an antiidiotypic monoclonal antibody to anti-estradiol receptor monoclonal Ab labeled with fluorescein isothiocyanate (FITC), (2) immunohistochemistry using monoclonal antihuman estradiol receptor antibody, labeled with strept. A-B immunoperoxidase, and (3) autoradiographic localization of estradiol using labeled (3H) 17 beta estradiol. In fetuses aged 10 weeks or older, intranuclear and perinuclear localization of ER was demonstrated by all methods, mainly amongst chondrocytes of the proliferating and higher hypertrophic zones of the epiphyses and in the cartilage of vertebral bodies. These data suggest that estrogen acts directly on chondrocytes of human fetuses through an ER-mediated mechanism.

The binding of estradiol labelled with 3 H or with external labels (isoluminol and horseradish peroxidase) to anti-estradiol—6-carboxymethyloxime—bovine serum albumin antibodies was compared. The external labels were coupled covalently to the steroid via homologous 6-carboxymethyloxime or heterologous 3- and 17-hemisuccinate bridges. Different factors and conditions influencing this binding and the slope and shape of the calibration graph were studied, including the type of label, the liquid- and solid-phase antibody system, the matrix effect and general incubation conditions, e.g., time and temperature. External homologous labels yielded the most sensitive calibration graphs and the lowest detection limits. Large differences in the binding of the label and in the shape of the calibration graph between the different antibodies were observed.

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.

Background: We demonstrated previously that phytoestrogens and vitamin D analogs like estradiol-17β (E2) modulate bone morphology in rat female model. Aim: We now analyze the effects of phytoestrogens, E2, selective E2 re ceptor modulators, and the less-calcemic analogs of vitamin D: JKF1624F2-2 (JKF) or QW1624F2-2 (QW) on fat content in bone marrow (BM) from long bones in ovariectomized female rats (OVX). Materials and methods: OVX rats were injected with treatments known to affect bone formation, 5 days per week for 2.5 month for analysis of fat content in BM. Results: In OVX young adults there is a decreased bone formation and a 10-fold increase in fat cells content in BM. Treatment with E2, raloxifene (Ral) or DT56a resulted in almost completely abolishment of fat cells content. Daidzein (D) decreased fat cells content by 80%, genistein (G) or biochainin A (BA) did not change fat cells content and carboxy BA (cBA) had a small but significant effect. JKF or QW did not affect fat cells content, whereas combined treatment of JKF or QW with E2 resulted in complete abolishment of fat cells content. These changes in fat cells content are inversely correlated with changes in bone formation. Conclusions: Our results demonstrate that adipogenesis induced by OVX is a reversible process which can be corrected by hormonal treatments. The awareness of a relationship between fat and bone at the marrow level might provide a better understanding of the pathophysiology of bone loss as well as a novel approach to diagnosis and treatment of postmenopausal osteoporosis.

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) βmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17β (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERβ-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

The ability of a monoclonal antiidiotypic antibody (clone 1D5) directed against the binding site of a monoclonal antiestradiol antibody to interact with the estrogen receptor (ER) was investigated. The following lines of evidence indicate that clone 1D5 has the capacity of mimicking the actions of estradiol, and recognizes ER: 1) in binding experiments, clone 1D5 inhibited the binding of [3H]estradiol to porcine cytosolic 32-kilodalton ER fragment in a dose-dependent manner; irrelevant antibody had no effect; 2) in sucrose gradient density analysis, clone 1D5 abolished the specific peak of the [3H] estradiol-ER complex in the 4S region; 3) in immunoprecipitation experiments, clone 1D5 interacted with unoccupied ER, but not with estradiol-occupied ER; 4) in direct immunofluorescence studies clone 1D5 stained the nuclei of cultured rat epithelial cells and recognized estrogen binding sites in nuclear cryostat sections prepared from human, rat, and mouse estrogen-responsive tissues; and 5) When clone 1D5 was injected to immature female rats, it caused 46% increase in uterine creatine kinase activity, suggesting that clone 1D5 may possess estrogenic like activity. Under the same experimental conditions, estradiol caused 58% increase in creatine kinase activity. Collectively, these results suggest that clone 1D5 interacts with the steroid binding site of ER. Therefore, clone 1D5 can serve as a tool in the study of function and structure relationship of ER and to detect changes of ER levels in target cells of various species.