Interaction of apolipoproteins A-I and E in the regulation of DNA, RNA, and protein biosynthesis in cultured rat hepatocytes.

Experiments on hepatocyte culture showed that apolipoprotein A-I-tetrahydrocortisol complex increases the rate of DNA, RNA, and protein biosynthesis measured by radioactive label incorporation. Apolipoprotein E acted as competitor of the apolipoprotein A-I-tetrahydrocortisol complex and abolished biological activity of the latter. We hypothesize that this mechanism of regulation plays an important role in processes of intracellular regeneration and proliferation.

[Features of interaction of complexes cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I with eukariotic DNA].

On primary culture of hepatocytes it is shown, that a complex cortisol-apolipoprotein A-I did not change rate of biosynthesis DNA and protein, whereas the complex tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) essentially raised rate of incorporation 3H-thymidine in DNA and 14C-leucine into protein. By a method of small-angle X-ray scattering it is shown, that appreciable interaction with eukariotic DNA is marked only in case of use of a complex THC-apoA-I, thus there is local fusion of DNA. The most probable region of interaction of the given complex with DNA is repetition (GCC)n the type, included in structure of many genes eukariot, including the human. It is synthesized oligonucleotid (duplex) of this type. It is shown, that at his interaction with complex THC-apoA-I there is a formation of more difficult complex, which breaks up with formation of complementary chains of oligonucleotides. The last also enter interaction with complex THC-apoA-I. It is given of kinetic this multiphasic process. Interaction of a complex cortisol-anoA-I with a duplex is less specific and does not result reduce in decay of the duplex and in formation of complementary oligonucleotides.

Tetrahydrocortisol-apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes.

Tetrahydrocortisol stimulates DNA and protein biosynthesis in hepatocytes only when it enters the complex with apolipoprotein A-I. Tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) complex specifically interacts with eukaryotic DNA isolated from rat liver. In the process of interaction, rupture of hydrogen bonds between the pairs of nitrous bases occurs with the formation of single-stranded DNA structures. In such state DNA forms complexes with DNA-dependent RNA-polymerase. The most probable site of binding the tetrahydrocortisol-apolipoprotein A-I complex with DNA is the sequence of CC(GCC)(n) type entering the structure of many genes, among them the structure of human apolipoprotein A-I gene. Oligonucleotide of this type has been synthesized. Association constant (K(ass)) of it with tetrahydrocortisol-apolipoprotein A-I complex was shown to be 1.66 x 10(6)M(-1). Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K(ass). It was assumed that in the GC-pairs of the given sequence tetrahydrocortisol itself participates in the formation of hydrogen bonds with cytosine, favoring their rupture with complementary base-guanine.

Effect of tetrahydrocortisol-apolipoprotein A-I complex on the secondary structure of eukaryotic DNA and its interaction with RNA-polymerase.

The in vitro effect of tetrahydrocortisol-apolipoprotein A-I complex on native adult rat liver DNA results in the formation of S1 nuclease sensitive fragments that are irregularly distributed throughout a genome. Low-angle X-ray scattering showed that after the interaction with the tetrahydrocortisol-apolipoprotein A-I complex, DNA can bind to RNA-polymerase with a high and dose-dependent cooperativity. This indicates that the effect of tetrahydrocortisol-apolipoprotein A-I complex on secondary eukaryotic DNA structure causes a local denaturation of the double helix, promoting high cooperativity of binding to RNA-polymerase. The reduced form of the hormone, tetrahydrocortisol, previously considered as an inactive metabolite, when complexed with apolipoprotein A-I, promotes a biological function similar to that of a transcription factor.

3Alpha,5beta-tetrahydrocortisol effect on outflow facility.

3Alpha,5beta-Tetrahydrocortisol (THF) was administered topically and intracamerally to ocular normotensive cynomolgus monkeys to determine whether it affects outflow facility. Monkeys received THF either topically at a dose of 2 x 5 microl drops of 300 microg/10 microl twice daily for 4 days (n = 4) or 3 times daily for 10 days (n = 4) with 10% DMSO as vehicle to the control eye, or intracamerally via 2 ml anterior chamber (AC) exchange of 30 microg/ml THF with vehicle, 0.1% DMSO, to the control eye followed by a second AC exchange using 300 microg/ml THF with vehicle to the control eye. Outflow facility was measured by a two-level constant pressure AC perfusion after administration of eye drops or after baseline outflow facility measurement and AC exchange with THF solution. The results showed no effect on outflow facility in normotensive cynomolgus monkeys.

[Interaction of tetrahydrocortisol-apolipoprotein A-I complex with eukaryotic DNA and with single-stranded oligonucleotides]. / Vzaimodeistvie kompleksa tetragidrokortizol-apolipoprotein A-I c éukarioticheskoi DNK i odnotsepochechnymi oligonukleotidami.

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC-ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (Ka) was 1.66 x 10(6) M-1. Substitution of THC with cortysol considerably decreases the Ka. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.

[Effect of tetrahydrocortisol-apolipoprotein A-I complex on RNA polymerase interaction with eukaryotic DNA and the rate of protein biosynthesis in hepatocytes]. / Vliianie kompleksa tetragidrokortizol-apolipoprotein A-I na vzaimodeistvie RNK polimerazy s éukarioticheskoi DNK i na skorost' biosinteza belka v gepatotsitakh.

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lypoproteins, cortisol, and lypopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) complex in macrophages, which display 5 alpha- and 5 beta-reductase activity and are constituents of nonparenchymal liver hepatocytes. Using the small-angle X-ray scattering technique, it was shown that the THC-apoA-I-eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.

Progestins suppress estrogen-induced expression of vascular endothelial growth factor (VEGF) subtypes in uterine endometrial cancer cells.

Vascular endothelial growth factor (VEGF) contributes to the early advancement of uterine endometrial cancers that conserve hormone dependency via angiogenic activity. This process prompted us to study sex steroidal suppression of VEGF expression in Ishikawa cells (a line of well-differentiated uterine endometrial cancer cells). Estrogen transiently induced VEGF subtype (VEGF165 and VEGF121) secretion from Ishikawa cells. Progestins (progesterone, medroxyprogesterone acetate (MPA) and 17 alpha-hydroxyprogesterone) suppressed the estrogen-induced events. In conclusion, progestins could suppress VEGF-related angiogenic potential, which contributes to tumor growth in the early stage of uterine endometrial cancers that conserve estrogen dependency.

The embryoid body as a novel in vitro assay system for antiangiogenic agents.

Tumor progression necessitates the induction of blood vessels that converge upon the tumor and enhance the diffusibility of oxygen and nutrients. Approaches to treat cancer by antiangiogenic therapy are therefore straightforward, and there is a great need for suitable in vitro systems to test antiangiogenic agents. In the present study, embryoid bodies (EBs) differentiated from totipotent mouse embryonic stem (ES) cells and cultivated using the spinner flask technique are introduced as an in vitro system for antiangiogenesis research. ES cells effectively differentiated endothelial cells within the three-dimensional tissue of EBs. The total area of capillary-like structures, which were positive for CD31 (platelet endothelial cell adhesion molecule, PECAM-1), was assessed by confocal laser scanning microscopy and image analysis of a series of optical sections. Endothelial differentiation occurred between Day 4-5 and Day 8 of EB development. Within 7 days, 100% of EBs contained capillary-like structures. Suramin, tamoxifen, tetrahydrocortisol, and a combination of tetrahydrocortisol and heparin were tested for their antiangiogenic capacity in the EB system and were found to efficiently inhibit endothelial differentiation. Diffusion studies of a 10-kd 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran and the fluorescent, amphiphilic agent doxorubicin in avascular and vascularized EBs revealed that the endothelial structures formed functional vessels that facilitated diffusion. The diffusion coefficient D for doxorubicin was 296 x 10(-9) cm2 s(-1) in vascularized 8-day-old EBs, ie, about 10-fold larger than in avascular 3-day-old EBs (18 x 10(-9) cm2 s(-1)) and EBs treated with suramin (14 x 10(-9) cm2 s(-1)), tamoxifen (13.5 x 10(-9) cm2 s(-1)), and tetrahydrocortisol/heparin (18.5 x 10(-9) cm2 s(-1)). Consequently, avascular EBs treated with antiangiogenic agents developed central necrosis, which was absent in vascularized EBs. Our findings indicate that EBs are a suitable in vitro model system to study the effects of antiangiogenic agents in a three-dimensional tissue context. Furthermore, EBs provide a unique model to investigate the diffusion of anticancer agents in a tissue in both the avascular and vascularized states.

Antiestrogenic compounds inhibit estrogen-induced expression of fibroblast growth factor family (FGF-1, 2, and 4) mRNA in well-differentiated endometrial cancer cells.

To clarify the effect of sex steroids on neovascularization in the growth, invasion and metastasis of endometrial cancer, the regulations of acid fibroblast growth factor (FGF-1), basic FGF (FGF-2) and hst-1 (FGF-4) mRNA expressions were studied in well-differentiated endometrial cancer cells under the influence of sex steroids. The levels of FGF-1 and 2 mRNAs in the well-differentiated endometrial cancer (Ishikawa) cells were significantly increased by estradiol. This increase was significantly inhibited by progestins (progesterone, medroxyprogesterone acetate [MPA] and 17 alpha-hydroxyprogesterone) and tamoxifen, but not by tetrahydrocortisol, hydrocortisone and danazol. The expression of FGF-4 mRNA was not altered by sex steroids. Therefore, estrogen might stimulate FGF-2 with FGF-1 secretion of endometrial cancer cells for neovascularization, and antiestrogenic compounds do inhibit estrogen-induced events.

Sex steroids regulate the expression of plasminogen activator inhibitor-1 and its mRNA in fibroblasts derived from uterine endometrium.

In order to clarify a role of stromal cells in sex steroidal neovascularization, plasminogen activator inhibitor (PAI)-1 [an inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)] and its messenger ribonucleic acid (mRNA) were analysed in fibroblasts derived from uterine endometrium as a model for endometrial stromal cells under the influence of sex steroids. The determinations were carried out by an enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction-Southern blotting, respectively. In the fibroblasts, either estradiol or progestogens (progesterone, medroxy progesterone acetate or 17 alpha-hydroxyprogesterone) induced expressions of PAI-1 and its mRNA, and their combination further increased their expression by approximately twofold. PAI-1 from endometrial stromal cells under the influence of sex steroids might contribute to endometrial neovascularization through its effect on endothelial cells in endometrial vessels.

Loss and restoration of immune privilege in eyes with corneal neovascularization.

PURPOSE: To delineate the time course for loss of immune privilege after induction of corneal neovascularization (NV), and to test whether treatment with angiostatic agents can restore the eye's capacity to induce anterior chamber-associated immune deviation (ACAID). METHODS: Corneal NV in murine eyes was induced by placement of intrastromal sutures. At different time points after NV induction, study eyes were initiated on a 10-day regimen of one of a variety of anti-inflammatory or angiostatic agents. After the completion of their treatment regimen, eyes were tested as to whether they could support ACAID. To test whether any observed effect on the delayed-type hypersensitivity response was because of a systemic absorption of the topically applied medication, certain animals had only their fellow eyes treated. RESULTS: Inflammatory corneal NV leads to the loss of immune privilege during the first week of the NV induction. Left untreated, these eyes remain incapable of supporting ACAID, even weeks after the initial corneal insult. However, when treatment with an anti-inflammatory agent is initiated during the first 2 weeks after the NV induction, these eyes show a restored capacity for ACAID induction, and this appears to be unrelated to any systemic effect of the treatment regimen. Treatment started at later time points is not capable of restoring the eye's normal capacity for inducing deviant immunity. CONCLUSIONS: Corneal neovascularization leads to loss of immune privilege in the anterior segment manifested as the inability to sustain ACAID. Moreover, topical angiostatic strategies can lead to restoration of immune privilege when instituted sufficiently early in the course of the neovascular response.

Inhibition of dexamethasone-induced cytoskeletal changes in cultured human trabecular meshwork cells by tetrahydrocortisol.

PURPOSE: To determine the cellular mechanism of action of the intraocular pressure (IOP) lowering steroid tetrahydrocortisol (THF). METHODS: Tetrahydrocortisol was evaluated for glucocorticoid antagonist activity using in vitro and in vivo assays. Systemically administered THF was evaluated for its ability to inhibit dexamethasone-induced body weight loss and systemic hypertension in rats. In vitro receptor antagonism was tested using the supernatant fraction of IM9 cells as the source of soluble glucocorticoid receptor in 3H-dexamethasone displacement binding assays. In addition, six different primary human trabecular meshwork (TM) cell lines were cultured for 0 to 14 days in the absence or presence of dexamethasone (10(-7) M) and/or THF (10(-6) to 10(-8) M). The effects of these steroids on the TM cytoskeleton were determined by epifluorescent microscopy and by transmission electron microscopy. RESULTS: Tetrahydrocortisol was unable to inhibit the dexamethasone (DEX)-induced systemic hypertension and decrease in body mass in rats and was unable to displace 3H-DEX from the soluble human glucocorticoid receptor. However, THF inhibited the DEX-induced formation of cross-linked actin networks in cultured human TM cells in a progressive and dose-dependent manner (IC50 = 5.7 x 10(-7) M). Dexamethasone caused changes in the TM cell microtubules that were reversed partially by concomitant treatment with THF. Tetrahydrocortisol alone appeared to increase microfilament bundling in TM cells. CONCLUSIONS: Tetrahydrocortisol was not a glucocorticoid antagonist at the level of the classical glucocorticoid receptor and did not appear to antagonize systemically mediated glucocorticoid activity in the rat. Tetrahydrocortisol inhibited DEX-induced changes in the TM microfilaments and microtubules. These results may explain partially the IOP lowering activity of THF because glucocorticoid-mediated changes in the TM cytoskeleton have been proposed to be involved in the generation of ocular hypertension.

beta-cyclodextrin tetradecasulfate/tetrahydrocortisol +/- minocycline as modulators of cancer therapies in vitro and in vivo against primary and metastatic Lewis lung carcinoma.

Tetrahydrocortisol, beta-cyclodextrin tetradecasulfate, and minocycline used alone or in combination are not very cytotoxic toward EMT-6 mouse mammary tumor cells growing in monolayer. Tetrahydrocortisol (100 microM, 24 h) and beta-cyclodextrin tetradecasulfate (100 microM, 24 h) protected EMT-6 cells from the cytotoxicity of CDDP, melphalan, 4-hydroperoxycyclophosphamide, BCNU, and X-rays under various conditions of oxygenation and pH. Minocycline (100 microM, 24 h) either had no effect upon or was additive with the antitumor alkylating agents or X-rays in cytotoxic activity toward the EMT-6 cells in culture. The combination of the three modulators either had no effect upon or was to a small degree protective against the cytotoxicity of the antitumor alkylating agents or X-rays. The Lewis lung carcinoma was chosen for primary tumor growth-delay studies and tumor lung-metastases studied. Tetrahydrocortisol and beta-cyclodextrin tetradecasulfate were given in a 1:1 molar ratio by continuous infusion over 14 days, and minocycline was given i.p. over 14 days, from day 4 to day 18 post tumor implantation. The combination of tetrahydrocortisol/beta-cyclodextrin tetradecasulfate diminished the tumor growth delay induced by CDDP and melphalan and produced modest increases in the tumor growth delay produced by cyclophosphamide and radiation. Minocycline co-treatment increased the tumor growth delay produced by CDDP, melphalan, radiation, bleomycin, and, especially cyclophosphamide, where 4 of 12 animals receiving minocycline (14 x 5 mg/kg, days 4-18) and cyclophosphamide (3 x 150 mg/kg, days 7, 9, 11) were long-term survivors. The 3 modulators given in combination produced further increases in tumor growth delay with all of the cytotoxic therapies, and 5 of 12 of the animals treated with the 3-modulator combination and cyclophosphamide were long-term survivors. Although neither tetrahydrocortisol/beta-cyclodextrin tetradecasulfate, minocycline, nor the three modulator combination impacted the number of lung metastases, there was a decrease in the number of large lung metastases. Treatment with the cytotoxic therapies alone reduced the number of lung metastases. Addition of the modulators to treatment with the cytotoxic therapies resulted in a further reduction in the number of lung metastases. These results indicate that agents that inhibit the breakdown of the extracellular matrix can be useful additions to the treatment of solid tumors.

Antiangiogenic agents potentiate cytotoxic cancer therapies against primary and metastatic disease.

The formation of a blood supply (angiogenesis) is critical to the growth of solid tumors. The naturally occurring steroid tetrahydrocortisol, the synthetic cyclodextrin derivative beta-cyclodextrin tetradecasulfate, and the tetracycline derivative minocycline have antiangiogenic activity. Tetrahydrocortisol and beta-cyclodextrin tetradecasulfate in a 1:1 molar ratio by continuous infusion over 14 days and minocycline administered i.p. over 14 days from day 4 to day 18 postimplantation of the Lewis lung carcinoma significantly increased the growth delay of the primary tumor after treatment with cis-diamminedichloroplatinum(II), melphalan, cyclophosphamide, Adriamycin, bleomycin, and radiation therapy administered in standard regimens. Addition of the antiangiogenic agents to treatment with the cytotoxic therapies not only reduced the number of lung metastases formed from the primary tumor but also reduced the number of large metastases. Five of 12 animals treated with the antiangiogenic modulators and cyclophosphamide were long-term survivors (> 120 days). Thus, antiangiogenic therapies can potentiate the efficacy of standard anticancer therapies.

A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1.

Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.

Intraocular hypotensive effect of a topically applied cortisol metabolite: 3 alpha, 5 beta-tetrahydrocortisol.

3 alpha, 5 beta-tetrahydrocortisol, previously considered an inactive metabolite of cortisol, was found to lower significantly the intraocular pressure (IOP) of rabbits made ocular hypertensive with dexamethasone alone or with threshold levels of dexamethasone plus 5 beta-dihydrocortisol. The ocular hypotensive effect appeared within 3-7 days after the metabolite was started and persisted through the duration of the experiments. The metabolite did not lower the IOP of ocular normotensive untreated animals. Thus, 3 alpha,5 beta-tetrahydrocortisol is a naturally occurring steroid antagonist, which may be of use in the treatment of primary open-angle glaucoma.